I.

Title

STANDARD OPERATING PROCEDURE FOR IMMUNOSEROLOGY,

HEMATOLOGY, CLINICAL CHEMISTRY, MICROBIOLOGY AND CLINICAL

MICROSCOPY AND PARASITOLOGY SECTIONS (OVERALL TITLE)

AUTHORS:

AYONQUE, JOHN NICOLO

AGBAYANI, AUBREY NOVIE

DALAYAP, KYNA CATHERINE

GADIAZA, SANDARA

GARCIA, GENESIS

EFFECTIVE DATE:

APRIL 17 , 2024

II. Table of Contents

II. Purpose

III. Scope

IV. Responsibilities

V. Materials

* A. Equipment

* B. Reagents
* C. Supplies

VI. Procedures

VII. Interpretation of Results

VIII. Reporting

IX. Quality Control

X. Safety Precautions

XI. References

(PAKISUNDAN PO TONG FORMAT NA ITO, THANK YOUU!)

III. Purpose

● Briefly state the objective of the SOP.

IV. Scope

● Define the applicability of the SOP.

V.Responsibilities

● Outline the roles and responsibilities of personnel involved in the procedure.

● This can include technicians, supervisors, and pathologists.

VI. Materials

● List all necessary materials, equipment, and reagents required for the

procedure.

● Include specific details like brand names, catalog numbers, and preparation

VII. Procedure

● Provide a detailed, step-by-step guide on how to perform the test.

 ● Use clear and concise language.

● Include quality control measures like positive and negative controls.

VIII. Interpretation of Results

● Explain how to interpret the test results based on observed agglutination

patterns.

● Include reference ranges or expected values when applicable.

IX. Reporting

● Describe how to document and report the test results.

● Specify the format for reporting, such as laboratory information system (LIS)

or paper forms.

X. Quality Control

● Outline the quality control procedures to ensure the accuracy and reliability of

the test results.

● Specify the frequency of performing controls and the acceptable ranges.

XI. Safety Precautions

● List potential hazards associated with the procedure and how to minimize

risks.

● Emphasize standard laboratory safety practices like wearing personal

protective equipment (PPE).

XII. References

● Include any additional references not listed earlier.

Haematology Section

Hematocrit (Hct) Testing

III. Purpose

● Hematocrit (Hct) testing is to provide guidelines for accurately determining

the proportion of red blood cells in whole blood. Hct testing is essential for

diagnosing and monitoring various hematological conditions, assessing

dehydration, evaluating response to therapy, and detecting blood disorders.

IV. Scope

● This SOP applies to all laboratory personnel involved in performing Hct

testing, including technicians, medical technologists, and pathologists. It

encompasses the entire Hct testing process, from specimen collection to

result interpretation, ensuring consistency, accuracy, and adherence to safety

protocols.

V.Responsibilities

● Technicians: Responsible for collecting blood specimens, preparing samples,

calibrating instruments, performing Hct assays according to established

protocols, and documenting results accurately.

● Medical Technologists:Supervise technicians, ensure compliance with SOPs,

troubleshoot technical issues, interpret Hct results, and communicate findings

to healthcare providers.
 ● Pathologists: Oversee Hct testing procedures, provide guidance on result

interpretation, verify critical results, and ensure quality assurance and

compliance with regulatory standards.

VI. Materials

A. Equipment:

● Hematocrit centrifuge

● Hematocrit reader

● Microhematocrit capillary tubes

● -Hematocrit sealant

● Hematocrit rotor

B. Reagents:

● Anticoagulant tubes (e.g., EDTA)

● Calibration controls

● Cleaning solutions

C. Supplies:

● Gloves

● Lancets

● Alcohol swabs

● Labelling materials

● Disposal containers
VII. Procedure

Preparation:

1. Ensure all equipment and reagents are at room temperature.

2. Label test tubes with patient identifiers.

3. Put on appropriate personal protective equipment (PPE), including gloves and

lab coat.

4. Blood Collection:

5. Collect venous or capillary blood using a sterile technique.

6. Fill a capillary tube with blood to the mark indicated, taking care to avoid air

bubbles.

Sealing the Capillary Tube:

7. Seal one end of the capillary tube with clay or sealing compound.

8. Ensure the sealed end is securely closed to prevent leakage.

9. Centrifugation:

10. Place the sealed capillary tubes in a hematocrit centrifuge, ensuring they are

balanced.

11. Centrifuge the tubes at [specified rpm] for [specified time] to separate the

blood components.

Reading the Hematocrit:

12. Carefully remove the capillary tubes from the centrifuge without disturbing the

sedimented blood.
 13. Measure the length of the packed red blood cells (RBCs) column and the total

length of the blood column using a hematocrit reader or microhematocrit tube

reader.

14. Calculate the hematocrit as the ratio of the length of the packed RBCs column

to the total length of the blood column, multiplied by 100.

Recording Results:

15. Record the hematocrit value in the patient's medical record, along with

relevant patient information and any pertinent notes.

VIII. Interpretation of Results

a. Explanation of Agglutination Patterns:

● Normal Results:

● In a normal sample, red blood cells (RBCs) will settle to the bottom of

the capillary tube, forming a distinct red layer with a clear plasma layer

above.

● The hematocrit value obtained from the packed RBCs column should

fall within the expected reference range.

Abnormal Results:

● Agglutination of RBCs may occur due to various factors such as

autoimmune disorders, transfusion reactions, or improper sample

handling.

● Agglutination can lead to inaccurate hematocrit measurements and

should be noted during result interpretation.

Reference Ranges or Expected Values:

 ● The reference range for hematocrit levels varies depending on factors

such as age, sex, and altitude.

● Typical reference ranges for adults are:

● Male: 40% to 54%

● Female: 36% to 48%

● Values outside of these ranges may indicate conditions such as

anemia or polycythemia.

interpretation Guidelines:

Normal Hematocrit:

● If the observed agglutination pattern is absent and the hematocrit value

falls within the reference range, the result is considered normal.

● No further action is required, and the result can be reported as within

normal limits.

Abnormal Hematocrit:

● If agglutination is observed or the hematocrit value falls outside the

reference range, further investigation is warranted.

● Repeat the test using a new blood sample to confirm the result and rule

out any pre-analytical errors.

● Consult with a healthcare provider to determine the underlying cause of

the abnormal result and appropriate management.

IX. Reporting

a. Documentation and Reporting:

● Patient Identification:

● Ensure proper patient identification by matching the test sample with the

patient's name, identification number, and any other relevant identifiers.

Test Results:

● Record the hematocrit value obtained from the test in the designated area of

the laboratory information system (LIS) or on paper forms.

● Include any relevant notes or comments regarding the sample condition or

test interpretation.

Quality Control Data:

● Document any quality control data, including control sample values,

calibration results, and any deviations from expected ranges.

● Ensure all quality control information is recorded accurately and maintained

for future reference.

Reporting Format:

● Use the designated format provided by the laboratory information system

(LIS) for electronic reporting.

● If reporting on paper forms, ensure the format includes fields for patient

information, test results, and any additional comments.

Verification:

● Verify the accuracy of the recorded test results and quality control data before

finalizing the report.

● Cross-check patient identifiers and test values to prevent errors in reporting.

Format for Reporting:

Laboratory Information System (LIS):

 ● Enter test results directly into the LIS using the provided interface.

● Follow the designated fields and format for entering patient information and

test values.

Paper Forms:

● Fill out paper forms with legible handwriting or typing.

● Include sections for patient demographics, test results, quality control data,

and any relevant comments.

● Ensure all required fields are completed accurately and completely.

X. Quality Control

a. Outline:

Daily Calibration:

● Calibrate the hematocrit centrifuge according to manufacturer

instructions to ensure accurate separation of blood components.

● Verify the calibration with standard reference materials or control

samples.

