THE PREPARATION AND STAINING OF BLOOD

• FILMS Examining a properly prepared and stained blood film constitutes the most important investigation in Haematology. It is performed for: Differential Leucocyte Counts (DLC) General assessment and verification of various cell counts Study of RBC morphology for classifying various anaemias Study of WBC morphology for diagnosing leukaemias and other WBC disorders Study of platelet morphology for diagnosing some platelet disorders

• Study of parasites found in plasma or RBCs (haemoparasites) Study of other defects like Rouleaux formations, agglutination, fragmentation RBC inclusions,

• WBC inclusions, • platelet clumps etc

THE STAINING OF BLOOD FILMS The stains most commonly used for the staining of blood films are Romanowsky Stains.These stains are composed of Azure B and Eosin Y. Azure B combines with anionic components of the cell, e.g. DNA and stain these blue,whereas Eosin Y combines with the cationic components, various proteins and stains them red. Then there occurs a stain-stain interaction.

This composition and mode of action allows Romanowsky Stains to reveal the subtle differences in shades of the staining and allows for a differential staining of granules.

The pH of the staining mixture is extremely important for the differential staining. An alkaline pH accentuates the basic dye staining.Therefore; an optimum pH is to be sought. A pH of 6.8 is recommended for the optimal staining of all components.

Romanowsky Stains

The four most commonly used are Stain Wright's Stain Leishman Stain Giemsa Stain Jenner's Stain

STAINING OF BLOOD FILM

• After preparation, the slide is stained to distinguish the cells from each other. Cytohaematology is the study of the cellular components of blood cells. Parasites may also be observed during microscopic examination of the blood smear.

• Leishman Stain and May-Grunwald-Giemsa Stain are the most-frequently used. The preparation & method of using these stains is described below:

LEISHMAN’S STAIN

• It is named after Sir William Boog Leishman from Scotland.

• Stain PreparationDissolve 0.15 g of leishman’s stain powder in 100

ml of absolute methanol.The stain is then filtered into stock bottle.Place at 500C for 15 minutes in water bath.Again filter into clean brown borosilicate glass

bottle and store in dark at room temperature.

LEISHMAN’S STAIN

• Staining Procedure• Prepare the blood film and air-dry it. • Keep it on a staining rack and completely cover it with the stain. • Leave it to stain for 2 minutes. • Pour the buffered water onto the slide about twice the amount

of the stain. • Mix by blowing gently through a pipette. Leave for 5-7 minutes.

Pour off the stain mixture. • Wash in the buffer, cleaning the underside of the slide with a

cotton swab or tissue paper.• Place vertically to drain and dry.

good practice

• A good practice is to initially make 2-3 bottles at one time. When one bottle is finished, it should be replaced with freshly prepared stain and left to mature. In the meantime, another bottle of stain is used. The required volume of stain for daily use should be filtered into a smaller dropping bottle every morning

GIEMSA’S STAIN

• It is named after Dr. Gustav Giemsa fromGermany.

Stain Preparation Transfer 0.3 g of Giemsa stain powder to a

motor and mix with 25 ml of glycerin thoroughly. This makes the stock solution.

GIEMSA’S STAIN• Staining Procedure1) Prepare blood film, air dry and place in a staining rack.2) Fix with methanol for 3 minutes.3) Dilute the stain 1 in 10 with distilled water or buffer

solution of pH 6.8.4) Cover the blood film with diluted Giemsa stain for 10

minutes.5) Wash with distilled water, dry and examine under

microscope.