Hematology 1

The field of Clinical Hematology deals with the study of the cellular components (red blood cells, white blood cells, and
platelets) and the hemostatic elements (proclotting factors and inhibitors of coagulation) in order to aid in the diagnosis and
monitoring of diseases of the blood or other conditions that affect these different components. Hematological tests, primarily
the Complete Blood Count, offers a wealth of information to the clinician regarding various physiological processes.

In this module, the students will be introduced to the field of Hematology. Basic knowledge of hematology and
hematological tests will be laid out as the foundation for future lessons.

1. Define correctly the terms related to Clinical Hematology.

2. Organize completely the different hematological methods of examination.
3. Recite and explain the universal precaution & quality assurance program for clinical hematology.
4. Explain and differentiate among the different methods of blood collection
5. Enumerate the different additives used in blood collection & describe how each additive works.
6. Design accurately the color-coding scheme used to identify the presence or absence of additives in blood collection
tubes, name the additive, laboratory departments & individual tests associated with various color-coded tubes.
7. Recite thoroughly the “order of draw” when collecting multiple tubes & explain why it is important.
 Hematology 1
Overview of Hematology
• Hematology is defined as the study of blood, its development, and diseases associated with it
• It includes the study of RBCs (erythrocytes), WBCs (leukocytes), platelets (thrombocytes), and hemostatic elements in
plasma in order to provide a differential diagnosis

Functions of the blood:

1. Respiratory
- refers to the delivery of gases to and from the tissues
- Oxygen attach to hemoglobin in RBCs for delivery from the lungs to the peripheral tissues.
- Carbon dioxide is also transported by the blood from the tissues for elimination by the lungs
2. Homeostasis
- refers to the regulation of certain balances in the body.
- The blood is involved in a number of certain homeostatic mechanisms such as pH homeostasis and temperature
regulation
3. Nutritive
- refers to the delivery of nutrients to the tissues. These nutrients are dissolved in the plasma.
4. Excretory
- refers to the delivery of waste products from the tissues to the excretory organs for elimination from the body.
Ex. Non-protein nitrogenous wastes (NPNs) such as urea and creatinine are dissolved in plasma and filtered by the
kidneys
 Hematology 1
5. Immunity
- refers to the body’s defense against infectious organisms (Bacteria, Fungi, Parasites, or Viruses)
- White blood cells populations in the blood are play a protective role against a group of microorganisms
- Neutrophils protect against most bacterial agents by engulfing these bacteria, and releasing its granular contents,
which have anti-bacterial action
- Monocytes protect against a wide range of infectious agents by performing phagocytosis
- Eosinophils protect against helminthic infections
- Lymphocytes are active against most viruses and its subpopulation of B-lymphocytes differentiate into plasma cells
to produce antibodies
6. Water Regulation
- the blood is involved in maintaining the water levels in the body. Water is excreted or reabsorbed into the blood as
a function of plasma osmolality, which is the measure of solute concentration of the blood. Increased plasma
osmolality promotes water reabsorption in the kidneys. Decreased plasma osmolality promotes water excretion.
7. Transportation of Hormones
- Hormones are secreted by endocrine glands. The targets of endocrine hormones are distant from its source. Thus,
the hormones need to be dissolved in the plasma to reach its target tissue/organ
8. Hemostasis
- refers to the mechanism by which blood flow to an injured blood vessel is arrested in order to prevent excessive
blood loss.
- This occurs as a function of platelets (forming a platelet plug) and hemostatic elements in plasma (responsible for
clot formation and dissolution).
 Hematology 1
Common Hematological Tests
1. Complete Blood Count (CBC) - Composed of a panel of tests to evaluate the formed elements in the blood
- the CBC results offer a wealth of information regarding the health status of an individual
- each component of CBC is associated with different conditions (similar to the components of urinalysis)

Components of CBC:
1.1. Cell counts (RBC count, WBC count, Platelet count) – determines the quantities of circulating blood cells
- increases or decreases in each of the cell counts may reveal the presence of a disease process
1.2. Hemoglobin determination - quantitative measure of hemoglobin, the protein in RBCs responsible for binding
oxygen. Low levels of hemoglobin impair oxygen delivery to the tissues and are most often associated with
anemia
1.3. Hematocrit testing - determination of the amount of red blood cells in whole blood (expressed as a relative
quantity; ratio of red blood cells to whole blood in a sample)
1.4. WBC differential count: details the relative counts of each white blood cell population (Neutrophils, Lymphocytes,
Monocytes, Eosinophils, and Basophils)
- each WBC population is expressed as the percentage of white blood cells
1.5. RBC indices – values of these indices aid in making a specific diagnosis of anemia:
1.5.1. Mean Cell/Corpuscular Volume (MCV) – average volume of red blood cells in a sample
1.5.2. Mean Cell/Corpuscular Hemoglobin (MCH) – average amount of hemoglobin in each red blood cell in a
sample
1.5.3. Mean Cell/Corpuscular Hemoglobin Concentration (MCHC) - average hemoglobin content for a specified
volume of red blood cells
1.5.4. Reticulocyte Distribution Width (RDW) – quantitates the spread of RBC sizes in a sample. The greater the
RDW, the greater is the difference in RBC sizes in the sample.
 Hematology 1
2. Peripheral Blood Smear (PBS) Examination - performed to analyze the morphologic characteristics of RBCs, WBCs, and
Platelets
- abnormal morphologic characteristics may indicate the presence of disease processes
- Example: the presence of agranular platelets may be associated with Gray Platelet Syndrome, which also affects platelet
function