Control Samples:

● Run control samples with known hematocrit values alongside patient

samples to monitor instrument performance.

 ● Use commercially available control materials or in-house prepared

control samples.

● Analyze control samples at the beginning of each testing session and

after any instrument maintenance or recalibration.

Negative Controls:

● Run negative controls (e.g., saline solution) to check for any

contamination or interference in the testing process.

● Ensure that negative controls show no significant hematocrit value.

Operator Training:

● Provide initial training to laboratory personnel on proper testing

procedures and quality control measures.

● Conduct regular competency assessments and refresher training

sessions to ensure proficiency.

b. Frequency and Acceptable Ranges:

Daily Calibration:

● Perform calibration procedures daily before testing patient samples.

● Acceptable Range: The calibration should align with expected values

within ±2% of the standard reference materials or control samples.

Control Samples:
 ● Analyze control samples at the beginning of each testing session and

after any instrument maintenance or recalibration.

● Acceptable Range: Control samples should fall within predetermined

acceptable ranges established by the laboratory's quality control

program.

Negative Controls:

● Run negative controls regularly to check for contamination or

interference.

● Acceptable Range: Negative controls should show no significant

hematocrit value.

XI. Safety Precautions

II. Potential Hazards:

Bloodborne Pathogens:

● Risk of exposure to bloodborne pathogens such as hepatitis B, hepatitis C,

and HIV during blood collection and handling.

Chemical Hazards:

● Exposure to hazardous chemicals used in the testing process, such as clay

for sealing capillary tubes and cleaning agents for equipment maintenance.

● Centrifuge Hazards:
 ● Potential risks associated with the operation of the centrifuge, including

mechanical hazards from rotating parts and the potential for sample spillage

or breakage.

Sharp Objects:

● Risk of needlestick injuries or cuts from handling sharp objects like capillary

tubes and lancets.

● Electrical Hazards:

● Potential electrical hazards from the operation of laboratory equipment such

as the centrifuge and hematocrit reader.

III. Minimizing Risks:

Personal Protective Equipment (PPE):

● Wear appropriate PPE including gloves, lab coat, and safety goggles or face

shield to protect against exposure to blood and chemicals.

● Use puncture-resistant gloves when handling sharp objects to minimize the

risk of needlestick injuries.

● Safe Work Practices:

● Follow standard laboratory safety practices, including hand hygiene, avoiding

mouth pipetting, and proper disposal of biohazardous waste.

● Handle all blood samples and chemical reagents with care to prevent spills

and contamination.

Centrifuge Safety:
 ● Ensure proper loading and balancing of the centrifuge rotor to prevent

imbalance and minimize the risk of sample spillage or equipment damage.

● Do not open the centrifuge lid until the rotor has come to a complete stop to

avoid injury from rotating parts.

● Handling Sharp Objects:

● Use caution when handling capillary tubes and lancets to avoid accidental

needlestick injuries or cuts.

● Dispose of used capillary tubes and lancets in puncture-resistant containers

designated for sharps disposal.

Electrical Safety:

● Inspect electrical cords and plugs for any damage before use and do not use

equipment with frayed cords or exposed wires.

● Follow proper procedures for powering on and off laboratory equipment to

minimize the risk of electrical hazards.

XII. References

● Smith, J., & Johnson, K. (Year). "Standard Operating Procedure for

Hematocrit Testing in the Clinical Laboratory." *Journal of Clinical Laboratory

Medicine*, Volume(Issue), Pages.

Total Hemoglobin (Hgb or Hb)

II. Purpose
The objective of this Standard Operating Procedure (SOP) for Total Hemoglobin

(Hgb or Hb) testing is to provide guidelines for accurately measuring the

concentration of hemoglobin in blood samples. Total Hgb testing is essential for

diagnosing and monitoring various hematological conditions, evaluating

oxygen-carrying capacity, assessing anemia, and guiding transfusion therapy.

III. Scope

This SOP applies to all laboratory personnel involved in performing Total Hgb testing,

including technicians, medical technologists, and pathologists. It encompasses the

entire Total Hgb testing process, from specimen collection to result interpretation,

ensuring consistency, accuracy, and adherence to safety protocols.

IV. Responsibilities

Technicians: Responsible for collecting blood specimens, preparing samples,

performing Total Hgb assays according to established protocols, and documenting

results accurately.

Medical Technologists: Supervise technicians, ensure compliance with SOPs,

troubleshoot technical issues, interpret Total Hgb results, and communicate findings

to healthcare providers.

Pathologists: Oversee Total Hgb testing procedures, provide guidance on result

interpretation, verify critical results, and ensure quality assurance and compliance

with regulatory standards.

V. Materials

A. Equipment:

● Hemoglobinometer or spectrophotometer

● Microcentrifuge

● Pipettes (various sizes)

● Hemolysis tubes

● Timer

● Incubator (if required)

● Safety equipment (lab coat, gloves, goggles, etc.)

B. Reagents:

● Hemoglobin standard solution

● Hemoglobin reagent (e.g., Drabkin's reagent)

● Diluent solution (if required)

● Lysis buffer (if required)

C. Supplies:

● Microcentrifuge tubes

● Pipette tips

● Test tubes or cuvettes

● Absorbent paper

● Labels

● Waste disposal containers

VI. Procedures

1. Prepare the hemoglobin standard solution according to manufacturer

instructions.

2. Label all tubes, cuvettes, and containers appropriately.

3. Collect the blood sample using a sterile technique and transfer it to a

hemolysis tube containing the appropriate lysis buffer if needed.

4. Centrifuge the sample at [specified rpm] for [specified time] to separate the

hemoglobin from cellular debris.

5. Transfer a measured volume of the hemolysate to a clean test tube or cuvette.

6. Add the hemoglobin reagent to the test tube or cuvette as per the

manufacturer's instructions.

7. Mix the contents thoroughly and allow them to stand for the specified

incubation period.

8. Measure the absorbance of the sample using the hemoglobinometer or

spectrophotometer at the appropriate wavelength.

9. Prepare a standard curve using the absorbance values of the hemoglobin

standard solutions.

10. Calculate the concentration of hemoglobin in the sample using the standard

curve.

11. Record the results and ensure proper documentation.

12. Dispose of all waste materials according to laboratory safety protocols.

VII. Interpretation of Results

Total Hemoglobin testing results based on observed agglutination patterns and

provides reference ranges or expected values for different demographic groups.

a. Interpretation Based on Agglutination Patterns:

 ● Normal Result: Absence of agglutination or clumping of red blood cells

indicates a normal level of hemoglobin.

● Abnormal Result: Presence of agglutination may indicate abnormal

hemoglobin levels or other blood disorders. Further investigation and

confirmatory tests may be necessary to determine the underlying cause.

b. Reference Ranges or Expected Values:

● Adult Male: [Specify range, e.g., 13.8-17.2 grams per deciliter (g/dL)]

● Adult Female: [Specify range, e.g., 12.1-15.1 g/dL]

● Child: [Specify range for relevant age group, e.g., 11-16 g/dL]

● Newborn: [Specify range for newborns, e.g., 14-24 g/dL]

VIII. Reporting

Documentation and Reporting of Test Results:

● Recording Test Results: After completing the Total Hemoglobin testing

procedure, record the obtained results accurately.

● Identification: Clearly label the test results with patient information, including

name, identification number, date and time of sample collection, and any other

relevant identifiers.

● Units: Specify the units of measurement for hemoglobin concentration (e.g.,

g/dL, g/L).

● Quality Control Data: If applicable, document any quality control measures

taken during the testing process, including calibration checks, control sample

results, and any corrective actions performed.

● Authorized Personnel: Ensure that only authorized personnel are involved in

documenting and reporting test results.

