Standard Operating Procedure (SOP) for Differential White Blood Cell (WBC) Count

Differential White Blood Cell (WBC)

The purpose of this SOP is to outline the procedure for performing a Differential White Blood Cell (WBC) count, which categorizes and quantifies the five major types of WBCs in a blood sample: neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

The levels of these cells in the blood increase based on the type of problem the body is facing.

For example, eosinophils typically rise during allergic reactions or parasitic infections. Therefore, a differential WBC count is used for diagnosing infections, allergies, and blood-related disorders.

 

Equipment and Materials

• EDTA anticoagulated blood sample (venous)
• Glass slides
• Microscope with oil immersion objective (100x)
• Staining reagents for Romanowsky stains (e.g., Giemsa stain, Field’s stain, Leishman stain, or Wright stain)
• Staining rack
• Immersion oil
• PPE (gloves, lab coat)
• Biohazard waste containers
• Microscope cleaning materials (lens paper, alcohol)

 

 

Specimen Collection

Venous Blood:

• • Collect 2-5 mL of venous blood into an EDTA anticoagulated tube.
  • Invert the tube gently to mix the anticoagulant and prevent clotting.

Preparing the Blood film

Making the Thin Blood Film:

• Prepare a thin blood film by placing a small drop of blood on a clean glass slide.
• Using a spreader slide at a 30-45° angle, spread the blood in a thin layer.
• Allow the film to air dry thoroughly.

Fixation:

• Fix the dry thin film by immersing it in absolute methanol for 30 seconds.
• Allow the slide to air-dry after fixation.

Staining with Field’s Stains:

• Immerse the fixed slide in Field’s Stain B (basic stain) for 30 seconds.
• Rinse the slide gently in buffered water for a few seconds to remove excess stain.
• Immerse the slide in Field’s Stain A (acidic stain) for 30 seconds.
• Rinse the slide again in buffered water for a few seconds to remove excess stain.
• Allow the slide to air-dry in a vertical position or blot it gently with absorbent paper.

 

Microscopic Examination

• Focus Under Low Power (40x):
  • Place the stained slide on the microscope stage.
  • Apply a drop of immersion oil to the slide, close to the tail region of the film.
  • Focus the slide using 40x objective to find the optimal area where the red cells are evenly distributed (this area is ideal for differential counting).
  • Switch to the 100x objective.
  • Begin scanning the slide using a systematic pattern.

• Counting WBCs:
  • Count the first 100 white blood cells and differentiate the five cell types as you go. (Using the differential counter).
    • Neutrophils: Multilobed nucleus with pale cytoplasm.
    • Lymphocytes: round nucleus with scant cytoplasm.
    • Monocytes: Kidney-shaped nucleus with gray-blue cytoplasm.
    • Eosinophils: Bilobed nucleus with large red/orange granules.
    • Basophils: Bilobed nucleus with large blue/purple granules.
  • Keep a manual tally of each cell type or use an automated differential counter.

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Calculation of Differential Count

Relative Count:

• • The results of the differential count are expressed as a percentage of each WBC type from the total 100 cells counted.

 

Absolute Count:

• Calculate the absolute number of each white cell type by multiplying the number of each cell counted (expressed as a decimal fraction) by the total WBC count.
• Example: If the percentage of neutrophils counted is 80% i.e., 0.80 when expressed as a decimal fraction and the total WBC count is 6 10⁹/l; Absolute neutrophil count is 0.80 X 8.6 = 6.9 10⁹/l.

 

Interpretation of Results

Normal Ranges

• Adults

1. • Neutrophils: 1.5–7.5 × 10⁹/L (40–75%)
   • Lymphocytes: 1.2–4.0 × 10⁹/L (21–40%)
   • Monocytes: 0.2–1.0 × 10⁹/L (2–10%)
   • Eosinophils: 0.02–0.6 × 10⁹/L (1–6%)
   • Basophils: 0.01–0.1 × 10⁹/L (0–1%)

• Children (2–6 years)

1. • Neutrophils: 1.5–6.5 × 10⁹/L (20–45%)
   • Lymphocytes: 6.0–8.5 × 10⁹/L (45–70%)
   • Monocytes: 0.1–1.0 × 10⁹/L (2–10%)
   • Eosinophils: 0.3–1.0 × 10⁹/L (1–6%)
   • Basophils: 0.01–0.1 × 10⁹/L (0.1–1%)

 

Abnormal Differential Counts:

1. • Neutrophilia: Elevated neutrophil count, indicative of bacterial infections or inflammation.
   • Lymphocytosis: Increased lymphocytes, commonly seen in viral infections.
   • Monocytosis: Increased monocytes, often seen in chronic infections and inflammatory conditions.
   • Eosinophilia: Elevated eosinophil count, suggestive of parasitic infections or allergic reactions.
   • Basophilia: Elevated basophils, rare but may indicate allergic responses or myeloproliferative disorders.

 

References

• Cheesbrough, M. District Laboratory Practice in Tropical Countries, Part 2, 2nd edition. Cambridge University Press, 2005.