staining of blood smears.pptx

hematology

1. Staining of blood smears By dr Abdiasis Omar Mohamed MBBS

2. Introduction Ehrlich was the first to use aniline dyes at first in sequence and latter as a premixed acidic – basic stains (neutral dyes). Jenner (1880) found that the precipitate formed when eosin and methylene blue are mixed could be dissolved in methyl alcohol to form a useful stain combining certain properties of both parent dye stuffs.

3. Romanowsky (1890) found that when old (ripened and therefore "polychromed") methylene blue solution is mixed with eosin and the precipitate dissolved in methyl alcohol, a stain results that has a wider range than Jenner’s stain staining cell nuclei and platelet granules (which Jenner’s mixture failed to stain).

4. Principle of staining Acidic dyes such as eosin unites with the basic components of the cell (cytoplasm) and hence the cytoplasm is said to be eosinophilic (acidic). basic stains like methylene blue are attracted to and combine with the acidic parts of the cell (nucleic acid and nucleoproteins of the nucleus) and hence these structures are called basophilic. Other structures stained by combination of the two are neutrophilic

5. Romanowsky stains in common use Wright stain In its preparation, the methylene blue is polychromed by heating with sodium carbonate. It is purchased as a solution ready to use or as a powder.

6. Staining Method Place the air-dried smear film side up on a staining rack (two parallel glass rods kept 5cm apart). Cover the smear with undiluted stain and leave for 1 minute. The methyl alcohol in the satin fixes the smear. When it is planned to use an aqueous or diluted stain, the air dried smear must first be fixed by flooding for 3-5 minutes with absolute methanol.

7. if films are left unfixed for a day or more, it will be found that the background of dried plasma stains pale blue and this is impossible to remove without spoiling the staining of the blood cells. Dilute with distilled water (approximately equal volume) until a metallic scum appears. Mix by blowing. Allow this diluted stain to act for 3-5 minutes.

8. Without disturbing the slide, flood with distilled water and wash until the thinner parts of the film are pinkish red.

9. Leishman Stain in its preparation, the methylene blue is polychromed by heating a 1 % solution with 0.5% sodium carbonate at 650 C for 12 hours after which a further ripening is allowed to proceed for 10 days before it is mixed with an equal volume of 0.1% eosin B.

10. Staining method The method is similar to that used in Wright’s stain except for step 3. With Leshman’s stain, dilution is effected with approximately two volume of distilled water to one volume of stain (the best guide is the appearance of a metallic scum).

11. Microscopic appearance of cells and cell components in Romanowsky-stained blood films (Films stained with either Wright or Leishman stains are pinkish in color when viewed with the naked eye): - Red cells - pink with a central pale area - Nuclei of leucocytes - blue to purple - Cytoplasmic neutrophilic granules - tan

12. - Eosinophilic granules - red orange each distinctly discernible - Basophilic granules - dark blue - Cytoplasm of monocytes - faint blue gray - Platelets - violet granules - Malaria parasites - sky blue cytoplasm and red purple chromatin

13. Giemsa stain Instead of empirically polychromed dyes, this stain employs various azure compounds (thionine and its methyl derivative) with eosin and methylene blue). This is an alcohol-based Romanowsky stain that required dilution in pH 7.1-7.2 buffered water before used. It gives the best staining of malaria parasites in thick films.

14. Staining of thick smears The stains used employ the principle of destroying the red cells and staining leucocytes and parasites. The method using Giemsa stain is satisfactory.

15. Method Cover the air-dried smear with a 1:10 diluted Giemsa using buffered distilled water at pH 6.8 as a diluent. Do not fix the films before staining. Leave the stain to act for 15-30 minutes. Do not fix the films before staining. 2. Wash with distilled water and air dry.

16. Panoptic staining Panoptic staining consists of a combination of a Romanowsky stain with another stain, e.g. Giemsa. This improves the staining of cytoplasmic granules and other bodies like nucleoli of blast cells. Popular methods are Jenner - Giemsa and May-Grunwald - Giemsa.