 ● Data Integrity: Maintain the integrity and confidentiality of patient data in

accordance with applicable regulations and institutional policies.

b. Reporting Format:

● Laboratory Information System (LIS):

● If your laboratory utilizes a Laboratory Information System (LIS), enter the test

results directly into the system.

● Input patient information and test results according to the LIS interface

requirements.

● Ensure that the LIS generates a report with all necessary information for

review and interpretation by healthcare providers.

Paper Forms:

● If paper forms are used for reporting, fill out the designated sections

accurately and legibly.

● Include patient demographics, test results, date and time of testing, and any

relevant remarks or comments.

● Ensure that the paper forms are securely stored and archived according to

institutional policies.

c. Distribution:

● Healthcare Providers: Transmit the test results promptly to the requesting

healthcare provider or physician.

● Medical Records: Ensure that the test results are accurately recorded in the

patient's medical records for future reference and continuity of care.

● Quality Assurance: Perform regular reviews of the reporting process to ensure

compliance with SOPs and regulatory requirements.

d. Retention of Records:

● Retention Period: Maintain records of Total Hemoglobin testing results in

accordance with institutional policies and regulatory requirements.

● Storage: Store electronic and paper records securely to prevent loss,

unauthorized access, or tampering.

IX. Quality Control

a. Outline:

● Daily Calibration: Calibrate the hemoglobinometer or spectrophotometer using

standard hemoglobin solutions to ensure accurate readings.

● Control Samples: Run control samples with known hemoglobin concentrations

alongside patient samples to monitor instrument performance and verify the

accuracy of results.

● Reagent Checks: Perform regular checks on hemoglobin reagents to ensure

they are within specified expiry dates and maintain their integrity.

● Operator Training: Train laboratory personnel on proper testing procedures

and ensure proficiency through regular competency assessments.

● Documentation: Maintain detailed records of quality control data, including

control sample values, calibration results, and any deviations from expected

ranges.

b. Frequency and Acceptable Ranges:

● Daily Calibration:

● Frequency: Perform calibration procedures daily before testing patient

samples.

● Acceptable Range: Ensure that the calibration curve aligns with expected

values within ±2% of the standard hemoglobin concentrations.

● Control Samples:
 ● Frequency: Run control samples at the beginning of each testing session and

after any instrument maintenance or recalibration.

● Acceptable Range: Control samples should fall within predetermined

acceptable ranges, typically established by the laboratory's quality control

program.

● Reagent Checks:

● Frequency: Check reagent integrity before each testing session and verify the

expiry date.

● Acceptable Range: Reagents should show no signs of contamination or

degradation, and their expiry dates should not have passed.

● Operator Training:

● Frequency: Provide initial training to new operators and regular refresher

courses for all personnel involved in hemoglobin testing.

● Acceptable Range: Operators should demonstrate proficiency in following

testing protocols and interpreting results accurately.

Documentation:

● Frequency: Maintain up-to-date records of quality control data for each testing

session.

● Acceptable Range: Document any deviations from expected ranges and

corrective actions taken to address them.

X. Safety Precautions

1. Handle all biological samples and reagents with care to avoid contamination.

2. Wear appropriate personal protective equipment (PPE) including gloves, lab

coat, and goggles.

3. Dispose of all biohazardous materials in designated waste containers.

4. Follow all laboratory safety guidelines and procedures.

XI. References

Smith, J., & Johnson, K. (Year). "Standard Operating Procedure for Total

Hemoglobin Testing in the Clinical Laboratory." Journal of Clinical Laboratory

Medicine, Volume(Issue), Pages.

IMMUNOSEROLOGY SECTION:

ABO GROUPING AND Rh D TYPING BY MICROTEST PLATE TEST

II. Purpose

Blood typing is a common test when blood transfusions and tissue transplants

are required, as well as during pregnancy. A blood type lab test identifies certain

inherited substances (antigens) that may be present on the surface of red blood cells

and classifies them into four common groups: A, B, AB, or O, and is known as the

ABO system.

In addition, a second system, the Rh system, determines if the red blood cells

are Rh-positive or Rh-negative. You are Rh-positive when you have the Rh factor. It

is important to know both the ABO and Rh types in that a mismatch is capable of

inducing an intense immunogenic reaction that can be fatal.

III. Scope
 This procedure applies to all those activities that are performed to determine

the correct ABO group and Rh D type of a donor and ensure the reliability of the

results. This procedure describes the method of detection of the presence or

absence of A, B & D antigens on red cells by using Anti-A, Anti-B and anti D antisera

(antibodies) against the corresponding antigens. Anti A & Anti B are monoclonal IgM

antibodies specific against A & B red cell antigen. Anti D is also monoclonal which

may be purely IgM or a blend of IgG and IgM (preferably). Reverse blood grouping

should always be run in parallel with forward ABO typing for group confirmation

Mismatch transfusion of ABO/D blood group can cause fatal transfusion reactions

and sensitization against transfused D positive antigens in Rh D negative individuals

especially in child bearing age females where it may cause haemolytic disease on

the newborn.

IV. Responsibilities

In the Immunohematology Laboratory following staffs are responsible for this

procedure:

❖ Technician is responsible to perform the ABO grouping and RhD typing of

donors

❖ Technologist is responsible to verify the results

❖ Medical Officer is responsible to supervise the procedure and to rule out any

blood group discrepancy by further workup

❖ It is the responsibility of all staff performing the ABO grouping and D typing to

ensure that quality controlled reagents and proper cell concentrations are

used.

V. Materials

A. Equipment
❖ Refrigerator to store samples and reagents at 2- 6 oC

❖ Dispensers (optional): Semi-automated devices for dispensing equal volumes

to a row of wells

❖ Microtest plate rotator

❖ Microtest plate readers (optional): Automated photometric devices that read

the results by the light absorbance in U-shaped bottom wells to differentiate

between positive and negative tests. The microprocessor component of the

reader interprets the reactions and prints the blood testing results

❖ Special plate carriers are required to fit common table-top centrifuges

❖ Magnifying glass for microtest plates

❖ Incubator

B. Reagents

❖ Anti A, Anti-B antisera

❖ 2-3%% suspension of group A1, B reagent red cells

❖ 6% Albumin Control Reagent ( Rh Control)

❖ Use only Anti-D reagents approved for use in microtest plate tests2

❖ Isotonic (0.9%) saline

C. Specimen

❖ Automated methods may require the use of samples drawn from donor into a

specific anticoagulant (K3-EDTA)

❖ Test red cells suspended in saline (2-3%)

 D. Miscellaneous

❖ Rubber teats for Pasteur pipettes

❖ Permanent Markers

❖ Timer

❖ Disposal box

❖ 2 plastic beakers

❖ Aluminium racks to hold sample tubes

VI. Procedures

RED BLOOD CELLS TESTING / FORWARD GROUP TESTING

1. Place 1 drop of anti-A, 1 drop of anti-B and 1 drop of donor‘s plasma/serumin

separate clean wells of a U-bottom microtest plate.

2. Add 1 drop of 2-3% saline suspension of red cells to each well containing

blood typing reagent.

3. Mix the contents of the wells by gently rotating the plate on the microtest plate

rotator.

4. Centrifuge the plate at the appropriate conditions established for the

centrifuge.

5. Resuspend the red cell buttons by gently manually tapping the plate or with

the aid of a mechanical shakerRead, interpret, and record results. Compare

red cell test results with those obtained in testing serum or plasma.

SERUM TESTING/REVERSE GROUP TESTING

1. Add 1 drop of serum or plasma under test to each well.

2. Add 1 drop of 2-3% suspension of A1, B and O reagent red cells to separate

clean wells of a U-bottom microtest plate.

3. Mix the contents of the wells by gently tapping the slides of the plate or on a

microtest plate rotator.

 4. Centrifuge the plate at the appropriate conditions established for the

centrifuge.

