PREPARATION OF BLOOD SMEAR for Microscopic examination

Microscopic examination of the peripheral

Microscopic examination of the peripheral:- blood is performed by preparing, staining and examining a thin film of blood. It provides valuable information on the status of blood cells and presence of parasitic elements. The blood film is the only permanent record of haematological investigations. Therefore, a well-made, well-stained smear is essential. It is a common practice to prepare at least two blood films from each specimen, one stained, and the other kept in reserve, to be stained if and when necessary.

Capillary blood is ideal for preparing a blood smear. A drop of blood is transferred to a slide or touch the slide to the site of fingerprick during blood flow. If venous blood is used, EDTA is the anticoagulant of choice because it retains the morphological features of blood cells and also prevents clumping of platelets.

PREPARATION OF BLOOD SMEAR:- For the correct interpretation of a blood film, the film must be prepared in a technically correct manner so that the cells are evenly distributed throughout the smear. An ideal blood film

 1. should be smooth and not interrupted by ridges and holes.

2. it should cover 1/2 to 3/4th of the area of the slide.

3. it should be thickest at the origin or 'head' (thick end), and gradually thin on forming a 'tail' (feather edge) without defined border at the end. (Fig. 4.1)

Preparation of a good blood smear is learnt by practice. The thickness and the length of the film will depend on the angle of the spreader, the size of the drop of blood and the speed at which the spreader is pushed forward.

It is essential to use a clean slide. A glass slide cleaned with alcohol and wiped dry gives the best results. A spreader, e.g. a margin-free slide with ground glass edges, should be used to make the film.

Technique:-Place a small drop of EDTA anticoagulated blood, about 2 mm in diameter, about 1 cm from one end of the slide. If blood is collected from a finger-prick, wipe away the first drop and place the second drop of blood on the slide by touching it with the slide.

Place the slide on a flat surface.Hold it down firmly at the opposite end with the thumb and the forefinger.

Quickly, place the spreader just in front of the drop of blood at 45° angle. Draw it back slightly to touch the drop of blood and to allow the blood to spread along the contact line. Push the spreader forward smoothly and rapidly, maintaining the contact between the slide and the spreader (Fig. 4.1).

The smear formed should be about 3-4 cm long, slightly thicker at the base or head and thin at the tail end without ragged tails. If the patient is anaemic, the spreader should be held more erect (at 60°), and blood spread more quickly to obtain a thicker smear. Even distribution of cells in the smear plays an important role in obtaining accurate results. Allow the smear to dry in air. Label the slide with patient's identification at the thick end with a lead pencil.

Romanowsky Stains:- Affinity of a cell or its constituents for a particular stain depends on its chemical nature and the pH of the medium. In an appropriate pH (e.g. 6.8) the acidic structures take up basic dyes (which are therefore, called basophilic) and the basic structures stain with acidic dyes (therefore, called acidophilic).

The Romanowsky stains containmethylene blue (a basic dye), eosin (an acidic dye) and polychrome methylene blue or methylene azure. Usin; this stain, the acidic cell components such as nucles DNA and cytoplasmic RNA are stained bluish pur ple with polychrome methylene blue, while basi components such as haemoglobin and granules i eosinophils are stained orange to pink with eosin Various types of Romanowsky stain which are i common use are Wright, Leishman or Giems stains.

Leishman Stain :-

Preparation Solution 1 

Methylene blue  1g

Sodium carbonate   100 ML

 (0.5 % aqueous solution)

Eosin (0.1 % aqueous solution) 100 ml.

Methyl alcohol 100 ML

Dissolve methylene blue in the sodium carbonate solution. Heat at 65°C for 12 hours, cool and allow the mixture to stand for 10 days. Add equal volume (100 ml) of eosin solution, mix well and allow to stand for 6 to 12 hours. Filter and collect the precipitate. Wash the precipitate with several changes of distilled water until no more colour is extracted. Dry the precipitate at 37°C. Grind to powder in a mortar.

Weigh 0.15 g of the powder and grind it in a mortar with methyl alcohol. Pour off the supernatant into a volumetric flask and add more methanol until all the powder is dissolved. Make up the volume up to 100 ml with methanol. Store in a dark tightly stoppered bottle and allow to stand for 24 hours before use.

Note:-The Leishman stain is also commercially available in powder and liquid forms.

Solution 2 Buffer solution (pH 6.8)

(a) Disodium hydrogen phosphate (anhydrous) M/15 solution: Dissolve 9.47 g of the powder per litre of distilled water.

(b) Potassium di-hydrogen phosphate (anhy. drous) M/15 solution. Dissolve 9.08 g per litre of distilled water. To prepare the buffer, mix 49.6 ml of solution (a) and 50.4 ml of solution (b). Check the pH.  

Staining technique

(i) Prepare a thin smear as described earlier.Dry the smear in air.

(ii) Cover the smear completely with Leishmanstain.

(iii) Stain for 1 to 2 minutes.

(iv) Dilute the stain with twice its volume of the buffer solution. The slide will almost be completely flooded.

(v) Stain for 10 minutes.

(vi) Wash the slide with the buffer solution.

(VII)Use tap water if not too acidic or alkaline.

 (viii) Drain and dry in air by keeping it in a slanting position.

Glemsa stain

Solution 

1 Azure II eosin 3 g

Azur II 0.8 g

Glycerol 200 ml

Methyl alcohol300 ml

Grind the two dyes together in a mortar. Mix the alcohol with glycerol in a 1 litre flask. Sprinkle the ground dye carefully over the surface of the alcohol-glycerol mixture and allow it to stand for 24 hours. Stir the mixture at intervals after 24 hours to ensure that all the dye is passed into solution. Store in a tightly stoppered dark bottle.

Solution 2 Buffer solution pH 7.0

M/15 disodium hydrogen phosphate (9.47 g per litre) 61.1 ml.

M/15 potassium dihydrogen phosphate (9.08 g per litre) 38.9 ml. Mix the two solutions Check the pH.

 Staining technique:-

(i) Prepare a thin blood film, dry in air and fix in methyl alcohol for 3 minutes.

(ii) Dilute one volume of solution 1 with nine volumes of solution 2.

(iii) Flood the slide with the stain and allow to act for 10-15 minutes.

(iv) Wash and differentiate with the buffer

(v) Drain, dry in air and examine microscopically.

MICROSCOPIC EXAMINATION OF THE BLOOD FILM:- If the blood film has been properly stained, it should appear light purple to the naked eye. A thorough examination of the blood smear should include the following steps:

A. Examination of the Blood Smear Under the Low Power (10x) Objective The purpose of this examination is

- To evaluate the quality of the blood film.

- To estimate roughly the red cell and the