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VETERINARY CLINICAL PATHOLOGY

VETS 3243
Lecture Notes, Case Reports, Practical Notes

UOS Coordinator Dr Rachael Gray

Professor Paul J Canfield Revised in 2010 by Rachael Gray 2010

PREFACE
The discipline of Veterinary Clinical Pathology (Laboratory Medicine) focuses on the laboratory investigation of disease for purposes of diagnosis and prognosis. Whilst Veterinary Clinical Pathology is taught as an independent unit of study, it is important to realise its connection to other disciplines and units of study for purposes of disease investigation. Consequently, this unit of study builds on Veterinary Pathology, Veterinary Microbiology, Veterinary Parasitology; integrates horizontally with Small Animal Medicine and Animal Disease taught within the same semester; and prepares students for both small animal practice and large animal health and production and clinical practice. Because of this, Veterinary Clinical Pathology should be studied in conjunction with these other disciplines for purposes of assessment. These notes are by no means a complete reference for clinical pathology. They are based on lectures and practical classes that are provided to Veterinary Science Students, and are meant to complement learning through formal interaction. They provide information not only on dogs and cats but also on horses and farm animals. Students will find the clinical pathology information on horses and farm animals useful when studying Equine Medicine, Ruminant Health and Production and Pig Health and Production. Moreover, since the notes provide a conceptual framework for the laboratory investigation of disease in all species, they have applicability to other species such as wildlife or laboratory animals. The lecture theory notes are supported by a series of case reports. An index is provided for both of these. There is no index for the practical notes, but a comprehensive table of contents exists. Undoubtedly there will be mistakes in the notes, and I would appreciate the reader's assistance in correcting the contents.

TABLE OF CONTENTS
LEARNING OUTCOMES (OBJECTIVES) FOR VETERINARY CLINICAL PATHOLOGY......................................................1 Introduction ........................................................................................................................................................................1 Generic Skills and Graduate Competences ....................................................................................................................1
1. Knowledge skills ......................................................................................................................................................................................1 2.Thinking skills ...........................................................................................................................................................................................1 3.Personal skills...........................................................................................................................................................................................1 4.Personal attributes....................................................................................................................................................................................1 5.Practical skills ...........................................................................................................................................................................................2

Further information on main learning outcomes..........................................................................................................................................3

What is Veterinary Clinical Pathology? ...........................................................................................................................2 Main Learning Outcomes ..................................................................................................................................................2

PRINCIPLES OF ASSESSMENT FOR VETERINARY CLINICAL PATHOLOGY ...................................................................5 Assessment Format...........................................................................................................................................................5 Summative assessment ........................................................................................................................................................5 Formative assessment..........................................................................................................................................................5 Grade Descriptors for Veterinary Clinical Pathology: ....................................................................................................5 Past Examination Question for Veterinary Clinical Pathology, with example of a model answer:............................8 Suggested Approach To Answering Examination Questions................................................................................................8 Model Answer for 2004 Examination Question ...................................................................................................................13 VETERINARY CLINICAL PATHOLOGY (LABORATORY MEDICINE).................................................................................17 Introduction ......................................................................................................................................................................17 Necropsy Technique and Collection of Specimens for Histopathology...................................................................................18 The Nature of Laboratory Tests, Problems for Interpretation and Reference Intervals............................................18 General Remarks on Sampling for Blood Biochemistry ..............................................................................................19 Specimen storage ...............................................................................................................................................................19 Haemolysis..........................................................................................................................................................................19 Gross Lipemia (and its relationship to laboratory detected hyperlipidemia)........................................................................20 Plasma/Serum Enzymology .............................................................................................................................................20 Factors affecting plasma levels of enzymes .......................................................................................................................20 Naming of enzymes ............................................................................................................................................................21 Combining enzymes to detect disease processes..............................................................................................................23 LABORATORY EVALUATION OF HEPATIC DISEASE........................................................................................................24 Terminology ......................................................................................................................................................................24 Introduction .......................................................................................................................................................................24 Summary of Tests............................................................................................................................................................25 Hepatocellular Damage (Degeneration and/or Necrosis) ...........................................................................................25 Cholestasis.......................................................................................................................................................................26 Bilirubin Metabolism and Analysis ................................................................................................................................27 Tests for Reduced Functional Hepatic Mass ................................................................................................................30 a) Bile acids........................................................................................................................................................................30 b) Blood ammonia determination .......................................................................................................................................30 c) Serum protein estimation ...............................................................................................................................................31 Other Laboratory Tests .........................................................................................................................................................32 A. Bromosulphthalein (BSP, sulfobromophthalein)........................................................................................................32 B. Cholesterol ................................................................................................................................................................32 C. Glucose .....................................................................................................................................................................32

LABORATORY EVALUATION OF URINARY TRACT DISEASE..........................................................................................33 Terminology .....................................................................................................................................................................33 Introduction ......................................................................................................................................................................33 Some of the Laboratory Tests used to Detect Renal Dysfunction..............................................................................34 Assessment of glomerular function .....................................................................................................................................34 Assessment of tubular function ...........................................................................................................................................36 Miscellaneous Biochemical Alterations that may occur in Various Forms of Renal Disease ..............................43

LABORATORY INVESTIGATION OF SOME DIGESTIVE TRACT DISORDERS ................................................................45 Terminology .....................................................................................................................................................................45 Exocrine Pancreatic Disorders.......................................................................................................................................45 Investigation of Colic in the Horse.................................................................................................................................46 Investigation of Problem Diarrheas ...............................................................................................................................47 1. Faecal examination (coprology) ................................................................................................................................47 2. Peritoneal fluid analysis - abdominocentesis (see under fluid analysis) ...................................................................48 3. Absorption tests.........................................................................................................................................................49 4. General haematological and biochemical testing......................................................................................................50 5. Endoscopy, exploratory laparotomy, intestinal washings and biopsy........................................................................50 Additional Notes on Malassimilation .............................................................................................................................51 LABORATORY INVESTIGATION OF ENDOCRINE DISORDERS, INCL. CALCIUM & PHOSPHATE DERANGEMENTS 52 Introduction ......................................................................................................................................................................52 Diabetes mellitus (DM) ....................................................................................................................................................52 Hyperinsulinism ...............................................................................................................................................................54 Hyperadrenocorticism.....................................................................................................................................................54 Hypoadrenocorticism (Adrenal Insufficiency - AI) .......................................................................................................55 Hypothyroidism................................................................................................................................................................56 Hyperthyroidism ..............................................................................................................................................................56 Introduction to Calcium and Phosphate Derangements..............................................................................................57 Persistent Hypocalcemia (decreased calcium on two or more occasions) ...............................................................57 Persistent Hypercalcemia (elevated calcium on two or more occasions) .................................................................58 Derangements of magnesium.........................................................................................................................................59 Hypermagnesemia ...........................................................................................................................................................59 Hypomagnesemia ............................................................................................................................................................59 WATER, ELECTROLYTES AND ACID/BASE BALANCE.....................................................................................................60 Introduction ......................................................................................................................................................................60 Hydration Status (Assessment of Total Body Water)...................................................................................................60 Electrolyte Status.............................................................................................................................................................60 Acid/Base Balance............................................................................................................................................................62 HAEMATOLOGY.....................................................................................................................................................................63 Haematological Disturbances and how they reflect Host-Pathogen-Environmental Factor Interactions...............63 Introduction to Haematological Investigation...............................................................................................................66 Some Common Haematological Terms .........................................................................................................................67 DISORDERS/RESPONSES OF THE ERYTHRON.................................................................................................................72 a) Dynamics of erythrocyte production..........................................................................................................................72 b) Anaemia .......................................................................................................................................................................73

i) Laboratory confirmation of anaemia..........................................................................................................................73 ii) Classifying the anaemia on the basis of mechanisms:..............................................................................................73 Some General Comments on Bone Marrow Examination............................................................................................77 Polycythemia....................................................................................................................................................................79 DISORDERS/RESPONSES OF THE LEUKOCYTES ............................................................................................................80 Introduction ......................................................................................................................................................................80 Leukocytosis ....................................................................................................................................................................80 Neutrophilia .....................................................................................................................................................................81 Physiological neutrophilia (leukocytosis).............................................................................................................................81 Corticosteroid induced neutrophilia (leukocytosis)..............................................................................................................82 Neutrophilia due to regenerative anaemia ..........................................................................................................................82 Neutrophilia related to inflammatory demand .....................................................................................................................82 Neutropenia ......................................................................................................................................................................85 Monocytes ........................................................................................................................................................................86 Eosinophils.......................................................................................................................................................................86 Basophils..........................................................................................................................................................................86 Lymphocytes....................................................................................................................................................................87 HAEMATOPOIETIC NEOPLASIA ..........................................................................................................................................88 Introduction ......................................................................................................................................................................88 Lymphoproliferative Disorders.......................................................................................................................................88 a) Lymphosarcoma (malignant lymphoma)........................................................................................................................88 b) Acute lymphoblastic leukemia (ALL)..............................................................................................................................89 c) Chronic lymphocytic leukemia (CLL)..............................................................................................................................89 d) Plasma cell myeloma (multiple myeloma)......................................................................................................................90 Myeloproliferative Disorders ..........................................................................................................................................90 BLEEDING DISORDERS (ABNORMALITIES OF HAEMOSTASIS) .....................................................................................93 Introduction to Haemostatis ...........................................................................................................................................93 Introduction to Bleeding Disorders................................................................................................................................94 Laboratory Evaluation of Bleeding Disorders...............................................................................................................94 a) Vessel wall defect ..........................................................................................................................................................95 b) Thrombocytopenia .........................................................................................................................................................95 c) Platelet function defect...................................................................................................................................................95 d) Clotting factor deficiency................................................................................................................................................96 e) Excessive fibrinolysis.....................................................................................................................................................97 Summary...........................................................................................................................................................................97 Laboratory results for some common bleeding disorders ...................................................................................................98 IMMUNODIAGNOSTICS (for diseases with an immune base immune-mediated diseases and immunodeficiency)...........................99 Introduction ......................................................................................................................................................................99 Autoimmunity.................................................................................................................................................................100 a) Coombs' test (direct)....................................................................................................................................................100 b) Rheumatoid factor (RF) ...............................................................................................................................................100 c) The antinuclear antibody test (ANA) ............................................................................................................................101 d) Direct and indirect immunofluorescence (IF) of tissue biopsies...................................................................................101 Immunodeficiency .........................................................................................................................................................101 DIAGNOSTIC CYTOLOGY AND BODY FLUID ANALYSIS ................................................................................................102 Diagnostic Cytology (Exfoliative Cytology; Cytopathology) .....................................................................................102

a) Solid tissue cytology ....................................................................................................................................................102 b) Diagnostic cytology as a component of body fluid analysis ..........................................................................................107 Body FLuid Analysis .....................................................................................................................................................107 a) Body cavity effusions ...................................................................................................................................................107 b) Synovial fluid analysis..................................................................................................................................................111 c) Cerebrospinal fluid evaluation......................................................................................................................................113 d) Analysis of airway and pulmonary lesions by respiratory washes (transtracheal aspirates, bronchoalveolar lavages) ...114 CASE REPORTS........................... 119 Introduction ............................ 119 CASE: 1......................... 123 CASE: 2......................... 125 CASE: 3......................... 127 CASE: 4......................... 129 CASE: 5......................... 132 CASE: 6......................... 134 CASE: 7......................... 136 CASE: 8......................... 138 CASE: 9......................... 140 CASE: 10....................... 142 CASE: 11....................... 144 CASE: 12....................... 146 CASE: 13....................... 148 CASE: 14....................... 150 CASE: 15....................... 152 CASE: 16....................... 154 CASE: 17....................... 156 CASE: 18....................... 158 CASE: 19....................... 160 CASE: 20....................... 162 CASE: 21....................... 164 CASE: 22....................... 167 CASE: 23....................... 169 CASE: 24....................... 171 CASE: 25....................... 173 CASE: 26....................... 175 CASE: 27....................... 177 CASE: 28....................... 179 CASE: 29....................... 181 CASE: 30....................... 183 CASE: 31....................... 185 CASE: 32....................... 187 CASE: 33....................... 189 CASE: 34....................... 191 CASE: 35....................... 193 INDEX ............................ 195

LECTURE NOTES

LEARNING OUTCOMES (OBJECTIVES) FOR VETERINARY CLINICAL PATHOLOGY

Introduction
The purpose of this section is to provide students with an idea of the capabilities they are expected to develop during, and the abilities required of them at the end of, VETS 3243 (Veterinary clinical Pathology). In plain English, this section focuses the student on what is important in Clinical Pathology and how this can be applied to the processes of diagnosis and treatment of disease that are essential for practising veterinarians.

Generic Skills and Graduate Competences

Before presenting learning outcomes directly linked to Veterinary Clinical Pathology, it should be mentioned that students are expected to develop certain generic skills during the unit of study. These generic skills are University of Sydney Policy (Academic Board, 1997). These generic skills will come into play during all teaching strategies employed in Veterinary Clinical Pathology and, indeed, are applicable to all units of study in Veterinary Science and in the University. They include:

1. Knowledge skills
Graduates should: a. have a body of knowledge in the field(s) studied; b. be able to apply theory to practice in familiar and unfamiliar situations; c. be able to identify, access, organize and communicate knowledge in both written and oral English.

2. Thinking skills
Graduates should: a. be able to exercise critical judgment; b. be capable of rigorous and independent thinking; c. be able to account for their decisions; d. be realistic self evaluators; e. adopt a problem solving approach.

3. Personal skills
Graduates should have: a. the capacity for and a commitment to life-long learning; b. the ability to plan and achieve goals in both the personal and the professional sphere; c. the ability to work with others.

4. Personal attributes
Graduates should: a. strive for tolerance and integrity; b. acknowledge their personal responsibility for their own value judgments; and their ethical behaviour towards others.

5.Practical skills
Graduates should a. be able to use information technology for professional and personal development; and, where appropriate, be able to: b. collect, correlate, display, analyse and report observations; c. apply experimentally-obtained results to new situations; d. test hypotheses experimentally; and e. apply technical skills appropriate to their discipline. These generic skills effectively apply to what is termed Day 1 Competences for Graduating Veterinarians. Day 1 competences are expected by the accrediting bodies of the Faculty of Veterinary Science (VSAAC; RCVS; AVMA) and include not only generic competences but also practical and knowledge competences. In terms of Veterinary Clinical Pathology, the RCVS has designated the following practical Day 1 competency: C1.6 Collect, preserve and transport samples, perform standard laboratory tests, and interpret the results of those generated in-house, as well as those generated by other laboratories (Commentary: new graduates are expected to have a working knowledge of tests to be undertaken include conditions relating to infectious & contagious diseases; alimentary system; respiratory system; circulatory system; urinary system; nervous system; endocrine system; mucucutaneous system; musculoskeletal system; trauma; poisoning; obstetrics; paediatrics; parturition; reproduction)

What is Veterinary Clinical Pathology?

Veterinary Clinical Pathology is also called Laboratory Medicine. This implies that it is a component of Veterinary Medicine. Veterinary Medicine encompasses the diagnosis and treatment of disease. Veterinary Clinical Pathology assists in the diagnostic process by providing laboratory information/evidence. This information or data is the result of biochemical, Haematological, cytological, pathological, microbiological and parasitological investigations. The sick animal is presented for assessment and the veterinary practitioner decides if the laboratory can provide information that will assist diagnosis. If this information is utilised/interpreted in a logical manner and with common sense, then the disease process (aetiology, host response and effect on host) can be better understood. Laboratory information may also be useful in monitoring an animals response to medical or surgical treatment of a disease. Since the course consists of lectures, tutorials and practical classes, there are several broad learning outcomes (or aims) for Veterinary Clinical Pathology. The first and second ones are most important and the written examination will be solely based on these. The third and fourth outcomes will provide the bulk of practical marks:

Main Learning Outcomes

1. At the end of this course of study, students will be expected to have the ability to identify alterations in laboratory data and to critically evaluate their significance for diagnosis and prognosis of disease in animals. 2. At the end of this course of study, students will be expected to have the capacity to think logically, critically and with common sense in relation to the role of Veterinary Clinical Pathology in Veterinary Medicine in providing assistance in diagnosis and prognosis of disease in animals.

3. At the end of the practical course, with respect to a range of simple laboratory tests commonly utilised in veterinary practice (in-house testing), students will be expected to: (a) understand the theory of operation for each test (b) have the ability to mechanically perform each test (c) be aware of the limitations and strengths of each test in order to know when to apply them for the purposes of diagnosis or prognosis of disease in animals. 4. At the end of the practical course, students will be expected to understand the workings of a pathology laboratory and have an awareness of the range of tests available to the veterinary practitioner.

Further information on main learning outcomes

1. At the end of this course of study, students will be expected to have the ability to identify alterations in laboratory data and to critically evaluate their significance for diagnosis and prognosis of disease in animals. 2. At the end of this course of study, students will be expected to have the capacity to think logically, critically and with common sense in relation to the role of Veterinary Clinical Pathology in Veterinary Medicine in providing assistance in diagnosis and prognosis of disease in animals. To achieve these linked learning outcomes, students will be exposed to animal case reports in lectures and tutorials and provided with a logical, stepwise approach to identifying and interpreting laboratory data alterations. Laboratory data include haematological values; biochemical values; urinalysis; faecal analysis; parasitological, microbiological and pathological results; fluid analysis; and cytological results. Students will not be expected to memorise information but will be expected to be able to retrieve that information efficiently from written sources and to apply it with common sense and understanding. Secondary or expanded learning outcomes of this process are: Students will develop a capacity to understand the limitations of reference intervals, and to utilise reference intervals to identify laboratory data alterations. Reference intervals are established for each laboratory test by collecting samples from clinically healthy animals. Students will be expected to critically evaluate laboratory data alterations in light of available clinical information. Clinical information includes history, clinical signs, the results of physical examination, and the results of non-laboratory diagnostic aids (e.g. image analysis) Students will be expected to reach a level of diagnosis from the available laboratory data and to be able to recognise the need for further clinical or laboratory testing in order to more specifically define the disease problem. 3. At the end of the practical course, with respect to a range of simple laboratory tests commonly utilised in veterinary practice (in-house testing), students will be expected to: (a) understand the theory of operation for each test (b) have the ability to mechanically perform each test (c) be aware of the limitations and strengths of each test in order to know when to apply them for the purposes of diagnosis or prognosis of disease in animals. Secondary or expanded learning outcomes of this process are: Students will be expected to revise the theory of operation behind simple laboratory tests which will be provided in the Veterinary Clinical Pathology Handbook and which have been provided in the practical components of earlier units of study (particularly Cell Biology, Animal Structure

and Function, Principles of Disease and Veterinary Pathology). Students will be expected to perform simple laboratory procedures relating to biochemistry (blood urea and glucose analysis), urinalysis, haematology (packed cell volume and total plasma protein determination, and differential leukocyte analysis on blood films), blood coagulation (fibrinogen determination and platelet assessment) and cytology (fine needle cell aspiration and preparation of smears). Students will be expected to be able to use a light microscope and to identify different blood cells, elements of urine and basic cell types in cytological smears. Students will be expected to understand the usefulness of these simple laboratory procedures in veterinary practice in relation to diagnosis and treatment of disease. Limitations and strengths of each test will be provided in the Veterinary Clinical Pathology Handbook or through written manufacturers information.

4. At the end of the practical course, students will be expected to understand the workings of a pathology laboratory and have an awareness of the range of tests available to the veterinary practitioner. Secondary or expanded learning outcomes of this process are: Students will be able to collect appropriate fluid and tissue samples for laboratory evaluation Students will be able to correctly label samples and fill out laboratory request sheets Students will be expected to be aware of the range of tests available from a commercial pathology laboratory. In particular, this refers to an awareness of the types of biochemical and Haematological tests available and how their application varies among animal species.

PRINCIPLES OF ASSESSMENT FOR VETERINARY CLINICAL PATHOLOGY

Assessment Format
Summative assessment
Intra-semester assessment consists of: 1. Three case report tutorials will each be marked out of 10% (then an average determined). The three practicals must be attended for students to be eligible for the full 10%. 2. Intra-semester examination of one case report in Week 8 - Monday 13th September This written assessment contributes 15% of the marks for this unit of study. It will be 45 minutes in duration and open book. Further details of this assessment will be provided at the start of the semester. A feedback session will be provided on this assessment to ensure that this assessment is both summative and formative. Formal examination period (November 2010): 3. Two hour written examination consisting of two case reports for analysis. This will be an openbook assessment and will contribute 75% of the marks for the unit of study.

Formative assessment
During lectures, students will be presented with unknown case reports. An approach to evaluation for these case reports will be provided beforehand. This approach is the same as used in tutorials, and is the one expected to be used in the written examinations. Students will be given time to evaluate the case reports before the lecturer analyses the case reports. Students will be given the opportunity to ask questions and to respond to questions during the analysis. Students are encouraged to work through the case reports in the Veterinary Clinical Pathology Handbook and to discuss these with the lecturer. Some practical sessions will be devoted to case report evaluations. This will be done in small groups. Oral presentation will be part of the analysis process.

Grade Descriptors for Veterinary Clinical Pathology PASS

Denotes satisfactory achievement of main learning outcomes. The student has demonstrated: 1. a basic capacity to recognize and evaluate alterations in laboratory data. To do this requires a basic capability of retrieving and understanding relevant information from selected information sources; and a basic capability to think logically and critically about the utilization of that relevant information (this will be assessed in a written examination on case reports; and during tutorials and presentations of case reports).

2. a basic capability to perform and to understand the usefulness of selected simple laboratory tests (this will be assessed by direct observation and questioning of the student in practical classes and tutorials). 3. a basic knowledge of the collection of biological samples for laboratory evaluation. This will involve a simple understanding of the range of biological samples that can be collected, and how they are collected (this will be assessed in a written examination on case reports; and during practical classes, tutorials and presentations of case reports).

CREDIT
This denotes work of predominantly good quality on all specified learning outcomes. The student has demonstrated: 1. a sound capacity to recognize and evaluate alterations in laboratory data. To do this requires an above basic capability of retrieving and understanding relevant information from selected information sources; and an above basic capability to think logically and critically about the utilization of that relevant information. The emphasis for grading will be more on evaluation rather than recognition of alterations of laboratory data, and a sound understanding of how this provides assistance in general disease diagnosis and prognosis. The student will have a good style (logical, well organized and clear) for written presentations of case reports. 2. a sound capability to perform and to understand the usefulness of selected simple laboratory tests. The emphasis of grading will be more on understanding the limitations and strengths of selected simple laboratory tests. 3. an above basic knowledge of the collection of biological samples for laboratory evaluation. The student will be aware of some of the problems related to collecting various biological samples, and how these affect results of the tests.

DISTINCTION
This denotes work of superior quality on all specified learning outcomes. The student has demonstrated: 1. a superior capacity to evaluate alterations in laboratory data. To do this requires a superior capability to think logically and critically about the utilization of that relevant information. The student will be expected to fully understand how laboratory information assists general disease diagnosis and prognosis. It is expected that the student will have a strong capability to suggest further laboratory testing and the utilization of other diagnostic aids in order to reach a diagnosis. The student will have a superior style (improved logicality, organization and clarity) for written presentations of case reports. 2. a sound capability to perform and a superior capability to understand the usefulness of selected simple laboratory tests. Students should be able to show evidence of additional reading/information on, and a strong knowledge of the limitations of, the selected simple laboratory tests. 3. a superior knowledge of the collection of biological samples for laboratory evaluation. Students will be aware of many of the important problems related to the collection of biological samples.

HIGH DISTINCTION
Denotes work of exceptional quality on all specified learning outcomes. The student has demonstrated: 1. an exceptional capacity to evaluate alterations in laboratory data. To do this requires an exceptional capability to think logically and critically about the utilization of that relevant information. The student will have read widely and be knowledgeable of current debates on the value of utilization of laboratory tests. This will be incorporated in the general interpretation of laboratory data and the discussion of further testing. The student will have an excellent style (exceptional logicality, organization and clarity) for written presentations of case reports. 2. a sound capability to perform and an exceptional capability to understand the usefulness of selected simple laboratory tests. Students should be able to show evidence of considerable reading/information on, and an excellent knowledge of the limitations and strengths of, the selected simple laboratory tests. 3. a superior knowledge of the collection of biological samples for laboratory evaluation. The student will have read widely and be aware of all the important problems of collecting various biological samples.

Past Examination Question for Veterinary Clinical Pathology, with example of a model answer:
Suggested Approach To Answering Examination Questions
Think about the information given before looking at the laboratory data. Ask yourself what can be deduced from the history, signalment and clinical findings: Is there an indication of time course? Is age, sex or breed important? What organs or tissues are involved? Is there an indication of the pathological process(es) occurring? Is there an indication of cause? Keep your deductions in mind when interpreting the laboratory data and thinking about further investigation 1. LIST/HIGHLIGHT Abnormalities (DETECT AND DESCRIBE) This is for your benefit to ensure you dont forget to discuss any abnormalities. Remember to use the book information on what is regarded as mild, moderate and marked alterations (see the beginning of the Section on Case Reports). 2. GENERAL INTERPRETATION (DEDUCE) Consider the reasons for abnormalities. Do not give complete lists of reasons but only those which could relate to the case. Relate to information (history etc) given to you in the question. You will use this information in reaching conclusions. This can be incorporated in your conclusions if your prefer (i.e. give a conclusion and then add your reasons for reaching it) 3. CONCLUSION(S)/FURTHER INVESTIGATION/IMPLICATIONS FOR MANAGEMENT? (DEDUCE) This is most important and MOST MARKS will come from this section. A. Conclusions These can be in the form of questions or statements (e.g. is the regenerativedisease due to blood loss or blood destruction? OR the animal has regenerative anaemia due to either blood destruction of blood loss) Think about whether there appears to be a main problem. Remember, there may be more than one. The main problem may be presented as organ/tissue dysfunction (e.g. liver disease), a general disturbance/pathological process (e.g. regenerative anaemia; inflammation) or possibly a specific disease (e.g. acute pancreatic necrosis in a dog). Can you explain all the abnormalities in light of the main problem(s)? Dont forget about what you deduced from the given animal and clinical information when you are reaching your conclusions. B. Further investigation Often you will need to investigate the case further. This should be logical and based on your conclusions reached from analyzing case history and laboratory data. It is important to prioritize investigation i.e. consider what you think is the main conclusion when suggesting further investigation.

A checklist for further investigation might include: Do you need to get more history, and if so what? Do you need to recheck some clinical signs or repeat physical examination? Do you need to undertake specialist clinical procedures (e.g. ECG, EMG) Do you need to undertake image analysis? What modality would be most useful? Do you wish to undertake more laboratory testing? Sometimes you may consider a response to treatment as part of your further investigation to reach a diagnosis, but this can sometimes be misleading unless you verify your response with further testing. So, you need to state how you would support any response to therapy approach. Sometimes you can sequence your further investigation on the basis of probable findings e.g. I wish to undertake hepatic ultrasound. If I find any masses on ultrasound I would wish to take an FNA or biopsy. C. Implications of the results for your approach to treatment and prognosis (i.e. management of the case while you are investigating it further) You may not wish to undertake any treatment while investigating the case further. In some situations you may wish to undertake treatment after asking for more tests (e.g. electrolyte analysis for possible fluid therapy).

REMEMBER, not all abnormalities may be explicable in light of the case. Be honest, if you dont know, say so BUT think about how you would find out. Also, put the abnormalities in perspective. Some mild abnormalities may not be very important for the case.

THE UNIVERSITY OF SYDNEY

FACULTY OF VETERINARY SCIENCE VETERINARY MEDICINE & CLINICAL PATHOLOGY VETS 4112 Paper 1 - Clinical Pathology

Semester 1, 2004 INSTRUCTIONS

This is an OPEN BOOK examination

Time allowed: 1 hour

Dont ignore the HISTORY AND OTHER writing your case report discussion

INFORMATION

when

Remember to include CONCLUSIONS, FURTHER INVESTIGATION, if appropriate, and IMPLICATIONS FOR MANAGEMENT for each case report discussion

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Semester 1 2004

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QUESTION 1 Lucy is a 14-year-old, 15.5 kg, female border collie. She has problems sitting and climbing stairs and has wobbly back legs. She is not eating as much as she used to and has lost 3kg in the past 2 years. She has had occasional loose stools all of her life but more frequently in the past 2 years. On examination she has a normal pulse, respiration rate 60/min and temperature 39.1C. She is thin, has a discharge from one eye and a crusty, scaly ear (she has had otitis externa before). Her bladder is large. She has a wide-based stance, reduced patellar reflexes and reduced proprioception in the right hind leg and is sore and stiff on palpation of the hips. As part of the initial work up blood and urine were taken for analysis. Comment on the results, especially in relation to REASONS FOR THE Abnormalities. What CONCLUSION(S) can you draw? What FURTHER INVESTIGATION is required? What IMPLICATIONS are there FOR MANAGEMENT of the animal?

DETECT, DESCRIBE, DEDUCE TEST AMYLASE IU/L ALP IU/L ALT IU/L CK IU/L Serum protein (biuret) g/L Albumin (BCG) g/L Globulins g/L Total cholesterol mmol/L Glucose mmol/L Urea mmol/L Creatinine mol/L Calcium mmol/L Inorganic phosphate mmol/L Sodium mmol/L Potassium mmol/L Chloride mmol/L Bicarbonate (TCO2) mmol/L Anion gap mmol/L SAMPLE 1704 3301 539 91 70 38.8 31.3 5.24 5.85 21.55 103 2.71 1.63 146.6 4.2 112.4 24.6 13.8 REFERENCE INTERVAL <1400 <110 <60 <200 50-70 23-43 27-44 1.4-7.5 3.3-6.4 3.0-10 40-120 2.1-2.9 0.8-1.6 137-150 3.3-4.8 105-120 18-24 15-25

QUESTION 1 CONTINUED NEXT PAGE

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Semester 1 2004 QUESTION 1 CONTINUED

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TEST SAMPLE REFERENCE INTERVAL Plasma appearance Clear Clear PCV L/L 0.35 .37-.50 Plasma protein g/L (refractometer) 78 55-75 Haemoglobin g/L 128 100-150 Erythrocytes x1012/L 5.3 5-7 MCV fl 66 60-75 MCHC g/L 366 300-350 MCH pg 24.2 20-25 Leukocytes x109/L 12.2 7-12 Neutrophils (seg.) x109/L 9.88 4.1-9.4 Neutrophils (band) x109/L 0.24 0-.24 Lymphocytes x109/L 0.98 .91-3.6 Monocytes x109/L 0.73 .2-.96 Eosinophils x109/L 0.37 .14-1.2 Basophils x109/L 0 0-.36 Platelets x109/L 925 200-600 Reticulocyte % (uncorrected) 0.2 0-1.5 Blood film: Some neutrophils hypersegmented. Some toxic granulation of neutrophils. Occasional macroplatelet
Urinalysis (cystocentesis)

Appearance: slightly cloudy PH: 6.0 Colour: pale yellow Glucose: -ve Specific gravity: 1.015 Ketones: -ve Protein (SSA): trace Blood: trace Centrifuged: not done Bilirubin: + Microscopic findings: moderate bacteria, some cellular debris, 10-20 White blood cells per high power field, 10 Erythrocytes per high power field. THIS IS THE END OF THE PAPER

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Model Answer for 2004 Examination Question

Assessment on signalment and history: Lucy has a history of chronic disease. Musculoskeletal/ neurological, digestive (wt loss, loose stools) and possibly respiratory (increased RR) abnormalities appear to be of main concern. In this old dog, the disease processes could be degenerative, neoplastic or inflammatory reasons; and there is a distinct possibility that there are multiple problems. Abnormalities in test results: There are marked elevations in ALP and ALT, and mild/marginal elevations in urea, AMS, inorganic phosphate and bicarbonate. There is a decreased anionic gap. In haematology, there are mild increases in TPP, MCHC and a mild decrease in PCV. Leukocytes are marginally increased, with very mild/ marginal neutrophilia. The neutrophils are hypersegmented and have toxic granulation. Platelets are increased with occasional megaplatelets viewed on the smear. Urinalysis reveals a specific gravity close to isosthenuria, with a trace of protein and 1+ bilirubin. WBC are present in significant numbers (pyuria) and high in proportion to RBC numbers (hematuria). There are moderate numbers of bacteria. Reasons for changes: The dog has significant cholestasis and hepatocellular damage. This combination might suggest hepatic disease. Another possibility is that the ALP elevation is unrelated to cholestasis and due to corticosteroid induction (e.g. hyperadrenocorticism). However, clinical signs are not highly supportive of HyperA (we do not have any information about the animal has been medicated with corticosteroids). The elevation of blood urea in combination with hyperphosphataemia and a specific gravity close to isosthenuria (in a dog with possible dehydration elevated TPP) might suggest a degree of renal disease. The increased AMS could be related to renal disease, but may be related to pancreatic/intestinal disease (chronic diarrhoea). The elevated bicarbonate is difficult to assess. Respiratory, intestinal, renal and hepatic disease could all be contributing to electrolyte and acid/base levels. The dog has a marginal non-regenerative anaemia which could be simply related to chronic disease. Leukocyte changes are minimal, but do not rule out ongoing mild inflammatory disease. The toxic neutrophils could be due to inflammation or metabolic disturbances. The hypersegmented neutrophils are often due to corticosteroid effects. The high platelets (with some megaplatelets) might be related to inflammatory disease. Intestinal disease could be contributing? The urinalysis shows a 1+ bilirubin, which at this sp gr could be significant and reflect the cholestasis. The trace of blood is related to mild hematuria. The pyuria is significant and probably related to the bacturia (a fresh cystocentesis sample should be free of bacteria) and suggests urinary tract infection. Considering the large bladder and likely neurological defects, the dog is likely to have cystitis. The ratio of WBC to RBC numbers is roughly 2:1 and suggests that the hematuria is related to inflammation rather than hemorrhage. Conclusions and further testing: The dog certainly has strong indications of multisystemic disease. Many could be due to old age (e.g. degenerative joint/spinal disease). The likely conclusions from the clinical pathology results are hepatic disease, renal disease and cystitis. Pancreatic/gut disease has not been assessed effectively. The possibility of respiratory disease has not assessed. Suggested further testing may include image analysis of the thoracic (lungs) and abdominal (pancreas, liver, gut, bladder, adrenals) cavities. A bile acid analysis could be done to further assess liver. A history of corticosteroid medication might negate any interest in the possibility of HyperA. Image

13

analysis of the spine and joints would be useful. The urine sample should be cultured and assessed for antibiotic sensitivity. Implications for management and prognosis?: Many of the changes are related to chronic disease, but the urinary tract infection should be treated as soon as possible. Further management really hinges on what is detected on image analysis. Whatever eventuates, it is likely that the prognosis should be guarded. (Postscript: Ultrasound of the abdomen revealed an irregularly-shaped liver with variable echogenicity. An ultrasound-guided fine needle cell aspirate revealed cells that could have been neoplastic (diagnosis of possible hepatoma/ hepatocarcinoma). The neoplastic cells were obtained from several sites suggesting widespread involvement of the liver. Ultrasonography of the kidneys revealed bilateral dilation of the renal pelvices and hyperechoic renal cortices. The dog was given a poor prognosis and euthanasia was recommended. Unfortunately, the owners returned to the referring veterinarian for euthanasia of the dog and a necropsy was not performed.)

14

GENERAL REFERENCES
Some of these references were utilised in the preparation of both the lecture and practical notes. They can be used as an adjunct to the notes. Some references cover all species. The preferred references are in bold. Archer RK. Jeffcott LB. Comparative Clinical Haematology. Blackwell Scientific Publications, Oxford, UK. 1st Edn, 1977. ISBN 0-632-00289-1. Baker R. Lumsden JH. Eds. Color Atlas of Cytology of the Dog and Cat. Mosby Inc., St Louis, USA. 1st Edn, 2000. ISBN 0-8151-0402-2. Cowell RL. Tyler RD. Meinkoth JH, DeNicola DB. Eds. Diagnostic Cytology and Haematology of the Dog and Cat. Mosby Elsevier, St Louis, USA. 3rd Edn, 2008. ISBN 978-0-323-03422-7. Cowell RL. Tyler RD. Eds. Diagnostic Cytology and Haematology of the Horse. Mosby Inc., St Louis, USA. 2nd Edn, 2002. ISBN 0-323-01317-1. Day MJ. Clinical Immunology of the Dog and Cat. Manson publishing Ltd London UK; 2nd Edn, 2008. ISBN:978-1-84076-098-9. Davidson M. Else R. Lumsden J. Eds. Manual of Small Animal Clinical Pathology. British Small Animal Veterinary Association, Cheltenham, UK. 1998. ISBN 0-905214-41-2. Eade SC. Bounous DI. Ed. Pratt PW. Laboratory Profiles of Equine Diseases. Mosby Inc., St Louis, USA. 1st Edn, 1997. ISBN 0-8151-1731-0. Feldman BF. Zinkl JG. Jain NC. Eds. Schalms Veterinary Haematology. Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, USA. 5th Edn, 2000. ISBN 0-683-30692-8. Harvey J. Atlas of Veterinary Haematology. WB Saunders Co., Philadelphia, USA. 1st Edn, 2001. ISBN 0-7216-6334-6. Hawkey CM. Dennett TB. Color Atlas of Comparative Veterinary Haematology. Iowa State University Press, Ames, Iowa, USA. 1st Edn, 1989. ISBN 0-8183-0449-3. Jain NC. Schalms Veterinary Haematology. Lea & Febiger, Philadelphia, Pennsylvania, USA. 4th Edn, 1986. ISBN 0-8121-0942-2. Kaneko JJ. Harvey JW. Bruss ML. Clinical Biochemistry of Domestic Animals. Eds. Academic Press Inc., San Diego, California, USA, 5th Edn, 1997. ISBN 0-12-396305-2. Latimer KS. Mahaffey EH. Prasse KW. Eds. Duncan & Prasses Veterinary Laboratory Medicine Clinical Pathology. Iowa State University Press, Blackwell Publishing Co., Ames, Iowa, USA. 4th Edn, 2003. ISBN 0-8183-2070-7. Meyers DJ. Coles EH. Rich LJ. Veterinary Laboratory Medicine. WB Saunders Co, Philadelphia, Pennsylvania, USA. 1st Edn, 1992. ISBN 0-7216-2654-8. Raskin RE. Meyer DJ. Eds. Atlas of Canine and Feline Cytology. WB Saunders Co., Philadelphia, USA. 1st Edn, 2001. ISBN 0-7216-6335-4. Reagan WG, Rovira ARI. DeNicola DB. Veterinary Haematology Atlas of common domestic species. Wiley Blackwell, Ames, Iowa. 2nd Edn, 2008. ISBN 978-0-8138-2809-1. Rebar AH. MacWillliams PS. Feldman BF. Metzger Jr FL. Pollock RVH. Roche J. Ed. Cann CC. A Guide to Haematology in Dogs and Cats. Teton NewMedia, Jackson Wyoming. USA. 1st Edn, 2002. ISBN 1-893441-48-2.

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Sodikoff CH. Laboratory Profiles of Small Animal Diseases. Mosby Inc., St Louis, USA. 3rd Edn, 2001. ISBN 0-323-00956-5. Thrall MA. Baker DC. DeNicola D. Fettman MJ. Lassen ED. Rebar A. Weiser G. Ed Troy DB. Veterinary Haematology and Clinical Chemistry. Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, USA. 1st Edn, 2004. ISBN 0-683-30415-1. Willard MD. Tvedten H. Turnwald GH. Small Animal Clinical Diagnosis by Laboratory Methods. WB Saunders Co, Philadelphia, Pennsylvania, USA. 3rd Edn, 1999. ISBN 0-7216-7160-8.

16

VETERINARY CLINICAL PATHOLOGY (LABORATORY MEDICINE)

Introduction
Clinical pathology covers the use of laboratory aids (histopathology, microbiology, parasitology, immunology, biochemistry, cytology and haematology) to assess the health, and/or detect a disease state, of an animal. Clinical pathology is often referred to as laboratory medicine because it is a component of the investigative process that leads to the diagnosis and treatment of medical conditions. Laboratory aids may be used to: a. assess breeding or racing performance b. assess the disease status of an animal that is to be transported c. aid a clinical diagnosis d. aid prognosis and treatment Laboratory aids are selected on the basis of clinical information (signalment, history, clinical signs and physical examination) and sometimes on the results of other diagnostic tests (e.g. image analysis). The type of test selected will depend also on whether you are dealing with the diagnosis of individual animal or group animal disease. Individual animal disease (mainly small animals and horses) relies on biochemistry and haematology primarily to detect organ or tissue dysfunction (although haematology can detect host response to tissue damage). Cytology, histopathology and microbiology assist in this process but in addition investigate aetiopathogenesis (the time course and development of disease; it includes causes and pathological processes [degeneration and necrosis; inflammation and repair; vascular disturbance; disorder of growth; and pigmentations/deposits]). Group animal disease (mainly production animals) relies less on biochemistry and haematology as necropsies on one or more dead animals usually provide significant information on aetiopathogenesis. Microbiology and toxicology are important for aetiological diagnosis in group animal disease and samples are commonly taken at necropsy. The use of biochemical and Haematological tests, especially for the diagnosis of individual animal disease, is not always necessary. If they are required, they are selected either to provide information that cannot be determined on clinical signs (i.e. none or few clinical signs available or the clinical signs are non-specific) or to investigate clinical possibilities. Clinical possibilities may involve either a specific disease entity or apparently unrelated clinical signs. In general, a combination of biochemical and Haematological tests is used to investigate clinical disease. Profiles (a broad range of biochemical tests and full Haematological investigation) are commonly used when investigating non-specific clinical signs or specific organ diseases. Whatever the reason for the use of laboratory tests, their interpretation must be done in light of history, signalment, clinical signs, physical examination and the results of other diagnostic tests. This is especially so for Haematological and biochemical tests which are rarely specific for particular diseases.

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Necropsy Technique and Collection of Specimens for Histopathology

Basically there are two common necropsy techniques: midline and flank. It is best to learn one basic technique and then adapt that to suit the animal and the suspected disease. Variations in technique usually occur due to size of the animal and the differing gastrointestinal tract (GIT). Samples for histopathology are usually placed in the fixative 10% neutral buffered formalin (10 parts formalin to 1 part tissue) in a proportion of about 1 volume tissue to 9 volumes fixative. The section of tissue will depend on whether it is biopsy or necropsy material. Samples are not supposed to be thicker than 1 cm but thicker samples may be taken if several slices (for penetration of the fixative) are made in the collected tissue or if the sample is to be further selected shortly after. Additional information on necropsy technique and collection of tissue for histopathology is provided in the handbooks for Principles of Disease and Veterinary Pathology (VETS 2013 and 3011).

The Nature of Laboratory Tests, Problems for Interpretation and Reference Intervals
Most haematological and biochemical tests are non-specific and are commonly used in combination to acquire meaningful information. Other laboratory aids are more specific but their interpretation still depends on the clinical information. Biochemical tests need to be varied depending on what species is being investigated. Species variation in interpretation of tests also exists. But perhaps the greatest difficulty in interpretation is due to the limitations of establishing a so called normal or reference interval (range) for a quantitative test within an animal population. The reference interval is usually determined from a clinically normal population of mature animals regardless of sex, breed and activity. It is expressed as the mean + 2 S.D., which essentially covers 95% of the population tested (this assumes that the distribution of results is Gaussian). In theory, individual laboratories should establish their own reference interval for a particular constituent but for several reasons they may not be willing to do so. Reference intervals quoted in texts can be used with reservation for haematology and end point biochemistries but should be used cautiously for kinetic assessment of enzymes. It is best for the laboratory to assess enzyme levels in 5-10 normal animals and use those results as a guide if proper statistical analysis cannot be done. There is always some overlap between results for a normal population and those from a diseased population for a particular biochemical or haematological constituent. The degree of overlap varies for different constituents but useful tests tend to have low levels of false positives and false negatives (i.e. a limited degree of overlap) (see figure following).

18

FIGURE:Valuesforatestforahealthyandasickgroupofanimals.Thegreytriangledenotesthesmalloverlapforthetwo groupsandsuggeststhatthistestmightbeusefultodelineatehealthyandsickanimals.Inthiscase,thedistributionofthe healthygroupofanimalscouldbeusedtoestablishthereferenceintervals(mean+2standarddeviations)

Because of these limitations, the reference interval should be used as a guide to abnormality rather than as an absolute indication. Values just outside (or inside) the reference interval need to be treated with care. This is when other available information is most important for interpretations. What can be said is that the more distant a particular value falls from the reference interval, the more likely it reflects a definite abnormality.

General Remarks on Sampling for Blood Biochemistry

What samples are required for particular tests will depend on the commercial laboratory being used. In general, both serum and plasma can be utilised with some exceptions (see practical notes on sampling for blood biochemistry): Serum is the fluid component derived from clotted blood. Plasma is the fluid component derived from whole blood containing an anticoagulant (i.e. still contains clotting proteins such as fibrinogen). Heparin (mostly used for biochemistry), EDTA (primarily used for haematology), citrate and oxalate are commonly used anticoagulants.

Specimen storage
How long a biochemical constituent remains stable in a sample varies with: a. temperature of storage e.g. many constituents are more stable if chilled (4 degrees Centigrade) or frozen ( -20 degrees Centigrade) b. what biochemical constituent it is e.g. some enzymes are particularly unstable once stored c. species e.g. some enzymes vary in stability between species The rule of thumb is to store at 4 degrees Centigrade unless the commercial laboratory instructs otherwise.

Haemolysis
Can interfere with the estimation of many biochemical constituents by: a. changing the concentration of the biochemical constituent (depends on the relative concentrations of the constituent within the plasma and within the erythrocyte) and b. interfering with the performance of the biochemical test (commonly due to free haemoglobin) To avoid haemolysis, carefully handle blood and separate the plasma/serum promptly from the erythrocytes if there is to be any prolonged delay in biochemical analysis e.g. overnight.

19

Gross Lipemia (and its relationship to laboratory detected hyperlipidemia)

Hyperlipidemia refers to a measured increase in plasma/serum lipid levels (i.e. the levels are above reference intervals for the laboratory). These can be triglycerides, cholesterol or phospholipids (usually, only the first two are measured). If the hyperlipidemia is caused by significant increases in triglyceride rich lipoproteins (e.g. chylomicra [chylomicrons] and very low density lipoproteins [VLDL]) then there can be a milky appearance to the serum or plasma (this may be a diffuse white cloudiness or just a plug of white material at the top of the serum or plasma the latter is related to chylomicra and can be accentuated by refrigeration at 4 degrees Centigrade). Historically, this gross milky appearance has been referred to as lipaemia (hyperlipaemia less commonly used in the horse the term hyperlipaemia syndrome is even more specific and refers to a disease of mainly obese ponies that commonly occurs during pregnancy and lactation), even though the term is a perhaps a little misleading. Gross lipaemia, apart from occurring in certain disease conditions e.g. diabetes mellitus), is a common finding in monogastric animals fed prior to collection of blood samples (postprandial lipaemia due to the presence of chylomicra). Gross lipaemia can affect the measurement of biochemical constituents by elevating optical density and thus interfering with colorimetric techniques of analysis. In addition, gross lipaemia can enhance the fragility of erythrocytes leading to haemolysis. To avoid excessive gross lipemia in monogastrics take blood samples after fasting (8 hours minimum).

Plasma/Serum Enzymology
Enzymes detected in the serum/plasma can be divided into: a. plasma specific. They are rarely measured (exceptions include glutathione peroxidase to detect selenium/Vitamin E deficiency in ruminants and cholinesterase reductions to detect organophosphate poisoning in a variety of species) b. plasma non-specific. They have no known function in the plasma and are present in the plasma at levels much lower than those that exist in tissues/cells. They can be divided into: i) enzymes associated with cellular metabolism. These can be divided further into organ specific and organ non-specific. Organ specific enzymes have relatively high levels in certain organs and therefore on release usually reflect damage to those organs e.g. ALT in hepatocellular damage in the dog or cat. Organ non-specific enzymes are usually in comparable levels in a number of tissues/organs e.g. AST in liver and skeletal muscle. Organ non-specific enzymes can be made more organ-specific by separating into isoenzymes (different forms of the same enzyme that perform the same function e.g. CK isoenzymes in striated muscle, gut and brain) ii) secretory enzymes. These may elevate in blood if there is damage to the cells producing the enzyme or there is blockage to the secretory route e.g. AMS from the pancreas

Factors affecting plasma levels of enzymes

These are incompletely understood. Consequently the effect of disease on certain plasma enzyme levels is not always predictable. a. Those affecting enzyme release i) alterations in cell membrane permeability or internal cell organisation. Those enzymes of low molecular weight (e.g. CK) and high solubility (cytoplasmic in situation e.g. LD, CK) are usually released more readily than others during changes in membrane alteration. ii) cell death. Usually marked increases in plasma enzyme levels occur, irrespective of molecular weight or enzyme situation iii) altered enzyme synthesis (induction e.g. ALP)

20

b. Those affecting enzyme removal i) circulatory impairment ii) obstruction of normal excretory route iii) increased catabolism of enzymes c. Those affecting measurement i) technique of the enzyme assay- varying techniques (especially kinetic reactions) can have marked effects on reference intervals ii) collection and storage of the sample. Enzymes are proteins which can be easily denatured by rough handling or by repetitive freezing and thawing iii) time of collection. Each enzyme will rise and fall at different rates after a disease process affects a particular organ

Naming of enzymes
The overall reaction catalysed by the enzyme is the basis of the name. All enzymes have a designated numerical code (4 numbers), an official name, a trivial name and an abbreviation. The latter 2 are used most widely in veterinary circles. The following is an example: trivial name: Lactic dehydrogenase, abbreviation: LDH (LD), numerical code: 1.1.1.27, official name: L lactate: NAD+ oxido-reductase

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TABLE:Someofthemorecommonenzymesusedindomesticanimals

ENZYMEUSAGEINDOMESTICANIMALS
A.GENERALDETECTION OFCELLDAMAGE B1.HEPATICDISEASE HEPATOCYTEDAMAGE Dog/Cat Horse Sheep/Cattle Pig AST(mainlymuscle/Liver),LD(presentinmostcells)forallspecies IDorGD(ARG) GDorOCT(ARG) OCT(ARG) ALT(ARG canbeused forallspecies butrarely done) ALP,GGT GGT, ALP (less GGT GGT commonlyused) CK,AST,LD(especiallyisoenzymeanalysis)forallspecies.NB.ithasbeen reportedthatcertainskeletalmusclediseasesinsomedogsandcatsmay causeelevationsofALT AMS notdone notdone AMS,LPS (other laboratory aidsmore useful) AMS?,ALP?otherlaboratoryaidsareofgreateruseforallspecies CK(inCSF)forallspecies,otherlaboratoryaidsofgreateruse otherlaboratoryaidsofgreateruse Otherlaboratoryaidsofgreateruse(AST,ICD,LDcanbemeasuredinthe synovialfluid,especiallyinthehorse) otherlaboratoryaidsofgreateruse(GGThasbeenmeasuredinurineindogs andcatstodetectrenalcelldamage) laboratoryconfirmationusuallyrequireshormonalassaysenzymesmay detectsecondaryorgandysfunction(e.g.diabetesmellitusmaycause elevationinliverenzymes)NBs.hyperadrenocorticisminthedogcaninducea specificisoenzymeofALP;hypothyroidisminthedogcanbecharacterisedby increasesinCK

B2.HEPATICDISEASE CHOLESTASIS C.CARDIAC/SKELETAL MUSCLECELLDAMAGE D.PANCREATICACINAR CELLDAMAGE

E.GUTDAMAGE F.CNSDAMAGE G.REPRODUCTIVE TRACTDAMAGE H.JOINTDISEASE I.RENALDAMAGE J.ENDOCRINE DISORDERS

ALPAlkalinePhosphatase,ALTAlanineTransaminase,AMSAmylase,ARGArginase,ASTAspartate Transaminase,CKCreatineKinase,GDGlutamateDehydrogenase,GGTGammaGlutamylTransferase, IDIditolDehydrogenase(oldnameSDHSorbitolDehydrogenase),ICDIsocitrateDehydrogenase,LD LactateDehydrogenase,LPSLipase,OCTOrnithineCarbamoylTransferase

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Combining enzymes to detect disease processes

Example: In the horse CK has been used in combination with AST to detect active damage in muscle. CK return to normal in about 2-4 days whereas AST may be elevated for up to 1 week.

CK AST
Level of enzyme

Time Single episode of muscle damage

23

LABORATORY EVALUATION OF HEPATIC DISEASE

Terminology
Jaundice (icterus) Cholestasis Hyperbilirubinemia
yellowing of the skin and other tissues due to deposition of bilirubin bile without motion increased circulating levels of bilirubin in the blood. Retention form refers to increases in unconjugated, regurgitation form refers to increases in conjugated and combined refers to increases in both signs of central nervous disease related to liver disease. In the dog and cat it is invariably related to congenital or acquired vascular shunting refers to altered (mostly increased) levels of specific proteins (mostly globulins) in the blood. They can include clotting proteins (e.g .fibrinogen), haptoglobin and C-reactive protein. They occur in response to a variety of acute/active degenerative/inflammatory conditions after feeding

Hepatic encephalopathy Acute phase reactants

Post prandial

Introduction
In individual animal disease initial laboratory evaluation of hepatic disease depends on biochemical analysis. Biochemical tests, (liver "function" tests) can detect 3 basic processes that can occur in liver disease: hepatocellular damage, cholestasis and reduced functional mass. Haematological analysis is usually of limited use in the diagnosis of liver disease but a cytological aspirate and/or biopsy can more specifically define the hepatic problem (i.e. investigate the aetiology as well as the pathogenesis). In group animal hepatic disease biochemical tests still have a role to play but histopathological, microbiological and toxicological examination of hepatic tissue taken at necropsy are of greater importance (many liver diseases in group animals, especially in farm animals, are caused by infectious agents and environmental poisons [including plants], which may be detected by gross and microscopic examination of the liver, and by microbiological and toxicological analysis). However, the significance of detected liver lesions must be determined in light of history, signalment, clinical signs and physical examination because not all may produce clinical disease (due to the large reserve capacity of the liver). Biochemical tests employed to detect liver disease are selected on the basis of what disease processes might be occurring. For example, significant hepatocellular damage is more likely to occur in acute or sub-acute disease rather than in chronic; reduced functional hepatic mass is more likely to be of importance in chronic disease; cholestasis can occur at any stage of liver disease. All the tests have limitations and consequently, they are commonly used in combination. Liver dysfunction may occur as a primary disease or secondary to another organ/tissue dysfunction

24

(e.g. endocrine abnormalities leading to secondary liver damage). In many cases biochemical tests cannot differentiate primary and secondary hepatic disease. Liver "function" tests can be affected by extrahepatic factors e.g. blood flow to the liver can influence the results for bile acid testing. By serial sampling, liver "function" tests can trace the course of a hepatic disease (e.g. the use of enzymes with different T1/2s to determine the persistence of hepatocellular damage). For convenience, liver "function" tests will be presented according to their main usage, i.e. detecting hepatocellular damage, cholestasis or reduced hepatic cell function. However, many tests will detect, and be affected by, more than one of these basic processes. Also, it is worthwhile remembering that a liver disease will often involve more than one of these basic processes.

Summary of Tests
PRINCIPALUSAGE ACTIVELIVERCELLDAMAGE CHOLESTASIS CHOLESTASIS,CELLDAMAGE REDUCEDFUNCTIONAL HEPATICMASS BIOCHEMICALTEST AST,ALT,LD,ID,ARG,OCT,GD,ICD(dependingon species) ALP,GGT(bothinducedenzymes) Bilirubinanalysis(total;unconjugated;conjugated) Bileacids*,Bromsulphopthalein(BSP)test*,blood ammonia/urea,serumproteins

*The BSP test and bile acid analysis will detect all three basic processes but are more commonly usedtodetectreducedfunctionalhepaticmassasothertestsarecheaperandsimplertodoforthe detection of hepatocellular damage and cholestasis. The BSP test is no longer used now, being largelyreplacedbybileacidanalysis.

Hepatocellular Damage (Degeneration and/or Necrosis)

For levels of serum enzymes to be elevated in liver disease, an active damaging process to a significant number of hepatocytes is required. This is more likely to occur in acute/sub-acute disease rather than in chronic disease. Elevated levels of enzymes may be seen with reversible degenerative changes as well as in cell necrosis. Consequently, the levels may not be an accurate assessment of prognosis. a) ALT - Reasonably specific in the dog and cat for hepatocellular damage although it may be elevated in certain muscle diseases in certain breeds of dogs and some cats (therefore, combine with CK if in doubt). It is present in the cytoplasm; therefore, even changes in cell permeability can increase levels. Consequently, elevated ALT does not necessarily mean cell death. In the dog, corticosteroids and some anticonvulsants may induce ALT. It is rarely used in farm animals (horses, cattle and sheep) due to limited elevations in hepatic disease, and the fact that elevations may also occur in muscle disease. ALT has been used in combination with arginase, which is liver-specific for many species, to detect progressive damage (arginase, returns to normal more quickly than ALT- in the dog, T1/2s of ARG and ALT are less than 12 hours and 40 to 60 hours respectively). b) ID - Iditol dehydrogenase (old name SDH-sorbitol dehydrogenase) - Reasonably liver specific in most farm animals but returns to normal rapidly after a non-progressive bout of hepatic damage.

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It is commonly used only for the horse. It can be used in the dog. c) d) e) f) OCT - Liver specific in most species. It is rarely used except in swine. GD - Reasonably liver specific in cattle and sheep. It can be used in the horse and dog. ARG - Liver specific in most species but not widely used. LD, AST, ICD They are not liver specific enzymes but can be used to detect hepatocellular damage if other organ diseases can be ruled out (especially muscle). They are commonly used if more specific enzymes are not available.

NOTE: Enzymes are often used in combination to detect hepatocellular damage as expected increased serum levels in hepatocellular damage do not always occur for a particular enzyme.

Cholestasis
The term cholestasis literally means "bile without motion". It refers to reduced excretion of bile which may occur due to hepatocellular damage (often interferes with the energy production required to excrete bilirubin into the bile canaliculi) or due to physical obstruction of intrahepatic or extrahepatic bile canals. The enzymes ALP and GGT are the most sensitive indicators of cholestasis and elevations are supposed to precede the onset of hyperbilirubinemia or significant bilirubinuria. In fact, cholestasis may never become severe enough to cause hyperbilirubinemia even though enzyme levels may become high. On the other hand, most cases of hyperbilirubinemia due to cholestasis are accompanied by high enzyme levels. Serum levels of ALP and GGT are high in neonatal puppies and kittens due to high levels in ingested colostrum. Consequently, measurement is of little use until about two weeks of age. ALP isoenzymes do exist for bone and gut. The gut isoenzyme has a very short half life and gut disease, therefore, causes little in the way of blood increases. Bone growth and diseases can cause 2-3X increases in blood ALP. ALP is the enzyme of choice in the dog and cat. In the dog, mild to marked increases can occur in cholestasis (e.g. 200 up to, and exceeding, 1000 IU/L). In the dog moderate to high levels of ALP may be present in the serum in cases of hyperadrenocorticism due to induction of a specific hepatic isoenzyme (may be in excess of 2000 IU/L). Exogenous corticosteroids in the dog also induce the specific isoenzyme but this normally takes a few days. The cat appears not to have the specific isoenzyme for corticosteroids. Moreover, in the cat the magnitude of elevation of ALP in cholestasis is much less than in the dog. This is due to the lower levels in hepatocytes and biliary epithelium and the shorter plasma T1/2 in the cat (6 hours compared to 72 hours in the dog). Consequently, any elevation in ALP in the cat, irrespective of the magnitude, is considered significant and warrants further investigation. In the dog, ALP can be induced by anticonvulsants, volatile anaesthetics and barbituates. ALP is rarely used in the horse but elevations can occur in cholestasis. GGT is more commonly used in the horse to detect cholestasis. ALP is not reliable in sheep and cattle and GGT is the enzyme of choice for cholestasis in farm animals. GGT can be used in the dog (questionable usage in the cat) to detect cholestasis and is not apparently affected by barbiturates or anticonvulsants. However, it is induced by corticosteroids in the dog. GGT in renal epithelium is only released in the urine on damage; therefore, increased urine levels may indicate tubular damage.

26

Bilirubin Metabolism and Analysis

This is a general discussion of bilirubin metabolism and will mention both hepatic and extrahepatic diseases that can cause derangements. When analysing bilirubin metabolism, serum bilirubin, urine bilirubin, urine urobilinogen and faecal bile pigments can be measured. Most emphasis is placed on the first two. Serum bilirubin levels can be visually assessed by the icterus index (II) technique (by visually assessing the colour of plasma or serum). Dogs and cats have an II less than 5. Horses have a value less than 15 but this can be up to 25 if the animal is on green feed or has a high PCV (0.40-0.55 L/L). The ox usually has less than 15 but can have levels up to 25 presumably due to serum carotenoid levels. The sheep and pig have an II of less than 5. The II technique is rarely used these days, but it is still worthwhile assessing the yellowishness of plasma or serum. Accurate assessment of serum bilirubin requires the Van den Bergh's test. This measures total bilirubin, but can be modified to measure the proportions of unconjugated to conjugated bilirubin (total bilirubin on its own is of limited use in determining the reason for hyperbilirubinemia, but there is some debate over the usefulness of measuring conjugated to unconjugated proportions). Bilirubin metabolism in the body in the dog (view diagram) can be applied to other domestic species, but with some minor variations (NB. birds form biliverdin instead of bilirubin from the breakdown of heme, but biliverdinemia due to hepatic disease is apparently rare in birds). Urine bilirubin levels provide an indication of blood levels of conjugated bilirubin. Only conjugated bilirubin is passed by the kidney. However, the male dog's kidney apparently has good ability to break down haemoglobin and to conjugate bilirubin. In addition, the dog has a low renal threshold to conjugated bilirubin. Consequently, bilirubinuria in the dog needs to be interpreted with care e.g. a one plus to two plus may be of little significance in a male dog or in concentrated urine over specific gravity of 1.030. Urine urobilinogen as a test has fallen out of favour. Increased urobilinogen can be seen in haemolysis and certain types of liver disease. Decreased or no urobilinogen can occur with disturbances of the enterohepatic circulation or as a chance finding in a normal animal. Faecal bile pigments are rarely measured. Increases may occur in haemolytic crises, decreases may occur in biliary obstructions. Traditionally, icterus (jaundice [yellowing of the tissues due to the presence of significant hyperbilirubinemia]) has been divided into 3 types: a. Haemolytic - is non-cholestatic and essentially involves an excess production due to haemolysis of erythrocytes. b. Hepatocellular - is partly due to non-cholestatic mechanisms (reduced cell conjugation) and partly due to intrahepatic cholestasis (reduced cell excretion due to lack of energy and blocked bile canaliculi primarily due to cell swelling). c. Obstructive - principally refers to post-hepatic obstruction of bile flow or obstruction to the larger bile ducts. The jaundice is completely due to cholestasis, at least in the earlier stages.

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FIGURE:BilirubinMetabolismintheDog
Other haemoproteins 15% Erythrocytes 85%

Free indirect reacting, unconjugated, water insoluble bilirubin in plasma (bound to protein for transport)

20%

80% recycled Direct acting, conjugated, water soluble bilirubin in the hepatocytes

Portal blood system

Most excreted in the bile (minor levels in the urine

In the intestine bacteria reduce conjugated bilirubin to compounds of similar structure called urobilinogens General circulation

10-15%

85-90%

Urobilinogens and conjugated bilirubin in urine

Further conversion and excreted in the faeces (faecal bile pigments)

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This classification system is useful but has limitations when being applied to specific domestic species. Consequently, another classification system has been developed that attempts to cover all possibilities: a. Retention hyperbilirubinemia This is characterised by unconjugated bilirubin as the predominant form in the blood. Bilirubinuria may or may not occur. Haemolytic crises are the main cause but a variety of extrahepatic and hepatic diseases can interfere with hepatic uptake and conjugation (in horse, cattle, sheep and swine anorexia associated with many diseases can give rise to retention hyperbilirubinemia. Jaundice may not develop. This cause of retention hyperbilirubinemia occurs irregularly and mildly in the dog and cat). b. Regurgitation (cholestatic) hyperbilirubinemia This is characterised by conjugated bilirubin as the predominant form. Bilirubinuria is usually present. Both intrahepatic and extraheptic obstruction to bile flow produce regurgitation hyperbilirubinemia. c. Combined hyperbilirubinemia This develops due to both retention and regurgitation methods; therefore, both conjugated and unconjugated bilirubin levels in the blood are increased. Bilirubinuria is usually present. This type of hyperbilirubinemia can occur in both acute and chronic hepatic diseases. In the dog and cat most hyperbilirubinemias are combined, with the conjugated bilirubin being anything between 20% and 60%. Pure retention can occur in haemolytic crises (although elevated conjugated bilirubin generally occurs in the later stages). In post-hepatic obstruction the hyperbilirubinemia can be mainly regurgitation although some unconjugated bilirubin is nearly always present. In the cat, bilirubinuria always means increased conjugated bilirubin in the blood stream. In the horse hyperbilirubinemia is commonly retention in type due to a variety of extrahepatic diseases inducing anorexia and causing significant increases in unconjugated bilirubin (rarely do you get jaundice with this cause of hyperbilirubinemia). Combined hyperbilirubinemia occurs in both hepatic disease and post-hepatic obstruction, but with the unconjugated bilirubin always predominating (possibly still due to the impact of anorexia). However, there is a greater percentage of conjugated bilirubin in primary cholestatic disease in the horse. Bilirubinuria always signifies increased conjugated bilirubin in the blood. In farm animals hyperbilirubinemia is commonly retention in type due to haemolytic crises and due to a variety of extrahepatic diseases inducing anorexia (especially in cattle). In ruminants, bilirubin elevations are inconsistent in hepatic disease and post hepatic obstruction and hence used sparingly; but can be interpreted similarly as in dogs and cats. It is best to combine bilirubin analysis with other tests for laboratory diagnosis of hepatic disease in ruminants. Naturally, this classification system requires measurement of total, conjugated and unconjugated bilirubin levels. When interpreting the proportions of conjugated to unconjugated it is also important to look at their total amounts (i.e. the magnitude of the rise).

29

Tests for Reduced Functional Hepatic Mass

a) Bile acids
Bile acids are synthesized by hepatocytes from cholesterol and then conjugated prior to excretion in the bile. They are stored and concentrated in the gall bladder during the interdigestive period. Upon feeding, cholecystokinin release occurs following gastric emptying. This stimulates gall bladder contraction and expulsion of bile into the duodenum. In the small intestine bile acids are necessary for fat assimilation. Most secreted bile acids are actively reabsorbed in the ileum (90% efficient) and returned directly to the liver via the portal system (i.e. an enterohepatic circulation similar to that for bilirubin exists which allows for about 90% of the bile acids to be extracted by the liver). The liver has a tremendous reserve capacity to produce bile acids and failure of bile acid synthesis is rare. Any process that impairs the hepatocellular, biliary or portal components of the enterohepatic circulation of bile acids results in increased serum levels. Consequently, serum levels of bile acids can be used to check for a wide variety of liver related diseases. Increased plasma levels can occur in hepatocellular damage, cholestasis, reduced functional hepatic mass and in portosystemic shunting, but the levels usually cannot be used to distinguish the type of disease. Decreased serum bile acids may occur in the final stages of end stage liver disease (only when greater than 90% of functional mass has gone). Serum bile acids can be measured prior to feeding (fasting levels) or 2 hours after feeding a high protein or fatty meal (post-prandial). Post-prandial levels can be significantly elevated compared to fasting levels in dogs with vascular shunting and cirrhosis (reduced functional mass). Consequently, serum bile acid analysis can be used instead of other tests currently used to detect these abnormalities (e.g. BSP or other dyes and NH3 tests). NB. bile acid analysis is unreliable in Maltese dogs in determining shunting or liver disease as healthy Maltese can have levels far in excess of reference intervals for other dogs.

b) Blood ammonia determination

NH3, produced by bacterial action in the gut, is converted by the liver to urea. Elevated blood NH3 may be due to: i) terminal hepatic insufficiency (i.e. significantly reduced functional hepatic mass) ii) circulatory bypass of the liver (i.e. intrahepatic or extrahepatic shunting - a common use for the test) iii) urea cycle enzyme deficiency iv) excessive vomiting v) shock In practice only (i) and (ii) are important in causing significant elevations of NH3. Urea is commonly low. In the dog, fasting blood NH3 levels (8-12 hrs after feeding) are normally elevated in significant shunting of blood around the liver (congenital or acquired due to hepatic disease). If chronic liver disease is unassociated with significant acquired shunting then fasting blood NH3 levels may be normal. The ammonia tolerance test (ATT-oral or rectal) can be performed and may produce marked increases in NH3 due to reduced functional hepatic mass in terminal hepatic insufficiency (5 x fasting blood NH3). In significant shunting, where there usually is a relatively high fasting level, 2-2.5 x fasting blood NH3 levels may occur with the ATT (these figures are for oral ammonia tolerance test). Ammonia and urea investigations should be done in tandem (as there is often an inverse relationship) and any suggestion of shunting should be supported by vascular studies.

30

Congenital shunts have been recorded in the dog, cat, horse and cow. They can produce what is termed hepatic encephalopathy. In ruminants and sometimes the horse, however, signs of hepatic encephalopathy can also occur in a variety of liver diseases (e.g. hepatic lipidoses, necrosis, chronic progressive hepatitis) not usually associated with significant vascular shunting. In these diseases it may be necessary to combine blood NH3 determination with other tests (e.g. in the horse liver biopsy is extremely useful) to determine the etiopathogenesis. NB. In dogs, because of the difficulty of performing the ATT and technical problems with NH3 analysis, bile acid analysis (fasting and post-prandial) may be used, with care, to detect congenital or acquired vascular shunting.

c) Serum protein estimation

Serum protein estimation (total and types of protein) can be useful in detecting a chronic hepatopathy. It is rarely of use in early hepatopathies (but there may be increased levels of globulins in acute hepatic disease. These increased proteins are usually acute phase reactants and include clotting proteins, haptoglobin, transferrin and many others). Mild hypoalbuminemia has been recorded in acute inflammatory disease of the liver but this is usually insignificant and probably related to the fact that albumin is a negative acute phase reactant (nb. T1/2 of albumin in the serum is about 8 days). In a chronic hepatopathy with marked reduction in functional hepatic mass a significant hypoalbuminemia may occur. The beta and gamma globulins may be increased but this depends on the disease process. Total serum protein may be decreased. Fibrinogen levels may be lowered in advanced hepatic insufficiency. Clotting factors (other than fibrinogen) may have reduced production in both acute and chronic liver disease (they have short plasma half lives). Rarely are they sufficiently suppressed to produce spontaneous hemorrhage (but they could contribute to a bleeding disorder with another disease process - see Bleeding Disorders). Decreased levels have been detected by modified clotting factor tests (OSPT, APTT). Serum protein analysis is commonly incorporated in general biochemical profiles, as abnormalities can occur in a variety of extrahepatic diseases. Usually, this involves direct measurement of total serum protein and serum albumin, with a derived value for total globulins. If abnormalities are detected through these simple biochemical methods, then serum protein electrophoresis can be performed to better delineate changes to albumin and the various classes of globulins (e.g. to detect reasons for hyperglobulinemia other than due to dehydration). Some specific proteins, apart from albumin, can be measured in the laboratory; and most of these are acute phase reactants. Blood levels of these proteins are often altered in acute degenerative/inflammatory conditions, although some can be increased in other disease situations. Most of these proteins increase but some, such as pre-albumin, albumin and transferrin, may decrease (called major negative acute-phase proteins) in response to acute degenerative/inflammatory conditions (transferrin may increase in acute liver disease). Major positive acute-phase proteins (i.e. those that increase in the blood) vary amongst species, but most are detected as alpha and beta globulins through serum protein electrophoresis. Fibrinogen (and some other non-enzymic clotting proteins) is useful in most domestic species, but especially in ruminants and horses. C-reactive protein is a major responder in dogs and pigs, serum amyloid A is useful in dogs, horses, cattle and birds, Haptoglobin is a major responder in farm animals, whilst TNF-alpha is supposedly useful in cats. There are many other positive acute phase proteins, and it is likely that their usage will increase in delineating disease processes (e.g. Porcine major acute protein; alpha 2-Macroglobulin in cattle).