5. Re-suspend the red cell buttons by manually tapping the plate or with the aid

of a mechanical shaker, read, interpret, and record results. Compare test

results on serum or plasma with those obtained in testing red cells.

Rh D GROUP TESTING

1. Place 1 drop of anti-D reagent into a clean well of the microtest plate. If the

reagent requires use of an Rh control, add 1 drop of the control to a second

well.

2. Add 1 drop of a 2-3% suspension of red cells to each well.

3. Mix the contents of the wells by gently tapping the slides of the plate or on a

microtest plate rotator.

4. Centrifuge the plate at the appropriate conditions established for the

centrifuge.

5. Re-suspend the red cell buttons by manually tapping the plate or with the aid

of a mechanical shaker. Examine for agglutination, read, interpret, and record

the results.

6. To enhance weak reactions, incubate negative tests at 37o C in the incubator

for 15 to 30 minutes and repeat steps 4 to 6.

VII. Interpretation of Results

❖ Agglutination in any well of red blood cells tests and agglutination or

haemolysis in serum test constitutes a positive test result. The expected

agglutination reaction for positive tests are 3+ to 4+.(cf. Annex 3)

❖ A smooth suspension of red cells after re-suspension of the cells button is a

negative test result.

❖ The interpretation of ABO group is as follows:

 ❖ If any of the following discrepancies occur, the Sample should be handed over

to the Medical Officer In charge (cf. SOP- 22).

There is a positive reaction in the reverse grouping with O cells

D- Control is positive Auto- control is positive

There is a discrepancy between the forward and reverse ABO blood grouping

There is a discrepancy between the results of the two wells for Rh D grouping

❖ Any discrepancy between results on cell and serum or plasma tests should be

resolved before an interpretation is recorded for the patient‘s or donor‘s ABO

group

VIII. Reporting

Presence (+) or absence (-) of agglutination/haemolysis in ABO grouping

Presence (+) or absence (-) of agglutination in Rh D Typing

Confirm the ABO cell grouping results with those obtained in serum/reverse grouping

and vice versa

All Rh-D-negative results must be retested with a weak D test (cf. BTS/SOP/TP/24a)

*Presence of “Weak D” in blood bank: As a donor he/she should be considered as

RhD-Positive
IX. Quality Control

Positive and negative controls must be run and documented each day of

testing. Before running controls, reagents and controls should be checked for

expiration date, lot number, and turbidity. Expired or contaminated controls and

reagents should not be used since they could give inaccurate or weak reactions.

Positive and negative controls may either be purchased from a manufacturer

or the lab may choose to use the ABO reagents used to type the blood. The reagent

A cells and B cells used for the reverse typing

can be used as the negative and positive

controls for Anti-A and Anti-B reagents. When

using one reagent for a control of another, both

reagents are simultaneously verified.

Controls for Anti-D can be purchased from the manufacturer or verified known

patients can be used. When performing Weak D (Du) testing, always confirm that the

AHG is not inactivated by checking for agglutination with antibody-coated Coombs

check cells

X. Safety Precautions

❖ Discard the alcohol swabs, lancet, cotton balls and toothpick after their use.

❖ Drop all the materials, including the glass slide into the biohazard disposal

container after observing the result.

XI. References

ABO and Rh Blood Typing. Lab Florida | Clinical Laboratory of Florida.

https://www.labflorida.com/internal/COLA/guides/elf27.pdf

Blood group test. (2023, August 11). VEDANTU.

https://www.vedantu.com/biology/blood-group-test
CROSS MATCH (SALINE/BOVINE-ALBUMIN/IAT)

II. Purpose

The purpose of the crossmatch is to detect the presence of antibodies in the

recipient against the red blood cells of the donor. These antibodies attach to the red

blood cells of the donor after transfusion. An incompatible transfusion can result in a

severe hemolytic anaemia and even death.

III. Scope

This procedure is applied for compatibility testing of all patients requiring

transfusion. There are two types of cross match, i.e. Major and Minor. Routinely

major cross match is done in which donor red cells are cross matched with patient

serum/plasma to detect incomplete antibodies in the patient serum/plasma (including

IAT phase). Minor crossmatch is done when a transfusion reaction is observed and

is done by taking patient red cells which cross match with the donor plasma to detect

antibodies in the donor plasma. Incompatible blood units should never be used for

transfusion.

IV. Responsibilities

It is the responsibility of the technician in the immunohematology laboratory to

perform compatibility testing to demonstrate ABO Incompatibility and document the

results. If any unexpected antibody is detected, the Medical officer should be

informed for further investigation.

V. Materials

A. Equipment

❖ Refrigerator to store samples & reagents at +20 to+60C

❖ Tabletop centrifuge

❖ Automated cell washer

 B. Reagents

❖ Polyspecific Antihuman globulin reagent (anti-IgG+anti-C3d)

❖ IgG sensitised control cells

❖ 0.9% saline

C. Specimen

❖ Patient‘s serum or plasma. Patient sample should not be older than 3 days

❖ Donor red cells acquired from the blood packs intended to be transfused

D. Miscellaneous

❖ Disposal box

❖ 2 glass beakers

❖ Aluminium racks to hold serum and coombs' tubes

VI. Procedures

SALINE ROOM TEMPERATURE IMMEDIATE SPIN

Saline room temperature is done to detect Major ABO incompatibility and

complete (IgM) antibodies/cold antibodies like M, N, S, P, Lewis, Lutheran, etc. This

crossmatch method can be done in emergency issues of blood (in emergency

situations).

1. Label a clean glass tube.

2. Prepare 3% red cell suspension of donor red cells (60 microliters of washed

red cells and 2 ml 0.9% normal saline) OR Prepare 5% red cell suspension of

donor red cells (100 microliters of washed red cells in 2 ml 0.9% normal

saline).

3. Centrifuge patient blood at 3400 rpm for 5 minutes to get clear serum.

4. Dispense 2 drops of patient serum into the labelled glass tube.

5. Add one drop of 3% or 5% donor red cell suspension to the tube containing

patient serum.

6. Centrifuge immediately at 3400 rpm for 15 seconds.

 7. Take out the tube gently.

8. Observe for haemolysis and then for agglutination by gently shaking the tube.

9. Grade and record results.

10. Always continue with the AHG phase, even in emergency situations, but in

this case blood packs can be released after this phase).

AHG/COOMBS PHASE

AHG/Coombs test is done to detect the presence of unexpected incomplete

(IgG) antibodies in patients blood which can cause destruction of the transfused

donor red cells. Coombs' phase should always be included in the cross match.

Continue with the tubes used in 5.1 saline room temperature immediate spin.

1. Optional: 2 drops of 22% Bovine Albumin can be added to this tube as

enhancement media.

2. Incubate the tube at 37 0C for 45 minutes for saline/IAT or for 30 minutes if

22% Bovine Albumin is added.

3. Take out the tube and centrifuge at 3400 rpm for 15 seconds, then observe

haemolysis and agglutination by gently shaking the tube.

4. If no agglutination/haemolysis is seen (grade and record results) then wash

the tube 3 times to remove unbound antibodies.

5. After the 3rd wash discard all saline/supernatant by gently tapping on tissue

paper.

6. Add 2 drops of polyspecific coombs reagent.

7. Mix & centrifuge immediately at 3400 rpm for 15 seconds.

8. Take out the tube gently, observe macroscopically for haemolysis and

agglutination by gentle shaking.

9. Grade and record results.

10. To all negative results add one drop of ―Coombs Control Cells‖ to validate

the results. (Dispense one drop of check cells to the negative result tube and
 centrifuge immediately at 3400 rpm. Take out the tube and gently disperse the

cell button, this time agglutination should be present at least 1+ or 2+. (The

free AHG/Coombs reagent in the test tube causes agglutination of the check

cells/sensitised cells). This validates that the Coombs reagent was working

properly and cells were properly washed after incubation).

