Romanowsky stains are used universally for routine staining of blood films.
Principle
The main components of Romanowsky stain are:
1) A cationic or basic dye such as azure B, which binds to anionic sites and gives a blue-grey colour to nucleic acids, nucleoproteins, granules of basophils, and weakly to granules of neutrophils.
2) An anionic or acidic dye, such as eosin Y, binds to cationic sites on proteins and gives rise to orange-red colour to hemoglobin, eosinophil granules. Among the Romanowsky stains, Giemsa is a good one, and Lieshman’s stain, is also a simple method that is especially suitable when a stained slide is urgently required.
Staining can be carried out in a jar or in a rack.
Jenner-Giemsa stain
1. Air-dried slides.
2. Fix in methanol for 2-3 minutes at room temperature.
3. Stain for 5 minutes in Jenner’s stain.
4. Tip off the stain
5. Stain in Giemsa stain freshly diluted with buffered distilled water (1:9) for 15 min.
6. Wash in running tap water and
7. Allow to air dry.
Lieshman’s stain
1. Air dry slides.
2. Flood the slides with Lieshman’s stain for 2 minutes; as the stain formulation includes methanol, this will fix the cells.
3. Dilute the stain with equal volume of buffered water.
. Leave the slide for 5-7 minutes. 4
5. Then wash it in a stream of buffered water until it has acquired a pinkish tinge (up to 2 minutes).
6. After that, the back of the slide has been wiped clean.
7. Set it up to dry.
Result
Staining characteristics of a correctly stained blood film
1) Cytoplasm is deep pink
2) Nuclei purple
3) Granules of neutrophils-fine purple Basophils are purple-black Eosinophils: orange red
4) Platelet purple
Sources of error
Error in film preparation
1) Holes in film- slides are contaminated with grease.
2) Film is too short and too thick- incorrect spreading angle.
3) Irregular spread with ridges and long tail-edge of the spreader is chipped.
4) Cellular degenerative changes: delay in fixing or methanol is contaminated with water.
Error in staining
1) Too much blue film: inadequate staining time. Too thick smear.
2) Too pink film: excessive time in buffer. Buffer ph is too low.
3) Blue background: blood anticoagulated in heparin-prolonged storage before fixation.
4) Stain deposit: stain solution not filtered.