3. Bleeding Time and Coagulation Tests - these are tests used to evaluate hemostatic functions of the blood:
3.1. Bleeding Time: general screening test for hemostatic abnormalities
3.2. Prothrombin Time: coagulation test used to evaluate the extrinsic pathway
3.3. (Activated) Partial Thromboplastin Time: coagulation test used to evaluate the intrinsic pathway
3.4. Thrombin Time and Reptilase Time: coagulation tests used to evaluate the Fibrinogen function
3.5. Stypven Time / Dilute Russel Viper Venom Time: coagulation test used to evaluate the common pathway

4. Reticulocyte Count - Reticulocytes are immature red blood cells. These are normally found in the blood in small numbers
- Evaluation of its count reveals the bone marrow’s response to hypoxia

5. Erythrocyte Sedimentation Rate (ESR) - This test is a measure of the rate of fall of red blood cells within a packed column
- Traditionally, this test was used as a screening test for the presence of inflammatory processes
 Hematology 1
Physical Characteristics of Blood:
1. Fluid in vivo (due to balance between proclotting factors 4. Reaction: slightly alkaline
and anticoagulants) – Arterial blood pH: 7.35 - 7.45
2. Volume: 4-6 L (normal adult volume) or 75-85 mL per – Venous blood pH: 7.38 - 7.48
kilogram of body weight 5. Osmolality: 280-295 mOsm/kg
3. Viscosity: Viscous (3.5-4.5 times thicker than water) 6. Average specific gravity: 1.055

Composition of Blood:
1. Cellular Components (Formed Elements):
1.1. Red Blood Cells (Erythrocytes)
1.2. White Blood Cells (Leukocytes)
1.3. Platelets (Thrombocytes)
2. Liquid Component (called Plasma or Serum)
- composed mostly of water
- dissolved elements include carbohydrates, proteins,
lipids, non-protein nitrogenous compounds (NPNs),
vitamins, electrolytes, trace elements, hormones, and
gases.
 Hematology 1