31

Other Laboratory Tests

A.

Bromosulphthalein (BSP, sulfobromophthalein)

The BSP test is rarely used as it has been replaced by bile acid analysis. It is primarily used as a research tool today. The BSP test measures hepatocyte functional integrity and hepatic blood flow. BSP is transported in the serum bound to albumin, is conjugated by the hepatocytes and excreted in the bile (75%- 25% is cleared by skeletal muscle and kidney). This is analogous to bilirubin hepatic metabolism. Consequently, in jaundice, the BSP test is limited by competition between BSP and bilirubin for excretory pathways. Bile acid determination has replaced the BSP test to a great extent. The BSP test involves some expense and difficulty. Consequently, simple tests, if available, are often used to define liver problems - cholestasis, ALP and bilirubin; active hepatocellular damage, ALT and other specific enzymes. But the BSP test may be used to confirm these conditions, and in the case of chronic hepatopathies with low levels of active damage and cholestasis, used to detect the conditions. In the horse (and cattle ) a BSP clearance rather than retention method is used. One gram of BSP is given intravenously and between 5-12 minutes later 2 heparinized samples are taken 4-6 minutes apart. The T1/2 is determined from the samples and should be between 2 and 3.7 minutes for the normal horse. The interpretation of the BSP test in the horse is similar to that in the dog and cat. Indocyanine Green is another substance that can be injected to assess liver problems. Unlike BSP, ICG is solely cleared by the liver, but the disadvantage of ICG is that it is more expensive than BSP.

B.

Cholesterol
Cholesterol is primarily produced by hepatocytes, intestinal epithelium, adrenal cortex and gonads. Cholesterol is utilised for the synthesis of bile acids. Hypercholesterolemia can occur in cholestatic conditions but may also occur in the nephrotic syndrome, acute pancreatitis, congenital hyperlipidemias and certain endocrinopathies (DM, hyperadrenocorticism, hypothyroidism). Increases may also occur post prandially. Increased blood levels of cholesterol alone will not cause gross turbidity of the serum or plasma. Decreased serum cholesterol can occur in hepatic shunting and in gut problems (Exocrine Pancreatic Insufficieancy, malabsorption, Protein Losing Gastroenteropathy).

C.

Glucose
In chronic liver disease there may be fasting hypoglycemia and prolonged post-prandial hyperglycemia.

*SEE CASE REPORTS 1-6

32

LABORATORY EVALUATION OF URINARY TRACT DISEASE

Terminology
Azotemia Uraemia
the presence of excessive amount of nitrogenous compounds in the blood a clinical syndrome occurring in kidney failure characterized by a variety of systemic and neurological signs. Numerous biochemical abnormalities exist of which azotemia is one the passage of an excessive quantity of urine a reduction in the quantity of urine produced suppression or arrest of urinary output continued inability of the kidney to produce either concentrated or dilute urine the constant secretion of urine of low specific gravity (below 1.008 and often as low as 1.001) a combining form denoting either condition of urine or the presence of a substance in urine e.g. hematuria, crystalluria, bacteriuria the presence of casts in the urine the presence of increased leukocytes (pus) in the urine

Polyuria Oliguria Anuria Isosthenuria Hyposthenuria -uria Cylindruria Pyuria

Introduction
The basis for understanding renal disease and the tests used to detect that disease depends on knowledge of normal structure and function. The primary function of the kidney is to maintain the volume and composition of the extracellular fluid. It does this by: a) regulating water levels b) elimination of metabolic waste products and certain foreign toxic substances c) regulating electrolyte and acid/base balance. Urine is the end result of these processes and is dependent on glomerular filtration, tubular function and interstitial integrity. The glomerular filtrate is an ultrafiltrate of plasma and has a specific gravity of 1.008-1.012. The rate of formation of the glomerular filtrate within a nephron (i.e. the glomerular filtration rate - GFR) is dependent on 2 factors: a) effective filtration pressure in the glomerulus which is equal to the blood pressure within the glomerular capillary minus the osmotic pressure of plasma protein and the back pressure within Bowman's capsule b) renal blood flow.

33

Total glomerular filtrate produced is dependent on the number of functioning glomeruli (nephrons). In the proximal tubules, 85% of the water and solids are reabsorbed. From that point onwards, the tubular fluid is either diluted or concentrated. Water resorption occurs primarily in the distal and collecting tubules and is dependent on the presence of ADH and a hypertonic interstitium (i.e. concentrating power of the kidney is dependent on the integrity of the tubules and interstitium, and the presence of ADH). The tubules are also important in acid/base balance and sodium retention. When evaluating urinary tract disease, examination of the urine is essential. Urinalysis will provide information on renal disease which may or may not be producing renal failure. Urinary tract disease lower than the kidney can in many circumstances be adequately assessed by urinalysis. Urinalysis is commonly combined with a variety of plasma chemical tests to detect renal disease and failure (e.g. Blood urea and creatinine). Acute and chronic renal failure will give rise to uraemia (the clinical condition related to renal failure). Azotemia, oliguria and isosthenuria characterise acute renal failure. The polyuric phase of chronic renal failure is commonly accompanied by loss of tubular concentrating ability (e.g. continued excretion of urine at less than a specific gravity of 1.030 for the dog, less than 1.035 for the cat [value not agreed on and some veterinary urologists would prefer less than 1.040 for the cat this may depend on the refractometer used and whether it has a separate scale for the cat], less than 1.025 for the horse and probably for the cow [value not agreed on, some suggest 1.030 for the horse whilst others suggest 1.020 for the horse and cow) but may or may not be accompanied by azotemia (e.g. in the dog isosthenuria usually has to be reached before significant azotemia is present. This is not always the case in the cat where azotemia can accompany values of 1.020 or less). In the oliguric/anuric phase of chronic renal failure azotemia and isosthenuria are present. The animal is invariably dehydrated. If the chronic renal disease primarily involves the glomeruli and has limited effects on the tubules it will be characterized by azotemia with variable loss of tubular concentrating ability. Commonly there is a significant proteinuria in glomerular disease (often 3-4 + on the urine dipstick). Renal disease progressing to renal failure (i.e. chronic renal disease/failure) is most common in the dog and cat, less common in the horse and rare in farm animals. In farm animals renal disease/failure is often acute and due to infections or poisons. Because a group of farm animals is normally affected, necropsy is often possible and this reduces the need for detailed biochemical or haematological investigations. However, the approach to diagnosis of renal disease/failure in farm animals is similar to that employed for companion animals when only individual animals are affected and they are valuable (e.g. suspected pyelonephritis in a stud or good milking cow).

Some of the Laboratory Tests used to Detect Renal Dysfunction

Assessment of glomerular function
Generally involves measurement of the glomerular filtration rate or related tests.

a)

Clearance tests

Renal clearance of a substance measures the amount of substance cleared from the plasma in a certain time. Urea, creatinine and mannitol are substances that can be measured. However, the clearance value is highly specific for glomerular function only if tubular reabsorption or excretion is minimal e.g. creatinine in the dog is minimally reabsorbed by the tubules; hence the endogenous creatinine clearance test is useful. Clearance tests are commonly used when renal disease is suspected but the animal does not appear to be in renal failure; that is, clinical signs may be vague or intermittent.

34

Urinalysis is also useful to investigate renal disease that has not yet progressed to renal failure. NB: Sulfanilate clearance test: Sodium sulfanilate is eliminated from the blood primarily by glomerular filtration. The test is suggested to be of value in the dog for detecting decreases in renal function before there is development of azotemia or concentration abnormalities.

b)

Blood concentration levels

These are less accurate than clearance tests in estimating glomerular function, but are easier to perform. i) Blood urea (sometimes called blood urea nitrogen). Urea is the end product of catabolism of protein and is formed in the liver. It is filtered by the glomeruli, but a certain percentage is reabsorbed by the proximal tubules. Consequently, blood urea levels are affected by hepatic function, protein metabolism, glomerular function and tubular function. Blood creatinine. Creatinine is formed in the metabolism of muscle. Its blood level is relatively constant, although it can be affected minimally by diet and exercise, and it is excreted by the glomeruli with minimal reabsorption by the tubules (nb. some may be secreted by tubules but this is usually limited). Consequently, it has been suggested that it is a better indicator of glomerular function than urea. It has been suggested that creatinine is more useful than urea in acute renal disease but it is probably wise to utilise both analytes when investigating suspected renal disease of any duration. As most renal failures in dogs and cats are chronic in form, both analytes are likely to be increased. A common method used for the measurement of creatinine (the kinetic Jaff reaction) is affected by substances in the serum (non-creatinine chromagens) which can cause false increases or decreases. Examples of non-creatinine chromagens include acetoacetic acid (ketone body), glucose, cephalosporin antibiotics and bilirubin (nb. hyperbilirubinemia may give false lows).

ii)

Nb. Elevations of urea and/or creatinine warrant a diagnosis of azotemia but don't necessarily mean that the animal is uraemic (i.e. in renal failure). However, all uraemic animals will be azotemic. Azotemia can be divided into prerenal, renal and post-renal in origin. Prerenal causes of azotemia include: i) increased protein intake or catabolism (mainly elevates urea) ii) reduced glomerular filtration rate e.g. shock, haemorrhage, congestive heart failure and dehydration (affects both urea and creatinine). Pre-renal azotemia is common in a variety of diseases. In small animals, the magnitude of urea elevation in prerenal azotemia is usually limited (rarely greater than 40 mmol/L) when compared to renal and post-renal causes. However, the problems are that any level of azotemia can be present in renal disease and that there may be concomitant pre-renal and renal azotemia. Values for urea can reach or pass 100 mmol/L in renal disease and post renal obstruction in the dog and cat. Renal azotemia occurs due to glomerular or tubular damage reducing glomerular filtration rate or total glomerular filtrate. Post-renal azotemia occurs due to urinary obstruction/retention interfering with glomerular filtration and tubular function via backpressure. In farm animals the same principles apply to azotemia. However, elevation of urea in renal

35

failure may not be as marked and consequently, there may be more overlap with pre-renal causes of azotemia. In the horse, blood urea levels in renal failure commonly vary between 18-80 mmol/L. In pre-renal causes of azotemia (especially GIT disturbances) urea levels can reach or pass 50 mmol/L. Consequently, in GIT disturbances in horses the level of pre-renal azotemia is often used for prognostic purposes. In cattle, prerenal causes of elevated urea are important and the levels overlap markedly with those for renal azotemia (10-100 mmol/L of urea in renal failure; 10-60 mmol/L of urea in other disturbances). In ruminants, the rumen has a limited capacity to utilize urea and this may lead to a 'tempering' effect on early elevation of urea with renal failure (the reason why creatinine is more useful in early or mild renal failure). It is unlikely to have an effect when renal failure is advanced.

c)

Urinalysis
Urinalysis may be helpful in determining glomerular dysfunction, e.g. haemorrhage or significant protein loss from glomeruli. However, urinalysis is more useful in detecting tubular dysfunction.

Assessment of tubular function a) b) c) Dye excretion tests - used rarely Blood urea level Tests based on water elimination and reabsorption (urine concentration tests)
e.g. water deprivation tests - generally used to detect concentrating ability in two situations: 1. in animals with polyuria, low specific gravity (<1.030 dog, <1.035 cat [some researchers suggest <1.040], <1.025 horse and cow [some researchers suggest <1.020 for the cow and horse]) and no azotemia 2. in animals with non-azotemic isosthenuria. ADH is released in response to the induced dehydration (usually 3% or more clinical dehydration) and its effect on urine concentration is assessed by determining urine specific gravity or plasma to urine osmolality. Suckling neonates have poor concentrating ability and the above figures do not apply. If the animal is azotemic and/or dehydrated then urine concentration tests are not warranted and could be dangerous. In dehydrated animals the finding of a specific gravity around the isosthenuric range usually suggests renal azotemia. If the urine is concentrated (i.e. above the values mentioned for water deprivation tests) then the animal probably has prerenal azotemia.

d)

Aberrations of acid/base balance

Renal tubular acidosis (RTA) syndrome can produce metabolic acidosis due to either defective bicarbonate reabsorption from the proximal tubules (Type I) or defective H+ excretion from the distal tubules (Type II). There is also a Type IV RTA, which involves defective secretion of potassium and hydrogen ions from the collecting ducts due to aldosterone deficiency or resistance.

e)

Urinalysis
Apart from its obvious uses in detecting renal disease and lower urinary and genital tract disease, it is useful also in analysing many extra-renal problems e.g. diabetes mellitus. Therefore, urinalysis will be presented in a form that covers its uses for all possible abnormalities.

36

Urinalysis involves the following procedures and should be done completely in the first instance as often the results are interrelated. i) ii) iii) iv) v) observation of physical properties estimation of solute concentration chemical analysis sediment examination optional examinations based on the results of the first 4 procedures

The way urine is collected will alter the results of urinalysis. Ideally, a 24 hour sample is necessary for accurate quantitative analysis, but in practice a single sample is analysed. Once the sample has been collected it should be analyzed quickly (within the first 2-3 hours - store at 4 degrees C if analysis cannot be done straight away).

A)
1)

Observation of Physical Properties

Volume - volume estimation ideally should be done on urine collected over a 24 hour period. This will allow the detection of increased volume (polyuria) or decreased volume (oliguria). Volume changes can be transient or persistent (pathological)*. Normally an indication of volume is gained from the measurement of specific gravity (or osmolality). The generalization is that low specific gravity means high volume (exception: acute renal failure, oliguric phase of chronic renal failure). *Transient polyuria (e.g. diuretic therapy, increased fluid intake, parental administration of fluids and administration of ACTH and/or corticosteroids) or pathological (e.g. pyometra, hyperadrenocorticism, diabetes insipidus, diabetes mellitus, polyuric phase of renal failure). Transient oliguria (e.g. decreased water intake, high environmental temperature, hyperventilation in the dog) or pathological (e.g. shock, fever, dehydration, urinary tract obstruction, oliguric phase of renal failure). 2) Colour - The normal colour of urine is dependent on the concentration of urochromes (amorphous pigments and metabolic waste products). Normal urine for most of the domestic species is varying shades of yellow or light amber (Note: horse's urine may turn brown on standing). Turbidity - Normal urine is clear to mildly turbid. Turbidity, if present, indicates crystals, cells, mucus and other formed elements in high concentrations. Turbidity may develop on standing of urine due to the precipitation of crystals or bacterial overgrowth. Odour - The normal odour of urine is derived from volatile organic acids. Abnormal odours include ammonia and ketone bodies.

3)

4)

If abnormal gross characteristics are detected then the rest of urinalysis should detect the reasons for the changes. If the gross characteristics are normal it does not mean that the urine is normal and the other components of urinalysis should still be performed.

37

B)

Solute Concentration
This gives an indication of the ability of the kidney tubules to dilute or concentrate excretory products (i.e. a true function test). In addition, an estimation of solute concentration is essential for the correct interpretation of levels of chemicals and cells. Solute concentration can be determined by measuring the specific gravity of urine. Specific gravity is dependent on the types of solutes, their numbers, molecular size and weights e.g. salts can affect specific gravity significantly because of their low molecular weight and large numbers. Specific gravity is measured today with a refractometer. If the urine is excessively turbid, it should be centrifuged prior to determining specific gravity (if not the specific gravity may be falsely elevated by 002-004 units). Solute concentration (as determined from specific gravity) is inversely related to urine volume in health and in many disease processes. Isosthenuria can be defined as continued excretion of urine at the specific gravity of the glomerular filtrate i.e. 1.008 - 1.012. A specific gravity reading of 1.008 - 1.012 from a single urine sample may indicate: 1) a chance finding in a normal animal (e.g. the dog kidney is able to dilute urine to 1.001, and concentrate urine to 1.060 [1.080 for the cat]) 2) renal failure. Unlike (1), repeated urine samples will have a specific gravity of 1.008 to 1.012 or close to it. Proteinuria or glucosuria may elevate the specific gravity reading (usually by 001-005 units depending on the levels). In renal failure with proteinuria this may take the reading out of the isosthenuric range. Hyposthenuria can be defined as the continued excretion of urine of low specific gravity i.e. less than 1.008. A low specific gravity may be found occasionally in a normal animal but if it is constant it points to such conditions as diabetes insipidus, psychogenic polydipsia and nephrogenic diabetes insipidus. At times the specific gravity may be in the isosthenuric range for these conditions. Beware, healthy suckling animals may have continued excretion or urine below 1.008! Rather than measure specific gravity for an estimation of solute concentration, the osmolality of urine can be determined. This appears to give a better estimation of the solute concentration as it is dependent only on the number of molecules rather than their molecular size or weight. Osmolality is expressed in milliosmoles and is measured by an osmometer (the measurement commonly involves the degree of freezing point depression which is directly proportional to solute concentration). Urine osmolality assessment is normally done in conjunction with plasma/serum osmolality to determine the concentrating ability of the kidney (the ratio of urine to plasma osmolality on single blood and urine samples will provide an accurate assessment of renal concentrating ability e.g. a plasma reading of 297 and a urine reading of the same indicates no concentration by the kidney). Osmometers, however, are expensive and specific gravity will continue to be used for estimation of solute concentration in veterinary practice.

38

C)

Chemical analysis of urine

Reagents strips and tablets are commonly used to detect chemicals in urine. They are screening tests and consequently have limitations. If more accurate measurement of a urinary chemical constituent is required, a 24 hour sample of urine should be collected. NB: Chemical constituents of urine are determined according to concentration. Increases in concentration may occur due to increases in total amount of the chemical constituent excreted (true elevation) or be due to decreased urinary volume which is indicated by an increased specific gravity reading (relative or false elevation). 1) Protein - Urine from a normal animal has minimal levels of protein (mainly albumin). Reagent strips are used as a screening test for protein as they are sensitive to albumin but they are less sensitive to globulins (which will include Bence Jones protein). Highly alkaline urine and urine with high haemoglobin or myoglobin may give false positives on the reagent strip. Consequently, if a positive is recorded on the reagent strip, it is best to check it by another method e.g. sulfosalicylic acid (SSA) precipitation (nb. all herbivores commonly have alkaline urine). SSA is less sensitive to albumin but detects all protein. The magnitude of proteinuria can be assessed relatively accurately by determining the ratio of urine protein to urine creatinine (UP/UC or UPC). Protein is usually assessed by a microprotein method. A UPC ratio less than 0.5 is usually considered normal, a ratio between 0.5-1.0 is minimal or of questionable significance, whilst values equal or greater than 1.00 indicate significant proteinuria. Proteinurias related to glomerular disease usually give the greatest UPC ratios. Proteinuria can be transient/physiological (e.g. exercise, oestrus) or pathological (e.g. cardiac disease, glomerular/tubular damage, lower urinary or genital tract inflammation). Physiological causes of proteinuria usually produce minimal elevations (rarely greater than 1+). NOTE: Haemorrhage: the protein strip may show a 1 to 3+ reading if the blood strip is showing a maximal reading. 2) Glucose - Glucose is passed by the glomerulus and usually completely reabsorbed by the tubules unless the renal threshold is exceeded (cats around 15-16 mmol/L, dogs and horses around 10-11 mmol/L, cattle around 5-6 mmol/L). The 2 main methods of glucose estimation are reagent strips utilizing glucose oxidase and the Clinitest reagent tablets utilising the copper reducing power of glucose and other sugars. The reagent strips can give false negatives in the presence of high ketone or ascorbic acid levels. The Clinitest may give false positives in the presence of antibiotics, salycylates and other sugars. Glucosuria can be transient (e.g. stress, heavy meal of carbohydrate related to transient hyperglycemia) or pathological (most related to persistent hyperglycemia). Transient glucosuria is particularly common in ruminants (as mentioned cows have a low renal threshold of about 5-6 mmol/L) and can occur not only in physiological changes but also in diseases that cause transient hyperglycemia (e.g. neurological diseases, bovine milk fever, ovine enterotoxemia). Transient glucosuria in the cat can occur due to excitement or stress. By the time it is identified the hyperglycemia may have disappeared.

39

In dogs, cats and horses glucosuria is usually related to diseases causing persistent hyperglycemia, and, therefore, is persistent itself. In the dog and cat diabetes mellitus is the common cause but in horses it can occur in both hyperadrenocorticism and diabetes mellitus. Glucosuria unrelated to hyperglycemia is uncommon but in the dog, cat and horse has been recorded related to specific tubular diseases. 3) Ketones - Blood ketones increase whenever there is an increase in lipolysis or an interference with lipogenesis of the TCA cycle (i.e. defective carbohydrate metabolism). Since the renal threshold is low for ketones, ketonuria is often detected before a significant ketonemia develops. Ketonuria in dogs and cats in commonly related to diabetes mellitus. It may be seen also in puppies and kittens that have been starved (rarely in adults). In ruminants ketonuria is non-specific and can occur in starvation, high fat diet, bovine ketosis, ovine pregnancy toxemia and in a variety of diseases that induce anorexia. Ketonuria is uncommon in the horse. Ketones in the urine can be detected by reagent strips (specific for acetoacetate) or by Acetest tablets (reacts with acetone and acetoacetate). 4) Bilirubin - Detected by the Ictotest or by the bilirubin strip which are mainly reactive with conjugated bilirubin and rather insensitive to unconjugated bilirubin. The significance of bilirubinuria has been discussed in the laboratory evaluation of liver disease. In all common domestic species except for the dog, bilirubinuria means conjugated hyperbilirubinemia. In the dog, especially the male, bilirubinuria is not highly specific for conjugated hyperbilirubinemia because of the low renal threshold and a renal capacity to conjugate and even breakdown haemoglobin to bilirubin. However, the presence of marked amounts of bilirubin in the urine of a dog usually means hyperbilirubinemia. pH - In the dog and cat the pH is normally 5.5-7.5. In the normal horse the pH is close to 8. Ruminants have a pH 7.4-8.4. Most young animals suckling, irrespective of species, will have acid urine. Urine pH must be measured promptly and is the result of renal regulation of blood pH. It should not be used alone to evaluate acid/base status as it is heavily influenced by diet, therapy and urinary tract disease. Aciduria occurs with a carnivorous diet, starvation, fever, metabolic acidosis and renal tubular acidosis. Occasionally, aciduria occurs with metabolic alkalosis because of the kidney concentrating more on correcting related electrolyte imbalances (hypochloremia and hypokalemia) and dehydration. This is called paradoxical aciduria, as it is unexpected, and can be seen in severe vomiting, gastric or abomasal reflux (the last two are so called internal vomiting in herbivores) where both HCl and other electrolytes (such as potassium) can be lost to the body. Alkaline urine may be due to a herbivorous diet, cystitis (depends on the type of bacteria present - urea splitting bacteria will commonly produce alkaline urine), urine retention, metabolic alkalosis or extended standing of urine before testing (bacterial action).

5)

40

6)

Blood - The reagent strip test is based on the peroxidase activity of haemoglobin or myoglobin. The test is more sensitive to free haemoglobin than to haemoglobin within the erythrocyte although the two are supposed to be able to be distinguished on the strip (dont rely on the strip for this - examine the sediment for erythrocytes). A positive test means haematuria, haemoglobinuria or myoglobinuria. Haematuria can be distinguished on sediment examination while the other 2 can be distinguished on ammonium sulphate precipitation and plasma colour (plasma needs to be saturated with haemoglobin i.e. obviously red, before haemoglobin appears in urine. This is not the case with myoglobin as it is a smaller molecule and more easily passed by the kidney). Haematuria indicates urogenital tract disease (especially if the sample has been voided), haemoglobinuria indicates intravascular haemolysis or lysed erythocytes (may occur at low specific gravities), and myoglobinuria indicates muscle necrosis.

D)

Sediment examination
What structures are present in the sediment will depend not only on disease but also on how the sample of urine is collected (i.e. voided, catheterisation or cystocentesis). Structures present in urine can be divided into 3 groups: Group 1 consists of those formed elements that are present in health in low numbers. Increased numbers of these structures may have little significance unless indicated by other information e.g. most epithelial cells, many types of crystals, lipid, sperm, mucus, fungi, bacteria. Group II, like group I, consists of formed elements present in health in low numbers. However, increased numbers commonly have significance e.g. casts, erythrocytes, leukocytes. Group III consists of formed elements of sediment that are not normally present in urine e.g. neoplastic epithelial cells, unusual crystals, unusual casts. Their presence in any numbers are significant. Below is information on elements that may be found in urine. Usually urine is concentrated by centrifugation prior to examination to ensure recognition of structures. In this laboratory 10mls of urine is centrifuged and the sediment is resuspended in about 0.5mls of supernatant. Other laboratories may centrifuge 5mls of urine and resuspend sediment in 0.5mls of supernatant: this will alter normal levels of cells presented below. Irrespective of the amount it is important to maintain a standard technique to ensure direct comparability of results. 1) Epithelial Cells *Renal epithelium. In the dog and cat, their numbers are not a reliable indication of renal disease unless they are in clusters and associated with casts. *Caudate epithelium. Primarily derived from the renal pelvis, ureter and prostatic urethra. Increased numbers may indicate disease of the areas mentioned if other indicators of disease are also present e.g. inflammatory cells. *Transitional epithelium. Principally from the bladder. Their significance is questionable. *Squamous epithelium. Derived from the lower urogenital tract and of little significance. They usually indicate contamination.

41

*Abnormal cells e.g. carcinoma cells. These are of obvious significance. 2) Erythrocytes Indicates haematuria if greater than 5 per high powered (x 40 obj) field in moderately concentrated urine. Leukocytes Increased numbers (i.e. greater than 3-5 per high powered [x 40 obj] field) in moderately concentrated urine indicates inflammation in the urogenital tract. In cows, it has been suggested that greater than 8 per high powered [x 40 obj] field is more appropriate for a voided sample. Beware, leukocytes tend to disintegrate in alkaline or hypotonic urines that are left to stand (even if stored at 4oC). Consequently, in some bacterial cystitides with highly alkaline urine there could be few leukocytes present. Additionally, some bacteria may cause direct lysis of leukocytes and lead to false low numbers. Remember, a voided sample of urine may have leukocytes from the genital tract as well as the urinary tract. Casts Casts are precipitates of protein formed in the distal convoluted tubules and the collecting tubules. They conform to the shape of the tubules and may contain any material present in the tubular lumen at the time of formation. A small number of casts, especially hyaline casts, may be found in normal urine (up to one per low powered [10x objective] field). The significance of cylindruria depends on the numbers and their types. However, in moderately concentrated urine greater that 1 cast per low powered (x 10 0bj) field is suggestive of active renal damage, decreased urine flow or renal perfusion, or a response to renal damage. Persistent cylindruria is more significant than transient cylindruria in identifying renal damage. *Hyaline casts. Increased numbers have been noted in mild renal tubular damage, fever conditions, and as a consequence of surgery or exercise. *Granular casts. When hyaline casts have refractile particles embedded in them, they are called fine or course granular casts. The granules were thought to be derived from disintegrating tubular epithelium and, therefore, indicate tubular degeneration and necrosis, but recently the granules were shown to be composed of fractions of various serum proteins. Therefore, granular casts have the same significance as hyaline casts. These are the most common type in dogs. *Waxy casts. They are thought to be derived from granular casts and indicate advanced renal disease. The presence of any waxy casts is significant. *Fatty casts. Granular casts with a high degree of fat. These are the common type in the cat. *Renal epithelial casts. Well preserved renal epithelium within the cast may indicate acute tubular damage. Any casts are deemed significant. Casts may also contain erythrocytes and leukocytes (any number are significant and suggest cell origin in the tubules), and may be stained with bilirubin.

3)

4)

42

5)

Lipid droplets Increased amounts may occur in obesity, high fat diet, hypothyroidism and diabetes mellitus. Lipid may be seen in normal dog and cat urine (very common in the cat). The lipid is derived from the renal tubular epithelium. Spermatozoa Fungi The yeasts and hyphae are nearly always contaminants. Significance will depend on the presence or absence of inflammation. Fungal cystitis can occur, mainly with Aspergillus spp, and should be accompanied by increased leukocytes. Bacteria The significance of bacteria in the urine will depend on determining an association with inflammation in the urogenital tract. Occasionally, leukocytes will not be obvious due to the destruction by bacterial toxins, low specific gravity or highly alkaline urine. Consequently, inappropriate bacteruria without pyuria should be investigated further. Cystocentesis should provide a sterile urine sample, whereas a voided urine sample will be invariably contaminated with bacteria from the lower urethra or genitalia Crystals Crystals are frequently found in urine and their precipitation is dependent on their concentration and the pH of the urine. They are usually of little significance but leucine/tyrosine/ammonium biurate (the last may occasionally occur in healthy dogs, especially Dalmatians) crystals may be indicative of chronic liver disease and/or portosystemic shunting, oxalates (mainly calcium oxalate in the monohydrate form) of ethylene glycol toxicity, sulphonamides of nephrosis, and bilirubin or haemoglobin crystals of acute haemolytic states or haemorrhage.

6) 7)

8)

9)

Miscellaneous Biochemical Alterations that may occur in Various Forms of Renal Disease
1. Non-regenerative anaemia. Commonly occurs in chronic renal failure due to a combination of factors including decreased erythropoietin action, toxic depression of bone marrow and enhanced red cell fragility. The degree of anaemia is extremely variable and can vary from mild to life threatening. Metabolic acidosis. This is due to the retention of acids (titration acidosis) and is dealt with in the section on acid-base inbalances. Metabolic acidosis is common in renal failure in dogs and cat and a little more variable in horses. In cattle, there is likely to be normal acid-base balance (as measured by total CO2 or Bicarbonate) or metabolic alkalosis because of rumen atony and HCl sequestration (internal vomiting). Similar mechanisms may operate for some cases of renal failure in dog, cat (excess external vomiting) and horse (excess internal vomiting), with normal acid-base balance the consequence. In those cases, there is still retention of so called uraemic acids, which can only be detected by determination of an increased anion gap (see later section on acid-base imbalances). Hypercholesterolemia. May be seen in certain renal diseases and conditions (e.g. the nephrotic syndrome), probably due to derangements of lipoprotein metabolism and or the presence of acute phase reactants (mainly lipoproteins).

2.

3.

43

4. 5.

Hyperamylasemia/hyperlipasemia. May be seen with high azotemia in uraemia in dogs. The reasons for elevation are not completely clear. Hyperphosphatemia. This is common in chronic renal failure in dogs and cats. Persistent hyperphosphatemia can lead to renal secondary hyperparathyroidism and variable effects on serum calcium levels (they may be decreased, normal or rarely increased). In horses, hyperphosphatemia is not common in renal failure except in young horses (usually less than 1 yo) where there might also be hypocalcemia; in fact, hypophosphatemia (or normal levels) is more common in chronic renal failure in the adult horse. In contrast to dogs and cats, hypercalcemia is not uncommonly seen in oliguric or anuric chronic renal failure in the adult horse (the adult horse's kidney may be more important in calcium excretion rather than retention in relation to calcium rich diets). In acute renal failure in the horse the blood calcium levels are commonly low to normal. In cattle, serum calcium levels are extremely variable in renal disease, but hypocalcemia is probably more common. Hyperphosphatemia is common in acute renal failure, but is rarely dramatic.

6.

Hyperkalemia. This is more commonly seen in acute renal failure and may be related to metabolic acidosis as well as renal shutdown. It is not as common in chronic renal failure, but can occur in the oliguric and anuric stages. In cats, hypokalemia can actually accompany chronic renal failure (possibly due to chronic metabolic acidosis, renal loss and decreased intake) and lead to a specific myopathy (mainly muscle weakness) if the potassium is extremely low. This syndrome has not been noted in other domestic species. Hypokalemia may also be seen in the recovery phase of acute renal failure due to kaliuresis. Cattle may get hypokalemia in renal failure due to decreased intake, but hyperkalemia is more common in acute renal failure. Hyponatremia and hypochloridemia may occur with renal failure, particularly in the horse and cow. Hypermagnesemia may occur in renal failure in monogastric animals and occasionally in ruminants.

7.

*SEE CASE REPORTS 7-13

44

LABORATORY INVESTIGATION OF SOME DIGESTIVE TRACT DISORDERS

Terminology
Malassimilation Maldigestion Malabsorption Diarrhea Melena Steatorrhea Creatorrhea Amylorrhea Lipemia Hyperlipidemia
Collective term for maldigestion and malabsorption abnormality resulting in the failure of normal digestive processes abnormality resulting in the failure of normal absorptive processes increased frequency of defecation and volume/fluid consistency of the faeces the discharge of faeces coloured black by altered blood. Usually indicates small intestinal bleeding increased amounts of fat in the faeces increased amounts of nitrogen (protein) in the faeces. In dogs and cats it is usually detected by increased amounts of undigested muscle in the faeces increased amounts of starch in the faeces milky appearance of serum or plasma caused by increased levels of triglyceride-carrying lipoproteins (e.g. chylomicrons) is an increase in serum or plasma lipids (triglycerides, cholesterol or phospholipids)

Exocrine Pancreatic Disorders

These can be divided into acute pancreatic necrosis (necrotizing pancreatitis) and pancreatic insufficiency. Pancreatic necrosis commonly presents as an acute abdominal problem while pancreatic insufficiency is a major cause of maldigestion. Pancreatic insufficiency will be discussed under problem diarrheas and malassimilation.

Pancreatic Necrosis
In the dog, acute pancreatic necrosis presents as a distinct entity and its cause is still poorly understood (although diet is an important factor). It has been recorded to a lesser extent in the cat and rarely for the horse. Most cases in the dog involve extensive necrosis and consequently, produce severe clinical signs. Lower grade pancreatic necrosis/inflammation can occur with limited clinical signs. The syndrome has also been called acute necrotizing pancreatitis due to the intense inflammation that relates to pancreatic and surrounding fat necrosis (this should not be confused with pancreatitis in most species which can occur due to a variety of known causes such as bacteria or parasites).

45

To confirm acute bouts of pancreatic necrosis in the dog and other species requires: a) Elevation of serum enzymes. In the dog and cat elevations of amylase and lipase often occur in pancreatic necrosis. Lipase appears more reliable. The elevations for both enzymes are not consistent and there are other causes of elevations. Consequently, the 2 enzymes are often used in tandem. In the dog they can rise within 1-3 hours of damage. The extent of the elevation depends on the degree of damage to the pancreas (low up to 3-4x for AMS; low up to 2-3x for LPS). A canine pancreas-specific lipase immunoassay may become the test of choice in the future. In the cat the enzymes are less reliable to confirm pancreatic necrosis/inflammation (this may be partly due to the fact that most cases are probably low grade). In the horse AMS elevations have been recorded in the few reports on acute pancreatic necrosis but, as in other species, need to be supported by other laboratory tests. The trypsin-like immunoreactivity (TLI) test (primarily used to detect pancreatic insufficiency) has been suggested as useful when investigating acute pancreatic necrosis in the dog and cat, but appears less useful than the canine pancreas-specific lipase immunoassay. Increased levels for TLI (e.g. >35 ug/L in the dog) may occur with acute pancreatic necrosis. Like all other tests, it is best used in combination. b) Other supportive laboratory findings for acute pancreatic necrosis (primarily reported in the dog but inconsistent): i) Hamatology. Stress reaction with a left shift; hamoconcentration ii) hyperlipidemia (fasting) iii) fluid imbalance (due to vomiting and loss of HCl) iv) pre-renal azotemia v) non-septic exudate in peritoneal cavity vi) transient, mild hyperglycemia vii) transient hypocalcemia

NOTE: Hepatocellular necrosis and cholestasis may occur secondarily to the acute pancreatic necrosis and these need to be investigated appropriately.

Investigation of Colic in the Horse

Colic (abdominal pain) in the horse is potentially lethal, has numerous causes and is a common condition primarily because of anatomical factors related to the arrangement of the gastrointestinal tract. Consequently, laboratory aids to diagnosis are extremely important to differentiate cause as well as to consider prognosis. Hamatocrit and total plasma protein determinations are important to support assessment of hydration status; blood gas analysis and electrolyte determination are important to detect metabolic acidosis or alkalosis (rapid deterioration in acid-base balance is a poor prognostic sign) or electrolyte disturbances, urea and creatinine determinations to primarily detect the degree of pre-renal azotemia; and serum enzyme determination (e.g. ALP may rise in complicated abdominal disease presumably due to release from intestinal cells; AST and CK will increase due to muscle damage related to rolling). Abdominal paracentesis is perhaps one of the more useful laboratory tests in investigating colic. In the horse, assessment of peritoneal fluid can be done even when an effusion is not present. It is a useful diagnostic tool for the investigation of abdominal disease, and reflects changes occurring on mesothelial surfaces and, to a lesser extent, in abdominal organs. The approach to interpretation of abdominal fluids is the same as for dogs and cats and will be discussed under the section of Body Fluid Analysis

46

Investigation of Problem Diarrheas

Problem diarrheas refer to persistent diarrheas that are refractory to simple treatment or correction. In the dog and cat primary small intestinal disease gives rise to voluminous, fluid faeces and is often divided into those diseases that produce significant steatorrhea and weight loss (malassimilation) and those diseases that are relatively non-steatorrheic. Primary large intestinal disease in small animals does not produce a profuse watery diarrhea and does not significantly interfere with assimilation. However, the faeces may be soft or slightly fluid and there is a marked increase in frequency of defecation. In the horse profuse, watery diarrhea is produced only when the large intestine is involved. Primary small intestinal disease may give rise to malassimilation but diarrhea is limited to a soft consistency of faeces ('dung pat' faeces). The reasons for these differences between the horse and small animals are related to differing functions of the large intestine. In the horse the large intestine is responsible for breakdown of plant material to absorbable substances; water is absorbed primarily along with the volatile acids derived from carbohydrate breakdown. Problem diarrheas in all species should be investigated in the laboratory by the following methods: 1. Faecal examination - used also in acute diarrheas 2. Peritoneal fluid analysis - normally limited to the horse (sometimes cattle) and applied in all types of diarrhea 3. Absorption tests - used for the detection of malabsorption 4. Haematological and serum biochemical tests - these provide supportive evidence for diagnosis e.g. low serum protein in a suspected protein-losing gastroenteropathy (PLG). 5. Exploratory laporotomy, intestinal washings and biopsy (mainly dog, cat and horse).

1. Faecal examination (coprology)

This is the starting point for the investigation of any gastrointestinal disturbance:

i) Gross characteristics
Involves examination for odour, colour, consistency, volume and presence of unusual material. In the dog and cat steatorrhea will commonly give a rancid odour (may be masked by blood and other faecal components). If blood is suspected of being present but is not obvious, then the Hematest can be employed.

ii) Microscopic examination a. Parasites (worms, protozoa) - these should not be overlooked as a cause of diarrhea,
especially in farm animals.

b. Faecal smears - stained for morphology. These may be useful in detecting large intestinal
inflammatory or neoplastic disease (cells) and for detecting certain protozoal organisms.

c. Faecal Occult blood blood in the faeces is not always obvious grossly, so if intestinal
bleeding is suspected then it is useful to test with the Hematest or something similar.

d. Examination for undigested/unabsorbed food particles - examine for the presence of

47

steatorrhea (undigested [unsplit] and unabsorbed [split] fat), creatorrhea (undigested muscle) and amylorrhea (undigested starch). They are screening tests for malassimilation and are best interpreted when compared to control smears of faeces from normal animals on the same diet. They are useful in the dog and cat for preliminary investigation of malassimilation. They are not of use in the horse with malassimilation (LI microbial activity degrades all the undigested/unabsorbed food particles).

iii) Measurement of Trypsin (protease) in faeces and Trypsin-like Immunoreactivity (TLI) in serum for exocrine pancreatic insufficiency (EPI)
In faeces, trypsin can be estimated by gelatin digestion methods or determined quantitatively by a dye binding technique (azoalbumin method). Trypsin activity varies between, and within, animals. Also, diet can have an effect on the trypsin level. A fresh stool is required for analysis as trypsin activity falls rapidly. It is best to check trypsin activity in faeces on 3-4 consecutive days, and take into account results from microscopic examination for undigested/unabsorbed food particles, before deciding on whether an abnormality exists (increased starch, undigested muscle and undigested [unsplit] fat suggests maldigestion commonly due to EPI). A more accurate assessment of pancreatic function can be determined by feeding the dog or cat n-benzoyl-l-tyrosol-p-aminobenzoic acid (BT-PABA). Chymotrypsin attacks the tripeptide to produce PABA which is absorbed and measured in the blood stream (or urine). This test apparently is not affected by malabsorption but is more difficult to perform than trypsin. Another test for EPI in the dog and cat, and currently the test of choice, involves a radioimmunoassay for trypsin-like immunoreactivity (TLI) in serum. The test requires a single fasting serum sample and detects trypsinogen plus other related substances which normally leak into blood from the pancreas. The test is species specific, so there are distinct dog and cat assays for suspected EPI. Dogs with EPI, even if already supplemented with pancreatic enzyme extract, are expected to have serum TLI levels of less than 2.5 g/L (normal range 5-35 g/L). This must be a fasting sample as feeding may increase the level of TLI in dogs with EPI into the low normal range. Increased levels of TLI are supposedly seen in dehydrated or dogs in renal failure due to reduced glomerular filtration, and in dogs with pancreatic inflammation (i.e. acute pancreatic necrosis - see that section). Once again, the test should be combined with other findings and tests for a definite diagnosis of EPI. EPI is most common in the dog but cases have been recorded in the cat. It is the major cause of maldigestion in small animals. In the horse EPI is rare and most cases of malassimilation are due to malabsorption involving the small intestine.

iv) Faecal culture

Usually a low return diagnostic procedure due to the presence of normal flora. More commonly done in horses and farm animals with prolific diarrhea (e.g. for Salmonella spp or Mycobacterium paratuberculosis in ruminants).

2.

Peritoneal fluid analysis - abdominocentesis (see under fluid analysis)

48

3.

Absorption tests
Absorption tests are often difficult and tedious to perform but do provide a more specific assessment of digestion and absorption of specific components of the diet.

i) Tests for carbohydrate digestion and absorption

In the dog and cat they were used to investigate malabsorption usually after simpler tests have been performed (e.g. smears for unabsorbed [split] fat), but today endoscopic biopsies are more likely undertaken. In the horse they are employed after faecal examination as initial tests to investigate malabsorption. a. Oral glucose tolerance test (OGTT) - for the dog after a 16 - 24 hour fast, 2g/Kg body weight of glucose is administered orally as a 20% solution. Blood samples are collected at 0, 30, 60, 90, 120 and 180 minutes and assayed for blood glucose. For the horse, glucose is administered via nasogastric tube at 1g/kg as a 20% solution. A normal response is accepted as an increase in blood glucose levels of more than 100% of the baseline levels after 1-2 hours This procedure measures the monosaccharide absorptive capability of the small intestine and can be used effectively as long as glucose metabolism has not been disturbed by other diseases. b. Xylose absorption test (XAT) - a similar test. Measures the monosaccharide absorptive capability of the duodenum and cranial jejunum. The XAT is often preferred to the OGTT in the horse as the latter test can be affected by endocrine controls and disturbances of glucose metabolism. Moreover, xylose is not normally found in equine blood. In the horse, food is withheld for 12 hours and 0.5g/kg of dxylose as a 10% solution is administered via nasogastric tube. For a normal horse, d-xylose should be detected in the blood at a level of 1.0 mmol/L or greater after 1-2 hours. Other carbohydrate absorption tests may test both digestion and absorption but are not commonly used e.g. lactose tolerance test, starch tolerance test, sucrose tolerance test.

ii) Tests for fat digestion and absorption (for dog and cat not horse)
a. Post-prandial lipemia - the animal is fasted and then fed 4 ml of corn oil/Kg body weight mixed with a small amount of dog food. Blood samples are taken at 2, 4 and 6 hours and the plasma/serum appearance compared with that of a sample taken prior to feeding. Plasma/serum should be clear in the fasting state and develop an obvious turbidity 2 - 6 hours after feeding (the test is normally done by collecting blood in EDTA and utilizing microhematocrit tubes for centrifugation). Nb: The determination of triglyceride levels in serum is better than a visual assessment of turbidity. This test measures the functional integrity of the entire fat digestion and absorption process. Nb: By adding a pancreatic enzyme supplement to the corn oil prior to administration, information can be gained on whether there is a lipase deficiency (and, therefore, the presence of EPI). Assessment for post-prandial lipemia may be done before performing such tests as faecal smears for indigested/unabsorbed food particles. However, the results are not always conclusive and other tests are usually needed to support the diagnosis of malassimilation. b. Fat balance determination - fat levels are determined in the feed and faeces over a certain

49

period of time e.g. 3 days. In the normal dog, 95-98% of ingested fat is absorbed. With malabsorption 80-90% is absorbed. With maldigestion 40-80% is absorbed. Fat balance studies are rarely done for obvious reasons. 4.

General haematological and biochemical testing

These are generally of limited use in investigating problem diarrheas but may detect changes that could be related to GIT diseases (e.g. eosinophilia associated with eosinophilic enteritis; anaemia associated with prolonged GIT bleeding). In protein losing gastroenteropathy (PLG), biochemical testing is necessary to confirm hypoproteinemia (all globulins and albumin usually depressed) and to determine if the gut is the source of loss. As it is difficult to determine protein loss from the gut other causes of hypoproteinemia are eliminated first (e.g. renal disease, hepatic failure and malnutrition). Since many of the causes of PLG can, under the right circumstances, produce malabsorption, appropriate testing will establish a diagnosis by implication (for example, it is highly unlikely in an animal with malabsorption that a hypoproteinemic state is produced by disease other than in the GIT). PLG can develop due to chronic GIT conditions. Such conditions as chronic gastric ulceration, chronic enteritis and colitis, eosinophilic gastroenteritis, intestinal neoplasms (e.g. cat lymphosarcoma), and lymphatic obstruction (e.g. lymphangiectasia) may give rise to PLG. Many of these diseases may give rise to a chronic uncomplicated diarrhea or malabsorption. Consequently, preliminary investigations must have a common basis (e.g. faecal examination). PLG is common in farm animals in relation to enteritis and parasitism, and can develop quickly in the horse with persistent diarrhea and inflammatory bowel disease. Serum folate and vitamin B12 (cobalamin) levels have been measured in cases of malassimilation in the dog. Folate is absorbed in the proximal small intestine while vitamin B12 is absorbed primarily in the ileum. Prolonged anorexia, small intestinal disease and EPI may produce reduced serum levels of folate and vitamin B12. Bacterial overgrowth, which may accompany small intestinal disease (or EPI), is supposed to reduce serum vitamin B12 (by binding and interfering with absorption) but increase serum folate (involves bacterial synthesis). This is not consistent, but can be used to investigate the possibility of bacterial overgrowth.

5.

Endoscopy, exploratory laparotomy, intestinal washings and biopsy

In some cases of chronic SI diarrhea with or without malabsorption, an endoscopic procedure or exploratory laparotomy is undertaken as other procedures have been unable to provide an exact diagnosis. A similar situation commonly occurs with PLG. Intestinal washings may be useful if a parasitic cause is suspected (e.g. giardiasis in dogs and cats). In cases of malabsorption it may be useful to remove tissue for histopathological examination from stomach, liver, duodenum, jejunum and ileum. In large intestinal disease the collection of biopsy material is a simpler procedure due to coloscopy/protoscopy. This procedure may be undertaken early on in the investigation of a diarrhea but should not precede simpler tests such as faecal examination. Large intestinal biopsies should include the full depth of the mucosa and preferably some submucosa.

50

Additional Notes on Malassimilation

Malassimilation, for simplicity, is commonly divided into maldigestion and malabsorption. To understand the significance of tests used in investigating maldigestion/malabsorption, knowledge of the normal mechanisms of digestion and absorption is necessary. It is essential to remember that there are complex interactions between organs (stomach, liver, pancreas, gut, lymphatics) in the process of digestion and absorption. Consequently, disease processes often affect both digestion and absorption in varying proportions (with obvious effects on the results of laboratory tests). Some generalisations can be made on disease processes that lead to maldigestion/malabsorption: a) b) diseases that affect the production of bile salts or the lymphatics have a principal effect on fat digestion and absorption. diseases that affect the pancreas or gut usually have an effect on fat, protein and carbohydrate metabolism. However, in most clinical cases in dogs and cats, the manifestations of carbohydrate and protein maldigestion/malabsorption are grossly overshadowed by those of fat maldigestion/malabsorption. in dogs and cats diagnosis is based on the detection of significant steatorrhea. EPI is the common cause of maldigestion in the dog but congenital or acquired deficiencies of the small intestinal brush border enzymes have been recorded (e.g. lactase). In cats, malabsorption due to small intestinal disease is more common than maldigestion due to EPI. in the horse most cases of malassimilation are due to malabsorption of SI origin. Steatorrhea is not a feature and diagnosis is primarily based on absorption tests. malassimilation is not well recognised in ruminants and pigs but chronic diarrheas with or without PLG are common and should be approached in a similar manner (usually investigations are undertaken when a group of animals are affected - e.g. certain parasites can cause PLG in cattle, sheep and pigs).

c)

d) e)

*SEE CASES 19,28,29

51

LABORATORY INVESTIGATION OF ENDOCRINE DISORDERS, INCLUDING CALCIUM AND PHOSPHATE DERANGEMENTS

Introduction
Information on laboratory investigation of endocrine disorders is available primarily for the dog and the cat, and to a lesser extent for the horse. In farm animals, endocrine disorders are of consequence only if they affect a significant proportion of the herd or flock. Most are dietary related affecting endocrine gland operation, but some could be heritable e.g. calcium homeostasis and the parathyroid gland; hypomagnesemia; intake of goitrogenic substances or inherited goiter. Many of the tests outlined in these notes, especially the hormonal assays, can be expected to change and develop in the future.

Diabetes mellitus (DM)

Diabetes mellitus (hypoinsulinism) is important in both the dog and cat. Diabetes mellitus has been recorded in the horse. This is commonly defined as a disorder of carbohydrate metabolism due to disturbance of the normal insulin mechanism. There may be an absolute or relative lack of insulin. In people DM is divided into insulin-dependent (IDDM - Type I), non-insulin dependent (NIDDM Type II) and secondary DM. IDDM usually develops in children and requires insulin treatment. NIDDM occurs in older people who are predisposed to abnormal interactions of insulin with peripheral tissue receptors. Delayed insulin secretion and impaired insulin action occur and obesity may trigger overt DM. These patients are normally managed by diet and oral hypoglycemic agents. Secondary DM develops because of predisposing disease such as pancreatitis, hyperadrenocorticism, acromegaly, prolonged administration of certain drugs or hormones (e.g. progestogens or glucocorticoids). These patients are treated for the underlying condition but still may require insulin treatment on a long term basis. Many of the causes of DM in the dog and cat are poorly understood. Some are likely to have a genetic basis, but it is well accepted that immune-mediated destruction of islets may occur for both dogs and cats (although the latter has been better documented for dogs). In addition, obesity, infection, pancreatitis and drugs (e.g. glucocorticoids in dogs; megestrol in cats) may predispose. As in people, DM can be loosely divided into IDDM (absolute lack of insulin) and NIDDM (relative lack of insulin; usually due to insulin resistance at tissues or a combination of this and reduced insulin production) based on the animals need for insulin (it should be emphasized that this classification is not strictly followed by all veterinary endocrinologists). IDDM, because it is characterised by beta cell destruction/Loss (immune-mediated destruction [most frequent cause of DM in the dog]; islet amyloid deposition leading to beta cell loss or destruction [common in the cat]), requires insulin treatment. It may be sudden in onset or take some time to develop clinically. NIDDM, as mentioned, is characterised by insulin resistance and, sometimes, dysfunctional beta cells. Consequently, insulin levels may be increased, normal or sometimes decreased. The distinction between IDDM and NIDDM is not always clear-cut as some affected animals (especially cats) may appear to fluctuate between the two during the course of clinical disease. In the dog, IDDM accounts for most cases and tends to occur in middle-aged or older dogs, and ketoacidosis can be a complication. A form similar to NIDDM does occur in the dog but is uncommon and tends to occur in juveniles. In the cat, most cases occur in middle-aged or older cats and more than half appear to be commonly responsive to insulin (i.e. most are IDDM). However, unlike the dog, NIDDM is more common and may account for 30-50% of cases of DM. These cats tend to be obese and resistant to the development of

52

ketosis. Insulin therapy may not be required in these cats and management involves weight reduction, dietary control and oral hypoglycemics. DM in cats (rarely in dogs) may fluctuate from being clinical to sub-clinical and is sometimes referred to as Transient Clinical Diabetes Mellitus. Basically, insulin is an anabolic and is especially important for glucose uptake and/or metabolism in specific organs (liver, skeletal muscle, fat). The actions of insulin in the body are antagonised by a variety of hormones and other substances ('diabetogenic factors'). They are responsible for diabetes mellitus due to a relative lack of insulin (although some reduced insulin production by the pancreas may be involved as well). Hormones that oppose the effects of insulin include glucagon, glucocorticoids, catecholamines, growth hormone, progesterone and thyroxine. In addition, such conditions as azotemia and obesity can be diabetogenic. In the dog and cat the hallmark of DM is persistent fasting hyperglycemia and glucosuria (glucosuria in the dog is uncommon in other conditions due to the high renal threshold [approximately 10-11 mmol/L]; cats may have transient glucosuria in stress but this is not consistent as the renal threshold can be up to 15-16 mmol/L). In these cases it may not be necessary to perform a glucose tolerance test to confirm DM. However, there are earlier/mild forms of DM that do not present with a severe hyperglycemia and glucosuria; and an intravenous glucose tolerance test can be performed to diagnose DM (a flat curve or low glucose disappearance co-efficient suggests glucose intolerance). In all the dogs with DM, the insulin response to the IV glucose tolerance test will determine whether there is an absolute or relative lack of insulin (i.e. if diabetogenic factors are involved). In the horse, although the disease is less common, DM can result from absolute or relative lack of insulin as well as being a secondary form. The most common reason appears to be Secondary DM due to Pituitary adenoma, commonly of the pars intermedia (i.e. pituitary dependent hyperadrenocorticism called Equine Cushings Disease; pituitary adenoma of the pars intermmedia may also give rise to Diabetes Insipidus). NIDDM has been recorded in relation to obese ponies. In most cases of IDDM some increase in ketone production will occur but normally this is controlled by peripheral utilisation of ketones. If this peripheral utilisation of ketones is impaired or exceeded then ketoacidosis may develop. Ketoacidosis appears to be a rare complication of NIDDM in dogs and cats. The test for ketones in urine is not sensitive for the main type of ketone produced early on in DM (hydroxybutyrate). Consequently, early ketonuria may be missed nb. in adult dogs (and probably cats) DM is about the only condition that will give rise to ketonuria; in suckling puppies and kittens ketonuria is sometimes seen with starvation. In the horse, ketonuria related to diabetes mellitus has been recorded, but its commonness in relation to IDDM, NIDDM and Secondary DM has not been distinguished (most likely due to Secondary DM). Other laboratory abnormalities that may occur in DM include hyperlipidemia (nearly all cases and due to increases in tryglycerides, cholesterol and free fatty acids gross lipemia, therefore, is commonly evident), increased liver enzymes (due to fatty change), increased PCV and total plasma protein (dehydration) and electrolyte loss (due to osmotic diuresis), and proteinuria (due to lower urinary tract infection and/or glomerular disease). Other laboratory abnormalities may occur in Secondary DM depending on the other disease (e.g. hyperadrenocorticism will give rise to its own set of laboratory abnormalities) Blood glucose levels can be monitored in treated DM by measuring glycosylated haemoglobin (HbA1) or fructosamine in the blood. HbA1 concentration reflects blood glucose concentrations over the previous 2-3 months while fructosamine levels are supposed to give an indication of blood glucose levels over shorter periods (normally 2-3 weeks).

53

Hyperinsulinism
This is caused by a productive islet cell tumour of the B cells. It is only of significance in the dog. An insulin-secreting neoplasm may be diagnosed by demonstrating a severe hypoglycemia at the time of appropriate clinical signs. However, in most cases confirmation requires the demonstration of an inappropriately elevated plasma insulin concentration at the time when plasma glucose is within or below the reference interval (use of the amended insulin to glucose ratio after fasting the dog).

Hyperadrenocorticism
This refers to the clinical syndrome produced by prolonged and excessive blood levels of glucocorticoids. It can be produced iatrogenically (long term corticosteroid administration), but commonly it is due either to adrenal tumours (usually unilateral) or to adrenal hyperplasia secondary to excessive pituitary stimulation (PDH). Hyperadrenocorticism is most common in the dog, uncommon in the horse and rare in the cat. In the dog and horse most cases of hyperadrenocorticism are PDH. PDH can result from hyperplasia or neoplasia of ACTH secreting cells in the anterior pituitary (and occasionally pars intermedia, especially common type in the horse in which it can lead also to decreased ADH secretion and Diabetes Insipidus, as well as predisposing to Secondary DM ). In the cat both PDH and adrenal tumours have been noted but they have not been well studied because of the rareness of hyperadrenocorticism. If hyperadrenocorticism is suspected clinically then laboratory confirmation requires: Routine laboratory tests (results are inconsistent and species dependent) - elevated ALP isoenzyme (dog) and other liver enzymes (e.g. GGT in the horse), elevated blood glucose (glucosuria in the horse, cat), white cell changes in the blood (a stress response), hypercholesterolemia (in small animals). Hormonal evaluation - this aims at confirming hyperadrenocorticism and differentiating PDH and adrenal tumours. Confirmation and then differentiation should always include correlation with clinical signs and other tests, as all hormonal tests have limitations.

To confirm hyperadrenocorticism: 1) Basal plasma cortisol level - in the dog there is some overlap of levels between normal and hyperadrenocortical dogs. Consequently, other tests may need to be used to confirm (ACTH response test and low dose dexamethasone test). It is also important to remember that basal plasma cortisol immunoassays may cross react with prednisolone and other exogenously-provided steroids (except dexamethasone) to give a false elevation. Horses with PDH have inconsistent increases in baseline cortisol. ACTH response (stimulation) test - plasma cortisol levels are determined pre- and post-ACTH (synthetic ACTH is utilised). In dogs and cats, the post-ACTH sample is taken at one hour in the dog and cat, and 2 and 4 hours in the horse. In normal dogs there is a 2-3x increase in cortisol after ACTH whilst many PDH dogs will show an exaggerated response (4x baseline levels) Nb. some endocrinologists prefer to deal with absolute values for cortisol rather than percentage increases, for example accept a baseline level of cortisol as 28-110 mmol/L and a post ACTH cortisol level for PDH cases as greater than 55 mmol/L for dogs. In the small number of PDH and for the large number of adrenal tumours (less than 50%) that have a normal response to exogenous ACTH and which have normal baseline cortisol levels, the low dose

2)

54

dexamethasone test can be used in the dog. Iatrogenic hyperadrenocorticism will have little or no response to exogenous ACTH administration, so it is the test of choice to confirm the condition. 3) Low dose dexamethasone suppression test - plasma cortisol levels are determined preand post-dexamethasone (usually 8 hours in the dog and cat). In normal dogs there is an extended drop in cortisol. In most cases of PDH and nearly all cases of adrenal tumours there will be no or little suppression of cortisol. One disadvantage of the test in dogs for confirmation is that many non-adrenal diseases may have no or little suppression of cortisol. Nb. The urine cortisol/creatinine ratio has been advocated as a screening test for hyperadrenocorticism (i.e. confirmation or rejection). Increased ratios will not only occur in hyperadrenocorticism but also in stressed dogs due to non-adrenal disease. To differentiate PDH from adrenal tumours: 4) High dose dexamethasone suppression test - plasma cortisol is measured pre- and posthigh dose dexamethasone (commonly 8 hours after). The difficulty with this test is to find the level of dexamethasone that suppresses PDH cases but has little effect on adrenal tumours. A small percentage of PDH dogs will not show adequate suppression (less than 50% of baseline cortisol) and may be misdiagnosed as adrenal tumours (but there is always image analysis. In the horse there is some contention as to whether it effectively identifies PDH cases. It is not commonly used though, because adrenal tumours are rare and there is little need to differentiate them from PDH. 5) Circulating endogenous ACTH - provides accurate differentiation of PDH and adrenal tumours but the test is more difficult to perform. In PDH in dogs, plasma ACTH is usually normal to increased; in adrenal tumours in dogs, plasma ACTH is usually decreased.

Hypoadrenocorticism (Adrenal Insufficiency - AI)

This occurs mainly in dogs due to deficient adrenal production of glucocorticoids and/or mineralocorticoids. Primary AI involves adrenal cortex destruction. Secondary AI is due to pituitary dysfunction. In the horse, there is a lack of substantive evidence for true AI, but it has been suggested as a cause of poor performance in racehorses and endurance horses. Laboratory findings in AI are extremely variable and depend on the degree of glucocorticoid and/or mineralocorticoid disturbance. i) Routine laboratory findings - Aldosterone lack leading to increased potassium and decreased sodium (a ratio of less than 23:1 for sodium to potassium is suggestive of AI in the dog, less than 18:1 is close to conclusive), white cell changes due to lack of glucocorticoids (e.g. lymphocytosis in some cases) and hypoglycemia, anaemia, hypercalcemia, pre-renal azotemia and acidosis. Hormonal assays - these will detect reduced glucocorticoids e.g. resting plasma cortisol, ACTH response test. Dogs with naturally occurring AI (either pituitary or adrenal based) will have little or no response to ACTH administration.

ii)

55

Measurement of plasma ACTH may be useful to differentiate primary nad secondary AI e.g. dogs with pituitary dependent (secondary) hypoadrenocorticism generally have decreased ACTH in the blood while those with the primary adrenal dependent form generally have increased levels.

Hypothyroidism
This is a commonly diagnosed endocrine disorder in the dog and is usually associated with immunemediated lymphocytic thyroiditis (tests for detecting autoantibodies against thyroglobulin, T3 and T4 are available) or idiopathic thyroid atrophy. Secondary hypothyroidism (due to a pituitary lesion) in the dog is uncommon. In the cat it usually occurs secondarily to treatment for hyperthyroidism but a familial form has been noted in Abyssinians. Hypothyroidism occurs rarely in the adult horse (and this has been poorly documented), but neonatal hypothyroidism has been documented in foals born to mares that have ingested excessive iodine during pregnancy. Laboratory diagnosis (mainly applies to the dog) requires: Routine laboratory tests non-regenerative anaemia, hypercholesterolemia, and elevated CK (all these changes are inconsistent e.g. about half of the cases have anaemia, about 80% hypercholesterolemia and about 10% increased CK). Hormonal assessment - most assays for thyroid function involve the measurement of T4 (T3 can be measured but has no distinct advantage over T4). The measurement of serum free T4 has been suggested as being superior to the measurement of serum T4; however, there are still limitations. 1) Baseline T4 (and/or T3) -a major problem in the dog is that 20% of euthyroid dogs have low levels of T4 (due to physiological variation, hypoproteinemia, drug therapy e.g. corticosteroids, hyperadrenocorticism, diabetes, chronic renal failure). TSH (thyrotropin) stimulation test - levels of T4 are determined pre- and post-TSH. Failure of response suggests hypothyroidism (most normal dogs show 3-4x increases in baseline levels, but there are still some euthyroid dogs with other illnesses that fail to respond to TSH). This test not only can be used to confirm most cases of hypothyroidism but also it may help differentiate primary and secondary hypothyroidism (secondary hypothyroidism -which is uncommon and usually due to pituitary lesions causing reduced TSH secretion - will respond to repeated doses of TSH but rarely to one).

2)

Hyperthyroidism
This occurs most commonly in the cat and is usually due to functional adenomas. In the dog , only a small group of thyroid tumours are functional, and not all of these will give rise to clinical hyperthyroidism. It can be confirmed in the laboratory by: Routine laboratory tests (variable) erythrocytosis (about 50% of cats), increased ALT and ALP (about 60% of cats), hyperphosphatemia. Hormonal evaluation - elevated resting T4 (in most cases but because of day to day fluctuation more than one sample may be needed), reduced increase in T4 in response to TSH (stimulation test).

*SEE CASES 31-34

56

Introduction to Calcium and Phosphate Derangements

The controls over serum calcium levels are complex. Changes in blood levels cause rapid alteration in the rate of calcium mobilisation from bone (via parathormone and thyrocalcitonin). Consequently, transient hypo- or hypercalcemias only become persistent if the control mechanisms are exceeded in capacity. In addition to bone mobilisation, blood calcium levels are controlled by gut absorption and renal excretion/retention. Vitamin D, phosphorous levels, thyrocalcitonin (from thyroid follicular cells C cells) and parathormone (from parathyroid gland) interact at all the key tissues to maintain blood levels. In contrast, blood levels of inorganic phosphorus are inexactly and primarily controlled by renal excretion/retention. Increased serum phosphorus will cause a rapid increase in renal excretion (therefore, persistent hyperphosphatemia will not occur until these control mechanisms are exceeded). Decreased serum phosphorus levels, however, are more difficult to correct as the kidney takes time to convert to phosphorus retention. Fortunately, persistent hypophosphatemia is an uncommon occurrence in disease (exceptions include hyperinsulinism, milk fever, in some forms of neoplasia and renal failure in the adult horse). Calcium can be measured by assessing total serum levels (unbound plus that bound to protein [mainly albumin]) or ionised levels. Ideally, ionised levels should be measured as these provide a direct indication of the calcium available for neuromuscular activity and for other body functions. If total serum calcium is measured, altered serum protein levels will have an impact. Many laboratories correct total serum calcium for the level of serum albumin (e.g. corrected calcium = measured calcium + [average albumin - measured albumin, the resultant value is then divided by 40]; where average albumin is 33 g/L for dog, 29 for cat and 30 for horse).

Persistent Hypocalcemia (decreased calcium on two or more occasions)

This can occur in: a) hypoalbuminemia (decreases the protein bound component - use correction formula provided) b) renal secondary hyperparathyroidism (decreased calcium is related to phosphorus retention but at times the calcium may be normal or increased mainly for the dog and cat) c) eclampsia in the bitch, mare or ewe d) parturient paresis (milk fever) in cattle e) hypomagnesemic tetany in cattle (75% of cases have hypocalcemia) f) acute pancreatic necrosis in the dog (50% of cases) g) nutritional hyperparathyroidism ( now less common; related to diets low in calcium and/or high in phosphorous) h) miscellaneous e.g. ethylene glycol toxicity, small intestinal enteropathies.

57

Persistent Hypercalcemia (elevated calcium on two or more occasions)

This can occur due to: a) pseudohyperparathyroidism (related to certain types of neoplasia and operates through multiple mechanisms, which vary with the type of neoplasm). Pseudohyperparathyroidism is seen more commonly in the dog and less so in the cat and horse. In lymphosarcoma (most common cause) and plasma cell myeloma (multiple myeloma) in the dog, cat and horse, hypercalcemia may result from osteolysis through release of a tumour factor (e.g. osteoclast-activating factor in multiple myeloma in the dog) and stimulation of release of parathyroid hormone because of paraprotein binding to ionised calcium. In the dog, anal sac adenocarcinoma is the second commonest neoplasm to cause hypercalcemia, in this case through release of PTH-related peptide. Hypercalcemia has also been reported in other carcinomas, and in some thymomas. b) primary hyperparathyroidism (rare) Hypophosphatemia commonly accompanies (a) and (b) as long as the hypercalcemia has not caused significant renal damage (then the phosphate begins to rise) c) d) e) f) hypoadrenocorticism (in dog - small percentage of cases) renal failure - hypercalcemia is more common in the adult horse than in small animals (see section on renal disease) osteolytic metastatic tumours of bones miscellaneous e.g. ingestion of certain plants.

In (c) to (f) hyperphosphatemia commonly accompanies the hypercalcemia; one exception is in renal disease in the horse where hypophosphatemia is the rule.
*

SEE CASE 35

58

Derangements of magnesium
Blood magnesium levels are primarily dependent on dietary intake, tissue requirements and renal excretion. They may play a role in regulating PTH release. Blood magnesium abnormalities are of greatest concern for ruminants, and are usually related to feed intake. In dogs, magnesium depletion may be related to insulin treatment for diabetic ketoacidosis.

Hypermagnesemia
Persistent hypermagnesemia is uncommon because the kidneys can readily excrete excess magnesium in the blood. Hypermagnesemia can occur due to: a) Renal failure in ruminants and horses b) Iatrogenic (related to treatment with magnesium-rich substances [e.g. magnesium rich laxatives or antacids] to patients already in renal failure.

Hypomagnesemia
Persistent hypomagnesemia is of greatest concern in calves, adult cattle and sheep. Hypomagnesemia may complicate the situation of hypocalcemia in cattle because of its role in regulating PTH release. Hypomagnesemia can occur due to: a) Rapid change in diet in cattle, which may be related to alterations in rumenal magnesium transport b) Calves fed milk only diets low in magnesium (hypomagnesemic tetany milk tetany) c) Feed deprivation in lactating ewes with twins (hypomagnesemic tetany) d) Adult cattle (mostly old cows) grazing cereal crops or lush grass-dominant pasture (hypomagnesemic tetany grass tetany). Grass tetany has a multifactorial aetiology and is incompletely understood.

59

WATER, ELECTROLYTES AND ACID/BASE BALANCE

Introduction
The laboratory can assess hydration status, electrolyte levels and acid/base disturbances for ill patients. This is more useful for management of the illness rather than diagnosis. However, some electrolyte and acid/base analysis do have implications for the diagnosis of some diseases (these will be discussed in the following sections).

