Ayder Comprehensive Specialized Document Number: ACSHL/ALS/SOP/5.

5-
Hospital Laboratory 003

Title: SOP For Manual Differential Copy No. Version: 1.0

Leukocyte count Test Page 1 of 10 Effective date: 01/Dec/2018

Ayder Comprehensive Specialized Hospital

Laboratory

Standard Operating Procedure

For
Manual Differential Leukocyte count Test

Compiler: Rahel Afewerki Signature & date:

Approver: Aregawi H/Slassie Signature & date:

Authority: Ayder Comprehensive Specialized Hospital Laboratory Authorized Date: 01/Dec/2018

NOTE: This is a CONTROLLED document for internal use only.
 Ayder Comprehensive Specialized Document Number: ACSHL/ALS/SOP/5.5-
Hospital Laboratory 003

Title: SOP For Manual Differential Copy No. Version: 1.0

Leukocyte count Test Page 2 of 10 Effective date: 01/Dec/2018

I. REVSION AND AMENDMENT

A. Annual Review of Document
Reviewed by: Approved by:
Revision No. Review Date
Name Signature Name Signature

B. Version Change History/Description

Version Effectiv Description of Version Change Name & Name &

. No. e Date Signature of Signature of
Reviewer approval

C. Amendment

Rev. Page Description of Amendment Amend Effective Name &

No. No ment Date Signature of
Date approval

Authority: Ayder Comprehensive Specialized Hospital Laboratory Authorized Date: 01/Dec/2018

NOTE: This is a CONTROLLED document for internal use only.
 Ayder Comprehensive Specialized Document Number: ACSHL/ALS/SOP/5.5-
Hospital Laboratory 003

Title: SOP For Manual Differential Copy No. Version: 1.0

Leukocyte count Test Page 3 of 10 Effective date: 01/Dec/2018

Contents
I. REVSION AND AMENDMENT..............................................................................2
A. Annual Review of Document................................................................................2
B. Version Change History/Description....................................................................2
C. Amendment...........................................................................................................2
1. Purpose............................................................................................................................4
2. Abbreviation....................................................................................................................4
3. Materials..........................................................................................................................4
4. Sample.............................................................................................................................6
4.1. Limitations:...............................................................................................................6
5. Special Safety Precautions...............................................................................................7
6. Quality Control................................................................................................................7
7. Procedure.........................................................................................................................7
8. Result Interpretation........................................................................................................8
8.1. Expected Values.......................................................................................................8
9. Limitations.......................................................................................................................8
10. Principle.........................................................................................................................8
11. Clinical Utility...............................................................................................................9
12. Reference......................................................................................................................9
Declaration.......................................................................................................................10

Authority: Ayder Comprehensive Specialized Hospital Laboratory Authorized Date: 01/Dec/2018

NOTE: This is a CONTROLLED document for internal use only.
 Ayder Comprehensive Specialized Document Number: ACSHL/ALS/SOP/5.5-
Hospital Laboratory 003

Title: SOP For Manual Differential Copy No. Version: 1.0

Leukocyte count Test Page 4 of 10 Effective date: 01/Dec/2018

1. Purpose
This Procedure provides instructions on how to differentiate white blood cells and
look at Morphology of white blood cells manually

2. Abbreviation
WBC- White blood cells
RBC- Red blood Cells
DIFF- Differential cell count
PLT- Platelet
QC- Quality Control
IQC-Internal Quality Control
EQA- External Quality Assessment
CBC- Complete blood cell count
Mono- Monocyte
Neut - Neutrophil
Eos - Eosinophil
Baso -Basophil
Gran-Granulocyte Absolute Count
%Gran-Granulocyte Percentage
%LYM-Lymphocyte count percentage
EDTA-Ethylene di tetra acetate.

3. Materials
Reagents
Romanowsky stains(Eg. May-Granuwald- Giemsa,
 Giemsa stain (powder)
 Absolute Methanol
 Glycerol
 Methyl Alcohol

Reagents preparation:

Authority: Ayder Comprehensive Specialized Hospital Laboratory Authorized Date: 01/Dec/2018

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Hospital Laboratory 003

Title: SOP For Manual Differential Copy No. Version: 1.0

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Giemsa powder ---------------------3.8 g

Glycerol (Glycerin) ------------------250ml
Methanol (Methyl Alcohol) ----------250 ml

Step Action
1 Weigh the Giemsa and transfer to a dry brown bottle of 500 ml
capacity which contains a few glass beads.
2 Using Dry Cylinder, Measure the methanol and add to the stain. Mix
well
3 Tightly stopper the bottle and shake the bottle for 2-3 minutes
4 By Using the same cylinder ,Measure the glycerol

Step Action
5 Add measured glycerol and continue shaking for 2-3 minutes at 30
minutes interval at least 6 times.
6 Keep the bottle for 2-3 days; shaking it 3-4 times a day
7 Label the bottle with name of reagent and date of preparation and
mark it flammable and Toxic, store at room Temperature in the
dark place.

Reagents stability and storage:

Giemsa stain solutions are stable for several months if it is kept at room
Temperature in the Dark.

Supplies
 Disposable gloves.
 Microscopic slides
 Syringe/Lancet
 Alcohol 70%
 Reagent bottles
 cotton swab
 Glass beads
 Pipette

Authority: Ayder Comprehensive Specialized Hospital Laboratory Authorized Date: 01/Dec/2018

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Hospital Laboratory 003

Title: SOP For Manual Differential Copy No. Version: 1.0

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 Rack

Equipments
 Microscope
 Diff counter
 Beam balance
 Cylinder

4. Sample

Sample type Amount Transport and Stability

required Storage
Anti coagulated Minimum of 6 µl Keep at room 8 hrs at room
Whole Blood or or one large drop Temperature. Temperature.
finger prick blood
blood.