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 Hematology 1
Differentiation between Plasma and Serum
Plasma Serum
Liquid part of anticoagulated and unclotted blood Liquid part of clotted blood
Hazy Clearer
All Coagulation Factors are present (Fresh Plasma): Clotting Factors that are consumed in Coagulation:
FI, FII, FV, FVII, FVIII:c, FIX, FX, FXI, FXII, FXIII, PK, HMWK FI / I (Fibrinogen)
FV / V (Proaccelerin)
FVIII:c / VIII:c (Anti-Hemophilic Factor A)
FXIII / XIII (Fibrin Stabilizing Factor)
Only residual amount (less than 20%) of FII / II
(Prothtrombin) is present
*F stands for Factor, the clotting factors are named as Roman numerals
 Hematology 1
Common Prefixes Used in Hematology Vocabulary
Prefix Meaning Prefix Meaning
a- / an- lack, without, absent, decreased micro- small
aniso- unequal, dissimilar myel(o)- (1) from bone marrow; (2) spinal cord
ante- before pan- all, overall, all-inclusive
crena- wrinkled phleb- vein
cyt- cell phago- Eat, ingest
dys- abnormal, difficult poikilo- Varied, irregular
erythro- red Poly- Many
ferr- Iron Pre- (pro-) Before
hemo- (hemato-) pertaining to the blood Pykno- Dense
hypo- beneath, under, deficient, decreased Reticulo- Netlike
hyper- above, beyond, extreme Schis- Split
iso- equal, alike, same Scler- Hard
leuk(o)- white Sidero- Iron
macro- large, long Spleen- Spleen
mega- large, giant Thrombo- Clot, thrombus
meta- (1) after, next; (2) change Xanth- Yellow
 Hematology 1
Common Suffixes Used in Hematology Vocabulary
Suffix Meaning Suffix Meaning
-algia Pain along a nerve -oma Swelling or tumor
-ase An enzyme -opathy Disease
-cide The killer of -osis (1) Abnormal increase; (2) disease
-crit To separate -penia Deficiency, decreased
-cyte Cell -phil(ic) Attracted to, affinity for
-ectomy Incision and removal -plasia (-plastic) Cell production or repair
-emia Blood -poiesis Cell production, formation, and
-it is Inflammation development
-lysis Destruction or dissolving -poietin Stimulates production
 Hematological terminologies
Hematology (Haematology) – the study of blood. It involves the study of blood cells and
coagulation. It includes the study of diseases associated with the blood, as well as the
reaction of the formed elements to the presence of disease.
Agglutination – clumping of cells
Aggregation – in blood coagulation, it is the clumping of platelets together in the formation of a
platelet plug
Anisochromia – variation in hemoglobin content of red blood cells
Anisocytosis – increased variation in size of red blood cells
Anticoagulant – substance that prevents clot formation; in vivo, includes natural anticoagulants
such as Heparin, Antithrombin III, and Proteins C and S; ex vivo, substances that are added
to a blood sample that inhibit clot formation either by binding with calcium, precipitation of
calcium, inhibition of thrombin, or removal of fibrin
Apoptosis – programmed cell death; process of ordered removal of organelles and cells
Arterial tap – process of blood collection by accessing an artery
Basophil – white blood cell morphologically characterized by coarse blue-black granules that
obscure the view of the nucleus; involved mainly in mediating allergic response
Blast – early stage of differentiation of a blood cell as it transitions from stem cell to a mature
cell. It is normally confined to the bone marrow and is the earliest recognizable stage of a
blood cell using Light Microscopy.
Blood Film – aka Peripheral Blood Smear; a stained smear of a drop of blood that, when
viewed through a microscope, produces additional morphologic information about the blood
cells
Bone Marrow – soft tissue found inside hollow bones responsible for production of blood cells.
Cellularity – expression of the amount of blood cells within the bone marrow
Chemotaxis – movement of white blood cells toward or away from the source of a chemical
gradient
Clotting factors – aka Proclotting Factors or Procoagulants; specialized proteins that, when
activated, form an interplay that effects coagulation
Coagulation – aka clot formation or clotting; process of clot formation through the interaction of
specialized proteins in plasma culminating in the conversion of fibrinogen to fibrin
Coagulopathy – hereditary or acquired abnormality of blood coagulation
Complete Blood Count (CBC) – test performed in the hematology laboratory that determines
red blood cell count, white blood cell count and differential count, platelet count, hemoglobin
concentration and hematocrit of a patient
Cytopenia – a reduction in number of one or more cell types in the blood
Deoxyhemoglobin – aka reduced hemoglobin; hemoglobin that is not carrying oxygen
Dyserythropoiesis – abnormal red blood cell development
Ecchymosis – (pl: ecchymoses); bruising caused by leakage of blood from blood vessels
EDTA – ethylenediaminetetraacetate / -tetraacetic acid; most common anticaoagulant used for
hematological studies, especially for CBC
Embolus – a blood clot that migrates through the blood stream and lodges into another vessel,
causing blockage of blood flow
Eosinophil – white blood cells morphologically characterized by bilobed nuclei and coarse
orange granules; involved mainly in anti-helminthic immune response and regulating allergic
response
Erythrocytes – aka red blood cells; cells that contains hemoglobin and carries oxygen through
the blood
Erythropoietin – glycoprotein hormone produced by the kidneys in response to tissue hypoxia;
targets red blood cell precursors in the bone marrow to stimulate proliferation and
maturation
Fibrinolysis – process of dissolution of the clot effected by the action of plasmin (fibrinolysin)
Hematoma – accumulation of blood in the tissues or cavities of the body
Hematocrit – relative expression of the amount of red blood cells in relation to the amount of
whole blood in a sample (in vitro) or total body fluids (in vivo)
Hematopoiesis / Hemopoiesis – involves the production, development, differentiation, and
maturation of blood cells in a blood-forming organ or tissue
Hemoglobin – oxygen-binding protein found within red blood cells
Hemolysis – inappropriate destruction of red blood cells
Hemorrhage – Excessive bleeding leading to leakage of blood from the vessels to the
surrounding tissues and brought about by a breakdown of hemostasis
Hemostasis – process of arresting blood flow to a site of vessel injury; also, the system that
keeps the blood in a fluid state under normal conditions
Hypoxia – lack of oxygen experienced by the tissues; physiologic stimulus for production of red
blood cells
Leukocytes – aka white blood cells; blood cells that are involved in the body’s immune
response
Lymphocytes – smallest of the white blood cells; morphologically characterized by a round
nucleus and scanty sky blue cytoplasm; involved in anti-viral immune response and antibody
production
Macrophages – Phagocytes found in tissues
Monocytes – white blood cells morphologically characterized by a round or irregularly-shaped
nucleus and agranular light blue cytoplasm; considered as professional phagocytes of the
blood; develop into macrophages in the tissues
Neutrophils – white blood cells morphologically characterized by multilobed nuclei and neutral
(lilac or purple) staining of the cytoplasm; involved mainly in anti-bacterial immune response
and inflammatory response
Oxyhemoglobin – hemoglobin with oxygen bound to it
Peripheral blood – blood that is contained within the circulatory system
Peripheral puncture – aka capillary puncture; process of blood collection via skin puncture
Petechiae – red pinpoint-sized hemorrhages of small capillaries in the skin or mucus
membranes
Phagocytosis – process of engulfment and destruction of foreign and unwanted material (such
as bacteria or senescent red blood cells)
Phlebotomy – process of blood collection
Plasma – liquid portion of unclotted blood or anticoagulated blood
Serum - liquid portion of clotted blood
Supravital stain – dyes employed in staining cellular elements while the cell is inits living state
(eg. New Methylene Blue, Brilliant Cresyl Blue)
Thrombocytes – aka platelets; cellular elements that are involved in promoting hemostasis
Thrombosis – inappropriate or pathological formation of a clot in an artery or vein
Thrombus – blood clot that usually develops in a deep vein of the body
Venipuncture – process of blood collection via intravenous access
Wright stain – a type of Romanowsky stain; most common stain used for studying blood cell
morphology