Hydration Status (Assessment of Total Body Water)

Dehydration commonly causes hypovolemia which will artificially raise most laboratory biochemical and hamatological values. However, it is PCV, TPP and urine specific gravity that respond significantly to hypovolemia; and, consequently, they are useful laboratory tests to assess hydration. Additionally, urea and creatinine are commonly increased by dehydration due to reduce renal perfusion (i.e. decreased glomerular filtration) and is the major reason for pre-renal azotemia. The measurement of osmolality of blood may provide an indication of changes to hydration status, but only when there is pure water loss. Sodium and chloride are the main contributors to osmolality and if their levels are disturbed in disease the osmolality may not be a good indicator of hydration status. Therefore, electrolyte analysis should always accompany the measurement of osmolality. Osmolality can be measured directly with an osmometer or indirectly by using a formula that utiliss levels of electrolytes and other solutes (e.g. mOsmol/Kg = 2[Na+ + K+]; mOsmol/Kg = 1.8[Na+ + K+] + glucose + urea [if urea or glucose is elevated]). Osmolality of blood is roughly 270-330, with some variation between species (e.g. the cat is normally 308-336). Hypo-osmolality is nearly always due to hyponatremia while hyperosmolality is commonly associated with hypernatremia. Hypo-osmolality, if developing quickly, can cause brain oedema and intravascular haemolysis due to rapid movement of water from the extracellular fluid (ECF) to the intracellular fluid (ICF). A similar situation may occur if hyperosmolality is corrected rapidly. Rarely do other molecules significantly change osmolality (an exception: glucose increases in DM may significantly alter osmolality). Urea is not an effective osmole in the body (i.e. does not add to tonicity) as it freely moves between ECF and ICF (therefore little effect on water movement) but does influence laboratory measurement of osmolality (hence its use in some of the formulae used to derive osmolality).

Electrolyte Status
Commonly Na+, K+ and Cl- are measured. Serum/plasma Na+ is a good reflection of total body Na+. Hyponatremia, normonatremia and hypernatremia may occur with dehydration, normal hydration and overhydration (any combination is possible). The common situations are: a) hyponatremia and decreased extracellular (ECF) water (hypotonic dehydration-hypo-osmol [except for DM where glucose increases may override the sodium loss] as electrolytes are lost in excess of water) e.g. DM (osmotic diuresis), diarrhoeas in horses, well developed hypoadrenocorticism [hypoaldosteronism], renal disease in many cattle. Hyponatremia and normal ECF water (normal hydration) e.g. salt deficiency in cattle; excess saliva loss in horses; sustained exercise in the dog or horse; early hypoaldosteronism

b)

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c) d) e)

normonatremia and decreased ECF water (isotonic dehydration - normally isosmol as electrolytes are lost at the same rate as water) e.g. vomiting, most diarrhoeas in small animals, exudation, hemorrhage, gut fluid sequestration. normonatremia with increased ECF water (edema - water and electrolytes are retained) e.g. congestive heart failure, liver disease in the dog. normonatremia and normal hydration - health

A word of warning, the examples listed above are inconsistent in their effects on sodium levels. Other combinations are less common and occur in particular conditions e.g. hypernatremia and normal hydration in salt poisoning in pigs. Changes in serum/plasma chloride commonly parallel changes in Na+ but with loss of gastric HCL hypochloridemia and normonatremia (or hypernatremia) may occur. Likewise, hyperchloridemia may occur with normonatremia due to bicarbonate loss in metabolic acidosis. Serum/plasma potassium levels are not always a good indicator of total body K+ as much of the K+ is intracellular. However, the circulating levels are important as they indicate the levels available for normal functions (e.g. neuromuscular activity). Serum K+ levels can be altered by internal shifts (extracellular to intracellular and vice versa) and/or by external factors (intake, loss). Commonly, if both internal and external factors are influencing blood levels of K+, the external factors are more important in determining the overall changes. Hyperkalemia due to internal shifts include: a) metabolic acidosis b) hyperosmolality c) tissue degeneration/necrosis d) insulin deficiency e) hamolysis (variable depending on species and breeds within species e.g. erythrocytes of horses and pigs are usually high in K+; in cattle and sheep moderate to low; in most breeds of dogs and cats low), the same effect can occur through high leukocyte or thrombocyte counts (through leakage) f) diarrhoea (mainly calves) Hyperkalemia due to external factors include: a) anuria (usually in acute renal failure or post-renal obstruction not as common in dogs or ruminants for the latter condition) b) hypoadrenocorticism (hypoaldosteronism) c) various drugs e.g. high doses of crystalline penicillin d) polyuric renal disease in the horse e) marked pleural or abdominal effusions in dogs and cats Hypokalemia rarely occurs due to internal shifts but can occur due to external factors such as: a) increased GIT losses (diarrhoea [especially horses] and persistent vomiting; in cattle abomasal sequestration may act the same as persistent vomiting) b) increased urinary loss (kaliuresis) e.g. polyuric renal disease, especially in cat and cow but not usually in the horse. c) decreased oral intake (e.g. anorexia due to disease; mainly in herbivores as they commonly have excess K+ in their diet and their kidneys are used to excrete. With a drop in intake of K+, the kidneys take a few days to adapt to K+ conservation). d) various drugs e.g. insulin, diuretics, amphotericin B e) profuse sweating in the horse f) prolonged exercise in the dog or horse

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Acid/Base Balance
The following is probably an oversimplification of a complex situation, but does provide a starting point to understanding the usefulness of acid/base investigation in the laboratory. Acid/base balance in the body is maintained by blood buffer systems (e.g. bicarbonate buffer), respiratory function (CO2 removal) and renal function (acid secretion, bicarbonate retention). In limited disturbances the blood buffers will keep the pH within the acceptable range (roughly 7.3 - 7.5). In more severe disturbances respiratory adaptation will occur quickly while renal adjustment will occur over a few days. Commonly acid/base disturbances are classified as metabolic, respiratory and mixed. Evaluation of respiratory disturbances requires blood gas analysis. The evaluation of metabolic disturbances commonly requires the determination of blood pH (alterations are called acidemia and alkalemia) and/or bicarbonate (alterations are called metabolic acidosis and metabolic alkalosis). In addition, by determining Na+, K+ and Cl- the anion gap (=[Na+ + K+] [Cl- + HC03-]) can be calculated and used to further elucidate forms of metabolic acidosis and mixed metabolic disturbances. The anion gap (AG) is based on the concept of electrical neutrality in the ECF i.e. anions = cations. In health most cations are Na+ and K+ whereas most anions are Cl- and HCO3. Unmeasured cations (e.g. Ca+, Mg+) are always exceeded by unmeasured anions (e.g. protein, organic acids, inorganic acids) i.e. Na+ + K+ > HCO3- + Cl- to maintain electrical neutrality. The difference is called the anion gap and indirectly measures the level of unmeasured anions. In health, the AG in dogs and cats is 15 to 25mmol/L and in other domestic animals about 10 to 20 mmol/L, but these values will vary from laboratory to laboratory.

FIGURE: Anion Gap based on concept of electrical neutrality in the blood stream and that unmeasured anionscommonlyexceedunmeasuredcations

In metabolic acidosis ([MAc] - lowered bicarbonate, commonly less than 20 mmol/L for most species, but probably a little lower for dogs and cats) the AG may be normal or increased. In MAc due to secretion (loss of HCO3 e.g. diarrhoea, excess loss of saliva in cattle, some cases of renal tubular acidosis) the AG will invariably be normal due to compensatory increases in Cl-. In MAc due to titration (acid excess and titration of bicarbonate e.g. uraemic acids in renal failure; lactic acidosis in shock, hypoxia, grain overload in ruminants, neonatal diarrhoea in calves, some cases of grain induced laminitis in horses ; diabetic ketoacidosis and ketosis in ruminants) the AG increases due to an increase in unmeasured anions (derived from acids). Bicarbonate decreases, but there is little need for chloride increases as the unmeasured anions have increased. In metabolic alkalosis ([MAk] raised bicarbonate, commonly above 25 mmol/L for most species, but usually not significant until closer to 30 mmol/L) the AG is normal to slightly increased. Most cases of MAk are due to vomiting (or abomasal reflux) and involve decreases in Cl- and compensatory increases in bicarbonate. In mixed MAc (mainly titration) and MAk a low serum Cl- and high AG commonly accompanies the close to normal HCO3 (e.g. vomiting and renal failure).

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HAEMATOLOGY
Haematological Disturbances and how they reflect HostPathogen-Environmental Factor Interactions
Haematology, the study of blood, has been traditionally approached from its usage in the diagnosis and treatment of disease. The reason why it can be approached in such a manner, is that the haematopoietic system has fundamental systemic roles through the export of its cellular components. The haematopoietic cells, primarily produced by bone marrow and a lesser extent by spleen and liver, interact with all body tissues through the circulatory system; hence, the circulatory system is a portal for viewing the haematopoietic system. Haematopoietic cells can be primarily divided into erythroid (erythrocytes) and myeloid (leukocytes) series. Megakaryocytes, which produce platelets, are another hematopoietic cell line. The erythrocytes and platelets can perform their major roles, namely gaseous exchanges for tissue cells and haemostasis, respectively, without leaving the circulatory system; but leukocytes have their primary roles after moving into tissues. Their primary roles are very much related to host defences, with leukocytes contributing to both innate and adaptive immunity through inflammation and repair. These roles can be better understood by taking a host-pathogen-environment interaction (HPEI) view of disease (Figure 1). Disease refers to an effect on the host because of detectable (clinical or subclinical) dysfunction or altered structure. There are four basic pathological processes that arise from or are present in disease: degeneration and necrosis, vascular disturbances, disorder of growth and pigmentations and deposits (Table 1). Pathogens are causes of disease. Although, pathogens have become to mean living agents of disease, particularly micro-organisms, by definition physical, chemical, heritable and immune-based causes can also be referred to as pathogens (Table 2). For pathogens to cause disease, there is a need for them to overcome host defences, which are commonly divided into innate and adaptive immunity. Inflammation and repair form part of that immunity after local and systemic damage due to an agent of disease and is regarded as the fifth major pathological process (Table 1). The external environment influences both the capacity of the pathogen to cause disease and the capacity of the host defences to resist disease. Hence, a changing environment can have both positive and negative impacts on the host. Environmental factors may act as direct pathogens (e.g. fire).

63

Agent of disease (pathogen) Effect on host (DISEASE)

Environment

Host defence/response
FIGURE1:Aconceptualframeworkfortheinteractionofhost,pathogenandenvironmentalfactors toproducedisease(effectonhost)

Table 1. Five basic pathological processes arise from HPEI: EFFECT ON HOST 1. Degeneration and necrosis 2. Disorder of growth a. Developmental abnormality b. Hyperplasia/hypertrophy, atrophy, metaplasia, dysplasia c. Neoplasia 3. Circulatory (vascular) disturbances 4. Pigmentation/deposit HOST DEFENCE 5. Innate and adaptive Immunity (includes adaptive behaviour, surface defence mechanisms and response to local and systemic damage [Inflammation and repair]) Nbs The key pathological process, sometimes time course, and the tissue affected are the bases for the morphological diagnosis (e.g. chronic dermatitis) there may be more than one pathological process occurring in a disease; however, one will predominate or be the reason for the presence of the others (e.g. degeneration and necrosis and inflammation may accompany neoplasia neoplasia is still most important and is the basis for the morphological diagnosis)

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Table 2. There are five main groups of causes of disease (i.e. aetiological agents; pathogens) 1. Living agents of disease a. Multicellular (e.g. metazoan such as nematodes; predators?) b. Micro-organisms (e.g. bacteria, protozoa, fungi, viruses, algae) 2. Physical agents (e.g. heat, motor vehicle accidents) 3. Chemical agents a. poisons/toxins b. metabolic disturbances c. nutritional deficiencies and excesses 4. Heritable diseases (genetic based) 5. Immune-based disease (i.e. host immunity is the major cause of the effect on the host through an excessive response e.g. immune mediated allergy, autoimmunity; lack e.g. immunodeficiency and immunosuppression). Immune-based diseases are often triggered by one of the other four groups of aetiological agents Nbs These form the basis for the name for the aetiological diagnosis. Once again, if more than one agent of disease is involved then the key aetiological agent may be determined as the initiating agent or perhaps the agent that has the most impact on the development of disease (as long as they can be identified). For example, immunemediated glomerulonephritis is named because most of the damage to the host is immune-based. However, the trigger may be living agents (e.g. viruses) or chemicals (e.g. certain drugs). An animal with an intussusception (physical blockage of the bowel) actually dies from invasion of living agents and through necrosis of the intestine releasing chemicals. Some diseases may be named after two agents of disease if both have made a significant contribution (e.g. a dog hit by a car, which has led to lacerations of the intestine and the development of septicaemia and death would have an aetiological diagnosis of trauma with secondary bacterial complications). Some agents of disease operate in more than one category (e.g. bacteria may produce toxins; nutritional deficiencies may cause immunodeficiency; genetic disease may express itself through metabolic disturbance or autoimmunity). This is part of the pathogenesis of the disease and raises the concept of agents operating in tandem to cause disease and perhaps death Environmental factors may operate directly as pathogens e.g. motor vehicle accidents (physical damage) Iatrogenic disease (disease induced through inappropriate treatment of disease) is commonly through chemical agents Idiopathic disease (unknown cause) will eventually be placed in one or more of the five known categories

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Why then is a fundamental understanding of HPEI useful for the analysis of circulating blood? Alterations in erythrocytes can be a reflection of disease agents affecting a wide ranges of tissues that lead to impairment of circulation, oxygenation and provision of chemicals (e.g. hormones, iron, protein) vital for effective erythrocyte production (effect on host due to H-P interactions); or due to disease agents that directly act on erythrocyte production or viability (H-P interaction). This is very much a host-pathogen interaction leading to abnormalities of erythrocyte production. Platelets (thrombocytes in non-mammalian species) are principally involved in vascular disturbances (primary haemostasis), but they also provide chemicals that can contribute to the innate immune (inflammatory) response (H-P interactions and effect on host). Leukocytes are fundamentally associated with both innate and adaptive immunity to pathogens and the subsequent tissue damage (Host response - the fifth basic pathological process; a H-P interaction for disease ). Neutrophils are a key cell for phagocytosis of microbes (mainly bacteria) and minor cell damage, monocytes are capable of becoming macrophages that have important roles in macromolecular phagocytosis (particulate cell debris, microbes and foreign material) and antigenpresenting to activate adaptive immunity. Lymphocytes are quintessential for adaptive immunity. B lymphocytes and resultant plasma cells that produce antibodies are responsible for the humoral arm of adaptive immunity, whilst T lymphocytes are responsible for cell-mediated adaptive immunity (host response to pathogens). Null Killer (NK) lymphocytes appear to play a role in innate immunity but can also be recruited in adaptive immunity. Bone marrow is regarded as a primary lymphoid organ as B lymphocyte differentiation originates and continues there into adulthood. Plasma cells are also present in adult bone marrow. Immature T lymphocytes are also produced in bone marrow during early development, but are then transported to the thymus for maturation and export to secondary lymphoid organs such as lymph nodes. Consequently, bone marrow has the potential, even in adulthood to produce both B and T lymphocytes. Eosinophils have a specific role to play in adaptive immunity on mucosal surfaces and to certain groups of pathogens. Usually, prolonged antigenic stimulation (e.g. such as through parasitic, fungal or protozoal) is required before blood levels of eosinophils are consistently elevated. Mast cells can produce chemicals that attract eosinophils in tissues. Basophilia commonly accompanies eosinophilia, probably because both relate to prolonged antigenic stimulation leading to the formation of Ab-Ag complexes. Haematological analysis of circulating blood, and sometimes bone marrow, should then be undertaken in the context of alterations providing vital information on the cause of disease, effects on the host, the host response and environmental influences (Figure 1). It is only through this awareness, and an understanding of how these factors interact, can haematological analysis be utilised to effectively assist diagnosis and treatment of disease. Although the following information is organised along traditional lines, there will be a continual reminder of how haematological disturbances shed light on HPEI, and how this understanding can be utilised in the diagnostic (interpretive) process by the veterinary clinician for the purposes of effective treatment.

Introduction to Haematological Investigation

Blood is studied either (1) to detect disease processes that are primarily affecting the hematopoietic system or (2) to detect disease processes that the hematopoietic system is indirectly affected by or reacting to (e.g. neutrophil response related to inflammation). For (2), it is only by combining the results of haematological analysis with other findings that they become useful e.g. the finding of a non-regenerative anaemia in a dehydrated dog with azotemia and isosthenuria supports a diagnosis of chronic renal failure.

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When analysing the hematopoietic system it is important to remember that there are four components: bone marrow (hematopoiesis); spleen and liver (phagocytic activity and hematopoiesis); peripheral circulation (main site of erythrocyte and platelet function); and tissues (main site of leukocyte action). Results of analysis of the peripheral blood component will be a reflection of what is occurring in all four components. In other words, peripheral blood analysis will provide information on the dynamic equilibrium between production, phagocytic activity and tissue utilization. However, at times the other components will need to be examined to provide a comprehensive evaluation of the problem (e.g. bone marrow analysis).

Some Common Haematological Terms

Acanthocytes:
Irregularly spiculated red cells with projections of varying length and position. Seen in derangements of lipid metabolism (e.g. certain liver diseases) and splenic disorders. They are reported to be common in haemangiosarcoma in the dog.

Anaemia:
1) 2) A reduction below the reference interval for the total circulating red cell mass. A decrease in the haemoglobin value, the packed cell volume, or the erythrocyte count below normal limits.

Anisocytosis:
Variation in the size of cells, usually of the erythrocytes.

Aplastic:
Cessation of blood cell formation.

Azurophil:
Applied to granules seen typically in the cytoplasm of cells of the lymphocytic and monocytic series and the progranulocyte (promyelocyte) of the granulocytic series. The term refers to an affinity for a particular dye and not to the colour of the granules (but the granules are commonly deep red).

Basophilia (basophilic):
Term that, when applied to a cell or cells of the erythrocytic series, indicates that the cell shows no trace of the characteristic haemoglobin colour and that the cytoplasm shows a strong affinity for basophilic dyes.

Basophilic stippling (punctate basophilia):

Erythrocyte that shows blue, basophilic granules and lines scattered throughout (on Romanowsky staining). The material is usually aggregates of ribosomes. Basophilic stippling can occur in lead poisoning and in intense regenerative anaemias (in the latter, the stippling often appears more distinct).

Blood dyscrasia:
An abnormal condition of the blood or the blood-forming organs.

Buffy coat:
The layer of leukocytes, platelets, and nucleated erythroid cells, if any, that collect immediately above the erythrocytes in sedimented or centrifuged whole blood.

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Burr cells:
See Crenation.

Codocytes:
See Target cells

Crenation:
Shrinkage of cells in a hypertonic solution with the formation of irregular margins and a number of regular spikes. Often seen when cells have been in prolonged contact with EDTA. Also seen in smears affected by dirty glassware, slow drying, extreme temperatures, and poor smearing technique. Crenation should be distinguished from echinocytes (burr cells) that occur in kidney failure (they usually have blunt projections).

Dacryocytes ("tear drop" cells):

Have an elongated point and commonly occur in myelophthisic anaemias and in myelofibrosis.

Disintegrated, smudge or basket cell:

Any cell of any series in which the cytoplasmic outline has been disrupted or the nuclear chromatin is no longer surrounded by a membrane. Usually caused by the smear technique but increased numbers may indicate aged or rapidly proliferating cells.

Dhle bodies:
Small (1-2 microns) round or oval, gray-blue bodies in the cytoplasm of neutrophilic leukocytes, thought to be due to incomplete utilisation of RNA during maturation of the cytoplasm. They may be due to toxicity or due to an administered drug affecting metabolism. Nb. domestic cats may contain small Dhle bodies in some of their neutrophils in health

Echinocytes:
See Crenation.

Elliptocyte:
Erythrocytes that are elliptical (rod shape) in wet or dry films. Also called ovalocytes. These may be seen in a myriad of unexplained anaemias. A hereditary form can occur in dogs. Elliptocytes are normal in Camelidae.

Granulocyte:
A leukocyte that contains specific cytoplasmic granules (neutrophils, eosinophils, basophils) regardless of the stage of differentiation.

Heinz bodies:
An intraerythrocytic mass of denatured globin, irregular in shape and appearing as refractile granules when out of focus (in unstained smears). The latter property is responsible for their being called erythrocyte refractile (ER) bodies. In stained smears, intraerythrocytic Heinz bodies present as clear areas near or bulging from the surface. In ruptured erythrocytes Heinz bodies often remain attached to the membrane and appear bluish and round. They are due to certain oxidant drugs and chemicals that cause haemolytic anaemias. Nb: E.R. bodies may be seen in 10% of feline erythrocytes in health (need to stain with 0.5% brilliant cresyl blue in normal saline or another supravital stain to visualise them effectively) and may increase in size and number in various diseases affecting cats (i.e. relatively non-specific). They are most significant when associated with haemolysis.

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Howell-Jolly bodies:
Small, round, densely staining bodies in the erythrocytes that are considered to be nuclear remnants and are usually eccentric in location. More frequently observed in regenerating anaemias and in splenic disorders (the spleen is responsible for removing them). Common in normal horse and cat blood.

Hypochromasia or hypochromatic:
Terms which, when applied to the microscopic appearance of a cell or cells of the erythrocyte series, indicate a significant decrease in density of the characteristic haemoglobin colour. This may be due to either thinness of the cell, decreased concentration of haemoglobin, or both. Normochromasia or normochromatic means normal haemoglobin colour.

Hypochromic:
An adjective describing a blood picture in which the erythrocytes have a saturation index (MCHC) and usually a colour index (MCH) below the reference interval (MCHC is the more important index). Normochromic refers to MCHC and MCH within the reference interval.

Leptocyte:
A thin erythrocyte of decreased volume in relationship to its diameter, often characterised by abnormality of shape. Observed in chronic debilitating diseases and anaemias. They are common in diseases that alter lipid metabolism (e.g. endocrinopathies, liver disease). In dogs, they commonly occur in lead poisoning. Target cells and folded cells are types of leptocytes. Target cells are also called codocytes.

Leukemia:
Neoplastic disease commonly arising in hematopoietic tissue (but not always) in which the type cells commonly appear in the peripheral blood.

Leukemoid:
Resembling leukemia by having a marked leukocytosis and/or having many immature leukocytes in the peripheral blood.

Lupus erythematosus cell:

A neutrophil that is distended by an intracytoplasmic, homogeneous, red-purple body and has the lobes of its nucleus compressed at the periphery of the mass. It is an uncommon occurrence but may occur in Systemic Lupus Erythematosus and sometimes other immune-mediated diseases.

Macrocyte:
An erythrocyte having a diameter exceeding that of the reference interval. Microcyte has a diameter below the reference interval.

Macrocytic:
An adjective describing a blood picture in which the erythrocytes have a volume index (MCV) above the reference interval. Microcytic refers to a MCV below the reference interval; while normocytic refers to one within the reference interval.

Macroplatelet:
Abnormally large platelets that may occur in abnormal production or increased turnover (i.e. a marrow response). Also called megaplatelets and giant platelets. Nb. Thrombocyte equals platelet. All non-mammalian species have thrombocytes.

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Myelofibrosis:
Displacement of bone marrow by fibrous tissue.

Myelophthisis:
Displacement or crowding out of bone marrow by abnormal cells or tissues. Myelofibrosis is a form of myelophthisis.

Platelet (thrombocyte):
A fragment of a megakaryocyte (mammal) involved in haemostasis. It is non-nucleated in the mammal.

Poikilocytosis:
Refers to an abnormality in shape of circulating erythrocytes. Commonly observed in chronic blood loss, iron deficiency and diseases that increase erythrocyte fragility. In many cats, marked poikilocytosis may occur in response to non-specific severe illness (especially liver diseases).

Poikilocyte:
An erythrocyte with an abnormal shape, not to be confused with distortion that results from faulty technique. Poikilocytes are seen in many anaemias. This term encompasses all misshapen erythrocytic forms, e.g. elliptocytes, dacryocytes.

Polychromasia, polychromatic, polychromatophilic:

Erythrocytes that show a faint bluish tint due to an admixture of the characteristic colours of haemoglobin and basophilic erythrocytic cytoplasm.

Polycythemia:
A state of the blood characterized by increased numbers of erythrocytes (therefore, commonly increased PCV and Hb levels).

- penia:
Combining form denoting deficiency of (i.e. reduced numbers).

- philia:
Combining form denoting affinity for (i.e. increased numbers).

Reticulocyte:
Any non-nucleated cell of the erythrocytic series containing RNA, which when supravitally stained with new methylene blue or brilliant cresyl blue will have discernable granules or a diffuse network of fibrils.

Rouleaux Formation:
The arrangement of erythrocytes in a column, with their flat surfaces facing, in which they appear as figures resembling stacks of coins. It is most common in the horse and cat.

Schistocytes (Schizocytes):
Are red cell fragments often seen in vascular disorders (e.g. D.I.C., haemangiosarcoma, vasculitis) due to collision with fibrin strands. Keratocytes (horn cells) are distorted cells that have 2 or more large points and probably form from blister erythrocytes; they too can occur in vascular disorders.

Sedimentation rate:
The rate at which the erythrocytes will fall in their own plasma in a given length of time.

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Siderocyte:
An erythrocyte in which blue granules, 1-20 or more in number, can be demonstrated to contain iron (haemosiderin) by the Prussian Blue reaction.

Spherocyte:
A spheroid erythrocyte of decreased diameter in relationship to its volume and having the microscopic appearance of a hyperchromatic microcyte, commonly seen in autoimmune haemolytic anaemias in abundance. They are supposed to form through phagocytic removal of part of their membrane. Occasional spherocytes can be seen in a variety of diseases associated with erythrocyte damage or enhanced activity of phagocytes. Spherocytes are more obvious in canine rather than feline smears because of the size of erythrocytes.

Stomatocyte:
Erythrocyte with a shape deformity in which there is a linear rather than a central area of pallor. May be seen in certain liver diseases and hereditary stomatocytosis of Alaskan Malamutes.

Supravital staining:
Staining of cells with a stain of low toxicity that will not cause death to the living cells, so that vital and functional process may be studied in the live cells (e.g. supravital stains show RNA in reticulocytes).

Target cells (codocyte):

An erythrocyte with a central rounded area of pigmentation surrounded by a clear zone, with a dense ring of cytoplasm about the periphery of the erythrocyte. They are also called bullseye cells. Nb. They are a form of leptocyte. Folded cells are other forms.

Thrombocyte:
See platelet.

Toxic neutrophil:
A neutrophil characterised by toxic granules, basophilia of the cytoplasm, or vacuoles. Doehle bodies can be loosely called a form of toxic change. Toxic neutrophils commonly suggest metabolic disturbances or infective processes.

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DISORDERS / RESPONSES OF THE ERYTHRON

Erythron:

In haematology, a concept of the peripheral blood erythrocytes and the erythropoieticbonemarrowasonefunctionalunit.

a)

Dynamics of erythrocyte production

Erythrocyte production occurs primarily in the bone marrow (BM) but other organs/tissues (especially the spleen and liver) are capable of production in times of stress in certain species (this is called extramedullary erythropoiesis). The release of erythrocytes from the BM is orderly: mainly normal erythrocytes are released (most abnormal cells are phagocytosed) while immature erythroid cells enter the peripheral circulation in limited numbers (mainly reticulocytes, but some nucleated erythroid cells). Those abnormal cells that do enter the circulation are quickly removed by phagocytes in the spleen and liver (mainly spleen). These phagocytes are also important in removing aging erythrocytes and for pitting the nuclei from released nucleated erythroid cells. Extramedullary hematopoiesis or increased phagocytosis of erythrocytes, if excessive, can cause enlargement of liver and spleen (hepatomegaly and splenomegaly), but splenomegaly is usually more obvious. In times of increased demand for erythrocytes the BM becomes 'leaky' in certain species and releases more immature erythroid cells into the circulation (reticulocytes, nucleated red cells). The release is usually orderly with a greater proportion of later immature cells. If extramedullary erythopoiesis is significantly contributing to erythroid numbers then the proportions of immature cell types may not be as orderly. Distinct disorderly release of immature erythroid cells occurs in many cases of erythroid neoplasia and in dysplasias of the BM (e.g. lead poisoning in the dog). In splenic disorders, it may appear that there has been disorderly release of immature erythroid cells as nucleated red cells may not be pitted (i.e. the nucleus removed by phagocytes) at the usual rate. In addition, there may be increased numbers of Howell-Jolly bodies (nuclear remnants). Erythropoiesis in the BM is under hormonal control mainly through erythropoietin. Other hormones can have an effect on erythropoiesis, usually by altering erythropoietin levels (e.g. thyroxine can enhance levels by altering cell requirements for oxygen; oestrogens can inhibit levels). Androgens may have a direct enhancing effect on erythropoiesis. The naming of erythroid cells is variable between countries. In all terminologies the mature red cell is called an erythrocyte while the immature non-nucleated red cell is called a reticulocyte. Nucleated erythroid cells vary in their terminology (e.g. normoblast, erythroblast etc). In bone marrow, erythroid cells can be divided into those that are capable of division and those that are simply capable of maturation. When the haemoglobin content reaches a critical level in the cytoplasm of a nucleated red cell division no longer occurs and the cell simply matures. One stem cell tends to produce about 16 mature cells in about 3-5 days (this is a generalisation and may not apply to all species). In times of demand this time period can be reduced in the BM and cells will be released earlier into the peripheral blood. Increased production requires increased stem cell input, which usually takes a few days. Erythrocytes have different life spans in the circulation depending on the species (e.g. dogs 110120 days, cats 70, horses 145, ox and sheep 150-160). This must be taken into consideration when assessing how long it takes for a significant anaemia to develop due to interference with bone marrow production. The erythrocytes age and are removed primarily by splenic macrophages, although some do rupture in the circulation. Antibody coating of aging erythrocytes may be important for macrophage destruction even in health.

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b)

Anaemia
When assessing an anaemia, variations in breed (for the species under investigation), sex and age must be taken into consideration, as all these will alter the accepted reference range for a species. For example, the packed cell volume (as determined by the microhematocrit method) for a normal kitten can be as low as 0.25 but in an adult cat it would rarely be below 0.30. In fact the young of most domestic species (especially pre-weaning) will have lower reference values for red cell measurements. In foals even the mean corpuscular volume will be lower than in an adult and has led to misdiagnoses of microcytic anaemia. In some species males have higher reference values than females, while training can enhance values in horses and greyhounds. Anaemia can be simply defined as a reduction in the circulating red cell mass as measured by reduced packed cell volume, haemoglobin concentration and red cell number below the reference interval. This will lead to decreased oxygenation of cells in tissues and a reduced capacity to remove carbon dioxide and transport it to the lungs for expiration. Depending on the level of anaemia, there may be clinical or sub-clinical disease related to cardio-respiratory compensation, and compromised tissue perfusion (effect on host). Anaemia may be the primary presenting complaint (agent of disease directly targets the erythron) or secondary to another disease process (i.e. an effect on the host leads to the development of anaemia e.g. chronic renal disease leading to anaemia through several mechanisms). To understand the etiopathogenesis of anaemia, knowledge of the controls and dynamic equilibrium of erythrocyte production and destruction is required. Some of this information has been presented before under (a) Dynamics of Erythrocyte Production. The following is a suggested approach to investigation of the problem of anaemia (suspected on clinical signs). The goal is to characterise anaemia so that a cause or causes (agent of disease acting directly [primary] or indirectly through another effect on host [secondary]) can be detected to allow effective treatment and control:

i)

Laboratory confirmation of anaemia

Packed cell volume (microhematocrit reading) in conjunction with the total plasma protein (to determine hydration status) measurement can confirm a diagnosis of anaemia. Haemoglobin estimation and the total erythrocyte count may support the laboratory confirmation of anaemia, but they are more useful in classifying the anaemia by determination of the red cell indices.

ii)

Classifying the anaemia on the basis of mechanisms:

1) 2) 3) 4) determine whether the anaemia is regenerativeor non-regenerative regenerative anaemias are due to either blood loss or haemolysis non-regenerative anaemias are due to either reduced or defective erythropoiesis further classification according to etiological agent (often difficult)

Nb: This is an over simplified classification as some agents of disease, either directly or indirectly through other effects on host, cause anaemia through a variety of mechanisms. For example: chronic renal failure in the dog produces anaemia via some haemolysis, some blood loss (e.g. in GIT) and some bone marrow depression; Equine Infectious Anaemia (EIA) produces anaemia primarily by haemolysis but other factors operate that can give rise to bone marrow depression (secondary). In both these cases the net effect can still be classified as either regenerative or nonregenerative on the basis of simple criteria, but variability occurs e.g. in chronic renal failure bone marrow depression, due to lack of erythropoietin, predominates leading to non-regenerative anaemia; in EIA some cases are regenerative but many are non-regenerative.

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1)

Establishment of a diagnosis of regenerative anaemia

A) In the dog and cat the hallmark of regeneration is an increased reticulocyte count (red cells that supravitally stain for RNA). This can be called an adaptive response by the host (i.e. part of the host response to anaemia; repair through regeneration). If the reticulocyte response is significant this will be accompanied by polychromasia, macrocytosis and, possibly, nucleated red cells in the peripheral blood film. After simple blood loss or haemolysis there is a lapse of 2-3 days before the reticulocyte response occurs. In the horse, even though the process of regeneration is similar to that in the dog and cat, reticulocytes do not occur in peripheral blood in health or in anaemias (except occasionally in intense regeneration in adult horses and even then there are low numbers; young foals may show some circulating reticulocytes in regeneration). Consequently, a diagnosis of regenerative anaemia has to rely on serial packed cell volumes (PCV) or bone marrow examination (where the regeneration is primarily occurring). In adult ruminants, reticulocytes do not occur in health but will be present in peripheral blood if intense haemorrhage or haemolysis has occurred. Consequently, any circulating reticulocytes suggest regeneration. However, the level of reticulocytosis does not always give an indication of the intensity of the regeneration, and in mild blood loss or haemolysis, reticulocytes may be absent. In ruminants with regenerative anaemia basophilic stippling is common. This can occur in dogs and cats but is uncommon. Another form of basophilic stippling occurs in lead poisoning, commonly in the dog - this is rarely related to an anaemia. Pigs respond with reticulocytosis in regenerative anaemia. In the adult dog 0-1.5% aggregate reticulocytes occur in health. This can be increased to over 10% in intense regeneration. This figure is an expression of the percentage of red cells in the circulation which are reticulocytes (500-1000 red cells are counted on a peripheral blood smear stained with a supravital stain such as brilliant cresyl blue) i.e. it is a relative figure. It should be corrected for the level of anaemia (and, if possible, maturation times [these are probably not really necessary and besides, only possible for the dog]). By obtaining a corrected reticulocyte count, an anaemia can be called a truly regenerative one. Another way of correcting is by multiplying the reticulocyte percentage by the erythrocyte count to get an absolute reticulocyte count. This is then compared to the reference intervals. Correction for the level of anaemia: Corrected reticulocyte count (%) = observed reticulocyte count x (Patient PCV/average PCV [0.45 for dog; 0.37 for cat]) A corrected reticulocyte count of greater than 1.5% for the dog (and 1% for the cat) is indicative of regeneration. Less than these values suggests an inadequate response by the bone marrow (i.e. the bone marrow is not responding adequately to restore the packed cell volume to its original level). Correction for the level of anaemia and maturation time (reticulocyte production index [RPI] in the dog: RPI = observed reticulocyte count x (Patient PCV/Average PCV) x (1/maturation) Maturation times for the dog - 1.5 days at a PCV of 0.35, 2 days at a PCV of 0.25, and 2.5 days at a PCV of 0.15. These are extrapolated from figures for humans. No values are apparently available for cats.

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A RPI greater than 2 is definitely regenerative, a RPI of 1-2 is an inadequate (sub-optimal) response by bone marrow (i.e. the packed cell volume will not be restored to its original level), while a RPI less than 1 is definitely non-regenerative. Some of the anaemias classified as regenerative on correction for the level of anaemia alone, may turn out to be sub-optimal by this correction method. Note: RPI has only been used in the dog, and then sparingly. It is usually acceptable just to correct the reticulocyte count for the level of anaemia. In the adult cat 0-1% aggregate reticulocytes occur in health. In addition punctate reticulocytes can occur in up to 10% of cells. Usually only aggregate reticulocytes are counted but large numbers of punctate reticulocytes may indicate regeneration even in the absence of aggregate reticulocytes. The reticulocyte count should be corrected for the level of anaemia (average PCV is taken as 0.37 L/L for an adult).

Absolute reticulocyte counts: This is becoming the correction of choice for dogs and cats. The upper level of the reference range for dogs is 0.075 (grey zone to 0.105) x 1012/L and for cats 0.060 (grey zone to 0.100) x 1012/L. If a dog has 4% reticulocytes and an erythrocyte count of 3.0 x 1012/L then the absolute reticulocyte value is 0.120 x 1012/L. This suggests regeneration. In the dog and cat, associated with reticulocytosis, there are related changes to red cell morphology and indices. B) Alterations in red cell morphology on the peripheral blood smear: When a Romanowsky stain (e.g. Giemsa, Diff Quik) is used on a peripheral blood smear, reticulocytes appear as large, bluish cells (macrocytes, polychromatophilic). Consequently, a blood smear in a regenerative anaemia exhibits anisocytosis and polychromasia. Nucleated red cells may also be presented in peripheral blood smears in a regenerative anaemia. However, if they occur unaccompanied by reticulocytes, a non-regenerative anaemia with abnormal marrow or a splenic disorder should be suspected. C) Alterations in the red cell indices. On the basis of mean cell volume (MCV), anaemias can be designated microcytic, normocytic or macrocytic. On the basis of mean cell haemoglobin concentration (MCHC) (and, to lesser extent, mean cell haemoglobin [MCH]) anaemias can be designated as hypochromic or normochromic. Regenerative anaemias, depending on the numbers of reticulocytes, are often macrocytic and hypochromic (depressed MCHC, normal or increased MCH). The changes in the red cell indices last as long as there is a regenerative response; for this reason, a regenerative anaemia is often referred to as a transitory or pseudomacrocytic anaemia. The majority of non-regenerative anaemias are normochromic and normocytic. However, a non-regenerative anaemia associated with myeloproliferative disorders in the cat may be macrocytic, while iron deficiency anaemia is usually microcytic and hypochromic (depressed MCHC and MCH).

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2)

Classification of regenerative anaemias

A) Blood loss anaemia (haemorrhagic anaemia) Primary blood loss anaemia may be caused by physical agents (e.g. trauma), living agents (e.g. blood-sucking agents such as fleas), chemicals and heritable disease (the last two mainly affecting blood clotting thereby leading to increased susceptibility to bleeding). It may also occur secondary to another disease (pathological) process such as degeneration and necrosis of tissues or neoplasia affecting the integrity of vessels or clotting mechanisms. The site of haemorrhage should be found before classifying the anaemia as blood loss. This may be difficult in internal hemorrhage, especially if continuous and low grade. Blood loss anaemia can be further classified depending on whether it is external or internal hemorrhage and acute or chronic. These factors affect the capacity for regeneration and, therefore, laboratory findings in blood loss anaemia e.g. external hemorrhage: loss of iron and plasma protein, if chronic may develop into an iron deficiency non-regenerative anaemia; internal hemorrhage: the components are mainly reabsorbed (about 1/3 of the erythrocytes are reabsorbed and 2/3 phagocytosed) and reutilised. In an acute episode of external blood loss, the PCV is normal initially due to equal loss of components, but splenic contraction may transitorily elevate PCV. After 2-3 hours and to about 48-72 hours, the blood volume is restored by the addition of interstitial fluid which dilutes the erythron leading to lowered PCV and erythrocyte count i.e. laboratory signs of anaemia. Increased erythroid production is usually evident after 2-3 days and should reach a maximum 7 days after the onset of haemorrhage i.e. reticulocytosis. The peripheral blood picture usually returns to normal 1-2 weeks following a single acute haemorrhagic episode. If the reticulocytosis persists longer than 2-3 weeks, continual bleeding should be suspected. In chronic external blood loss the anaemia develops slowly and hypovolemia is not seen. The PCV can reach quite low levels before clinical signs of anaemia become obvious because of adaptive mechanisms by the animal. The animal can develop hypoproteinemia and low body iron, which can cause the regenerative response to develop into a nonregenerative one. B) Accelerated erythrocyte destruction can occur as intravascular haemolysis or as extravascular haemolysis (mainly due to splenic macrophage activity). In a haemolytic anaemia, destruction usually occurs in both sites but one predominates. In haemolytic anaemia there is usually a marked regenerative response (greater than for blood loss) as iron and other components from the red cells can be reutilised quickly (availability of iron decides the magnitude of the regenerative response). If destruction is of a significant magnitude, haemoglobinemia or hyperbilirubinemia may develop. Diseases causing principally intravascular haemolysis usually occur as per-acute or acute episodes wth haemoglobinemia a predominant feature. Hyperbilirubinemia and icterus will occur if the haemolysis is of sufficent duration and excessive. Neutrophilia, sometimes with left shift (see Disorders/responses of Leukocytes) is common in haemolytic anaemias in the dog, but the mechanisms for the neutrophilia are poorly understood. Diseases principally causing extravascular haemolysis (i.e. phagocytosis by the mononuclear phagocytic system especially in the spleen) are usually more chronic with an insidious onset. Haemoglobin is not present in the blood or urine and hyperbilirubinemia occurs only in a minority of cases. Depending on the cause, some of these diseases can ultimately produce a non-regenerative anaemia due primarily to bone marrow depression (the mechanisms are not fully understood). In other cases the regenerative response may

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adequately compensate for the destruction, thereby producing a normal PCV (compensated haemolytic anaemia). The blood smear and bone marrow should show the regenerative response. Causes (agents of disease) of haemolysis include living agents (e.g. haemobartonellosis), chemicals (e.g. acute copper poisoning), physical (e.g. microangiopathic haemolytic anaemia fibrin strand damage to circulating erythrocytes), genetic (e.g. pyruvate kinase deficiency) and immune-mediated (e.g. auto-immune haemolytic anaemia). Whether these agents cause primarily intravascular or primarily extravascular haemolysis depends on the degree of damage to the erythrocyte. Many agents can cause both intravascular and extravascular haemolysis, but one usually predominates. Some agents can predominantly cause direct haemolysis (e.g. acute, high dose copper poisoning in sheep) whilst others may predominantly cause sub-lethal damage to the erythrocyte membrane (e.g. some blood parasites such as Anaplasma spp) which predisposes to splenic macrophage scavenging and destruction.

3)

Classification of non-regenerative anaemias

Non-regenerative anaemias due to reduced erythropoiesis are usually slow in onset and have a chronic clinical course (the rate of reduction commonly relates to the life span of the circulating erythrocytes). The agent of disease may be causing some blood loss or destruction, but depression of bone marrow erythropoiesis is more important and produces the overall non-regenerative anaemia. In other cases the non-regenerative anaemia is due to defective erythropoiesis rather than reduced erythropoiesis. These are less common and, depending on the cause, can have a variable time course. Whether reduced or defective erythropoiesis, BM examination is warranted when investigating non-regenerative anaemias particularly of undetermined origin. A classification for, and some causes of, nonregenerative anaemia are provided under the General Comments on Bone Marrow Examination.

Some General Comments on Bone Marrow Examination

Bone marrow is the primary site for haematopoetic production. Consequently, bone marrow examination allows assessment of production of all cell lines. When peripheral blood examination reveals the presence of persistent unexplained leukopenia, non-regenerative anaemia, thrombocytopenia, or abnormal cell types, bone marrow examination should be considered. Bone marrow examination can often confirm a leukemia and help differentiate it from a leukemoid reaction in the dog. In the horse, bone marrow examination is useful also in establishing a diagnosis of regenerative anaemia (although serial PCVs are more commonly done). A number of deficiencies/Limitations exist for bone marrow examination. They include the difficulty of obtaining representative samples of bone marrow, the problem of peripheral blood 'contaminating' bone marrow, the lack of a method for quantitative evaluation of cellular elements in bone marrow and inter-individual variability in differentiating various bone marrow cell types. Bone marrow smears are usually in the form of 'squash' preparations and are stained with a

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Romanowsky stain (e.g. Giemsa, commercial Diff Quik). Bone marrow particles are identified under a lower power objective (4-10x) and megakaryocyte numbers assessed (commonly 5-10 in a particle per 10x objective is considered normal for the dog). Individual nucleated cell types are differentiated under the oil objective (100x). A minimum of 500 cells are differentiated and a myeloid (granulocytes) to erythroid ratio (M:E) obtained. This term expresses the proportion of total granulocytes to nucleated erythroid cells. For healthy animals, the following M:E ratios apply: Dog M:E of 0.75-2.53:1 (average 1.25:1) Cat M:E of 0.6 -3.9:1 (average 1.6:1) Horse M:E of 1.1 -10.2:1 (average 2.43:1) Specific notes on bone marrow examination and M:E ratios: a) Authors very greatly in the values presented for M:E ratios. The above figures are a guide only. They are derived from Schalms Veterinary Haematology (4th Edition) and supported by our laboratorys findings. b) Within the myeloid and erythroid cell lines it is useful to differentiate the maturity of cells and to divide them into proliferating and non-proliferating pools. In health, approximately 80% of granulocytes should be in the non-proliferating pool (mainly metamyelocytes onwards - no division, just maturation), and approximately 90% of erythroid cells should be in the nonproliferating pool (mainly intermediate to late normoblasts [nucleated erythroid cells]). c) The M:E ratio should not be interpreted without knowledge of peripheral blood cell counts. The M:E in regenerative anaemia is usually reduced (i.e. marked increase in erythroid series, normal to slight increase in myeloid series) with an increase in the proportion of erythroid cells in the proliferating pool (greater than 10%). d) The M:E in non-regenerative anaemias is variable.

Non-regenerative anaemias can be classified as follows: i) Reduced (hypoproliferative) erythropoiesis In domestic animals, causes of non-regenerative anaemia commonly tend to be in this category. Normocytic normochromic anaemia; normal neutrophil and platelet production; increased M:E (hypocellular erythroid) A common type 1) Anaemia of erythropoietin lack - chronic renal disease and certain endocrinopathies. 2) Anaemia of chronic disease (inflammatory or neoplastic) - iron is unavailable for erythropoiesis due to sequestration into macrophages (availability of iron determines the intensity of regeneration). Other factors may operate depending on the cause. The anaemia is mild to moderate and may become hypochromic, microcytic in the later stages. A common cause of mild anaemia, especially in farm animals. Note: a mild to moderate anaemia can occur in acute inflammatory disease (e.g. within 4-5 days after abscess induction in a cat) and appears to be primarily due to accelerated erythrocyte destruction and utilisation of iron by the bacteria. The accelerated destruction is possibly due to antibody coating of erythrocytes and destruction by macrophages. This mechanism may also contribute to anaemia in chronic disease. 3) FeLV-associated non-regenerative anaemia (FIV may also produce something similar). 4) Immune mediated red cell aplasia (intramedullary destruction of erythroid cells).

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Normocytic, normochromic anaemia; decreased neutrophil and platelet production; M:E difficult to determine due to too few cells: 1) Aplastic anaemia - certain chemicals and a range of therapeutic drugs (effects on neutrophil and platelet production seen earlier as these cells have shorter life spans). 2) Myelophthisic anaemia - replacement of BM with abnormal cells and/or fibrous tissue (myelofibrosis). 3) FeLV induced anaemia. 4) Feline infectious panleukopenia virus induced anaemia (inconsistent late finding in the disease). Normocytic, normochromic anaemia; increased production of neutrophils; increased M:E 1) Granulocytic leukemia. 2) Feline infectious panleukopenia virus in recovery. ii) Defective (hypercellular) erythropoiesis The M:E is usually reduced because erythroid precursors are numerous in the bone marrow. Abnormal size, haemoglobinisation and/or asynchronous nuclear-cytoplasmic maturation may characterize the erythroid precursors. Microcytic, hypochromic anaemia; Variable M:E due to variable effects on myelopoiesis 1) Iron, pyridoxine and copper deficiences. Macrocytic, normochromic anaemia; M:E usually reduced 1) Erythremic myelosis or erythroleukemia. NB: lead poisoning in dogs gives rise to defective erythropoiesis but rarely gives rise to anaemia and is usually diagnosed without the examination of bone marrow.

*SEE CASES 21-24

Polycythemia
Polycythemia refers to an increased red cell mass i.e. the PCV, total erythrocyte count and haemoglobin concentration are increased. Polycythemia can be divided into: a) Spurious or relative polycythemia An increased PCV but the red cell mass is normal e.g. haemoconcentration through dehydration or shock; splenic contraction in a young horse or cat. b) Absolute polycythemia An increased PCV due to an increased red cell mass (i.e. increased erythropoiesis). Plasma volume and protein are normal. Absolute polycythemia may be regarded as primary (myeloproliferative disorder of unknown etiology) or secondary (due to increased erythropoietin production). Secondary polycythemia may be appropriate (e.g. in chronic hypoxia) or inappropriate (e.g. erythropoietin producing tumour of the kidney).

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DISORDERS / RESPONSES OF THE LEUKOCYTES

Introduction
Leukocytes have fundamental roles to play in the innate and adaptive immune responses to agents of disease (host response), but like all cells can be affected by degenerative processes and disorders of growth (primarily neoplasia). The numbers and the appearance of leukocytes can be easily examined by taking a sample of peripheral blood, which can be supported by bone marrow examination. Peripheral blood levels of leukocytes are a reflection of bone marrow supply (occasionally extramedullary leukopoiesis in the spleen and liver), tissue demand and their distribution within the circulation (marginal or circulating pool). This particularly applies to neutrophils (Figure 1). Only the circulating pool of leukocytes is measured in peripheral blood.

FIGURE1:Leukocytes(neutrophils,eosinophilsandmonocytes)canmovebackwardsandforwardsbetween thecirculatingpool(CP)andthemarginalpool(MPcapillarybeds).Thisismainlyimportantforneutrophils, wherethepoolsarereferredtoasmarginalandcirculatingneutrophilpools(MNPandCNP,respectively). The storage pool of bone marrow is an important supply of leukocytes to the CP. Extramedullary hematopoiesiscancontributetotheCPwhennecessary.Mostneutrophilsarelostinthetissues.

Leukocytosis
Note: the evaluation of peripheral blood levels of leukocytes is determined on absolute values rather than relative values (i.e. the percentages). The main exception to this is the ratio of the relative values of segmented to band neutrophils in determining whether a left shift exists. Another exception is perhaps the neutrophil to lymphocyte ratio used in farm animals. Leukocytosis per se is not of significance; what is important is the reason for leukocytosis: is it due to

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neutrophilia (most common cause in dogs, cats, horses and pigs), lymphocytosis (contributes more so in ruminants), eosinophilia, basophilia or monocytosis? The detection of leukopenia must be treated in the same manner; that is, the cell types involved must be determined. In the dog, cat and horse leukopenia is usually due to neutropenia, although lymphocytopenia may contribute. In ruminants, lymphocytopenia can be the reason for leukopenia, either alone or in conjunction with a neutropenia.

Neutrophilia
Neutrophils are produced in the bone marrow from a precursor cell that is capable, under the right stimuli of also producing monocytes. Neutrophil production is regulated by granulopoietin, which is produced by a variety of cells primarily in response to depletion of the bone marrow storage pool (there are about 4-5 days supply of mature neutrophils stored in the bone marrow). Granulopoietin stimulates neutrophil production and replenishes the storage pool. Some bacterial products stimulate granulopoietin production. Neutrophil release from the BM storage pool can be promoted by a plasma factor (called either leukocytosis inducing factor or neutrophil releasing factor). The factor's production is stimulated by bacterial products and neutropenias. The factor causes a rapid neutrophilia and a depletion of the bone marrow storage pool (this in turn causing granulopoietin release). If the process continues (e.g. continued tissued demand), then immature neutrophils appear in the circulation. Neutrophils survive about 10 hrs in the circulation where they are distributed between a measurable circulating neutrophil pool (CNP) and a non-measurable marginal neutrophil pool (MNP - slow moving cells normally adjacent to small vessel walls, especially in capillary beds). Most neutrophils are lost in tissues or secretions/excretions (see Figure 1). Neutrophilia may occur in response to physiological reasons, release of corticosteroids, tissue (inflammatory) demand and regenerative anaemias. Changes to the numbers of other cell types may help to differentiate the types of neutrophilia. In most situations neutrophilia due to inflammatory demand is of importance (indicating a host response to either a disease agent or to damaged tissue); other causes of neutrophilia merely interfere with its detection. Basically, the mechanisms for neutrophilia are: a) b) mobilisation of the cells in the MNP increased rate of release from the storage department (due to leukocytosis inducing factor).

(a) and (b) are the reasons for rapid neutrophilia (few minutes to 2-3 days) c) increased neutrophil production (controlled, in part, by granulopoietin). Increased stem cell input will take 3-5 days before changes appear in the CNP. Increased effective granulopoiesis (additional divisions) takes 3 days before changes appear in the CNP.

Physiological neutrophilia (leukocytosis)

Physiological leukocytosis (principally due to neutrophilia) may occur in such situations as parturition, lactation or after eating (e.g. 1 hr post-prandial in the dog), but the significant increases occur due to release of epinephrine in response to exercise, fear or excitement. Physiological leukocytosis is thought to be a pro-active protective host response, but does not always prove useful. Physiological leukocytosis due to excitement supposedly only occurs in healthy animals and is

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common in the young cat and horse (levels of leukocytes can reach 20-25 x 109/L), in which both neutrophilia and lymphocytosis occur. The neutrophilia occurs because of shift of cells from the MNP to the CNP. In the cow eosinopenia appears to occur in physiological leukocytosis.

Corticosteroid induced neutrophilia (leukocytosis)

This response commonly occurs in sick animals with severe illness (this is usually seen in acute illness, but may occur during intense episodes of recurrent or chronic disease, or in terminal disease). It is often produced by pain or temperature extremes (e.g. fever) and is more common in the dog than in the cat and horse. In the horse excessive (over-exertion) muscular activity can produce typical changes, whilst in cattle a variety of conditions (e.g. ketosis, milk fever, dystocia, GIT disturbances) have been recorded as producing corticosteroid induced changes. In pigs and cattle strenuous exercise and difficult parturition may cause corticosteroid induced changes. Corticosteroid induced neutrophilia has as its basis protection for the host, but often this goes awry because of the alterations to other leukocytes. The neutrophilia is accompanied by lymphocytopenia (lymphopenia), eosinopenia and, in some species (e.g. dog and inconsistently in the cat), by monocytosis. In the horse and cow monocytopenia is apparently more common in acute stress. This is often followed by a later monocytosis. A left shift for the neutrophilia does not occur unless there is another disease process causing inflammatory demand. Lymphocytopenia is rarely dramatic in horses, whilst eosinopenia is not easy to detect in cattle. The neutrophilia occurs due to a shift from the MNP to CNP, decreased migration from peripheral blood and release from the BM storage pool. Lymphocytopenia (may just be low normal) occurs due to a redistribution of lymphocytes in the short term (held in the lymphoid tissue) and a selective lymphocytolysis in the long term. Eosinopenia is probably due to a shift from the CP to the MP and an inhibition of bone marrow release (eosinopoiesis in BM still continues and a rebound eosinophilia may occur if the animal recovers). Monocytosis is probably due to a shift from the MP to the CP. In the dog total leukocytes can reach 40 x 109/L or greater, but, commonly, levels are between 15-25 x 109/L. In the cat values of 30 x 109/L or greater have been recorded, while in the horse levels are usually around 20 x 109/L. Cattle may show up to 18 x 109/L total leukocytes. These are guidelines only and assessment must be supported by appropriate changes in levels of individual cell types. Nb. Hyperadrenocorticism in dogs will produce similar changes in white cells. However, in long term hyperadrenocorticism the magnitude of the neutrophilia will be diminished.

Neutrophilia due to regenerative anaemia

Neutrophilia commonly accompanies both haemorrhagic and haemolytic anaemia if there is moderate to intense regeneration. The response is more pronounced in haemolytic anaemia, in which neutrophilia with left shift may occur (this has been primarily detected in the dog). The mechanism for the neutrophilia is not well understood

Neutrophilia related to inflammatory demand

This usually occurs due to purulent inflammation due to infection, but can occur due to tissue damage (i.e. degeneration and necrosis) related to another disease process e.g. neoplastic necrosis; pancreatic necrosis. The degree of neutrophilia and left shift present in the peripheral blood depends on: the degree of tissue demand for neutrophils the reserves of neutrophils available in the MNP and BM storage pool the ability of BM to increase production.

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In some types of inflammation (e.g. cystitis, catarrhal enteritides) where there is limited tissue demand, neutrophilia may not be necessary as the existing circulating levels of neutrophils can cope. As tissue demand for neutrophils increases variable levels of neutrophilia develop due to a shift from the MNP and release from the BM storage pool. The level of neutrophilia depends on the balance between BM production and storage, and the loss of neutrophils from peripheral blood into the tissues (see Figure 1). For example, in some purulent diseases an initial neutropenia may occur due to demand exceeding supply (i.e. more neutrophils are moving into tissues than are moving into the CNP). In good prognostic cases this is quickly followed by neutrophilia due to enhanced production and release in the CNP. A left shift (the release of an increased proportion of immature neutrophils into the peripheral circulation) will occur in tissue demand if the BM storage pool becomes depleted for any length of time and granulopoietin levels increase. The left shift will disappear as soon as increased production restores storage pool numbers. If a left shift doesn't accompany a neutrophilia it doesn't mean that tissue demand is not occurring (the BM storage pool may not have been depleted or it may have recovered), but it is possible also that the neutrophilia is due to another reason such as corticosteroid release (this is more likely in acute, intense illness i.e. clinical signs will provide invaluable information for differentiation). In acute onset, intense inflammation there is likely to be a corticosteroid-induced component to the inflammatory demand neutrophilia (e.g. acute pancreatic necrosis in the dog). This will only be determined by detecting lymphocytopenia and eosinopenia.

What is classified as a left shift?

There is a normal proportion of immature to mature neutrophils in the peripheral blood. As the proportion of immature cells increases (due to demand causing depletion of the bone marrow storage pool) then the likelihood of a left shift developing exists. When the proportion drops less than 1:16-18 for the dog, 1:10-12 for the cat and 1:12-16 for the horse (there is great variation in the literature for these values and they should be used with caution) then it is definite that a left shift has developed. Proportions just above these, however, should not be taken to mean that a left shift is not present or developing. Serial samples are useful in deciding whether a left shift is developing if the ratio is around or just above its upper limit. Ruminants rarely have immature neutrophils circulating in health, therefore, their presence indicates a left shift without the need for ratios. Nb. In the dog, and possibly in the cat, it is accepted by many haematologists that greater than 1 x 109/L bands is definitely a left shift irrespective of the ratio. In the horse, some are happy to accept greater than 0.3 x 109/L bands as indicating a left shift. Consequently, it is not clear cut. Perhaps it is best to use the ratios when values for bands are below those designated acceptable levels for left shifts? Common sense is the answer. It is important to remember, also, that the lack of a left shift does not rule out inflammatory demand!

What constitutes a favourable prognosis (in relation to neutrophil numbers and types in the peripheral blood)?
Neutropenia in the early stages of overwhelming demand cannot always be regarded as a poor prognostic sign. However, the persistence of neutropenia, especially without the development of a left shift, is a poor prognostic sign, as it indicates bone marrow failure and a compromise of an important part of innate immunity. Morphological changes can occur in neutrophils related to inflammatory demand. These are loosely called 'toxic changes' and most probably interfere with neutrophil function. Toxic changes include:

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a)

Dhle (Doehle) bodies (bluish angular cytoplasmic inclusions). These are related to disturbances of neutrophil maturation and occur in many cases of increased neutrophil production. Although they can be taken as an indication of mild toxemic change, they may occur unrelated to toxemia e.g. certain drugs can cause them. Cytoplasmic basophilia and vacuolation. This represents a more severe manifestation of toxemia and often occurs in severe infections and intense inflammation (e.g. pancreatic necrosis in the dog). Cytoplasmic granulation (purplish). Prominent purplish granulation of neutrophils in the horse can indicate toxemia.

b)

c)

Persistent toxic changes to neutrophils, especially after the initiation of treatment, in conjunction with low CNP neutrophil levels, may suggest a poor prognosis.

What levels of neutrophilia can accompany tissue demand?

In dogs any level is possible but most total WBC counts are within 10-50 x 109/L (corticosteroid induced WBC counts are usually less than 25 x 109/L but can occur higher). In certain purulent infections (e.g. pyometra) the count may be between 50-100 x 109L and would commonly be accompanied by an extreme left shift (i.e. myelocytes, metamyelocytes start to circulate in the peripheral blood). This is called a 'leukemoid response' as it may resemble chronic neutrophilic (granulocytic) leukemia. In cats, total WBC levels in inflammatory demand rarely exceed 35 x 109/L (corticosteroid induced total WBC levels are usually less than 30 x 109/L). The frequency of left shift parallels that in the dog. In horses total WBC levels in inflammatory demand are similar to those that occur in the cat (corticosteroid induced total WBC levels are usually less than 20 x 109/L). In adult horses left shifts in inflammatory demand are uncommon and rarely spectacular. Most cases of inflammatory demand are reflected as simple neutrophilia. In foals, left shifts are more common especially in purulent infections. In the dog, cat and horse, as mentioned, a neutropenia may develop in early intense inflammatory demand, and may take a few days to become a neutrophilia. Gram-negative bacteria causing overwhelming sepsis and endotoxemia commonly produce early neutropenia (horses are particularly sensitive to small amounts of endotoxin). A rebound neutrophilia is likely later. In adult cattle, early neutropenia is the rule in inflammatory demand (as reserve levels of neutrophils in the MNP and bone marrow storage pool are low). Therefore, in addition to endotoxemia related to Gram-negative bacteria, a wide range of acute purulent inflammations commonly induce early neutropenia in cattle. This is commonly accompanied by a left shift and lymphocytopenia (thereby causing a leukopenia). Toxic changes in neutrophils are commonly observed but, as mentioned, are only a poor prognostic sign if they persist. Although the adult ox appears to have low reserves of neutrophils to combat purulent infections (hence the early neutropenia), the BM responds quickly (hence the moderate left shift) and by 2-3 days neutrophil levels may be starting to recover. The left shift normally persists as long as the inflammatory demand persists, while total WBC levels (due to neutrophilia and a return to normal lymphocyte levels) can reach up to 30 x 109/L or greater in long standing cases (commonly levels of total WBCs in inflammatory demand will be in a range up to 20 x 109/L; but corticosteroid induced leukocytosis is rarely prominent due to the lymphocytopenia). Young calves may have inflammatory demand leukocyte changes similar to dogs and cats. This is presumably due to differing reserve levels of neutrophils.

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In fibrinous, non-purulent inflammation cattle may show little or no neutrophil response and high plasma fibrinogen levels may be the only laboratory sign of inflammatory disease. Consequently, fibrinogen levels (in association with total protein estimation to exclude a false elevation due to dehydration) have been used in many animal species to indicate inflammatory or degenerative disease. Fibrinogen is an 'acute phase reactant' protein (and the major clotting protein) and rises rapidly in a variety of diseases. It appears to be the main protein that alters erythrocyte sedimentation rate. It has been well used in ruminants (and less so in horses). In cattle, total serum or plasma protein to fibrinogen ratios of 10 to 1 or less indicates a significant increase in fibrinogen and suggests disease (i.e. it is a non-specific indicator). Nb: In dogs and cats, fibrinogen levels may be elevated in inflammatory/degenerative disease; but other acute phase reactant proteins (e.g. haptoglobin, Creactive protein) are more commonly used to detect inflammatory or degenerative disease. In pigs, neutrophilia due to inflammatory demand is of moderate intensity when compared to the dog and cat but may be missed due to the wide reference range. Left shifts are common in purulent infections and may be accompanied by total WBC levels of up to 100 x 109/L, but most are between 20-35 x 109/L. Pigs, like cattle, may develop little neutrophil response in non-purulent inflammation and fibrinogen may be of use to indicate inflammation.

Neutropenia
Neutropenia may occur through a variety of mechanisms. These include reduced survival, reduced production, increased ineffective granulopoiesis, unknown mechanisms, and sequestration. Reduced survival (or increased utilisation) neutropenia occurs when destruction of mature neutrophils or excess tissue utilisation exceed bone marrow supply e.g. immune-mediated neutropenia, overwhelming sepsis, bovine acute purulent diseases. Recovery to a neutrophilia usually takes a few days. Reduced production neutropenia may be transient or persistent and is usually due to an agent of disease directly affecting cell production. Transient causes include certain acute infectious diseases such as feline infectious panleukopenia, canine adenovirus. As with reduced survival, rebound neutrophilia can occur. Persistent causes of neutropenia (usually indicating bone marrow failure) have a poor prognosis and may be due to myelophthisis, drug toxicity or irradiation. Increased ineffective granulopiesis produces neutropenia by failure of release of cells from the bone marrow, intramedullary destruction, and arrest of granulocyte maturation (e.g. myelophthisis, feline leukemia virus). It has a poor prognosis. Some neutropenias develop through unknown mechanisms in a variety of infectious (mainly viral) diseases in the acute stages (e.g. canine distemper, equine infectious anaemia, sporadic bovine encephalomyelitis). Commonly, they are transient. Sequestration neutropenia (pseudoneutropenia) is due to a sudden and transient margination of neutrophils. It lasts for a few hours and is followed by rebound neutrophilia e.g. common in endotoxemia and anaphylactic shock. It should be obvious from the preceding discussion that some infections may cause neutropenia by a variety of mechanisms and that many are transient.

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Monocytes
Monocytes are formed in the bone marrow, are transported in the circulation and are capable of being transformed into macrophages to complement fixed tissue macrophages when there is increased demand for macro-molecular phagocytosis (innate immunity). They have additional roles to play in adaptive immune responses (antigen-presenting cells; effector cells when activated by cytokine release from T lymphocytes). Monocytes, like the neutrophils, may be unevenly distributed in the circulation (i.e. there are marginal and circulatory pools: MP and CP). Monocytosis may occur in intense stress due to corticosteroid release (primarily in the dog; it is variable in the cat), in disorders characterised by tissue demand for phagocytosis of macromolecular molecules, in conditions in which cellular immunity is potentiated, and in neutropenic states related to granulocytic hypoplasia (probably a compensatory mechanism). In the horse and cow monocytopenia is apparently more common in acute stress. This is often followed by a later monocytosis. Monocytopenia can occur in endotoxemia. Monocytopenia is not of consequence in itself and may be difficult to detect in the individual animal as reference ranges for most species have low limits.

Eosinophils
Bone marrow storage of eosinophils is minimal and cells are usually in the circulation for a short time. Eosinophils are attracted by and inhibit chemical mediators released from mast cells (principally histamine). They are also parasiticidal. Eosinophils have weak bactericidal properties. Eosinophilia can occur in response to histamine release or antigenic stimulation via sensitized T lymphocytes (may be involved in stimulation of eosinopoiesis). Consequently, eosinophilia can occur in hypersensitivities especially involving skin, lung, GIT and the reproductive tract. Prolonged parasitic contact can induce eosinophilia. Localised eosinophilic lesions do not necessarily produce blood eosinophilia. Some eosinophilias are quite marked and inexplicable (hypereosinophilic syndrome). Eosinophilia has been seen during oestrus in a number of species, especially the bitch. Eosinophilia may also accompany some neoplastic conditions (e.g. mast cell tumours, some lymphosarcomas) Eosinopenia is commonly associated with corticosteroids but it can be caused by adrenaline release, and it can occur in acute infections by additional mechanisms independent of corticosteroids. With corticosteroid release, blood histamine levels are reduced and mast cell regranulation is inhibited. This causes eosinophils to probably move to the marginal pool and possibly to remain in the bone marrow. Prolonged release of corticosteroids reduces bone marrow eosinophil production.

Basophils
Basophils contain many mediators of inflammation and release these in response to immune complexes on their surface (i.e. in adaptive immunity) and to various chemicals and physical agents. The reasons for basophila are poorly understood but it is accepted that basophilia usually accompanies eosinophilia as in some parasitic and allergic diseases (both adaptive immune responses). Basophilia may occur without eosinophilia (e.g. in horses), but is rare. Basopenia is not of significance.

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Lymphocytes
Lymphopoiesis mainly occurs in the lymphoid tissue but some production continues in the bone marrow (especially for B lymphocytes). Lymphocytes when stimulated transform into lymphoblasts which proceed to prolymphocytes and then to T effector cells, B lymphocytes or memory lymphocytes. Activated B lymphocytes are the source for plasma cells. Lymphocytes are the most important cells for adaptive immunity. In circulating blood the majority of lymphocytes are of the T cell variety (there is some variation between species). Plasma cells, lymphoblasts and prolymphocytes are rarely seen in peripheral blood. However, transformed lymphocytes (`immunocytes' or activated hyperbasophilic lymphocytes) of both the B and T cell lines are seen in peripheral blood and their numbers can increase in periods of antigenic stimulation. Blood lymphocyte numbers are most often influenced by effects on cell recirculation. Mild lymphocytosis may occur in physiological leukocytosis, chronic infections and hypoadrenocorticism. It does not have to accompany lymphoid hyperplasia due to antigenic stimulation, but is common in prolonged situations. Lymphopenia (lymphocytopenia) can be due to corticosteroid-induced redistribution of recirculating lymphocytes, but can be related also to acute systemic infection (usually viraemias). Recirculating lymphocytes are entrapped in lymph nodes by the presence of antigenic material. Other causes of lymphopenia are less common and include T cell deficiencies and loss of lymphocyte rich lymph. Lymphoid leukemia may show circulating small lymphocytes but more often lymphoblasts and prolymphocytes are the predominant cell types (see notes under hematopoietic neoplasia.)

*SEE CASES 14, 16-20, 25,26

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HAEMATOPOIETIC NEOPLASIA
Introduction
Haematopoietic neoplasia can be divided into lymphoproliferative and myeloproliferative forms. Lymphoproliferative disorders involve either proliferation of lymphocytes or plasma cells. Commonly, this leads to infiltration and enlargement of organs or tissues. Tissues affected vary but if BM is involved then a leukemic manifestation is likely (circulating neoplastic cells). Myeloproliferative disorders usually present as leukemias as the BM is always involved. Lymphoproliferative disorders are more common than myeloproliferative disorders in most species. In farm animals, myeloproliferative disorders are rare.

