STAINING TECHNIQUES OF BLOOD FILMS IN HAEMATOLOGY

Optimal results are obtained by fixing and staining immediately after the blood film is completely air-dried. Fixation of blood films before staining is recommended, although many laboratories have a practice of staining immediately after air-drying of blood films. This procedure usually yields acceptable results. methanol, is essential for good staining and presentation of cellular detail.

However, if slides cannot be stained immediately, fixation in methanol is necessary within 4 hours, but preferably 1 hour after air-drying; otherwise, the plasma will cause gray/blue background effects. Staining and fixing solutions must be as free of water as possible (<3%) to prevent morphological artifacts.

If manual staining procedures are used, it is recommended that slides be immersed in reagent-filled (Coplin) jars rather than covering slides with staining solution because formation of precipitate by evaporation may occur. If staining is performed under extremely hot conditions, care must be taken to prevent evaporation during staining, eg, by performing staining in a closed jar or in a closed petri dish. Automated staining devices have their own specific staining procedures that are provided by the manufacturers. Users may modify these procedures to satisfy local requirements for cell staining. Staining protocols vary between laboratories, and no generally accepted routine staining method or result is available.

ICSH has published a “reference” staining method for blood films based on purified azure B and eosin Y solutions (ICHS, 2010). As a general rule for judging the quality of a stained blood film, the laboratory must ensure that all cell types in a blood film can be identified reliably by the staining procedure. This rule applies equally to manual and automated methods. Blood films are typically stained by Romanowsky dyes (consisting of a variety of thiazines and eosins) (Henry, 1998). A number of methods have been described and include the following: Wright, Wright-Giemsa, May-Grünwald-Giemsa, and Leishman., giemsa, as described above.

STAINING TECHNIQUE USING LEISHMANN STAIN:

1.      Prepare a thin smear as described earlier. Dry the smear in air

2.      Cover the smear completely with Leishman stain

3.      Stain for 1 to 2 minutes

4.      Dilute the stain with twice its volume of the buffer solution

5.       Wash the slide with the buffer solution. Use tap water if not too acidic or alkaline.

6.      Drain and dry in air by keeping it in a staining position.

STAINING TECHNIQUE USING GIEMSA STAIN:

1.      Prepare a thin blood film as discussed earlier and dry in air

2.      Fix in methanol for 3 minute

3.      Dilute one volume of the solution (Azure II eosin, Azure II, glycerol, methyl alcohol) with 9 volumes of the buffer solution.

4.      Flood the slide with the stain and allow to act for 10-15 minutes

5. Wash and differentiate with the buffer solution. Drain, dry in air and examine microscopically.

METHODS OF BLOOD FILM READING IN HAEMATOLOGY

There are two major methods employed while reading blood films in haematology. They are:

·        Battlement Method: In battlement method, the blood film is examined systematically by being traversed three fields along the edge, two fields up, two fields along and two fields down. This sequence is continued until a minimum of 100 white cells have been counted.

·        Longitudinal method: in the longitudinal method, the different types of white cells are counted in one complete longitudinal strip of the film. If less than 100 cells are counted, a second strip should be similarly enumerated (Baker et al., 2009).