Manual of basic techniques for a health laboratory - libdoc.who.int

9. Haematology 2999.10 Preparation and staining of thin blood films9.10.1 PrincipleA thin blood film is prepared by spreading a small drop of blood evenly on a slideso that there is only one layer of cells.Thin blood films are stained with Romanowsky stains, which contain essentialazure B and eosin dyes.The Romanowsky stains most widely used include:● Leishman stain, which is used alone.● May–Grünwald stain, which is used with Giemsa stain.● Giemsa stain, which can be used alone or together with May–Grünwald or Jennerstain.● Field stains A and B, which are prepared in water unlike the above-mentionedstains, which are made up in methanol. Field stains are used for both thin andthick blood films.The Romanowsky stains prepared in methanol can be used to fix thin films beforebeing diluted on the slides to stain the films. Better results are obtained by fixingfirst with methanol, then staining with pre-prepared diluted stains, as describedbelow.After staining, blood films are used for:— determining leukocyte type number fractions (see section 9.13)— detecting abnormal erythrocytes (see section 9.10.4)— identifying certain parasites (see section 4.7)— estimating the number of thrombocytes (see section 9.14).9.10.2 Materials and reagents●Microscope● Microscope slides (should be well washed and, if necessary, cleaned with ethanolor ether using a piece of soft cloth (Fig. 9.63))● Spirit lamp or Bunsen burner● Glass spreader (see below)● Blood lancet● Two glass rods, either over a sink or over a staining tank● Measuring cylinder, 50 ml or 100 ml● Beakers or bottles containing clean tap water● Wash bottle containing buffered water (reagent no. 15)●●Interval timerRack for drying slides● Field stain (reagent no. 25)● Giemsa stain (reagent no. 29)● Leishman stain (reagent no. 34)● May–Grünwald stain (reagent no. 38)● EDTA dipotassium salt, 10% solution (reagent no. 22)●●Methanol70% Ethanol or ether.