This document provides information on methods for performing a complete blood count (CBC), including white blood cell (WBC) count, corrected WBC count, and differential leukocyte count (DLC). The WBC count involves using a counting chamber, pipettes, and diluting fluids to count WBCs under a microscope. The DLC involves making a blood smear, staining it, counting different types of WBCs, and reporting results as relative or absolute counts. Normal ranges are provided for WBC subtype percentages and counts.

1. SHAH SUNIL KUMAR
(BOND KING)

2. A. WBC count (Hemocytometry method/ Microscopic
method/ Manual method)
 Counting chamber
 Pipets
 Diluting fluids
1. Counting chamber
- improved neubauer

3. W W
Area= 1mm square
W W

4. 2. Diluting fluids
 2-3% glacial acetic acid (gentian violet dye)
 1% HCL
 Turk’s solution (methyl violet dye )
- Diluting fluid easy to prepare, must be
isotonic, must be carciogenic (most toxic substance
in 100 LL the benzene)
- Hypotonic soln ( WBC) , Isotonic soln (RBC)
- Only hemolyse mature RBC
- Disadvantage of diluting fluid – nucleus in
cytoplasm.(i.e. immature RBC will not hemolyse so
counted WBC)

5. Short stem
-WBC bulb is smaller than RBC
-Upper calibration = 11
bead
-Constant volume of pipet = 11-1 =
bulb 10
-Volume of bulb is 10 times greater
than volume of bulb
Long
stem

6. Procedure
 Suck the blood up to 0.5 mark.
 Suck diluting fluid to 11 mark.
 Shake the pipet for 2 minute.
 Discard first few drops.
 Charge the counting chamber – 3 minute for settling
down.
 Count the WBC in 4 WBC square using LPO.
 Compute for total WBC count.

7. Computation
 WBC count = nos. of WBC counted x DCF x VCF
 DCF = vol. of bulb 10/0.5 =20
amount of blood sucked
 VCF = volume desired (1) 1/ 1x0.1x4 = 2.5
area x depth of counting chamber x nos. of
square used.
Normal value
 5000 – 10,000 / cumm or 5-10 x 10^9/L.

8. B. Corrected WBC Count
 Done when there is high WBC count and there are
more than 10 nucleated RBC per 100 WBC in the
blood smear
A = B x C A = corrected WBC count
C+D B = uncorrected WBC count
C = constant (100)
D = nos. of nucleated RBC > 10
 Nucleated or immature RBC will not hemolyse by
hypotonic used in WBC as diluting fluids.
 In WBC count ,mistakenly immature RBC is counted
thinking WBC.

9. C. Differential Leukocyte Count (DLC)
 Expression in % the relative number of the types of
WBC.
 4 general steps
- blood smear preparation
- staining
- counting
- reporting

10. 1. Blood smear preparation
 Wedge method – 2 glass slide method(most
commonly used)
 Beacon’s method – coverslip and glass slide method
 Erlich’s method- 2 coverslip method

11. Procedure (wedge method)
 A drop of blood is applied near one end of the
slide using capillary tube.
 The spreader is drawn back into the drop of blood
and held until blood has spread across its width.
 The spreader at an angle of 40- 45 degree is
pushed steadly along the slide to produce a
thin, even film of blood.

12. Method of drying a smear
 Air dry
 Use of low flame
 Use of oven
 Immersion in methyl alcohol for 1-2 minutes.
- Drying a smear (up – down) i.e. Reverse because in
blood smear transaction of blood should be thinner
to thicker.

13. Prerequisite for proper blood smear
1. Slide and spreader should be clean
2. Size of the drop of blood
3. Smearing should be done quickly.
4. Angle and pressure.
Types of smear
1. Thick - parasite counting
2. Thin – cell counting

14. 2. Staining
 Romanowsky group of stain
(basic stain – methylene blue , acidic stain –
eosin, neutral stain – mixture of acid and basic stain
– cytoplasm)
* eosinophilic as granules found in cytoplasm
 Wright’s – most common
 Leishmann’s – urgent
 Giemsa’s – produce more delicate staining
 Jenner–giemsa
 May – Grunwald – giemsa (best method)
* Wright stain and 50 drops buffer – Metallic
greenish sheen.

15. Proper staining reaction
 Neutrophils – dark blue nucleus ; lilac pink granules.
 Eosinophils - dark blue nucleus; blue black granules
.
 Lymphocyte - dark blue nucleus; sky blue
cytoplasm.
 Monocyte – faint blue nucleus ; faint blue gray
cytoplasm.
* Robin- egg blue – sky - blue cytoplasm.
- platelets – pale lilac blue
- RBC – pinkish buff to orange.
- bacteria – blue

16. Counting (scanning of smear) only used one method
for entire
 Two field meander
 Four-field meander
 Strip method
 Exaggerated battlement
 Crenellation (most commonly used )

17. Reporting
A. Relative count-gives the number of WBC type per
100 WBCs.
nos. of specific WBC x 100 = %
100
N.R.
 Neutrophil - 51- 67% (bacteria)
 Lymphocytes – 25-33% (virus)
 Monocyte – 2-6% (bacteria)
 Eosinophil – 1-4%
 Basophil – 0-1%

18. B. Absolute count – gives the number of specific WBC
type per cumm.
Relative count x WBC count = / cumm
(most informative method)
N.R.
 Neutrophil – 1,600 -7,260 /cumm
 Lymphocyte – 960 - 4,400/cumm
 Monocyte – 180 - 800 /cumm
 Eosinophil – 45 - 440/cumm
 Basophil – 45 -110/cumm