VII. Interpretation of Results

POSITIVE RESULT: Haemolysis/Agglutination of red cells / Mixed Field is

incompatible crossmatch.

NEGATIVE RESULT: No Haemolysis / No Agglutination of red cells is compatible

crossmatch.

NOTE:

❖ All steps should be done immediately

❖ Never use plastic tubes for cross match as it adsorbed IgG antibody which

can lead to false negative results

❖ Haemolysed patient blood samples should not be used. If there is haemolysis

going on in the patient then monitor the size of cell button after incubation at

37 0C by centrifugation at 3400 rpm and the supernatant colour should be

matched with the original blood sample. If the colour of the supernatant

becomes darker then the original sample it means haemolysis had occurred

during incubation at 370C

❖ Shaking should be done gently

❖ Haemolysed bag should not be selected for cross match

❖ Use clean glasswares

❖ Use all reagents according to the manufactures advice

Limitations
The saline/enzyme cross match will not:

❖ Detect incomplete antibody

❖ Ensure normal donor’s red blood cell survival

❖ Detection of antibodies connected to low level presence of antigens (as with

heterozygous expressed blood groups like Fya /Fyb )

VIII. Reporting

IX. Quality Control

Quality control of blood supply and transfusion includes collection,

documentation, storage, serological testing, administration and follow-up. The

provision of serologically compatible blood requires meticulous monitoring of

grouping, screening and crossmatching techniques.

X. Safety Precautions

To minimise the chance of an adverse reaction during a transfusion, health

care professionals take several precautions. Before starting the transfusion, usually

a few hours or even a few days beforehand, the person is cross-matched with the
donor blood (not done for transfusions of plasma or platelets). In cross-matching,

blood bank personnel mix a small amount of blood from the donor and the recipient

to ensure there is no reaction.

After double-checking labels on the bags of blood that are about to be given

to ensure the units are intended for that recipient, the health care professional gives

the blood to the recipient slowly, generally over 1 to 4 hours for each unit of blood.

Because most adverse reactions occur during the first 15 minutes of the transfusion,

the recipient is closely observed at first. After that, a nurse checks on the recipient

periodically and must stop the transfusion if an adverse reaction occurs.

XI. References

Standard Operating Procedures For Blood Transfusion Services, Punjab. Welcome to

Punjab Blood Transfusion Authority | Punjab Blood Transfusion Authority.

https://pbta.punjab.gov.pk/system/files/SOP%20for%20Blood%20bank.pdf

HEMATOLOGY SECTION:
CLINICAL CHEMISTRY SECTION:

Basic Metabolic Panel (BMP) Standard Operating Procedure (SOP)

II. Purpose

The Basic Metabolic Panel (BMP) is a group of blood tests used to assess general

health and screen for potential medical conditions. This SOP outlines the procedures

for performing a BMP analysis in the Clinical Chemistry section of the laboratory.

This SOP establishes a standardized approach for performing BMP analysis on

serum or plasma specimens, ensuring accurate and reliable results.

III. Scope

This SOP applies to all laboratory personnel involved in analyzing BMPs, including
phlebotomists collecting blood samples, laboratory technicians performing the
analysis, and technologists interpreting and reporting results.

IV. Responsibilities

● Phlebotomist: Responsible for proper blood collection, specimen labeling,

and timely transport to the laboratory.
● Laboratory Technician: Performs the BMP analysis according to this SOP,
including instrument calibration, quality control checks, and sample analysis.
● Laboratory Technologist: Reviews and interprets BMP results, verifies
accuracy, and releases them to the Laboratory Information System (LIS).

V. Materials

A. Equipment

● Laboratory centrifuge
● Automated chemistry analyzer
● Pipettes (calibrated)
● Refrigerated centrifuge
● Refrigerator for specimen storage

B. Reagents

● Commercially prepared test kits or reagents for electrolytes (Na, K, Cl), BUN,
Creatinine, Total Protein, Albumin, ALP, and Glucose (specific to the
chemistry analyzer)
● Quality control materials (controls with known values for each analyte)
● Disposable cuvettes or reaction cartridges (compatible with the analyzer)
C. Supplies

● Tourniquet
● Blood collection tubes (serum or plasma separator)
● Aliquot tubes
● Waste disposal containers
● Personal Protective Equipment (PPE) like gloves, lab coat, safety glasses

VI. Procedures

1. Specimen Collection and Handling

● Follow standard phlebotomy procedures for blood collection.
● Use serum or plasma separator tubes for BMP analysis.
● Centrifuge the blood sample according to laboratory protocol to
separate serum/plasma.
● Transfer serum/plasma to labeled aliquot tubes.
● Store unanalyzed specimens refrigerated until testing.
2. Instrument Calibration and Quality Control
● Perform regular calibration of the chemistry analyzer as per
manufacturer's instructions.
● Analyze quality control materials before running patient samples.
● Document QC results and take corrective actions if necessary.
3. Analysis
● Allow the analyzer to reach operating temperature.
● Load cuvettes or cartridges specific to BMP analytes onto the analyzer.
● Using calibrated pipettes, transfer serum/plasma and reagents into
cuvettes/cartridges as per manufacturer's instructions.
● Initiate the automated BMP analysis program on the analyzer.
● The instrument will measure and calculate final concentrations for each
analyte.
4. Data Analysis and Reporting
● Review results for outliers or values exceeding the reference range.
● Compare results with QC data to ensure test accuracy.
● Verify and release final BMP results to the LIS for physician access.

VII. Interpretation of Results

● A qualified laboratory professional will interpret BMP results in conjunction

with other clinical findings and patient history.
● Reference ranges for each analyte are provided for guidance (refer to Section
IX).
● Deviations from the reference range may indicate potential health problems
and require further investigation.
VIII. Reporting

● Finalized BMP reports are released electronically through the LIS to

authorized healthcare providers.
● Critical results requiring immediate attention are communicated promptly to
the healthcare provider.

IX. Quality Control

● Regular calibration and preventive maintenance of the chemistry analyzer are

essential.
● Commercially prepared quality control materials are analyzed with each batch
of patient samples to monitor test accuracy and precision.
● Corrective actions are taken if QC results fall outside the acceptable range.

X. Safety Precautions

● Follow Universal Precautions when handling all specimens and potentially

infectious materials.
● Wear appropriate PPE like gloves, lab coat, and safety glasses.
● Dispose of biohazardous waste according to laboratory safety guidelines.
● Handle reagents according to the manufacturer's safety data sheets (SDS).

XI. References

● Tietz Textbook of Clinical Chemistry and Molecular Diagnostics

Liver Function Tests (LFTs) Standard Operating Procedure (SOP)

II. Purpose

This document outlines the standard operating procedure (SOP) for performing liver
function tests (LFTs). The purpose of this SOP is to ensure the consistent and
accurate performance of LFTs in a clinical laboratory setting. Standardized
procedures promote reliable results that aid in the diagnosis and monitoring of liver
disease.

III. Scope

This SOP applies to all personnel involved in performing LFTs, including

phlebotomists, laboratory technicians, and pathologists.

IV. Responsibilities
 ● Phlebotomist: Responsible for proper patient identification, blood collection,
and specimen labeling.
● Laboratory Technician: Performs the LFT assays according to the outlined
procedures, ensuring proper handling and analysis of specimens.
● Pathologist: Reviews and interprets LFT results, integrating them with clinical
findings for diagnosis and patient management.