Lymphoproliferative Disorders
a) Lymphosarcoma (malignant lymphoma)
This is a malignant solid neoplasm of lymphocytes or precursor cells (i.e. affecting lymphoid organs or infiltrating other tissues of organs). Benign neoplasms of lymphocytes have not been recognised in animals, so many are willing to drop the malignant from malignant lymphoma and just use lymphoma to designate the condition. Lymphosarcoma may become leukemic in the later stages (figures range from 25-60% for the dog and up to 25% for the cat). Lymphoid leukemia can occur on its own (primary acute and chronic lymphoid leukemia see below), but usually, at least in the cat and dog, occurs in combination with lymphosarcoma. Anaemia is not uncommon in dogs with advanced lymphosarcoma and may be related to immune-mediated destruction and/or chronic disease. Hypercalcemia and a gammopathy may also be detected in dogs with lymphosarcoma. Solid masses can occur in variable sites in lymphosarcoma and this forms the basis of an anatomical classification (e.g. nodal; abdominal; cranial mediastinal; miscellaneous; primary leukemia without solid masses). Nodal in association with involvement of internal organs is common in the dog (called multicentric). The cranial mediastinal form is more commonly seen in the cat, but the most common form in the cat appears to be alimentary. The horse has a low incidence of lymphosarcoma, which is predominantly multicentric with high involvement of abdominal (alimentary tract mainly) tissues. If external lymph nodes are involved then there is usually marked enlargement as opposed to mild enlargement which may occur in myeloproliferative and primary lymphoid leukemias. Lymphosarcoma, particularly affecting lymph nodes, can be classified on architectural pattern of the neoplastic cells (diffuse or nodular). Lymphosarcoma can also be classified according to the predominant cell type (e.g. lymphoblast, prolymphocyte, lymphocyte), and in relation to the main immunophenotype (e.g. B or T cell). Immunophenotyping in people is important for prognosis but, at this stage, immunophenotyping, on its own, appears to have limited prognostic value for the dog and cat. This may alter with increasing sophistication of testing and with increased treatment. There are several classification systems for lymphosarcoma in animals. These include the National Cancer Institue Working Formulation (based on architectural pattern and cellular morphology), the Updated Kiel System (based on architectural pattern, cellular morphology and immunophenotyping) and the Revised European-American Classification of Lymphoid Neoplasms (REAL based on cellular morphology, immunophenotyping and sometimes cytogenetics). The first two, have been better utilised so far (especially for canine tomours) and classify lymphosarcomas into low, intermediate and high grade forms.

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In the cat some cases of lymphosarcoma appear to be associated with Feline Leukemia Virus (FeLV) or Feline Immunodeficiency Virus (FIV), but the numbers vary from country to country. FIV could well have its impact through altering the immune response rather than directly causing transformation of lymphocytes. If viruses are involved in the transformation, anaemia commonly accompanies the disease (may be either regenerativeor non-regenerative). It has been reported from overseas that FeLV negative lymphosarcomas are more likely to be abdominal (alimentary) in form and not be associated with anaemia. Heritable factors may exist for some forms of feline lymphosarcoma Treatment of lymphosarcoma in dogs and cats has developed dramatically and several successful protocols of chemotherapeutic agents are available. In horses, lymphosarcoma has variable cell morphology and the multicentric form is commonly B cell in origin (although they may be associated with many mature T lymphocytes). Subcutaneous and alimentary forms are also usually composed of B lymphocytes, whilst the mediastinal form is composed of neoplastic T lymphocytes. Lymphocytosis and leukemia are uncommon, but a monoclonal gammopathy has been recorded in the multicentric form. Anaemia (due to either immunemediated destruction or chronic disease) is often the most common laboratory finding and may be the presenting complaint. In cattle lymphosarcoma presents as two main forms: sporadic and enzootic. Sporadic lymphosarcoma tends to affect animals less than four years of age. A sporadic multicentric form occurs in calves, a sporadic mediastinal form in yearlings and a sporadic cutaneous form in young cattle. Enzootic bovine lymphosarcoma (EBL) tends to affect older animals and, commonly, several animals in one herd are affected. EBL is commonly multicentric in form, involves B lymphocytes, and is transmissible as a retrovirus has been incriminated (Bovine Leukemia Virus). Persistent lymphocytosis can precede EBL for several years, but a true leukemic manifestation (abnormal lymphoid cells usually in high numbers) occurs in less than 30% of affected cattle.

b) Acute lymphoblastic leukemia (ALL)

ALL can occur as a primary leukemia or as a manifestation of late stage lymphosarcoma. It is not always easy to distinguish these two conditions in terminal disease, but the lack of solid tissue masses should suggest the leukemia is a primary manifestation. Immunophenotyping has shown many are B cells, but T and NK (natural killer) cell types have also been recorded for dogs and cats. ALL is commonly rapid in onset, causes myelophthisis and usually presents with low to medium increases in circulating lymphoblasts.

c) Chronic lymphocytic leukemia (CLL)

CLL tends to occur in older animals and most occur in dogs. The time course is months to years and enlargement of lymph nodes may occur in the later stages so as to make it difficult then to decide if this is a primary leukemia or a form of small cell lymphosarcoma with a leukemic manifestation. Most CLLs are T cells, but B cell forms with monoclonal gammopathy have been recorded. Circulating levels of neoplastic lymphocytes can vary from 10-100+ x 109/L Large granular lymphocyte (LGL) leukemia is a distinct form, whose presentation is similar to CLL. LGL lymphosarcoma also occurs. LGLs are cells with disctinct, often large, azurophilic granules in the cytoplasm. In humans they are thought to be related to either activated cytotoxic T lymphocytes (T cell LGL) or natural killer (NK) cells (NK cell LGL). In dogs with LGL neoplasia they appear mostly to be T cell LGL, whilst in cats both T cell LGL and NK cell LGL have been implicated. In horses, both LGL leukemia and lymphosarcoma (mainly affecting gut) have been reported, with T cell LGL being implicated.

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d) Plasma cell myeloma (multiple myeloma)

This is a malignant neoplasm of older animals derived usually from a single clone of plasma cells and their precursors. Solid masses usually develop in bones or visceral tissues, and the neoplastic cells often produce an immunoglobulin or immunoglobulin sub unit in excess. Most cases have been recorded in the dog but any animal species may be affected. Dogs can respond successfully to chemotherapy. NB: plasmacytoma - refers to a benign neoplastic mass of plasma cells. It is rare in most domestic animals, but skin plasmacytomas are common for the dog. Laboratory findings in plasma cell myeloma may include: a) non-regenerative anaemia (30% of cases in dogs due to myelophthisis) b) circulating plasma cells (20% of cases in dogs) c) hyperglobulinemia (majority of cases in the dog) Hyperglobulinemia is the most consistent finding. It most commonly presents as a narrow-based peak either in the beta or gamma zone of the electrophoretogram (hence the name monoclonal gammopathy occasional biclonal forms have been reported). The abnormal globulin is often referred to as a paraprotein or M-component. If the paraprotein is a light chain of an immunoglobulin it may be passed in the urine (Bence-Jones protein). If the globulin produced is IgM (macroglobulinemia) or polymerized IgA, then the hyperviscosity syndrome can occur (can lead to a bleeding diathesis). Derangements of haemostasis may also occur because of thrombocytopenia (related to myelophthisis) and paraprotein binding to platelets (thrombocytopathy). Hypercalcemia in the dog may result from osteolysis through release of a tumour factor and stimulation of release of parathyroid hormone because of paraprotein binding to ionised calcium. NB: Occasionally lymphosarcoma may produce a hyperglobulinemia which may be a polyclonal, biclonal or monoclonal gammopathy. Hypercalcemia through pseudohyperparathyroidism (see section under derangements of calcium metabolism for mechanisms) is probably more common in lymphosarcoma than in multiple myeloma.

Myeloproliferative Disorders
Medically, myeloproliferative disorders are far less common than lymphoproliferative disorders, and include a variety of non- and pre-neoplastic conditions as well as neoplastic conditions. In veterinary medicine little is known about myeloproliferative disorders other than those that are obviously neoplastic. However, a pre-neoplastic condition called Myelodysplastic syndrome (MDS) is recognised most commonly for the cat (can be FeLV positive), and infrequently in the dog and horse. Myelodysplastic syndrome is characterised by less than 30% blasts in BM, dyserythropoiesis (abnormal maturation and morphology) and cytopenia affecting more than oen cell line. Invariably MDS progresses to AML. Myeloproliferative disorders are all characterised by BM hyperplasia and a variable degree of myelophthisis. Consequently, there is variable disturbance of all BM cell types. The neoplastic cells will be circulating and commonly in high numbers. However, at times they may be low in number or even absent (so called aleukemic or subleukemic forms occurs because the neoplastic cells, for some reason, are released from BM spasmodically or not at all). Variable infiltration of systemic organs (spleen, liver, lymph nodes) will occur. Acute and chronic forms are recognised (this also applies to primary lymphoid leukemia). Solid tumours (chloromas) are rare. Myeloproliferative disorders are more common in the cat in which some are associated with FeLV.

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However, even in cats they are becoming less common. They are occasionally seen in the dog, but are rare in other domestic species. Most appear to respond poorly to chemotherapeutic agents, although chronic leukemias have a better prognosis (that also includes CLL). 1. Acute myeloid leukemia (AML) is recognised primarily in cats and dogs and develops rapidly and is invariably fatal because of myelophthisis and related effects. The system of classification is based on the human French-American-British Group (FAB) classification, but there are some unclassifiable and hybrid forms in animals. AML is diagnosed when >30% of the nucleated cells in bone marrow are blasts and the total non-erythroid population of bone marrow cells is >50%. a) b) Acute undifferentiated leukemia (AUL). This term is used when it is difficult to classify the cells. Included in this category are the previously described cases in cats called reticuloendotheliosis. Myeloblastic leukemia (This was originally called acute granulocytic leukemia.). This form is divided into two subtypes: myeloblastic leukemia without differentiation (AML-M1 90% of non-nucleated cells in BM are blasts, with less than 10% differentiating into neutrophilic or eosinophilic promyelocytes); and myeloblastic leukemia with differentiation (AML-M2 3090% of non-nucleated cells are blasts, with more than 10% differentiating into promyelocytes and later forms of granulocytes [neutrophils, eosinophils and, rarely, basophils). Eosinophilic differentiaton is rare in the cat and non-existent in the dog. Basophilic differentiation is extremely rare for all species. In the cat some cases are FeLV positive (some are possibly related to FIV but more research is required to confirm this - this virus may allow a variety of Haematopoietic malignancies to develop through immunosuppression or produce them directly). In the cat, acute granulocytic leukemia may be the terminal stage of a disease that has progressed from another form such as erythroleukemia. In both the dog and cat, acute granulocytic leukemia is commonly accompanied by non-regenerative anaemia and thrombocytopenia with bizarre platelets. c) d) Promyelocytic leukemia (AML-M3). This form is extremely rare and has only been reported in the pig. Myelomonocytic leukemia (AML-M4). This is the most common form of AML in dogs, cats (some are FeLV positive) and horses. Both neutrophilic and monocytic cell lines are affected (both come from the bipotential precursor blast) and myeloblasts and monocytoblasts comprise more than 30% of the nucleated BM cells. More than 20% of the nucleated cells of BM are differentiating neutrophilic and monocytic cells. Monocytic leukemia (AML-M5). This can be further typed as M5a (more than 80% of nucleated cells in BM are monoblasts or promonocytes) and M5b (30-80% nucleated cells in bone marrow are monblasts and promonocytes, with prominent monocyte differentiation also). In the dog this form may progress to M4 (and possibly M1-3 for the granulocytic line) Erythroleukemia (AML-M6). This is characterised by a mixture of erythroid precursors and myeloblasts/monoblasts in BM (usually greater than 30% of all nucleated cells). If erythroid precursors exceed 30% of the nucleated cells in bone marrow, then AML-M6Er is used instead of M6. Originally, M6Er was designated erythremic myelosis and in cats was often FeLV positive. In cats, M6 can sometimes progress to M1 or M2. Megakaryoblastic leukemia (AML-M7). This has been recorded in both the dog and cat (FeLV positive in the cat) and more than 30% of all nucleated cells in BM are megakaryoblasts. Abnormal forms circulate. Myelofibrosis is common.

e)

f)

g)

Nb. Another form of megakaryocytic neoplasia involving mainly the platelets is called Essential Thrombocythemia and is really a chronic myeloproliferative disorder.

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2. Chronic Myeloproliferative disorders In contrast to CLL, these disorders are rare. They are characterised by blasts being less than 30% of all nucleated cells in the BM. MDS is sometimes included in this group.The group includes polycythemia vera (primary erythrocytosis or essential erythrocytosis described in cats, dogs, cattle and horses), chronic granulocytic (myelogenous) leukemia (described in cats, dogs and horses), chronic eosinophilic leukemia (described in cats only, but there is the problem of differentiating this from the hypereosinophic syndrome in cats and dogs), chronic basophilic leukemia (has been described in dog and cat, but rare), essential thrombocythemia (rare, but seen in dogs and cats), chronic myelomonocytic leukemia, and chronic monocytic leukemia. 3. Mast cell leukemia. In the dog this can be a primary haematopoietic neoplasm, distinct from mastocytomas. Circulating mast cells apparently occur with advanced forms of mastocytoma (sometimes called mastocytosis), but this is distinct from primary mast cell leukemia. In the cat, splenic mast cell tumours have a systemic mastocytosis (sometimes called a leukemic manifestation).

*SEE CASES 27 & 35

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BLEEDING DISORDERS (ABNORMALITIES OF HAEMOSTASIS)

Introduction to Haemostatis
Disorders of haemostasis can lead to either bleeding (haemorrhage) or thromboemobolism (hypercoagulation). This section will focus mostly on bleeding disorders. To understand the causes and investigation of bleeding disorders it is important to have a general knowledge of haemostatis. The following is a simplistic view of the complex topic of haemostatis, but should suffice for the investigation of bleeding disorders. Haemostatis depends upon a complete interaction among vascular endothelium, platelets and clotting (coagulation) factors. In normal haemostatis vascular injury leads to platelet adherence (temporary haemostatic plug), which in turn promotes clotting. The result is a fibrin clot that stabilises the platelet plug. At the same time of activation of clotting there is activation of fibrinolysis that aims to remodel and remove formed clots to maintain vascular channels. Endothelial cells normally form a non-thrombogenic surface, but when damaged they release substances that activate clotting. Platelets adhere to exposed sub-endothelial collagen upon endothelial damage. Upon adherence and aggregation, platelets cause further platelet activation and release non-enzymic clotting factors associated with their membranes. Platelet phospholipid in the membrane accelerates the intrinsic pathway of clotting while other substances are released from the platelets that contribute to the formation of a stable clot. Platelets also play an essential role in inflammation, through the release of vasoactive substances and the production of cytokines, and tissue repair. Platelets appear to provide a factor that `nourishes' endothelial cells. In prolonged or extreme thrombocytopenias endothelial disturbances can lead to petechiation. Platelet production is under control of a hormone named thrombopoietin which enhances platelet production upon reduced platelet mass. Coagulation and fibrinolytic systems are intricately involved as well as having complex interactions with platelets and endothelial cells. Coagulation basically involves sequential factor activation, which yields thrombin, an enzyme that enhances conversion of fibrinogen to fibrin. Clotting activation can occur via an intrinsic or extrinsic pathway. Both activate a common pathway which gives rise to fibrin. The intrinsic pathway can be activated in vivo by endothelial cell damage, collagen exposure, pyrophosphate from platelets and endotoxin. The extrinsic pathway is activated in vivo by the release of tissue thromboplastin (Factor III), which is a lipoprotein present in most cells. The extrinsic system accelerates the whole process of blood clotting and is probably the initiator of coagulation in most disease. In many disease situations, it is likely that both the intrinsic and extrinsic pathways are activated. Most clotting factors are produced by the liver and can be divided into enzymic and non-enzymic types. Enzymic factors are usually circulating in inactive forms (FVII is the exception) and activated forms are inhibited by natural inhibitors. This is necessary as enzymic factors are not consumed by the clotting reaction. Enzymic factors II, VII, IX and X are dependent on Vitamin K for production by the liver. The level of the enzyme determines clotting ability. Therefore, partial deficiencies lead to partial loss of clotting ability. Non-enzymic factors (Calcium, Factor V, VIII and fibrinogen) primarily become associated with platelet membranes and are localised during haemostatis by platelet aggregation. With clotting these factors are consumed. Partial deficiencies of most of these factors do

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not interfere with clotting as only small amounts are required. Therefore, almost complete absence is required for clotting defects (although clotting factor tests may be altered if fibrinogen is less than 0.5 g/L). Factors V, VIII and fibrinogen are all 'acute phase reactants' and rapid increases can occur in a variety of inflammatory or neoplastic diseases. Factor VIII is a complex factor which is composed of 2 main components: Factor VIII:C (procoagulant activity - most important in the intrinsic pathway of coagulation) and Factor VIII:vW (von Willebrand's Factor, also called Factor VIII:related antigen). Factor VIII:vW is actually produced by endothelial cells. It is absorbed by circulating platelets (like most non-enzymic factors) and is neccessary for their adherence to collagen. Circulating naturally occurring anticoagulant proteins downregulate the coagulation cascades by inhibiting the procoagulant proteins of the intrinsic, extrinsic and common pathways i.e. there is a balance between anticoagulant and procoagulant proteins. This ensures some control of the degree of haemostatis occurring during vascular damage. The major anticoagulant protein is Antithrombin III, a protein produced in the liver and which requires heparin to operate at endothelial surfaces in order to bind to and inactivate thrombin. Antithrombin III (AT III), because of its low molecular weight, can be lost in forms of renal disease (e.g. nephrotic syndrome), protein losing enteropathy and severely ischemic bowel disorders in horses. Inherited or acquired (e.g. DIC) deficiencies of Antithrombin III can predispose to increased risk of thrombosis (hypercoagulability). Consequently, there are veterinary tests for AT III. Other important naturally occurring anticoagulants include Protein C, Protein S and alpha 2 macroglobulin. Protein C is a vitamin K-dependent protein and can also decrease in acquired hypercoagulable disorders.

Introduction to Bleeding Disorders

A bleeding disorder is characterised by uncontrollable/inexplicable hemorrhage because of altered haemostatis. This excludes hemorrhage through trauma that is controlled by surgical intervention because, apart from the damage to vessels, there is normal haemostatis. Bleeding disorders are characterised by the occurrence of petechiation, ecchymoses and haematomas. Petechiation often occurs on mucous membranes and, therefore, can give rise to epistaxis, haematuria and melena. What type of haemorrhage occurs will depend on what component of the haemostatic process has been disturbed. Vessel wall defects, platelet problems and vW factor deficiency will commonly give rise to petechiation and ecchymoses (all involved in the initial platelet plug primary haemostatis). General clotting factor deficiencies will commonly give rise to internal tissue haematomas or massive bleeding into cavities (involved in producing the fibrin plug that stabilises the temporary platelet plug secondary haemostatis). Laboratory investigation of bleeding disorders needs to take into consideration the clinical signs, especially the type of haemorrhage. The following provides basic information on laboratory tests.

Laboratory Evaluation of Bleeding Disorders

Bleeding disorders can involve one or more of the following: a) Vessel Wall Defect (Uncommon) b) Thrombocytopenia c) Platelet Function Defect d) Clotting Factor Deficiency e) Excessive Fibrinolysis

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a) Vessel wall defect

Laboratory evaluation aims at determining platelet numbers and function and coagulation factors (and possibly fibrinolysis). Vessel wall defects are more difficult to investigate, and because of their rarity, at least in small animals, are usually considered after other causes have been eliminated. Clinical signs are important to decide on what tests to perform. As mentioned, if petechiation, small ecchymoses or bleeding from mucous membranes predominate then thrombocytopenia or platelet function defects should be considered (von Willebrand's disease due to an inherited absolute deficiency of Factor VIII:vW will also cause similar clinical signs. Rarely does it interfere with the intrinsic clotting pathway unless it is completely absent). Vascular defects will also cause petechiation. If massive internal bleeding or external haemorrhage is occurring unrelated to injury then clotting factor defects should be considered. In some diseases more than one component of haemostatis may be affected and this will give rise to a variety of clinical signs (e.g. disseminated intravascular coagulation [DIC]).

b) Thrombocytopenia
Thrombocytopenia is a common cause of a bleeding disorder. Platelet numbers are usually assessed by direct manual counting, although they can be indirectly assessed on peripheral blood films (e.g. in the dog less than 3-4 platelets per oil objective field is considered a significant thrombocytopenia). From manual counts, dogs have platelet numbers greater than 200 x 109/L (cat > 300 x 109/L, horses > 100 x 109/L), but values less than these do not cause spontaneous (as opposed to bleeding after trauma) haemorrhages unless either the value reaches 25-50 x 109/L or there are associated platelet function defects. If values are above these levels (say 50 to 100 x 109/L) and spontaneous bleeding is occurring then other defects may be working in conjunction with the thrombocytopenia. At levels below 100 x 109/L (and especially 75 x 109/L) bleeding with light trauma is possible without other defects in haemostatis being present (Nb. healthy greyhounds can have levels between 100-200 while cavalier King Charles spaniels may occasionally have levels below 100 x 109/L without clinical disease). Macro (mega, giant) platelets have more activity than normal sized platelets (overall activity depends on platelet mass rather than platelet numbers); therefore, lower numbers of these types of platelets can occur without predisposing to spontaneous bleeding. Causes of thrombocytopenia include collection artefact (i.e. platelet aggregation in the tube), disorders of production (e.g. oestrogen and other drug [e.g. phenylbutazone] toxicity, any cause of myelophthisis), distribution (splenic disorders may cause sequestration but usually not dramatic drops in numbers), utilisation (e.g. DIC) and destruction (e.g. immune mediated; many drugs cause shortened life spans for platelets). Macroplatelets (also called shift platelets) are prominent in peripheral blood and megakaryocytes increase in bone marrow when there is either excessive utilisation or destruction of platelets. When platelet numbers drop below 10 x 109/L the Activated Clotting Time test will usually be affected as it relies on adequate platelet phospholipid to be present. The Activated Partial Thromboplastin Time and the One Stage Prothrombin Time tests will not be interfered with by absolute thrombocytopenia as an exogenous source of platelet phospholipid (or its equivalent) is added (but they will be affected in vivo). Primary immune-mediated thrombocytopenia may occur alone or in combination with other immune-mediated diseases. The anti-PF3 test is an indirect assay for anti-platelet antibodies. Platelet numbers and function are affected (the antibodies coat the platelets thereby allowing destruction of some and interfering with function of others).

c) Platelet function defect

Thrombocytopathies may be congenital (rare in domestic animals) or acquired (drug induced e.g. non-steroidal anti-inflammatory agents, or secondary to other diseases e.g. chronic liver or

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kidney disease, myeloproliferative disorders, SLE). As a consequence, platelet dysfunction is not uncommon. However, the level of dysfunction may not be severe enough to cause clinical signs of spontaneous haemorrhage. Platelet function assessment is difficult but tests include:

i)

Bleeding time (BT

This is determined from a standard incision made in mucous membrane (buccal mucosal bleeding time) or hairless skin. BT is prolonged in thrombocytopenia, platelet function defects, some cases of von Willebrand's disease, severe fibrinolysis and vessel wall defects. BT is a measurement of small vessel haemostatis which does not require clotting factors in the first instance.

Nb. deficiencies of clotting factors will not prolong BT but because there is no fibrin stabilisation of the platelet plug re-bleeding may occur a few hours later.

ii)

Clot retraction
A contractile protein, thrombasthenin, is produced by platelets and is responsible for strengthening the haemostatic plug by pulling the fibrin strands taut. This leads to clot retraction in vivo and in vitro. Failure of clot retraction (i.e. serum separation) in vitro (the basis of the clot retraction test) can be due to platelet function defects, thrombocytopenia and lack of fibrinogen. This test is rarely done.

iii)

Others
These are sophisticated laboratory tests that either detect defects of platelet adhesion or aggregation. Usually they are not asked for unless the other causes of a bleeding disorder have been excluded.

d) Clotting factor deficiency

These can be initially assessed by the Activated Clotting Time (ACT). The test measures the time required for fibrin clot formation in non-anticoagulated blood in vitro. It is prolonged in deficiencies or inhibition of intrinsic or common clotting factors. The test requires a standardised technique and has low sensitivity (the factor deficiency must be less than 5% of the normal level to prolong ACT). Greater sensitivity is provided by the citrated plasma clotting tests. They include: Activated Partial Thromboplastin Time (APTT) One Stage Prothrombin Time (OSPT) Thrombin Clotting Time (TCT) Other citrated plasma clotting tests are used once a problem has been detected e.g. specific factor analysis (add specific factors and re-perform APTT or OSPT). The results of all tests should be compared to the results for control animals.

Activated Partial Thromboplastin Time (APTT)

APTT measures the time required for fibrin clot in recalcified (calcium is bound by citrate and is essential for clotting), fresh, citrated plasma after addition of a contact activator and phospholipid in vitro. The APTT yields the same, but more precise, information as clotting time (CT) i.e. it measures the intrinsic and common pathways of clotting factors (which includes fibrinogen). Note the APTT

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is prolonged only when the deficient factor is less than 30% of the normal level i.e. haemophiliac carriers having 40-60% of the normal level of factor VIII are not detected by APTT. Fibrinogen of less than 0.5 g/L may also prolong APTT. Fibrinogen, factors VIII and V may increase in inflammatory disease (acute phase reactants) leading to a shortened APTT.

One Stage Prothrombin Time (OSPT)

OSPT measures the time required for Fibrin clot formation in recalcified, fresh, citrated plasma after addition of tissue thromboplastin in vitro (tissue thromboplastin activates extrinsic system of clotting factors in vivo). A prolonged OSPT occurs in Factor VII deficiency (extrinsic clotting factor) or in common system factor deficiencies (factors need to be less than 30% of normal level). Fibrinogen of less than 0.5 g/L may also prolong OSPT.

Thrombin Clotting Time (TCT)

TCT measures the time required for fibrin clot formation in recalcified, fresh, citrated plasma after addition of thrombin (activated factor II that converts fibrinogen to fibrin). The TCT measures the common pathway. By adding excess thrombin, the TCT can be modified to measure fibrinogen. It is one of the more sensitive ways of detecting decreases in fibrinogen concentration. A crude method of detecting changes in fibrinogen concentration (especially increases) is by measuring the difference between total serum and plasma protein or by direct measurement after heat precipitation of fibrinogen in a microhematocrit tube (see practical notes).

e) Excessive fibrinolysis
Fibrinolysis is activated at the same time as coagulation by plasminogen activators released from injured endothelium. Plasmin acts locally on the clot to degrade fibrin and factors V and VIII. Fibrin (fibrinogen) degradation products (FDP) are normally removed by macrophages but if there is excessive fibrinolysis, excess fibrin (Fibrinogen) degradation products can inhibit clot formation (affect platelet plugging and fibrinogen polymerisation). FDP can be measured and the test is mainly used to detect increased levels in DIC (increases also occur in severe internal bleeding and in thrombotic disease such as acute pulmonary thrombosis). D-dimer is a specific by-product of plasma degradation of cross-linked fibrin (i.e. one type of FDP) and can be measured in dogs to detect DIC or pulmonary thrombosis.

Summary
All the tests for bleeding disorders have inadequacies but if they are used in combination they can help determine the likely cause. Vascular defects and platelet function defects are most difficult to assess, but fortunately they are rare as primary causes of bleeding disorders. At the University of Sydney a platelet count, fibrinogen estimation, OSPT and APTT are used to initially assess a bleeding disorder. This helps differentiate common disorders such as pure thrombocytopenia, DIC, Vitamin K deficiency or antagonism [coumarin poisoning], and hereditary factor deficiency. Hepatic insufficiency may contribute to a bleeding disorder but usually the other effects of hepatic insufficiency are more important. On the basis of the results of these tests more specific testing can be undertaken. History is always very useful in differentiating causes of bleeding disorders. The following table shows the likely laboratory results for some of the more common bleeding disorders.

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Laboratory results for some common bleeding disorders

Platelet APPT count Thrombocytopenia Decrease Normal Platelet function defect Normal Normal Thrombocytopathy) Vit K antagonism or Normal Extended deficiency Heritablefactor Normal Variable deficiency (often normal forFVIII:vW) AcuteDIC Hepaticinsufficiency Decrease Extended Normal Extended OSPT Normal Normal Extended Fibrinogen Normal Normal Normal FDP Normal Normal Normal Normal

Normal Variable unless (often normal for primary deficiency FVIII:vW) Extended Decrease Extended Decrease (variable)

Increase Normal

Hereditary coagulation disorders in domestic animals are well documented, especially in the dog. They are not common in the cat and, in all species; they are probably not as important as other causes of bleeding disorders in the population as a whole. In the dog, haemophilia A (F VIII:C), haemophilia B (F IX) and von Willebrand's disease (FVIII:vW) are the more common hereditary factor deficiencies. Breed dispositions do occur and this may be useful in diagnosis. Disseminated intravascular coagulation (DIC) can occur in a variety of conditions characterised by extensive tissue or vessel damage (trauma, surgery, neoplasia, infections). The damage leads to widespread activation of clotting mechanisms as well as fibrinolysis (consumptive coagulopathy), which eventually becomes self-perpetuating. Initial activation may be due to the release of factors, such as tissue thromboplastin (or a thromboplastin like substance) from neoplastic cells or inflammatory altered moncytes, directly into the blood stream. The initial effect is widespread microvascular thrombosis (also aided by decreased levels of anticoagulants such as Antithrombin III and Protein C; and impaired fibrinolysis) which can give rise to tissue hypoxia and dysfunction, as well as incitement of systemic inflammation through the release of inflammatory mediators. Eventually, the consumptive coagulopathy may give rise to widespread bleeding because of the lack of platelets, clotting factors and increased fibrinolysis. In dogs, bleeding is commonly seen in clinical DIC, but is less common in cats and horses. In those species, clinical DIC often presents with nonspecific signs related to tissue hypoxia due to the microvascular (small and medium vessels) thrombosis. DIC also varies in degree and, consequently, can give variable laboratory test results. In the severe cases there is thrombocytopenia, prolonged APTT and OSPT, decreased fibrinogen and increased FDP and D-dimer. Schistocytes (and occasional keratocytes and spherocytes) are common on peripheral blood films due to fibrin strand damage in small vessels. Sub-clinical DIC is probably more common than clinical DIC. It may only be detected by FDP/D-dimer analysis or by the examination of peripheral blood films for schistocytes.

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IMMUNODIAGNOSTICS (for Diseases with an Immune Base Immune-Mediated Diseases & Immunodeficiency)
Introduction
In immune mediated (related) diseases most of the damage done to the tissues is due to the action of antibodies, complement and effector cells (i.e. the hosts immune response). The agent (antigen) triggers the immune response but often does limited direct damage itself. Hypersensitivities have been loosely referred to as immune mediated disease but most immune mediated disease relates to so called autoimmunity. Hypersensitivities are also called allergies and they describe a variety of reactions that produce inadvertent significant tissue damage (i.e. the immune response to the foreign antigen causes inflammation which secondarily damages tissue to the extent that clinical signs are produced - any immune response to a foreign antigen will cause some secondary tissue damage but it is usually limited). Autoimmunity causes direct tissue damage because of a reaction (usually involving antibodies) to attached antigens regarded as foreign by the hosts immune system. In the past few years researchers have come to realise that cases of autoimmunity may not only be due to the production of true autoantibodies (primary) against normal tissue antigens (self-antigens), but also due to the production of antibodies directed against an antigen (foreign) which is bound to tissues or cells (secondary). The mechanisms of immune reactions in autoimmunity are the same as occur in hypersensitivity reactions and in normal immune responses which aim to remove foreign antigens with minimal tissue damage. Types I, II and III mechanisms are antibody mediated while Type IV is mediated by T lymphocytes. However, for these mechanisms to operate effectively, there often needs to be cooperation between humoral factors and T lymphocytes. Type I involves the synthesis and binding to mast cells and basophils of IgE (reaginic or homocytotropic antibody) in response to exposure to allergens. Degranulation of the bound cells leads to the clinical signs (e.g. bronchoconstriction in allergic bronchitis). This is a common type of hypersensitivity in skin, gut, gastrointestinal tract and urogenital tract (anywhere Mast cells are in abundance). Systemic anaphylaxis, canine and feline respiratory and gastrointestinal allergies, bovine allergic rhinitis and equine urticaria are all examples of Type I hypersensitivities. Type I can also operate in conjunction with other mechanisms in certain hypersensitivities (e.g. canine flea allergy has elements of both Type I and Type IV reactions). Kits for measuring IgE are available. Type II reactions are mediated by complement fixing antibodies (usually IgG and IgM and called cytotoxic antibodies) directed to cell surface antigens. Autoimmune haemolytic anaemia and neonatal isoerythrolysis are examples of Type II reactions. Drug and parasitic antigens attached to cell surfaces (e.g. Haemobartonellosis and erythrocytes) will initiate a Type II reaction. Pemphigus in dogs may involve a Type II reaction. Glomerulonephritis due to autoantibodies against glomerular basement membrane is another example of a Type II reaction. Type III reactions are mediated by immune complexes formed from antigen and antibody (often IgG). Sometimes the antigen is a pathogenic agent, other times it is a self-antigen. The immune complexes, when large enough, lodge in glomeruli and small blood vessels. Complement is activated and local neutrophilic inflammation occurs. This causes tissue damage and dysfunction (vasculitis in various

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organs or generalised and glomerulonephritis). Examples of Type III mediated disease include glomerulonephritis in a wide range of domestic species, dermatitis due to immune complex-mediated vasculitis and systemic lupus erythematosus (SLE). Type IV reactions are mediated by sensitised T lymphocytes. These in turn release a number of cytokines that directly or indirectly (often via macrophages) lead to death of tissue cells. Type IV may be involved in canine flea allergy and in canine lymphocytic thyroiditis.

Autoimmunity
It is not always possible to specifically define an autoimmune disease but available tests should allow the detection of immune mediated disease (e.g. a polyarthritis can be assessed as being immune mediated but it is not always possible to determine whether this is rheumatoid arthritis, systemic lupus erythematosus or some other form). To diagnose autoimmune disease, history and clinical examination are essential. Laboratory investigation can be divided into two parts: the first part relies on the use of routine laboratory tests to establish which organs or tissues are involved and that the disease is inflammatory (and, therefore, likely to be immune mediated): a) b) c) Haematology (general organ/tissue assessment) Biochemistry (general organ/tissue assessment) Urinalysis (specifically for glomerulonephritis), joint fluid analysis etc

Once these tests are done, it may be worthwhile to look specifically at immunoglobulin. d) examination of humoral immune function: i) serum protein electrophoresis ( globulins) ii) immunoelectrophoresis of immunoglobulins iii) single radial immunodiffusion (immunoglobulin quantities) iv) examination of the complement cascade v) examination of cell mediated immunity Once these tests have suggested that the disease is immune mediated then the second part of laboratory investigation can be undertaken. This involves the use of specialised immunodiagnostic tests to more specifically define the problem.

a) Coombs' test (direct)

This test aims at detecting antibodies or complement on the surface of circulating erythrocytes or red cell precursors present in the bone marrow. The test is used to diagnose autoimmune haemolytic anaemia (AIHA) which can occur as a primary condition or as a component of a multisystem autoimmune disease. It may also occur secondary to a wide range of non-specific diseases e.g. neoplasia such as lymphosarcoma. Titres of antibodies in AIHA are highest for the primary form of the disease. Autoimmune thrombocytopenia may accompany AIHA (called Evans Syndrome), but it can also be a distinct entity in itself. AIHA occurs in the dog, cat, horse and cow but is more common in the dog. Haemolysis can occur intravascularly or extravascularly and the formation of spherocytes is common. Before performing the Coombs' test evidence for a haemolytic anaemia should be obtained.

b) Rheumatoid factor (RF)

This is an IgM (or IgG) autoantibody directed against the Fc of IgG that has been altered. The resulting complex fixes complement whose activation leads to cartilaginous damage (rheumatoid

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arthritis RA). RF detection aids in the diagnosis of rheumatoid arthritis, although RF may be present in many autoimmune disorders and RF negative rheumatoid arthritis has been documented. RA diagnosis depends on characteristic radiological changes, synovial fluid changes as well as on the presence of RF. In the dog 40-75% of cases of RA have RF.

c) The antinuclear antibody test (ANA)

This test has a non-specific usage in the diagnosis of autoimmune disease but is essential for the confirmation of systemic lupus erythematosus (SLE). The basis of the test is the detection of antibody directed against various nuclear proteins. This test requires specific antisera. In the dog ANA occurs in a low percentage of the normal population but at a low titre. Autoimmune diseases other than SLE may have low titres of ANA (hence its non-specific usage). The ANA test has replaced the LE cell test (40% of SLE cases will be negative for LE cells). SLE has only been positively diagnosed in the dog and laboratory rodents, although probable cases have been reported in the cat.

d) Direct and indirect immunofluorescence (IF) of tissue biopsies

This is useful to detect various autoantibodies to various cell types, e.g. autoimmune skin disease such as pemphigus vulgaris; glomerulonephritis. Direct IF is supposedly better than indirect IF. Instead of immunofluorescence to detect the autoantibodies, tissue sections can be labelled by an immunoperoxidase method: autoantibodies present in tissues will then stain brown when viewed with a conventional light microscope.

Immunodeficiency
Immunodeficiency may be heritable or acquired and involve defects of one or more arms of the immune system (commonly cell mediated and humoral defects). The dog and horse are most commonly affected by immunodeficiency. Immunodeficiency should be suspected in recurrent cases of increased susceptibility to low grade pathogens or to unusual pathogens, and poor response to therapy. Laboratory assessment of humoral function (Ig quantitation, lymph node biopsy, quantitation of circulating B cells) cell mediated immune function (total blood lymphocytes, lymph node biopsy, blastogenesis), complement levels and phagocytic function (e.g. as in cyclic haematopoiesis in the Collie dog) should be undertaken if appropriate.

*SEE CASE 21

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DIAGNOSTIC CYTOLOGY AND BODY FLUID ANALYSIS

Diagnostic Cytology (Exfoliative Cytology; Cytopathology)
Diagnostic cytology is the examination of individual cell details for the purpose of diagnosis and prognosis. Although more information can usually be gained from the examination of tissues (histopathology) rather than individual cells, cytology does have distinct advantages over histopathology: a) Cytological samples are cheaper and easier to obtain b) Cytological samples are simpler to process. Consequently, the result from a cytological preparation is quickly obtained. c) Agents of disease are sometimes`easier to detect because of better resolution at 100 x magnification Cytological preparations may be stained with blood stains, e.g. Giemsa, Diff Quik. Commonly, a diagnosis on the basis of cytological data is limited and broad. Histopathological examination usually needs to be done to support the cytological findings (by biopsy or at necropsy), and is the only way that tissue architecture (including margins of a lesion) can be examined.

a) Solid tissue cytology

i) Tissue imprints/scrapings are useful for examining accessible soft tissues (in the living animal, from biopsy material, from necropsy material). The surface of the tissue is blotted free of debris and blood and a slide touched onto the exposed surface. The imprint allows examination of individual cell details, cell relationships (if the imprint is performed carefully) and examination for the presence of etiological agents. In firm tissues or in sites inaccessible to imprints (e.g. conjunctiva) tissue scrapings can be done. The tissue is cleaned as for an imprint and then material collected on the `scraper' is then gently spread over a slide (buttering) or transferred to a slide and spread as for a peripheral blood film. Fine needle cell aspirates are principally of use in the living animal (examination of internal and external solid/fluid masses). The cellular material is aspirated with a fine gauge needle (usually 21-23) and syringe (2 ml or 5 ml commonly) and then placed on a slide and gently smeared (as a squash preparation or as a for a peripheral blood film).

ii)

Solid tissue cytology is approached the same way as histopathology: detect, describe and deduce (the three Ds for diagnosis). Since cytological smears are often examined under the 100x objective, the focus for description is cellular detail, although both acellular material and living and non-living agents of disease may be detected. The interpretation is based firstly on the five basic pathological processes (degeneration and necrosis; inflammation and repair; vascular disturbance; disorder of growth; and pigmentation/deposit). In most cases, the lesions are proliferative (hyperplastic or neoplastic i.e. a disorder of growth), inflammatory or degenerative (the last including pigments and deposits) (see Table 1). Further differentiation will depend on cytological and acellular features. Neoplastic lesions can often be differentiated into benign or malignant epithelial, mesenchymal (connective tissue) and round cell types. Solid tissue cytology is particularly useful for detecting and identifying round cell neoplasms (e.g. mastocytoma, lymphosarcoma, benign cutaneous histiocytoma). Inflammatory lesions may be differentiated into acute and chronic (including granulomatous) processes. Cytology is also useful for detecting agents of disease. These may be physical (e.g. foreign bodies), living (e.g. acid fast bacteria, toxoplasmal organisms, chlamydial organisms) or chemical (e.g. calcification of tissue).

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An example of the use of solid tissue cytology would be to investigate lymphadenomegaly. A fine needle cell aspirate of an enlarged lymph node may be able to distinguish among benign lymphoid hyperplasia, lymphadenitis, lymphosarcoma and metastatic neoplasia.

1. Primary degenerative lesions

These include those lesions that are cystic in nature (e.g. epidermal cyst, renal pseudocyst or cyst), those that are due to deposits of minerals or pigments (e.g. calcium in calcinosis circumscripta, haemosiderin in haemorrhages, silicon or asbestos in lung macrophages), and those that are due to damage (e.g. physical trauma or toxic substances).

2. Inflammatory lesions
Inflammatory lesions are characterised primarily by the presence of inflammatory cells or by the presence of infectious agents (e.g. bacteria, viruses, fungi). Cytology is extremely useful in detecting infectious agents and may at times provide positive identification without further investigation; for example the detection of Distemper virus or chlamydial inclusions in conjunctival smears. However, in most instances, further investigation (e.g. culture, molecular studies) is required to positively identify the infectious agent. Lesions caused by infectious agents often feature large numbers of inflammatory cells. Occasionally, as in some cases of cryptococcosis or clostridial hepatitis, few inflammatory cells are present. These lesions are still classified as inflammatory. Not all inflammatory lesions are caused by infectious agents. Other causes include foreign material (e.g. sterile penetration wound; injected irritant) or endogenous irritants (e.g. ruptured epidermal cyst and release of keratin; bile or urine escape into the abdomen). Inflammatory lesions, apart from being characterised by the presence of their cause, can be further classified by the types of inflammatory cells present. This can provide clues to the cause of the problem. Neutrophils indicate acute inflammation in response to damage or to infective agents such as bacteria. Neutrophils can also form a component of ongoing ('chronic') inflammation if the chemotactic factor (such as bacteria) is persistent in the lesion (active chronic inflammation). The term purulent exudate refers to a neutrophil predominance and is often further classified as septic (bacteria are present, especially within neutrophils or non-septic (other causes such as fungi, sterile irritants or immune-mediated disease). Based mainly on nuclear morphology, neutrophils can be described as lytic or nonlytic (degenerate and non-degenerate). The nucleus of lytic neutrophils appears faded and can have ragged or distorted edges. Lytic neutrophils develop under the influence of toxins and are often interpreted as indicating the presence of bacteria. Lytic neutrophils, however, can also be seen in some viral infections (feline infectious peritonitis) or as a result of exposure to chemical toxins as in bile peritonitis or uroperitoneum. Not all septic exudates feature lytic neutrophils. The term degenerate is often used interchangeably with lytic. Eosinophils commonly accompany lesions which have a strong immune component and which usually take some time to develop (e.g. response to some parasites, allergies). They may also be present in lesions involving mast cells (e.g. mastocytoma in dogs; inflammatory lesions involving the skin, respiratory tract, urogenital tract or gastrointestinal tract) or other neoplastic cells (paraneoplastic phenomenon due to the production of chemicals that stimulate eosinopoiesis and/or attract eosinophils). Some predominantly eosinophilic inflammatory lesions are of unknown cause (e.g. the eosinophilic granuloma complex in cats, idiopathic pulmonary infiltrates with eosinophils in dogs).

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Macrophages (also called histiocytes) are important for the phagocytosis of macromolecular material. They usually form a component of chronic inflammatory lesions and are enlisted by a demand for phagocytosis and by lymphocyte derived cytokines. Macrophages can also be present very early on in an inflammatory reaction if the need for phagocytosis is great e.g. in the face of much tissue damage or foreign debris. This is especially so in certain organs or tissues such as the lower airways. The predominance of macrophages, especially large, aggregated or multinucleate macrophages, in an inflammatory lesion may suggest a granulomatous response. This is a special form of chronic inflammation defined by a particular histopathological arrangement of macrophages that may be caused by certain agents of disease such as foreign debris, certain fungi, protozoa, multicellular parasites and bacteria (e.g. staphylococcal, mycobacterial, nocardial organisms). Many of these agents of disease can also cause a pyogranulomatous response in which both neutrophils and macrophages are found in significant numbers. Lymphocytes and plasma cells are present in lesions due to adaptive immune stimulation and commonly accompany prolonged inflammation. However, in some skin lesions, such as allergies, they can accumulate quickly. While their presence suggests an immune component to the inflammation, it does not automatically mean that the immune response is the primary cause of the problem (i.e. autoimmunity or hypersensitivity). Fibroblasts are normally present in chronic inflammation (therefore, also in granulomatous inflammation) and in healing wound. Be careful, reactive fibroblasts can be quite pleomorphic and can overlap in appearance with neoplastic spindle cells.

3. Proliferative (hyperplastic/neoplastic) lesions

Because hyperplastic and neoplastic lesions tend to produce lumps and bumps that are relatively easy to sample, this is the most common category to be diagnosed by cytology. As mentioned previously, inflammation can accompany many tumours and can be the more prominent feature. Neoplasms can cause damage to surrounding tissue and many tumours have a high cell turnover. Both these processes produce factors that are chemotactic for inflammatory cells. Examples include the intense inflammation that commonly accompanies a squamous cell carcinoma (the irritant nature of free keratin also contributes to the inflammation); the nonseptic exudates that accompany neoplastic cells exfoliating into the pleural, pericardial and peritoneal cavities; septic inflammation due to clostridial infection of necrotic liver tumours and septic peritonitis secondary to a perforating neoplasm of the gastrointestinal tract. If tumour necrosis is extensive, the neoplastic cells can be difficult to find or identify. Experienced cytopathologists realise this and often warn the practitioner with the phrase 'suspect underlying neoplasia as a cause'. Much has been written about diagnosing neoplasia by cytology, especially in relation to certain tumours such as mast cell neoplasia. The suggested approach involves a logical, stepwise approach using general principles. It allows identification of the neoplastic process and often leads to diagnosis of the specific neoplasm. Questions to be asked, in order of importance, are: 1. Is the lesion primarily proliferative (disorder of growth) and, if so, is it hyperplastic, benign or malignant? Proliferative lesions can be hyperplastic or neoplastic, benign or malignant. Differentiation can be extremely difficult and is not always straightforward based on nuclear and cytoplasmic features. It is probably not critical or possible to distinguish between hyperplastic and benign neoplastic processes on cytology. Detection of malignancy, however, is highly desirable but, again, not always possible. The hallmarks of malignancy are anaplasia, invasion, and metastasis. The presence of

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invasion into surrounding tissue can only be assessed on gross and histological examination. Metastases can be detected by imaging and cytology e.g. aspiration of lymph nodes, organ masses. Anaplasia, which refers to the fact that the neoplastic cells are poorly differentiated and, consequently, highly variable in appearance, can be assessed cytologically. Anaplasia is a common feature of malignancy, but does not have to be present. Some malignant neoplasms can have relatively 'benign' cellular features (i.e. the cells look like well differentiated origin cells and show only slight nuclear and cytoplasmic variation). A common example would be some thyroid carcinomas in the dog. Even the cells from their metastases can look relatively well differentiated and be in a glandular pattern! Despite this limitation, many tumours can be designated as malignant on cytological features. Although it is difficult to make rules for cells that break rules to become established and spread, it can generally be said that the more bizarre the cells appear the more likely are they to be malignant (exceptions plasmacytomas, fibroblasts). Cytological features that are most likely to indicate malignancy include: numerous and abnormal mitoses, cell and nuclear pleomorphism, cytoplasmic basophilia, enlarged or bizarre nucleoli, and multinucleation (especially with variably sized nuclei). If you suspect neoplasia from the history but your cytological sample is not supportive, dont disregard your suspicion. Re-aspirate if the answer is not clear cut. Neoplastic cells may be difficult to detect, especially if there has been blood dilution, tissue necrosis or masking by inflammatory cells as mentioned previously. 2. Are the predominant cells round, spindle, or epithelial? These are the three basic cell groups responsible for proliferative lesions and are classified according to cell shape and embryonic origin. Round cells include all haematopoietic cells (including lympho-histiocytic cells) and mast cells. As suggested, the cells are round in shape. They usually show little tendency to aggregate in smears (unless the smear is thick) and are of embryonic mesoderm origin. Spindle cells suggest a mesenchymal (embryonic mesoderm) origin (i.e. all connective tissues, muscle, vessels etc). Not all spindle cells are tapering, some can appear rounded or angular. While spindle cells typically occur as individual cells in smears, some mesenchymal neoplasms exfoliate well to produce epithelial-like aggregates of cells e.g. haemangiopericytomas. Epithelial tumours are derived from ectoderm or endoderm. Cells vary from square to columnar, and at least some should be found in groups in smears. Some specific tumours derived from neuroectoderm (melanocytes and Schwann cells) can present in either spindle or epithelial forms. Malignant melanocytes may even present as round cells. 3. Can the cell types be subcategorised? Subcategorisation depends on the detection of specific arrangements of cells or the production of a cell product. For example, if the epithelial cells are in a duct or acinus arrangement or if they are in a solid clump but have an intracellular product, then they are identified as glandular. Intracytoplasmic melanin will suggest a melanotic tumour while intracytoplasmic, purple granules could suggest mast cell neoplasia.

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TABLE 1: Approach to interpretation of cytological smears (based on predominant pathologicalprocess)

Predominantpathological process Primarydegenerative changes Mainfeaturefor characterisation cysts(e.g.renalcyst) Usefulnessofcelltype Basisforfurther characterisation

Primaryinflammatory lesions(i.e.notsecondary toprimarydegenerative lesionsorneoplastic conditions)

damage(e.g.burn, trauma) depositsandpigments (e.g.calcinosis circumscripta) Onpredominantcell type

neutrophils(purulent inflammationsepticdueto bacteria;nonsepticdueto othercauses) eosinophils(strongimmune componentinthe inflammation) macrophages(histiocytes) epithelioidcellsand multinucleatesmayindicate granulomatous inflammation lymphocytesandplasma cellsindicatechronic immunestimulation Epithelial Spindle

Hyperplasticandneoplastic lesions

Onmaincelltype

Round

variableshape(some mesodermaland neuroectodermaltumours)

glandularorductularin arrangement squamousdifferentiation patternandacellular depositsmayallow differentiation mastocytoma, lymphosarcoma, haematopoietictumours, histiocytictumours melanomasshouldhave cytoplasmicpigment; osteosarcomasshouldbe producingosteoid

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b) Diagnostic cytology as a component of body fluid analysis

Cytological preparations for fluids can be obtained by direct smearing of the fluid or by centrifuging the fluid and smearing the sediment. Fluids of an inflammatory nature should be placed in anticoagulant (e.g. EDTA) as clotting interferes with smear preparation. Interpretation of the cell smears will depend on the site of origin of the fluid and will be discussed under body fluid analysis. Nb. Cells deteriorate rapidly in body fluids. If a delay is envisaged before the sample reaches the laboratory then smears should be made directly from the fluid prior to despatch.

Body FLuid Analysis

a) Body cavity effusions
This is mainly referring to abdominal and thoracic fluids but there is applicability to pericardial and other cavities) An effusion refers to a pouring out of any fluid into a body cavity or tissue, i.e. excess fluid. By assessing gross characteristics and determining protein levels (by refractometer or by the chemical Biuret method), total nucleated cell count, and differential cell count (on a smear). Abdominal and thoracic cavity fluid is commonly assessed when there is an effusion. In cattle and horses, abdominal cavity fluid may be assessed as part of an abdominal cavity disease investigation even when increased fluid (an effusion) is not present. Effusions can be divided into:

i)

Pure transudates
These may occur in hypoproteinemic states due to lowered plasma osmotic pressure and in some forms of chronic liver disease in the dog. They are really normal body fluid in excess (therefore low protein and low cells). These are non-inflammatory and uncommon.

ii)

Modified transudates
These are still non-inflammatory in origin. They are modified by the addition of protein or cells (but limited compared to values for exudate), e.g. ascites due to chronic passive congestion, neoplasia, chylothorax. These are very common. Nb. A process may start off as a pure transudate and, with time, progress to a modified transudate and even to a non-septic exudate (once serum protein components, neoplastic cells etc start to accumulate they may create chemotaxis for phagocytes, thereby causing secondary inflammation). Consequently, it may be difficult at times to separate some modified transudates from early non-septic exudates due to variable alterations of protein and cells. Chronic heart failure in the dog tends to cause thoracic and abdominal effusions that quickly pass from pure transudates to modified transudates (erythrocytes are common in the fluid). Occasionally they may progress to non-septic exudates. Chronic cardiac disease in the cat can cause thoracic effusions, which are commonly classified as chylous (i.e. chylothorax).

iii)

Chylous effusions
These most commonly occur in the thorax (but can be present in the abdomen) and are assumed to be due to leakage from major or minor lymphatics. Consequently, chylous effusions are characterised by many small lymphocytes and high triglycerides (higher values than in plasma).

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A variety of conditions can give rise to chylothorax (e.g. neoplastic interference to lymphatic flow, trauma to lymphatics, heartworm, idiopathic), but the most common cause in the cat is chronic heart disease. In contrast, chylous effusion in the thorax of the dog is less common than in the cat and is rarely associated with chronic heart disease. Instead, it is most commonly due to thoracic duct compression (rarely rupture), but may occasional occur in other diseases affecting lymphatics. Most chylous effusions are milky white but exceptions do occur. In addition, some milky white fluids may be pseudochylous. These are extremely rare. They are turbid fluids that can be caused by exfoliating neoplasms or tissue inflammation. With cell breakdown large amounts of cholesterol build up in the fluid. However, the milky colour of the fluid is not due to fat but due to cell debris and chemical complexes). To differentiate chylous from pseudochylous effusions, it is best to support gross and cytological findings with simultaneous analyses of triglycerides and cholesterol in both effusion and plasma (or just effusion and determine the ratio). Chylous effusions will have high triglycerides and low cholesterol effusion levels when compared to plasma levels; and vice versa for pseudochylous effusions.

iv) 1)

Exudates (high protein and high total nucleated cell numbers) non-septic exudate
This type of effusion either develops from a modified transudate or is due to direct inflammation caused by irritants that usually produce few toxic changes in neutrophils, e.g. sterile foreign bodies, ruptured urinary bladder (this more commonly causes a modified transudate), bile peritonitis, Feline Infectious Peritonitis (FIP- it has been suggested that this condition gives rise to a modified transudate rather than a non-septic exudate as cell counts are often limited. However, since both cells and protein are increased, and it is known to be an inflammatory process, it is more appropriate to call it a non-septic exudate).

2)

septic exudate
This type of effusion is the product of inflammation caused by a wide variety of microbes that are toxic to neutrophils (principally causing karyolysis due to interference with water balance). Values for protein and total nucleated cells are high compared to the other categories.

v)

Haemorrhagic effusion (recent or long standing)

Recent haemorrhage will be characterised by clear supernatant on centrifugation and platelet clumps on examination of the smear. With time, the supernatant will become red-brown or yellow due to degradation of erythrocytes. A smear will show erythrophagocytosis and/or haemosiderin in macrophages.

It is important to remember that the basis for the classification of body fluid effusions depends upon protein estimation, total nucleated cell count and differential cell count on a smear. Values are provided for abdominal fluid analysis (probably can be applied to pleural and pericardial fluid analysis with care): Refer to the following table. Values provided by texts for protein estimations by refractometer and total nucleated cell numbers for the different categories may vary from these. Consequently, the following values should be used as guides and not be regarded as absolute.

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TABLE: Characteristics of body fluid effusions for the dog, cat and horse (mostly applies to peritoneal fluid)
Gross characteristics Totalproteing/L (refractometer) Puretransudate* clear/colourless(S); clear/paleyellow(H) <25 Chylous effusion variablebutoftencloudy oftenturbid whiteorpink 2550(S);2530(H) >25(inaccurate duetopresence ofthefat) 3005000(S);5000 variablebut 12,000(H) usually500 5000 asforpuretransudatebut Small increasednonlytic(non lymphocytes usually degenerate)neutrophils predominate andsomeerythrocytes andreactivemesothelial cells Modifiedtransudate Nonseptic exudate Cloudy 3070 Septicexudate markedcloudiness 3070 Haemorrhagic effusion cloudyredorbrown variablebutusually high variablebutusually high mixedperipheral bloodcellsplus macrophagesand mesothelialcells

Totalnucleatedcell 5001500(S);1500 count(x106/L) 10,000(commonly <7,500)(H) Differentialcell monocytes, count(smear) lymphocytesandnon lytic(nondegenerate) neutrophils

5000100,000(S); >12,000(H) mainlynonlytic (nondegenerate) neutrophils,some erythrocytesand mesothelialcells

5000100,000(S); >12,000(H) mainlylytic (degenerate) neutrophils, microbes,some macrophagesand erythrocytes

*alsoappliestovaluesfornormalabdominalfluid H=horse;S=dogandcat;ifnotstatedappliestoboth

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NOTES ON BODY FLUID EFFUSIONS i) Horse abdominocentesis: The procedure can be done even though significant effusion may not be present (e.g. in evaluation of GIT disturbances, colic). In horses, lactate levels in peritoneal fluid may be analysed in conjunction with plasma levels to assess abdominal problems. In healthy horses lactate levels in peritoneal fluid are usually lower than in plasma. With abdominal crisis lactate levels in peritoneal fluid exceed those in plasma (especially when there is gut hypoperfusion [e.g. strangulation] or sepsis), and the wider the gap, the poorer the prognosis (lactate levels are not used for diagnosing abdominal catastrophes, just for prognosis and response to therapy). Nb. General notes on the use of blood lactate in the horse: Blood lactate (an end product of anaerobic glycolysis) can be measured in the horse as part of a general health assessment. Physiological hyperlactaemia can occur with strenuous exercise, but blood levels are rapidly removed by the liver and kidney. Pathological hyperlactemia occurs when oxygen delivery to tissues is inadequate to meet their demands (tissue hypoxemia due to hypovolemia leading to tissue hypoperfusion e.g. acute blood loss and severe anaemia; plasma volume depletion; septic/endotoxic shock; heart failure), and lactate production exceeds blood buffering and liver/renal clearance (e.g. the commonest reasons are severe liver disease, malignancy, sepsis and endotoxemia, and excess catecholamine release). Blood lactate elevations are, therefore, non-specific and may be elevated in a wide range of diseases. However, levels can be useful in prognosis and response to therapy, especially for estimating inadequacy of tissue perfusion. At the University Veterinary Teaching Hospital Camden, normal lactate concentrations (for L-lactate, which is the mammalian cell form) measured in whole blood in adult horses are <2.0 mmol/L. Normal levels in newborn neonatal foals <36 hours of age are slightly higher at <2.5 mmol/L. Pathological elevations in blood lactate between 2.0-5.0 mmol/L (termed hyperlactatemia) are regarded as mild, moderate between 5.0-7.0 mmol/L (termed lactic acidosis), and severe when >7.0 mmol/L (strenuous exercise is an exception, when under physiological conditions blood lactate levels reach greater than 20.0mmol/L). ii) Pathological haemorrhage needs to be differentiated from iatrogenic hemorrhage. If blood vessel penetration has occurred at the time of abdominocentesis then the blood is often streaky. Splenic penetration will produce a high PCV and many lymphocytes. Erythrocytes can accompany modified transudates and exudates (via diapedesis), but will never reach the levels experienced in haemorrhage due to rhexis. iii) In cattle abdominocentesis has been used to diagnose peritonitis. Total nucleated cells and protein levels, however, are not as useful as the differential. In healthy animals protein levels, as determined by the refractometer, are up to 30 g/L while total nucleated cells are up to 10,000 x 106/L (10 x 109/L). Eosinophils are commonly high in normal peritoneal fluid (>30%) while neutrophils are usually less than 50% (nb. healthy cows, less than two weeks post-partum may have a nucleated cell count greater than 10,000 x 106/L, while either neutrophils or eosinophils may predominate in the differential). In peritonitis, protein levels vary from 20 g/L upwards (top of around 60) while total nucleated cells vary from 1,000 x 106/L upwards (top can be over 200,000). However, more importantly, the proportion of neutrophils increases while the proportion of eosinophils drops. The presence of appropriate bacteria and high fibrinogen levels support the diagnosis of peritonitis. iv) Neoplastic effusions have an extremely variable appearance. Diagnosis is dependent on examination of the cytological smear and identification of neoplastic cells. Malignant neoplasms which commonly exfoliate into body cavities include mesotheliomas, adenocarcinomas and lymphosarcoma. If neoplastic cells have not exfoliated, then the effusion can present as a modified transudate, chylous effusion, an exudate or as a haemorrhagic effusion.

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b) Synovial fluid analysis Introduction

Except for septic arthritis, synovial fluid analysis rarely provides a specific diagnosis. What it does do is to provide information that allows placement of the joint problem into a general category (e.g. degenerative, inflammatory). When this is combined with other physical, radiological and laboratory findings a specific diagnosis may be obtained. Synovial fluid is a dialysate of plasma to which mucus is added by synovial cells as the plasma diffuses through the synovial membrane into the joint cavity.

Laboratory evaluation of synovial fluid Normal gross characteristics i)

1) volume: 0.01 - 1 ml for the dog (less in the cat, more in horse) 2) colour: colourless (may be light yellow in the horse) 3) transparency: clear 4) viscosity: noticeably viscous (due to hyaluronic acid content) 5) mucin content: determined by the mucin clot test: One ml of synovial fluid is mixed with 5 ml of 2.5% acetic acid and left for at least 1/2 hour. Normal synovial fluid, which is high in mucin, produces a tight ropy clot in a clear solution (good mucin clot). A fair mucin clot is a soft mass in a slightly turbid solution. A poor mucin clot is a small friable mass in a turbid solution. A very poor mucin clot is a few flecks in a turbid solution.

ii)

Normal cytological characteristics

A total nucleated cell count is determined from synovial fluid collected in EDTA. A differential cell count is determined from a smear of synovial fluid (preferably made directly from withdrawn synovial fluid). The dog has a total nucleated cell count of less than 3000x106/L (this depends on the joint sampled) while a horse usually has less than 500x106/L. In health, 90% of the nucleated cells are lymphocytes and monocytes.

iii)

Other characteristics (investigated when indicated by disease)

e.g. Protein levels (mainly of use in the horse- although not completely accurate, the refractometer can be used to estimate total protein in a healthy horse the value is usually 10-15 g/L), glucose levels (in health, are often equal or slightly higher than in the serum), certain enzymes (e.g. AST, ALP).

When collecting synovial fluid, place some in an EDTA tube and some in a plain tube (sterile if you suspect septic inflammation). Make a smear immediately from withdrawn fluid. Often in the dog and cat not enough fluid is obtained to do all tests. Priority should be given to the smear (for differential cell count) and a total nucleated cell count, unless the history indicates otherwise

Changes in synovial fluid characteristics with disease:

Refer to table on following page. Basically, synovial fluid changes due to disease are categorised as acute degenerative/traumatic, low grade/Long term degenerative or primary inflammatory (non-septic or septic). Note: lytic (degenerate) neutrophils, which are common in septic inflammation anywhere, are sometimes not obvious in septic synovial fluid due to the apparent protective nature of chemicals present. Neoplasms involving joints and causing synovial fluid changes are uncommon.

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TABLE: Synovial fluid analysis showing changes in disease Volume Colour Turbidity Fibrinclot Viscosity Mucin Totalnucleatedcells Differentialcellcount Acutedegenerative/ traumaticconditions Increased normaltodiscoloured(red) slighttomarked veto+ve Variable fairtopoor slighttomoderate erythrocytes,neutrophils Lowgrade/Longterm degenerativeconditions normaltoincreased normaltodiscoloured(red brown) normaltoslight ve normaltoslight normaltofair normaltoslightincrease macrophages(cartilage fragmentsmaybeseen) Primaryinflammatory (nonseptic) increased discoloured slighttomarked +ve decreased fairtoverypoor Primaryinflammatory (septic) increased discoloured marked +ve decreased poortoverypoor

moderatetomarkedincrease mainlyneutrophils(nonlytic(nondegenerate)fornon septic,usuallylytic(degenerate)forseptic)plus erythrocytes(viadiapedesis),macrophagesand lymphocytes(microbesforseptic)

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c) Cerebrospinal fluid evaluation Introduction

Cerebrospinal fluid (CSF) is produced partly by diffusion from plasma and partly by active secretion from the choroid plexi and ependymal linings. In the dog and cat, CSF is collected usually from the subarachnoid space at the atlanto-occipital articulation. In the horse and dog the subarachnoid space at the lumbosacral articulation can be used.

Laboratory evaluation of CSF i) Physical Examination

1) Colour In the healthy dog, cat and horse CSF is colourless. Xanthochromia (yellowing of the CSF) may occur in a jaundiced animal or in an animal that has had previous haemorrhage in the CSF. Red CSF may indicate recent haemorrhage (pink to orange supernatant) or haemorrhage at the time of collection (clear supernatant). A grey-green CSF may indicate suppuration. Turbidity Normal CSF is clear. A turbid CSF suggests excess cells (turbidity is only observed when the cell count is in excess of 500x106/L) or perhaps protein or bacteria. Coagulation Normal CSF doesn't clot. Clotting may be due to inflammation or haemorrhage.

2)

3)

ii)

Total nucleated cell count

CSF is collected in an EDTA tube (in case of risk of clotting) and in a plain serum tube (for protein estimation). If infection is suspected some CSF should be collected in a sterile tube. The total nucleated cell count (usually done on the EDTA tube) has to be performed quickly as there is rapid degeneration of cells (usually within a few hours). Pleocytosis (an increase in the number of nucleated cells in CSF-in health: the healthy dog, cat and horse have counts usually 5 x 106/L or below; 5-8 is a grey zone for increases) may be due to trauma, primary inflammatory conditions, toxic conditions or neoplasia. Pleocytosis requires the performance of a differential cell count (if total cell count is greater than 500x106/L then a smear should be made directly from the CSF, if the total count is less than 500x106/L then ideally a smear should be made of the sediment of CSF after unit centrifugation).

iii)

Differential cell counts

In practice, smears should be made at the time of collection, irrespective of total nucleated cell count. This is because cells in CSF deteriorate rapidly. In a normal sample most cells are small lymphocytes (around 60-70%) and monocytes/macrophages (20-30%). In pyogenic infections the cells are mostly lytic (degenerate) neutrophils. In haemorrhage the cells are mostly non-lytic (non-degenerate) neutrophils. In viral, uremic, some chronic bacterial and toxic conditions, lymphocytes and monocytes usually predominate. Neoplastic cells may be seen in meningeal tumours.

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iv)

Protein estimation
Normal CSF has a low level of protein (less than 0.3 g/L for dog, 0.2 g/L for cat and 0.67 g/L for horse). Most of the protein is albumin. Increases in CSF protein may occur in a variety of inflammatory conditions (primary or secondary) and principally involve increases in globulins. To detect increased levels of protein, either total protein (ponceau S method) or globulin (Pandy test) levels can be determined (urine protein reagent strips will provide an indication of increased protein).

General comments on disease affecting CSF

The greatest alterations to CSF usually occur with meningeal disease. Nervous parenchymal disease may cause minimal changes to the CSF. CSF analysis is an aid to diagnosis and requires to be analysed in conjunction with history, clinical signs and other diagnostic procedures performed. Commonly, CSF changes are categorised as degenerative (e.g. disc disease), inflammatory (subcategories include non-suppurative, suppurative and granulomatous depending on proportions of cells present) and neoplastic (primary or metastatic). Sometimes, infectious agents may be detected on the cytological smear (e.g. cryptococcal and toxoplasmal agents).

*SEE CASES 14-16, 25, 26, 30

d) Analysis of airway and pulmonary lesions by respiratory washes (transtracheal aspirates and bronchoalveolar lavages)
Respiratory washes for airway and pulmonary disease can be performed in any animal species, but is commonly done in the dog, cat and horse

Sampling techniques
Collection of cells from the tracheal and bronchoaveolar surfaces can be done in a number of ways. Bronchoaveolar lavage (BAL), via a bronchoscope or catheter, provides the best cellular samples of the lower respiratory tract (alveolar spaces and smaller airways). Occasionally, oropharyngeal contamination may occur by this method (usually related to contamination from the endotracheal tube). Transtracheal aspiration (TTA) will usually provide sterile samples for microbiological investigation but cell retrieval, especially from the alveolar spaces, may be limited.

Oropharyngeal contamination of respiratory washes

This will be recognised by the presence of squamous epithelium (often with associated bacteria). Specific oropharyngeal bacteria, such as Simonsiella sp, may be present. This will jeopodise the investigation of a possible infectious process affecting the lower respiratory tract. Moreover, any neutrophils present may be related to oropharyngeal inflammation rather than lower respiratory inflammation.

Types of lining cells and other structures present in respiratory washings

Cellular features will vary depending on whether the sample has been taken by TTA or by BAL. BAL: will contain a greater proportion of alveolar macrophages and lower airway epithelial cells, and will not usually have oropharyngeal contamination.

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Ciliated columnar epithelium The majority of tracheal and bronchial cells will be ciliated columnar cells. They have small round to oval nuclei situated at the end of the cell not displaying the plate of cilia. Cilia are often lost in the washing process and will be found free: they can be mistaken for filamentous bacteria.

Mucosecretory (goblet) cells These are large oval to bulging, elongated cells containing numerous large deep-pink granules in their cytoplasm.

Bronchoalveolar cells These refer to the small, round to square cells, often present in clusters. Their nuclei are usually round and they have moderate amounts of blue-grey cytoplasm. Some of these cells are basal cells while others may be lining cells from the lower airways. Some may be pneumocytes just becoming macrophages.

Alveolar macrophages Alveolar macrophages are usually large with obvious cytoplasmic vacuoles or ingested material (e.g. blue granules of haemosiderin, black-brown granules of carbon). There are always some alveolar macrophages present in washes and, rather than indicate disease, may simply indicate that the sample is truly representative of alveolar spaces. Of course, increased numbers, their appearance and the presence of specific inclusions may indicate disease. Mucus This is commonly present in washes and will be pink to light blue. Excessive and prolonged secretion of mucus will occur in a variety of chronic respiratory conditions (inflammatory or neoplastic). In these circumstances the mucus often stains a darker blue. In addition there may be dark blue to purple spirals of inspissated mucus called Curschmann's spirals. Inflammatory cells Inflammatory cells (neutrophils, eosinophils, mast cells and lymphocytes) may be present in normal respiratory washes but they are usually in low numbers (e.g. less than 5% for neutrophils, only occasional mast cells). An exception is eosinophils in cats where levels of up to 15% can be normal.

Commonly diagnosed airway and pulmonary conditions on the basis of respiratory washes
Many of these are simply divided into pathological processes, the main ones being inflammatory, vascular or neoplastic. Acute (active) inflammation In the dog, cat and horse this will be characterised by high levels of neutrophils and lesser numbers of alveolar macrophages. In some cases this will be related to bacterial infection (with appropriate intracellular bacteria septic inflammation). At other times there will no bacteria visible, nor will any be cultured. This type of sterile (non-septic) inflammation may accompany neoplastic conditions but can also be idiopathic (even though some appear to respond to antibiotics in dogs and cats!).

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Active inflammation can be present in chronic conditions of the respiratory tract. Septic inflammation is often a sequellae of viral pneumonia.