17. Jenner-Giemsa method Dry the films in the air then fix by immersing in a jar containing methanol for 10-20 minutes. For bone marrow films leave for 20-25 minutes. Transfer the films to a staining jar containing Jenner's stain freshly diluted with 4 volumes of buffered water and leave for 4 minutes.

18. Transfer the slides without washing to a jar containing Giemsa stain freshly diluted with 9 volumes of buffered water pH 6.8. Allow to stain for 7-10 minutes. Transfer the slides to a jar containing buffered water, pH 6.8; rapidly wash in 3 or 4 changes of water and finally allow to stand undisturbed in water for 2-5 minutes for differentiation to take place Place the slides on end to dry.

19. May- Grünwald -Giemsa Dry the films in the air then fix by immersing in a jar containing methanol for 10-20 minutes. For bone marrow films leave for 20-25 minutes. Transfer the films to a staining jar containing MayGrünwald's stain freshly diluted with an equal volume of buffered water and leave for 15 minutes.

20. Transfer the slides without washing to a jar containing Giemsa's stain freshly diluted with 9 volumes of buffered water pH 6.8. Allow to stain for 10-15 minutes. Transfer the slides to a jar containing buffered water, pH 6.8; rapidly wash in 3 or 4 changes of water and finally allow to stand undisturbed in water for 2-5 minutes for differentiation to take place. Place the slides on end to dry.

21. Field's stain Field’s stain was introduced to provide a quick method for staining thick films for malaria parasites. It this water-based Romanowsky stain is composed of two solutions, Field’s stain A and Field’s stand B. It is buffered to the correct pH and neither solution requires dilution when staining thick films.

22. When staining thin films, Field’s stain B requires dilution. Compared with Giemsa working stain, Field’s stains are more stable. They stain fresh blood films, well, particularly thick films. The rapid technique is ideally suited for staining blood films from waiting outpatients and when reports are required urgently

23. Thin film Field’s staining technique Required - Field’s stain A - Field’s stain B, diluted 1 in 5 Buffered pH 7.1-7.2 water

24. Method Place the slide on a staining rack and cover the methanol-fixed thin film with approximately 0.5ml of diluted Field’s stain B. Add immediately an equal volume of Field’s stain A and mix with the diluted Field’s stain B. Leave to stain for 1 minute. The stain can be easily applied and mixed on the slide by using 1ml graduated plastic bulb pipettes.

25. 3. Wash off the stain with clean water. Wipe the back of the slide clean and place it in a draining rack for the film to air-dry

26. Thick film Field’s staining technique Required - Container of fields’ stain A - Container of Field’s stain B - Two containers of clean water (need not be buffered)

27. Method Holding the slide with the dried thick film facing downwards, dip the slide into Field’s stain A for 5 seconds. Drain off the excess stain by touching a corner of the slide against the side of the container. Wash gently for about 5 seconds in clean water. Drain off the excess water. Dip the slide into Field’s stain B for 3 seconds. Drain off the excess stain.

28. Wash gently in clean water. Wipe the back of the slide clean and place it upright in a draining rack for the film to air-dry.

29. Problems in staining Excessively blue stain Causes: too thick films, prolonged staining, inadequate washing, too high alkalinity of stain or diluent Appearance: erythrocytes-blue green, nuclear chromatin-deep blue to black, granules of neutrophils-deeply stained and appear large and prominent. Correction: preparing films with ideal thickness, reducing staining time, using less stain and more diluent, prolonging washing, adjust pH of buffer or prepare a new batch of stain.

30. Excessively pink stain Causes: insufficient staining, prolonged washing, too high acidity of the stain or buffer (exposure of stain or buffer to acid fumes). Appearance: erythrocytes-bright red or orange, nuclear chromatin-pale blue, granules of eosinophils-sparkling brilliant red Correction: prolonging staining time, reducing washing, preparing a new batch of stain.

31. Precipitate on the film Causes: unclean slides, drying during the period of staining, inadequate washing of slide at the end of the staining period Correction: use clean slides, cover the smear with generous amount of the stain, wash the slide until thinner parts of the film are pinkish

32. THE END