estimation of total leukocyte count (TLC):

APPARATUS

Neubauer’s chamber. WBC pipette (Thomas diluting pipette), diluting fluid cover slip, pricking needle (blood lancet) and spirit swab.

METHODS:

There are two methods of calculating the WBC count:

•  Electronic counting method(Computerized method).

•  Visual method.

WBC diluting fluid (Turk’s fluids):

• Gentian violent 1%: lml, stains nucleic of WBCs.
• Galacial acetic acid: 3ml to destroy RBCs and platelets (destroys cell membrane).
• Distilled water: To make 100 ml.

PRINCIPLE:

Diluted specimen of blood with stained WBCs is loaded on to haemocytometer and the cellular elements are counted. The product of the number of cells calculated per cu mm diluted blood and the dilution factor gives the number of cells/mm3 in undiluted blood. Inbrief white blood cells are counted in diluted blood and actual count is calculated by multiplying it by dilution factor.

PROCEDURE:

• Take 3-5 ml Turk’ fluid in a watch glass.
• Prick the ring finger of left hand after cleaning it with spirit swab.
• Wipe off first drop of blood Suck next drop of blood in WBC pipette exactly up to 0.5 mark, taking care that there should be no air bubble. If excess blood has been drawn, remove it by touching the pipette on cotton swab or tissue paper very carefully.
•  Wipe off the blood sticking around the tip of the pipette with cotton swab or tissue paper.
• Now suck the Turk’s fluid in the pipette up to mark 11.
• Hold the pipette in between palms and mix the blood with diluting fluid by rolling it gently or place your thumb and index finger over open tips and vigorously gorousl rotate pipette back and forth at right angle to axis of pipette for 30-45 seconds. Y

• Focus the Neubauer’s chamber under low power and charge the chamber after discarding 2-3 drops of fluid from the pipette in the same way as in RBC count. Put tip of the pipette at angle of 45. The diluted blood (white blood cells) will flow in by capillary action to charge the chamber.
• Let the cells settle for 3-5 min, and keep the Neubauer’s chamber again under low power of microscope and count WBC in outer four corner squares. The white blood cells are counted in 64 smaller squares by applying Thomas rule.
• During counting WBC enter the number of cells counted in different squares in the squares drawn on a paper .

CALCULATIONS:

Dilution factor: 0.5 part of blood mixes in total 10 part of mixture in bulb (9.5 parts diluting fluid). Fluid present in the stem (1.0 part) does not take part in mixing. This is why 2-3 drops of fluid is discarded before charging.

64 small sized squares contain cells = x

1 small sized squares contain cells =x/64 Side of one small square =1/4 mm

Area of one small square =1/4 x 1/4 1/16 mm2

Depth= 1/10 mm

Volume of one small square =1/16 x 1/10 1/160 mm3

Volume 1/160 mm3 contains= x/64 cells Volume mm3 contains = x/64 cells x

= 10x/4

since dilation is 1:20 10x/4×20 = 50x

This is total no of white blood cells in undiluted blood

PRECAUTIONS:

• All the precautions are observed as in RBC count.
• There should not be a difference of 15 cells in four corner square counting in highest and lowest total number of cells.
• Exact measurement should be done upto the mark 0.5 mark.
• There should be no bubble in column of blood or fluid mixture in the pipette during suction.

VARIATION IN WBC COUNT:

leucocytosis: Physiological: Severe muscular exercise, pregnancy, newborn babies and food. intake.

Pathological: Acute pyogenic infection, e.g. abscess, pneumonitis and appendicitis and appendicitis, Tonsillitis. Leucopenia: Pathological: Typhoid, parazyphoid, bone marrow depressing drugs (chloramphenicol etc).