Allergic or parasitic (hypersensitivity) inflammation This will commonly be characterised by significant numbers of eosinophils. Mast cells and neutrophils will be present in variable numbers. Often, many of the eosinophils and mast cells will degranulate, thereby producing a granular background. Parasites, such as lungworm or microfilaria, may be visible in the washes. Chronic Obstructive Airway Disease in horses is regarded as a hypersensitivity. However, the presence of increased numbers of eosinophils is variable, and may just be a feature of early disease. Most features are consistent with those seen for chronic respiratory disease

Mycotic (fungal) inflammation Eosinophils may also be a prominent component of mycotic (fungal) inflammation due to similar hypersensitivity mechanisms which occur in allergies or parasitic infections. However, the inflammatory profile is more variable depending on the agent and the duration of the disease. Both purulent and pyogranulomatous responses may occur. Yeasts may be present in Cryptococcus neoformans infection, while fungal hyphae may be present in Aspergillus spp infection. Multinucleated macrophages may be present but are not specific for this type of infection. They can also occur in foreign body reactions and be present non-specifically in any form of chronic lung disease. Pulmonary haemorrhage This is not usually a specific feature and can occur in most conditions affecting the lungs. However, it can be a prominent feature in trauma, heart failure, infarction (e.g. heartworm), bleeding disorders, certain types of neoplasia and certain poisons (e.g. paraquat toxicity). Erythrophagocytosis by alveolar macrophages will be visible in acute haemorrhage, while haemosiderin-laden macrophages are more of an indication of past or chronic haemorrhage. Exercise-Induced Pulmonary Haemorrhage (EIPH) occurs in horses as a consequence of strenuous exercise. TTA, and especially BAL will support the diagnosis. In the early stages erythrophagocytosis will be prominent, while haemosiderin-laden macrophages will develop and remain for weeks after the event. Chronic respiratory disease The features of chronic respiratory disease are variable and depend on the cause (i.e. these are nonspecific findings that may be related to a wide variety of causes). Mucin and increased numbers of macrophages are common features but are not highly specific. Goblet cell and general epithelial hyperplasia are common if there is airway involvement. There may be squamous metaplasia with chronic irritation to lining cells.

Primary and secondary neoplasia

Neoplasia of the lungs cannot always be diagnosed on respiratory washes. There is more chance of diagnosing primary epithelial lung tumours (commonly adenocarcinomas) than metastatic tumours. Fine needle aspiration or biopsy of the pulmonary tissue is usually required to diagnose neoplasia. Consequently, most of the information on pulmonary neoplasia has been presented in the next part. Primary and metastatic neoplasia are rare in the horse.

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CASE REPORTS

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CASEREPORTS

Introduction
The following case reports are primarily derived from clinical cases presented to the University Veterinary Teaching Hospitals. The clinical pathology for the case reports was performed by the Veterinary Pathology Diagnostic Services laboratory in the Faculty of Veterinary Science. They are meanttocomplementtheprecedinglecturenotes. Thecasereportsareorganisedintwoparts: 1. Information on the animal, history, clinical signs and physical examination findings, and clinical pathologyresults 2.Casereportanalysis Case reports form the basis for the written form of assessment in Veterinary Clinical Pathology. These cases are designed to provide information about specific conditions as well as allowing studentsto testthemselvesoncasereportanalysis.Theanalysispartforeachcaseisplacedona separate page. It is suggested that the readers attempt their own case report analysis first before readingtheprovidedanalysis.Notethatthestyleofanalysishasbeenmodifiedforsomefromthe formexpectedintheexamination.Thesuggestedexamination/assessmentstyleforinterpretation isgivenbelowinsummarisedform.Pleasealsoseetheexaminationquestionandmodelanswer under ASSESSMENT as there is more detailed discussion of approach to answering case report examinationquestions. Casereportanalysisshouldbeorganizedinto: (a) (b) detectionandhighlightingofabnormalities general interpretation of the abnormalities, keeping in mind any information (history, clinical signs, physical examination) provided for the animal. In other words, general interpretationmustbeappropriateforthecaseinhand integrationofallthedatatoreachconclusion(s)oraspecificdiagnosis.Inmanysituations it is not possible to reach a specific diagnosis until further investigation is undertaken. Sometimes only several general conclusions can be drawn from the data, and further investigation is required to follow several lines. Think medicine when considering further investigation i.e. dont restrict investigation to laboratory testing, include image analysis, exploratory laparotomy etc. A consideration of the implications of the conclusions for managementofthecaseshouldbeincludedinananswer

(c)

Thisstyleemphasisesthat(a)and(b)providethebasisfor(c),anditis(c)thatisthemostimportant; i.e. conclusions, further investigation and implications for management are the key outcomes of laboratoryinvestigation.Remembertoassesstheclinicalinformationprovidedfortheanimal,asthis islikelytogivecertaincluestothediseaseproblem. Pleasenotethatforthefollowingcasereportsoftenonlytheresultsofrelevantbiochemicaltests areprovided.Theresultsofhaematologyareusuallyprovidedinfull. Interpretationofclinicalpathologyresultsisassistedbythefollowinginformationonassessmentof thelevelsofbiochemicalandHaematologicalincreasesindisease.Thetablecanbereferredtowhile attemptinginterpretation.Thetableprovidesanindicationofwhatincreasesordecreasesinsome

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ofthemorecommonlyusedanalytesareneededtocallachangemild,moderateormarked.They are subjective (i.e. based on the authors experience) and based on values detected by the VeterinaryPathologyDiagnosticServiceslaboratoryatTheUniversityofSydney.Theequinevalues were determined from the University Veterinary Teaching hospital Camden. They do not apply to valuesfromotherdiagnosticlaboratories.ThevaluescanbeusedfortheExaminationinVeterinary ClinicalPathology.

Remember:
1.Thedegreeofchangeinthevalueoftenindicatestheseverityofthediseaseprocess,butthere areexceptions(e.g.moderatetomarkedincreasesinCKcanoccurinexerciseaswellasdisease). 2.Someoftheanalytesarenonspecificandchangesmaybecausedbyseveraldiseaseprocessesin several organs or tissues (e.g. amylase can be increased in GIT, renal and exocrine pancreatic disease). 3.Thesedonotapplytocertainanalytesforsomebreedsofdogs.Thisisbecausethebreedshave eitherdifferentreferenceintervalsordifferentresponsesindiseasee.g.bileacidsinMalteseterriers are of little use in disease as reference intervals are extremely large; greyhounds and some other sighthoundshavemuchlowerreferencelevelsfortotalleukocytesandneutrophils(canbeaslowas 34 x 109/L for total leukocytes), and sometimes increases in disease may be muted compared to otherbreeds. Analyte CKIU/L Mildchange Increases 201800; 4001,000(H) Increases 14011800(D&C) Increases 301500(D) 101300(C) Increases 111500(D); 51100(C); 260400(H) Increases 61200(D&C) Increases 8.320(D); 3650(H) Increases 4.820(H) Moderatechange 8012000; 10012000(H) 18012800(D&C) 501800(D) 301500(C) 5011000(D); 101200(C); 401600(H) 201500(D&C) 2130(D); 5170(H) 2150(H) Markedchange >2000* >2800(D&C) >800(D) >500(C) >1000(D); >200(C); >600(H) >500(D&C) >31(D); >71(H) >50(H)

AmylaseIU/L LipaseIU/L

ALPIU/L

ALTIU/L GGTIU/L

GLDHIU/L

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SerumProteing/L

BileAcids(fasting) mol/L TotalBilirubinmol/L

Glucosemmol/L

Creatininemol/L

Ureammol/L

Potassiummmol/L

PCVL/L

TotalPlasmaProtein g/L

Increases 7180(D); 7480(C); 7780(H) Decreases 4549(D); 4553(C); 5360(H) Increases 1125(D); 615(C) Increases 8.220(D); 3.620(C); 5175(H) Increases 6.58.0(D); 6.715(C); 6.48.0(H) Increases 121180(D); 181220(C); 150200(H) Increases 10.125(D); 10.825(C); 8.325(H) Increases 4.95.2(D); 4.75.2(C); 5.15.5(H) Decreases 3.04.8(D); 3.54.6(C); 2.52.7(H) Decreases 0.30.36(D); 0.20.29(C); 0.25.31(H) Increases 7685(D); 7990(C); 8590(H)

8190 3544; 4552(H) 2650(D); 1650(C) 21100; 76100(H) 8.115(D&H); 15.120(C) 181500(D); 221500(C); 201500(H) 25.140

>90

<34; <45(H) >50 >100

>15(D&H); >20(C) >500

>40

5.37.0; 5.67.0(H) 2.52.9(D); 2.53.4(C); 2.22.4(H) 0.20.29(D); 0.15.19(C); 0.20.24 86100(D); 91100(C&H)

>7.0(deathat10 12forD&C) <2.5; <2.2(H) <0.20(D&H); <0.15(C) >100

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Leukocytesx109/L

Neutrophilsx109/L

Plateletsx109/L

Increases 12.120(D); 14.120(C); 13.118.0(H) Decreases 57(D); 68(C); 46(H) Increases 9.418(D); 10.118(C); 7.014.0(H) Decreases 3.04.0(D); 3.03.7(C); 2.02.5(H) Decreases 101200(D); 101300(C); 6079(H)

20.150(D); 20.140(C); 18.130.0(H) 3.04.9(D); 3.05.9(C); 3.03.9(H) 18.135(D); 18.130(C); 14.125(H) 2.02.9(D&C); 1.51.9(H) 50100(D&C); 4059(H)

>50(D); >40(C); >30(H) <3.0

>35(D); >30(C); >25(H) <2.0(D&C); <1.5(H) <50(D&C); <40(H)

*Thevaluesapplytodog,catandhorseunlessotherwisestated D=dog;C=cat;H=horse

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CASE1
ANIMAL:3yearoldmaleBeagle. PRESENTINGCOMPLAINTS:Severeweightloss,inappetenceandoccasionalvomitingfor4months. Thedoghadhadahistoryofrecurrentboutsofvomitingandacuteabdominalpainbeforethefour months.Radiographsduringoneepisodesuggestedhepatomegaly. LABORATORYRESULTS

BIOCHEMISTRY AmylaseIU/L LipaseIU/L ALPIU/L ALTIU/L ASTIU/L CKIU/L GGTIU/L Serumprotein(biuret)g/L Albumin(BCG)g/L Globulinsg/L Serumprotein(refract.)g/L Albumin(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L Bileacids(fasting)mol/L Totalbilirubinmol/L Unconjugatedbilirubinmol/L Conjugatedbilirubinmol/L URINALYSIS(voidedsample) Appearance Cloudy Colour Yellow Specificgravity 1.026 Protein Trace *NDnotdone

SAMPLE ND* ND 165 48 ND ND ND ND ND ND 41 17.3 4.7 10.7 8.3 45 32 0 32

REFERENCEINTERVAL <1400 <60 <110 <60 <40 <100 0.68.2 5070 2343 716 5070 2339 716 916 412 <10 1.25.1 1.25.1 1.25.1

PH Glucose Ketones Blood Bilirubin

6 ve ve 4+ 4+

Microscopicfindings:erythrocytes5060perHPF,leukocytes23perHPF,manystruvitecrystals

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INTERPRETATIONOFLABORATORYFINDINGS Theinformationprovidedsuggestsachronicdiseasethatcouldinvolvethedigestivetract,especially theliver. (a) Detected laboratory abnormalities: mild elevation of ALP, mild to moderate conjugated hyperbilirubinemia, hypoproteinemia due to decreases in albumin and alpha globulins, moderate elevationofbileacids,haematuria,hyperbilirubinuria. (b)&(c)generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The animal has evidence of cholestasis (mild elevation of ALP, mild to moderate conjugated hyperbilirubinemia and hyperbilirubinuria) but no hepatocellular damage (normal ALT). This in combination with a moderate elevation of bile acids (will increase in a wide variety of liver conditions) suggests that the hypoproteinemia may be due to liver disease (conclusion). Hypoalbuminemiainliverdisease(decreasedproduction)usuallymeanschronicdiseaseandhepatic failure (conclusion). This was supported by the history. Further investigation could have been an ultrasoundguidedfineneedleaspirationoftheliver Inendstageliverdiseasethereisoftenadecreaseinallproteinproduction.Gammaglobulinsare notnormallydecreased(productionisbyplasmacells)andinmanycasesisincreasedduetoantigen stimulation.Thebetaglobulinsinthiscasewerenotdecreased,whichisalittleunusual,butcould beduetothefactthatsomeimmunoglobulinsweredetectedinthiszoneonelectrophoresis.The causeofthehaematuriawasnotdeterminedandisprobablynotimportant.However,itcouldhave beeninvestigatedfurtherthroughimageanalysisandcollectionofurinebycystocentesis. Implicationsformanagement:Theownersweregivenapoorprognosisfortreatmentandelected foreuthanasia. FINALDIAGNOSIS:Hepaticfailure(cirrhosis).Confirmedatnecropsy

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CASE:2

ANIMAL:7yearoldfemaleneuteredPersiancat. PRESENTING COMPLAINTS: Jaundice and depression for about 3 weeks. Previous history was unhelpful. There had been occasional vomiting and the cat had probably lost weight. The cat appearedtobedehydratedonpresentationandwasverythin. LABORATORYRESULTS

BIOCHEMISTRY AmylaseIU/L ALPIU/L ALTIU/L Totalbilirubinmol/L Unconjugatedbilirubinmol/L Conjugatedbilirubinmol/L

SAMPLE ND 213 567 263 57 206

REFERENCEINTERVAL <1400 <50 <60 2.53.5 <3.5 <3.5

HAEMATOLOGY SAMPLE REFERENCEINTERVAL Plasmaappearance Yellow Clear PCVL/L 0.28 0.300.45 Plasmaproteing/L 86 5978 Haemoglobing/L 98 80140 12 Erythrocytesx10 /L 6.7 610 MCVfl 42 4045 MCHCg/L 350 310360 9 Leukocytesx10 /L 7.8 814 9 Neutrophils(seg.)x10 /L 6.2 3.810.1 9 Neutrophils(band)x10 /L 0 00.4 9 Lymphocytesx10 /L 1.1 1.67.0 Monocytesx109/L 0.2 0.10.6 Eosinophilsx109/L 0.3 0.21.4 9 Basophilsx10 /L 0 00.2 Bloodfilm:moderatepoikilocytosisandanisocytosisoferythrocytes Plateletsx109/L ND 300700 Reticulocyte%(uncorrected) 0.3 01 URINALYSIS(cystocentesis) Appearance Cloudy PH Colour Yellow Glucose Specificgravity 1.050 Ketones Protein Trace Blood Bilirubin Microscopicfindings:manytransitionalcellsandmuchlipid 6 ve ve ve 4+

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INTERPRETATIONOFLABORATORYFINDINGS The information provided suggests ongoing disease. The jaundice could be prehepatic, hepatic or posthepatic. (a)Detectedlaboratoryabnormalities:Markedhyperbilirubinemia(approximately80%conjugated), moderate elevations of ALP and ALT, mild nonregenerative, normocytic, normochromic anaemia, hyperproteinemia,mildlymphocytopenia,hyperbilirubinuria. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Thecathasevidenceofhepatocellulardamageandcholestasis.ThelevelofALPissignificantforthe cat but would probably mean little in a dog. The hyperbilirubinemia is of a combined form (both retention and regurgitation forms are elevated) but significant regurgitation (cholestasis) exists. Since the hepatocellular damage is moderate, the cholestasis was interpreted as being primarily intrahepatic. Thehyperbilirubinuriaismarkedand,unlikethedog,isadirectreflectionofthelevelsofconjugated bilirubin in the blood stream. The anaemia is nonspecific and possibly related to the chronic disease.Poikilocytosiscanbecommoninavarietyofseverediseasesincertainbreedsofcatandis relatively nonspecific. However, it is reported more commonly in liver disease. The hyperproteinemia was possibly due to dehydration (mild dehydration was noted) but hyperglobulinemiacouldnotberuledout. Theinitialconclusionwassimplyhepatopathy.Furtherinvestigationwouldrequireimageanalysis andcytological/histologicalsamplesfromaffectedtissue.Theownerselectedforeuthanasia. POSTSCRIPT AND DIAGNOSIS: Necropsy revealed a pancreatic carcinoma which had caused bile duct obstruction. On reflection, the hepatocellular damage probably occurred secondarily to the marked and prolonged cholestasis but part could have been due to the nonspecific metabolic derangementsthatcanceroccasionallycauses.

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CASE3
ANIMAL:7yearoldmaleSiamesecat. PRESENTING COMPLAINTS: Depression, vomiting of greater than 5 days duration. This cat was presentedvastlyoverweight.Therewasnoprevioushistoryofproblems. LABORATORYRESULTS

BIOCHEMISTRY AmylaseIU/L ALPIU/L ALTIU/L Totalbilirubinmol/L Unconjugatedbilirubinmol/L Conjugatedbilirubinmol/L Ureammol/L HAEMATOLOGY Plasmaappearance PCVL/L Plasmaproteing/L Haemoglobing/L Erythrocytesx1012/L MCVfl MCHCg/L Leukocytesx109/L Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Bloodfilm:normal Plateletsx109/L Reticulocyte%(uncorrected)

SAMPLE ND 230 987 30 15 15 4 SAMPLE Yellow 0.26 64 87 6.0 43 334 26 24 0.3 1.3 0.4 0.13 0 ND ND

REFERENCEINTERVAL <1400 <50 <60 2.53.5 <3.5 <3.5 7.210.7 REFERENCEINTERVAL Clear 0.300.45 5978 80140 610 4045 310360 814 3.810.1 00.4 1.67.0 0.10.6 0.21.4 00.2 300700 01

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INTERPRETATIONOFLABORATORYFINDINGS Theinformationprovidedpointstoamyriadofdiseasesthatmightcausevomitinginanoverweight cat(e.g.diabetes,pancreatitis,hepatitis,GITdisease). (a)Detectedlaboratoryabnormalities:ModerateelevationofALP,moderatetomarkedelevationof ALT, mild combined hyperbilirubinemia, mild normocytic, normochromic anaemia, leukocytosis primarilyduetoaneutrophiliawithoutleftshift,lymphocytopeniaandeosinopenia. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The cat had significant hepatocellular damage with some cholestasis. The combined hyperbilirubinemia(albeitlow)supportedtheinterpretationofactivehepatopathy(conclusion).The anaemiawasassumedtobenonregenerative(nopolychromasiaonthebloodfilm),butcouldnot beexplainedintermsoftheapparentshortclinicalcourse.Theleukocytechangeswereconsidered tobeduetostress(corticosteroidinduced).Furtherinvestigationcouldhaveinvolvedimageanalysis andultrasoundguidedfineneedlecellaspiration.Bileacidanalysismayhavebeenuseful. Liver disease was confirmed by image analysis, which showed diffuse hepatomegaly. However, furtherinvestigationwasnotundertaken. DIAGNOSISANDPOSTSCRIPT:Theanimalsconditionworsenedanditdieddespitefluidtherapy.At necropsytheanimalhadseverehepaticsteatosisandnecrosis.Theconditionofhepaticsteatosisis welldocumentedinoverfatcatsbutthepathogenesisispoorlyunderstood.Commonlyitpresents as a chronic condition leading to clinical signs of hepatic failure. However, occasionally severe necrosisdevelopsleadingtodramaticclinicalsigns.Onreflection,themildanaemiacouldhavebeen relatedtotheliverdisease.

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CASE4
ANIMAL:9yearoldmaleSchnauzerdog. PRESENTING COMPLAINTS: Long history of spasmodic inappetence, polydipsia and polyuria. Last fourdayscollapsedandvomiting.Presentedjaundicedanddehydrated. LABORATORYRESULTS

BIOCHEMISTRY ALPIU/L ALTIU/L Totalbilirubinmol/L Unconjugatedbilirubinmol/L Conjugatedbilirubinmol/L Totalcholesterolmmol/L Glucosemmol/L Ureammol/L Sodiummmol/L Potassiummmol/L

SAMPLE 479 190 170 45 125 2.3 4.7 30.7 144 3.8 SAMPLE Yellowpink 0.34 62 127 5.3 64 373 89 70.2 0.4 3.3 14.7 0.4 0 1

REFERENCEINTERVAL <110 <60 1.25.1 1.25.1 1.25.1 1.47.5 3.36.4 310 137150 3.34.8 REFERENCEINTERVAL Clear 0.370.50 5575 100150 57 6075 300360 712 4.19.4 00.24 0.93.6 0.21.0 0.141.2 00.4 01.5 0.11.0 24

HAEMATOLOGY Plasmaappearance PCVL/L Plasmaproteing/L Haemoglobing/L Erythrocytesx1012/L MCVfl MCHCg/L Leukocytesx109/L Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Bloodfilm:normal Reticulocyte%(uncorrected) Eosinophils(directcount)x109/L Fibrinogeng/L

URINALYSIS(catheterised) Appearance Cloudy PH 5 Colour Yellow Glucose ve Specificgravity 1.015 Ketones ve Protein ve Blood 2+ Bilirubin 4+ Microscopicfindings:23erythrocytesperHPF,23leukocytesperHPF,23granularcastsperLPF

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INTERPRETATIONOFLABORATORYFINDINGS Theinformationprovidedsuggestschronicdiseasethatmayhavereachedacrisisorendpoint.The polyuria and polydipsia might suggest renal, endocrine or liver disease. The developed jaundice couldberelatedtoprehepatic,hepaticorposthepaticdisease. (a)Detectedlaboratoryabnormalities:Mildnonregenerativeanaemia,markedleukocytosisdueto neutrophilia (without left shift) and monocytosis, mild elevation in ALP, mild elevation in ALT, marked hyperbilirubinemia (approximately 70% conjugated), moderate azotemia (only urea), a specific gravity close to the isosthenuric range, possible haemoglobinuria and maximum bilirubinuria. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The animal has marked cholestasis but minimal hepatocellular damage. Bilirubin analysis shows a combined type but with regurgitation predominating. The 4+ bilirubinuria reflects the hyperbilirubinemiabut,unlikemostotherspecies,isnotpurelyreflectiveofthelevelsofconjugated bilirubin(dogsapparentlyhavesomecapacitytodegradehaemoglobintobilirubin,andtoconjugate bilirubin).Theseresultssuggestaprimarycholestaticdisease,whichcouldbebiliary(extrahepaticor intrahepatic)insite(conclusion). Theanaemiaisprobablyrelatedtochronicdisease.ThefindingofanelevatedMCHCsuggestseither laboratoryerror(technicalerrororinterferingsubstances)orhaemolysispriortooraftercollection. The leukocyte changes may reflect an intense continuing inflammatory demand. The lack of a left shiftmightbeduetobonemarrowproductionadequatelyadapting totheincreaseddemand(i.e. thebonemarrowstoragepoolhasbeenreplenishedovertime).Themarkedmonocytosiscouldbe due to demand for macromolecular phagocytosis. Although there is no lymphocytopenia or eosinopenia,corticosteroidreleasemaybecontributingtotheneutrophilia(theclinicalsignssuggest aperiodofintenseillness). Azotemiaincombinationwithclinicaldehydrationandasp.gr.closetoisosthenuriasuggestsrenal failure(conclusion).Thisappearsinadditiontothehepaticdisease.The2+bloodonthestripand yetlackofsignificanterythrocytesinthesediment,suggestseitherfreehaemoglobinormyoglobin. The differentiation usually requires specific chemical testing but in this case history appears to excludethepossibilityofmuscledamage.Assumingthatthebloodishaemoglobin,thismaybedue tohaemoglobinemia(usuallytheplasmahastobesaturatedbeforethehaemoglobinisfiltered)or duetobreakdownoferythrocytesintheurinerelatedtodelaysintestingorextremesofpHorsp. gr.Thelatterismorelikely.However,consideringthePCV,itisprobablynotofgreatsignificance. Further investigation might have been aimed at investigating the probable liver/renal disease. Diagnostic imaging through radiographs or ultrasonographs would have been useful. Further tests wouldhavebeendependedonthefindingsofimageanalysis. Theimplicationsformanagementsuggestedapoorprognosis. DIAGNOSISANDPOSTSCRIPT:Theownerelectedeuthanasiaandatnecropsythedoghadextensive cholangiohepatitisandalargehepaticabscess.Inaddition,therewasevidenceofnephrosis.With these liver changes it is unusual not to get a greater elevation of ALT. However, other laboratory changesareconsistentwiththesefindings.Theanaemiaprobablydevelopedduetoacombination of factors including increased turnover of erythrocytes and unavailability of iron stored in macrophages. Thelaboratoryfindingofrenalfailure(lackofconcentratingability)wassupportedbythedetection

130

of nephrosis. This probably developed secondarily to the liver diseases (nb. in older dogs, it is commontohavesomechronicrenaldiseasewhichisnotaproblemunlessotherdiseasedevelops. Thentherenaldiseasemaybepushedtowardsrenalfailure.Thismayhavebeensuspectedinthe presentcasebutwasnotso). Nodefiniteconclusionscanbedrawnonthesuspectedhaemoglobinemiaandhaemoglobinuria.Itis possible that some haemolysis was occurring related to toxic or metabolic disturbances caused by thehepaticabscess(clostridialorganismswereisolatedfromtheabscess).

131

CASE5
ANIMAL:8yearoldSydneysilkiedog. PRESENTINGCOMPLAINTS:Longperiodsofvomiting.Nowjaundicedanddepressed. LABORATORYRESULTS

BIOCHEMISTRY AmylaseIU/L LipaseIU/L ALPIU/L ALTIU/L Totalbilirubinmol/L Unconjugatedbilirubinmol/L Ureammol/L Creatininemol/L Sodiummmol/L Potassiummmol/L Chloridemmol/L Bicarbonate(TCO2)mmol/L

SAMPLE 3340 ND 942 64 154 0 103 860 147 3.8 71 ND

REFERENCEINTERVAL <1400 <60 <110 <60 1.25.1 1.25.1 310 40120 137150 3.34.8 105120 1724

132

INTERPRETATIONOFLABORATORYFINDINGS Theinformationhashighlightedvomitingandjaundiceinamiddleageddog.Thiscouldberelatedto liverdisease,pancreaticdiseaseorGITdisease. (a) Detected laboratory abnormalities: Moderate elevation of ALP, unconvincing change in ALT, markedhyperamylasemia,markedhyperbilirubinemia,markedazotemia(bothureaandcreatinine elevated)andhypochloridemia. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The dog has significant cholestasis which is reflected in the ALP and bilirubin levels. The hyperbilirubinemiaistotallyregurgitationinorigin,whichcouldpointtolargebiliaryvesselorpost hepatic obstruction. The marked azotemia could be due to both prerenal and renal factors, but there is not enough information to identify all the factors. Vomiting, if causing significant loss of fluid,willelevatebothureaandcreatinineduetodecreasedrenalbloodflow.Thehypochloridemia probablyhasdevelopedbecauseofthevomiting. The hyperamylasemia could be due to pancreatic necrosis or possibly due to small intestinal obstruction (amylase is known to elevate in cases of intestinal obstruction which cause marked vomiting). Becauseofthelimitedtestsperformed,itisnotpossibletobedefiniteaboutinterpretation(i.e.no strongconclusions.Posthepaticobstruction,possiblyduetoswellingcausedbypancreaticnecrosis, wasconsideredandcouldhavebeenfurtherinvestigatedbyimageanalysis. DIAGNOSIS AND POSTSCRIPT: At necropsy, surprisingly, an acquired pyloric stenosis (obstructive jaundice) was detected. Extension of the disease process into the proximal part of the duodenum possiblyinterferedwithbileoutflow.Thepancreasappearednormalbutthisdoesnotruleoutsome interferencewithpancreaticductoutflow.Thekidneyshadmildinterstitialnephritis.

133

CASE6
ANIMAL:8montholdSydneysilkiedog. PRESENTINGCOMPLAINTS:Poorgrowthandvagueneurologicalsignsrelatedtolackofawareness. LABORATORYRESULTS

BIOCHEMISTRY ALPIU/L ALTIU/L Bileacids(fasting)mol/L Bileacids(postprandial)mol/L NH3(fasting)mol/L NH3(postNH4Cl)mol/L Ureammol/L URINALYSIS(voided) Appearance Cloudy Colour Yellow Specificgravity 1.012 Protein ve Microscopicfindings:manytransitionalcells

SAMPLE 229 50 65 350 234 591 2.7

REFERENCEINTERVAL <110 <60 <10 <25 050 5090 310

PH Glucose Ketones Blood Bilirubin

6 ve ve ve Trace

134

INTERPRETATIONOFLABORATORYFINDINGS The information suggests possibly a congenital problem that is producing neurological signs. This couldmeanprimaryneurologicaldiseaseorsecondaryneurologicalsignsduetosystemicdisease. (a)Detectedlaboratoryabnormalities:MildelevationofALP,markedelevationsofammoniaandbile acids.Urinespecificgravitywithintheisosthenuricrangeandatracebilirubin. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Thefindingofanelevatedfastinglevelofammoniaincombinationwithalowlevelofureasuggests a lack of conversion by the liver. Post NH4Cl levels supports this lack of conversion and suggests shunting(conclusion).Thebileacidvaluesalsosupportthispossibility.ThelackofchangeinALTand theminimalchangeinALParenotuncommoninportosystemicshunting.Furtherinvestigationwill normallyinvolvediagnosticimagingandvascularstudies. The urine is unremarkable but commonly, ammonium biurate crystals may be present in urine in casesofshuntingandincertainotherhepatopathies.Thetraceofbilirubinhasnosignificanceand thesp.gr.readingmaybeachancefinding. DIAGNOSIS AND POSTSCRIPT: Hepatic encephalopathy due to portosystemic shunting. A congenitalportosystemicshuntwasconfirmedthroughimageanalysisandtreatedsurgically.

135

CASE7
ANIMAL:8yearoldmaleBullterrierdog. PRESENTINGCOMPLAINTS:Prolongedhistoryofpolydipsiaandpolyuria,nownoteatingordrinking. Itappeareddehydratedonpresentation. LABORATORYRESULTS

BIOCHEMISTRY Ureammol/L Creatininemol/L HAEMATOLOGY Plasmaappearance PCVL/L Reticulocyte%(uncorrected)

SAMPLE 27 600 SAMPLE Clear 0.27 0.5

REFERENCEINTERVAL 310 40120 REFERENCEINTERVAL Clear 0.370.50 01.5

URINALYSIS(voided) Appearance Clear PH 6.5 Colour Yellow Glucose ve Specificgravity 1.013 Ketones ve Protein Trace Blood ve Bilirubin ve Microscopicfindings:occasionaltransitionalcell,1leukocyteperHPF

136

INTERPRETATIONOFLABORATORYFINDINGS Theinformationsuggestsaprolongeddiseaseproducingpolyuriaandpolydipsia.Renal,hepaticor endocrineconditionsshouldbeconsidered. (a)Detectedlaboratoryabnormalities:Markedazotemia,moderatenonregenerativeanaemia,close toisosthenuricsp.gr. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: A close to isosthenuric reading for sp.gr. in combination with azotemia and probable dehydration suggests renal failure (conclusion). A nonregenerative anaemia is commonly found in association withchronicrenalfailureandoccursduetoavarietyofmechanisms(lackoferythropoietinmost important, enhanced haemolysis and direct bone marrow depression). The history supports a diagnosis of chronic renal failure. Further investigation might include diagnostic imaging of the kidneysandelectrolyteanalysis(couldhaveimplicationsformanagement). DIAGNOSIS:Chronicrenalfailure.

137

CASE8
ANIMAL:AgedHolsteincow. PRESENTING COMPLAINTS: Acute onset of depression, listlessness, anorexia, unwilling to drink and haematuria. The cow appeared mildly dehydrated on examination and was reluctanttomove. LABORATORYRESULTS

BIOCHEMISTRY Serumprotein(biuret)g/L Glucosemmol/L Ureammol/L Creatininemol/L Sodiummmol/L Potassiummmol/L Chloridemmol/L HAEMATOLOGY Plasmaappearance PCVL/L Plasmaproteing/L Leukocytesx109/L Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Bloodfilm:normal

SAMPLE 80 7.4 24 400 110 2.3 48 SAMPLE Clear 0.29 97 9.7 7.8 0.3 1.0 0.5 0.1 0

REFERENCEINTERVAL 6875 2.54.2 711 88117 132152 3.95.8 97111 REFERENCEINTERVAL Clear 0.240.46 7080 412 0.64.0 00.1 2.57.5 00.9 02.4 00.2

URINALYSIS(voided) Appearance Cloudy PH 7 Colour Amber Glucose 2+ Specificgravity 1.016 Ketones ve Protein 3+ Blood 4+ Bilirubin ve Microscopicfindings:greaterthan50erythrocytesperHPF,34granularcastsperHPF

138

INTERPRETATIONOFLABORATORYFINDINGS Theinformationprovidedsuggestsacuteseveredisease.Thehaematuriacouldbecausedbyurinary orgenitaldisease. (a)Detectedlaboratoryabnormalities:Hyperproteinemia,mildhyperglycemia,moderateazotemia, hyponatremia,hypokalemia,hypochloridemia,leukocytosisduetoneutrophilia(probableleftshift), lymphocytopenia,lownormaleosinophils,haematuria,proteinuriaandglucosuria. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Thecowhasazotemiawithasp.gr.closetoisosthenuria(itisprobablytrulyisosthenuricasprotein andglucosefalselyelevatethespecificgravityreading).Consideringthecowwasnotdrinkingand mildly dehydrated, this suggests renal failure (conclusion). The presence of casts suggests that perhapsthehaematuriaisrelatedtorenaldiseaseratherthantolowertractabnormalities.Allthese findings,withthehistory,pointtoacuterenalfailure(conclusion). Theproteinuriamayberelatedtorenaldisease.However,whenthediagnosticstripforbloodshows amaximum4+oftentheproteinstripispositivetovariabledegrees.Theglucosuriaisrelatedtothe hyperglycemia as the renal threshold for bovine animals is low (about 5.5 6 mmols/L). Hyperglycemia in cattle is common and transient, and commonly due to corticosteroid or catecholamine release. In this case, corticosteroid release is supported by the leukocyte changes (neutrophiliaandlymphocytopenia,butnotapossibleleftshift)(conclusion). Allelectrolytesarelow.Hyponatremiaandhypochloridemiaarecommoninrenaldiseaseincattle. The hypokalemia is most likely due to decreased intake and increased loss (kaliuresis but the history did not provide evidence for polyuria). The hypochloridemia is most likely due to gastrointestinal stasis and abomasal loss of HCl (a parallel can be drawn with vomiting in monogastrics),whichcanoccurinawidevarietyofconditionsinruminants. Totalplasmaproteinandtotalserumproteinaredistinctlydifferent.Thedifference(17g/L)canbe assumed to be primarily fibrinogen (although two different methods were used to measure the proteinsandthismaybepartlythereasonforthedifference).Fibrinogenisan'acutephasereactant' protein which rises nonspecifically in a wide range of degenerative and inflammatory diseases. Increases are larger and more consistent in farm animals than in dogs and cats. In this cow the increasecanbeassumedtoberelatedtoacuterenaldisease.Nb.indehydratedanimalsallproteins arefalselyelevatedthisincludesfibrinogen.Tocorrectforthisandtodetermineifatrueincrease infibrinogenhasoccurred,theratiooftotalplasmaproteintofibrinogenshouldbedetermined.A ratiooflessthan10to1(e.g.9to1)definitelyindicatesincreasedfibrinogen.Thisisthecaseinhand wheretheratioisroughly5.6to1. Thecowdiedandatnecropsyacuterenalnephrosiswasdiagnosed.Thecausewasassumedtobe aningestedtoxin. DIAGNOSIS:Acuterenalfailure.

139

CASE9
ANIMAL:1yearold,femaleDSHcat. PRESENTINGCOMPLAINTS:Weightlossandgeneralisedoedemaoverthreeweeks. LABORATORYRESULTS

BIOCHEMISTRY Serumprotein(refract.)g/L Albumin(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L Totalcholesterolmmol/L HAEMATOLOGY Plasmaappearance PCVL/L Plasmaproteing/L Haemoglobing/L Erythrocytesx1012/L MCVfl MCHCg/L Leukocytesx109/L Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Bloodfilm:normal

SAMPLE 50 8.1 21.4 10.1 10.4 6.0 SAMPLE Clear 0.24 58 85 5.0 48 354 14.2 7.9 0 5.4 0.3 0.4 0.2

REFERENCEINTERVAL 5473 2430 921 815 923 1.93.9 REFERENCEINTERVAL Clear 0.300.45 5978 80140 610 4045 310360 814 3.810.1 00.4 1.67.0 0.10.6 0.21.4 00.2

URINALYSIS(voided) Appearance Cloudy pH 7.0 Colour Lightyellow Glucose ve Specificgravity 1.016 Ketones ve Protein 3+ Blood ve Bilirubin ve Microscopicfindings:12leukocytesperHPF,1fattycastperHPF,muchlipid

140

INTERPRETATIONOFLABORATORYFINDINGS Theinformationsuggestssubacutetochronicillnessinayoungcat.Thegeneralisedoedemacould beduetohypoproteinemia(reducedintake,decreasedproductionbyliver,increasedlossesthrough GITorkidney)orincreasedhydrostaticpressure(e.g.heartfailure). (a) Detected laboratory abnormalities: mild hypoproteinemia, marked hypoalbuminemia, marginal increase in alpha globulins, hypercholesterolemia, anaemia, possible hyperfibrinogenemia (difference between total plasma protein and total serum protein both measured by the refractometerinthiscase),increasedMCV,mildleukocytosis,mildbasophilia?,poorconcentrating abilityandproteinuria. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: A significant decrease in the albumin suggests that this could be the primary cause of the generalised oedema (although other factors are probably contributing to the oedema). It appears that the main reason for low albumin is proteinuria. This would point to nephrotic syndrome. Glomerular disease in the cat is more likely to give rise to the nephrotic syndrome (conclusion). Azotemiamightbeexpectedwiththisbutunfortunatelyureaorcreatininewerenotanalysed.The low urine sp. gr. (close to isosthenuric range), if persistent, could indicate tubular dysfunction (conclusion). Theanaemiacouldbeduetorenaldisease(conclusion),butunfortunatelyithasnotbeenclassified. ThehighMCVdoesnotmakesenseandispossiblyduetolaboratoryerror(FeLVmaybeapossibility for macrocytic anaemia). The leukocytosis is not of significance as the individual leukocyte groups havenotrisen(themarginalbasophiliaisnotofrelevance). Derangements of lipid metabolism often occur in nephrotic syndrome in man. Apparently, diminishedplasmaoncoticpressurestimulateshepaticlipoproteinsynthesis,althoughurinarylossof plasma protein factors regulating lipoprotein synthesis or catabolism may also play a role. This is reflectedinhypercholesterolemiaandlipoproteinpeaksintheproteinelectrophoretograph.Inthe dog and cat hypercholesterolemia occurs inconsistently in the nephrotic syndrome but other lipid changeshavenotbeeninvestigated. Inthepresentcasehypercholesterolemiaispresentinthecat.Inman,lipiduriaisalsocommonin thenephroticsyndromebutsincethecatnormallyhaslipidintheurinethisisnotuseful(nb.fatty castsarethecommontypefoundincats).Themildincreaseinalphaglobulinsmaybeduetothe presence on acute phase reactant proteins that rise in a wide variety of inflammatory and degenerative conditions. However, lipoprotein may also be contributing (one would have to do individualproteinanalysistobesure).Hyperfibrinogenemiasupportsthepresenceofdegenerative or strong inflammatory disease (increases in fibrinogen in the cat and dog related to these conditions are not as common as in farm animals and horses). However, the leukogram seems to ruleoutsignificantinflammationortissuedamage(i.e.wearepresentwithinconsistentfindings). Further investigation could include diagnostic imaging, fine needle aspiration and biopsy. Implications for management could depend on the findings of biopsy; however, the oedema and lowproteinwillneedtobemanaged. DIAGNOSIS:Nephroticsyndrome.

141

CASE10
ANIMAL:12monthold,femaleLabradordog. PRESENTING COMPLAINTS: Depression, vague abdominal pain and frequent micturition of about twoweeksduration.Thedoghadpossiblecystitisontwooccasionsoverthelastsixmonths LABORATORYRESULTS

BIOCHEMISTRY Ureammol/L HAEMATOLOGY Plasmaappearance PCVL/L Plasmaproteing/L Haemoglobing/L Erythrocytesx1012/L MCVfl MCHCg/L Leukocytesx109/L Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Bloodfilm:normal

SAMPLE 17.1 SAMPLE Clear 0.42 80 136 7.1 59 323 29.5 23.3 2.8 1.2 2.2 0 0

REFERENCEINTERVAL 310 REFERENCEINTERVAL Clear 0.370.50 5575 100150 57 6075 300360 712 4.19.4 00.24 0.93.6 0.21.0 0.141.2 00.4

URINALYSIS(voided) Appearance Cloudy pH Colour Yellow Glucose Specificgravity 1.022 Ketones Protein Trace Blood Bilirubin Microscopicfindings:abundantbacteriaand60leukocytesperHPF

6 ve ve ve ve

142

INTERPRETATIONOFLABORATORYFINDINGS Theinformationsuggestslowerurinarytractdisease,butthedepressionandpainisalittleunusual. (a) Detected laboratory abnormalities: mild azotemia, mild hyperproteinemia, erythrocytes just above reference interval, MCV just below reference interval, leukocytosis primarily due to neutrophiliawithleftshift(ratiooflessthan1to10definiteleftshiftinthedogiftheratioisless than 1 to 1618 or the total band count is greater than 1 x 109/L), monocytosis, absolute eosinopenia,pyuriaandbacteruria. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: From the urinalysis it can be deduced that inflammation is occurring somewhere in the urogenital tract (conclusion). The trace protein may be significant at this relatively low sp. gr., but the leukocytesarethekey.Presumablytheinflammationisduetoabacterialinfection.Azotemiaand neutrophilia with a left shift are more likely to occur in either renal or uterine disease as cystitis rarely causes these changes (conclusion). The fact that TPP is elevated could suggest some haemoconcentration but since there is no evidence of clinical dehydration, an increase in a particular protein must also be considered (e.g. gamma globulins or fibrinogen). If the animal was dehydrated then the fact that there is azotemia and a less than optimal concentration of urine wouldmorelikelypointtothekidneyasbeingthesiteofdisease(conclusion).Theyoungageofthe dogwouldgoagainstthepossibilityofendometritis/pyometron. Themonocytosis,absoluteeosinopeniaandlownormallymphocytescouldsuggestthatsomeofthe neutrophilia is due to stress (i.e. inflammatory demand is on top of this). The high erythrocytes (relativetoPCVandHb)couldbealaboratoryerror.Consequently,theMCVislow. Further investigation could involve diagnostic imaging of the urinary tract. Any detection of renal involvement has implications for management. Obviously, the urine should be cultured and an appropriateantibioticregimedeterminedasthishasimplicationsformanagement. DIAGNOSISANDPOSTSCRIPT:Thedogdiedandatnecropsyabilateralpyelonephritiswasfound. However, further histological investigation showed that the inflammation probably started in the bladder(i.e.cystitiswasalsopresent).

143

CASE11
ANIMAL:5yearoldfemalecrossbreddog. PRESENTINGCOMPLAINTS:Redurineofoneweekduration. LABORATORYRESULTS

URINALYSIS(voided) Appearance Cloudy pH 5.5 Colour Amber Glucose ve Specificgravity 1.056 Ketones ve Protein 2+ Blood 4+ Bilirubin ve Microscopicfindings:greaterthan200erythrocytesperHPF,515leukocytesperHPF

144

INTERPRETATIONOFLABORATORYFINDINGS Red urine in a middleaged female dog provides little information, but haematuria or haemoglobinuriashouldbeconsidered. (a)Detectedlaboratoryabnormalities:proteinuriaandpositivebloodstripduetohaematuria. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The dog has haematuria, which was the cause of the positive blood strip (the strip will detect erythrocytes,freehaemoglobinandmyoglobin).Theproteinuriamaybeduetoleakagefromvessels (this level of red cells would suggest some rhexis of vessels lower levels can occur just with diapedesise.g.30perHPF)orduetointerferencefromthebloodstripithasbeenshownthatany reactioncanoccurontheproteinstripifthereactiononthebloodstripismaximali.e.4+. Thelevelofleukocytes,althoughabovethereferenceinterval(roughly25leukocytesperHPF)are probablyrelatedtothebleeding(i.e.theconclusionissimplybleeding).Bydeterminingtheratioof leukocytes to erythrocytes, one can get a reasonable idea whether the increased leukocytes are relatedtofrankhaemorrhageorduetotrueinflammatorydemand.Inthedog,circulatingbloodhas aleukocytetoerythrocyteratioofoneto200400orgreater.Inmostfluidse.g.peritonealfluid,a ratiooflessthanoneto200issuspiciousoftrueincreasesinleukocytesbuttobecertaintheratio should be below one to 50. In urine, normal levels of leukocytes should be subtracted before the ratioisdetermined.Inthepresentcasesubtractionleavesbetween5and10leukocytes.Theratio cannotbeaccuratelydeterminedastheerythrocytesaregivenasbeinggreaterthan200perHPF. However,itishighlyunlikelythattheratiowillbelowenoughtobesignificant. Thecauseofthehaematuriacouldbefurtherinvestigatedthroughdiagnosticimagingandbiopsyof the bladder. It may be worthwhile to collect a urine sample by cystocentesis. Detection of an underlyingcausewillhaveimplicationsformanagement. DIAGNOSISANDPOSTSCRIPT:Thecauseofthehaematuriawasnotdeterminedforthisdogandit clearedupwithouttreatmentorfurtherinvestigation,andwithoutasignificantanaemiaoccurring. Itwaspresumedthatthebleedinghadoccurredinthebladderandwasidiopathic.

145

CASE12
ANIMAL:8yearoldfemaleneuteredLabradordog PRESENTINGCOMPLAINTS:Redurineoftendaysduration LABORATORYRESULTS

URINALYSIS(voided) Appearance Cloudy PH 6 Colour Red Glucose ve Specificgravity 1.039 Ketones ve Protein 3+ Blood 4+ Bilirubin ve Microscopicfindings(uncentrifuged):greaterthan200erythrocytesperHPF,greaterthan 100leukocytesperHPF

146

INTERPRETATIONOFLABORATORYFINDINGS Red urine in an aged female dog provides little information, but haematuria or haemoglobinuria shouldbeconsidered. (a)Detectedlaboratoryabnormalities:Haematuria,pyuria. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Although the chemical analysis of the urine shows similar results to case 12, the sediment shows distinctdifferences.Thereweresomanycellsthatitwasnotnecessarytoconcentratetheurine1in 20.However,thisdoesnotinterferewiththedeterminationoftheleukocytetoerythrocyteratio.In this case it could be as low as 1 to 2. Thus true inflammatory demand (conclusion) is present somewhereintheurinarytract(thedoghasbeenspayed).Thefactthatthelevelsoferythrocytes are so high suggests that some of the bleeding is occurring through rhexis rather than through simpleinflammatorydiapedesis.This,forexample,couldberelatedtourinarycalculiinthebladder ortoneoplasiacausingsecondaryinflammationandhaemorrhage.Occasionallysevereinfectionand inflammationwillcauserhexisleadingtohighlevelsoferythrocytes(alltheseareconclusions). Further investigation could involve diagnostic imaging and biopsy of the bladder. Culture of the urine (perhaps on a cystocentesis sample) should be considered. Determining the cause of the inflammationandbleedingwillhaveobviousimplicationsformanagement. DIAGNOSISANDPOSTSCRIPT:Radiographsofthisdogfoundbladdercalculi(analysedasstruvite) andthedogwastreatedforcystitisandthecalculi(i.e.haematuriaduetocystitisrelatedtourinary calculi).

147

CASE13
ANIMAL:4montholdfemaleThoroughbredfoal PRESENTINGCOMPLAINTS:Depression,swollenjoints,pyrexiaforoveroneweek LABORATORYRESULTS

HAEMATOLOGY SAMPLE REFERENCEINTERVAL Plasmaappearance Clear Variable PCVL/L 0.23 0.320.52 Plasmaproteing/L 64 5884 Haemoglobing/L 78 110190 12 Erythrocytesx10 /L 6.7 812.5 MCVfl 34 4149 MCHCg/L 339 300360 Leukocytesx109/L 39 6.013 9 Neutrophils(seg.)x10 /L 31 2.57 9 Neutrophils(band)x10 /L 2 00.2 9 Lymphocytesx10 /L 4.5 1.65.4 9 Monocytesx10 /L 1.3 00.7 9 Eosinophilsx10 /L 0.2 0.21 9 Basophilsx10 /L 0 00.4 Bloodfilm:vacuolationofneutrophils,someHowellJollybodiespresent Fibrinogeng/L 10 14 Othertests:
STIFLEFLUIDANALYSIS

Appearance: Protein(g/L): Totalnucleatedcells (x106/L): Erythrocytes(x106/L): Differential:

slightlycloudy,yellowgreen(usuallyclearandlightyellow) 45(usuallylessthan20) 19,100(usuallylessthan3,000) 32,000(usuallynone) 97%neutrophilswithintracellularbacteriaand3% macrophages(usuallymostcellsarelymphocytesor monocyteswithneutrophilsbeinglessthan10%)

E.colirecoveredfromjointfluid

148

INTERPRETATIONOFLABORATORYFINDINGS In a young foal with pyrexia and multiple joint effusions, an infectious arthritis would be a top consideration. (a) Detected laboratory abnormalities: Anaemia, decreased MCV, leukocytosis due to neutrophilia and mild monocytosis, absolute eosinopenia, toxic neutrophils (vacuolation of cytoplasm), hyperfibrinogenemia,andsepticexudateinthestiflejoint. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Thefoalhasanaemia,whichaccordingtoreferencevaluesismicrocytic.However,foalsbetween19 monthsoftenhavelowMCVs(meanisactuallyaround34.5forthoroughbredsfoalsofthisage)i.e. theMCVisactuallynormalforthisagefoal.Alwaysrememberthattheyoungofmostspecieswill have different reference intervals for many analytes when compared to adult values (it requires a quick check of the texts!). The anaemia cannot be further categorised without serial sampling or bonemarrowexamination(unwarrantedatthisstage). Theleukocytechangesareprobablypartlyduetostressbutthereappearstobemoreoccurring.The neutrophilia is quite high for stress alone (levels for total leukocytes rarely exceed 20.0x 109/L in casesofstressinadulthorses)andthisinconjunctionwithadefiniteleftshift(seebelow)andtoxic changes to neutrophils suggest an inflammatory demand component (conclusion). This is possibly supportedbytheincreasedfibrinogen. The joint fluid shows increased cells (which are predominantly neutrophils with intracellular bacteria)andincreasedprotein(asmeasuredbytherefractometeracrudemethodfordetermining protein in joint fluid). This indicates a septic exudate (conclusion) (nb. septic exudates in most cavitiesarecharacterisedbylytic(degenerate)neutrophils.However,thisisrarelythecaseinjoint fluidduetotheapparentprotectivenatureofsynovialsecretions). Onreflection,theanaemiawasprobablysecondarytotheinflammation(enhancedhaemolysisand utilisation of iron by bacteria). Although the ratio of bands to segmented neutrophils is 1 to 15.5 (greyzoneforaleftshiftinhorse),someveterinarianswouldbehappytodefinitelycallthisaleft shiftastherearemorethan0.3x109/Lbands. Further investigation requires culture and determination of an appropriate antibiotic regime. The findingofasepticexudatehasobviousimplicationsformanagement.Onemaydecideonantibiotic treatmentbasedonpastexperiencewhilewaitingforthelaboratorytoprovideinformationonthe microorganismsinvolved. DIAGNOSISANDPOSTSCRIPT:GeneralisedsepticarthritisduetoEscherichiacoli.

149

CASE14
ANIMAL:10yearoldfemaleWeimeraner PRESENTINGCOMPLAINTS:Depression,swollenjoints(bothstifles,elbows,carpiandtarsi),pyrexia ofgreaterthanoneweeksduration LABORATORYRESULTS STIFLEFLUIDANALYSIS

Appearance: Protein(g/L): Totalnucleatedcells (x106/L): Erythrocytes(x106/L): Differential:

slightlycloudy,colourlessandclear 30(usuallylessthan25) 3,200(usuallylessthan2,900) 1,000(usuallynone) 77%neutrophils,20%monocytesand3%macrophages (usuallymostcellsarelymphocytesormonocyteswith neutrophilsbeinglessthan10%)

Nobacteriarecoveredfromjointfluid

150

INTERPRETATIONOFLABORATORYFINDINGS Theinformationprovidedsuggestspolyarthropathyinanageddog.Thiscouldbeinflammatoryand bothinfectiousandnoninfectious(e.g.immunemediated)causesneedtobeconsidered. (a)Detectedlaboratoryabnormalities:Nonsepticexudateinthestiflejoint. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The dog had multiple joints affected. Inflammatory joint disease is commonly divided into septic (bacterial) and nonseptic. Nonseptic forms can include infectious agents such as mycoplasmal organisms, fungal agents (Cryptococcus neoformans) and protozoal organisms but these are rare. Mostnonsepticformsarenoninfectious.Theseareclassifiedasimmunemediated(autoimmune) or nonimmunemediated and generally involve a number of joints. Nonimmunemediated types are uncommon and may be due to crystal deposition (e.g. certain forms of calcium) or, more commonly, due to haemoarthrosis due to a bleeding disorder. Immunemediated types include erosive forms (e.g.s rheumatoid arthritis in the dog, polyarthritis in the greyhound), proliferative types(e.g.chronicprogressivepolyarthritisinthecat)andnonerosiveforms(e.g.ssystemiclupus erythematosus, polyarthritis/polymyositis syndrome, and idiopathic polyarthritis). The idiopathic typesofimmunemediatedarthritisareprobablyrelatedtoimmunecomplexdeposition,whichmay occur due to infections or neoplasia elsewhere in the body. There appears to be a particular associationbetweenvariousgastrointestinaldiseasesandthistypeofarthritis. Inthiscasethenonsepticpolyarthritiswasconsideredtobeimmunemediatedbecauseofthelack of bacteria, but no extrajoint disease was detected (conclusion). There would be little need for further investigation, but one could do haematology (to detect inflammatory demand), a rheumatoidfactoranalysisandanantinuclearantibodytest. DIAGNOSISANDPOSTSCRIPT:Thedogwasconsideredtohaveimmunemediatedpolyarthritis.The dogrespondedtoantiinflammatorymedication.

151

CASE15
ANIMAL:7yearoldmaleJapaneseChindog PRESENTING COMPLAINTS: Respiratory distress, abdominal effusion slowly developing over a month.Thehearthadadistinctvalvemurmur. LABORATORYRESULTS

HAEMATOLOGY SAMPLE Plasmaappearance Clear PCVL/L 0.30 Plasmaproteing/L 49 Haemoglobing/L 104 12 Erythrocytesx10 /L 4.7 MCVfl 64 MCHCg/L 347 9 Leukocytesx10 /L 21.6 9 Neutrophils(seg.)x10 /L 19.5 Neutrophils(band)x109/L 0.1 9 Lymphocytesx10 /L 0.8 9 Monocytesx10 /L 1.1 9 Eosinophilsx10 /L 0 9 Basophilsx10 /L 0 Bloodfilm:slightpolychromasia,1NRBCper100leukocytes Othertests:
PERITONEALFLUIDANALYSIS

REFERENCEINTERVAL Clear 0.370.50 5575 100150 57 6075 300360 712 4.19.4 00.24 0.93.6 0.21.0 0.141.2 00.4

Totalprotein: Nucleatedcells: Erythrocytes: Smear:

19g/L(normal<25g/L) 5000x106/L(normal<500x106/L) 40,000x106/L(nonenormally) 70%nonlytic(nondegenerate)neutrophils,30% mononuclearcells(normallymainlymononuclears)

152

INTERPRETATIONOFLABORATORYFINDINGS Theprovidedinformationcouldindicateacardiacrelatedabdominaleffusion. (a) Detected laboratory abnormalities: Mild anaemia, mild hypoproteinemia, leukocytosis due to neutrophilia and mild monocytosis, lymphocytopenia, absolute eosinopenia and a modified transudativefluid. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Theidentificationoftheperitonealfluidasamodifiedtransudatecouldsupportadiagnosisofheart failureinthedog(conclusion).Oftenthefluidstartsoffasapuretransudatebutquicklybecomesa modifiedtransudate,andmayprogresstoanonsepticexudate.Theerythrocytesarepresentdueto diapedesisrelatedtoincreasedhydrostaticpressureoveraprolongedperiodoftime. Theanaemiahasnotbeenclassifiedbutinchronicheartfailureiscommonlynonregenerative.The slight polychromasia and presence of a normoblast suggest attempts at regeneration in this case. The mild hypoproteinemia may possibly be due to chronic disease and interference with liver function. However, it is possible that the levels of individual classes of proteins are still within reference intervals i.e. the hypoproteinemia is not of significance. The leukocyte changes are consistentwithstressi.e.corticosteroidrelease.Thisiscommoninthedogrelatedtointenseillness. Possiblythisdoghasreachedacrisisinthediseaseprocesswhichhasledtoenhancedcorticosteroid release(conclusion). Furtherinvestigationrequiresdiagnosticimagingandelectrocardiography. DIAGNOSISANDPOSTSCRIPT:Cardiacimagingdetectedvalvularlesions(endocardiosissuspected). The diagnosis was heart failure due to advanced endocardiosis. The animal was given a poor prognosisandtheownerselectedeuthanasia.

153

CASE16
ANIMAL:4yearoldmaleSiamesecat. PRESENTING COMPLAINTS: Depression, pyrexia and abdominal effusion. The cat had suddenly returnedhomeafterathreedayabsence. LABORATORYRESULTS

BIOCHEMISTRY Ureammol/L Creatininemol/L Sodiummmol/L Potassiummmol/L Chloridemmol/L

SAMPLE 9.3 137 131 4.5 103

REFERENCEINTERVAL 7.210.7 98180 147156 44.6 115130 REFERENCEINTERVAL Clear 0.300.45 5978 80140 610 4045 310360 814 3.810.1 00.4 1.67.0 0.10.6 0.21.4 00.2

HAEMATOLOGY SAMPLE Plasmaappearance Clear PCVL/L 0.39 Plasmaproteing/L 73 Haemoglobing/L 131 12 Erythrocytesx10 /L 8.6 MCVfl 45 MCHCg/L 333 9 Leukocytesx10 /L 25.1 9 Neutrophils(seg.)x10 /L 21.4 Neutrophils(band)x109/L 1.4 9 Lymphocytesx10 /L 0.9 9 Monocytesx10 /L 1 9 Eosinophilsx10 /L 0.2 9 Basophilsx10 /L 0 Bloodfilm:toxicgranulationoftheneutrophils Othertests:
PERITONEALFLUIDANALYSIS

Totalprotein: Nucleatedcells: Erythrocytes: Smear:

57g/L(normal<25g/L) 64,100x106/L(normal<500x106/L) 20,000x106/L(nonenormally) 90%lytic(degenerate)neutrophils(withintracellularbacteria), 10%macrophages(normallymainlymononuclears)

Culturerevealedmixedanaerobicbacteria

154

INTERPRETATIONOFLABORATORYFINDINGS The information of pyrexia and abdominal effusion in a four yearold cat could suggest infectious disease(althoughpyrexiaisnotexclusivetoinflammatorydisease). (a) Detected laboratory abnormalities: Moderate leukocytosis due to neutrophilia (with left shift) and monocytosis, lymphocytopenia, toxic granulation of neutrophils, hyponatremia, hypochloridemiaandsepticexudatewithintheperitonealcavity. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Theperitonealfluidanalysisindicatesincreasedproteinandnucleatedcells.Sincemostofthecells arelytic(degenerate)neutrophilsandcontainbacteria,adiagnosisofsepticexudatecanbemade (conclusion).Thiswasmostlikelytheresultofacatbiteorapenetrationwound(conclusion).The erythrocytesareprobablythereduetodiapedesis. Onewouldexpectthecattobestressedi.e.increasedcorticosteroidrelease,andthisisreflectedin the lymphocytopenia and probably part of the neutrophilia. The low normal eosinophils and increased monocytes may also be a reflection of this (although monocytosis is not as common in stressasinthedog).A1to15ratioofbandtosegmentedneutrophilsmayindicatealeftshift(one can'tbecertainuntiltheratiodropsbelow1to1012butforthiscat1to15maybeadequate,but asmentionedsomeveterinarianswouldautomaticallyacceptthisasaleftshiftasthereareover1x 109 /L bands. The monocytosis may also be due to inflammatory demand for macromolecular phagocytosis as stress induced monocytosis is not as common in the cat as in the dog. The toxic changesintheneutrophilsareareflectionofthebacterialinfection. Thehyponatremiaispossiblyduetotheextensiveeffusionandredistributionofsodium.Thedropin chlorideoftengoeshandinhandwithadropinsodium.Thelackofazotemiainthiscaseisunusual but is possibly a good prognosis (the development of shock with subsequent reduction of renal bloodflowisacommonreasonfordecentincreasesinureaandcreatinineinprerenaldiseases). Further investigation would require culture and determination of appropriate antibiotic therapy. One may place the animal on a broad spectrum antibiotic (and probably drain and flush the abdomen)whilewaitingforlaboratoryanalysis(implicationsformanagement). DIAGNOSIS:Bacterialperitonitis(mixedmicroorganisms).

155

CASE17
ANIMAL:6yearoldThoroughbredmare. PRESENTING COMPLAINTS: Depression and pyrexia after foaling. Possible increase in abdominal fluid. LABORATORYRESULTS

BIOCHEMISTRY Ureammol/L Creatininemol/L Sodiummmol/L Potassiummmol/L Chloridemmol/L

SAMPLE 43 656 134 5.5 92

REFERENCEINTERVAL 3.78.2 87149 132150 2.85.0 99110 REFERENCEINTERVAL Variable 0.320.52 5884 110190 612 4149 300390 613 2.57 00.2 1.65.4 00.7 0.21 00.4

HAEMATOLOGY SAMPLE Plasmaappearance Clear PCVL/L 0.54 Plasmaproteing/L 75 Haemoglobing/L 197 12 Erythrocytesx10 /L 12.3 MCVfl 44 MCHCg/L 364 Leukocytesx109/L 8.1 9 Neutrophils(seg.)x10 /L 3.7 9 Neutrophils(band)x10 /L 0 9 Lymphocytesx10 /L 3.5 9 Monocytesx10 /L 0.9 9 Eosinophilsx10 /L 0 Basophilsx109/L 0 Bloodfilm:moderategranulationofneutrophils

156

INTERPRETATIONOFLABORATORYFINDINGS Thelimitedinformationcouldsuggestagenitalproblempostfoaling. (a) Detected laboratory abnormalities: Polycythemia, absolute eosinopenia, possible toxic changes toneutrophils,moderateazotemia,hyperkalemiaandmildhypochloridemia. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The polycythemia is most likely due to haemoconcentration (despite the TPP still being within the referenceinterval)relatedtofluidimbalances.Inthiscasewehaveeosinopeniaandpossibletoxic granulationofneutrophils.Thelattercouldbeduetotoxaemiaorinfection(conclusion). The moderate azotemia is related to the catabolic state of the animal and the expected derangementsinrenalbloodflow.Inhorses,itisacceptedthatthehigherthevalues,thepoorerthe prognosis (i.e. an indicator of the development of shock). The hyperkalemia could be related to possibleacidbaseimbalance(metabolicacidosismaycauseashiftofpotassiumfromcellsintothe plasma in exchange for excess H+) and widespread cell damage (due to the infection and related tissuedamage).ThemilddropinchloridemayberelatedtothelownormallevelofNa+(i.e.thisNa+ level may be low for this particular horse). Both could be due to the peritoneal effusion and redistribution. Furtherinvestigationwillinvolvegenitalexaminationandperitonealeffusionanalysis(ifaruptured uterus is suspected). It will also involve bacterial culture and appropriate antibiotic therapy if infectionisidentified(implicationsformanagement). DIAGNOSIS AND POSTSCRIPT: Ruptured uterus and subsequent peritonitis diagnosed on further investigation.Thehorsediedduringearlytreatment. The lack of leukocyte changes is unusual in this case. A stress response (neutrophilia, lymphocytopeniaandeosinopenia),aresponsetoendotoxemia(neutropeniaandlymphocytopenia thehorseisparticularlysensitivetosmallamountsofendotoxinproducedbycertainGramnegative rods) or a response to infection (early neutropenia due to over whelming demand, lymphocytopenia due to entrapment of lymphocytes within lymph nodes, eosinopenia due to mechanismsunrelatedtocorticosteroidrelease;laterneutrophiliawithleftshift,althoughleftshift developmentisnotascommonintheadulthorseasitisindogsandcatsorruminants)wouldbe expected.

157

CASE18
ANIMAL:2yearoldmaleLabradordog. PRESENTINGCOMPLAINTS:Vomitingandabdominalpain,forabout4daysduration. LABORATORYRESULTS

BIOCHEMISTRY AmylaseIU/L LipaseIU/L ALPIU/L ALTIU/L Ureammol/L Sodiummmol/L Potassiummmol/L Chloridemmol/L HAEMATOLOGY Plasmaappearance PCVL/L Plasmaproteing/L Haemoglobing/L Erythrocytesx1012/L MCVfl MCHCg/L Leukocytesx109/L Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Bloodfilm:Doehlebodiesinneutrophils

SAMPLE 7500 850 1202 170 9 149 3.8 103 SAMPLE Clear 0.57 83 201 8.9 64 352 15.8 12.8 1.7 0.5 0.8 0 0

REFERENCEINTERVAL <1400 <60 <11 <60 310 137150 3.34.8 105120 REFERENCEINTERVAL Clear 0.370.50 5575 100150 57 6075 300360 712 4.19.4 00.24 0.93.6 0.21.0 0.141.2 00.4

158

INTERPRETATIONOFLABORATORYFINDINGS Acutevomitingandabdominalpainmightsuggestacutehepatopathy,acutepancreaticnecrosis,GIT inflammationorobstruction,peritonitis/splenitis,orlesslikelyacutenephropathy. (a) Detected laboratory abnormalities: Marked hyperamylasemia, marked hyperlipasemia, marked elevationofALP,mildelevationofALT,mildhypochloridemia,polycythemia,mildhyperproteinemia, mildleukocytosisduetoneutrophilia(withleftshiftratiolessthan1to10andtotalvaluegreater than1x109/L),lymphocytopeniaandabsoluteeosinopenia. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Themarkedhyperamylasemiaandhyperlipasemiaaresuggestiveofpancreaticnecrosisorperhaps highintestinalobstruction(conclusion).Thepresenceofthestressleukogramincombinationwith true inflammatory demand (left shift) would support the possibility of pancreatic necrosis. The marked cholestasis and mild hepatocellular damage could be explained by bile duct obstruction throughpancreaticswellingandmildtoxemia. The polycythemia appears to be spurious due to haemoconcentration (probably through vomiting andshock).Thehypochloridemiaisprobablyduetovomiting.Thesechangesarecommonin,butnot specificforpancreaticnecrosis.Thelackofaprerenalazotemiaisunusualinpancreaticnecrosis. In summary, most of the laboratory changes can be explained by the animal having pancreatic necrosis (conclusion). Further investigation could involve diagnostic imaging (especially ultrasonography). DIAGNOSIS AND POSTSCRIPT: Acute pancreatic necrosis. It responded well to fluids and symptomatictherapy.

159

CASE19
ANIMAL:2yearoldfemalegreyhound. PRESENTINGCOMPLAINTS:Painandreluctancetomoveafterrace. LABORATORYRESULTS

BIOCHEMISTRY ASTIU/L CKIU/L Serumprotein(refract.)g/L Albumin(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L Ureammol/L Sodiummmol/L Potassiummmol/L Chloridemmol/L

SAMPLE 213 5207 78 43.1 7 11.4 16.5 65 116 4.8 77

REFERENCEINTERVAL <40 <100 5070 2339 716 916 412 310 137150 3.34.8 105120 REFERENCEINTERVAL Clear 0.370.50 5575 100150 57 6075 300360 712 4.19.4 00.24 0.93.6 0.21.0 0.141.2 00.4

HAEMATOLOGY SAMPLE Plasmaappearance Clear PCVL/L 0.67 Plasmaproteing/L 77 Haemoglobing/L 274 12 Erythrocytesx10 /L 11.7 MCVfl 66 MCHCg/L 356 Leukocytesx109/L 18.7 9 Neutrophils(seg.)x10 /L 14.6 9 Neutrophils(band)x10 /L 0 9 Lymphocytesx10 /L 0.75 9 Monocytesx10 /L 3.3 9 Eosinophilsx10 /L 0.1 9 Basophilsx10 /L 0 Bloodfilm:hypersegmentationoftheneutrophils

160

INTERPRETATIONOFLABORATORYFINDINGS Theinformationistoolimited,butcouldsuggesttraumathroughracing. (a) Detected laboratory abnormalities: Mild elevation of AST, marked elevation of CK, mild hyperproteinemia due to elevations in both albumin and globulins, marked azotemia (urea only), hyponatremia, hypochloridemia, elevated PCV, Hb and erythrocytes (polycythemia), leukocytosis (moderatetomarkedfor agreyhound astheycommonlyhavelowerreferenceintervals) dueto a neutrophiliaandmonocytosis,lymphocytopeniaandeosinopenia. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The significantly elevated CK after a race suggests muscle damage. Of course CK will elevate after any muscular activity, and sometimes quite high, but in this case the elevation is accompanied by other changes which point to true rhabdomyolysis (pain and reluctance to move after the race) (conclusion).TheASTisonlymildlyelevatedbutoftenthistakeslongertorisethanCKafterdamage (theadvantageofusingenzymesintandem). The dog appears to be haemoconcentrated (elevated red cell parameters and TPP), which is supported by the protein electrophoresis (albumin could only be increased due to haemoconcentration,i.e.dehydration).Thereisastressleukogram.Thelevelofneutrophiliaisquite significantforagreyhoundastheytendtohavelowercirculatinglevelsinhealthcomparedtoother dogbreeds,andtheirresponsetodiseaseismorelimited.Hypersegmentationofneutrophilsoccurs due to aging in the circulation as corticosteroids released in stress have inhibited the escape of neutrophilsintothetissues. Thedogissignificantlyazotemicwhichcouldbeprimarilyduetoprerenalfactors(tissuebreakdown and derangements of renal blood flow) but renal disease cannot be excluded without urinalysis (conclusion). The low sodium is difficult to explain but may be related to renal disease. Chloride changes often follow sodium changes. The upper normal potassium could be related to metabolic acidosisthroughtissuebreakdownorrenaldisease,ordirectlyduetoreleasefromdamagedcells. All these changes support a diagnosis of paralytic rhabdomyolysis. Further investigation through urinalysis would have been useful in determining the presence of free myoglobin (positive on the blood strip) and the extent of possible kidney damage which has been recorded with extensive rhabdomyolysis(socalledmyoglobinuricnephrosis). DIAGNOSIS:Paralyticrhabdomyolysis.