References:
Keohane, E. M., Smith, L. J., and Walenga, J. M. (Eds). (2016). Rodak’s Hematology:
Clinical Principles and Applications (5th ed.). SG: Elsevier Pte Ltd.
McPherson, R. A. and Pincus, M. R. (Eds). (2012). Henry’s Clinical Diagnosis and
Management by Laboratory Methods (22nd ed.). SG: Elsevier Pte Ltd.
Moore, G. W., Knight, G., and Blann, A. D. (2010). Fundamentals of Biomedical Science:
Haematology. Oxford, NY: Oxford University Press
Turgeon, M. L. (2012). Clinical Hematology: theory and procedures (5th ed.). Baltimore,
MD: Lippincott Williams and Wilkins
Stiene-Martin, E. A., Lotspeich-Steininger, C. A., and Koepke, J. A. (1998). Clinical
Hematology: principles, procedures, correlations (2nd ed.). Philadelphia, PA:
Lippincott-Raven Publishers
 Hematology 1
Specimen Collection and Specimen Considerations in Hematology
1. Patient Identification
- considered as the most critical step in Specimen collection
- Identify the patient by asking the patient to state his/her full name (including middle name); if the patient cannot
speak, verify the patient’s identity from the relative, attending physician, or nurse
- Whenever possible, verify a 2nd patient identifier (eg. Date of Birth / Social Security Number) for a positive patient
identification

2. Physiologic factors affecting test results:

Factor Effect/s
2.1. Posture Shift in posture from supine to erect may cause an increase in the levels of Lipids, Enzymes, and Proteins
2.2. Diurnal Rhythm In the afternoon, decreased levels of ACTH and cortisol and increased levels of eosinophils may be seen
2.3. Stress Causes an increase in WBC counts, FI, FV, FVIII:c, and FXIII
2.4. Exercise Causes an increase in WBC and Platelet counts, Creatine Kinase (CK), and Lactate Dehydrogenase (LD)
2.5. Diet After a fatty meal, increased Alkaline phosphatase (ALP) may be seen, false increase in hemoglobin
using the cyanmethemoglobin method
2.6. Smoking Increases cortisol and WBC counts; Chronic smoking may increase RBC count, hemoglobin, and
hematocrit
 Hematology 1
3. Sources of blood for Hematology testing:

3.1. Skin Puncture / Peripheral Puncture – source of peripheral blood (contains a mixture of capillary blood from the
capillaries, venous blood from the venules, arterial blood from the arteries, and interstitial and intercellular fluids)
- used when only small quantities of blood sample are required
3.1.1. Indications of Skin Puncture:
3.1.1.1. Infants/neonates/pediatric patients
3.1.1.2. Geriatric patients
3.1.1.3. Adults who are/have:
3.1.1.3.1. extremely obese;
3.1.1.3.2. a history of thromboembolism, stroke, Disseminated Intravascular Coagulation/Coagulopathy (DIC), or
Transient Ischemic Attack (TIA); or
3.1.1.3.3. extensive burns
3.1.1.4. If the examination requested necessitates skin puncture (bleeding time, micro methods of clotting time)
3.1.2. Recommended sites for skin puncture:
3.1.2.1. Distal portion on the palmar surface of the 3rd or 4th digits of the non-dominant hand
- the middle and ring fingers are usually the least used and are less calloused compared to the thumb
and index fingers.
3.1.2.2. For infants, the plantar surface of the foot/sole (heel portion).
(see shaded portion on the right)

www.slideshare.net/fatehiaawny/types-of-punctures
 Hematology 1
3.1.2.3. Traditionally, the free margin of the earlobe was used since there was less tissue juice and there were less
nerve endings causing less pain. This is no longer recommended due to less capillary access.
3.1.3. Important considerations when performing skin puncture:
3.1.3.1. Depth of puncture should be 2-3mm for adults; <2mm for infants and small children
3.1.3.2. Do not milk or squeeze the site of puncture to avoid contamination with interstitial fluids
3.1.3.3. Do not puncture cyanotic, swollen, inflamed, or edematous sites
3.1.3.4. You may warm the site by massage, warm compress, or warm water bath prior to disinfection
3.1.4. Order of Draw for Skin Puncture:
3.1.4.1. Tube for blood gas analysis
3.1.4.2. Slides
3.1.4.3. EDTA microcollection tubes (must be filled before other microcollection tubes to ensure adequate blood
volume for testing)
3.1.4.4. Other microcollection tubes with anticoagulants
3.1.4.5. Serum microcollection tubes