V. Materials

A. Equipment

● Centrifuge
● Spectrophotometer
● Automatic pipettes
● Incubator (if required for specific assays)
● Quality control materials

B. Reagents

● Reagents for each LFT test (e.g., alanine aminotransferase (ALT), aspartate
aminotransferase (AST), alkaline phosphatase (ALP), bilirubin, albumin)

C. Supplies

● Blood collection tubes

● Tourniquet
● Alcohol swabs
● Sharps container
● Personal protective equipment (PPE)

VI. Procedures

1. Patient Preparation
● Instruct the patient to fast for a specific timeframe before the test, if
required by the specific LFTs being performed.
● Document any medications or supplements the patient is taking, as
some can affect LFT results.
2. Blood Collection
● The phlebotomist will identify the patient using two identifiers (e.g.,
name and date of birth).
● Apply a tourniquet and locate a suitable vein for blood collection.
● Disinfect the venipuncture site with an alcohol swab.
● Perform venipuncture and collect blood into the appropriate tubes
according to the tests ordered.
 ● Properly label the blood collection tubes with patient identification
information and test name(s).
● Release the tourniquet and dispose of sharps in a designated sharps
container.
3. Specimen Processing
● Centrifuge the blood collection tubes as per the manufacturer's
instructions to separate serum from red blood cells.
● Transfer the serum to appropriately labeled test tubes for analysis.
4. LFT Assays
● Laboratory technicians will perform the specific LFT assays according
to the manufacturer's instructions and established laboratory protocols.
● This may involve using automated analyzers or performing manual
assays.
● Proper controls should be run concurrently to ensure the accuracy of
the tests.
5. Data Analysis
● The instrument will generate readouts for each LFT test.
● The laboratory technician will record the results in the laboratory
information system (LIS).

VII. Interpretation of Results

● The pathologist will review the LFT results in conjunction with the patient's
clinical history and other laboratory findings.
● Elevated or abnormal LFT values may indicate liver damage or disease. The
specific pattern of abnormalities can help narrow down the cause.
● The pathologist will interpret the results and provide a report for the ordering
physician.

VIII. Reporting

● LFT results are reported to the ordering physician electronically or through the
LIS.
● The report should include the individual LFT values, reference ranges, and
any interpretive comments by the pathologist.

IX. Quality Control

● Regular quality control procedures are essential to ensure the accuracy and
consistency of LFT results.
● This includes running control materials with each batch of tests to monitor for
errors or variations.
● Routine calibration and maintenance of equipment should also be performed
according to the manufacturer's recommendations.
X. Safety Precautions

● Standard laboratory safety precautions should be followed at all times.

● Wear appropriate PPE, such as gloves, lab coat, and safety glasses.
● Properly handle and dispose of biohazardous materials.
● Be aware of the potential hazards of working with bloodborne pathogens.

XI. References

● National Institutes of Health: https://www.nih.gov/) - Liver Diseases

● American Association for the Study of Liver Diseases: https://www.aasld.org/ -
Diagnostic Tests for Liver Disease
MICROBIOLOGY SECTION:

Gram Stain Standard Operating Procedure (SOP)

II. Purpose

This SOP outlines the procedure for performing a Gram stain, a rapid and differential

staining technique used to classify bacteria into two broad categories: gram-positive

and gram-negative.

III. Scope

This SOP applies to all personnel in the microbiology laboratory responsible for

performing Gram stains on various clinical specimens.

IV. Responsibilities

● Technologist/Technician: Performs the Gram stain procedure, interprets

results, and documents findings.

 ● Supervisor: Ensures adherence to this SOP, reviews and approves results,

and addresses any discrepancies.

● Pathologist: Provides final interpretation of results in the context of patient

clinical information.

V. Materials

● Microscope slides

● Staining reagents:

○ Crystal violet solution (prepared according to manufacturer's

instructions)

○ Gram's iodine solution

○ Safranin solution (prepared according to manufacturer's instructions)

○ Decolorizing agent (acetone-alcohol solution, commercially available)

● Bunsen burner or alternative heat source

● Inoculating loop

● Bibulous paper

● Disinfectant wipes

● Personal protective equipment (PPE): lab coat, gloves, safety glasses

VI. Procedure

1. Specimen Preparation:

○ Prepare a smear of the specimen on a clean microscope slide.

○ Allow the smear to air dry completely.

○ Heat fixation: Carefully pass the slide through the flame of a Bunsen

burner two to three times to heat-fix the smear (avoid overheating).

 2. Crystal Violet Staining:

○ Flood the slide with crystal violet solution for 60 seconds.

○ Rinse briefly with tap water.

3. Gram's Iodine Application:

○ Flood the slide with Gram's iodine solution for 60 seconds.

4. Decolorization:

○ Tilt the slide and slowly add decolorizing agent drop-by-drop until the

runoff becomes almost clear (approximately 15 seconds).

○ DO NOT over-decolorize.

5. Counterstaining:

○ Flood the slide with safranin solution for 30 seconds.

○ Rinse briefly with tap water.

6. Air-drying:

○ Allow the slide to air dry completely.

7. Microscopic Examination:

○ Examine the slide under oil immersion objective (100x magnification).

○ Observe for bacterial morphology (cocci, bacilli) and gram reaction

(purple for gram-positive, pink for gram-negative).

VII: Quality Control:

● Include a positive control (known gram-positive and gram-negative

organisms) with each batch of stains.

 ● Expected results: Staphylococcus aureus (gram-positive) and Escherichia coli

(gram-negative) should show characteristic staining patterns.

VIII. Interpretation of Results

● Gram-positive bacteria: Stain purple due to the retention of the crystal

violet-iodine complex in their thick peptidoglycan cell wall.

● Gram-negative bacteria: Stain pink due to the loss of the crystal violet-iodine

complex in their thinner cell wall and subsequent counterstaining with

safranin.

● Mixed bacterial flora may be present in some specimens.

● An inconclusive result may necessitate repeating the stain or performing an

alternative diagnostic test.

IX. Reporting

● Document the Gram stain results in the laboratory information system (LIS) or

on a paper requisition form.

● Report the presence of gram-positive, gram-negative, or mixed bacterial flora.

● Notify the supervisor of any unexpected or inconclusive results.

X. Quality Control

● Perform positive and negative controls with each batch of stains.

● Monitor the expiration dates of all reagents.

● Regularly calibrate the microscope.

● Document all quality control procedures.

XI. Safety Precautions

 ● Wear appropriate PPE (lab coat, gloves, safety glasses) at all times.

● Handle all specimens with care as potentially infectious.

● Dispose of contaminated slides and materials in designated biohazard waste

containers.

● Disinfect work surfaces and equipment after use.

● Follow standard laboratory safety protocols for handling chemicals and

biological materials.

XII. References

● Reference for Gram stain technique: e.g., Baron S, et al. Baron’s Medical

Microbiology. 9th ed. McGraw-Hill; 2019

Standard Operating Procedure (SOP) for Potassium Hydroxide (KOH)

Preparation

III. Purpose

This Standard Operating Procedure (SOP) outlines the proper techniques for

performing a KOH examination of clinical specimens for the detection of fungal

elements, such as yeast and pseudohyphae.

IV. Scope

This SOP applies to all laboratory personnel performing KOH examinations on

various clinical specimens, including skin scrapings, nail clippings, sputum, vaginal

swabs, and oral swabs.

V. Responsibilities

● Technician: Performs the KOH examination according to this SOP,

documents the procedure, and reports the results.

● Supervisor: Reviews the technician's work, ensures adherence to the SOP,

and addresses any discrepancies.

● Pathologist: Interprets the KOH examination results in conjunction with

clinical information and may recommend additional testing.

VI. Materials

● Sterile microscope slides

● Sterile Pasteur pipettes

● Cover slips

● 10% KOH solution (prepared with KOH flakes and distilled water)

● Disposable gloves

● Safety glasses

● Waste disposal container

● Microscope with brightfield illumination

VII. Procedure

1. Preparation:

○ Don appropriate personal protective equipment (PPE) including gloves

and safety glasses.

○ Label a clean microscope slide with the patient's identification

information.

2. Specimen Processing:
 ○ Mix the specimen container thoroughly to ensure even distribution.

○ Using a sterile pipette, transfer a small drop (approximately 10

microliters) of the specimen onto the center of the slide.

