WBC

METHODOLOGIES I
SHAH SUNIL KUMAR
(BOND KING)

WBC count (Hemocytometry method/ Microscopic

method/ Manual method)
. Counting chamber
. Pipets
. Diluting fluids
1. Counting chamber
- improved neubauer
A.

Area= 1mm square

2. Diluting fluids
2-3% glacial acetic acid
(gentian violet dye)
1% HCL
Turks 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)

Short stem
bead
bulb

Long
stem

-WBC bulb is smaller than RBC

-Upper calibration = 11
-Constant volume of pipet = 11-1 =
10
-Volume of bulb is 10 times greater
than volume of bulb

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.

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.

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.

C. Differential Leukocyte Count (DLC)

Expression in % the relative number of the types of
WBC.
4 general steps
- blood smear preparation
- staining
- counting
- reporting

1.
.

.
.

Blood smear preparation

Wedge method 2 glass slide method(most
commonly used)
Beacons method coverslip and glass slide method
Erlichs method- 2 coverslip method

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.

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.

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

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
Wrights most common
Leishmanns urgent
Giemsas produce more delicate staining
Jennergiemsa
May Grunwald giemsa (best method)
* Wright stain and 50 drops buffer Metallic
greenish sheen.

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

Counting (scanning of smear) only used one method

for entire
Two field meander
Four-field meander
Strip method
Exaggerated battlement
Crenellation (most commonly used )

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%

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