Preparation and staining methods for blood and bone marrow films

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Chapter 4 Preparation and staining methods for blood and bone marrow films

Preparation of blood films on slides

Blood films should be made on clean glass slides. Films made on coverglasses have negligible advantages and are unsuitable for modern laboratory practice. Films may be spread by hand or by means of an automated slide spreader, the latter being either a stand-alone instrument or a component of an automated blood cell counter.

Manual Method

Blood films can be prepared from fresh blood with no anticoagulant added or from ethylenediaminetetra-acetic acid (EDTA)-anticoagulated blood. Heparinized blood should not generally be used because its staining characteristics differ from those of EDTA-anticoagulated blood. Good films can be made in the following manner, using clean slides, if necessary wiped free from dust immediately before use. Slides should measure 75 × 25 mm and be approximately 1 mm thick; ideally, they should be frosted at one end to facilitate labelling, but these are more expensive.

First, make a spreader from a glass slide that has a smooth end. Using a glass cutter, break off one corner of the slide, leaving a width of about 18 mm as the spreader. A spreader can be used repeatedly unless the edge becomes chipped, but it must be thoroughly cleaned and dried between films.

Place a small drop of blood in the centre line of a slide about 1 cm from one end. Then, without delay, place a spreader in front of the drop at an angle of about 30° to the slide and move it back to make contact with the drop. The drop should spread out quickly along the line of contact. With a steady movement of the hand, spread the drop of blood along the slide. The spreader must not be lifted off until the last trace of blood has been spread out; with a correctly sized drop, the film should be about 3 cm in length. It is important that the film of blood finishes at least 1 cm before the end of the slide (Fig. 4.1).

The thickness of the film can be regulated by varying the pressure and speed of spreading and by changing the angle at which the spreader is held. With anaemic blood, the correct thickness is achieved by using a wider angle, and, conversely, with polycythaemic blood, the angle should be narrower.

The ideal thickness is such that on microscopy there is some overlap of red cells throughout much of the film’s length (see p. 30). The leucocytes should be easily recognizable throughout most of the film. With poorly made films the leucocytes will be unevenly distributed, with monocytes and other large leucocytes being pushed to the end and the sides of the spread. An irregular streaky film will occur if the slide is greasy, and dust on the surface will cause patchy spots (Fig. 4.1).

The films should be allowed to dry in the air. In humid conditions the films may be exposed to a current of warm air (e.g. from a hairdryer), but this should be in a microbiological safety hood.

Fixing Blood Films

To preserve the morphology of the cells, films must be fixed as described on p. 59. This must be done without delay, and the films should never be left unfixed for more than a few hours. If films are sent to the laboratory by post, it is essential that, when possible, they are thoroughly dried and fixed before dispatch.

Bone Marrow Films

The method for preparation of films of aspirated bone marrow is described on p. 126. They should be made without delay. Films must be thoroughly dry before they are fixed or artefactual changes will occur. At least one film should be fixed for a Perls’ stain on the initial bone marrow aspirate of each patient, and, if necessary, films should be fixed in the appropriate fixatives for special staining (Chapter 15); others should be fixed and stained with a Romanowsky stain as described later. Crushed bone marrow particles and touch preparations from trephine biopsy specimens can be stained in the same manner.

Staining blood and bone marrow films

Romanowsky stains are used universally for routine staining of blood films, and satisfactory results can be obtained. The remarkable property of the Romanowsky dyes of making subtle distinctions in shades of staining, and of staining granules differentially, depends on two components: azure B (trimethylthionin) and eosin Y (tetrabromo-fluorescein).1,2

The original Romanowsky combination was polychrome methylene blue and eosin. Several of the stains now used routinely that are based on azure B also include methylene blue, but the need for this is debatable. Its presence in the stain is thought by some to enhance the staining of nucleoli and polychromatic red cells; in its absence, normal neutrophil granules tend to stain heavily and may resemble ‘toxic granules’ in conventionally stained films.3

