Preparation and staining of

peripheral blood smear

Preparation of blood film

• Making an idel slide is backbone of Hematology ……….

• Wedge Technique• Preparation of blood film is a fundamental

technique in haematology and done for differential leukocyte count and observing red cells morphology.

Requirements

• SpreaderSelect a glass microscope slide or use a cover

slip with at least one smooth end as spreader.The edge must be wiped carefully and dried

before and after each use, and the slide must be discarded if the spreading edge becomes chipped.

Requirements

• Clean Slides It is essential to use clean, dry, dust free slides. Boxes of clean grease-free slides may be available. If not available then proceed as follows. Leave the slides overnight in a detergent solution.

Then wash thoroughly in running tap-water, dry with a clean linen cloth. Before use, wipe the surface with methylated spirits(95% ethanol) or methanol and dry with a clean cloth.

Procedure (Thin Blood Film)1) Place a drop of blood in the centre line of slide 1 cm from one

end.2) Place the spreader in front of glass slide at an angle of 30o.3) Move the spreader back, so that it touches the blood drop.

Blood will spread along the edge of spreader by Capillary action.

4) Push the spreader forward along the length of slide by rapid but straight movement.

5) Then allow the smear to dry in the air before staining.6) When dry, patient’s name, number and other identifications

may be written on the slide with a diamond marker or pencil.

Procedure (Thick Blood Film)

• These are widely used in the diagnosis of Haemoparasites, particularly malarial parasites. Generally the blood films should be made about 10 times thickness of normal smears.

1) Place a drop of blood on a clean slide and spread it with the corner of another slide until the printed matter is just visible through the smear.

2) Allow to air-dry and label the slide properly.

Sources of Errors

1) Film extending to end of slide: blood drop too large.

2) Short thin film: blood drop too small.3) Irregular spread with ridges and long tail:

Edge of spreader dirty or chipped; dusty slide.

4) Holes in film: Slide contaminated with fat or grease.

Sources of Errors

5) Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water.

6) Irregular leukocyte and platelet distribution, especially in tail: poor film-making technique.

7) Film too short and too thick: spreader held at incorrect angle

8) Film extends to edge of slide: spreader too wide or not positioned correctly

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’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 Procedure1) Prepare blood film, air dry and place in a staining

rack.2) Cover the smear with stain and leave for 2

minutes. The methanol fixes the smear.3) Pour distilled water on the slide about twice the

amount of stain until a metallic scum appears. Allow this diluted stain to act for 5-7 minutes. During this time differentiation takes place.

Staining Procedure continued…….

4) Without disturbing the slide, flood with distilled water and wash until thinner parts of the film are pinkish red.

5) If distilled water doesn’t give adequate differentiation, a buffer solution of pH 6.8 can also be used.

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.