161

CASE20
ANIMAL:8yearoldfemaleneuteredcrossbredcat. PRESENTING COMPLAINTS: Motor car accident (broken pelvis) one week before presentation. Checkup. LABORATORYRESULTS

HAEMATOLOGY SAMPLE REFERENCEINTERVAL Plasmaappearance Clear Clear PCVL/L 0.26 0.300.45 Plasmaproteing/L 76 5978 Haemoglobing/L 93 80140 12 Erythrocytesx10 /L 5.8 610 MCVfl 45 4045 MCHCg/L 358 310360 9 Leukocytesx10 /L 25.1 814 9 Neutrophils(seg.)x10 /L 20.4 3.810.1 Neutrophils(band)x109/L 0 00.4 9 Lymphocytesx10 /L 3.3 1.67.0 9 Monocytesx10 /L 0.5 0.10.6 9 Eosinophilsx10 /L 0.9 0.21.4 9 Basophilsx10 /L 0 00.2 Bloodfilm:moderateanisocytosisandpolychromasia,3NRBCper100leukocytes Reticulocyte%(uncorrected) 9.2 01

162

INTERPRETATIONOFLABORATORYFINDINGS (a) Detected laboratory abnormalities: Mild anaemia, leukocytosis due to neutrophilia without left shift,reticulocytosis. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Thisisaregenerativeanaemia.Thebloodchangesarecharacteristicofbloodlossaboutoneweek previously(maximumresponseusuallyoccursat79days)(conclusion).Thepresenceofimmature cells within the circulation (reticulocytes) produces the anisocytosis and polychromasia. The circulating NRBCs (normoblasts) are accompanying the regenerative response, and are in the right proportion for an orderly response. They are more common in the regenerative response to haemolytic anaemia. The high normal MCV may be a reflection of the level of circulating reticulocytes (larger cells). The reticulocyte count, 9.2%, because of its magnitude doesn't really needtobecorrectedtoensurethattheresponseistrulyregenerative.Fortheexercise,correction forthelevelofanaemiawillgiveavalueof6%.Theabsolutelevelofreticulocytesis0.530x1012/L (RRupto0.100x1012/L)Theneutrophilresponseislikelytoberelatedtotheintenseregenerative anaemiabutsomecouldbeduetotruetissuedemand(despitetheabsenceofaleftshift)relatedto the pelvic injuries. The total plasma protein is now normal due to increased production and replacement. Furtherinvestigationoftheanaemia,atthisstage,isprobablynotwarranted. DIAGNOSIS:Bloodlossanaemia.

163

CASE21
ANIMAL:3yearoldmaleCockerSpaniel. PRESENTINGCOMPLAINTS:Depressionandweaknessstarted5daysearlier.Nowjaundiced. LABORATORYRESULTS

BIOCHEMISTRY ALPIU/L ALTIU/L Totalbilirubinmol/L Unconjugatedbilirubinmol/L Conjugatedbilirubinmol/L

SAMPLE 409 238 203 19 184

REFERENCEINTERVAL <110 <60 1.25.1 1.25.1 1.25.1

HAEMATOLOGY SAMPLE REFERENCEINTERVAL Plasmaappearance Yellow Clear PCVL/L 0.21 0.370.50 Plasmaproteing/L 77 5575 Haemoglobing/L 80 100150 12 Erythrocytesx10 /L 2.5 57 MCVfl 84 6075 MCHCg/L 380 300360 Leukocytesx109/L(correctedforNRBC) 25.1 712 9 Neutrophils(seg.)x10 /L 17.5 4.19.4 9 Neutrophils(band)x10 /L 2.6 00.24 9 Lymphocytesx10 /L 0.7 0.93.6 9 Monocytesx10 /L 4.0 0.21.0 9 Eosinophilsx10 /L 0.3 0.141.2 Basophilsx109/L 0 00.4 Bloodfilm:markedanisocytosisandpolychromasia,30nucleatederythroidcells(NRBC) per100leukocytes,manyspherocytes Reticulocyte%(uncorrected) 10.2 01.5 Othertests: Coomb'stestwaspositive(endpoint1/32i.e.highestdilutionofserum showingagglutination).

164

INTERPRETATIONOFLABORATORYFINDINGS The limited information suggests a relatively intense, recent disease. The jaundice points to pre hepatic,hepaticorposthepaticdisease. (a)Detectedlaboratoryabnormalities:Moderateregenerativeanaemia,neutrophiliawithleftshift, lymphocytopenia, monocytosis, spherocytes, positive Coomb's test, mildly elevated ALP and ALT, hyperbilirubinemia(predominantlyconjugated). (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The anaemia is regenerative on the basis of a high reticulocyte count. Correction for the level of anaemiagivesavalueof4.8%(.21/.45[asaveragePCV]x10.2),correctionforbothlevelofanaemia anderythroidmaturationtime(takenas2daysforthisPCV)givesavalueof2.4(>2isregardedas trulyregenerative,avalueof12indicateslessthanoptimumregenerationwhileavaluelessthan1 is considered nonregenerative. The absolute reticulocyte count is 0.255 x 1012/L (RR less than 0.105 x 1012/L). The polychromasia and anisocytosis are a reflection of the high numbers of circulatingreticulocytes(polychromatophilicmacrocytesinasmearstainedbyGiemsaor'DiffQuik'). These large cells, because of their significant numbers, have raised the MCV above reference interval,andthiswillbemaintaineduntilreticulocytosissubsides.Normallyinintenseregenerative anaemiatheMCHCwillbedepressedduetothefactthatthereticulocytesareimmatureandhave toolittleHbfortheirsize(thesocalledpseudomacrocytic,pseudohypochromicresponseinintense regenerative anaemia). However, in this case the MCHC is actually increased above reference interval.AhighMCHCisnotpossibleaserythrocytescannotbeoversaturatedwithHb.Therefore, thevalueisduetolaboratoryerrororduetothepresenceoffreeHb(asinhaemolyticstatesmost likelyinthiscase). The large number of circulating nucleated erythroid cells (NRBC) is appropriate in regenerative anaemiawhenreticulocytosisismarked.Thisismorelikelytooccurinhaemolyticratherthanblood loss regenerative anaemias as iron is more easily reutilised (conclusion). The presence of spherocytes (dense cells without a central pallor which occur because of the pinching off of damaged or antibodycoated surface membrane by macrophages) and the positive Coomb's test suggestthatthehaemolyticanaemiaisimmunemediated(conclusion).Inthiscase,becauseofthe hightitreintheCoomb'stestandthelackofotherdiseaseprocessesthatmaygiverisetoantibody coatingoferythrocytes(e.g.erythrocyteparasites,drugsetc)itislikelythattheproblemisprimary immunemediated(autoimmune)(conclusionandfinaldiagnosis). Thetotalleukocytecounthasbeencorrectedforafalselyhighcountcreatedbynucleatederythroid cellsbeingincludedincountingbytheautomaticcellcounter.Normally,thetotalleukocytecountis not corrected unless the circulating nucleated erythroid cells reach 5 per 100 leukocytes (total leukocyte count x 100/100 + no. of circulating nucleated erythroid cells per 100 leukocytes). The neutrophilia,lymphocytopeniaandmonocytosis(eosinophilsarewithinreferenceintervalbutatthe lowerend)couldbeinterpretedasstressinduced(theanimalwasstillintenselyill).However,there is a left shift to the neutrophilia, which suggests true inflammatory demand for part of the neutrophilia.Thisisprobablyinresponsetothehaemolyticanaemia.Intensehaemolyticanaemias commonlycauseleftshifts,especiallyinthedog,buttheexactmechanismsarepoorlyunderstood. Nonspecific stimulation of the macrophage system may be involved as may be released products fromcellbreakdown.Partofthemonocytosisinthiscasemaybedirectlyrelatedtothehaemolytic anaemia. ALP elevation suggests mild cholestasis, ALT elevation suggests mild hepatocellular damage (fatty change for example). Both of these could be related to hepatic hypoxia created by acute onset of anaemia.Themarkedlyhightotalserumbilirubinistobeexpectedinhaemolyticanaemiasthatare

165

predominantly intravascular. Free Hb produced by intravascular erythrocytic destruction is quickly convertedtobilirubin.Intheearlystagesmostofthebilirubinisunconjugatedbutastimegoeson an increase in conjugated may occur. In this case the conjugated levels are marked. This may be partlyduetosomecholestasiscausedbyhepatocyteswellingbutismostlikelytobeduetosimple regurgitationrelatedtoenhancedproduction. Thesupportdiagnosisinthiscasewasstraightforward.Theprognosisfortheautoimmuneanaemia appears good as regeneration is substantial. However, the process of destruction appears to be continuing (the high MCHC probably means free HB through present erythrocyte destruction) and treatmentneedstobecontinued. Further investigation is really related to following the course of the disease and response to therapy.Thiswouldbedonethroughroutinehaematology. DIAGNOSIS:Autoimmunehaemolyticanaemia.

166

CASE22
ANIMAL:5yearoldfemalepony. PRESENTINGCOMPLAINTS:Weakness,rapidrespiration,darkurineandjaundice.Noticedaftera3 dayperiodwhenthehorsewasleftinapaddock. LABORATORYRESULTS

BIOCHEMISTRY Totalbilirubinmol/L Unconjugatedbilirubinmol/L Conjugatedbilirubinmol/L Ureammol/L

SAMPLE 160 154 6 16

REFERENCEINTERVAL 050 06.8 3.450 3.78.2 REFERENCEINTERVAL Variable 0.320.52 5878 130190 6.512.5 4149 300390 6.013 2.57 00.2 1.65.4 00.7 0.21 00.4

HAEMATOLOGY SAMPLE Plasmaappearance Redyellow PCVL/L 0.10 Plasmaproteing/L 82 Haemoglobing/L 47 12 Erythrocytesx10 /L 2.1 MCVfl 47 MCHCg/L 470 9 Leukocytesx10 /L 17.1 9 Neutrophils(seg.)x10 /L 14.6 9 Neutrophils(band)x10 /L 0.2 Lymphocytesx109/L 1.4 9 Monocytesx10 /L 0.9 9 Eosinophilsx10 /L 0 9 Basophilsx10 /L 0 Bloodfilm:manyghosterythrocytesandHeinzbodies URINALYSIS(voided) Appearance Cloudy PH Colour Red Glucose Specificgravity 1.021 Ketones Protein 4+ Blood Bilirubin Microscopicfindings:02erythrocytesperHPF

6.0 ve ve 4+ ve

167

INTERPRETATIONOFLABORATORYFINDINGS Theprovidedinformationsuggestsanacuteonsetdiseasethathasgivenrisetojaundice,weakness and red urine (Hb, Mb or haematuria?). Considerations would be prehepatic, hepatic and post hepatic causes of jaundice. If the red urine is directly related to jaundice then prehepatic haemolysismightbeareason. (a) Detected laboratory abnormalities: Marked anaemia, hyperproteinemia, elevated MCHC, leukocytosis due to neutrophilia, lymphocytopenia, eosinopenia, ghost cells and Heinz bodies, markedserumhyperbilirubinemia(primarilyduetoincreasedunconjugated),mildazotemia(urea), possiblehaemoglobinuria. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Theresults(thelowPCV,highMCHC[throughthepresenceoffreeHB],haemoglobinuriaandhigh unconjugated bilirubin) suggest that the pony has had a recent intravascular haemolytic crisis (conclusion).Someoftheincreaseinunconjugatedbilirubinisprobablyduetoanorexia.Ghostcells (i.e. ruptured erythrocytes) and Heinz bodies (denatured Hb) suggest haemolysis through Hb denaturationmechanisms.Drugsandplantscouldbecauses(conclusion). Thehyperproteinemiais possiblypartlyspurious,perhapsrelatedtofreeHb elevating thereading on the refractometer. However, some haemoconcentration can't be ruled out. The leukocyte changescanbeexplainedbycorticosteroidrelease(monocytosisrarelyoccursinhorses).Themild azotemia is probably related to protein catabolism and reduced renal blood flow through any haemoconcentration.ThepositiveurinarybloodstripisassumedtobeduetofreeHbasthereisno indication of muscle damage and intact erythrocytes are few in the sediment. The 4+ protein is probablyrelatedtothe4+blood.Nobilirubinisbeingpassedasitisalmostallunconjugatedinthe blood(onlyconjugatedispassedintheurineofmostdomesticspeciesthedogisanexception). Furtherinvestigationwouldinvolveobtainingmorehistoryfromtheownerabouttheenvironment and the past history for the horse. Did it have access to poisonous plants? Had it access to chemicals? DIAGNOSIS AND POSTSCRIPT: Heinz body haemolytic anaemia. On further questioning of the owner, it was found that the horse had been given phenothiazine (anthelmintic) prior to being releasedinthepaddock.Ifgivenahighenoughdose,itcouldhavecausedthehaemolysis.Thehorse diedadaylater.

168

CASE23
ANIMAL:4yearoldmalecrossbreddog. PRESENTINGCOMPLAINTS:Weightloss,inappetenceanddepressionforaperiodofthreeweeks. LABORATORYRESULTS

HAEMATOLOGY SAMPLE REFERENCEINTERVAL Plasmaappearance Clear Clear PCVL/L 0.46 0.370.50 Plasmaproteing/L 87 5575 Haemoglobing/L 150 100150 12 Erythrocytesx10 /L 6.7 57 MCVfl 69 6075 MCHCg/L 326 300360 Leukocytesx109/L(correctedforNRBC) 13.4 712 9 Neutrophils(seg.)x10 /L 8.7 4.19.4 9 Neutrophils(band)x10 /L 0 00.24 9 Lymphocytesx10 /L 1.9 0.93.6 9 Monocytesx10 /L 2.5 0.21.0 9 Eosinophilsx10 /L 0.3 0.141.2 9 Basophilsx10 /L 0 00.4 Bloodfilm:8nucleatederythroidcells(NRBC)per100leukocytes,moderatenumbersof targetcells,basophilicstipplingoferythrocytes Reticulocyte%(uncorrected) 2 01.5 Othertests Urinarydeltaaminolevulinicacid(ALA)analysis:190(referenceinterval<38)ataurinary specificgravityof1.030(i.e.moderatelyconcentratedurine)

169

INTERPRETATIONOFLABORATORYFINDINGS The provided information provides little in the way of clues for specific diseases or even what organs/tissuesmaybeinvolved. (a)Detectedlaboratoryabnormalities:Mildleukocytosisduetomonocytosis,basophilicstipplingof erythrocytes,moderatetargetcells(leptocytes),circulatingnucleatederythroidcells,mildlyelevated reticulocytecount(intheabsenceofanaemia),elevateddeltaALA. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The moderately elevated urinary delta ALA is suggestive of lead poisoning (conclusion and diagnosis).DeltaALAelevatesinurinewhenthereisinterferencewithporphyrinmetabolism(asin heme synthesis for erythrocytes). Lead is capable of doing this as it interferes with several of the enzymatic steps. Blood lead levels would have given an indication of recent exposure to lead but wouldnothaveindicatedpastexposure(leadisquicklystoredintissuesandbloodlevelswilldrop). DeltaALAisanindicationoftheeffectofincreasedbodylead,whetherobtainednoworinthepast. Basophilic stippling of erythrocytes, the presence of target cells and the occurrence of circulating nucleatederythroidcellsintheabsenceofanaemiasupportthediagnosisofleadpoisoning.These aretheviewedeffectsofalterederythropoiesiswhichleadproducesthroughseveralmechanisms. These changes are not always present in lead poisoning and moreover, may occur in other derangementsoferythropoiesis. Basophilicstipplingcanoccurinintenseregenerativeanaemias,especiallyinruminantsandpigs.In dogsandcatsitrarelyoccursanditsappearanceisslightlydifferenttothatinleadpoisoning.The presenceofcirculatingnucleatederythroidcellswithalowreticulocytecountand/orintheabsence oftheappropriatelevelofanaemiaindicatesinappropriate(defectiveordisorderly)erythropoiesis andcanoccurinbonemarroworsplenicdisorders(e.g.myelofibrosis,leukemia,hemangiosarcoma). Target cells are a form of leptocyte that have a large surface area for their volume. They may develop through membrane lipid derangements such as in certain types of hepatic disease or endocrinopathies(e.g.hypothyroidism). Themildmonocytosisinthiscaseisdifficulttoexplain.Itdoesnotseemtobeofimportanceinthe diagnosis. Furtherinvestigationmightinvolvefurtherquestioningoftheownersaboutpossibleaccesstolead. Ifingestionofsolidlead(e.g.fishingsinker)isconsidered,thenitmightbeusefultoradiographthe abdomen.Abloodleadanalysismightbeconsidered.Thiswillbeincreasedduringacuteingestion andabsorption,butmaynotbemuchalteredifmostoftheleadhasbeendepositedin boneand other tissues (past exposure). The diagnosis of lead poisoning has direct implications for management.Whiledeterminingthesourceoflead,theanimalcouldbetreatedwithleadchelating agents. DIAGNOSISANDPOSTSCRIPT:Leadpoisoning.Onfurtherquestioning,theowneradmittedthathe hadbeenrenovatinganoldhomeandtheremayhavebeenthepossibilityofaccesstoleadbased paint.Thedogrespondedwelltotreatment.

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CASE24
ANIMAL:OneyearoldmaleBurmesecat. PRESENTINGCOMPLAINTS:Respiratorydistress,coughingandelevatedtemperaturefor3weeks. LABORATORYRESULTS

BIOCHEMISTRY Serumprotein(refract.)g/L Albumin(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L

SAMPLE 78 30.9 17.7 9.8 19.6

REFERENCEINTERVAL 5473 2430 921 815 923

HAEMATOLOGY SAMPLE REFERENCEINTERVAL Plasmaappearance Clear Clear PCVL/L 0.29 0.300.45 Plasmaproteing/L 84 5978 Haemoglobing/L 95 80140 12 Erythrocytesx10 /L 6.7 610 MCVfl 44 4045 MCHCg/L 328 310360 Leukocytesx109/L 29.3 814 9 Neutrophils(seg.)x10 /L 23.6 3.810.1 9 Neutrophils(band)x10 /L 3.1 00.4 9 Lymphocytesx10 /L 2.3 1.67.0 9 Monocytesx10 /L 0.1 0.10.6 9 Eosinophilsx10 /L 0.2 0.21.4 Basophilsx109/L 0 00.2 Bloodfilm:toxicgranulationandDoehlebodiesinneutrophils Othertests: Transtrachealaspirate:Cytologyrevealednumerousalveolarmacrophagesandclustersof lytic(degenerate)neutrophils.ApuregrowthofStaphylococcusintermediuswasobtained fromthefluid. TheanimalwaspositiveforFelineLeukemiaVirus(antibodytestonserum).

171

INTERPRETATIONOFLABORATORYFINDINGS Respiratorydistressandcoughingcouldsuggestaprimaryrespiratorydiseaseaffectingairwaysand pulmonaryalveoli;butunderlyingcardiacdiseaseshouldalsobeconsidered.Thepyrexiamayoccur withanysevereinflammatoryordegenerativedisease,butismostcommonlyrelatedtoinfectious diseases. (a) Detected laboratory abnormalities: Septic exudate from respiratory tract, borderline anaemia (on PCV alone), mild hyperproteinemia, leukocytosis due to neutrophilia with left shift, toxic granulationandDoehlebodiesinneutrophils. (b) & (c) General interpretation, conclusions, further investigation and implications for management: The transtracheal aspirate findings support a diagnosis of pneumonia (conclusion and diagnosis). Alveolar macrophages suggest that the inflammation has reached the level of the pulmonaryalveoliandisnotjustasimpletracheitisorbronchitis.TheisolationofS.intermediusis unusualasthispartoftheskin'snormalflora.Itisobviouslyactingasanopportunisticpathogenbut howitgotintotherespiratorytractisdifficulttoanswer.Theanimalisprobablyhaemoconcentrated (elevated total protein), which could mean that the cat probably has a true mild anaemia. The anaemiacouldgoalongwithinflammatorydisease(bacterialutilisationofironor,inthelongterm, ironsequestrationbymacrophages),butsincethecatisFeLVpositive,thepossibilityofdirectbone marrow depression by the virus cannot be excluded. The neutrophilia with left shift is consistent with an inflammatory process of 3 weeks duration. The neutrophil response is not entirely satisfactoryasthebacteriaareobviouslyhavinganeffectonneutrophilfunction(toxicgranulation and Doehle bodies), but at least the bone marrow is responding well. If the changes in the neutrophilspersistwithtreatmentthismaybeapoorprognosticsignbuttheyaretobeexpectedin severe bacterial disease in the early stages. The Doehle bodies are an indication of altered neutrophilmaturationandsuggesttoxemiaortheeffectsofcertaindrugs(i.e.theyarenotspecific for toxemia). It should be remembered that in the cat small 'Doehle' bodies may occur in a small numberofneutrophilsinhealth.ThereforeonlylargeDoehlebodiesinasignificantnumberofcells (say30%plus)areconsideredindicationofalteredneutrophilfunction. Both the plasma and serum proteins would have been measured by refractometer. Consequently, thevaluescanbedirectlycomparedandthedifference(indirectmeasurement)isusuallyfibrinogen. In this case, it is elevated (6 g/L normally 24 g/L). Fibrinogen is an acute phase reactant and elevations may be seen in inflammatory or, less so, acute degenerative disease. This occurs more consistentlyinruminantsandhorsesthanindogsandcats.Otheracutephasereactantproteinsare being developed as tests for inflammatory disease in the dog and cat (see notes under liver diseases).Iftheserumproteinis measured bythe chemical method(Biuret) thenitisnotdirectly comparabletoplasmaproteinmeasuredbyrefractometerandfibrinogenmustbedeterminedbya differentmethod(seepracticalnotesonHaematology). Thecat'spneumoniamaywellbearesultofimmunecompromise.Thefindingofanopportunistic pathogen and the fact that the cat is FeLV positive could well indicate this fact (conclusion). Implicationsformanagementareasforanybacterialpneumonia.Furtherinvestigationisprobably notrequiredonceappropriateantibiotictherapyisinitiated,butrepeathaematologymaybeuseful to ensure that the inflammatory demand and toxic changes are diminishing (i.e. good prognostic signs).Presumably,diagnosticimaginghadbeenperformedatthesametimeasthepulmonarywash wasundertaken. DIAGNOSISANDPOSTSCRIPT:Bacterialpneumonia.Theownerwaswarnedofthepossibilitythat thecatmightbepredisposedtounusualinfectionsinthefuture.Thecatrecoveredfromantibiotic treatmentandsupportivetherapy.

172

CASE25
ANIMAL:OneyearoldfemaleBurmesecat. PRESENTINGCOMPLAINTS:Fever,depression,peritonealandpleuraleffusions. LABORATORYRESULTS

BIOCHEMISTRY Serumprotein(refract.)g/L Albumin(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L

SAMPLE 77 32.2 10.3 8.9 25.6

REFERENCEINTERVAL 5473 2430 921 815 923

HAEMATOLOGY SAMPLE REFERENCEINTERVAL Plasmaappearance Clear Clear PCVL/L 0.16 0.300.45 Plasmaproteing/L 85 5978 Haemoglobing/L 53 80140 12 Erythrocytesx10 /L 3.4 610 MCVfl 51 4045 MCHCg/L 331 310360 Leukocytesx109/L 25.5 814 9 Neutrophils(seg.)x10 /L 20.4 3.810.1 9 Neutrophils(band)x10 /L 1.3 00.4 9 Lymphocytesx10 /L 2.5 1.67.0 9 Monocytesx10 /L 0.7 0.10.6 9 Eosinophilsx10 /L 0.5 0.21.4 Basophilsx109/L 0 00.2 Bloodfilm:moderatepolychromasia,oneNRBCper100leukocytes Othertests:
PERITONEALFLUIDANALYSIS

Totalprotein: Nucleatedcells: Erythrocytes: Smear:

60g/L(normal<25g/L) 3000x106/L(normal<500x106/L) 600x106/L(nonenormally) 78%nonlytic(nondegenerate)neutrophils,22%mononuclear cells(includingreactivemesothelialcells)(normal:mainly mononuclears)

173

INTERPRETATIONOFLABORATORYFINDINGS Bodycavityeffusionsandpyrexiainayoungcatmightsuggestinfectiousdisease.FelineInfectious Peritonitis is one such cause (i.e. pattern recognition), and will need to be considered in investigation. (a)Detectedlaboratoryabnormalities:Moderateanaemia,mildhyperproteinemia,leukocytosisdue to neutrophilia with left shift, hyperglobulinemia, hyperfibrinogenemia, peritoneal fluid is a non septicexudate. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: ThelaboratorychangessuggestmultiorganinflammatorydiseasethatcouldbeconsistentwithFIP (conclusionandspecificdiagnosis). The cat has an anaemia which has not been investigated further. Changes on the smear (polychromasia,nucleatederythroidcellcouldpossiblyindicateregenerationbutthiswouldneedto be confirmed with a reticulocyte count. In FIP the anaemia can be quite variable. Inflammatory diseasetendstogiveamildtomoderatenonregenerativeanaemia.Theneutrophiliaissuggestive ofinflammatorydemand.Aleftshiftispresent,consideringthattotalnumbersofbandsexceed1x 109/L. Thecathashyperfibrinogenemia(8g/Ldifferencebetweentotalserumproteinandtotalplasma protein, both measured by the refractometer) and hyperglobulinemia which are suggestive of inflammatorydiseasewithastrongimmuneresponse.Theglobulinincreasewasbroadbasedonthe EPG (i.e. increase in a variety of gamma globulins). Fibrinogen increases can occur in a variety of degenerativeandinflammatoryconditionsbutaremoreconsistentinfarmanimals.Fibrinogenisan 'acute phase reactant' which is produced and stored by the liver. Therefore, rapid increases can occur.InFIP,fibrinousinflammationisafeature,possiblyduetothevasculitis. TheperitonealfluidchangesareconsistentwithFIP.Anextremelyhighprotein(muchofthiswould begammaglobulins)incombinationwithasubduedincreaseinnucleatedcellsishighlysuggestive ofFIPinthecat.OnthebasisofincreasedproteinandnucleatedcellsIhavecalledthisanexudate (nonseptic). However, some pathologists might prefer to call it a modified transudate because of thelimitedelevationsthatcanoccurinthenucleatedcellsinsomecases.Thisdifferenceinopinion isnotimportant.Itisimportanttorealisethatthechangesindicateaninflammatoryprocessofnon bacterialorigin.Thereactivemesothelialcellsaremorelikelytobeseeninmodifiedtransudatesand nonsepticexudates. FurtherinvestigationrequiresconfirmationforthecoronavirusthatcausesFIP.Thereareserological tests, but many of these have poor specificity (overlap with the intestinal coronavirus of cats that causeslittledisease).Coronavirusantigendetectioncannowbedoneoncytologicalandhistological preparations. ManagementofFIPisdifficultandmostaffectedcatsareeuthanised.Ifthecatisfromacatteryor multicathousehold,thenitisimportanttodevelopaplanofmanagementfortheowners. DIAGNOSIS AND POSTSCRIPT: Feline Infectious Peritonitis. Because this case occurred before the development of the coronavirus antigen detection test, confirmation of the disease was not obtained.NecropsyconfirmedtypicalFIPlesionsofvasculitisandpyogranulomatousinflammation.

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CASE26
ANIMAL:6yearoldmaleChihuahua. PRESENTING COMPLAINTS: Generalised lymphadenomegaly, hepatomegaly, splenomegaly, pale mucousmembranes. LABORATORYRESULTS

HAEMATOLOGY SAMPLE REFERENCEINTERVAL Plasmaappearance Clear Clear PCVL/L 0.13 0.370.50 Plasmaproteing/L 52 5575 Haemoglobing/L 45 100150 12 Erythrocytesx10 /L 1.7 57 MCVfl 76 6075 MCHCg/L 346 300360 Leukocytesx109/L(correctedforNRBC) 203 712 9 Neutrophils(seg.)x10 /L 7.9 4.19.4 9 Neutrophils(band)x10 /L 0 00.24 9 Lymphocytesx10 /L 187 0.93.6 9 Monocytesx10 /L 3.1 0.21.0 9 Eosinophilsx10 /L 0 0.141.2 9 Basophilsx10 /L 0 00.4 Bloodfilm:manylymphoidcellswereblasts,neutrophilswerehypersegmented, poikilocytosisandmildpolychromasiaoferythrocytes Reticulocyte%(uncorrected) 2 01.5

175

INTERPRETATIONOFLABORATORYFINDINGS Thepalemucousmembranescouldsuggestanaemiaorshock.Thegeneralisedlymphadenomegaly, hepatomegaly and splenomegaly could occur with widespread infiltrative disease of either inflammatoryorneoplasticnature. (a) Detected laboratory abnormalities: Marked nonregenerative anaemia, hypoproteinemia, leukocytosisduetolymphocytosis,circulatinglymphoblasts,hypersegmentedneutrophils. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Thediagnosisoflymphoidleukemia(circulatingneoplasticlymphoidcells)isobviousfromthehigh level of lymphocytes and the fact that significant numbers of lymphoblasts are circulating (usually there are none) (conclusion and specific diagnosis). The nonregenerative anaemia (corrected for thelevelofanaemiathereticulocytevalueislessthan0.6%;correctedforthelevelofanaemiaand maturation time, i.e. the Reticulocyte Production Index, the value is less than one; absolute reticulocyte count is 0.035 x 1012/L) isprobably due to a combination of factors but bone marrow replacementbytheproductionofneoplasticlymphoidcells(myelophthisis)islikelytobeimportant. Hypersegmentedneutrophilsareindicativeofretentionwithinthecirculationthatcanoccurdueto thereleaseofcorticosteroidsinstress.Thehypoproteinemiaispossiblyduetoneoplasticcachexia andpossiblyhepaticdysfunction(theliverandspleenareofteninfiltratedincasesofleukemiaof anycelltype). Lymphoidneoplasiainthedogoftenpresentsassolidmassesinorgansandenlargedlymphnodes (hence the name lymphosarcoma). A lymph node cell aspirate or biopsy should confirm the diagnosis(furtherinvestigation)butoftenbloodexaminationisundertakentoassistprognosisand treatment.Theleukemiacouldbefurtherinvestigatedthroughbonemarrowexamination. Ananimalwithlymphosarcomaandthatisleukemicandwithamarkednonregenerativeanaemiais lesslikelytorespondwelltochemotherapybecauseofthebonemarroweffectsandtheadvanced stage; however, whether treatment is undertaken will depend on the owners (implications for management). Lymphoid leukemia can develop in an animal with lymphosarcoma or it can be an entityinitselfaseitheracutelymphoblasticorchroniclymphocyticleukemia. DIAGNOSIS AND POSTSCRIPT: Lymphosarcoma and lymphoid leukemia. The owner elected for euthanasia.

176

CASE27
ANIMAL:2yearoldmaleSpringerspaniel. PRESENTINGCOMPLAINTS:Diarrhoea,inappetenceandweightlossof6weeksduration. LABORATORYRESULTS

BIOCHEMISTRY Serumprotein(refract.)g/L Albumin(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L Calciummmol/L HAEMATOLOGY Plasmaappearance PCVL/L Plasmaproteing/L Haemoglobing/L Erythrocytesx1012/L MCVfl MCHCg/L Leukocytesx109/L Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Bloodfilm:normal Othertests:

SAMPLE 17 8.8 2.9 4.7 0.6 1.6 SAMPLE clear 0.49 23 162 7.4 66 331 14.8 11.2 0 3.1 0 0.5 0

REFERENCEINTERVAL 5070 2339 716 916 412 2.12.9 REFERENCEINTERVAL Clear 0.370.50 5575 100150 57 6075 300360 712 4.19.4 00.24 0.93.6 0.21.0 0.141.2 00.4

Serumtrypsin/trypsinogenlikeImmunoreactivity(TLI)g/L:12(referenceinterval535)
FAECALANALYSIS

Trypsin(reallyallfaecalproteases):5Azoalbuminunits(referenceinterval>7) Blood:present Smearsforundigested/unabsorbedfoodparticles:noincreaseinstarch,undigested muscleorundigestedfat(unsplit,primarilytriglycerides);largeincreaseinunabsorbedfat (split,primarilyfreefattyacids)

177

INTERPRETATIONOFLABORATORYFINDINGS Chronic diarrhea with significant weight loss could suggest malassimilation associated with the intestinaldisease. (a) Detected laboratory abnormalities: Marked hypoproteinemia, elevated Hb and erythrocyte count, mild leukocytosis due to neutrophilia, monocytopenia, all protein fractions decreased, hypocalcemia,decreasedtrypsinlevel,faecalblood,increasedunabsorbedfat. The dog has a significant hypoproteinemia (all classes), which in this case is most likely due to an enteropathy(faecaltestsandtheclinicalsignssuggestthis)(conclusion).Endstageliverdiseasecan causeahypoproteinemiaaffectingmostproteingroups,althoughgammaglobulinsarenotusually affected and may actually be increased. Severe starvation may cause a hypoproteinemia. Kidney disease may cause hypoproteinemia but this commonly involves only albumin (globulins are generallytoolargetobelostbytheglomeruliortubules).Infactsomeglobulinsmayincreaseinthe blood. Thehypocalcemiainthiscaseispossiblyduetolossofproteinboundcalciumthroughdamagedgut (mostkitsdetecttotalcalciumwhichincludesproteinboundandfreeionisedforms). Increasedunabsorbedfatinthefaecessuggestsanenteropathyasmostcasesofmalabsorptionare duetoentericdisease(conclusion).Theslightlydecreasedtrypsinactivityhasnosignificanceinthis case as the smears for food particles do not suggest maldigestion. This is also supported by the normal TLI (less than 2.5 g/L is diagnostic for maldigestion due to EPI). The TLI has replaced the faecaltrypsintestformaldigestionduetoEPI.Thepositivefaecalbloodmayindicatemildleakage associatedwithagutdisease.However,thetestisnotterriblyreliableasitcangivefalsepositives due to the presence of other substances having peroxidase activity. The mild neutrophilia in the absenceofotherwhitecellchangesmaysuggestlimitedinflammatorydemand,perhapsrelatedto gut disease. The monocytopenia has no significance in any circumstances. The elevated Hb and erythrocyte count can be best explained by haemoconcentration. Commonly, nonregenerative anaemia occurs in chronic bowel diseases but perhaps this one has not been occurring for long enough. In summary, the persistent diarrhoea and evidence of malabsorption suggest that the hypoproteinemiaisduetogutdisease(mainconclusion). Further investigation could involve diagnostic imaging, intestinal absorption tests, endoscopic examinationandgutbiopsiesThehypoproteinemiacouldbeinvestigatedbyinjectingproteinbound toradioactivechromium,andcheckingforlossesinthefaeces. DIAGNOSIS AND POSTSCRIPT: Malabsorption and protein losing enteropathy due to severe inflammatoryboweldisease(IBD).IBDisapoorlyunderstood,probablymultifactorialconditionof dogsandcatsand,dependingonintensity,cancauseavarietyofclinicalsignsrelatingtodysfunction ofthesmallintestine.

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CASE28
ANIMAL:OneyearoldfemaleneuteredGermanshepherd. PRESENTINGCOMPLAINTS:Weightloss,normaltoincreasedappetite,softvoluminousfaeces. Graduallydevelopingover3months. LABORATORYRESULTS BIOCHEMISTRY

Trypsin/trypsinogenlikeImmunoreactivity(TLI)g/L:1.5(referenceinterval535)
FAECALANALYSIS

Trypsin(reallyallfaecalproteases):2.6Azoalbuminunits(referenceinterval>7) Blood:absent Smearsforundigested/unabsorbedfoodparticles:noincreaseinstarchorunabsorbedfat (split,primarilyfreefattyacids),Largeincreasesinundigestedmuscleandundigestedfat (unsplit,primarilytriglycerides).

179

INTERPRETATIONOFLABORATORYFINDINGS Significantweightlossandvoluminousfaeces,especiallyinananimalthathasnormaltoincreased appetite,couldsuggestmalassimilation.Thefactthatitisayoungdogmightsuggestcongenitalas wellasacquireddisease. (a) Detected laboratory abnormalities: Low faecal proteolytic (trypsin) activity, low TLI, increased undigestedmuscleandfat. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The results are highly suggestive of maldigestion (conclusion). Lowered TLI, faecal trypsin and increased unsplit fat in the faeces are the most important results and suggest Exocrine Pancreatic insufficiency(EPI)asacauseofthemaldigestion(conclusionanddiagnosis).Resultsforundigested starchandmuscleareextremelyvariabledependinginthediet.Ifpossibleitisbesttoanalysefaeces fromahealthydogplacedonthesamediet.Incasesofsuspectedmaldigestionwherecontradictory results between trypsin levels and smears for food particles occur (i.e. low trypsin and normal smears) repeat testing must be employed. This is because low trypsin values (less than 7) will occasionallybedetectedinhealthydogs.TheTLIhasreplacedtheneedforfaecaltrypsinanalysis. ThisdoghadclassicstoolsforEPI,i.e.rancid,softbutsemiformedandvoluminous.IncasesofEPI (themostcommoncauseofmaldigestion)thefaecesarerarelyfluid.Fluiddiarrhoeaismorelikelyto occurwithanenteropathy,withorwithoutmalabsorption. Furtherinvestigationmightinvolveultrasonographyofthepancreas. The laboratory results have direct implications for management as pancreatic extract and diet controlarewarranted DIAGNOSISANDPOSTSCRIPT:Exocrinepancreaticinsufficiency(EPI).Becauseofitsyoungageand the fact that it was a German shepherd, pancreatic hypoplasia was considered best fit (pattern recognition).TheanimalrespondedtotherapyforEPI.

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CASE29
ANIMAL:6yearoldmalecrossLabrador. PRESENTINGCOMPLAINTS:Depression,fitting,ataxia LABORATORYRESULTS CEREBROSPINALFLUIDANALYSIS

Grosscharacteristics clearandcolourless(normal) Protein 0.54g/L(referenceinterval<.4g/L) Cells erythrocytes9x106/L;nucleatedcells47x106/L(referenceinterval fornucleated<8x106/L,noerythrocytesnormallyseen) Differential 65%lymphocytes,35%monocytesandmacrophages(normal usuallyamixtureofmononuclearcelltypes)

181

INTERPRETATIONOFLABORATORYFINDINGS Theataxiaandfittingcouldsuggestprimaryneurologicaldisease,butmetabolicdisordersorpoisons shouldprobablybeconsideredalso. (a)Detectedlaboratoryabnormalities:Elevatedproteinandcellswithincerebrospinalfluid. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: ThechangesinCSFaresuggestiveofchronic(nonsuppurative)inflammation(conclusion).Themild increase in protein is significant as the concentrations of constituents are strictly controlled. This levelofproteincanonlybedetectedbymicroanalyticmethods(e.g.ponceauredS),buttheprotein stripontheurinarydipstickmaygivearoughguidetolevels(candetectvaluesabove.1g/L).The increaseinproteinisprobablyduetoinflammatoryleakagebutinsomecasesofdistemper,protein increases can occur without increases in cells (so called albuminocytologic dissociation this can also occur in some tumours and in vascular lesions i.e. protein leakage apparently unrelated to inflammation).Thelevelofincreasednucleatedcellsisquitesignificantandisnotgreatlyinfluenced bythefactthaterythrocytesarepresent(i.e.iatrogenicbleedingdoesnotusuallyelevatelevelsof nucleated cells until erythrocyte values exceed 30, and not significantly until values exceed 500 x 106/Linthesecasesincreasesinnucleatedcellsareduetoincreasedneutrophils).Thedifferential suggestschronicity(immuneresponsecellsandmacrophages)butitisimportanttorememberthat thesecellsarenormallypresentinlownumbersinCSFinhealth. CSF changes do not occur in all cases of neurological disease. Diseases affecting the meninges or adjacentlayersaremorelikelytocausechanges.Interpretationisbasicallyasforotherfluids. Furtherinvestigationwouldbetodeterminespecificcausesofnonsuppurativeinflammation.One might consider culture of CSF for protozoal or fungal organisms (although not detected in routine examinationandusuallycausingapyogranulomatouscellresponse).Serologicaltestsareavailable fortoxoplasmal,neosporalandcryptococcaldisease. DIAGNOSIS AND POSTSCRIPT: Necropsy revealed severe meningoencephalomyelitis with viral inclusions.ThiswassuggestiveofCanineDistemper.

182

CASE30
ANIMAL:9yearoldfemaleneuteredminiaturecolliedog. PRESENTING COMPLAINTS: Polydipsia, polyuria and weight loss for some months. Now collapsed withvomitinganddiarrhoea. LABORATORYRESULTS Bloodsamplemarkedlylipemic.

BIOCHEMISTRY ALPIU/L ALTIU/L Triglyceridesmmol/L Totalcholesterolmmol/L Glucosemmol/L Ureammol/L

SAMPLE 135 95 5.3 13.65 42.7 27.6

REFERENCEINTERVAL <110 <60 0.52.0 1.47.5 3.36.4 310

URINALYSIS(voided) Appearance Cloudy pH 5.0 Colour Yellow Glucose 2% Specificgravity 1.051 Ketones 2+ Protein Trace Blood ve Bilirubin Trace Microscopicfindings:hyalinecasts5060perLPF,finegranularcasts1015perLPF,much lipid,abundanttransitionalepithelium.

183

INTERPRETATIONOFLABORATORYFINDINGS Thepolyuriaandpolydipsiacouldoccurwithrenal,hepaticorendocrinediseases.Thefactthatitis now vomiting and severely unwell could suggest a terminal event or a totally unrelated disease processtotheonethatcausedpolyuria/polydipsia.Ifthefirstpossibilityisconsidered(thismakes moresenseOccamsRazor:thatthefewestpossibleassumptionsaretobemadeinexplaininga thing),thenchronicrenalfailureanddiabetesmellituswithdevelopedketoacidosisaremostlikely. (a) Detected laboratory abnormalities: Mildly elevated ALP and ALT, marked hypertriglyceridemia, moderate hypercholesterolemia, marked hyperglycemia, moderate azotemia, ketonuria, glucosuria andcylindruria. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Laboratorydiagnosisofdiabetesmellitusrequirestheidentificationofpersistenthyperglycemiaand glucosuria(2ormoresamples).However,inthiscasethemarkedelevationofbloodglucoseishighly suggestive of diabetes (conclusion and diagnosis). The glucosuria is to be expected as the renal threshold(about11mmols/Lforthedog)hasbeenexceeded.Thehyperlipidemiacommonlyoccurs andisduetoderangementsofcarbohydratelipidmetabolismandtheinhibitionoflipoproteinlipase activity. Elevations in triglyceride are commonly marked whilst elevations in cholesterol are more variable.ALTandALPminimalelevationsareprobablyduetofattychangeintheliverorjustdueto theseverityofillness. The presence of ketones in the urine and the low pH are highly suggestive that this dog is ketoacidotic(inanadultdogorcatketonuriararelyaccompaniesotherdiseases)(conclusion).Thisis probably contributing to the azotemia as would be the vomiting and inappetence. Although cylindruria (increased casts) may suggest tubular damage, the high specific gravity rules out a concentrating problem for the kidney (i.e. adequate function of the tubules, interstitium and adequatereleaseofADH).Perhapsthecylindruriaindicatesaperiodofdecreasedrenalbloodflow duetohaemoconcentrationfollowedbythefirstvoidingofurine?Abundanttransitionalepithelium maysupportthis.Thetraceproteinandbilirubinareofdoubtfulsignificanceinthiscaseandatthis concentration level. Lipiduria is a reflection of changes in tubular cells and can occur in healthy animals.Inthiscaseitwouldbeexpected. Mostcasesofadvanceddiabetesmellitusarerelativelyeasytodiagnosebutearliercasesaremore difficult.Bloodglucoselevelsmaynotbemarkedlyelevated,andelevationsmaybeinconsistent.In thosecases,anintravenousglucosetolerancetest(furthertesting)maybenecessarytosupportthe diagnosis of diabetes mellitus (a flat curve or low glucose disappearance coefficient suggests glucoseintolerance).Inalldogswithdiabetesmellitus,theinsulinresponsetothetolerancetestwill helpdecidewhetherthereisanabsoluteorrelativelackofinsulin(i.e.insulindependentdiabetes mellitus[IDDM]ornoninsulindependentdiabetesmellitus[NIDDM]). ImplicationsformanagementareforcontrollingtheketoacidosisandthenstabilisingtheDM DIAGNOSIS AND POSTSCRIPT: Diabetes mellitus with ketoacidosis. The dog was treated successfully andrecovered.Theanimalrequiredlongterminsulinasmost casesofDMinthedog areIDDM.MostcausesofIDDMareobscure.

184

CASE31
ANIMAL:8yearoldfemalepoodle. PRESENTINGCOMPLAINTS:Prolongedhistoryoflethargy,dry,shortenedcoat,obese,noncycling. LABORATORYRESULTS

BIOCHEMISTRY CKIU/L Totalcholesterolmmol/L HAEMATOLOGY Plasmaappearance PCVL/L Plasmaproteing/L Haemoglobing/L Erythrocytesx1012/L MCVfl MCHCg/L Leukocytesx109/L Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Reticulocyte%(uncorrected) Bloodfilm:markednumbersoftargetcells Othertests:

SAMPLE 135 16.8 SAMPLE Lipemic 0.28 Lipemia interfered Lipemia interfered 4.5 62 ND 26.6 22.3 0.3 2.9 1.1 0 0 2.1

REFERENCEINTERVAL <200 1.47.5 REFERENCEINTERVAL Clear 0.370.50 5575 100150 57 6075 300360 712 4.19.4 00.24 0.93.6 0.21.0 0.141.2 00.4 01.5

BaselineT4:8nmol/L(referenceinterval2040nmol/L)

185

INTERPRETATIONOFLABORATORYFINDINGS The clinical signs suggest a chronic disease that might be affecting more than body system (skin, genital tract) Obesity is a little unusual in chronic disease related to neoplasia, degeneration or inflammation(inappetenceanddirecteffectsofthepathologicalprocessoftenleadtocachexia).A metabolic/endocrinedisturbancemightbeconsidered(e.g.hypothyroidism). (a)Detectedlaboratoryabnormalities:MildelevationofCK,moderatehypercholesterolemia,non regenerative mild to moderate anaemia, leukocytosis due primarily to neutrophilia, mild monocytosisandabsoluteeosinopenia,targetcells,lowbaselineT4. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The history is suggestive of hypothyroidism and changes in nonspecific tests support this (conclusion and diagnosis). The mild elevation in CK has been recorded in a proportion of hypothyroid dogs and is apparently due to a myopathy. Hypercholesterolemia can occur in hypothyroidism due to decreased utilisation and reduced plasma clearance through decreased lipoprotein lipase activity. The visible lipemia present in this case is probably due to increased triglyceride within circulating lipoproteins as increased cholesterol rarely gives visible turbidity. Lipemia and the composition of the lipids are extremely variable in hypothyroidism. The non regenerativeanaemia(correctedforthelevelofanaemia,thevalueis1.3,andtheRPIislessthan one)islikelyduetointerferencewitherythropoietinproductionoractivity.Thewhitecellchanges are likely due to stress. This is uncommon in low grade chronic disease unless a clinical crisis has occurred.Targetcellscommonlyoccurwhenthereisderangementofplasmamembranestructure. Diseases that cause derangements of lipid metabolism may cause target cell production (e.g. liver diseases,endocrinopathies). All these changes are consistent with hypothyroidism, but the problem is that their occurrence in hypothyroidism is inconsistent. Therefore, the performance of specific hormonal assays is often necessary. Baseline T4 is an appropriate test in the first instance (T3 measurement has no real advantage over T4). However, a problem is that 20% of euthyroid dogs can show low levels of T4 (physiologicalvariation,hypoproteinemia,certaindrugs,insomecasesofhyperadrenocorticism).If thisisthecasethenaTSHstimulationtestcanbeperformed(furtherinvestigation).Ariseofless than3xthebaselinelevelabout4hoursafteradministrationofTSHissuggestiveofhypothyroidism. Inthecasediscussed,thehistory,changesinnonspecifictestsandthelowbaselinevalueforT4are enoughtobecertainofthepresenceofhypothyroidism. DIAGNOSIS AND POSTSCRIPT: Hypothyroidism. The dog was successfully treated for hypothyroidism.

186

CASE32
ANIMAL:7yearoldfemaleneuteredcorgi. PRESENTINGCOMPLAINTS:Anorexiaandweightloss,occasionalvomiting,possiblydehydrated. LABORATORYRESULTS

BIOCHEMISTRY Ureammol/L Sodiummmol/L Potassiummmol/L Chloridemmol/L HAEMATOLOGY Plasmaappearance PCVL/L Plasmaproteing/L Haemoglobing/L Erythrocytesx1012/L MCVfl MCHCg/L Leukocytesx109/L Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Bloodfilm:normal

SAMPLE 31 122 7.1 91 SAMPLE Clear 0.35 68 119 5.9 59 340 12.6 6.3 0 4.9 0.6 0.8 0

REFERENCEINTERVAL 310 137150 3.34.8 105120 REFERENCEINTERVAL Clear 0.370.50 5575 100150 57 6075 300360 712 4.19.4 00.24 0.93.6 0.21.0 0.141.2 00.4

187

INTERPRETATIONOFLABORATORYFINDINGS Theclinicalsignsarevagueandoflittlehelpinpinpointingspecificorgan/tissuedisease (a)Detectedlaboratoryabnormalities:Mildanaemia,borderlineleukocytosisduetolymphocytosis, moderateazotemia,hyponatremia,hyperkalemia,hypochloridemia. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Many of the results could be consistent with hypoadrenocorticism (conclusion and diagnosis). Vague clinical signs often accompany hypoadrenocorticism. The changes are related to variable decreasesinaldosteroneand/orcortisol.Consequently,thelaboratoryfindingsarevariable.Inthis casethehyponatremiaandhyperkalemiaarehighlysuggestiveofalackofaldosterone(conclusion). The ratio of approximately 17 to 1 is well below the 23 to 1 that some workers accept as being unequivocal evidence for hypoaldosteronism (although some think 2318 to 1 is a grey zone). The changes are due to urinary loss of sodium (the chloride loss follows this) and urinary retention of potassium.SomegastrointestinaldisturbancesmayaffecttheNa:Kratioinasimilarmannerwhich maycreatedifficultyindiagnosisassomehypoadrenocorticaldogsmaypresentwithdiarrhoea. The azotemia is consistent with hypoaldosteronism (80% of cases) and is due to prerenal factors such as protein catabolism, hypovolemia mainly due to electrolyte disturbances and subsequent reduced renal perfusion. Some animals may have impaired tubular concentrating ability with a specificgravitylessthan1.025.Thisoftenmeansthatprimaryrenalfailureisoneofthediagnoses under consideration. We are unable to comment on this case and further investigation for renal diseasemaybewarranted. The mild anaemia has not been categorised but is normocytic, normochromic and possibly worse thanitseemsiftheanimalishaemoconcentrated.Inhypoadrenocorticism,amildnonregenerative anaemia can occur due to cortisol lack (conclusion), which is an essential hormone for erythropoiesis.Thelymphocytosisinasickanimal,whichpossiblyshouldbestressed,isunusualand a further indication that there may be a lack of cortisol release. In some cases of adrenal insufficiencyeosinophiliamayalsooccur. All these laboratory changes are inconsistent in adrenal insufficiency (e.g. up to 26% of dogs with confirmedadrenalinsufficiencycanhavenormalelectrolytelevels).Therefore,itisoftennecessary tomeasure baselinecortisollevelsand levelsafterexogenousACTHadministrationtoconfirmthe diagnosis of adrenal insufficiency (further investigation). Of course, if it is a primary lack of aldosterone then the values may be normal but most cases of primary adrenal insufficiency do involveabnormalitiesofbothcortisolandaldosterone. DIAGNOSIS AND POSTSCRIPT: Hypoadrenocorticism (adrenal insufficiency) was confirmed by measuringbaselinecortisollevels(low).Theanimalwastreatedwithcorticoidreplacers.

188

CASE33
ANIMAL:11yearoldfemaleScottishterrier. PRESENTINGCOMPLAINTS:Polyuria,polydipsia,potbelliedandbilaterallysymmetricalalopecia. LABORATORYRESULTS

BIOCHEMISTRY ALPIU/L ALTIU/L Totalcholesterolmmol/L Glucosemmol/L Sodiummmol/L Potassiummmol/L HAEMATOLOGY Plasmaappearance PCVL/L Plasmaproteing/L Haemoglobing/L Erythrocytesx1012/L MCVfl MCHCg/L Leukocytesx109/L Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Bloodfilm:normal Eosinophils(directcount)x109/L URINALYSIS(voided) Appearance Clear Colour Lightyellow Specificgravity 1.014 Protein ve Microscopicfindings:littleseen

SAMPLE 4905 63 10.2 4.9 153 3.9 SAMPLE Clear 0.46 80 168 7.2 64 365 15.6 13.9 0.5 0.3 0.9 0 0 .006

REFERENCEINTERVAL <110 <60 1.47.5 3.36.4 137150 3.34.8 REFERENCEINTERVAL Clear 0.370.50 5575 100150 57 6075 300360 712 4.19.4 00.24 0.93.6 0.21.0 0.141.2 00.4 0.11.0

PH Glucose Ketones Blood Bilirubin

6.5 ve ve ve Trace

189

INTERPRETATIONOFLABORATORYFINDINGS Thedogprobablyhasmultiorgandiseasethathasgivenrisetopolyuria/polydipsia,symmetricalskin changesandpossibleabdominalenlargement.Anumberofdiseasescouldcontributetoindividual clinical signs, but the clinical signs in combination make it highly likely that this breed of dog (predisposed) has hyperadrenocorticism. This pattern recognition is something that increases in importanceforveterinariansastheybecomemoreexperienced.However,fortheinexperiencedit may be more prudent to consider likely organ/tissue dysfunction from the clinical signs and other informationforfurtherinvestigationratherthanrelyonpatternrecognition.Ifpatternrecognitionis utilised, then it is important for the proposed diagnosis to be fully supported and confirmed by furtherinvestigation. (a) Detected laboratory abnormalities: Marked increase in ALP, moderate hypercholesterolemia, mild hypernatremia, mild hyperproteinemia, mild leukocytosis due to neutrophilia without left shift, lymphocytopenia,eosinopenia(ondifferentialanddirectcount),aspecificgravityreadingclosetoisosthenuria. (b) & (c) General interpretation, conclusions, further investigation and implications for management: All these laboratory changes could be consistent with hyperadrenocorticism (conclusion and diagnosis). Elevations of ALP occur due to activation of a specific ALP liver isoenzyme are common in hyperadrenocorticism. In this case the value is marked; in other cases values can be absent or minimal. Occasionally, mild elevations of ALT occur which could be partly related to corticosteroid induction and partly due to altered hepatocyte metabolism. The hypercholesterolemiareflectsalteredcarbohydratelipidmetabolism(rarelyistherelipemiadueto increased triglyceride unless there is concomitant diabetes mellitus). The mild hypernatremia and hyperproteinemiaarepossiblyrelatedtohaemoconcentrationduetopolyuria.Thespecificgravity reading may also be a reflection of persistent polyuria and resultant medullary washout. In some cases renal disease cannot be ruled out without a gradual water deprivation test (gradual to counteractmedullarywashout)(furtherinvestigation).Lowspecificgravityreadingsarenotalways the case in hyperadrenocorticism, despite the polyuria. The normal glucose level is occasionally encountered in hyperadrenocorticism but more commonly the level is mildly increased. In small animals with hyperadrenocorticism, the value of glucose is not expected to surpass the renal threshold(whichisrelativelyhighfordogsandcats).Ifmarkedhyperglycemiaandglucosuriaoccurs then one should consider the possibility of concomitant diabetes mellitus (this has been primarily recordedintheUSA;itappearsnottobeascommonhere).Themildneutrophilia,lymphocytopenia andeosinopeniaareallconsistentwiththeeffectsofcorticosteroids(stressresponse).However,in prolonged release of corticosteroids, such as in hyperadrenocorticism, often the magnitude of neutrophiliadrops.Themostusefulandconsistentleukocyteabnormalityseemstobeeosinopenia. Thiscanbeassessedonthesmearbutitismoreappropriatetododirecteosinophilcounts.Often serialeosinophilcountsareemployedtomonitortheeffectoftreatmentoncorticosteroidrelease. Because all these routine tests are inconsistent and relatively nonspecific, confirmation of clinical diagnosisusuallyrequiresdirectevaluationofthepituitaryadrenalaxisusingplasmacortisollevels (furtherinvestigation).Oftensinglemeasurementsofbasalcortisollevelsarenotgoodenoughas there can be considerable overlap between normal and hyperadrenocortical dogs. This is due to episodic release of cortisol and the effects of stress on sick dogs with normal adrenal function. Consequently, dynamic testing of the pituitaryadrenal axis is necessary to confirm (e.g. ACTH responseTestand/orlowdosedexamethasonesuppressiontests)andtohelpdifferentiateadrenal tumours from pituitary dependent hyperadrenocorticism (PDH) (highdose dexamethasone suppressiontest).Itshouldbenotedthatlowresting(basal)T4canoccurinhyperadrenocorticism, butaTSHresponsetestwillusuallyshowanormalthyroidgland. DIAGNOSISANDPOSTSCRIPT:Hyperadrenocorticism.DynamictestingsuggestedPDH.

190

CASE34
ANIMAL:7yearoldmalecrossbreddog.

PRESENTING COMPLAINTS: Prolonged history of polyuria, polydipsia. The dog had pale mucous membranes,lethargyandinappetence.Splenomegalyandhepatomegalyweredetected.

LABORATORYRESULTS

BIOCHEMISTRY ALPIU/L ALTIU/L Serumprotein(biuret)g/L Albumin(BCG)g/L Globulinsg/L Glucosemmol/L Ureammol/L Calciummmol/L Inorganicphosphatemmol/L

SAMPLE 80 55 58 30 28 5.0 34 3.1 4.2

REFERENCEINTERVAL <110 <60 5070 2343 2744 3.36.4 310 2.12.9 0.81.6

HAEMATOLOGY SAMPLE REFERENCEINTERVAL Plasmaappearance Clear Clear PCVL/L 0.20 0.370.50 Plasmaproteing/L 60 5575 Haemoglobing/L 68 100150 12 Erythrocytesx10 /L 3.1 57 MCVfl 64 6075 MCHCg/L 340 300360 9 Leukocytesx10 /L 5.8 712 Neutrophils(seg.)x109/L 2.0 4.19.4 Neutrophils(band)x109/L 0.8 00.24 9 Lymphocytesx10 /L 2.7 0.93.6 9 Monocytesx10 /L 0.3 0.21.0 9 Eosinophilsx10 /L 0 0.141.2 9 Basophilsx10 /L 0 00.4 Bloodfilm:1NRBCper100leukocytes,mostofthelymphoidcellsareblasts Plateletsx109/L 390 200900 Reticulocyte%(uncorrected) 0.2 01.5

URINALYSIS(voided) Appearance Clear Colour Lightyellow Specificgravity 1.019 Protein ve Microscopicfindings:littleseen

pH Glucose Ketones Blood Bilirubin

6.0 ve ve ve Trace

191

INTERPRETATIONOFLABORATORYFINDINGS The polyuria and polydipsia could be due to renal/hepatic/endocrine disease. The pale mucous membranes could suggest anaemia. The spleno/hepatomegaly could be related to infiltrative disease.Atthisstage,theredoesnotseemtobeaspecificpatternemergingtoexplainalltheclinical signsinthischronicallyilldog. (a) Detected laboratory abnormalities: Moderate nonregenerative anaemia, mild leukopenia, moderate neutropenia with left shift, eosinopenia, circulating lymphoblasts (leukemia), moderate azotemia(urea),hyperphosphatemiaandhypercalcemia. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: Because the animal was leukemic (circulating lymphoid blasts), it was assumed that the non regenerative anaemia and neutropenia was primarily due to myelophthisis (conclusion and diagnosis). This was confirmed by bone marrow aspirates that showed numerous neoplastic lymphoidcellsandfewimmaturemyeloidorerythroidcells(furtherinvestigation).Theappearance of the immature erythroid and myeloid cells in the circulation was inappropriate but probably occurredduetodisruptionofthebonemarrowarchitecture.Thehepatomegalyandsplenomegaly were assumed to be due to neoplastic lymphoid infiltration. The clinical signs, nonregenerative anaemia, leukopenia and leukemic manifestation suggested that the prognosis was poor (implicationformanagement). Inaddition,thepresenceofhypercalcemiaandsuspectedrenaldisease(clinicalsigns,azotemiaand poorly concentrating urine) were further poor prognostic signs (conclusion and implications for management).Hypercalcemiacanoccurincasesoflymphoidneoplasia,andoccasionallyinmultiple myeloma. It is thought to be primarily due to the production of substances that enhance bone resorption (parathormonelike protein, prostaglandins, Vitamin Dlike steroid and osteoclast activatingfactorhencethenamepseudohyperparathyroidsm).Othertumourscanproducesimilar substancesinthedogsuchasapocrineglandadenocarcinomaoftheanalsacs.Commonly,inorganic phosphateisnotelevatedincasesofpseudohyperparathyroidismunlessthereissomeinterference in renal outflow. This appears to be the case in this dog and is probably due to the deposition of calciuminthekidneyinterstitiumandtubules.Sometumourscausehypercalcemiabymetastasising tothebonesandcausingosteolysis.Thismaybebydirectactionorbyactivatingosteoclasts.Itisstill referred to as pseudohyperparathyroidism. In these situations the inorganic phosphate may be elevated. Nb. Hypercalcemia may be transient or persistent. Only persistent hypercalcemia is important; therefore,ideallytwoormoreelevatedlevelsshouldbedetected. DIAGNOSIS AND POSTSCRIPT: The dog was euthanised rather than given chemotherapy The diagnosis of pseudohyperparathyroidism and hypercalcemic nephropathy associated with lymphosarcoma and lymphoid leukemia was confirmed at necropsy and subsequent histopathology.Abdominallymphnodeswerealsoaffected.

192

CASE35
ANIMAL:7yearoldmaleBoxer. PRESENTING COMPLAINTS: Lethargy, lack of exercise tolerance and inappetence in excess of one month. On presentation, the dog had pale mucous membranes, possible splenomegaly and hepatomegaly. LABORATORYRESULTS

HAEMATOLOGY SAMPLE REFERENCEINTERVAL Plasmaappearance Clear Clear PCVL/L 0.19 0.370.50 Plasmaproteing/L 50 5575 Haemoglobing/L 72 100150 12 Erythrocytesx10 /L 2.9 57 MCVfl 65 6075 MCHCg/L 379 300360 9 Leukocytesx10 /L(corrected) 58.5 712 9 Neutrophils(seg.)x10 /L 43.9 4.19.4 9 Neutrophils(band)x10 /L 5.4 00.24 9 Lymphocytesx10 /L 4.1 0.93.6 9 Monocytesx10 /L 4.8 0.21.0 Eosinophilsx109/L 0.3 0.141.2 9 Basophilsx10 /L 0 00.4 Bloodfilm:moderatepolychromasia,moderateacanthocytesandschistocytes (schizocytes),10NRBCper100leukocytes Plateletsx109/L 100 200900 Reticulocyte%(uncorrected) 5 01.5

193

INTERPRETATIONOFLABORATORYFINDINGS Achronicillnesswithhepato/splenomegalycouldsuggestinfiltrativedisease(neoplasticorchronic inflammatory).Thepalemucousmembranesandlackofexercisetolerancesuggestanaemia. (a) Detected laboratory abnormalities: Regenerative anaemia; mild hypoproteinemia; increased MCHC; marked leukocytosis (the leukocyte levels are always corrected for circulating nucleated erythroidcells[normoblasts]ifthenumbersexceed5per100leukocytes)duetoneutrophiliawith left shift, lymphocytosis and monocytosis; circulating normoblasts (NRBC), acanthocytes and erythrocytefragments(schistocytes);mildtomoderatethrombocytopenia. (b)&(c)Generalinterpretation,conclusions,furtherinvestigationandimplicationsformanagement: The regenerative anaemia (corrected reticulocyte count for the level of anaemia was 2.1%) was assumedtobeduetoblooddestructionratherthanbloodlossduetothepresenceofthecirculating nucleatederythroidcellsandthestrongneutrophiliawithleftshift(conclusion).Thelymphocytosis and monocytosis were considered the result of antigenic stimulation and erythrocyte destruction occurringoveraperiodoftime.Thepresenceofmoderatenumbersofschistocytesusuallyindicates a microangiopathic haemolytic process (fibrin strands laid down in small vessels damage erythrocytesintransit).Thiscanoccurinvasculitis,disseminatedintravascularcoagulation(dueto numerousmicrothrombiformationinsmallvessels),somesplenicdisordersandabnormalvascular proliferation (e.g. vascular tumours) (conclusion). Acanthocytes also occur in splenic and microangiopathicdiseases.Theyareoftencommoninhaemangiosarcoma(possiblediagnosis).The high MCHC was considered not to be due to laboratory error, but due to the presence of free haemoglobin(althoughthiswasnotdetectedgrosslyinplasma)relatedtolowgradehaemolysis. The low plasma protein did not fit in with blood destruction (more with blood loss), but was consideredtobeduetochronicdisease,inappetenceandprobableliverdisease. Further investigation could involve diagnostic imaging of the spleen and liver. Fine needle cell aspirationorbiopsymightbeconsidered. DIAGNOSIS AND POSTSCRIPT: Haemangiosarcoma of the spleen. The animal was investigated further by radiography and ultrasound. Multiple masses were detected in liver and spleen. A tentativediagnosisofneoplasiawasgiventotheownersandthedogwaseuthanased.Necropsyand histopathology revealed metastatic haemangiosarcoma. Haemangiosarcoma is one of the few diseases that commonly gives high levels of circulating nucleated erythroid cells. Other diseases include lead poisoning, erythroid neoplasia, other bone marrow disorders, occasional splenic disorders. In contrast to common causes of haemolytic anaemia, where the levels of nucleated erythroid cells are always proportional to the levels of reticulocytes, these diseases may give disproportionallevels.Inhaemangiosarcoma,forexample,theanaemiamaybenonregenerativeor regenerative, but circulating nucleated erythroid cells are common. This is probably due to the variable effects of this disease (it may cause some internal bleeding and/or it may cause bone marrowdepression).

194

INDEX

Abdominocentesisbovine,110 Abdominocentesisequine,110 Acid/basebalance,62 Acid/basedisturbances metabolicdisturbances,62 ActivatedClottingTime(ACT),96 ActivatedPartialThromboplastinTime(APTT),96 acutemyeloidleukemia,91 Acutephasereactants,94 Adrenalinsufficiency hormonalassays,55 RoutineLaboratoryfindings,55 Ammonia(blood),30 ammoniatolerancetest,30 fasting,30 Amylorrhea,45 Anemia,73 nonregenerative,73 regenerative,73 Anemiaofchronicdisease,78 Aniongap(AG),62 Antinuclearantibodytest(ANA),101 Anuria,33 Aplasticanemia,79 APTT,96 Autoimmunehemolyticanemia(AIHA),100 Autoimmunity,100 Azotemia,33 postrenal,35 prerenal,35 renal,35 Basophila,86 Basophilia,81 Basophilicstippling,74 Basophils,86 Bileacids,30 fasting,30 postprandial,30 Bilirubin,28 Fecalbilepigments,27 Urinebilirubin,27 Urineurobilinogen,27 bilirubinmetabolism,27 Bilirubinuria,40 Bleedingdisorder clottingfactordeficiency,96 excessfibrinolysis,97 plateletfunctiondefect,95 Thrombocytopenia,95 vesselwalldefect,95 BleedingDisorders,94

Bleedingtime(BT),96 BodyFLuidAnalysis,107 Bonemarrowexamination,77 M:E,78 Bonemarrowstoragepool,81 BSP,32 Horseandcattle,32 Calcium hypercalcemia,58 hypocalcemia,57 CalciumandPhosphateDerangements,57 Casts,42 cholestasis,26 chronicmyeloproliferativedisorders,92 Chylous,107 Chylouseffusions,108 Circulatingneutrophilpool(CNP),81 Clotretraction,96 Clottingfactors,93 Colic Abdominalparacentesis(horse),46 Corticosteroidinducedneutrophilia(leukocytosis), 82 Creatinine,35 CSFprotein,114 Cylindruria,33,42 DiagnosticCytology(exfoliativecytology,102 Ddimer,97 Defective(hypercellular)erythropoiesis,79 Diabetesmellitus,52 Diabetesmellitus(DM) intravenousglucosetolerancetest,53 ketones,53 persistenthyperglycemiaandglucosuria,53 Diagnosticcytology Solidtissuecytology,102 Diarrhea,45,47 dogandcat,47 horse,47 Disseminatedintravascularcoagulation(DIC),98 Dhlebodies,84 Electrolytestatus chloride,61 Na+,60 potassium,61 Enzooticbovinelymphosarcoma(EBL),89 Enzyme ALP,26 ALT,25 ARG,26 AST,26

195

GD,26 GGT,26 ICD,26 ID,25 LD,26 OCT,26 Enzymes,20 Eosinopenia,86 Eosinophilia,81,86 Eosinophils,86 Erythron,72 Exocrinepancreaticinsufficiency(EPI),48 extramedullaryhematopoiesis,72 exudates nonsepticexudate,108 septicexudate,108 Exudates,108 Eythrocyteproduction,72 FecalExamination,47 Fibrin(fibrinogen)degradationproducts(FDP),97 Fibrinogen,85 Fibrinolysis,97 Fineneedlecellaspirates,102 Folate,50 Glucosuria,39 Granulopoietin,81 hematologicalterms glossary,67 Hematopoieticneoplasia,88 Hematuria,41,42 Hemoglobinemia,76 Hemoglobinuria,41 Hemostasis normalmechanisms,93 Hepaticencephalopathy,31 Hereditarycoagulationdisorders,98 Hydrationstatus osmolality,60 hyperadrenocorticism urinecortisol/creatinineratio,55 Hyperadrenocorticism,54,82 Hormonalevaluation,54 PDHandadrenaltumourdifferentiation,55 Routinelaboratorytests,54 Hyperbilirubinemia,76 cat,29 Combined,29 dog,29 farmanimals,29 horse,29 Regurgitation(cholestatic),29 Retention,29 Hypercalcaemia casereport,192

Hypercalcemia,90 Hyperkalemia,61 hyperlipidemia,20 Hypermagnesemia,59 Hyperthyroidism,56 Hypoadrenocorticism(adrenalinsufficiencyAI), 55 Hypokalemia,61 Hypomagnesemia,59 Hyposthenuria,33,38 Hypothyroidism,56 Icterus,27 Icterus(Jaundice) Hemolytic,27 Immunemediated(related)diseases,99 Immunodeficiency,101 IndocyanineGreen,32 Insulinoma(hyperinsulinism),54 Isosthenuria,33,38 Ketonuria,40 lactate,blood,110 Leftshift,83 Leukemoidresponse,84 Leukocytosis,80 Leukopenia,81 lipemia,20 Liverdisease Cholesterol,32 Clottingfactors,31 Glucose,32 Lymphaticleukemia,88 Lymphocytes,87 Lymphocytosis,81,87 Lymphopenia(lymphocytopenia),87 Lymphoproliferativedisorder Acutelymphoblasticleukemia,89 chroniclymphocyticleukemia,89 lymphosarcoma,88 LymphoproliferativeDisorders,88 Malabsorption,45,49 Fatbalancedetermination(dogandcat),49 Oralglucosetolerancetest(OGTT),49 Postprandiallipemia(dogandcat),49 Xyloseabsorptiontest(XAT),49 Malassimilation,45 Maldigestion,45 Marginalneutrophilpool(MNP),81 Mastcellleukemia,92 Metabolicacidosis,62 Metabolicalkalosis,62 Monocytes,86 Monocytosis,81,86 Mucinclottest,111 Myelofibrosis,79

196

Myelophthisicanemia,79 Myeloproliferativedisorders,90 Myoglobinuria,41 Neoplasticeffusions,110 Nephroticsyndrome Hypercholesterolemia,43 Neutropenia,85 inflammatorydemand,83 Neutrophilia,81 Neutrophiliaduetoregenerativeanemia,82 Neutrophiliarelatedtoinflammatorydemand,82 Neutrophils toxicchanges,83 Nonregenerativeanemia,75,78 defectiveerythropoiesis,77 reducederythropoiesis,77 Nucleatedredcells,75 Oliguria,33,37 OneStageProthrombinTime(OSPT),97 Osmolality,38 OSPT,97 Pancreaticnecrosis,45 amylase,46 caninepancreaticspecificlipaseimmunoassay, 46 lipase,46 Pancreaticnecrosis(acutepancreatitis) dog,46 phosphorus hyperphosphatemia,57 PhysiologicalNeutrophilia(leukocytosis),81 Plasmacellmyeloma(multiplemyeloma),90 Pleocytosis,113 Polycythemia,79 Polyuria,33,37 Proteinlosinggastroenteropathy(PLG),50 Proteinuria,39 UrinaryProteintoCreatinineRatio,39 Pseudochylouseffusion,108 Pseudomacrocyticanemia,75 Redcellindices,75 Reduced(hypoproliferative)erythropoiesis,78 ReferenceInterval,18 Regenerativeanemia,74 Bloodloss,76 hemolytic,76 reticulocytecount,74 Renalclearancetests,34 creatinine,34 Sodiumsulfanilate,35 Renalfailure acute,34 chronic,34

farmanimals,34 Hyperamylasemia,44 hypercalcemia,44 Hyperkalemia,44 Hyperphosphatemia,44 hypokalemia(cats),44 hyponatremiaandhypochloridemia,44 hypophosphatemia,44 Metabolicacidosis,43 Nonregenerativeanemia,43 Renaltubularacidosis,36 Respiratorywashes,114 Reticulocytecount correction,74 Reticulocytes aggregate,74 Reticulocytosis anisocytosisandpolychromasia,75 RheumatoidarthritisRA,101 Rheumatoidfactor(RF),100 Serumprotein,31 Serumproteins acutephasereactants,31 Fibrinogen,31 globulins,31 hypoalbuminaemia,31 SLE LEcelltest,101 Steatorrhea,45 Synovialfluid,111 Normalcytologicalcharacteristics,111 Normalgrosscharacteristics,111 Othercharacteristics,111 SynovialFLuidAnalysis,111 Synovialfluidchangesduetodisease,111 Systemiclupuserythematosus(SLE),101 TCT,97 ThrombinClottingTime(TCT),97 Thrombocytopathies,95 Thrombocytopenia,95 Tissueimprints,102 Trypsinlikeimmunoreactivity(TLI),48 Urea,35 Uremia,33 Urinalysis,34,36 PhysicalProperties,37 Sedimentexamination,41 SoluteConcentration(specificgravity),38 UrinepH,40 Aciduria,40 VitaminB12,50 VonWillebrand'sdisease,95 Xanthochromia,113

197

PRACTICAL NOTES

199

200

t y

V e

t e

r i n a r

c i

e n

VETERINARY CLINICAL PATHOLOGY

VETS 3243
Practical Notes and Information on Techniques

Developed by:

Professor Paul J Canfield & David LP Griffin (Professional Officer) 1988, revised 2009

201

PREFACE
These notes have been designed for veterinary undergraduates but will have application for

laboratory medicine in veterinary practice. Only simple laboratory techniques are described, with some theory given where appropriate. The practical notes complement the previously provided lecture notes but can also stand on their own. Although most of the laboratory techniques are standard and will change little with time, others will need to be upgraded. This revision does occur regularly, but sometimes inadvertently certain laboratory techniques are the

overlooked.

