total count

TOTAL LEUKOCYTE COUNT

Dr.Gopala Krishnan.B.Yadav

It is a blood test to measure the number of white blood cells

It is a blood test to measure the number of white blood cells

Normal range

category Range / microlitre

adults 4000-10,000

birth 10,000-25,000

1-3yr 6000-18,000

4-7yr 6000-15,000

8-12yr 45000-13,500

Alteration in number and type of leukocytes is often associated with disease process & hence the count is informative

LEUCOCYTOSIS

Infections Malignancy like leukemia Severe emotional or physical stress Pregnancy

LEUCOPENIA

Infectionsbacterial-(typhoid,paratyphoid)viral(hepatitis)protozoa(malaria)

Some cases of leukemia Primary bone marrow depression

(Aplastic anaemia) Secondary bone marrow depression

(drugs,radiation) anaemia ( megaloblastic)

LETS COUNT…

SPECIMENS

Double oxalated or EDTA blood Capillary blood

REQUIREMENTS

Microscope Hemocytometer Wbc diluting fluid

HEMOCYTOMETER

Hemo: blood Cyto: cell Meter: measurement/counter

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

It includes:a) Neubauer’s slideb) Cover slipc) RBC pipetted) WBC pipette

NEUBAUER’S SLIDE

It is the name given to a thick glass slide . In the centre of the slide, there is an H- shaped groove. On the two sides of the central horizontal bar, there are scales for counting the blood cells The depth of the scales is 1/10mm or 0.1mm.

NEUBAUER’S CHAMBER

Neubauer’s slide with a cover slip over it, is called a Neubauer’s chamber

Four corner squares are meant for WBC counting.

Total = 64 small squares

WBC PIPETTE

It has a white bead It has graduations upto mark 11 Size of bulb is smaller Size of lumen is larger

WBC DILUTING FLUID:

It is prepared as follows: a) Glacial acetic acid: 2.0 ml b) 1 % (w/v) gentian violet: 1.0 ml c) Distilled water: 97 ml

PRINCIPLE

The glacial acetic acid lyses the red cells while the gentian violet slightly stains the nuclei of the leukocytes.

The blood specimen is diluted 1:20 in a WBC pipette with the diluting fluid.

The cells are counted under low power of the micro scope by using a counting chamber.

The number of cells in undiluted blood are reported per cu mm (µl) of whole blood

PROCEDURE

Draw blood up to 0.5 mark of a WBC pipette.

Carefully, wipe excess blood outside the pipette by using cotton.

Draw diluting fluid up to 11 mark. Mix the contents in the pipette and

after five minutes by discarding few drops, fill the counting chamber and allow the cells to settle for two to three minutes.

FOCUSING

4X to see the general formation of slide.

10X for WBC counting

COUNTING RULE

Do not count cells touchingBottom lineRight line This is to avoid double counting.

WBC COUNTING

DILUTION FACTOR

0.5 part of blood is mixed in 10 parts of fluid

So, 1 part of blood is in 20 parts of fluidThus, dilution factor for WBC counting is

20.

CALCULATION OF THE VOLUME OF WBC SQUARES

Length of one small square = 1/4mm Width of one small square = 1/4mm Depth of one small square = 1/10mm Volume of one small square = 1/4 x 1/4

x 1/10 = 1/160mm³

WBC COUNTING

Total no. of WBCs in 64 smallest squares = X No. of WBCs in one smallest square = X/64 Volume of one smallest square = 1/160mm³ No. of WBCs in 1/160mm³ = X/64 No. of WBCs in 1mm³ = X/64 x 160 No. of WBCs in 20 times diluted blood = X/64

x 160 No. of WBCs in undiluted blood = X/64 x 160

x 20/mm³ = X x 50/mm³