The blood specimen to be used for differential leucocyte count is capillary blood or EDIA anti-coagulated blood.
There are three major steps involved in differential cell count:
1. Preparation of blood smears
2. Staining of blood
3. Staining of smear
4. Microscopic examination
Preparation of Blood Smear:
Take a clean grease free slide. Obtain capillary blood directly from the finger or EDTA anti-coagulated blood. Place it on a corner of a slide. Take a spreader (Spreader is a slide with sharp edges).
Touch the edge of the spreader to blood drop on slide. Slightly push it backward so that, the drop is spread evenly to the edge of the spreader. Now, spread blood with the help of a spreader across clean grease free slide.
The angle between spreader and slide should be about 45°. With a quick movement, push the spreader towards the other end of the slide. Blood film should not be too thick. It should be 1 cm. from the edge of slide and 5 mm. in width.
Staining of Blood:
Blood cells have different structures, which take different stains. Some are basophilic; others are acidophilic while some cells accept neutral stain. Therefore, the stain used is combination of these three. Such stains are called as ‘Romanowsky stain’.
The three different types of Romanowsky stains are commonly in use.
i. Leishman stain
ii. Giemsa’s stain
iii. Wright’s stain.
Each of the stain contains acidic stain, basic stain and buffer solution. Generally, methylene blue or touline are basic stains, and Eosin, Azure-I, Azure-II are the acidic stains in use.
It is advisable to stain a slide soon after preparation of blood smear.
Staining of Smear:
Leishman stain is the most common and cheapest of all stain.
i. Leishman stains powder – 0.15 gm.
ii. Methyl alcohol – 100 ml.
Leishman stain crystals are grounded in a glass mortar. This powder is first dissolved in few ml of methyl alcohol, and then the remaining quantity of alcohol is added, so that the entire volume becomes 100 ml. Pour the stain in a clean dry bottle, close it well. Do not open it or filter it within 3 weeks. After 3 weeks it is ready for use.
Blood films are placed in a staining tray. The dry blood film is then covered with stain. The stain should be evenly distributed over the entire slide. After 1 min. add distill water or buffer solution (Sodium- potassium phosphate buffer at pH 6.8) to the slide.
The distilled water / buffer solution should be carefully mixed with the stain. Keep it as it is for about 7 to 8 min. Then wash the slide with distill water to remove excess of stain. Air dry the film and observe under oil immersion objective of microscope.
First examine stained blood smear under, low power objective. Note the background colour and distribution of cells.
In an ideal staining smear, three zones can be identified:
1. Thick area or head of smear
2. The central area is body
3. At the end of smear is tail region
Choose the portion of the smear in the body region, slightly before the tail end.
Observe the slide under oil immersion objective by putting a drop of oil over the slide.
Examine the slide in tail region using OIO and move the slide as shows in Figure 7.2.
Count each type of white cell observed. Record the observations either on a piece of paper in a tabular form or on a cell counter. The cell counter has different keys for different types of WBC. Continue the counting till 100 cells are counted. This will give the average number of white cells.
With Leishman stain, the cells are observed as:
Purple coloured nuclei with pink cytoplasm.
Cytoplasm is faint pink, nucleus is purple and granules are orange red.
Granules stain dark blue with purple nucleus.
Pink cytoplasm with purple colour nucleus.
Dark blue nucleus with light blue cytoplasm.
Violet coloured granules.
7. Red cells:
Wright stain is very similar to Leishman stain.
a. Wright stains powder – 0.3 gm.
b. Glycerol – 3 ml.
c. Methyl alcohol – 97 ml.
Left this solution as it is for a week.
Cover the slide with stain for 5 to 10 min. Take care that the stain does not dry. Now add equal amount of buffer solution/ distilled water to the slide. Allow it to react for 2 to 3 min. Then wash the slide with water, and air dry it. Now observe under microscope.
Wright stain gives the same colour reaction, as that of Leishman stain.
This is one of the best stains for malarial and other blood parasite. It is also satisfactory as a routine blood stain.
i. Giemsas stain powder – 0.75 gm.
ii. Glycerin – 25 ml.
iii. Methyl alcohol – 75 ml.
Place 75 ml methyl alcohol and 25 ml glycerin in a beaker. Put in a water bath and add 0.75 gm of dry Giemsa’s power. Warm up to 60°C. Stir with a glass rod. Filter the solution. This is a stock solution. For use, mix 1 ml of stock solution of stain with 4 ml of methyl alcohol.
Cover the slide with 15 drops of stain for 1 min. Add 30 drops of distilled water, mix well and allow to stand for 5 min. Wash with water, air dry the smear, and observe under OIO.
1. Nuclei of Neutrophil: Reddish purple colour
2. Eosinophlic granules: Red to orange colour
3. Basophilic granules: Blue colour
4. Lymphocyte: Dark blue nucleus with light blue cytoplasm
5. Platelets: Violet to purple granules
The basic use of DLC is to identify changes in distribution of white cells. These changes are related to specific infection, like bacterial, viral, parasitic, leukemic, etc. Different clinical terms are used for the different changes in normal values of the cells.
Thus, Differential Leucocyte Count gives us the idea about distribution of different proportions of white cells.