Consequently,

readers assistance is required in detecting errors or flaws in the presented techniques.

202

TABLE OF CONTENTS
SAFETY IN THE LABORATORY .............................................................................................205 SUMMARY OF PRACTICAL CLASSES....................................................................................207 ACTIVITIES 1, 3 & 5 CASE REPORT ANALYSIS TUTORIALS ...................................................208 ACTIVITY 2 PRACTICAL CLASS - HAEMATOLOGY ..................................................................220 ACTIVITY 4 PRACTICAL CLASS A. BIOCHEMISTRY ..............................................................224 ACTIVITY 4 PRACTICAL CLASS B. URINALYSIS ...................................................................225 ACTIVITY 6 PRACTICAL CLASS - CYTOLOGY.........................................................................227 REQUEST SHEETS AND COLLECTION OF SAMPLES FOR CLINICAL PATHOLOGY .....................229 1 INTRODUCTION ..........................................................................................................................229 2 REQUEST SHEETS .....................................................................................................................229 3 COLLECTION OF SAMPLES .......................................................................................................229 4 WHAT TO DO IF TAKING SPECIMENS FOR LABORATORY EXAMINATION AFTER HOURS..236 5 INFORMATION ON MEASUREMENT UNITS AND ENZYMES....................................................238 6 CONVERSION OF OLD ENZYME UNITS TO INTERNATIONAL UNITS (IU) .............................239 7 TEMPERATURE CONVERSION FACTORS FOR ENZYMES TO 37C ......................................239 8 CONVERSION FACTORS............................................................................................................240 9 REFERENCE RANGES FOR BIOCHEMISTRY.........................................................................242 10 LOCATION OF CLINICAL PATHOLOGY CLASSROOM AND VPDS LABORATORIES IN MCMASTER B14 ..243 HAEMATOLOGY PRACTICAL NOTES.....................................................................................245 1 INTRODUCTION ..........................................................................................................................245 2 SOME ANTICOAGULANTS .........................................................................................................245 EDTA ...........................................................................................................................................245 Heparin ........................................................................................................................................245 Sodium Citrate .............................................................................................................................245 3 ERYTHROCYTE ANALYSIS ........................................................................................................246 a) The Microhaematocrit (PCV) ..................................................................................................246 b) Haemoglobin Estimation Using the Cyanmethaemoglobin Method ........................................246 c) Erythrocyte Count - (Unopette) ...............................................................................................247 d) Erythrocyte Indices .................................................................................................................248 e) Reticulocyte Count..................................................................................................................249 f) Coombs Antiglobulin Test for Autoimmune Haemolytic Anaemia (AIHA) ..................................250 a) Leukocyte Count (Unopette method)......................................................................................251 b) The Peripheral Blood Smear (Film) ........................................................................................251 c) Total Eosinophil Count (Direct Method) ..................................................................................256 4 LEUKOCYTE ANALYSIS.............................................................................................................251 a) Leukocyte Count (Unopette method)......................................................................................251 b) The Peripheral Blood Smear (Film) ........................................................................................251 5 LABORATORY EVALUATION OF HAEMOSTASIS .....................................................................257 a) Bleeding Time.........................................................................................................................257

203

b) Clotting Time...........................................................................................................................258 c) Clot Retraction ........................................................................................................................259 d) Fibrinogen Estimation .............................................................................................................260 e) Prothrombin Time (ProT, PT or OSPT)...................................................................................261 f) Partial Thromboplastin Time (PTT)..........................................................................................262 g) Platelet (Thrombocyte) Count.................................................................................................263 h) Fibrin (fibrinogen) degradation products including D-dimer .................................................265 6 MISCELLANEOUS PROCEDURES .............................................................................................266 a) Bone Marrow Examination......................................................................................................266 b) Cross-matching Blood for Transfusion....................................................................................267 c) Blood typing for cats and dogs................................................................................................268 d) The Blood/Erythrocyte Sedimentation Rate (BSR:ESR).........................................................269 e) The Lupus Erythematosus (LE) Cell Test (For Systemic LE) .................................................270 7 REFERENCE RANGES FOR HAEMATOLOGY...........................................................................271 8 THE HAEMOCYTOMETER ..........................................................................................................272 U R I N A L Y S I S ................................................................................................................277 1 INTRODUCTION ..........................................................................................................................277 2 OBSERVATION OF PHYSICAL PROPERTIES ...........................................................................277 3 ESTIMATION OF SOLUTE CONCENTRATION...........................................................................277 4 CHEMICAL ANALYSIS.................................................................................................................277 5 SEDIMENT EXAMINATION..........................................................................................................279 C Y T O L O G Y ...................................................................................................................281 1 SOLID TISSUE CYTOLOGY (SURFACE LESIONS, BIOPSIES, AUTOPSY MATERIAL)...........................281 a) Tissue impression (imprint). ................................................................................................281 b) Tissue scraping. ..................................................................................................................281 c) Fine needle aspirate............................................................................................................281 2 FLUID CYTOLOGY (CSF, synovial, body cavity effusions, tracheal aspirate, fluid masses)........283 3 VAGINAL CYTOLOGY .................................................................................................................283 4 SEMEN ANALYSIS ......................................................................................................................284 F A E C A L A N A L Y S I S ...................................................................................................285 1 INTRODUCTION ..........................................................................................................................285 2 GROSS EXAMINATION ...............................................................................................................285 3 MICROSCOPIC EXAMINATION ..................................................................................................285 4 HEMATEST - FOR OCCULT BLOOD...........................................................................................287 5 EXAMINATION FOR TRYPSIN (PROTEASE) .............................................................................287 6 FAECAL STAIN ............................................................................................................................288 U R I N A R Y C A L C U L I ...................................................................................................289 1 INTRODUCTION ..........................................................................................................................289 2 PRACTICAL EXAMINATION OF CALCULI ..................................................................................291 a) Physical examination ..........................................................................................................291 b) Chemical Analysis (Based on the Merck Urinary Calculus Analysis) ................................292

204

SAFETY IN THE LABORATORY

Safety is important in the laboratory. It is designed to protect you, other members of your class, and staff. It forms part of your education in being an effective practitioner. If you injure yourself in practice through poor safety procedures then you lose time and often income! If learnt now, it should carry over into your professional life.

Personal safety is your own responsibility

The regulations in this laboratory are:

1 2 3 4 5 6 7 8

Laboratory coats to be worn at all times; gloves to be worn when handling biological samples No smoking, drinking or eating in laboratory and/or corridor Adequate protective footwear i.e. non slip, and fully enclosed footwear. No pipetting by mouth Washing of hands after practical classes No improper use of safety or fire fighting equipment No horseplay or reckless behaviour in the laboratory Never run in the laboratory or along corridors. Care in handling materials a) All material should be treated as infective b) Needles to be sheathed at all times c) Other sharp objects to be disposed of after use d) Broken glassware disposed of e) Used slides disposed of All material should be labelled correctly Do not leave bags in corridors where people can trip over them Care with bunsen burners and heating plates Care with water near electrical equipment Care with animal handling Report all accidents to the member of staff

9 10 11 12 13 14

Some of these sound trivial, but they all play a role in protecting yourself from serious injury and disease. Should the fire alarms sound, the building is to be evacuated immediately. The member of staff present will show you how to leave. Do not panic!

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Personal safety is your own responsibility. Check that any glassware that you are to use is not broken or cracked. Long hair should be tied or held back. By this time, you should all have had tetanus and hepatitis injections and necessary boosters. Also, you should have had a Mantoux test and, if negative, a B.C.G.

General Laboratory Safety Rules

Never adopt a casual attitude in the laboratory and always be conscious of the potential hazards. Always wear eye protection when in the laboratory area. Always exercise care when opening and closing doors and entering or leaving the laboratory. Always use a fume cupboard, fume cabinet or glove box when working with highly toxic, volatile or odoriferous substances. Do not work in isolation in a laboratory, ensure that at least a second person is with in call. Do not store food or drink in a refrigerator which is used to store laboratory materials. Regard all substances as hazardous unless there is definite information to the contrary. Never undertake any work unless the potential hazards of the operation are known as precisely as possible, and the appropriate safety precautions are adopted. Take additional care when carrying any potentially hazardous substances. Maintain the minimum required quantities of hazardous substances in the laboratory work area. Wash skin areas which come in contact with chemicals, irrespective of concentration. Keep all fire escape routes completely clear at all times. Ensure that all safety equipment remains accessible to the laboratory personal at all times. Clean up spills immediately.

206

SUMMARY OF PRACTICAL CLASSES

SEMESTER 6 Practical Classes/Tutorials
One two hour session per week: Tuesdays 11am-1pm, Weeks 1-12 inclusive Half the students per session, divided into three groups for three separate activities (Groups A-F) Group allocations will be posted on the Clinical Pathology Sydney eLearning site on Monday of Week 1. Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Activity1 GroupA D B E C F Activity2 B E C F A D Activity3 C F A D B E Activity4 GroupD A E B F C Activity5 E B F C D A Activity6 F C D A E B

Topic and site for Activity Activity 1: Tutorial (case report analysis) - Seminar room 223, Level 2 Gunn Building B19 Activity 2: Haematology Practical Class CP Lab Room 141, McMaster Building B14 Activity 3: Tutorial (case report analysis) Seminar room 353, Level 3 Gunn Building B19 Activity 4: Biochemistry/Urinalysis Practical - CP Lab Room 141, McMaster Building B14 Activity 5: Tutorial (case report analysis) Seminar room 223, Level 2 Gunn Building B19 Activity 6: Cytology Practical/Tutorial Level 4 Lab A, Gunn Building B19

Note: The three case report tutorials will each be marked out of 10% (thenanaveragedetermined).Thethreepracticalsmustbeattendedfor studentstobeeligibleforthefull10%(seesummativeassessment).

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ACTIVITIES 1, 3 & 5 CASE REPORT ANALYSIS TUTORIALS

INTRODUCTION TO INTERPRETATION OF LABORATORY RESULTS The interpretation of laboratory results is an integral part of the diagnostic process utilised in clinical medicine. Diagnosis of disease, in its simplest form, relies on the three Ds: Detect, Describe, Deduce. The first two Ds are objective and rely on an understanding of normal structure and function (therefore, able to detect abnormalities) and a development of descriptive powers. The last D, namely deduce, is partly subjective as it is influenced by ones past knowledge and experiences, but relies heavily on an integrative, sequential and common sense approach to interpreting abnormalities. Evidence-based interpretation (support for all conclusions, hunches and suspicions) can diminish the subjective impact on the deductive process. In a sense, the diagnostic process is akin to a detective story with clues being utilised effectively to reach evidence-based convictions. Every practicing veterinarian has a bit of the vetective in them and searches for clues that can be integrated to reach a diagnosis. Laboratory tests are just one group of diagnostic aids available to the practicing veterinarian. Their usefulness is enhanced by careful choice based on clinical detection and deduction and by careful interpretation based on all other available information of the case. The three Ds equally apply to the use and interpretation of laboratory tests. It is a logical process based on the detection and description of laboratory abnormalities, and then an interpretative (deductive) process drawing on all other information available to you about the case. The interpretive process is commonly one starting from the general then working to the specific (i.e. think of general disease processes before considering specific diseases). Remember also, laboratory results should not be interpreted in isolation of other case information. Once laboratory results are interpreted, then there is often a need for further investigation to support the conclusions. This may lead to complex further investigations, such as MRI to support abnormalities detected on CSF analysis, or simple further investigations, such as returning to the owner and asking them whether the animal had exposure to chemicals that may have contributed to the liver disease detected by elevations in liver enzymes. The plan for further investigation must be primarily based on the main conclusions. Apart from assisting in drawing conclusions and a plan for further investigation, laboratory results often have implications for management of the case while undertaking further investigation, for example detected electrolyte disturbances for fluid therapy. This session introduces the concepts behind the use of laboratory tests in the diagnostic process and will provide a technique to detect, describe and deduce. It will also emphasise the use of laboratory test interpretation in planning for further investigation and disease management while doing so. SUGGESTED TECHNIQUE FOR INTERPRETATION OF LABORATORY RESULTS AND LINKAGE WITH THE OVERALL DIAGNOSTIC PROCESS Think about the information given before looking at the laboratory data. Ask yourself what can be deduced from the history, signalment and clinical findings: Is there an indication of time course? Is age, sex or breed important? What organs or tissues are involved? Is there an indication of the pathological process(es) occurring? Is there an indication of cause?

208

Keep your deductions in mind when interpreting the laboratory data and thinking about further investigation 1. LIST/HIGHLIGHT ABNORMALITIES (DETECT AND DESCRIBE) This is for your benefit to ensure you dont forget to discuss any abnormalities. Remember to use the information on what is regarded as mild, moderate and marked alterations (see the beginning of the Section on Case Reports). 2. GENERAL INTERPRETATION (DEDUCE) Consider the reasons for abnormalities. Do not give complete lists of reasons but only those which could relate to the case. Relate to information (history etc) given to you in the question. You will use this information in reaching conclusions. This can be incorporated in your conclusions if your prefer (i.e. give a conclusion and then add your reasons for reaching it)

3. CONCLUSION(S)/FURTHER INVESTIGATION/IMPLICATIONS FOR MANAGEMENT? (DEDUCE) This is most important and MOST MARKS will come from this section. A. Conclusions These can be in the form of questions or statements (e.g. is the regenerative disease due to blood loss or blood destruction? OR the animal has regenerative anaemia due to either blood destruction of blood loss) Think about whether there appears to be a main problem. Remember, there may be more than one. The main problem may be presented as organ/tissue dysfunction (e.g. liver disease), a general disturbance/pathological process (e.g. regenerative anaemia; inflammation) or possibly a specific disease (e.g. acute pancreatic necrosis in a dog). Can you explain all the abnormalities in light of the main problem(s)? Dont forget about what you deduced from the given animal and clinical information when you are reaching your conclusions. B. Further investigation Often you will need to investigate the case further. This should be logical and based on your conclusions reached from analysing case history and laboratory data. It is important to prioritise investigation i.e. consider what you think is the main conclusion when suggesting further investigation. A checklist for further investigation might include: Do you need to get more history, and if so what? Do you need to recheck some clinical signs or repeat physical examination? Do you need to undertake specialist clinical procedures (e.g. ECG, EMG)? Do you need to undertake image analysis? What modality would be most useful? Do you wish to undertake more laboratory testing? Sometimes you may consider a response to treatment as part of your further investigation to reach a diagnosis, but this can sometimes be misleading unless you verify your response with further testing. So, you need to state how you would support any response to therapy approach.

209

Sometimes you can sequence your further investigation on the basis of probable findings e.g. I wish to undertake hepatic ultrasound. If I find any masses on ultrasound, I would wish to take an FNA or biopsy. C. Implications of the results for your approach to treatment and prognosis (i.e. management of the case while you are investigating it further). You may not wish to undertake any treatment while investigating the case further. In some situations you may wish to undertake treatment after asking for more tests (e.g. electrolyte analysis for possible fluid therapy).

REMEMBER,notallabnormalitiesmaybeexplicableinlightofthecase. Be honest, if you dont know, say so BUT think about how you would find out. Also, put the abnormalities in perspective. Some mild abnormalitiesmaynotbeveryimportantforthecase.

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CASE1:3yearoldmaleneuteredDSHcatcalledMory
BACKGROUND
Moryhadbeenobtainedbyhisownersasakitten.Therewere2othercatsinthehousehold both of whom were healthy. Mory had been neutered at 6 months of age and had been vaccinatedregularlyagainstFIE,FCVandFHV.Hehadfreeaccessoutsideandwasknownto beahunter.Hewasfedtinnedcatfood.

HISTORYOFILLNESS
Threemonthsbeforereferraltheownershadnoticedswellingofhislowerlimbs,faceand ventralabdomen.Thishadresolvedfollowingtreatmentwithfrusemide(adiuretic).Some weightlosshadbeennoticedsincethenandhewasinpoorcondition.Theswellinghadnow recurredandhewasreferredatthisstage.

CLINICALANDPHYSICALEXAMINATIONFINDINGS
He was bright but in poor condition, thin with a dull coat. Rectal temperature was 38.6 degreesCandheartrate160bpm.Pittingsubcutaneousoedemawaspresentaffectingthe distallimbs,faceandventralabdomen.Hisabdomenappearedtobeslightlydistendedand therewasasuspicionofafluidthrill.

LABORATORYRESULTS

Haematology
TEST Hb PCV RBC Platelets WBC Neutrophils Lymphocytes Monocytes Eosinophils TEST Totalproteins Albumin Globulins Urea Creatinine ALT ALP Sodium Potassium Calcium Cholesterol RESULT 48 0.23 7.3 310 27.8 24.7 1.7 1.1 0.3 UNITS g/L L/L X1012/L X109/L X109/L X109/L X109/L X109/L X109/L REFERENCEINTERVAL 80140 0.300.45 5.510 300700 814 3.7610.8 1.67 0080.56 0.161.4

SerumBiochemistry
RESULT 60.9 16.0 44.9 13.7 207 4 21 150 4.1 1.8 9.7 UNITS g/L g/L g/L Mmol/L mol/L IU/L IU/L mmol/L mmol/L mmol/L mmol/L REFERENCEINTERVAL 5473 1938 2651 7.210.7 90180 <60 <50 147156 4.04.6 1.752.6 1.93.9

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Urinalysis(cystocentesis)
Appearance:slightlycloudy Colour:darkyellow Urinespecificgravity:1.050 Protein:34+ pH:7.0 Otherchemistriesondipsticknegative Urinarysediment:occasionaltransitionalepithelialcellsandlownumberoftriplephosphate crystals Urinecreatinine:6.4mmol/L(=72.4mg/dl) Urineprotein(microproteinassay):8.84g/L(884mg/dl) UP/UCratioisover12(8.84/6.4Xbyconversionfactorof8.839ifworkinginSIunits)

212

CASE2:5yearoldmaleOldEnglishSheepdogcalledOscar

BACKGROUND
Oscar had been obtained as a puppy. The owners had no other pets. He was regularly vaccinated.Hisdietconsistedlargelyofcannedfoodsupplementedwithtablescraps.

HISTORYOFILLNESS
Oscars owners noticed that he had become less interested in exercise over the previous two weeks and become very reluctant to walk more than a short distance. He was quite lethargic.

CLINICALANDPHYSICALEXAMINATIONFINDINGS
Oscarappearedreasonablybrightbuthismucusmembraneswerepale.Rectaltemperature was38.2degreesCandheartrate135bpm.HispulsewasstrongandCRTunder2seconds. Asystolicmurmurwasaudibleloudestovertheleftapex.Heappearedtoresentabdominal palpationalthoughpaincouldnotbelocalised.

LABORATORYRESULTS

Haematology
TEST Hb PCV RBC MCV MCHC Platelets WBC Neutrophils BandNeutrophils Lymphocytes Monocytes Eosinophils Nucleatedredblood cells RESULT 74 0.21 2.6 80 352 220 24.6 19.3 0.6 3.4 0.7 0.3 0.4 UNITS g/L L/L x1012/L FL g/L x109/L x109/L x109/L x109/L x109/L x109/L x109/L x109/L REFERENCEINTERVAL 100150 0.370.55 57 6075 300350 200600 712 4.069.36 00.24 0.913.6 0.210.96 0.141.2 00.1

Bloodfilm:moderatepolychromasiaandmarkedanisocytosis.Therearenucleated erythroidcellsandsomebasophilicstipplingofpolychromatophiliccells.Someerythrocytes lackcentralpallor,butthismaybepartlyartefactual.

SerumBiochemistry
TEST Totalproteins Albumin Globulins Urea Creatinine ALT ALP RESULT 78.8 33.6 45.2 8.3 161 89 143 UNITS g/L g/L g/L mmol/L mol/L IU/L IU/L REFERENCEINTERVAL 5070 2343 2744 110 40120 <60 <110

213

GGT Totalbilirubin Sodium Potassium

5 5 146 3.8

IU/L mol/L mmol/L mmol/L

0.68.2 1.28.1 137150 3.34.8

Urinalysis(voided)
Appearance:clear Colour:darkyellow Urinespecificgravity:1.045 Proteins:Trace(0.13g/L) pH:6.5 Bilirubin:+ Blood:ve Glucose:ve Ketones:ve Urinarysediment:12leukocytesperHPF,occasionalsquamousandtransitionalcells.

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CASE3:9yearoldmaleneuteredLabradorcalledPoacher

BACKGROUND
Poacher had been obtained as a puppy. The owners had no other pets. He was regularly vaccinated.Hisdietconsistedlargelyofcannedfoodsupplementedwithtablescraps.

HISTORY
Three days before presentation to our clinic, Poacher became anorectic and started vomiting 23 times every day. The vomit had a liquid consistency, a yellow colour and containednofoodmaterial.

CLINICALFINDINGS
Poacherwasmarkedlyjaundicedbutbright.Rectaltempwas38.0degreesCandheartrate 120bpm.

LABORATORYRESULTS

Haematology
TEST RESULT UNITS g/L L/L x1012/L FL g/L x109/L x109/L x109/L x109/L x109/L x109/L x109/L x109/L Hb 161 PCV 0.45 RBC 6.87 MCV 65.1 MCHC 334 Platelets 341 WBC 10.8 Neutrophils 5.62 BandNeutrophils 0.11 Lymphocytes 2.27 Monocytes 1.08 Eosinophils 1.73 Basophils 0 Bloodfilm:unremarkable TEST Totalproteins Albumin Globulins Urea Creatinine ALT ALP GGT Cholesterol Totalbilirubin Amylase Lipase RESULT 75.9 38.2 37.7 3.2 91 535 2065 85 11.5 49 2756 400 REFERENCE INTERVAL 100150 0.370.55 57 6075 300360 200600 712 4.069.36 00.24 0.913.6 0.210.96 0.141.2 00.36

SerumBiochemistry
UNITS g/L g/L g/L mmol/L mol/L IU/L IU/L IU/L mmol/L mol/L IU/L IU/L REFERENCE INTERVAL 5070 2343 2744 110 40120 <60 <110 0.68.2 1.47.5 1.28.1 <1400 <60

215

216

CASE4:Adult(1315yrs)geldedmalethoroughbredhorsecalledTrojan

BACKGROUND
Trojanhadbeenownedfortwoyears.Beforethattimehehadhadvariousowners.Hewas boughtforrecreationalridingontheweekends,butinthepastsixtoninemonthshadbeen reluctanttotrotorgallop.

HISTORY
Trojan had been increasingly losing weight for more than 3 months and the owner had noticedadecreaseinfoodintakeandanincreaseinwaterintake.Trojanwaskeptinalarge paddockwithoneotherhorsethatwasingoodhealth.

CLINICALFINDINGS
Onexamination,Trojanwasinpoorconditionwithalossofmusclemassandadrycoat.The horse could have possibly been dehydrated with slightly sunken eyes. Vital signs (HR. Temperature,RR)werewithinreferenceintervals.

LABORATORYRESULTS

SerumBiochemistry
TEST ALPIU/L GGTIU/L Serumprotein(refract.)g/L Albumin(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L globulins(EPG)g/L Glucosemmol/L Ureammol/L Creatininemol/L Calciummmol/L Inorganicphosphatemmol/L Sodiummmol/L Potassiummmol/L Chloridemmol/L RESULT 96 18 63 27.1 11.3 15.1 9.6 5.6 98 1398 3.7 2.1 118 4.8 86 REFERENCEINTERVAL <210 5.622 5376 2836 813 815 714 3.36.1 3.66.5 110170 2.83.4 12.3 131147 2.14.8 99108

Haematology
TEST Plasmaappearance PCVL/L Plasmaproteing/L Hemoglobing/L Erythrocytesx1012/L MCVfl MCHCg/L Leukocytesx109/L RESULT Clear 0.38 66 135 6.4 59 355 11.9 REFERENCEINTERVAL Variable 0.320.53 5578 130160 811 4149 300360 6.512

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Neutrophils(seg.)x109/L Neutrophils(band)x109/L Lymphocytesx109/L Monocytesx109/L Eosinophilsx109/L Basophilsx109/L Bloodfilm:normal

9.6 0 2.1 0.2 0 0

2.57 00.2 1.65.4 00.7 0.21 00.4

Urinalysis
(voidedsample)
Appearance Cloudy pH Colour Brown Glucose Specificgravity 1.014 Ketones Protein 1+ Blood Bilirubin Microscopicfindings:muchmucinandcalciumcarbonatecrystals 7.3 ve ve ve ve

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TUTORIAL STRATEGY for approach to the technique for interpretation of laboratory results and linkage with the overall diagnostic process:
1. Work individually to look at clinical information and identify laboratory abnormalities (about 5-10 minutes) 2. Pair up and discuss possible reasons for laboratory abnormalities (about 10-15 minutes) 3. Form two groups and see if you can reach some (A) conclusions, (B) what further investigation may be necessary and, (C) any implications for management of the case (about 15-20 minutes). One person should keep a record of this. 4. The two groups come together and a scribe from each writes the main conclusions (in order of importance), any further investigation and any implications for case management onto the whiteboard. Everyone will read and reflect on the other groups statements before discussing any differences. Hopefully, a consensus will be reached and a plan agreed on by the end of this session (about 20-30 minutes).

219

ACTIVITY 2 PRACTICAL CLASS HAEMATOLOGY

1. Demonstration of PCV, TPP and peripheral blood smear preparation. Demonstration of automated haematology analyser. 2. Perform microhaematocrit on sample. (Check buffy coat for platelets and leukocytes.) 3. Determine total plasma protein (TPP). 4. Make blood smear and stain with "Diff-Quik". 5. Perform differential leukocyte count on your smear and also note number of normoblasts per 100 leukocytes as well as the morphology of the erythrocytes, leukocytes and platelets. 6. Calculate absolute values from differential and provided leukocyte count. 7. Calculate erythrocyte indices from provided erythrocyte count and haemoglobin, and own PCV. 8. Clean microscope and work area. 9. Check your results against the laboratory's results. N.B. Total cell counts you will be supplied with values of leukocytes, erythocytes and platelets for the practical class Staining Smears Diff-Quik Fix over 30 secs in methanol, then 5 one second dips in each of the two following stains, rinse in tap water. Do not rinse between stains. Giemsa Fix for 2 mins. with stain (1 vol). Add 1 vol buffer, mix then allow to stain for 7 mins. Rinse with tap water.

220

HAEMATOLOGICALRESULTSHEETFORACTIVITY2PRACTICALCLASS
Animal.............................. Age..............Sex................ I.D.................................... Results:

221

MICROHAEMATOCRITSCALE

222

HAEMATOLOGICALREFERENCEINTERVALS
VeterinaryPathologyDiagnosticServices FacultyofVeterinaryScience UniversityofSydney PCV Plasma protein Haemoglobin Erythrocytes MCV MCHC MCH Fibrinogen Leukocytes Neutrophils Lymphocytes Monocytes Eosinophils Basophils Platelets Reticulocytes Theremaybemarkedvariationsinnormalvaluesduetoage,sex,breedanduse.Theseare crudeapproximationsonly.Theyarenotnecessarilyapplicabletoresultsfromother laboratories.
*

L/L g/L g/L x10 /L fl g/L pg g/L x10 /L Seg.cells/10 /L

9 9 9 12

DOG 0.370.55 5575 100150 57 6075 300350 2025 24 712 4.069.36 0.913.6 0.210.96 0.141.2 00.36 200900 01.5

CAT 0.300.45 5978 80140 610 4045 310350 1317 13 814 3.7610.08 00.42 1.67.0 0.080.56 0.161.4 00.14 300700 01.0

HORSE (adult) 0.320.52 5884 110160 811 4149 300360 1316 24 6.013.0 2.476.96 00.24 1.65.4 0.00.72 0.160.96 00.36 100300 0

bandcells/10 /L 00.24 cells/10 /L cells/10 /L cells/10 /L cells/10 /L x10 /L %

9 9 9 9 9

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ACTIVITY 4 PRACTICAL CLASS

A. BIOCHEMISTRY
1) 2) 3) 4) Perform glucose estimation using the dextrostix reagent strips. Perform urea estimation using the azostix reagent strips. Perform glucose estimation using the glucometer or equivalent. Demonstration of biochemical analysis using equipment available in practice such as Vetscan and Vetest.

BIOCHEMICAL ANALYSIS RESULT SHEET FOR ACTIVITY 4

Animal............................ Age................................. Sex.................................. I.D. ................................

Results: GLUCOMETER.......................................... DEXTROSTIX............................................. AZOSTIX.....................................................

Comments: ...................................................................................................... .................................................................................................... ...................................................................................................... .....................................................................................................

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ACTIVITY 4 PRACTICAL CLASS

B. URINALYSIS
See theory and practical notes on urinalysis. 1. Perform biochemical analysis on urine using: Multistix 10SG for 1) Glucose 2) Ketones 3) Blood 4) PH 5) Protein 6) Bilirubin Use refractometer for specific gravity (dont rely on the Multistix). Use SSA to confirm protein on the Multistix 2. Examine centrifuged sediment

225

URINALYSISRESULTSHEETFORACTIVITY4PRACTICALCLASS
Animal........................... Age.............................. Sex.............................. I.D............................. Results:
URINE ROUTINE Appear Colour Sp.Gr Protein MICROSCOPIC Catheterised ANALYSIS EXAMINATION Voided Cystocentesis

Biochemical pH Glucose Ketones Blood Bilirubin CENT

UNCENT

Comments:
....................................................................................................................... ....................................................................................................................... ....................................................................................................................... .......................................................................................................................

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ACTIVITY 6 PRACTICAL CLASS CYTOLOGY

See cytology theory and practical notes. A demonstration will be provided of techniques to collect, prepare and stain cytological samples. 1) Perform imprints, scrapings and fine needle aspiration on tissue supplied 2) Make smears 3) Stain by Diff Quik 4) View slides

227

CYTOLOGICALRESULTSHEETFORACTIVITY6PRACTICALCLASS

Comments:
....................................................................................................................... ....................................................................................................................... ....................................................................................................................... .......................................................................................................................

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REQUEST SHEETS AND COLLECTION OF SAMPLES FOR CLINICAL PATHOLOGY

1 INTRODUCTION
The way samples are collected, and in what, will vary depending on the laboratory and what tests they employ. Therefore, familiarisation with individual laboratory requirements is an essential prerequisite to the collection of samples. Private laboratories will provide you with this information and do not expect you to remember it but do expect you to review it when taking samples. The list below is a summary of what request sheets and samples are required for tests performed by the Veterinary Pathology Diagnostic Services (VPDS) in The Faculty of Veterinary Science. Although many samples are acceptable, preferences are indicated. VPDS provides a service to external practices but also for the University Veterinary Teaching Hospital at Sydney (UVTHS). UVTHS employs the AIS (Animal Intelligence System) online package for tracking and retrieving animal cases.

2 REQUEST SHEETS
All request sheets are to be clearly labelled with the UVTHSs AIS animal number (no name) and the owners name (no number) and the registry number. The UVTHS label covers all this signalment. At present there is a single request sheet for most of the tests except for endocrine tests. Case information should be given in the space provided on the request sheets when interpretation is required. It is important that the clinician's initials are on the request sheet as well as your own. This is for two main reasons, firstly, the sample may be inappropriate and will need to be recollected e.g. unfortunate clotting of a sample for haematology; secondly, the results may need to be communicated immediately if one or more values are outside the reference range and the animal requires immediate attention. Reference intervals (normal values or range) are expressed with the results in the AIS report. These apply to this laboratory only, although they can be used as a rough guide for figures received from other laboratories. Reference intervals vary between laboratories due to the type of test performed, the conditions of the test and the way the test is performed.

3 COLLECTION OF SAMPLES
A result is only as good as the material that is presented to the laboratory. Use the correct type of container for the analyte to be examined. Do not go by the colour of the top but by the worded label of the container. Make sure that the container is mixed quickly and gently after collection. Specimens should reach the laboratory as quickly as possible and may not be processed the same day unless received by 12.30 pm. The specimen(s) with the request form(s) should be left on the bench in the VPDS Clinical Pathology laboratory in the McMaster Building.

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All containers, tubes and swabs (two from each site, one for gram stain, one for culture) must be labelled with the animals AIS number and the owner's name (i.e. 51165; Mr MacGregor: not Peter, Flopsy, Mopsy or Cottontail). During out-of-hours, many specimens need to be processed without delay. This processing should be done by the intensive care/weekend duty students. In particular, blood smears should be made immediately and heparin blood samples should be centrifuged within two hours and plasma removed into a plain blood tube. These samples are stored in the pharmacy or near the blood-gas machine in the refrigerator. The sample(s) must then be taken to Clinical Pathology the following morning. For details regarding processing required for samples collected after-hours (see Section 4).

Samples and Safety

Often syringes with needles attached are sent off to the Clinical Pathology Laboratory for processing. While it is highly desirable for the needle to be removed to avoid needle stick injuries, some samples, as in the case of Fine Needle Aspirates, may contain vital diagnostic material still in the needle. For this purpose special plastic containers have been provided for the transport of these potential dangerous items. Please use these containers for the transport of these items. These containers can also be used for the transport of slides as well to prevent the slides falling onto the ground or getting wet. Please note that this procedure, of leaving needles on syringes, is not acceptable in sending samples to outside pathology laboratories and in some case they may refuse to accept the sample. To help with diagnosis the laboratory offers some profiles. Basic small animal profile (Profile A) (70016 Biochem Profile A) Amylase, Alkaline Phosphatase (ALP), Alanine Aminotransferase (ALT, GPT), Creatine Kinase (CK, CPK), Protein, Albumin, Globulins, Cholesterol, Glucose, Total Bilirubin, Creatinine, Urea, Calcium, Phosphate, Sodium, Potassium, Chloride, Bicarbonate. Alimentary small animal (Profile B) (70017 Biochem Profile B) Amylase, Lipase, ALP, ALT, Urea. Pre-anaesthetic profile (Profile F) (70021 Biochem Profile F) ALP, ALT, Albumin, Glucose, Creatinine, Urea. Kidney/Renal Profile (70136 Profile Renal/Kidney) two different ones Albumin (Kidney only), Creatinine, Urea, Calcium, Phosphate, Potassium (Renal only). Liver Profile (70144 Profile Liver) Alkaline Phosphatase (ALP), Alanine Aminotransferase (ALT, GPT), Protein, Albumin, Globulins, Cholesterol, Total Bilirubin, Urea, VSP-D Comprehensive Diagnostic (70118 Biochem Profile VSD) Amylase, ALP, ALT, Protein-Total, Albumin, Globulins, Bilirubin-Total, Glucose, Creat., Urea, Ca, Phosphorus, Sodium, Potassium. VSP-P Prep profile II (70119 Biochem Profile VSP) ALP, ALT, Protein-total, Glucose, Creat, Urea.

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Individual information on each type of analyte is continued below:

BloodBiochemistry
InHeparin InFluoride (plasma) oxalate(plasma) Amylase X(P) X ALP X X(P) ALT X X(P) AST X X(P) CPK X X(P) GGTP X(P) X Lipase X X Bilirubin X(P) X TotalProtein X(P) X SPE X Cholesterol X(P) X Glucose X X X(P) Urea X X Creatinine X X BileAcids X Calcium X X InorganicPhosphate X X Sodium X X Potassium X X Chloride X X Magnesium X X Triglycerides X(P) X T4 X(P) X Phenobarbital X X Iron X X UIBC X Fructosamine X X X X Hydroxybutyrate LDH X X UricAcid X X Ammonia HeparinCollectonIce (Contactlaboratorypriortosamplecollection) XAcceptablesamplePPreferredbyourlaboratory Analyte Serum

For glucose estimation, samples in fluoride oxalate are preferred (fluoride is an enzyme poison and prevents glycolysis). The samples have to be presented to the laboratory promptly so that the plasma can be removed quickly from the cells. For ammonia estimation, the blood should be collected in anticoagulant (contact laboratory one type of reagent kit requires lithium heparin, while the other requires EDTA) and placed in a beaker of ice, and transported to the laboratory promptly so the plasma can be removed quickly from the cells. It is preferred that the anticoagulant container is chilled prior to collection.

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Urinalysis
Urine containers supplied to the UVTHS should be used. It is best if the sample is collected fresh at the UVTHS, than brought in by the owner. If not collected in the UVTHS please mark on the sheet. ALA (delta-aminolevulinic acid) -approx. 5 ml urine General examination -approx. 10 ml urine (smaller samples accepted) General exam comprises: specific gravity, dipstick chemistry and sediment microscopy Urinary Protein/Creatinine Ratio - approx 2ml urine

Faecal analysis
Stools should be placed in the provided urine containers. For special requests in parasitology, please contact that section. General exam -appox. 5 g fresh faeces Occult blood -approx. 1 g fresh faeces Protase only -approx. 1 g fresh faeces General exam comprises: microscopy for undigested fat, starch and muscle.

Haematology
For WBC and RBC Counts, PCV, Hb and TPP estimation, blood collected in EDTA (ethylenediamine tetra-acetic acid. Synonyms include versenate, versene, sequestrene) is preferred. It is important that the correct amount of blood be placed in the tube i.e. the tube is filled to the calibrated mark. Too much blood and the anticoagulant will not be effective and a clot will form, too little blood and the EDTA will affect the smear morphology. The tube is gently mixed and checked for any clotting. Peripheral blood smears can be made from blood collected in EDTA; however, for the best preservation of morphology, smears should be made directly from freshly collected blood. The anticoagulant Heparin is not acceptable for making blood smears as it interferes with the staining and morphology of leukocytes. For microfilarial examination, blood collected in Heparin is preferred as it does not interfere with the enzymatic procedure that is employed to differentiate Dipetalonema and Dirofilaria microfilarae by the acid phosphatase test. (refer to your parasitology notes) For coagulation studies (e.g .fibrinogen, prothrombin time and partial thromboplastin time), the anticoagulant 3.8% sodium citrate is used (contact the laboratory for this anticoagulant). D Dimer (for fibrinogen degradation products) requires plasma or whole blood that has been treated with an anticoagulant. Blood Grouping requires an EDTA sample. The requirements for other tests can be obtained by contacting the VPDS clinical pathology laboratory.

Bacteriological samples
All containers and swabs (two from each site - one for Gram stain, one for culture) must be labelled with unit number and owners name. All specimens must be collected in an appropriate manner (see relevant sections in 2nd and 3rd year Principles of Disease and Veterinary Microbiology handbooks) and handled so that specimen viability is maintained and so that samples do not spill or discharge into the environment.

Ifindoubtpleasecontactthebacteriologicalsectionofthelaboratory.

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a. Urine Samples, CSF and Other Liquids

Place in sterile, screw-capped containers which are kept upright and do not leak. Transport to the laboratory as quickly as possible. If a delay of greater than 2 hours occurs, store at 50C.

b. Pus, Synovial Fluid

These are best transported in a syringe from which all the air has been expelled. Remove the needle from the syringe and cap effectively to prevent leakage. If bottles are used, tape lids to prevent leakage.

c. Solid Material
Place in a small screw-capped bottle, with a swab moistened with sterile saline, if transport is required. Seal edge of Petri dish if transport is directly from surgery. If some distance from the laboratory, place tissue into the transport medium of a swab container (NB. Some commercial transport swabs have a plastic swab case filled with transport medium). d. BS (Blind Screen) Urine Culturing This is a crude test to determine the possibility of bacteria being present in urine. This does not provide for the identification and antibiotic sensitivity testing if an infection is present. Nor is it a substitution for the normal sediment examination of urine, nor does it include a gram stain. Sample - The sample must be a cystocentesis sample. The minimum amount is 1 ml. e. LCAT This is for assessment of cryptococcal antigen. This requires a minimum of 1ml of serum after clotting.

Virology
FeLV and FIV - Serum 1ml but Plasma (EDTA or Lithium Heparin) can be used

FIP Testing
Definitive diagnosis of FIP currently relies on histopathology and immunohistochemistry or its adaptation into immunofluoresece. Immunohistochemistry uses a monoclonal antibody against Feline Coronavirus to identify macrophages infected with the mutant form of this virus (known as FIP virus) within tissues. VPDS offers different options for the analysis of effusions (immunofluoresence) or tissues (immunohistochemistry) With effusions, it should be submitted as quickly as possible to reduce false negatives.

Cytology
Cytology should be submitted to the laboratory within three hours of preparation or, once airdried on a glass slide, dip in absolute ethanol or methanol for 15-60 seconds. CSF * - EDTA and sterile plain tubes. FNA - Smear, and/or syringe with needle still on and covered. Fluids - EDTA and sterile plain tubes. Smears - At least two smears of each site. *To be taken to the laboratory as soon as possible

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Other Special Examinations

Cross match - Recipient - EDTA and plain tubes. - Donor(s) - EDTA, plain and lithium heparin tubes.

Blood Grouping - EDTA sample Bone marrow * - Bone marrow on watch glass in EDTA solution. Calculi Semen * Immunology - Calculi in sterile container. - Semen in calibrated glass container. - contact laboratory or refer to UVTHS handbook.

* To be taken to the laboratory immediately after collection.

Histopathology
Biopsy These should be submitted with the request form to the Clinical Pathology laboratory and given to the laboratory staff. During out-of-hours the specimen and request form are placed in the Clinical Pathology In Tray in the Treatment Room. Place the sample in 10% buffered formalin (in the ratio of 1 volume of sample to 10 volumes of formalin). Using a pencil, write the owners name and the UVTHS animals identity number clearly and neatly on a piece of white cardboard and place the cardboard inside the container. FROZEN SECTIONS - If frozen sections are required, notify the histopathology laboratory so that the cryostat can be chilled down to operating temperature. This will allow the tissue to be processed as soon as it is brought to the histopathology laboratory. The request form is taken to the Clinical Pathology laboratory for processing. Generally, needle aspirates and crush preparations taken for intra-operative cytology are more useful than frozen sections at the UVTHS. Post Mortem Please remember that no post mortem examination can be carried out unless the owners permission has first been obtained. All bodies must be accompanied by request forms which are completed in full as described above. The necessity for a full report of the relevant history and clinical signs is stressed. Before any animal is destroyed, the students should consult the pathologist to determine the time at which euthanasia would be preferred and whether special procedures are desired (e.g. ante mortem perfusion of the eye). The body is taken to the cold room of the post mortem room and the request form is immediately taken to Clinical Pathology. Identity tags are available in the cold room and should be filled in and attached to the animal. If no labels are available use a white cardboard label with a UVTHS printed label attached to it and attach it to the cadaver. Place on the correct rack in the PM Cold Room These racks are all labelled for each purpose. During out-of-hours, the body, appropriately labelled, is placed in the post mortem cold room and the request form is placed in the Clinical Pathology In Tray in the Treatment Room.

Whenthecadaverisfinishedwith,itisplacedinthecorrectbinfordisposal.

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External Analytes
VPDS will provide a service for the following analytes to be tested at other laboratories on the provision of the following requirements. Blood lead Digoxin, phenobarbitone Von Willebrand or clotting factors Cholinesterase Trypsin Like Immunoreactivity (TLI) Folate, Vitamin B12,Bromide, FIP - 5ml blood in EDTA (do not centrifuge) - 5ml in lithium heparin - 4.5ml blood in 0.5ml 3.8% sodium citrate and separated with plastic pipettes into plastic tubes - 5ml blood in EDTA or Lithium Heparin Do not separate - 10ml blood with no anticoagulant - 10ml blood with no anticoagulant

Samples should be labelled with client's name, AIS animal number and date and accompanied by a pathology request form with the client's name and clinic unit number,clinician'sname,date,nameoftestrequested. Endocrine
These have a yellow Endocrine Request form in UVTHS. Samples should be labelled with clients name, the animals AIS number and date and accompanied with a yellow endocrine request form with the clients name and AIS number, clinicians name, date and name of test requested. Please contact the VPDS lab in the McMaster Building to organise PTH and Catecholamines. All anticoagulant blood collected for hormone analysis should be separated and plasma frozen within 1 hour of collection (15 mins for an accurate PTH value). Please take separated plasma/serum samples, unseparated clotted blood tubes and/or urine samples to the VPDS lab in the McMaster Building. If there are any problems please ring Dot Lewis on 13718 or 17456. ACTH response test (2x Cortisol at 0 and 1h) Cortisol -5ml in lithium heparin or 5ml clotted blood -5ml in lithium heparin or 5ml clotted blood

Dexamethasone suppression test -5ml in lithium heparin or 10ml clotted blood Canine - (3x Cortisol at 0,4 and 8h,) Equine (2 or 3 samples 0, (15) and 19h) UCCR (at 0h) - 5ml Urine

Conversion
Analyte Cortisol OldUnit g/dl NewUnit nmol/L ConversionFactor Old>New 27.59

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WHAT TO DO IF TAKING SPECIMENS FOR LABORATORY EXAMINATION AFTER HOURS

(This has more relevance for the practice situation than to the UVTHS) Ensure that the specimens are labelled prior to storage. Initial processing of specimens is essential to diminish the risk of erroneous results. SAMPLES FOR BIOCHEMISTRY Separate the serum*/plasma from the red cells and store at 4C (if overnight delay) or at 18C (longer delays) in the refrigerator.
*

allow the sample to clot properly before separation

ROUTINE FULL BLOOD COUNTS - Make a peripheral blood film (we prefer 2), label it, and leave at room temperature. - Perform PCV and TPP determination. - Leave rest of sample in the refrigerator (4C). For other specialist haematology tests contact the laboratory for advice.

MICROFILARIA CHECK - Store heparin sample at 4C.

URINE FOR URINALYSIS - The results of urinalysis can alter for delays of more than 4 hours, even if the urine is stored at 4C. Out of hours samples should be examined by the collector. Any difficulty with interpretation can be followed up on subsequent samples.

URINARY ALA - Acidify the urine (a few drops of glacial acetic acid) and store at 4C. i.e.. 100L/10mls

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FAECES Fresh stools should be: 1. 2. frozen (-18C) for TRYPSIN analysis chilled (4C) for stains for undigested/unabsorbed food particles and for routine parasitology.

CELL ASPIRATES - Smears should be made, dried, fixed in alcohol, preferably methanol, and left at room temperature.

FLUID ANALYSIS - A smear for cytology should be made, dried, fixed in alcohol, preferably methanol, and left at room temperature. Store the sample at 4C. N.B. CELLS DETERIORATE RAPIDLY IN BODY FLUIDS (CAVITIES, SYNOVIAL, CEREBROSPINAL) AND PROLONGED STORAGE (e.g. OVERNIGHT) MAY CAUSE FALSE LOW TOTAL CELL COUNTS. IN ADDITION BACTERIA WILL PROLIFERATE AND ALTER THE RESULTS.

HISTOPATHOLOGY - Place the sample in 10% buffered formalin. (In the ratio of 1 volume of sample to 10 volumes of formalin) Label with pencil on cardboard inside the container.

POST MORTEMS - Place the labelled sample at 4C in the cold room.

BACTERIOLOGY SWABS AND FLUIDS Make a smear, dry and leave at room temperature. Store the moist swab (if not moist add sterile saline) or fluid at 4C. N.B. BACTERIAL FLORA CAN ALTER SIGNIFICANTLY WITH PROLONGED DELAYS Theabovesuggestionswilldiminishbutnot removethepossibilityoferroneousresults. Wherepossible,avoidtakingsamplesoutofhours. Ensure that the specimens are labelled prior to storage.

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5 INFORMATION ON MEASUREMENT UNITS AND ENZYMES

In this laboratory most test results are expressed in S.I. units ("Systeme Internationale") or derivatives of such, which is a system of metric units. The litre (L) is the unit of volume used in clinical pathology but is outside the SI units. Enzymes are expressed in IU (international units) per litre. One international unit of enzyme (IU) is that amount which catalyses the transformation of one micromole (mol) of substrate per minute under defined conditions. Most chemical substance concentrations are expressed in moles or fractions of moles per litre. Although not necessary when using VPDS, the old units of measurement and their conversion factors to the new units will be given (Many Veterinary Clinical Pathology texts are using the old units - particularly North American texts). See page 16 for conversion factors.

Enzyme Terminology
ALP AMS ALT Alkaline phosphatase alpha Amylase Alanine transaminase. Synonym is (S)GPT [(Serum) Glutamic-pyruvic transaminase] Asparate (transaminase) (aminotransferase) Synonym is (S)GOT [(Serum) Glutamic-oxalo acetic transaminase] Creatine phosphokinase. Synonym is CK (Creatine kinase) Gamma glutamyl transpeptidase 2 Hydroxybutyrate dehydrogenase (Not done in this laboratory) (LD) Lactate or Lactic dehydrogenase (Not normally done in this laboratory) Lipase Sorbitol dehydrogenase (Synonym ID - Iditol dehydrogenase) (Not done in this laboratory)

AST

CPK GGT HBDH LDH

LPS SDH

(NB. Before the standard international units (IU) were accepted, numerous units of measurement were employed. They cannot be easily converted to the new units.)

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6 CONVERSION OF OLD ENZYME UNITS TO INTERNATIONAL UNITS (IU)

These are for enzymes measured at 37C and expressed as IU/L. Enzyme Amylase A.L.P. A.L.T./A.S.T. C.P.K./G.G.T.P. L.D.H. Lipase S.D.H. OldUnit Somogyi/Caraway HugginsRussell StreetClose RocheDyeunit uKatal BesseyLowryBlock Bodansky KingArmstrong (U.S.A.Phenolphthaleinphosphate) (KindKing uKatal BabsonTransac Karmen ReitmanFrankel SigmaFrankel WroblewskiLaDue uKatal uKatal Wacker Wroblewski uKatal RoeByler CherryCrandall SigmaFrankrl ConversionFactor 1.85 5.7 5.7 1.59 60 16.7 5.4 3.55 7.10 60 1.0 0.483 0.483 0.483 0.483 60 60 0.483 0.483 60 16.7 278 0.0167

7 TEMPERATURE CONVERSION FACTORS FOR ENZYMES TO 37C

Enzyme Amylase A.L.P. A.L.T. A.S.T. C.P.K. G.G.T.P. L.D.H. (Lactate) (<2mmpyr) (>2mmpyr) Resultat25C Unknown 1.63 2.00 2.10 2.35 1.70 2.50 2.15 2.74 Resultat30C 1.8 DEAbuffer1.29,AMPbuffer 1.35 1.40 1.48 1.50 1.35 1.64 1.55 1.69

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8 CONVERSION FACTORS
Biochemistry
Analyte Albumin Bilirubin(total, free conjugated) Protein (total,EPG) Cholesterol Glucose Urea OldUnit g/dl mg/100ml(dl) NewUnit g/L mol/L ConversionFactor Old>New 10 17.103

g/dl mg/dL mg/dL mg/dL BloodUreaNitrogen (BUN)inmg/dL mg/dL mg/dl mg/dL mEq/L mg/dL mEq/L mg/dl mEq/L mg/dl mEq/L mg/dl mEq/L g/dL mg/ml mg/dl mg/dl mg/dl g/dl ng/ml g/ml g/dl

g/L mmol/L mmol/L mmol/L mmol/L(Urea) mol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L mol/L mmol/L mmol/L mmol/L mmol/L nmol/L nmol/L mol/L nmol/L

10 0.0259 0.0555 0.1665 0.3561 88.402 0.0114 0.2495 0.5 0.323 1 0.435 1 0.256 1 0.282 1 0.588 12.5 0.411 0.0595 0.067 27.6 1.28 4.3 12.9

Creatinine Triglycerides Calcium Inorganic Phosphate Sodium Potassium Chloride Bicarbonate Ammonia Bromide Magnesium UricAcid Xylose Cortisol Digoxin Phenobarbital T4

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Haematology
Parameter OldUnit NewUnit Conversion Factor Old>New 0.01 10 10 0.621 1 1 10 1

PCV(packedcellvolume) TPP(Totalplasmaprotein) Hb(Haemoglobin) ErythocyteCount MCV(meancorpuscularorcell volume) MCHC(meancellhaemoglobin concentration) MCH(meancellhaemoglobin) BSR(ESR) (bloodorerythocytesedimentation rate) Leukocytecount NB. milli (m) = 10-3 micro () = 10-6 pico (p) = 10-12

% g/dL g/dL g/dL 106/L(mm 3 ) m3(3) g/dL 2g (picogram) mm1/2mm lhr 103/L

L/L g/L g/L mmol/L 1012/L fl (femtolitre) g/L pg Thesameno conversion 109/L nano (n) = 10-9 femto (f) = 10-15

Reference: SI Units in Clinical Pathology Printed by the Health Commission of New South Wales. These are based upon World Health Organization (1977) The SI for the Health Professions. WHO, Geneva.

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9 REFERENCE RANGES FOR BIOCHEMISTRY

VeterinaryPathologyDiagnosticServices TheFacultyofVeterinaryScience TheUniversityofSydney Unlessstated,thesevalueshavebeendeterminedintheVPDSlaboratory ANALYTE/ENZYME AMYLASE ALP ALT AST CK GGT LDH Lipase Bilirubintotal conjugated unconjugated Protein,total AlbuminBCG Cholesterol Glucose Urea Creatinine Ammonia(fast) BileAcids(fast) BileAcids(pfeed) ELECTROLYTES Calcium Magnesium InorgPhosp. Sodium Potassium Chloride Bicarbonate SPE ProteinAlb SPECIAL* BSP DeltaALA FaecalTryp. *mostarenowobsolete UNIT IU/L IU/L IU/L IU/L IU/L IU/L IU/L IU/L mol/L mol/L mol/L g/L g/L mmol/L mmol/L mmol/L mol/L mol/L mol/L mol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L mmol/L g/L g/L g/L g/L T2or%ret mol/L Azo.Alb.U DOG <1,400 <110 <60 <40 <200 0.68.2 <105 <60 1.25.1 1.25.1 1.25.1 5070 2343 1.47.5 3.36.4 3.610.0 40120 050 010 <25 2.12.9 0.730.98 0.81.6 137150 3.34.8 105120 1824 2339 716 916 412 <5%(2hr) <38 >7.0 CAT <1,400 <50 <60 <40 <200 110 <105 2.53.5 05.0 2.58.0 5473 1938 1.93.9 3.66.6 7.210.7 90180 050 05 <15 1.752.6 0.821.23 1.32.3 147156 4.04.5 115130 1721 2430 921 815 923 HORSE <260 <50 <400 <400 <36 <250 050 (nonfasting) 06.8 3.450 6076 2938 1.73.1 4.46.3 3.78.2 87149 060 020 2.83.4 0.731.23 0.801.77 132150 2.84.8 99108 2028 2836 813 815 714 2.03.7m

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10 LOCATION of Clinical Pathology Classroom and VPDS Laboratories in the McMaster Building B14

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244

HAEMATOLOGY PRACTICAL NOTES

1 INTRODUCTION
These notes, which are not intended to be intensive or extensive, present some simple manual haematological techniques that can be used to evaluate common haematological disturbances. For convenience, the techniques are categorised according to their usefulness in erythrocyte analysis, leukocyte analysis, coagulation analysis and in miscellaneous problem analysis. Some techniques, however, have general applicability to haematological analysis.

2 SOME ANTICOAGULANTS
Ideally, dry anticoagulants of known proportions should be used to minimize dilution, artefacts and alteration of staining properties. No one anticoagulant is entirely satisfactory.

EDTA - disodium or dipotassium salt of ethylenediamine tetra-acetic acid

(synonyms: sequestrene, versenate or versene) EDTA acts by chelating calcium ions required in the clotting mechanism. At a concentration of 1 mg per ml of blood, it is the anti-coagulant of choice for routine blood counts and smears. NB. excess EDTA, i.e. 2 mg per ml of blood, will cause shrinkage of the red blood cells and a significant decrease in PCV. So be careful to fill the tube to the graduated level only. With EDTA, thrombocyte (platelet) counts and cell counts may be performed up to several hours after the collection of blood. Refrigerated EDTA blood samples are suitable for cell counts, PCV and haemoglobin estimations up to 24 hours post-collection.

Heparin
Heparin prevents coagulation by interfering with the conversion of prothrombin to thrombin and with the action of thrombin on fibrinogen. Heparin is naturally occurring and found abundantly in the liver. It does not alter erythrocyte cell size, even in excess; but it does interfere with the staining of leucocytes (the cells appear hazy). This is due to its strong affinity for basic dyes. Heparin is used at a concentration of 10-50 international units per ml of blood.

Sodium Citrate
Sodium citrate is used for some coagulation studies at the rate of one part of 3.8% aqueous solution to 9 parts of blood. Citrate removes the calcium which is essential for clotting.

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3 ERYTHROCYTE ANALYSIS
a) The Microhaematocrit (PCV)
This makes use of high speed centrifugation, using capillary tubes, which ensures complete packing of erythrocytes in a minimum time. The speed of microhaematocrit centrifuges range from 8000 to 16,000 r.p.m. Method: After filling capillary tubes to 66-75% capacity by capillary attraction, the opposite end is sealed either by a small flame or by plasticine (preferred). The sealed tube is centrifuged for the requisite time - 2 minutes at 16,000 r.p.m. or 5 minutes at 8,000 r.p.m. - to give three distinct layers: (i) (ii) the mass of erythrocytes at the bottom called the packed cell volume (PCV) a mixed layer immediately above the erythrocytes, composed of leukocytes, platelets and other nucleated cells, called the buffy coat. The top white layer of the buffy coat is composed primarily of platelets. The leukocyte layer is immediately below the platelet layer and is usually an opaque dark red. This layer is difficult to visualise unless there are increased numbers of leukocytes. the top layer composed of plasma. The microhaematocrit value is determined by use of a reader supplied with the microhaematocrit centrifuge and is expressed as a fraction microhaematocrit value (PCV) = red cell volume (litres) total volume of blood (litres)

(iii)

Notes: The microhaematocrit reading is a good indication of the presence of anaemia or polycythaemia as long as the total plasma protein (TPP) concentration is determined - usually by refractometer. For instance, dehydration (TPP above normal units), by reducing plasma volume, may mask an anaemia or produce a spurious or relative polycythaemia. The colour of the plasma layer provides useful information. For instance, it may depict the presence of jaundice (icterus), lipaemia or haemolysis.

b) Haemoglobin Estimation Using the Cyanmethaemoglobin Method

The reagent used is called Drabkins reagent. Reagent: Place 500 mls of distilled water in the 1 litre volumetric flask. Add Sodium bicarbonate 1.00 gm Potassium cyanide 0.05 gm Potassium ferricyanide 0.02 gm Dissolve the contents, and make up to the 1 litre mark with distilled water. Principle: Haemoglobin is oxidised to methaemoglobin by ferricyanide, and the methaemoglobin is converted into the stable cyanmethaemoglobin by the addition of KCN. The absorbance of cyanmethaemoglobin is measured at 540 nm, where cyanmethaemoglobin exhibits a broad absorbance peak. Because of this broad peak it is acceptable to use a colorimeter (such as the unimeter) using a yellow-green filter (e.g. Ilford 625).

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Method: 0.02 ml (20l) of blood is added to 5 ml of reagent and mixed by inversion. The solution is then stood at room temperature for 10 minutes, then the absorbance is read at 540 nm against a reagent blank. This absorbance is then converted to grams of haemoglobin per litre from a standard curve. The curve is constructed using a commercially available standard. The unimeter method is the same as this except different amounts of reagent and sample are used.

c) Erythrocyte Count - (Unopette)

This method uses a pre-measured diluent reservoir of 1.99 ml (normal saline with sodium azide as a growth inhibitor) and a disposable 10 l micropipette. A haemocytometer is also required (see appendix 1). Note: Azide is dangerous The protective shield for the pipette is used to puncture the diaphragm of the reservoir. NB. Discard shield after this step. i) ii) iii) iv) v) The pipette is filled with well mixed whole blood and the excess wiped off. The reservoir is squeezed and the pipette placed onto the reservoir and locked in. The index finger is placed over the top of the pipette and the pipette rinsed with diluent by releasing then squeezing the reservoir several times. The finger is removed and the apparatus is mixed by inversion. The pipette is turned around and used as a dropper to fill the haemocytometer.

NB. A few drops of diluent are discarded before filling the chamber. Five of the 25 intermediate squares in the central large square are counted (4 corners and centre). Both ruled areas are counted using 40 x objective and the mean is used in the calculation. Calculation: count 100 x 1012/Litre

(see haemocytometer notes for more detail) Notes: Haemoglobin estimation and the erythrocyte count are usually not essential for establishing the presence of anaemia or polycythaemia but can provide supportive evidence. Also, they allow the determination of red cell indices which may be useful in categorising an anaemia.

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d) Erythrocyte Indices
These determine the relationships between the erythrocyte count, haemoglobin concentration, cell volume and cell thickness. 1. Mean corpuscular volume (MCV)

This is the average volume of a single cell expressed in femtolitres (fl- a femtolitre is 10-15 of a litre). It is obtained from the following formula: MCV (fl) = PCV (L/L) x 1000 (expressed as a whole number) Erythrocyte count (x1012/L)

2.

Mean corpuscular haemoglobin (MCH)

This measures the mean or average haemoglobin content of a cell in picograms (pg - a picogram is 10-12 of a gram). In some older and American texts it is refered to as the color index. MCH (pg) = Haemoglobin (g/L) (expressed to one decimal place) Erythrocyte count (x 1012/L)

3.

Mean corpuscular haemoglobin concentration (MCHC)

This measures the ratio of the weight of the haemoglobin to the volume of the erythrocyte, expressed in grams per litre. It takes into account the variation in size of erythrocytes, which is often present in anaemias. In some older and American texts it is referred to as the saturation index. MCHC (g/L) = Haemoglobin (g/L) PCV (L/L) (expressed as a whole number)

Notes: On the basis of MCV, anaemias can be designated macrocytic, normocytic or microcytic. On the basis of MCHC, and to a lesser extent MCH, anaemias can be designated hypochromic or normochromic. Regenerative anaemias (see explanation under reticulocyte count) are often macrocytic (due to the large numbers of reticulocytes) and, although the MCH is normal or increased, hypochromic due to lowered MCHC. However, these changes in red cell indices, if they do occur, are transient and return to normal on restoration of the red cell mass. For this reason, regenerative anaemias are often referred to as transitory or pseudomacrocytic anaemias. True macrocytic anaemias may be found in non-regenerative anaemias associated with myeloproliferative disorders in the cat (due to elevated levels of nucleated red blood cells). Iron deficiency anaemia, a non-regenerative anaemia, is usually microcytic and hypochromic. However, the majority of non-regenerative anaemias are normocytic and normchromic.

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e) Reticulocyte Count
Reticulocyte stain: Brilliant cresyl blue Sodium citrate Normal saline 1.0 gm 0.4 gm 100.0 mls

Dissolve the stain in saline, add sodium citrate, mix and filter. The stain should always be filtered before use. Method: Equal quantities (i.e. 2 drops) of well mixed blood (in EDTA) and stain are mixed in a test tube and allowed to stain for approximately 20 minutes. If the animal is severely anaemic (e.g. PCV 0.10) then 3 quantities of blood to 1 quantity of stain is used. A blood film is prepared in the usual manner and can be counterstained with Giemsa/Diff Quik although it is not advisable as the reticulocyte stain may be removed. Select an area of the smear where there is a monolayer of cells and count the number of reticulocytes. The mature erythrocytes are usually yellow-green while the reticulocytes contain dots or strings of blue staining material (residual cytoplasmic RNA). The reticulocyte count is performed, under an oil immersion lens, by differentiating at least 1000 red blood cells (sometimes it may be possible to count only 500) and expressing the result as a percentage of the erythocytes. If the reticulocyte count is to be expressed as a percentage, ideally it should be corrected for the reduced PCV present in anaemia (fewer erythrocytes are present to dilute reticulocytes released from the bone marrow). Corrected reticulocyte % = observed reticulocyte % x patient's PCV normal PCV

For this calculation the "average" PCV for a) Dogs is 0.45, b) Cats is 0.37. (In medical science, the reticulocyte percentage is corrected also for variation in maturation times of erythroid cells. However, maturation times have not been adequately determined in most animals (except dog - see lecture notes). The reticulocyte % can also be expressed as an absolute value: Absolute reticulocyte count(x 1012/L) = observed reticulocyte % X erythrocyte cell count. This is becoming the correction of choice for dogs and cats. The upper level of the reference interval for dogs is 0.075 (grey zone to 0.105) x 1012/L and for cats 0.06 (grey zone to 0.100) x 1012/L. If a dog has 4% reticulocytes and an erythrocyte count of 3.0 x 1012/L then the absolute reticulocyte value is 0.120 x 1012/L. This suggests regeneration. Notes: In all domestic species, except the horse, the reticulocyte count is an indication of whether the anaemia is regenerative (i.e. an adequate bone marrow response to anaemia). In regenerative anaemia the marrow responds, from 3 days after the onset of anaemia, by elevating the numbers of circulating immature erythrocytes (primarily reticulocytes but some may be nucleated red blood cells).

249

The dog has small numbers of circulating reticulocytes in health (0-1.5%). The cat has a reticulocyte percentage of 1.4 - 10.8% in health. The marked variation in reticulocyte numbers in the cat is partly due to the fact that two main types of reticulocytes exist. The aggregate type (strands or clumps of reticulum) is similar to other species and high values indicate current erythropoietic activity. The punctate type (dotted reticulum) may occur in large numbers in health (9-10%) and are increased in regenerative anaemias. They remain increased at least two weeks after the aggregate count has returned to normal; therefore, they can indicate a bone marrow response as much as four weeks previously. (In our laboratory we count only the aggregate type in cats which are 0-1.0% in health). The adult horse in health and in anaemia rarely has reticulocytes in the peripheral circulation (foals may develop reticulocytosis in response to anaemia); therefore, the response of bone marrow has to be assessed from changes in serial microhaematocrits and from, if required, bone marrow aspirate examination.

f) Coombs Antiglobulin Test for Autoimmune Haemolytic Anaemia (AIHA)

The basis of the Coombs test is the detection of antibodies or complement directed against the patient's own erythrocytes by the addition of species specific antiglobulin (or anticomplement) and the development of agglutination. The Coombs test forms the basis for all antibody detection and cross-matching tests. 1. Direct Coombs Test.

This detects antibodies attached to the erythrocyte membrane and is the method of choice for AIHA. A clotted sample is obtained from the patient and a control animal. Equal volumes of 0.1 ml of cell suspension and antiglobulin are combined in test tubes, set up in and incubated at 37C (and at 22C and 4C if cold agglutinins suspected) for 15 minutes. After incubation, the tubes are centrifuged at 1500 r.p.m. for 15 seconds and then gently shaken to check for agglutination. NB. it is best to use serial dilution of antiglobulin to distinguish between false and true positives. The test requires appropriate negative controls i.e. cells minus anti-globulin must be included for comparison. 2. Indirect Coombs Test (rarely used for AIHA)

This determines whether autoantibodies are in the patient's serum and have an affinity for a determinant on the red cells of normal dogs. Washed saline cell suspension are prepared from normal dogs. The cells are sensitised by combining equal volumes of cells suspension with the patient's serum and incubating for 30 minutes at 37C. The cells are then washed several times to remove unbound protein. The species specific anti-globulin is then added to the washed sensitised cells and incubated again for 15 minutes at the various temperatures. The samples are then centrifuged and checked for agglutination. This indirect Coombs test may be used in cross-matching (modified) and detecting neonatal isoerythrolysis.

