To determine the leukocyte count of your blood.
The number of WBCs in a known volume of diluted blood is counted and from this the number of WBC is
in 1 cubic millimeter of undiluted blood is calculated.
Hemocytometer with WBC pipette, diluting fluid, compound microscope, sterile lancet, watch glass, rectified spirit, cotton.
Diluting fluid: Ideal diluting fluid should dilute the blood, destroy RBC5 but not WBC5 and stain the nucleus of WBC5. Diluting fluid used isTurk’sfluid.
Composition: Gentian violet - 0.025gm
Glacial acetic acid - 2cc
Distilledwaterupto - lOOmI
Gentian violet stains the nucleus of WBC5. Glacial acetic acid destroys the RBC5 by hemolysis.
This is a bulb pipette having a long stem with a capillary bore and a pointed tip. The bulb contains a white bead inside. A small rubber tube provided with a mouth piece is connected to the small narrow portion, above the bulb for sucking blood and fluid into the pipette. The pipette has three markings on in it, 0.5 mark in the middle of the stem, 1 mark at the junction between stem and bulb, and 11 mark above the bulb. The total volume of the pipette is 11 parts, of which one part is in the stem and 10 parts in the bulb. The column of diluting fluid contained in the stem of the pipette does not enter into the dilution (i.e. 11-1 = 10), so that the blood sucked upto 0.5 mark will have a dilution of 0.5 in 10 or 1 in 20.
Other uses of WBC pipette:
1. Cell count in C.S.F. 2. Sperm count
3. Absolute Eosnophil count 4. R.B.C. count in severe anaemia
Clean the counting chamber and cover slip with water, dry it and mount it on the mechanical stage of the microscope. Focus any one of the four corner squares under the low power. Take a small quantity of the diluting fluid in a watch glass. See that the pipette is clean and dry.
Clean the finger tip and allow to dry. Using a sterile lancet make a sufficiently deep prick, so that blood flows freely. Discard the first drop, which may contain tissue fluid. Suck in blood upto the 0.5 mark of WBC pipette followed by the diluting fluid upto the mark 11 from the watch glass by keeping the pipette in vertical position. Allow the blood and diluting fluid to mix well by rolling the pipette horizontally in between the palms.
The counting chamber is charged by taking all precautions, using a small drop of the diluted blood at the tip of the pipette by just touching the edge of the cover slip on the chamber. Keep the counting chamber undistrubed for 2-3 mts, on the stage so that the cells settle down on the ruled area. Then count the cells under low power. Draw a chart of the squares and enter the number of cells in each square while counting. Precautions:
1. Counting chamber, pipette, fingertip and lancet should be clean and dry.
2. No air bubble should be present in pipette or counting chamber.
3. There should not be any overflowing in the chamber
4. The cells touching the left and lower borders should be counted. Those touching the right and upper borders should be omitted. This is to prevent counting the same cells twice.
5. Blood should not be allowed to clot in the pipette.
6. Cells should not be moving and should be more or less equally distributed.
Let the number of cells counted in 64 squares be “N”
Numberof cells in 1 square isN
Side length of 1 square = 1/4mm
Area of 1 square = 1/16mm2
Depthoffluidfilm = 1/10mm
So Volume of fluid in 1 square 1/16x1/1O =1/160mm3
Number of cells in 1/160 cu.mm of diluted blood = N
Number of cells in cu.mm of diluted blood = Nx160
Dilution factor= 1/20
No. of cells in 1 cu..mm of undiluted blood =Nxl6Ox 20 = N x 50 cells/cu.mm
Normal value :4,000- 11,000 cell/cubic millimeterof blood
An increase in W.B.C. count is called leucocytosis
A decrease in W.B.C. is called leucopenia
1 . After exercise
2. During emotional outbursts
3. After meals
4. Pregnancy- Return to normal after delivery
5. At birth
6. Diurnal variation
1. Infections: Acute pyogenic bacterial infections e.g-Acute tonsillitis, Acute appendicitis
2. Acute haemorrhage Starts within few hours of haemorhage
3. Trauma:mainly surgical operations, fractures etc.
5. Collagen disorders :Acute phases of rheumatoid arthritisLeukemias:
These are diseases of unknown etiology characterised by uncontrolled abnormal and wide spread proliferation of leucocytic cells of the body which infiltrate the bone marrow and other body tissues. This proliferation is usually accompanied by the appearance of immature leucocytes in peripheral blood which are morphologically abnormal.Broadly classified into acute and chronic leukemias.
When the count is below 4,000 cells/cu.mm.
a) Bacterial e.g:Typhoid, Paratyphoid, Brucellosis
b) Viral e.g:Hepatitis, Measles, Rubella
2. All types of overwhelming infections:
Miliary tuberculosis, Septicaemia, fatal cases of pneumonia.
3. Drug induced e.g: Chloramphenicol, Oxyphenbutazone, Indomethacin.
1. What are the important differences between leucocytosis and leukemia and leukamoid reaction?
2. What is the diagnostic and prognostic significance of Total Leucocyte Count?
3. How can you differentiate WBC from other artifacts?
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