3.2. Venipuncture – source of venous blood; preferred when large volumes of blood are required for testing.
- preferred sites of puncture are the veins in the antecubital area: median cubital vein (vein of choice), cephalic
vein, and the basilic vein
3.2.1. Important considerations when performing venipuncture:
3.2.1.1. Angle between the skin and the needle: 15 degrees
3.2.1.2. Tourniquet application:
3.2.1.2.1. Distance from the venipuncture site should be 3-4inches (7.5-10cm)
3.2.1.2.2. Length of time should be at 1 minute (maximum)
 Hematology 1
-Prolonged tourniquet application causes hemoconcentration, hemolysis, and shortened coagulation
time.
-Release the tourniquet once blood enters the hub.
3.2.1.2.3. A phlebotomist must never puncture the patient more than twice. After the second attempt, the
phlebotomist must endorse to a more senior or more experienced staff.
3.2.1.2.4. Standard needle for phlebotomy: 21G
3.2.1.2.5. Most common needle lengths: 1.0 and 1.5 inches
3.2.1.2.6. Common causes of hemolysis:
3.2.1.2.6.1. Prolonged tourniquet application
3.2.1.2.6.2. Moisture or contamination on blood collection tubes
3.2.1.2.6.3. Using needles with too small bores
3.2.1.2.6.4. Excessive agitation during mixing of samples
3.2.1.2.6.5. Introduction of bubbles (frothing) during blood collection.

4. Additives
Additive Color Code Notes
Antiglycolytic Agents (MOA: inhibits glycolysis)
Sodium Gray Top Preserves glucose for up to 3 days
Fluoride For Blood glucose and blood alcohol level determinations
Anticoagulant used: Potassium Oxalate
Number of Inversions: 8
Lithium Gray Top Preserves glucose for up to 24 hours
Iodoacetate For Blood glucose and blood alcohol level determinations
Anticoagulant used: Potassium Heparin
Number of Inversions: 8
 Hematology 1
Clot Activators – accelerates clotting of samples
Glass tube or Red Top Used for STAT serum determinations
silica particles Number of Inversions: 5
Thrombin Orange Top MOA: Cleaves Fibrinogen into Fibrin
Used for STAT serum determinations
Number of Inversions: 5
Separator Gel – inert material that undergoes a temporary change in viscosity during the centrifugation process.
Thixotropic Gel Gold Top Number of Inversions: 5
Anticoagulants – prevent the blood from clotting
EDTA Lavender Top Optimal anticoagulant concentration: 1.5-2.2mg / mL of blood
MOA: chelates calcium
Used for routine hematological studies (most commonly used anticoagulant for
Hematology testing) – CBC, Reticulocyte Count, modified Westergren method of ESR
Important considerations:
• K2EDTA, K3EDTA, LiEDTA, or Na2EDTA can be used (K2EDTA is the most preferred)
but never use Na3EDTA
• Insufficient EDTA (overfilled tube) - effect: Presence of clots (or microclots)
• Excessive EDTA (underfilled tube)
effects: falsely decreased Hematocrit and ESR, degenerative changes in WBC, and
falsely increased MCHC and platelet count
• When platelet clumping (platelet aggregation) is encountered when examining the
smear, recollect a blood sample using 3.2% sodium citrate and repeat the platelet
count
Number of inversions: 8
Heparin Green Top Optimal anticoagulant concentration: 15-20 units / mL of blood
MOA: inhibits thrombin
 Hematology 1
Anticoagulant of choice for Osmotic Fragility Testing and Blood Gas Analysis
Also used for STAT Chemistry determinations
Three formulations:
1. Ammonium Heparin
2. Sodium Heparin
3. Lithium Heparin – causes the least interference in chemistry testing; most
commonly used anticoagulant for plasma and whole blood chemistry assays.
Important considerations:
• Lithium Heparin must not be used for lithium determinations
• Sodium Heparin must not be used for sodium determinations or electrolyte panel
• Ammonium Heparin must not be used for ammonia level determinations
• Heparin induces cellular clumping, especially of platelets, causing a false increase in
WBC count, and falsely decreased platelet count
• Not to be used for blood smear preparation because it causes morphologic
distortion of platelets and WBCs as well as causes background staining with
Romanowsky stains such as Wright’s and Giemsa
• Not to be used for coagulation studies because it inhibits Thrombin
Number of inversions: 8
3.2% sodium Light blue top Critical ratio of blood to anticoagulant: 9:1
citrate MOA: binds to calcium in an unionized form
Used for coagulation tests (eg. PT and PTT/APTT)
Important consideration: Forceful mixing and excessive inversions can activate
platelets and shorten clotting times
 Hematology 1
4.1. Order of draw:
Blood Collection Tube Number of inversions Color Code
Blood Culture Tubes (SPS) 8 Yellow Top
Tubes for Coagulation Testing 3-4 Light Blue Top
(3.2% sodium citrate)
Serum Tubes 0 Red – Glass (no additive)
5 Red – Plastic (clot activator)
Heparin Tubes 8 Green Top
EDTA Tube 8 Lavender Top
Sodium Fluoride Tubes 8 Gray Top