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4 LEUKOCYTE ANALYSIS
a) Leukocyte Count (Unopette method)
The Unopette method employing a 20 l capillary tube, haemocytometer counting chamber and Unopette reservoir containing 1.98 ml of diluent mixture (glacial acetic acid 3%, concentration made up in distilled water). The protective shield for the pipette is used to puncture the diaphragm of the reservoir. NB. Discard shield after this step. i) ii) iii) iv) The pipette is filled with well mixed whole blood and the excess wiped off. The reservoir is squeezed and the pipette placed onto the reservoir and locked in. The index finger is placed over the top of the pipette and the pipette rinsed with diluent by releasing then squeezing the reservoir several times. Let the apparatus stand for 10 minutes, then mix thoroughly, convert the Unopette to the dropper assembly and fill the haemocytometer counting the chamber (ideally, both chambers should be filled and counted, and the average cell number per chamber used.)

Using 100 x magnification (i.e. 10 x objective) count the leukocytes in all nine large squares of the haemocytometer counting chamber (see appendix). Calculation: Count + 10% x 109/Litre 10 (see haemocytometer appendix for more detail) Notes: Total white blood cell count indicates the presence of leukocytosis or leukopenia. In the dog, cat and horse, leukopenia/leukocytosis are usually due to changes in the number of neutrophils. The total white blood cell count is essential for determining the absolute values of leukocyte types seen on the peripheral blood smear.

b) The Peripheral Blood Smear (Film)

1. Preparation of a peripheral blood smear.

Using chemically clean slides, free from dust, films may be made in the following manner: A small drop of blood (ideally the smear should be made directly from withdrawn blood rather than blood placed in anticoagulant) is placed on the slide 1 cm from the end. The spreading slide is placed at an angle of 30-45 degrees to the slide and then moved back to make contact with the drop. The drop should then spread out quickly along the line of contact of the spreader with the slide. The moment this occurs, the film should be spread by a smooth forward movement of the spreader. Films are then rapidly air dried. It is essential that the spreader should have an absolutely smooth edge and should be narrower in breadth than the slide on which the film is to be made. If the edge is rough, films with ragged tails, containing many leukocytes, result.

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The angle of the spreader determines the thickness of the film, i.e. the greater the angle, the thicker the film. The ideal thickness is such that there is some overlap of red cells throughout much of the smear's length with separation and lack of distortion towards the tail of the film. 2. Staining of a peripheral blood smear.

Flood the slide with blood Giemsa and leave to fix for 2 minutes. Dilute and mix the Giemsa with an equal quantity of water (pH7). Leave to stain for 7 minutes. Rinse the slide in tap water and allow to dry. Blood Giemsa: Giemsa Methanol (A.R.) Glycerine 4 gm 1000 ml 15 ml

Blood Giemsa is placed in an incubator at 37C for approximately 1 week before use. The solution is mixed daily while incubating. 3. Staining the smear for Haemoplasma (originally called Haemobartonella) spp.

The blood smear is fixed for 4 minutes in methanol. It is then stained in a coplin jar in 6% stock Giemsa in tap water overnight. The coplin jar is flooded with water to remove the Giemsa. The slide is then dried, rinsed in methanol, rinsed in tap water, redried and examined. Stock Giemsa: Giemsa Glycerine Methanol (AR) 7.6 gm 250 ml 750 ml

The solution is treated as for blood Giemsa Notes: 1. 2. A commercial stain ("Diff Quik" Lab-Aids Pty Ltd) can be used for 2 and 3. Mycoplasma (Haemobartonella) canis and Mycoplasma spp. in cats are usually found on the erythrocytes and may take one of several forms including chains of cocci, rods, bows and rings, or it may appear as a single coccus. 4. Examination of the Peripheral Blood Smear if the leukocytes are well distributed. A bad film will show uneven distribution, with cells collecting at the margins or in the tail of the smear; if there is a rouleaux formation; if there is red cell agglutination; if there are leukocyte clumps; if there are platelet or fibrin clots; if there are any extracellular parasites present.

Under low power note: (i)

(ii) (iii) (iv) (v) (vi)

Under the oil objective (100x) note: (i) Leukocyte Morphology

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Note the presence of any primitive or immature leukocytes e.g. myelocytes, metamyelocytes. Note the presence of any atypical or abnormal leukocytes, e.g. atypical mononuclears, plasma cells. Note if the neutrophils show any toxic granulation, vacuolation and any inclusions. For the appearance of cell types please consult posters (supplied) and text books. Once the slide has been scanned under high power, a differential leukocyte count can be performed, with the oil lens, using the modified battlement technique (takes in account that larger cells tend to collect at the extreme margins of the film while lymphocytes are usually more centrally placed). Commence counting at one edge of the slide, traverse across the slide to the other margin, move 4-5 horizontal edge fields then reverse the traverse (towards the tail).

In the differential leukocyte count, an attempt is made to determine the percentage distribution of the various leukocyte types in peripheral blood. The procedure is subject to considerable error, since an extremely small portion of the total number of leukocytes is observed. This error can be decreased by counting a large number of cells: at least 100 cells for every 10 x 109/L cells in the total white blood cell count. From the results each leukocyte type is expressed as a percentage of the total count. However, the important value for each leukocyte type is the absolute number per litre of blood. Absolute value for a leukocyte type = total leukocyte count x percentage of leukocyte type. The absolute values determine the following: the presence of neutrophilia, neutropenia, eosinophilia, eosinopenia, lymphocytosis, lymphopenia and monocytosis. However, the presence of a left shift can be determined from the percentage values for the segmented and immature neutrophils e.g. the dog has a normal ratio of > 16-18 segmented neutrophils to 1 immature form; for a left shift to be definitely present, the ratio would have to be below that normal ratio. For instance, 60% segmented neutrophils and 5% band neutrophils. The ratio is 12:1, thus a left shift is presumed to be present. For dogs and cats, it is accepted that bands greater than 1.0 x 109/L indicate a left shift. The ratio of bands to segmented is only used for values less than 1.0 x 109/L. If there are any nucleated red cells present in the smear, they are usually recorded either as numbers per 100 WBCs counted or as an absolute value derived from the initial leukocyte count. If there are over 5 nucleated red cells per 100 WBCs present, the total leukocyte count should be corrected before determining the absolute values for leukocyte types. Corrected total = leukocyte count initial total leukocyte count x 100 [ 100 + nucleated RBCs (per 100 WBCs)]

253

Leukocyte description
ThistableismodifiedfromoneinOutlineofVeterinaryClinicalPathologybyMaxineM. Benjamin,2ndEdition,TheIowaStateUniversityPress.1964 MORPHOLOGYOFBLOODCELLSUSINGROMANONSKYTYPESTAINS
CELL 1.GRANULOCYTES a.Myeloblast b.Progranulocyte c.Myelocyte d.Metamyelocyte (Juvenile) e.Bandcell(Stab) f.Segmented Neutrophil Size (microns) 1520 1420 1018 1018 Shape Roundoroval Roundoroval Roundoroval Indentedoval, resembling kidneyorbean Curvedwith parallelsides Raggedor dividedinto lobes 23lobes NUCLEUS Staining Chromatin Quality Light Finely purple reticulated Purple Slightly coarse Purple Fairlycoarse Deep Coarse purple Purplish blue Purplish blue Purplish blue Paleblue Coarse Coarse Nucleoli 25Pale Blue 13 Relative Amount Small Small Moderate Large CYTOPLASM Colour Granules Deepblue Blue Bluishpink Pink Fewazurophilic Startdifferentiating Neutrophilic, eosinophilic,basophilic Neutrophilic, eosinophilic,basophilic Violetorabsentin animalblood Brightred,Horsevery large,Dogvarying sizes,Catrodshaped Bluishblack,coarsein horseandcattle, moderateindogand cat

1015 1015

Large Large

Pink Faintpink

Eosinophil

1015

Coarse

Large

Bluishpink

Basophil

1015

Outlinecovered withgranules

Indistinct

Moderate

Bluishpink

2.LYMPHOCYTES a.Lymphoblast

1018

Roundoroval

b.Prolymphocyte

1018

Ovalorsl. indented Round,ovalor sl.indented Roundoroval Irregular Round, indented,band orlobed Round Round Round Pyknotic, fragmented,or partially extruded

c.Lymphocyte

618

3.MONOCYTES a.Monoblast b.Promonocyte c.Monocyte

1418 1418 1218

Lt. reddish purple Deep reddish blue Deep purplish blue Deep purple Purple Pale purple Purple Purple Deep purple Dense purple

Fine

12Pale blue Occasional

Small

Deepblue

Finetosl Coarse.

Large

Darkto med.blue Skyblue

Fewazurophilic

Largecoarse clumps Fineretic. Fineretic. Finestrands 12 01

Smallto moderate Moderate Moderate Large

Fewlargeazurophilic

Deepblue Grayblue Grayor grayblue Deepblue tograyblue Blue Bluetopink Pink

Fewfinelilac Fineazurophilic

4.ERYTHROCYTES a.Rubriblast b.Prorubricyte c.Rubricyte

1419 1015 812

Fine stippled Coarse Definite Solid

Usually12 Usually1

Small Moderate Moderate Moderate

d.Metarubricyte 710 (LateNormoblast)

e.Erythrocyte 48 5.THROMBOCYTES 24 (Platelets)

Total Total

Pink Paleblue

Azurophilic

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(ii)

Erythrocyte Morphology

Note any variation in size (anisocytosis) and the presence of microcytes, macrocytes. Note any variation in shape (poikilocytosis) and the presence of target cells, burr cells (acanthocytes) and spherocytes. Note any variation in colour (polychromasia) and the presence of hypochromatic or polychromatic (polychromatophilic) cells. Note the presence of erythrocyte inclusions e.g. Howell Jolly bodies, punctate basophilia (basophilic stippling) and parasites. NB. E.R. bodies (Heinz bodies) can be stained with 0.5% brilliant cresyl blue in normal saline. Reticulocytes, in the Giemsa stained peripheral blood smear, will appear blue and will be larger than the mature erythrocytes. Accordingly, a blood smear from a regenerative anaemia will show anisocytosis and polychromasia. The presence of nucleated red cells in the blood smear may also occur in regenerative anaemias. However, if in an anaemia, nucleated red cells appear in the blood unaccompanied by anisocytosis and polychromasia, a nonregenerative anaemia associated with abnormal marrow should be suspected. ErythrocyteMorphologynumberofcellsperoilfieldof200250erythrocytes ABNORMALITY Anisocytosis Dog Cat Horse Polychromasia Dog Cat Horse Poikilocytosis Allspecies Hypochromasia Allspecies Codocytes Dogsonly Spherocytes Allspecies* Echinocytes Allspecies SLIGHT 710 58 13 12 12 rarely observed 825 12 12 12 12 MODERATE 1120 920 410 310 315 26150 310 36 310 310 MARKED >21 >20 >10 >11 >15 >150 >10 >6 >10 >10

Acanthocytes,schizocytes,keratocytes,elliptocytes,dacryocytes,depranocytes, stomatocytes Allspecies 12 35 >5 *spherocytesarenoteasilyidentifiedinspecieswithsmallerythrocytes(e.g.cats, ruminants)

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(iii)

Platelets

Note their size and numbers. See coagulation section g (ii) for indirect count methods.

c) Total Eosinophil Count (Direct Method)

Sample: Collect blood in EDTA. Total count should be performed within a few hours. Stain: 2% Eosin Y (aqueous) Acetone (A.R.) Distilled water 5 ml 5 ml 90 ml

Prepare on day of use. This fluid is said to be stable for 2-3 weeks but it is best to prepare fresh. Method: Dilute well mixed blood 1 in 10 with stain and mix for no more than 30 seconds. Fill haemocytometer, allow to settle, then count all 9 squares on both sides. Calculation: Total no. of eosinophils counted = Count x 109/Litre 180 NB. To check count, a smear should be made and stained by Giemsa and examined. The absolute eosinophil count determined by this indirect method should approximate or be slightly greater than the direct count. If there is doubt the direct method should be repeated. The total eosinophil count is of importance in evaluating adrenocortical activity. For instance, eosinopenia is often present hyperadrenocorticism. There is a Unopette available for direct counting of eosinophils.

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5 LABORATORY EVALUATION OF HAEMOSTASIS

Haemostasis is dependent on vascular integrity, platelet function and numbers, and clotting factors. Consequently, laboratory evaluation of haemostasis involves the determination of these components. Although there are many tests available, these notes concentrate on the simpler and more commonly used coagulation tests. It is advisable to provide samples from control (normal) animals along with the test sample.

a) Bleeding Time
The interval between the occurrence of injury to the blood vessel and the cessation of bleeding is called the bleeding time. The test may be performed on the skin of the ear, the nose, the pad of the foot, or the inside of the lip.

Method: A thin coat of petroleum jelly is spread over the area, and a moderately deep puncture is made with a number II Bard Parker blade so that the blood flows freely. Do not use pressure. Drops of blood are absorbed onto a filter paper at 30 second intervals, and the time at which bleeding stops is noted. Normal bleeding time for most animals is 1-5 minutes. Notes: Bleeding time is an estimation of vascular integrity and platelet function and numbers, and is not dependent on clotting factors. Therefore, prolonged bleeding times occur in thrombocytopenia, platelet function defects and vessel wall defects. Von Willebrands factor deficiency (allows platelet adhesion) will also cause an extended bleeding time. A normal bleeding time occurs in health and in deficiencies of clotting factors.

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b) Clotting Time
This test measures the time required for fibrin clot formation in non-anticoagulated blood in vitro.

Method: A 5 ml syringe is rinsed with normal saline solution immediately before use. 3 ml of blood is withdrawn from a vein. The blood is divided into 1 ml lots and is transferred to 3 clean glass test tubes (8 mm bore). The tubes are maintained in a water bath at 25-37C. After 3 minutes, the tubes are tilted at 30 second intervals, starting with tube 1; when this clots, i.e. when the tube can be inverted without blood flowing out, the time is recorded. Clotting times are noted for tubes 2 and 3. The average time for coagulation in all 3 tubes is then determined. Normal values: 3-13 minutes for the dog 8 minutes for the cat 4-15 minutes for the horse

Notes: Clotting (coagulation) time is not dependent on vascular integrity or platelet function and numbers (as long as glass tubes are used). Clotting in vitro is primarily due to activation of the intrinsic pathway of clotting factors by direct contact with glass. However, in siliconized or plastic tubes, platelet factors are important in activating the intrinsic pathway of clotting factors). Also, clotting time is not affected by the extrinsic system of clotting factors as this system is activated only by the release of tissue thromboplastin from injured vascular endothelium. Prolonged clotting times occur in *deficiencies or inhibition of intrinsic or common pathways of clotting factors. Normal clotting times occur in health, thrombocytopenia, platelet function defects and vessel wall defects.
*

the deficiency must be less than 5% of the normal level before clotting time is extended.

258

c) Clot Retraction
Clot retraction in vitro is primarily a function of platelets, although the fibrinogen content of the blood does have an influence.

Method: 5 ml of blood is placed into a clean, dry glass test tube (with no added anticoagulant) and incubated at 37C. A normal clot will retract markedly within 2-4 hours and by the end of 24 hours will be a compact mass.

Notes: The above is purely a screening test and more quantitative methods are available. Clot retraction is impaired primarily by platelet function and number defects. It may be impaired in afibrinogenaemia and anaemia. When fibrinolysis is very active, the fibrin clot may be dissolved as quickly as it is formed, and clot retraction will obviously be impaired.

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d) Fibrinogen Estimation
Method: A 2 microhaematocrit tubes are filled with blood containing EDTA and centrifuged for 5 minutes as would routinely be done for determining packed cell volume. The plasma protein level is determined from a tube by using the refractometer. The second tube is placed in a water bath at 56C for 3 minutes. This will precipitate fibrinogen, which is removed from the plasma by further centrifugation for 5 minutes. The total plasma protein of the sample in the heated tube is determined with the refractometer. Fibrinogen concentration is calculated by subtracting the protein value of the plasma in the heated tube from that of the unheated tube. Method B (Millar Method - suggested method)) A microhaematocrit tube is filled with blood containing EDTA and centrifuged for 5 minutes as would routinely be done for determining packed cell volume. The tube is placed in a water bath at 56C for 3 minutes. Making sure that all the fluid is immersed. This will precipitate fibrinogen, which is packed on top of the buffy coat by further centrifugation for 5 minutes. The microhaematocrit tube is then attached to a slide so that measurements can be made of the different interfaces in the tube using the microscope's scale and its vernier. If the microscope does not have a scale, an ocular micrometer eyepiece can be used. For this calculation the interface between the buffy coat and the precipitated fibrinogen is called A. The interface between the precipitated fibrinogen and the serum is called B. The base of the meniscus of the plasma (now serum) is called C. The length of the column of the precipitated fibrinogen (AB) is measured in relation to the original length of the column of plasma (AC). Fibrinogen concentration (g/L) is calculated from the formula:

(B A) 100 (C A)

Normal fibrinogen (Factor I) levels: 2-4 g/L for the dog 1-3 g/L for the cat 2-4 g/L for the horse

Notes: (These methods are limited in their ability to detect reduced levels. Other more sensitive methods are available to detect reduced fibrinogen.) Fibrinogen (Factor I) levels are increased in a wide variety of inflammatory and tissue destructive conditions. They can be decreased in certain chronic liver diseases, D.I.C. and genetic disorders. Fibrinogen levels can have an effect on the results of certain coagulation tests.

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e) Prothrombin Time (ProT, PT or OSPT)

Measures the time required for fibrin clot formation in recalcified, fresh, citrated plasma after addition of tissue thromboplastin (which should be absent if the sample was taken correctly) in vitro i.e. it measures the extrinsic (tissue) and common pathways' clotting factors. Sometimes referred to as one stage prothrombin time (OSPT)

Methods: Add 4.5 ml whole blood (control and patient) to 0.5 ml of 3.8% (0.13M) trisodium citrate. Centrifuge and remove the plasma. Place the plasma and simplastin in a bath for 5 minutes set at 37C (plasma can be stored at 4C for a maximum of 4 hours before being used in the test). Label 3 control and 3 patient serological tubes. Add 0.2 ml of Simplastin to each tube. Add 0.1 ml of plasma (control and patient) to each tube and time the clot formation. Determine the average clotting time for the control and patient and determine any significant difference. Reference range values: 7-12 seconds in the dog 7-10 seconds in the cat 10-15 seconds in the horse

Notes: A prolonged PT occurs in deficiencies or inhibitions of extrinsic and common system clotting factors, and in heparin inhibition (if heparinized blood had been given by transfusion). A normal PT occurs in deficiencies or inhibitions of intrinsic system clotting factors and in health. With automation doing most of the coagulation work in larger laboratories and point of care instruments becoming more common in smaller laboratories and practices, there was some form of standardisation required in the measurement of the coagulation times. Some instruments measure when the clot first starts to form while others wait till the clot is fully formed. To overcome this and other abnormal determinations from instruments and reagents, International Normalised Ratios (INR) were determined. With the PT test, this is based upon International Sensitivity Indexes (ISI) (supplied by the reagent manufacturers) and Mean Normal Prothrombin Time (MNPT) (supplied by the instrument maker). You should be aware that some laboratories report the result as INR. The calculation for the INR is found in the reagent insert with a table for ISI values.

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f) Partial Thromboplastin Time (PTT)

Measures the time required for fibrin clot formation in recalcified, fresh, citrated plasma after addition of a contact activator in vitro (which activates the intrinsic [intravascular] pathways of clotting factors). The PTT yields the same, but more precise, information as clotting time, i.e. it measures the intrinsic and common pathways of clotting factors.

Method: 4.5 ml of whole blood (from a control animal as well as the patient) is placed into tubes containing 0.5 ml of 3.8% trisodium citrate and mixed. The sample is then centrifuged and the plasma removed. Label 3 control and 3 patient serological tubes. Pipette 0.1 ml of Platelin Plus Activator into the tubes and place in a 37C water bath for 2 minutes. Add 0.1 ml of the appropriate plasma to the tubes and incubate 5 minutes. Transfer 0.1 ml of 0.025M calcium chloride to each tube and simultaneously time for clot formation. Determine the average clotting time for control and patient, and any significant difference. Reference range values: 12-25 seconds for the dog 12-25 seconds for the cat 37-54 seconds for the horse

Note: The PTT is prolonged only when the deficient factor is less than 30% of the normal level. The PTT is very low in birds and reptiles.

262

g) Platelet (Thrombocyte) Count

I. DIRECT METHOD Platelet counting fluid: Ammonium oxalate 1.0 gm Brilliant Cresyl Blue 0.1 gm Distilled water 100.0 mls i.e. 20 drops of 0.5% brilliant cresyl blue in 1% ammomium oxalate. (Filter prior to using).

Method: A normal red cell dilution of 1 in 200 of well mixed whole blood in the fluid is made. It is mixed for approximately 5 minutes before being used to fill the haemocytometer counting chamber. The haemocytometer counting chamber is then placed in a moist chamber and left for about 15 minutes to allow the platelets to settle. Using the 40x objective, platelets in the entire centre 1.0mm2 area of both counting chambers of the haemocytometer are counted; this gives the number in 0.2ul. Counting is best carried out using a phase contrast microscope if possible. Make sure the objective is lined up the correct condenser. Calculation: Number of platelets = total platelets counted X 200 X 5 X 106 (x 109) per litre i.e. count x 109 /Litre Also, platelets may be counted using a special Unopette chamber. The leukocyte dilution is prepared, the leukocytes counted, and then the platelets counted and calculated. The direct method of counting platelets is plagued with difficulties. Platelets are small and are often confused with other particles such as dust and debris. Also, they may disintegrate or agglutinate, making counting difficult. Hence, direct platelet counts are often poorly reproducible. Reference range values (using the direct method of counting): 200-900 x 109/L for the dog 300-700 x 109/L for the cat 100-300 x 109/L for the horse

Notes: A moistened chamber may be produced from a petri dish with two sticks placed in the base (to support the haemocytometer) with a piece of moistened blotting paper. If using phase contrast the haemocytometer should have a flat base.

263

II. INDIRECT METHODS To cross check or estimate platelet numbers, the indirect methods can be employed. These methods have clinical application only when more sophisticated methods are not available. A. From the stained blood film, under oil immersion, the number of platelets per 100 leukocytes can be estimated. Calculation: platelets(x 109/L) = number of platelets per 100 leukocytes x total leukocyte count B. When using the Olympus microscope with the 100X oil immersion lens, an estimate of the number of platelets, in the peripheral blood smear, can be made by taking a average of 5 scattered fields and referring to the table below. This mainly works for the dog, and when there is not a severe anaemia present (upsets the relativity). This is an estimate only because the count does vary on the type of microscope, objective size and quality, and the ocular size and quality. Number 5 10 15 20 25 30 35 40 EstimateX109/L 103 200 298 396 494 592 690 780

Notes: The platelet count assesses the presence of thrombocytopenia e.g. less than 200 x 109 per litre for the dog. However, spontaneous haemorrhages (thrombocytopenia purpura) will not usually occur until the count is much lower than the minimum e.g. below 50-70 x 109 per litre for the dog, or there is a concomitant platelet defect. Thrombocytopenia may occur due to marrow damage, splenic disorders (stores a third of the platelets at any one time), accelerated use e.g. D.I.C. (disseminated intravascular coagulation), and immune-mediated destruction e.g. idiopathic thrombocytopenia purpura. Thrombocytopenia due to marrow damage is accompanied by decreased numbers of megakaryocytes, thrombocytopenia due to other causes usually has normal to increased numbers of megakaryocytes.

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h) Fibrin (fibrinogen) degradation products including D-dimer present in cross-linked fibrin degradation products
During blood coagulation, fibrinogen is converted to fibrin by the action of thrombin. The resulting fibrin monomers polymerize to form a soluble network of non cross-linked fibrin. This fibrin gel is then converted to cross-linked fibrin by thrombin activated Factor XIII to form an insoluble clot. Once the insoluble clot is formed, fibrinolysis is activated. This requires the activation of plasmin, which is the major clot-lyzing enzyme. Plasmin facilitates the conversion of both fibrinogen and fibrin to degradation products. Those degradation products from cross-linked fibrin contain D-dimer. Consequently, measurement of D-dimer levels is very useful to determine the extent of fibrinolytic activity. Fibrinolytic activity is likely to be enhanced in any state of hypercoagulability (Disseminated Intravascular Coagulation [DIC]; thrombolytic disease such as in pulmonary thromboembolism). Consequently levels of D-dimers are enhanced (even in cases of sub-clinical DIC). A number of tests have been developed to measure fibrin (fibrinogen) degradation products, but the more-specific D-dimer tests appear to be the present ones of choice. A simple specific canine D-dimer test kit (Agen Canine D-dimer Test), based on rapid immunochromatography utilising specific murine monoclonal antibodies against D-dimer, will detect elevated levels (negative or positive result). A specific cat one is likely to be developed if demand becomes high.

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6 MISCELLANEOUS PROCEDURES
a) Bone Marrow Examination
Bone marrow samples can be collected from the iliac crest, femur, rib and sternum. The site will depend on the species and size of the animal. (i) Making the smear

Methods of aspiration are available in most texts. As bone marrow rapidly clots, a sample is usually withdrawn in a syringe washed out with EDTA (make an aqueous solution of 2 ml from a vial with dried EDTA normally used for blood collection). Only a small amount of bone marrow should be aspirated (stop when it enters the main barrel of the syringe) and transferred to a watchglass. The bone marrow particles (white flecks) are carefully aspirated with a pasteur pipette so as to leave behind as much of the peripheral blood as possible. They are transferred to a cleaned glass slide, gently pushed into a mound and then squashed between two slides ("T" or "L" method). The pressure of squashing will vary between individual but it is usually mild to moderate. A correctly made bone marrow smear should be elliptical and have any peripheral blood present around the periphery of the squashed bone marrow particles. The slide is rapidly air dried, fixed in methanol and stained routinely with Giemsa but may be stained with Prussian blue (for iron) and peroxidase (for granulocytes). (ii) Examination of the Stained Smear

The degree of cellularity can be assessed under low power. Under oil immersion, a differential cell count can be performed. The most reliable results are obtained in the tail area of the smear, where 500-1000 cells are counted. From the differential cell count: the myeloid to erythroid (M:E) ratio (expresses the proportion of total granulocytes to nucleated erythroid cells), the proportions of myeloid and erythroid cells in the proliferating pools (promyelocytes and myelocytes for myeloid, usually less than 20%; pronormoblasts and early normoblasts for erythroid, usually less than 10%), and the proportion of megakaryocytes and other cell types can be determined. M:E ratios: 1.2:1 for the dog 1.5:1 for the cat 1.1-10.20:1 for the horse

Notes: Bone marrow examination is useful for: 1. 2. 3. 4. Investigating non-regenerative anaemias in most species. In the horse it can be useful for investigating all anaemias. Establishing or confirming the presence of a myeloproliferative disorder. Investigating a platelet disorder. Investigating a myelophthisic disorder.

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b) Cross-matching Blood for Transfusion

Saline Cross-Match (dog) This is the simplest method available. Although it is not as sensitive as the antiglobulin test, reagents are readily available whereas for the antiglobulin method they are hard to obtain and very expensive. a) Samples The sample of choice is clotted blood. From this sample both serum and cells may be obtained. After the sample has clotted it should be centrifuged to separate both serum and the cells. The serum is stored at 4C. The clotted blood should not be refrigerated until the serum and cells have been removed. The serum is only stable for up to 24 hours due to loss of complement. It is best to use fresh serum, but plasma must not be used as anticoagulants are anti-complement. Red cells are teased from the clot and used within 24 hours. If there is any difficulty in obtaining cells from the clot then cells from the EDTA sample may be used. Cells obtained in such a manner should be washed three times in buffered normal saline before use. If there is no alternative, cells collected in ACD (Acid Citrate Dextrose) may be used and these are stable for several days at 4C. b) Technique The clotted blood sample is centrifuged and the serum removed. The clotted samples need to be completely clotted - if the request is urgent, then the clotting process may be speeded up by placing the samples in the water bath at 37C. After the serum is removed a little normal saline is added to the clot and the clot broken up with a glass rod to free the cells. Some of the freed red cells are placed into a test tube and centrifuged at 200G for one minute. Take care that there is no clots in the cells. The saline is replaced and the process repeated twice more. The saline on the last wash is removed and a 3% suspension of red cells is made in LISS (1 drop of cells and 32 drops of LISS ). If LISS is not available saline may be used. Both donor and recipient samples are treated in the same manner. Duplicates of each of the following tubes are prepared. 1) 2) 3) 4) Saline Control Auto-Control Donor Test Donor Control - 1 drop Recipient Cells + 2 drops saline (LISS) - 1 drop Recipient Cells + 2 drops Recipient Serum - 1 drop Donor Cells + 2 drops Recipient Serum

- 1 drop Recipient Cells + 2 drops Donor Serum

Tubes 3 and 4 are repeated for each additional donor. One set of tubes is placed in a 37C water bath and the other left at room temperature for 30 minutes. The room temperature should be in the range of 20-24C , preferably closer to 24C as below 20C the test is affected by cold agglutinins. It is best to run at both temperatures as the literature is in conflict as to the ideal temperature and until this is determined it is best to cover all possibilities. At the end of 30 minutes the tubes are centrifuged for 1 minute at 750 G (slow speed) and then examined for haemolysis and/or macro-agglutination. Negative tubes should then be examined for micro-agglutination. If any doubt exists with the result then the blood is declared incompatible.

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Notes: Care must be taken not to confuse rouleaux for agglutination. The donor dogs are commonly checked for microfilaria prior to use. All animals having a second transfusion must be cross-matched. Breeding bitches that have already had litters should be cross-matched using the antiglobulin test as well as the saline cross-match. The horse is cross-matched using the antiglobulin test or the haemolysin test. The cat is commonly blood typed for transfusions since the saline cross-match often produces inadequate results.

c) Blood typing for cats and dogs

Blood typing for dogs and cats is now available for the veterinary practitioner and has, in many cases, replaced the need for cross matching. It is recommended that all cats undergoing a transfusion be typed as there may be high levels of circulating isoantibodies to incompatible blood groups. Dogs undergoing a transfusion for the first time usually do not have significant levels of isoantibodies to incompatible blood groups. These usually develop after the initial transfusion. Consequently, typing of dog erythrocytes is optional for first transfusions but essential for second and subsequent transfusions. RapidVet-H (Feline) (Kansas State University and dms/agrolabo products ag, Neuhausen am Rheinfali, Switzerland): is intended for use to classify cats as blood group Type A, Type B or Type AB. This is one blood group system in the cat, with the majority of cats possessing the A antigen. One third of the cats possessing A antigen have naturally occurring, low-titre, anti-B antibody. All Type B cats have naturally occurring, highly titred anti-A antibody. Type AB cats are rare and do not have or develop anti-A or anti-B antibodies. Cats with B erythrocytes will develop a marked anaphylactic and haemolytic response to Type A blood because of their natural, high titre anti-A antibodies. Cats with A erythrocytes and natural, low titre anti-B antibodies will exhibit only a mild reaction when transfused with Type B blood for the first time (but significant haemolysis can occur over a period of time). Cats with AB erythrocytes do not usually develop transfusion reactions. The kit assay is based on the agglutination reaction that occurs when an erythrocyte which contains either a Type A, Type B or a Type AB antigen on its surface membrane interacts with lyophilized specific antisera present on the test card. Whole blood collected in EDTA and test diluent are added to the Auto-agglutination Saline Screen well, the Patient Type A well and the Patient Type B well and mixed. The test card is rocked until agglutination occurs. The Patient Type A well agglutinates if the cat is Type A, the Patient Type B well agglutinates if the cat is Type B. Both wells agglutinate if the cat is AB. There are limitations to the test which are discussed in the information sheet. RapidVet-H (Canine DEA 1.1) (Kansas State University and dms/agrolabo products ag, Neuhausen am Rheinfali, Switzerland): is intended for use to classify dogs as DEA 1.1. Eight specific antigens have been identified on the surface of canine erythrocytes. The internationally accepted canine blood group system, the DEA= (Dog Erythrocyte Antigen), is based on these antigens. The antigens are DEA 1.1-1.8. DEA 1.1 and 1.2 are the most significant. Both are highly antigenic but DEA 1.1 is the primary lytic factor in canine transfusions. Dog erythrocytes with DEA 1.1 have the greatest potential to stimulate formation of isoantibodies. Thus most reactions resulting from the transfusion of incompatible cells occur when DEA 1.1 positive blood is given to DEA 1.1 negative

268

recipients. DEA 1.2 and 1.7 may produce minor transfusion reactions. Greyhounds are particularly low in DEA 1.1, 1.2 and 1.7 antigens and, therefore, are useful donors. About 40% of dogs are DEA 1.1 positive. A DEA 1.1 positive dog can receive both DEA 1.1 positive and negative blood. A dog that is DEA 1.1 negative should not receive DEA 1.1 positive blood. The kit assay is based on the agglutination reaction that occurs when an erythrocyte which contains a DEA 1.1 antigen on its surface membrane interacts with a murine monoclonal antibody proven specific to DEA 1.1 which is lyophilized on the test card. Whole blood collected in EDTA and test diluent are added to the Auto-agglutination Saline Screen well. Positive Control fluid is added to the DEA 1.1 Positive Control well. Whole blood collected in EDTA is added to the Patient Test well and mixed. The test card is rocked until agglutination occurs. The Positive Control should agglutinate. If the Patient Test agglutinates then the animal is DEA 1.1 positive. There are limitations to the test which are discussed in the information sheet.

d) The Blood/Erythrocyte Sedimentation Rate (BSR:ESR)

When blood containing an anti-coagulant is allowed to stand in a tube in a vertical position, the red cells will gradually settle, leaving the plasma as a clear supernatant. The rate of settlement over a specified time is known as the sedimentation rate.

Method: The Wintrobe method entails the use of a tube, in which 10 cm are graduated from the base in mm. The bore is 2.5 mm to 3 mm and the volume approximately 0.7 ml. The tube is filled to the 10 cm mark with blood and allowed to stand vertically for one hour, for the dog and cat, at which time a reading is taken of the sedimentation of the cells (for the horse, readings are taken at 10, 20, and 30 minutes). The test should be set up within 2 hours of collection of the sample (in EDTA). As variations in temperature affect the sedimentation rate, care should be taken to ensure that the test is performed under standard conditions (usually 22-27C). The rate of sedimentation is obtained by reading from the top of the blood plasma to the top of the layer of sedimented erythrocytes in mm. Ideally, the result should be corrected for PCV as the ESR varies inversely with PCV. Significant ESR difference = observed ESR - anticipated ESR at that PCV (read from a table)

Notes: The usefulness of the ESR has been questioned. The ESR is a non-specific reaction which does not aid differential diagnosis. Even a normal ESR does not exclude the possibility that a disease process exists. Today, it is only utilised by a few equine veterinarians. Elevated ESR may be found in infections, skin alterations, tissue injury or destruction and pregnancy. A depressed ESR may occur when there are significant numbers of abnormal or immature red cells, and when there is a low total plasma protein e.g. chronic liver disease, poor nutrition.

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e) The Lupus Erythematosus (LE) Cell Test (For Systemic LE)

The LE cell test is based upon the principle that a substance (LE factor) in the gamma globulin fraction of plasma of affected patients reacts with the nuclei of injured leukocytes. A nucleoprotein transformed by the presence of the LE factor acquires chemotactic properties and attracts phagocytes, which are usually segmented neutrophils. Consequently, the phagocytes ingest the nuclei. The final result, that is the LE cell, is a neutrophil (less commonly a monocyte or eosinophil) that has ingested a mass of nuclear material. The ingested nuclear material is the size and shape of a lymphocyte nucleus and is homogenous. The ingesting leukocyte has a peripherally displaced nucleus. Care must be taken to differentiate LE cells from Tart cells (monocytes containing foreign nuclei that retain some characteristic nuclear structure). Forty per cent of SLE cases will be negative for LE cells. Consequently, the Anti-nuclear Antibody (ANA) test is replacing the LE cell test.

Method 1: One ml of the patient's blood is added to a test-tube containing 4 glass beads. This is then shaken vigorously to prevent clotting and rotated to 33 r.p.m. at room temperature for 30 minutes. (This provides the necessary trauma to leukocytes as LE factor appears to be incapable of acting upon healthy living leukocytes). The tube is then placed at 37C for 10-15 minutes after which the contents are transferred to a haematocrit tube. Buffy coat films are made after centrifugation. The films are air dried and stained by Romanowsky method (Giemsa).

Method 2: Freshly drawn blood is permitted to clot and remain at room temperature for two hours. The serum and clot are pressed through a fine wire mesh screen into a petri dish. A wintrobe haematocrit tube is filled with mashed clot and centrifuged at 2000 G for 10 minutes. The supernatant is discarded and films made from the buffy coats then stained as for Method 1. NB. For both methods, slides should be examined for at least 15 minutes before a negative report is given.

270

7 REFERENCE RANGES FOR HAEMATOLOGY

VeterinaryPathologyDiagnosticServices TheFacultyofVeterinaryScience TheUniversityofSydney

PCV Plasma protein Haemoglobin Erythrocytes MCV MCHC MCH Fibrinogen Leukocytes Neutrophils Lymphocytes Monocytes Eosinophils Basophils Platelets Reticulocytes
*

L/L g/L g/L x10 /L fl g/L pg g/L x10 /L Seg.cells/10 /L

9 9 9 12

DOG 0.370.55 5575 100150 57 6075 300350 2025 24 712 4.069.36 0.913.6 0.210.96 0.141.2 00.36 200900 01.5

CAT 0.300.45 5978 80140 610 4045 310350 1317 13 814 3.7610.08 00.42 1.67.0 0.080.56 0.161.4 00.14 300700 01.0

HORSE (adult) 0.320.52 5884 110160 811 4149 300360 1316 24 6.013.0 2.476.96 00.24 1.65.4 0.00.72 0.160.96 00.36 100300 0

bandcells/10 /L 00.24 cells/10 /L cells/10 /L cells/10 /L cells/10 /L x10 /L %

9 9 9 9 9

Theremaybemarkedvariationsinnormalvaluesduetoage,sex,breedanduse.Theseare crudeapproximationsonly.Theyarenotnecessarilyapplicabletoresultsfromother laboratories.

271

8 THE HAEMOCYTOMETER
The improved Neubauer haemocytometer is used here (see Figure 1). A special optical plane coverslip must be used which is thicker than an ordinary coverslip. The depth between the coverslip and the ruled area is 0.1 mm. The ruled area is 9 mm2 and is divided into nine large squares. Each of the four large corner squares is divided into 25 smaller squares. The central large square is divided into 400 tiny squares arranged in 25 groups of 16 by triple boundary lines. There are two ruled areas per haemocytometer separated by a moat. When filling a haemocytometer the clean coverslip is placed on the clean haemocytometer and the diluted sample run in each side. The chamber must be filled quickly (i.e. reduce error in distribution) and must fill the raised area completely without the sample flowing into the moat. When counting, those cells touching or lying on the upper or left boundaries of the squares are included while those on the right and lower boundaries disregarded (or vice versa). For cell counts it is the usual routine for both chambers to be counted and the mean count used for the calculation. The total volume of the ruled area is 0.9 l (mm3) with each large square 0.1 l. From these values and the dilution factor it is a simple matter to calculate the cells per litre of blood. For Leukocytes (when using Unopettes) this is: count x 10 x D.F. x 106/Litre 9 10 corrects to one l. 9 D.F. (Dilution factor) = 100 106 brings the total volume to one litre A simple calculation although not totally correct is: count + 10% x 109/Litre 10 For Erythrocytes the volume counted is 5/25 of 0.1l (i.e. 1/ 50l). The calculation is thus: count x 50 x D.F. x 106/Litre if D.F. = 200 then Put simply count x 1012/Litre 100 The same approach may be used for all other cells counts using the haemocytometer. count x 50 x 200 x 106

272

FIGURE1

273

274

MICRO HAEMATOCRIT SCALE

275

276

URINALYSIS
1 INTRODUCTION
Urinalysis should always be performed when a urinary tract disorder is suspected. Also, it is useful in investigating and confirming many extra renal problems. Urinalysis should be performed completely as the results are often interrelated, e.g. the chemical detection of blood and the microscopic examination of sediment for the presence of erythrocytes. The laboratory procedure for urinalysis in VPDS is a standard one and involves:

2 OBSERVATION OF PHYSICAL PROPERTIES

For this, 10 ml of urine (well mixed as for all analyses) is placed in a centrifuge tube and examined for clarity (appearance), colour, odour and foam. Before the urine is sent to the laboratory, a note is made of the volume collected so that the results can be interpreted in relationship to that volume.

3 ESTIMATION OF SOLUTE CONCENTRATION

Generally this involves the determination of specific gravity by using the refractometer. If the urine is markedly turbid it should be cleared by centrifugation as cells, crystals and large macromolecules refract a disproportionate amount of light when compared to their true effect on specific gravity. Remember protein and glucose (see later and lecture notes) will also elevate urine specific gravity up to about 4 units. An estimation of solute concentration, by determining specific gravity, is essential before interpreting the results of chemical and microscopic examination.

4 CHEMICAL ANALYSIS
Generally this involves reagent strips and tablet methods, which are semi-quantitative and as such, are really only useful as screening tests. Instructions for these tests are apt to change from time to time. Therefore, the instructions that accompany the test should be read carefully. Multistix 10 SG: The reagent strips containing multiple squares are dipped into the urine and read at specific times, glucose 30 seconds later, bilirubin 30 seconds later ketones 40 seconds later, blood 60 seconds later pH & Protein 60 seconds later (The squares for specific gravity, urobilinogen, nitrite and leukocytes are of little use) NB. Care is to taken on removing the strips from the urine sample so that excess does not interfere with the different reagent squares.

277

The pH square may be confirmed by pH paper as an alkaline buffer in the protein test strip may flow over onto the pH strip and interfere with the result. If the test for protein records a trace or above, the salicylsulphic acid test is performed (add 1 volume of supernatant of centrifuged urine to 2 volumes of salicylsulphonic acid in a tube. Assess the cloudiness and precipitate). The Ames Multistix test depends upon pH for reaction and sometimes an alkaline pH will give false positives due to quaternary ammonium salts. Result negative Trace + ++ +++ ++++ Appearance no change mild cloudiness moderate cloudiness cloudiness and fine precipitate cloudiness and moderate precipitate cloudiness and heavy precipitate Approximate value <0.05 gms/L 0.20 0.50 2.00 5.00 10.00 gms/L

If there is any doubt to the actual value or a more accurate value is required, then the urinary protein can be determined by analytical (Spectrophotometerical) methods. If the test for glucose records a trace or above, or if it is negative and the test for ketone bodies is positive, the Clinitest can be performed (to a round bottom tube, 5 drops of urine and 10 drops of water are added; a clinitest tablet is added; after the effervescence has stopped, the tube is shaken and colour compared to that on a chart). Clinitest is less sensitive for glucose, sensitive to other reducing substances, and is not affected by ketones. It may give false positives with large amounts of ascorbic acid which may also depress the colour development in the Multistix The ketone reagent square can be double checked by the Acetest. Place a tablet on clean white paper after resealing the bottle. Place one drop of urine on the tablet. After 30 seconds compare colour of tablet to colour chart provided. False positives can be caused by L.dopa, BSP and large quantities of phenylketones. Blood - large amounts of ascorbic acid may inhibit the reaction and cause false negatives. Myoglobin causes false positives and some contaminating oxidizing agents can give false positives. Results are expressed as per the label on the Multistix bottle, except for protein which is explained above and glucose and ketone if clinitest and acetest are used. Ictotest: A test for bilirubin. (sometimes it is difficult to read the bilirubin reagent square). Ten drops of urine are dropped onto an absorbent test mat. A reagent tablet is placed on the mat, one drop of water is placed on the tablet, then after 5 seconds another drop of water is added so as to overflow onto the absorbent mat. After 60 seconds the colour of the mat around the tablet is noted. Results are expressed as; negative - no colour change, or orange is a negative. positive - Any formation of a purple colour is a positive for bilirubin.

278

5 SEDIMENT EXAMINATION
10 ml of urine is centrifuged for 5 minutes at 400-500G. The supernatant is removed by inverting the tube. The sediment is mixed with the remaining supernatant (about 0.5 ml) and examined (if the urine is already turbid, concentrated, centrifugation may not be necessary to examine the sediment). One drop of sediment is placed on one end of a slide and two drops on the other end. To the one drop of sediment add one drop of either toluidine blue, nile blue or new methylene blue, mix and overlay with a cover slip. Add a coverslip to the other end of the slide. The unstained preparation should be examined first, preferably under low power with reduced light (drop the condenser and close the iris diaphragm. Objects will appear refractile). Casts are examined under low power (10 x objective), > 1 in a moderately concentrated urine is usually considered abnormal. Cells are examined and counted (average of 4-5 scattered H.P. fields) under dry high power (40 x objective). Erythrocytes >5 per high power field is suggestive of haemorrhage, leukocytes >3 per high power field is suggestive of active inflammation (moderately concentrated urine may be up to 5 if voided urine). Other elements, such as crystals, fat droplets, etc., should be noted. The stained preparation should be used to further identify structures seen in the unstained preparation.

279

280

CYTOLOGY
1 SOLID TISSUE CYTOLOGY (surface lesions, biopsies, autopsy material)
a) Tissue impression (imprint)
Useful only for soft tissues that are exposed (biopsies, ulcerated skin lesions, and necropsy material). The surface must be cleaned of all debris and as much blood as possible. The tissue can be touched onto the cleaned slide or vice versa. Two or three impressions can be placed on the same side.

b)

Tissue scraping

Useful for exposed, hard as well as soft tissues (e.g. connective tissue tumour) and poorly accessible areas (e.g. conjunctiva). Once again, the surface must be cleaned. Scrapings can be done with a variety of instruments to suit the tissue (e.g. spatula, scalpel blade). The surface is gently scraped a few times and the material transferred to a cleaned slide. The material can be squashed between 2 slides or smeared (as for a peripheral blood smear) using a slide or the scraping instrument.

c)

Fine needle aspirate

Useful for non-ulcerated surface masses or internal organs/masses in the living animal. The size of the needle used will vary depending on the site and the species but commonly a 21-23 gauge is used. Aspiration must be done carefully so as not to cause excess haemorrhage. Once blood appears in the syringe stop aspirating. It is important to aspirate from several sites in the mass so as to acquire a representative sample. The material, if ample, can be dropped on a slide after removing the needle from the syringe and smeared as for a peripheral blood smear or squashed between 2 slides. Occasionally, little material is obtained via aspiration and most is in the hub of the needle. To obtain a smear from this situation either: (a) disengage the needle and fill the syringe with air. Reconnect the needle and blow the contents onto the end of a slide. A smear is then made as for a peripheral blood smear or as for a squash preparation. (b) Disengage the needle and aspirate some saline in the syringe. The saline can be used to remove the contents from the needle which can then be smeared. The disadvantage of this method is that the contents are diluted.
*

ACYTOLOGICALSMEARSHOULDBEAMONOLAYERANDHAVEMINIMAL DAMAGETOTHECELLSIFITISTOBEDIAGNOSTIC.

281

CYTOPATHOLOGY suggested approach to diagnosis when examining a solid tissue cytological preparation 1.Detect 2.Describe 3.Deducegeneraltothespecific
Detect, Describe (select an area that is a monolayer with minimal damage; avoid thick areas) Low power (4x, 10x and 20x objectives) more for scanning to detect the right area; little description o Areas of cellularity are they different in appearance, if so, note down? o Acellular areas? o Unusual colours? o Blood contamination? High Power (100x oil objective; 40x objective can only be used with coverslip) for most description o Appearance of constituent cells (nuclear and cytoplasmic detail) o Appearance of any inflammatory or abnormal cells (beware of artefactual changes to damaged cells) o Appearance of any acellular material o Can you detect agents of disease? o Is there foreign material present? Deduce (on the basis of description and clinical information) What basic pathological processes are occurring? o Degeneration and necrosis o Inflammation o Disorder of growth (e.g. neoplasia) o Vascular disturbance (usually impossible to determine and included under degeneration) o Pigments/deposits The first three are most important. Often there is more than one basic pathological process occurring so: Which pathological process is most important (basis for first part of morphological diagnosis)? If it is degeneration/necrosis then further questions are: Is it a primary lesion (cyst, haemorrhage)? Could it be related to any inflammation present? Could it be secondary to a disorder of growth? If it is inflammatory then further questions are: Is it acute (active), chronic or both (cell types determine this)? If chronic, is it granulomatous? Can you detect a cause? o Physical (e.g. foreign material) o Pathogen o Chemical (unlikely to detect cytologically, but history may suggest) o Immune-mediated? o Genetic (history might suggest predisposition)

282

If it is a disorder of growth then further questions are: What cell type is it? Round, epithelial or spindle? Is it hyperplasia/benign neoplasia? Is it malignant (on cellular detail and clinical information)? Cause is debatable, but familial predisposition (genetic), chemical, physical and pathogens may be considered on all information

2 FLUID CYTOLOGY
(CSF, synovial, body cavity effusions, tracheal aspirate, fluid masses)
The material is usually aspirated if the fluid has a high content of cells, smears can be made directly from the fluid; if the cells are low to moderate in content, the fluid* should be first centrifuged (400-500G) and the plug of cells resuspended in a small amount of fluid. If cell counts are to be performed, the latter method is done after those procedures. For both, it is best to smear as for a peripheral blood smear after some of the fluid has been taken up in a microhaematocrit tube.
*

THEFLUIDSHOULDBEPLACEDINEDTATOPREVENTPOSSIBLECLOTTING

NB. Cells in fluid deteriorate rapidly; therefore smears have to be made quickly. ALLCYTOLOGICALPREPARATIONSHAVETOBEFIXEDANDSTAINED.IFTHESOLUTIONS ARETOBEMETHANOLANDGIEMSATHENTHESLIDESSHOULDBERAPIDLYAIRDRIED FIRST(LIKEWISEFOR"DIFFQUIK").
*

3 VAGINAL CYTOLOGY
Samples are obtained from the anterior vagina either using a cotton swab stick or blunt spatula. If a cotton swab stick has been used it is gently rolled over the surface of a cleaned slide and the smear fixed and stained either by the Giemsa method or with Shorr stain. For the dog, the following characteristics are noticed during the different stages of the oestrous cycle. 1. Anoestrus - Non-keratinized round epithelial cells (parabasal and some intermediate). Minimal debris. Neutrophils may be present but less than in dioestrus. Occasional erythrocytes. Pro-oestrus - keratinized epithelia begin to appear (superficial cells with or without nuclei). Neutrophils decrease. Erythrocytes usually present. Debris and bacteria present. Oestrus - nearly all epithelial cells are superficial, keratinized. Debris minimal. Neutrophils are absent until the final one or two days. Erythrocytes are absent after the first couple of days. Dioestrus (metoestrus) - abundance of neutrophils. Reappearance of round nonkeratinized cells (parabasal and intermediate cells).

2.

3.

4.

For the cat, epithelial cell types are similar to those seen in the dog. In the follicular stage (pro-oestrus and oestrus) there is a gradual increase in the proportion of anucleated superficial cells (keratinized) and a drop in intermediate and parabasal cells. Erythrocytes may be seen. In the luteal phase (dioestrus), anucleated superficial cells disappear and intermediate and parabasal cells increase. In anoestrus intermediate and parabasal cells predominate. Neutrophils may be seen.

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4 SEMEN ANALYSIS
Fertility examination of the male involves semen examination. The results of semen examination vary with the method of collection. Basically, semen examination aims to evaluate the concentration, morphology and viability of the spermatozoa. Seminal fluid as such is of secondary importance. Despite species differences, laboratory methods of semen analysis are standardised and include the following: a. Gross examination - volume, colour, density, foreign material. i. ii. Volume. Graduated collecting vessels should be used. Colour, density. Semen varies from watery white to milky to creamy depending on the concentration of spermatozoa (e.g. creamy is equivalent to greater than 2 million per l). Certain bulls deliver semen of yellowish-green colour. Foreign material. Semen should contain only spermatozoa and occasional leukocytes. Wave motion. This refers to mass activity of the sperm and is a reflection of both the concentration and motility of sperm. Dog sperm has no or limited wave motion. Motility. The percentage of sperm motile is assessed on a warmed stage of a microscope. Live-dead differentiation. This is done by using selective stains such as eosin-nigrosin. Spermatozoal morphology. Generally in semen with adequate concentration and motility, the majority of spermatozoa will have normal morphology. Certain abnormalities will appear in a proportion of spermatozoa even in normal semen. Consequently, it is important to recognise not only the type of abnormalities but also the number of spermatozoa affected.

iii. b.

Microscopic examination. i.

ii. iii. iv.

Primary abnormalities reflect disturbances of spermatozoal production and include head abnormalities, mid-piece abnormalities and coiled tails. Secondary abnormalities reflect disturbances during storage of the spermatozoa in the epididymis or during the process of ejaculation. These include loose normal heads (i.e. tail-less heads), detachment of Galea Capitus, proximal droplets and bent tails. c. Spermatozoa concentration. This is done by direct cell counting in a chamber.

TABLE:Minimumstandardsforsemen Volume (mls) Bull 2 Stallion 50100 Boar 100 Ram 1 Dog 5 *NotAvailable Concentration (l) 50,000 10,000 10,000 100,000 10,000 Motility Primary >50% >50% >50% >50% >50% <15% <15% <15% <10% <15% Secondary abnormalities <15% <6% NA* NA NA

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FAECAL ANALYSIS
1 INTRODUCTION
Faeces, although unpleasant to examine, can provide important information about digestive tract disorders. To obtain the maximum amount of information from faeces, examination of a fresh stool is essential, especially as certain chemical characteristics may change with aging. Rectal samples may be necessary for certain parasitological tests. All tests should be interpreted in light of the diet of the animal. The technique of faecal examination employed in the Veterinary Clinical Pathology Laboratory at The University of Sydney involves the following:

2 GROSS EXAMINATION
Note the amount, colour and odour of faeces. Examine for worms, blood and unusual foreign material.

3 MICROSCOPIC EXAMINATION
a. b. Examination for Parasites (Metazoan parasites and Protozoa). (See Parasitology course). A faecal smear can be stained with Giemsa /Diff Quik and examined for the presence of cells, microorganisms, etc. For instance, the presence of many neutrophils may indicate active inflammation of the colon or rectum. Chemical examination (The values presented are applicable to the dog and probably to the cat).

c.

Add a small amount of faeces, about as big as a match-head, to each of 4 slides and emulsify with a drop of saline (not necessary if the stool is liquid). (i) for starch: Add a few drops of Lugol's iodine to the emulsified faeces on one slide and mix. Examine under low power (10X objective) for blue-black starch granules, with the condensor fully open. Greater than 3 granules per field (count a few scattered fields and determine the average) is considered abnormal but normal dogs and cats can show higher numbers at times. for protein (muscle fibres): Add a few drops of 1% alcoholic eosin to the emulsified faeces on one slide and mix. Examine first under low power (10X objective) with the condensor slightly dropped and closed to find the fibres and then use high power (40X objective) for the presence of muscle with visible cross striations. NB. Lugol's iodine will stain undigested muscle fibres.

(ii)

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(iii)

for fat: Fat in the faeces may be in the form of neutral fats, fatty acids and soaps (the last two are called split fats). To determine the component proportions, add a few drops of Sudan IIIA (alcoholic stain) to the emulsified faeces on one slide, and a few drops of Sudan IIIB (acetic acid stain) to the emulsified faeces on the other slide, and mix. Sudan IIIA will stain the neutral fats, which are in the form of droplets and plaques, an orange-red. Fatty acids, which are in the form of droplets, plaques and needles, and soaps, which are in the form of plaques, needles and crescents, are colourless with Sudan IIIA. Normal faeces have less than 10 droplets, smaller than 10 m, per high powered field (40 x objective). NB. This test is inconsistent. On heating the Sudan IIIB faecal mixture (warm the slide over a bunsen burner until it is just too hot to touch), much of the fat will melt and stain as deep orange globules, i.e. both the neutral fat and free fatty acids will stain. The soaps are melted on heating because of the presence of the acetic acid, but do not stain. When the slide cools the orange droplets of fatty acids transform to needles and spicules. Colourless droplets of soaps also transform into needles and spicules. Neutral fat droplets remain unchanged. In normal faeces less than 10 droplets, smaller than 20 m, are usually visible per high powered field. 0.5% Nile Blue is another stain that can be used for faecal fat. It stains droplets and plaques of neutral fat red and plaques of fatty acids blue-violet (all the rest are colourless). Although the results are not always easy to interpret, generally (at least for the dog) fat maldigestion leads to a greater increase in neutral fats rather than split fats, while fat malabsorption increases split fats more than neutral fats. Ideally, the results should be supported by fat absorption tests.

Results expressed as: Starch Normal <3 + ++ +++ ++++ r = rounded wd = well digested

Protein r/wd

Fat-unsplit <10 droplets of <10m

Fat-split <10 droplets of<20m

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4 HEMATEST - FOR OCCULT BLOOD

Will react with myoglobin, haemoglobin or dietary substances having peroxidase-like activity. This is not a very good test and may be later replaced.

5 EXAMINATION FOR TRYPSIN (PROTEASE)

Diseases of the exocrine portion of the pancreas may cause a reduction in the output of trypsin into the faeces. Little or no trypsin in the faeces mean severe exocrine pancreatic deficiency. However, normal animals may have low trypsin on single sampling; multiple samples are, therefore, sometimes necessary to confirm a case of exocrine pancreatic insufficiency. A simple screening test that can be employed for the detection of trypsin involves the use of x-ray film (a gelatin digestion test). One gram of well-mixed faeces is emulsified with 9 ml of 1% sodium carbonate in a centrifuge tube to give a 1/10 dilution. From the 1/10 dilution, after it has settled, 1/20, 1/100, 1/200, 1/1000, 1/2000 dilutions are prepared. A square of x-ray film is divided into smaller squares and 1 drop of sodium carbonate (blank) and 1 drop of each dilution of faeces are placed in separate squares. An alternative method is the placement of strips of x-ray film directly into tubes containing the different dilutions. The x-ray film is incubated at 37C for 30-60 minutes (or room temperature for 2-4 hrs). The x-ray film is allowed to harden (place in a refrigerator for 5 minutes) and washed with a gentle stream of water. The titre of trypsin is designated as the highest dilution showing complete digestion of the gelatin (i.e. when the blue celluloid backing becomes totally visible). NB. instead of a square of x-ray film, individual strips can be substituted and immersed in the tubes containing the dilutions of faeces - 'tube gelatin digestion test'. Interpretation: The titre of trypsin should be interpreted in light of the findings of the microscopic examination for undigested food particles. A titre of 1/100 or greater with minimal undigested food particles suggests that the animal does not have exocrine pancreatic insufficiency. A titre of less than 1/100 with abnormal numbers of undigested food particles (starch/muscle/neutral fats) is suggestive of exocrine pancreatic insufficiency. A titre of less than 1/100 but with minimal undigested food particles is inconclusive, and the faeces should be retested at least three times before making a decision. NB. A normal dog can have a trypsin titre from 1/20 to 1/2000 (may be lower). More sophisticated tests for the detection of faecal trypsin are available but restricted to laboratories. In addition, for the dog and cat, tests for the presence of trypsin-like immunoreactivity in the serum are available in laboratories, and which do not have the limitations of trypsin.

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6 FAECAL STAIN
Lugols Iodine Iodine crystals Potassium iodide Distilled water 1.0 gm 2.0 gm 100 mls

Grind solids together then dissolve in water.

Eosin Eosin yellow (alcohol & water sol.) 70 % ethanol Dissolve stain, then filter. 0.5 gm 100 mls

Sudan lllA (alcoholic) Sudan 111 95 % ethanol about 0.5 gm 45 mls

Heat in a water bath (not over flame) to 30-35C to dissolve stain then filter.

Sudan lllB (acetic acid sol.) Sudan 111 95 % ethanol Acetic acid(glacial) Mix then filter 0.2 gm 10 mls 90 mls

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URINARY CALCULI
1 INTRODUCTION
Calculi are solid concretions formed within ducts and hollow organs. Their physical presence may cause obstruction to the flow of secretions or excretions. There are several types of calculi; urinary, gall, intestinal, salivary and pancreatic. All/most are comprised of substances that are normally excreted. There are 3 main groups: 1) Simple - single constituent. 2) Mixed - two or more constituents - most common type. 3) Foreign body - rare usually start the formation i.e. nucleus such as fibrin. Urinary calculi (uroliths) may occur in the renal pelvis, ureter, bladder and urethra. They usually consist of an organic matrix and minerals, and if there is more than one main constituent they are referred to as mixed calculi. The type of constituent in a calculus is dependent upon 2 main factors: i) The species: urinary calculi in the dog and cat are commonly composed of MgNH4PO4.6H2O (MAP; struvite; "triple phosphate"). The horse has urinary calculi commonly composed of calcium carbonate with magnesium phosphate as a minor constituent. The pH of the urine: Certain types of calculi are more likely to occur in acid or alkaline urine, principally due to the effect on solubility of their constituents. However, some calculi occur in both acid and alkaline urine (e.g. struvite calculi). Urinarycalculimorelikelytobefoundin: Species Dog Cat Horse AcidUrine Oxalate Cystine UricAcid/Urates Cystine Oxalate UricAcid Silica (SiliconDioxide) AlkalineUrine CalciumCarbonate&Phosphate Struvite(MAP) AmmoniumUrate CalciumCarbonate&Phosphate Struvite(MAP) OtherPhosphates MagnesiumPhosphate CalciumCarbonate&Phosphate Struvite(MAP)

ii)

289

Other factors that are involved with urinary calculus formation include: i) Increased urinary output of one or more of the insoluble constituents

For example, in the dog phosphate stones may be associated with elevated excretion of calcium. Cystinuria or increased urinary levels of urates (especially in the Dalmation where there is an inherent breed failure to resorb urates present in the glomerular filtrate) may lead to calculus formation. However, concentrations of minerals in excess of their solubility don't always mean stone formation. Although all the reasons for this are not clear, it may be due in part to the colloidal properties of urinary proteins and polysaccharides stabilising large concentrations of minerals in solution and/or due to the presence of substances that form soluble complexes with the insoluble constituents of calculi, e.g. citrate stabilising calcium ions, thereby interfering with the formation of calcium phosphate/carbonate calculi. Hyperparathyroidism may cause enhanced deposition of calcium. ii) Infection

Bacterial degradation, principally of urea, leads to elevation of urinary pH. iii) Urinary stasis

Urinary stasis allows bacterial growth and the retention of substances that could act as nuclei for stone formation. Both ii) and iii) operate when there is interference to urine outflow. iv) Reduced water intake

Means concentrated urine in a healthy animal i.e. a supersaturated state is reached. This is common with calcium oxalate but a nucleus is still required. v) Nutrition

Ruminant urolithiasis is thought to be largely nutritional in origin. Vitamin A deficiency has been implicated in urate calculi in Dalmations. Not all the factors involved with urinary calculus formation are known. And it is quite obvious that those factors which are recognised, are not fully understood. Most probably, urinary calculus formation is dependent on a combination of factors rather than any single one.

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2 PRACTICAL EXAMINATION OF CALCULI

The constituents of urinary calculi can be determined using simple methods. The results may be useful in treating the condition, e.g. acidifying the urine for struvite (MAP) calculi.

a)
i)

Physical examination
Shape and form of urinary calculi With distilled water, wash the calculus free of debris. Dry and examine. Many bladder calculi have characteristic physical appearances depending on their constituents but there is overlap, therefore, other types of analyses may be necessary at times, e.g. heat, chemical analysis. Chemical analysis is always necessary for mixed calculi. Phosphate Calculi May be: (i) a large single calculus with a coral like surface e.g. struvite, calcium phosphate or a mixture of phosphates. (ii) numerous, variably sized, tetrahedral, smooth stones e.g. struvite. NB. Struvite (MAP) calculi in the cat are usually in the form of "sand", microcalculi. (iii) small, spherical stones which may be impossible to distinguish from cystine stones by naked eye examination. Phosphate calculi are usually white or cream and have a chalk-like appearance when broken i.e. the softest when broken. Phosphate calculi commonly occur in alkaline pH when there is infection of the urinary tract.

Cystine Calculi These are usually small, spherical, cream to yellow, smooth stones and waxy in appearance. Occasionally they may be brownish yellow to light green. Oxalate Calculi These are usually small. The dihydrate form of calcium oxalate calculi may have large shelflike projections from its surface. Because of this rough surface, the calculi may be blood stained. Other forms of oxalate usually produce smooth calculi. Oxalate stones are variable in colour, may be brittle and have obvious crystals when crushed. Calcium oxalate is the hardest stone and therefore is not so easily crushed. It is usually the chief constituent of the small stones from the renal pelvis.

Urate Calculi These are usually in the form of grey to yellow, highly polished spherical stones which when broken consist of thin concentric crystal layers.

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Carbonate Calculi Carbonate calculi are dull white to red-brown, chalky and usually hard. They often occur as large, spherical to ovoid calculi, having a coral-like surface. ii) Heat The calculus is split and checked to see if it is homogeneous. If different layers are apparent all should be analysed. Some of the stone is ground using a mortar and pestle. The ground stone is then burnt in a flame. Organic elements will tend to burn and disappear. Inorganic elements will remain. NB. nearly all calculi have some organic components. If there is some smoke and a "burnt protein" (sulphurous) smell, the calculus is probably composed of cystine. If there is a tongue-like flame and a small residue, it could be oxalate. If there is an ammonia smell and a small residue, it is probably composed or urates. MAP (struvite) calculi burn very little and usually leave a grey fused mass. The organic residues may be used for chemical analysis but as certain inorganic compounds may change form during heating, it is probably best to use freshly ground stone.

b)

Chemical Analysis (Based on the Merck Urinary Calculus Analysis)

i) ii) iii) iv) Grind sample of calculus very finely and mix well. Transfer a full white spatula to the red boat; add 5 drops of Reagent 1 (Conc. Sulphuric Acid) and stir to achieve complete dissolution. Foaming indicates carbonate. Transfer sample solution in the boat into the plastic graduated beaker, which is filled to one third with distilled water. Make up to 50 mls with distilled water, and stir several times with the boat. To carry out individual analyses, fill the reaction vessels with sample solution up to the 5ml calibration mark (except for magnesium which requires only 1ml of sample solution). Calcium (5ml). Add 2 drops of Reagent 2 (27% Sodium Hydroxide) while shaking. Then add 1 white spatula full of Reagent 3 (Calcon carboxylic acid) and add Reagent 4 (Titriplex III solution) drop by drop until the colour of the solution changes from red to blue. Count the drops required: the number of drops required, multiplied by 5, gives the percentage of calcium content. vii) Oxalate (5ml). Add while shaking, 2 drops of Reagent 5 (Borate Buffer solution), 3 drops of Reagent 6 (Ferric Chloride solution), and 3 drops of Reagent 7 (Sulphosalicylic acid). After 2 minutes, carry out colour comparison on the colour chart. viii) Ammonium (5ml). Add, while shaking, 3 drops of Reagent 8, (Potassium mercury iodide), and 3 drops of Reagent 2 (27% Sodium Hydroxide). Carry out colour comparison on the colour chart.

v)

vi)

292

ix)

Phosphate (5ml). Add, while shaking, 5 drops of Reagent 9 (Ammonium molybdate) and 5 drops of Reagent 10 (Reduction solution). After 5 minutes carry out colour comparison on the colour chart.

x)

Magnesium (1ml). Pipette 1 ml of sample solution into a reaction vessel and make up to the 5ml calibration mark with distilled water. Add, while shaking, 10 drops of Reagent 11 (Buffer solution) and 10 drops of Reagent 12 (Dye solution). After 1 minute, carry out colour comparison on the colour chart.

xi)

Uric Acid (5ml). Add 3 drops of Reagent 13 (Molybdate phosphoric acid), shake and allow to stand for 2 minutes; then add 2 drops of Reagent 5 (Borate buffer solution) and shake. Within 10 seconds, carry out colour comparison.

xii)

Cystine (5ml). Add, while shaking, 10 drops of Reagent 14 (Ammonia solution) and 1 red dosingspoonful of Reagent 15 (Reducing agent). After 1 minute, add 1 black dosingspoonful of Reagent 16 (Sodium nitroprusside) and shake. After 30 seconds, carry out colour comparison on the colour chart.

293

Veterinary Historical Notes

Arthur William Turner OBE DVSc DSc 1900-1989

Arthur Turner was born, educated and worked for most of his career in scientific research in Melbourne. He graduated BVSc (Hons) in 1923 at the University of Melbourne where he was appointed to the teaching staff of the Veterinary school. During that year he qualified for the higher degree of MVSc for research built on the work of Dr HE Albiston on infectious necrotic hepatitis of sheep (black disease). In 1924, he became Walter and Eliza Hall Fellow in Veterinary Science. He was awarded a Rockefeller Foundation Fellowship in 1926 and studied at the Pasteur Institute in Paris. There he received training and inspiration such as Dr JA Gilruth had received at that Institute at a similar age. In Paris, he continued his studies on black disease and ultimately developed an effective vaccine for the disease, which was responsible for heavy losses of sheep in eastern Victoria and southern New South Wales. In 1928, before returning to Australia to accept an invitation to join the staff of the newly-formed Animal Health Division of the Council for Scientific Research and Industry, he spent a period at the Institute of Animal Pathology at the University of Cambridge. On his return, Gilruth, who was Chief of the division of Animal Health, appointed Turner as Officer in Charge of the new laboratory at Oonoonba, Queensland. Here he was introduced to an environment where contagious bovine pleuropneumonia (CBPP) was enzootic and where nutritional and protozoan diseases of cattle required investigation. He established the foundation for a lifetime interest in CBPP and his studies on the causal organism, serological diagnosis, vaccination and the general pathology of the disease were invaluable in this country and overseas. In 1930, Turner was awarded the degree of DVSc for his outstanding research on black disease by the University of Melbourne, where he was subsequently awarded the degree of DSc. Returning to Melbourne in 1935, where Dr LB Bull had succeeded Gilruth as Chief of the Division of Animal Health of CSIRO was headed by Dr LB Bull at Parkville, Turner was appointed Officer in Charge of the Parkville laboratory. Always irked by the interruption of his research by administrative matters, Turner transferred the position to Dr TS Gregory and, until his retirement in 1960, spent his time on his first love, research. He earned a unique place in the estimation of veterinarians throughout Commonwealth for his achievements. He was a Foundation Fellow of the Australian Academy of Science and a Foundation Life Fellow of the Australian College of Veterinary Scientists. He was awarded the Gilruth Prize and the Syme Prize, and was a Fellow of the AVA. RI Taylor March 2002
References Albiston HE. Obituary. Aust Vet Assoc News, 6 June 1990, p N223. Citation for the Gilruth Prize Aust Vet J 1958;34:262-264. Schedvin CB Shaping science and industry, a history of Australia's Council for CSIR 1926-1949. Allen & Unwin, Sydney. 1989.pp 137-139, 141-142. University Veterinary Publications

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