There are a number of causes of variation in staining. One of the main factors is the presence of contaminants in the commercial dyes and a simple combination of pure azure B and eosin Y might be considered preferable to the more complex stains because this ensures consistent results from batch to batch.1,4,5 However, in practice, absolutely pure dyes are expensive, and it is sufficient to ensure that the stains contain at least 80% of the appropriate dye.6 Among the Romanowsky stains now in use, Jenner is the simplest and Giemsa is the most complex. Leishman’s stain, which occupies an intermediate position, is still widely used in the routine staining of blood films, although the results are inferior to those obtained by the combined May–Grünwald–Giemsa, Jenner–Giemsa, and azure B–eosin Y methods. Wright’s stain, which is widely used in North America, gives results that are similar to those obtained with Leishman’s stain, whereas Wright–Giemsa is similar to May–Grünwald–Giemsa.

A pH to the alkaline side of neutrality accentuates the azure component at the expense of the eosin and vice versa. A pH of 6.8 is usually recommended for general use, but to some extent this depends on personal preference. (When looking for malaria parasites, a pH of 7.2 is recommended to see Schüffner’s dots.) To achieve a uniform pH, 50 ml of 66 mmol/l Sörensen’s phosphate buffer (see p. 622) may be added to each litre of the water used in diluting the stains and washing the films.

The mechanism by which certain components of a cell’s structure stain with particular dyes and other components fail to do so depends on complex differences in binding of the dyes to chemical structures and interactions between the dye molecules.7 Azure B is bound to anionic molecules, and eosin Y is bound to cationic sites on proteins.

Thus, the acidic groupings of the nucleic acids and proteins of the cell nuclei and cytoplasm of primitive cells determine their uptake of the basic dye azure B, and, conversely, the presence of basic groupings on the haemoglobin molecule results in its affinity for acidic dyes and its staining by eosin. The granules in the cytoplasm of neutrophil leucocytes are weakly stained by the azure complexes. Eosinophilic granules contain a spermine derivative with an alkaline grouping that stains strongly with the acidic component of the dye, whereas basophilic granules contain heparin, which has an affinity for the basic component of the dye. These effects depend on molar equilibrium between the two dyes in time-dependent reactions.2 DNA binds rapidly, RNA more slowly, and haemoglobin more slowly still; hence the need to have the correct azure B to eosin ratio to avoid contamination of the dyes and to stain for the right time. Standardized stains and staining method have been proposed (see p. 61).

The colour reactions of the Romanowsky effect are shown in Table 4.1; causes of variation in staining are given in Table 4.2.

Table 4.1 Colour responses of blood cells to Romanowsky staining

Cellular component Colour
Chromatin Purple
Nucleoli Light blue
Erythroblast Dark blue
Erythrocyte Dark pink
Reticulocyte Grey-blue
Lymphocyte Blue
Metamyelocyte Pink
Monocyte Grey-blue
Myelocyte Pink
Neutrophil Pink/orange
Promyelocyte Blue
Basophil Blue
Promyelocyte (primary granules) Red or purple
Basophil Purple black
Eosinophil Red-orange
Neutrophil Purple
Toxic granules Dark blue
Platelet Purple
Other inclusions  
Auer body Purple
Cabot ring Purple
Howell–Jolly body Purple
Döhle body Light blue

Table 4.2 Factors giving rise to faulty staining

Appearances Causes
Too blue Incorrect preparation of stock, eosin concentration too low
Stock stain exposed to bright daylight
Batch of stain solution overused
Impure dyes
Staining time too short
Staining solution too acid
Film too thick
Inadequate time in buffer solution
Too pink Incorrect proportion of azure B:eosin Y
Impure dyes
Buffer pH too low
Excessive washing in buffer solution
Pale staining Old staining solution
Overused staining solution
Incorrect preparation of stock
Impure dyes, especially azure A and/or C
High ambient temperature
Neutrophil granules not stained Insufficient azure B
Neutrophil granules dark blue/black (pseudo-toxic) Excess azure B
Other stain anomalies Various contaminating dyes and metal salts
Stain deposit on film Stain solution left in uncovered jar
Stain solution not filtered
Blue background Inadequate fixation or prolonged storage before fixation
Blood collected into heparin as anticoagulant