4.2. Other blood collection tubes:

Blood Collection Tube Anticoagulant Uses
Yellow Top Sodium polyanethol sulfonate Blood culture
Acid Citrate Dextrose Blood bank tests, paternity testing,
HLA testing, DNA testing
Tan Top (certified to contain less than K2EDTA Lead Determination
0.01 ug/mL of Lead)
Royal Blue Top (contains very low levels K2EDTA Trace Elements
of trace elements) Nutritional Chemistry
White Top K2EDTA with Gel Separator Molecular Tests
Black Top 3.8% Sodium citrate Westergren method of ESR
determination
Pink Top (with special crossmatch label K2EDTA Blood Bank Test (Crossmatching)
approced by AABB)
 Hematology 1
5. Sample preparations used in the Hematology Laboratory:
5.1. Whole blood: prepared by collecting blood into a tube with an anticoagulant. Whenever the tests require whole blood,
the sample must be well mixed prior to aliquoting. Used for Osmotic Fragility Testing and ESR testing
5.2. Plasma: prepared by collecting blood into a tube containing an anticoagulant, followed by centrifugation mixing
5.2.1. Platelet-Poor Plasma (PPP): contains less than 10,000 platelets/uL and is prepared by a hard/heavy spin
(centrifugation at 1500-2000G for 10 minutes). Used for coagulation tests.
5.2.2. Platelet-Rich Plasma (PRP): contains 200,000-300,000 platelets/uL and is prepared by a light/soft spin:
centrifugation at 50-100G for 10 minutes. Used for platelet function tests (platelet aggregometry)
5.3. Serum: prepared by collecting blood into a tube containing no anticoagulant. The blood is allowed to clot and then
centrifuged. The liquid portion is used for Prothrombin consumption test.
5.4. Blood Smear: Prepared by spreading a drop of blood over the surface of a slide. The smear is allowed to dry and stained
then examined microscopically. Used for Peripheral blood smear examination, WBC Differential Count and Reticulocyte
Count.
5.5. Buffy Coat Smear: Prepared in the same manner as a blood smear but makes use of the buffy coat for smearing. Used
for WBC Differential counting in leukopenic patients (WBCs are concentrated in the buffy coat), studies of abnormal
cell morphology (eg. Lupus Erythematosus cell or LE cell), and diagnosis of blood-borne parasitic infections.
5.6. Defibrinated blood: Blood is collected in a flask with glass beads or paper clips. The flask is continuously agitated until
a clot forms on the surface of the beads or clips. The clot is removed and the part that remains is defibrinated blood.
Used for special hematological tests.
5.7. Fresh blood as it comes from skin puncture: used for bleeding time and micro methods of clotting time.
 Hematology 1
References:
[1] Aceron, Z. B. (unpublished). Lecture notes
[2] Bain, B.J. (2011). Collection and handling of blood, In C. Jury, et. al. (Eds.) Dacie and Lewis’ Practical
haematology (pp 1-9). China: Churchill Livingstone
[3] Benett, S.T., et. al. (Eds.). (2007.) Laboratory hemostasis: A practical guide for pathologists. New York, NY:
Springer Science+Business Media, LLC
[4] Dinglasan, R. J. A. R. (unpublished). Review notes
[5] Henry, J.B. (2012). Preanalysis. In K.W. Sanford & R.A. McPherson (Eds.) Clinical diagnosis and management
by laboratory methods (pp 24-36). Singapore: Elsevier Pte. Ltd.
[6] Hillyer, C.D., et. al. (2009). Transfusion medicine and hemostasis: Clinical and laboratory aspects. New York,
NY: Elsevier
[7] Rodak, B.F., et. al. (2012). Laboratory evaluation of hemostasis. In G.A. Fritsma (Ed.) Hematology: Clinical
principles and correlations (pp 734-764) Singapore: Elsevier Pte. Ltd.
[8] Tricore Reference Laboratories (2011). Preparation of platelet poor plasma for coagulation testing. Retrieved
from http://www.tricore.org/Healthcare-Professionals/Test-Information/Testing-Protocols/Preparation-
of-Platelet-Poor-Plasma-for-Coagulatio
[9] Turgeon, M.L. (2012). Clinical hematology: theory and procedures. Baltimore, MD: Lippincott Williams and
Wilkins
[10] Zhou, L. & Schmaier, A.H. (2005). Description of procedures with the aim to develop standards in the field.
American Journal of Clinical Pathology, 123, 172-183. DOI: 10.1309/Y9EC63RW3XG1V313
 Specimen Considerations in Hematology 7. Gender: differences of reference values also exist
between men and women.
Specimen collection is a preanalytic variable that may have
serious implications in laboratory testing. According to Non-physiologic interferences.
studies done in recent years, 32-75% of testing errors occur
In vivo: Tobacco Smoking.
during the preanalytic phase.
Tobacco smokers generally have high blood concentrations
Pre-Collection variables. of carboxyhemoglobin, plasma catecholamines, and
Physiologic factors influence laboratory determinations. cortisol. These hormonal changes also result in lower
These include: eosinophil counts and higher neutrophil and monocyte
1. Diurnal variation: most commonly encountered counts. Chronic smoking leads to increase Hgb, RBC count,
when testing for hormones, ACP, and urinary MCV, and WBC count.
excretion of electrolytes. In vitro
2. Exercise: Physical activity may have both long- Collection-Associated Variables:
term and transient effects on the patient. Exercise 1. Hemolysis: mechanical hemolysis may occur as a
may activate coagulation, fibrinolysis and result of:
platelets, but such activation is due to increased a. difficult extraction,
metabolic activity and returns to normal (pre- b. needle bore size too small,
exercise) levels soon after cessation of exercise. c. pulling a plunger too fast,
3. Diet: An individual’s diet may greatly affect d. improper transfer of blood to the tubes,
laboratory test results, notably for blood chemistry e. vigorous shaking or mixing of tubes, or
determinations. f. performing venipuncture without allowing the
4. Stress: mental and physical stress has been alcohol to dry.
implicated in production of ACTH, cortisol, and 2. Hemoconcentration/Hemodilution: can occur due
catecholamines. Total cholesterol has been to positional changes. Such problems can be
reported to increase with stress while HDL-chole avoided by allowing the patient to sit in a supine
has been observed to decrease. Hyperventilation position 15-20 minutes prior to drawing blood.
increases WBC count. Tourniquet application must also not exceed one
5. Posture: supine or upright position of the patient (1) minute to avoid concentrating the blood. When
during specimen collection should be taken note of using anticoagulants, the order of draw,
as this could affect laboratory tests. Upright appropriate blood to anticoagulant ratio, and
position of the patient decreases plasma volume, adequate mixing should be observed.
thus there is an increase in plasma concentrations Non-Collection-Associated Variables:
of proteins. Hemoglobin concentrations of 1. In vivo hemolysis: may be due to hemolytic
patients may decrease after bed rest in the anemias or hemolytic transfusion reactions,
hospital. among others. In vivo hemolysis may be
6. Age: Generally, four age groups have been differentiated from collection-associated
defined: Newborn, Childhood to Puberty, Adult, hemolysis by observing the serum or plasma from
and Elderly Adults. Most tests have different subsequent repeat collections.
reference values with regard to each age group. 2. Lipemia: may be observed shortly after a high-fat
With some tests, the differences are more meal or due to high-fat diet.
pronounced with each age group, compared to 3. Icterus: may be observed in patients with high
others. concentrations of bilirubin (≥2.5 mg/dL)