3. KOH application:

○ Without touching the specimen droplet, dispense a similar-sized drop

of 10% KOH solution directly next to it on the slide.

4. Mounting:

○ Gently tilt the slide to bring the two droplets together and mix them

slightly.

○ Carefully place a coverslip over the mixed droplet, avoiding air bubbles.

5. Microscopic Examination:

○ Allow the preparation to sit at room temperature for a few minutes to

facilitate clearing of cellular debris.

○ Observe the preparation under a microscope using the low power (10x)

objective to locate the area of interest.

○ Switch to the high power (40x) objective and systematically scan at

least 10 fields of view.

6. Positive Control (Optional):

○ Include a slide prepared with a known fungal element (e.g., Candida

albicans) as a positive control to verify proper KOH solution

functionality and observer technique.

7. Negative Control:
 ○ Include a blank slide with a drop of 10% KOH solution and a coverslip

as a negative control to ensure the absence of fungal elements in the

reagents.

8. Documentation:

○ Record the findings in the laboratory information system (LIS) or on a

designated report form.

○ Document the presence or absence of fungal elements (yeast or

pseudohyphae) and their morphology (if observed).

VIII. Interpretation of Results

● Positive Result: Visualization of budding yeast cells or filamentous structures

consistent with pseudohyphae suggests a fungal infection.

● Negative Result: Absence of fungal elements in the KOH preparation does

not necessarily rule out fungal infection. Negative results may require further

investigation with additional diagnostic tests.

Note: KOH examination is a rapid screening test and should be interpreted in

conjunction with clinical presentation and other diagnostic modalities.

IX. Reporting

● Report the KOH examination results in the laboratory information system (LIS)

or on a designated report form, including the presence or absence of fungal

elements.

● If fungal elements are identified, describe their morphology (e.g., budding

yeast cells, pseudohyphae).

X. Quality Control
 ● Perform positive and negative controls with each batch of KOH examinations.

● The positive control should demonstrate the expected morphology of the

fungal element used.

● The negative control should be free of any fungal elements.

● Regularly monitor the expiration date of the 10% KOH solution and prepare

fresh solution when necessary.

● Perform proficiency testing as required by the laboratory's accreditation

program.

XI. Safety Precautions

● KOH is a caustic substance and can cause skin and eye irritation. Wear

gloves and safety glasses at all times while handling KOH solution.

● Dispose of used slides and pipette tips in a designated biohazard waste

container.

● Decontaminate work surfaces with a disinfectant solution after use.

● Follow standard laboratory safety practices for handling potentially infectious

materials.

XII. References

● Indian Council of Medical Research:

https://main.icmr.nic.in/sites/default/files/guidelines/Standard_Operating_Proc

edures_Mycology_1stEdition.pdf Standard Operating Procedures - Mycology.

● Centers for Disease Control and Prevention:

https://www.cdc.gov/labtraining/docs/job_aids/routine_microscopy_procedure

s/KOH-Procedure.docx KOH Procedure.

CLINICAL MICROSCOPY AND PARASITOLOGY:

PURPOSE:

To ensure consistency, accuracy, and quality in laboratory practices related to

diagnosing diseases. By following SOPs, laboratories can minimize errors, maintain a
safe working environment, and enhance the reliability of diagnostic outcomes.

SCOPE

The applicability of a Standard Operating Procedure (SOP) for clinical microscopy and
parasitology can be defined as its relevance and necessity in the clinical laboratory
setting for specific tasks, processes, or activities related to the diagnosis and
identification of parasites in clinical samples. The SOP should provide clear
instructions and guidelines for the preparation, staining, and examination of blood
films, as well as quality assurance measures for ensuring accurate and reproducible
results.

RESPONSIBILITIES

The roles and responsibilities of personnel involved in the standard operating

procedure (SOP) for clinical microscopy and parasitology encompass various tasks
and functions to ensure the quality and accuracy of diagnostic procedures.

1. Health Care Providers (Physicians, Midlevel Practitioners, Dentists):

○ Perform patient testing under a CLIA Certificate for Provider-Performed
Microscopy (PPM) procedures.
○ Follow recommended practices outlined in the SOP for conducting
microscopic examinations during patient visits.
2. Medical Technologists:
○ Properly use, care for, and maintain microscopes in the clinical
laboratory setting.
○ Understand the mechanism of microscopes and troubleshoot issues
systematically.
○ Implement a maintenance program that includes cleaning lenses,
calibrating light sources, managing sample stages, and conducting
quality control checks.
○ Adhere to laser safety guidelines and wear appropriate lab attire when
working with microscopes.
3. Microscopists:
○ Prepare, stain, and examine blood films accurately to detect, identify,
and quantify malaria parasites.
○ Differentiate between parasite species and accurately estimate parasite
density.
○ Receive extensive training and experience to achieve proficiency in
microscopy techniques.
 ○ Follow the SOP to ensure the correct preparation and examination of
blood films for accurate and reproducible results.
4. Laboratory Personnel:
○ Adhere to standard operating procedures for diagnosing diseases of
public health importance at intermediate and peripheral levels.
○ Ensure that laboratory practices are consistent, accurate, and aligned
with quality assurance guidelines.
○ Implement SOPs for microscopy procedures to maintain high standards
in preparing and staining blood films and examining slides accurately.

MATERIALS

The necessary materials, equipment, and reagents required for the procedure in the
standard operating procedure for clinical microscopy and parasitology include:

1. Giemsa-stained blood film

2. Non-drying Immersion Oil for microscopy (Type A)
3. Compound microscope with 100x objective and 10x paired oculars
4. Cell counter with multiple tallies (piano type)
5. Calculator, handheld
6. Slide tray
7. Lens paper
8. Malaria registry book or results record book
9. Patient result form
10. Pens
11. Light microscope with 20 and 100X objectives
12. Fluorescence microscope with filters for fluorescein isothiocyanate (FITC)
examinations
13. Slides (multiwell printed microscope slides, standard glass slides, or frosted
glass slides)
14. Pasteur pipettes
15. Cotton Swabs
16. 4x4 inch gauze pads
17. Disposable conical pap er cups
18. Stool-preservative mixture
19. Para-Pak preserved samples
20. Formalin
21. MERIFLUOR® C/G kit
22. Saline
23. Iodine
24. Petroleum jelly and paraffin in a 1:1 ratio
25. Cotton tip swab
26. Hot sealant
27. Applicator stick
28. Positive microscope slides
29. Reference material (plates, photographs, digital images)
30. Staining reagents (depending on the staining method used)
PROCEDURE

Preparation of Microscope:

○ Calibrate the microscope before examination begins.

○ Ensure that the microscope is equipped with a 100x objective and 10x
paired oculars.
○ Adjust the light source optimally by looking through the ocular and the
x10 objective (low power).

Preparation of Wet Mount:

○ Obtain a microscope slide and the stool specimen.

○ Take a small amount of the specimen and place it on a microscope slide.
○ If the stool specimen is still somewhat solid, add a drop or two of saline
to the specimen and mix.
○ Ideally, two smears can be prepared on one slide, of which one can be
stained with iodine.
○ Thickness of the wet mount should be as illustrated in Figure A on the
right.
○ If desired, the coverslip(s) can be sealed with a preparation of petroleum
jelly and paraffin in a 1:1 ratio.
○ Systematically scan the entire coverslip area using the 10× objective as
illustrated in Figure C on the right.

Preparation of Stained Slide:

○ Use 3 × 1 slides to prepare permanent stained slides.

○ If the specimen is unpreserved, prepare a thin even smear of the
material by streaking the material back and forth on the slide with an
applicator stick.
○ If necessary, dilute feces with saline.
○ After the staining process is complete, systematically examine the
smear microscopically utilizing the 100× oil objective.
○ Examine at least 200 to 300 oil immersion fields.
○ Report protozoa seen as either trophozoites and/or cysts as applicable.

