Dr.Sidra Qaiser

Introduction:

In the differential leucocyte count (DLC) the percentage of each type of white blood cells in the total leucocyte population is determined. Each type of white cells, performs a different function in the battle against infections and each type of infection yields a different white cell picture in the blood. The morphology and staining characteristics of each type is peculiar and is responsible for the in specific typing.

Arneth Count

The age of the neutrophils can be determined by the number of lobes of its nucleus. The cells having less number of lobes are younger whilst with more lobes are relatively older. The maximum number of lobes is five in general.

Requirements:

Clean glass slides, microscope, pricking needle, cotton swab, dropper, glass rod, cedar wood oil, blow pipe and Leishman's stain comprising of

 Leishman’s powder = 0.15 gmMethyl alcohol (acetone free) = 100 cc

This stain contains both basophilic and eosinophilics (acidophilic) staining components and thus helps to discriminate among the granules of the differential leucocytes.

Procedure:

Preparation of blood filmSterilize the pricking needle and finger pulp

with spirit swab and then prick the finger, discard the first drop of blood. Now apply the slide inverted over the drop of the blood on the finger so that some blood sticks to the slide. With the help of spreader slide at an angle of 45° spread the blood drop quickly with a constant movement to prepare a thin film. Allow the slide to become dry in air. Prepare two or three blood film slides.

Staining of blood film

Place the slides on the glass rod stand and pour 20 -30 drops of leishman's stain to cover the slide having blood film and allow for 2 minutes. Now add distilled water in order to dilute it, avoid the spilling over.

Now wait for 10 minutes. Gently blow through the blow pipe for mixing of distilled water and Leishman's stain. Extra Leishman's stain is now rinsed by pouring the water slowly on the slide until the colour becomes pink. Dry the slide in the air.

Counting for DLC

Choose an area where the film is made quite clear. The three parts of a blood film include head, body and tail. The body of the film is the best area for cell-counting. Place two drops of cedar wood oil over the slide The slide is put under the oil immersion lens in contact with cedar wood oil Now move the slide slowly forward and backward from right to left for focusing and identification of different types of WBCs Count at least 100 cells to determine the percentage of DLC.

 

Precautions:

The blood film should be thin and smooth. The slides used must be clean. The staining and blowing should be as gentle

as possible.The lens and slide contact must be carefully

guarded to avoid the breaking of slides.