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SPECIMEN COLLECTION d. Platelet aggregometry tests (using platelet rich
plasma++)
Anticoagulants used in the hematology laboratory: e. Platelet counts when platelet satellitism or
1. EDTA/versene/sequestrene: chelates calcium clumping is encountered
K2EDTA is the preferred form due to its solubility, 3. Heparin: accelerates antithrombin action to inhibit
followed by K3EDTA (or Li2EDTA), and Na2EDTA. thrombin formation; inhibits thromboplastin
Na3EDTA is not recommended due to its high pH. Use 15-30 units per mL of blood.
1.5-2.2 mg EDTA per mL of blood is the Uses:
recommended ratio. a. RBC count
Uses: b. RBC parameters
a. Cell counts (RBC, WBC, Platelets) c. Osmotic fragility
b. RBC parameters (Hgb, Hct) Disadvantages:
c. RBC indices (MCV, MCH, MCHC) a. Causes agglutination of WBC
d. ESR determination b. Causes aggregation of platelets
e. Peripheral blood smear* c. Not recommended for coagulation tests
f. Differential count* because it affects all stages of coagulation
Disadvantages: d. Produces a blue background with Wright’s
a. Not recommended for coagulation testing due stain
to interference of the anticoagulant with Fibrin e. Expensive
formation and instability of FV and FVIII:c in 4. Oxalate: chelates calcium
EDTA. May be used as Na2C2O4, Li2C2O4, or a mixture of
b. Using old (>2 hrs) blood will cause K2C2O4 and (NH4) 2C2O4.
morphological changes: K2C2O4-(NH4) 2C2O4 mixture (in a 2:3 ratio) is also
i. Crenation of RBCs known as double oxalate, balanced oxalate, Paul-
ii. Vacuolization of WBCs Heller’s fluid, or wintrobe fluid. It is prepared as a
iii. Formation of artifacts and crystals and mixture because K2C2O4 causes shrinkage of RBCs
phagocytosis of these crystals while (NH4) 2C2O4 causes swelling of RBCs.
iv. Nuclear changes in WBCs (eg. separated Uses:
PMN nuclei) a. Coagulation Testing
v. Disintegration of platelets
b. Lithium oxalate is used to prevent clotting of
c. May cause platelet satellitism occasionally bloody body fluids
2. Citrate: chelates calcium c. Double oxalate can be used for:
May be 0.105M (3.13%) or 0.109M (3.2%)
 RBC count
trisodium citrate (Na3C6H5O7•2H2O) in a 9:1 blood-
 RBC parameters
to-anticoagulant ratio. 0.129M (3.8%)
 ESR determination
concentrations are no longer recommended
because it affects coagulation test results.
Uses:
*smear has to be prepared within 2 hours of collection to
a. Standard Westergren method of ESR: blood to prevent morphological changes in the blood cells.
anticoagulant ration is 4:1 +Platelet-poor plasma (PPP) contains <10,000
b. Coagulation testing (using platelet poor platelets/μL and is prepared by heavy spin:
plasma+) centrifugation at 1500-2000g for 10 minutes.
++Platelet-rich plasma (PRP) contains 200,000-300,000
c. Modified Westergren method of ESR for blood
platelets/µL and is prepared by light spin: centrifugation
collected with EDTA at 50-100g for 10 minutes.