4.1. Limitations:
Hemolysed sample, High volume of Anti-Coagulant, Inadequate volume of
samples i.e. less than 2/3

5. Special Safety Precautions

 All materials contaminated with patient specimen, reagent and controls should be
handled as potentially infectious and hazardous.
 Refer to Clinical Laboratory Safety Manual ACSHL/ALS/MAN/5.2-001 for
safety precaution procedures.

Authority: Ayder Comprehensive Specialized Hospital Laboratory Authorized Date: 01/Dec/2018

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 Ayder Comprehensive Specialized Document Number: ACSHL/ALS/SOP/5.5-
Hospital Laboratory 003

Title: SOP For Manual Differential Copy No. Version: 1.0

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6. Quality Control
Control preparation:
Check the quality of stain with the WBC count and staining reaction.
Note: Repeat counts on selected slides on subsequent days because this will give an
indication of the range in the variation of the results (include note on reagent
Quality)
-WBC Should have blue nucleus with a lighter staining cytoplasm.

7. Procedure
Step Action
1 Make a thin smear in a microscopic slide
2 Fix the slide with absolute methanol for 10-20 seconds
3 Dry the slides completely
4 Drop a Giemsa (diluted with 9 part of distill water with 1 part of
Giemsa stain) to a slide until it covers the smear.

Step Action
5 Stain/wait for 10 minutes.
6 Wash then allow it to stand undisturbed for 2 minutes.
7 Air Dry the slides
8 Examine the smear by using 10x objective to scan check
9 Then focus and count the cells by using 100x objective
10 Count a total of 100Leukocytes and record the number of each
type of leukocyte seen as percentage.
11 Report the percentages, Number and Morphology of Cells
counted.

8. Result Interpretation
 Percentage of leucocytes types
 Morphologic Reports:
 Report the presence of any parasites as well as bacteria, besides this also any
 Immature cell type should be reported.

8.1. Expected Values

Authority: Ayder Comprehensive Specialized Hospital Laboratory Authorized Date: 01/Dec/2018

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Hospital Laboratory 003

Title: SOP For Manual Differential Copy No. Version: 1.0

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Analyte Reference Range Units

Male Female
Neut 50-75 50-75 %
Lymph 25-40 25-40 %
Mono 3-7 3-7 %
Eos 0-7 0-7 %
Baso 0-2 0-2 %

9. Limitations
 Poorly made smear
 Dirty glassware
 Incorrect buffer PH
 Films prepared from old blood etc.

10. Principle

A drop of blood is spread on a slide and then fixed, stained, and examined under the
microscope. In this way, red blood cells, leucocytes, and platelets may be studied. The
Romanowsky stains contain eosin Y, which is an acidic anionic dye, as well as azure B
and other thiazine dyes, which are basic cationic dyes. When these Dyes are diluted in
buffered water, ionization occurs. Eosin stains the basic components of blood cells; for
example, haemoglobin stains pink-red, and other methylene blue–derived dyes stain the
acidic components of the cells. Nucleic acids and nucleoproteins stain various shades of
mauve-purple and violet, the granules of basophils stain dark blue-violet, and the
cytoplasm of monocytes and lymphocytes stains blue or blue-grey. The staining reactions
of Romanowsky stains are pH dependent, which is why the stains are diluted in buffered
water with a specific pH.

11. Clinical Utility

• Elevated white blood cell count may mean infection.

• Decreases in white blood cell count may occur with disease
Progression or may Indicate bone marrow suppression from ARV therapy.

Authority: Ayder Comprehensive Specialized Hospital Laboratory Authorized Date: 01/Dec/2018

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Hospital Laboratory 003

Title: SOP For Manual Differential Copy No. Version: 1.0

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Total lymphocyte count: When a patient is on ARV therapy, a decrease in

absolute lymphocyte count may reflect bone marrow suppression from treatment.
A total lymphocyte count of <1,200/mL has been correlated with a CD4 count of
<200 cells/mL. However, the total lymphocyte count alone should not be used in
Asymptomatic patients when deciding whether to start ARV therapy.
• An increase in Neutrophil may be due to an acute bacterial infection or
Hematological malignancies such as myeloid leukemia.
• An increase in Eosinophil may be due to a parasitic infection or an allergic
Reaction.
• An increase in lymphocytes may be due to viral infections or chronic infections

12. Reference

1. Central Board of Health (CBOH). 2003. Hematology Standard Operating

Procedures for Hospital Laboratories Level III. Lusaka, Zambia: Mipal Printers.
2. Lewis, S. M., B. J. Bain, and I. Bates (eds.). 2001. Dacie and Lewis Practical
3. Hematology. 9th edition. New York: Churchill Livingstone.
Coast provincial general hospital ART program standard operating procedures
March/2005, Kenya

Declaration
I, the undersigned laboratory personnel, certify that I am conducting every steps of the
procedures incorporated in this SOP after a prior reading.

Authority: Ayder Comprehensive Specialized Hospital Laboratory Authorized Date: 01/Dec/2018

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Title: SOP For Manual Differential Copy No. Version: 1.0

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Authority: Ayder Comprehensive Specialized Hospital Laboratory Authorized Date: 01/Dec/2018

NOTE: This is a CONTROLLED document for internal use only.