Practical Hematology Manual

Components of Normal Adult Blood

How to collect blood sample to do the Complete blood count (CBC)

When blood come in contact to external surfaces (tube), it will Normally clot as follow

We need to prevent this blood clotting to be able to proceed through the intended CBC,To do this we have to add anti-coagulants (chemicals that prevents the blood clotting

Separation of Components

Plasma = Less Dense

Hematocrit“Packed Cells”

More Dense

Platelets / WBC’s

Hemacytometer

• The hemacytometer counting chamber is used for cell counting.

• It is constructed so that the distance between the bottom of the coverslip and the surface of the counting area of the chamber is 0.1 mm.

• The surface of the chamber contains two square ruled areas separated by an H-shaped moat.

Hemo: blood

Cyto: cell

Meter: measurement/counter

Thus, it is an instrument used to count the blood cells.

It includes:)aNeubauer’s

slide)bCover slip

)cRBC pipette)dWBC pipette

haemocytometer chamber

Thoma white pipette

Rubber sucking tube

Hemacytometer

• Each scale is 3mm wide and 3mm long.• The whole scale is divided into 9 big squares.• Each primary square is 1mm long and 1mm width• Each primary square is further divided into 16 secondary square

(central 25 secondary square )• Each secondary square is further subdivided into 16 tertiary square

Principle of WBCs count

Free-flowing capillary or well-mixed anticoagulated venous blood is added to a diluent) at a specific volume in the diluting pipette.

The diluent lyses the erythrocytes but preserves leukocytes and platelets. The diluted blood is added to the hemacytometer chamber.

• Sample:– EDTA- anticoagulated blood or capillary blood is preferred.

• WBCs count diluting fluid :•May be one of the following:

• Acetic acid 1% (v/v) in distilled water.• HCL 1% (v/v) in distilled water.• Turks' solution which is formed of:

» Glacial acetic acid 1 ml » Crystal violet 1 ml » 100 ml distilled water.

Principle

Equipment1. White blood cells count diluting fluid

2. WBCs pipette

3. Hemacytometer and coverslip

4. Microscope

5. Lint-free wipe

6. Alcohol pads

For WBC counting

0.5 part of blood is mixed in 10 parts of fluid

So, 1 part of blood is in 20 parts of fluid

Thus, dilution factor for WBC counting is 20.

ProcedureCarefully charge hemacytometer with diluted blood by gently squeezing sides of reservoir to expel contents until chamber is properly filled.

•4X to see the general formation of slide.

•10X for WBC counting•40X for RBC counting

FOCUSING

Adjust the microscopic objective lens 40X for RBC counting

Counting Rule

• Cont RBCs in the L shape method

• Do not count cells touching–Top line–Right line This is to avoid

double counting.

WBC COUNTING

Total no. of WBCs in 4 Primary squares = X

Mean No. of WBCs in Primary squares (1 mm2) = X/4

(diluted)

X/4 x20 = No. of WBCs in 1 mm2 of undiluted blood

X/4 x20 x 10= No. of WBCs in 1 mm3 of undiluted blood

No. of WBCs in 1mm³ = 1 µl = X x 50/mm³

While X is the No of WBCs counted in the 4 primary

square• Normal Values:

• 4500 -11000/mm3 or µL

Disturbance of Leukocytes Number)

• Leucopenia :WBCs < 4000

• Leukocytosis : WBCs >11000

• Neutrophilia:

• Lyphoctosis

• Leukemia. Uncontrolled production of WBCs

can be caused by cancerous growth . Leukemia,

is characterized by WBCs > 30000/ul