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 Disadvantages: 3. Adults who are/have:
a. Causes agglutination of WBC  Extremely obese
b. Causes aggregation of platelets  History of thromboembolism, stroke, or DIC
c. Produces the same morphologic changes seen  Extensive burns
when using old EDTA-anticoagulated blood. 4. Examination requested necessitates skin puncture
(bleeding time, micro methods of clotting time)
Order of draw:
 blood culture tubes (yellow) Indications of venipuncture: all examinations except those
 coagulation tubes ctg. citrate (blue) that are indicated by skin puncture, especially when a large
 serum tubes with or without gel separator amount of blood is needed for testing
 heparin tubes with or without gel separator
(green) Patient considerations during venipuncture:
 EDTA tubes (lavender) 1. Identification
 Fluoride tubes (gray) - Ask the patient to state his/her COMPLETE
name. (Do not volunteer the patient’s name)
Sources of blood for Hematology testing: - Verify the name of the patient with the wrist
1. Skin / Peripheral puncture – source of peripheral tag
blood (contains a mixture of blood from venules, - If the patient only speaks a foreign language or
arterioles, capillaries, as well as interstitial and cannot speak, verify the patient’s name with
intercellular fluids) the companion, nurse or the doctor.
- Finger (ring or middle finger): least used 2. Isolation restrictions
- Ear lobe (free margin): less nerve endings a. Isolation – for patients with contagious
: less tissue juice disease.
: no longer recommended due to less capillary - Everything is left in the patient’s room
access after obtaining the specimen (gloves,
- Heel: for newborns and infants gown, mask, etc.)
- Big toe: for infants b. Reverse Isolation – for patients who are
 depth of puncture should be 2-3 mm immunocompromised
 do not milk the site to avoid contamination - Nothing should be left in the patient’s
with tissue juice room after obtaining the specimen
 do not puncture cyanotic, swollen, inflamed, 3. Note the time of extraction. WBC count is noted
and edematous sites. to be increased in the morning while RBC count,
 Warm the site by massage, warm compress, or Hemoglobin, and Hematocrit are decreased.
warm water bath 4. If both arms are receiving intravenous (IV)
2. Venipuncture – source of venous blood infusion,
- Median cubital vein a. ask the doctor or nurse to stop infusion for at
- Cephalic cubital vein least 2 minutes. Discard the first tube of blood
- Basilic cubital veins collected.
b. collect below the IV line. Discard the first tube
Indications of skin puncture: collected.
1. Infants/neonates/pediatric patients c. collect from the lower extremities, if and only
2. Geriatric patients if, the patient is not diabetic.

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 5. Respect the patient’s rights: Complications of venipuncture:
a. Right to refuse extraction 1. Hematoma
b. Right to confidentiality 2. Hemoconcentation
- confidentiality of results 3. Hemolysis
- confidentiality of identity 4. Syncope
c. Right to be informed
- as to purpose of testing
- as to results of tests

References:
[1] Bain, B.J. (2011). Collection and handling of blood, In C. Jury, et. al. (Eds.) Dacie and Lewis’ Practical haematology (pp
1-9). China: Churchill Livingstone
[2] Benett, S.T., et. al. (Eds.). (2007.) Laboratory hemostasis: A practical guide for pathologists. New York, NY: Springer
Science+Business Media, LLC
[3] Henry, J.B. (2012). Preanalysis. In K.W. Sanford & R.A. McPherson (Eds.) Clinical diagnosis and management by
laboratory methods (pp 24-36). McPherson, R.A. & Pincus, M.R. (Eds.) Singapore: Elsevier Pte. Ltd.
[4] Hillyer, C.D., et. al. (2009). Transfusion medicine and hemostasis: Clinical and laboratory aspects. New York, NY: Elsevier
[5] Rodak, B.F., et. al. (2012). Laboratory evaluation of hemostasis. In G.A. Fritsma (Ed.) Hematology: Clinical principles
and correlations (pp 734-764) Singapore: Elsevier Pte. Ltd.
[6] Tricore Reference Laboratories (2011). Preparation of platelet poor plasma for coagulation testing. Retrieved from
http://www.tricore.org/Healthcare-Professionals/Test-Information/Testing-Protocols/Preparation-of-Platelet-Poor-
Plasma-for-Coagulatio
[7] Turgeon, M.L. (2012). Clinical hematology: theory and procedures. Baltimore, MD: Lippincott Williams and Wilkins
[8] Zhou, L. & Schmaier, A.H. (2005). Description of procedures with the aim to develop standards in the field. American
Journal of Clinical Pathology, 123, 172-183. DOI: 10.1309/Y9EC63RW3XG1V313

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