UV Fluorescence Microscopy Procedure:

○ The demonstration of Cyclospora oocysts in wet preparations is greatly

enhanced by using UV fluorescence microscopy.
○ Cyclospora oocysts exhibit intense blue color when observed under a
fluorescence microscope (UV excitation filter set at 330-385 nm).
○ Under bright-field (differential interference contrast or DIC) microscopy,
Cyclospora oocysts appear as refractile spheres (8-10 µm) with a
distinct oocyst wall.

Quality Control Measures:

 ○ Positive microscope slides as well as reference material (plates,
photographs, digital images) should be available by the workstation to
compare morphological details and organisms.
○ For malaria diagnosis, a quality assurance (QA) system for malaria
microscopy is needed to ensure good practices related to sample
collection, staining, examination, recording and reporting and
cross-checking of results both in the central and peripheral diagnostic
facilities.
○ Training and supervision of the microscopists and laboratories are also
important aspects of ensuring quality microscopy services.

INTERPRETATION OF RESULTS

In the case of the Direct Agglutination Test (DAT) for Leishmania parasites, a serum
can be considered positive if at a dilution ≥ 1:3,200, agglutinated promastigote cells
are visible by naked eye as a blue mat in the microtiter plate wells; absence of
agglutination is visible as a neat blue spot in the bottom of the microtiter plate wells.
The diagnostic performance of DAT is high in all VL-endemic regions with overall
sensitivity and specificity of 95% (93-97%) respectively, irrespective of the Leishmania
species causing VL ( L. donovani or Leishmania [ L.] infantum).

In microscopy examination of red blood and yeast cell agglutination, the sample
volume, concentration of CV-IIL, and incubation time play a significant role in the
observation of agglutination patterns. High concentrations of CV-IIL give large
agglutinates clearly visible even with the naked eye on the microscope glass slide,
but high concentrations of CV-IIL in combination with a low amount of the yeast cells
give less stable agglutinates.

In urine microscopy, there is no set normal range for cells in urine, as it is not
possible to account for all patient types or conditions. The interpretation of urine
microscopy results should be based on the clinical context and the presence or
absence of abnormal cells or casts.

REPORTING

1.
2. Documentation Process:
○ Record Keeping: Maintain detailed records of all tests performed,
including patient information, specimen details, test procedures, and
results.
○ Verification: Ensure that all results are verified for accuracy before
documentation.
○ Date and Time: Document the date and time when the test was
conducted and when the results were obtained.
○ Quality Control: Include information on quality control measures
undertaken during the testing process.
3. Reporting Process:
 ○ Format: The format for reporting test results can vary based on the
laboratory's practices. Common formats include electronic reporting
through Laboratory Information Systems (LIS) or paper forms.
○ Laboratory Information System (LIS):
■ Electronic Reporting: Enter test results directly into the LIS for
automated reporting and storage.
■ Patient Identification: Ensure accurate patient identification
details are entered to match the results with the correct
individual.
■ Result Interpretation: Provide clear and concise interpretations of
the test results.
○ Paper Forms:
■ Manual Entry: Write down the test results on designated paper
forms.
■ Legibility: Ensure that the results are legible and clearly written
to avoid misinterpretation.
■ Signature: Sign and date the paper form to authenticate the
results.
4. Content of the Report:
○ Test Results: Clearly state the results of the test, including any
observed agglutination patterns or other relevant findings.
○ Reference Ranges: Compare the results to established reference ranges
or expected values to determine normalcy.
○ Interpretation: Provide an interpretation of the results based on the
observed agglutination patterns and reference ranges.
○ Quality Control: Include information on the quality control measures
followed and any positive or negative controls used during the testing
process.

QUALITY CONTROL

1.
2. Positive and Negative Controls:
○ Frequency: Positive and negative controls should be included with each
test run.
○ Purpose: Positive controls help validate the test's ability to detect weak
positive reactions, while negative controls ensure the absence of false
positives.
○ Acceptable Ranges: Positive controls should be close to the cut-off
value of the test, and negative controls should consistently show no
reaction.
3. Live Control Organisms:
○ Frequency: Live control organisms with predictable reactions should be
used to verify stains, reagents, and media regularly.
○ Purpose: These controls help ensure that the laboratory equipment and
materials are functioning correctly.
○ Acceptable Ranges: The reactions of live control organisms should
align with expected outcomes based on established standards.
 4. Regular Assessment of Equipment and Supplies:
○ Frequency: Regularly assess the quality and functionality of equipment,
reagents, and supplies.
○ Purpose: Ensures that high-quality work is maintained by using reliable
equipment and consumables.
○ Acceptable Ranges: Equipment should meet minimum standards, and
supplies should be of the specified quality to produce accurate results.
5. Documentation and Record-Keeping:
○ Frequency: Continuous documentation of quality control measures and
corrective actions.
○ Purpose: Provides a record of quality control processes and facilitates
continual improvement of the laboratory quality system.
○ Acceptable Ranges: Records should be maintained according to
regulatory requirements and best practices.
6. Incubator and Equipment Maintenance:
○ Frequency: Regular maintenance and monitoring of incubators,
microscopes, refrigerators, and other equipment.
○ Purpose: Ensures that equipment is functioning correctly and maintains
the integrity of test results.
○ Acceptable Ranges: Equipment should be calibrated and maintained
according to manufacturer guidelines.

SAFETY PRECAUTIONS

Potential hazards associated with clinical microscopy and parasitology procedures

include exposure to infectious agents, chemical hazards, physical hazards, and
ergonomic hazards.

1. Infectious Agents: Handling clinical specimens may expose laboratory

personnel to infectious agents, such as bacteria, viruses, and parasites. To
minimize risks:
○ Use appropriate personal protective equipment (PPE), such as gloves,
lab coats, and safety goggles.
○ Implement proper handling and disposal procedures for infectious
materials.
○ Follow standard microbiological practices, such as hand hygiene and
disinfection of work surfaces.
○ Use biosafety cabinets or other engineering controls to contain
infectious agents during procedures.
2. Chemical Hazards: Clinical microscopy and parasitology procedures may
involve the use of hazardous chemicals, such as stains, fixatives, and
disinfectants. To minimize risks:
○ Use appropriate PPE, such as gloves and safety goggles, when
handling hazardous chemicals.
○ Follow proper handling, storage, and disposal procedures for
hazardous chemicals.
○ Use appropriate ventilation, such as fume hoods, when working with
hazardous chemicals.
 ○ Use chemical safety data sheets (SDS) to understand the hazards and
safe handling procedures for chemicals.
3. Physical Hazards: Clinical microscopy and parasitology procedures may
involve physical hazards, such as sharp objects, electrical hazards, and
radiation. To minimize risks:
○ Use appropriate PPE, such as gloves and safety goggles, when
handling sharp objects.
○ Use electrical safety practices, such as grounding and insulation, when
working with electrical equipment.
○ Use appropriate shielding and radiation safety practices when working
with radiation sources.
4. Ergonomic Hazards: Clinical microscopy and parasitology procedures may
involve prolonged periods of sitting, standing, or repetitive motions. To
minimize risks:
○ Use ergonomic equipment, such as adjustable chairs and microscope
stands, to minimize strain and fatigue.
○ Take regular breaks and stretch to avoid muscle strain and fatigue.

REFERENCES

CDC - DPDx - Diagnostic Procedures - Stool Specimens. (n.d.).

https://www.cdc.gov/dpdx/diagnosticprocedures/stool/microexam.html

Parasitology. (2010, August 1). https://doi.org/10.1128/9781555817435.ch9

Interpretation of laboratory results. (n.d.).

https://acutecaretesting.org/en/articles/interpretation-of-laboratory-results