STAINING

staining

1. Stains used in hematology Dr. Ajit kumar singh MD (Laboratory Medicine) Chittranjan National Cancer Institute , Kolkata

2. ROMANOWSKY STAINS Staining : it is a biochemical technique of adding specific dye to a substrate ( DNA , proteins , lipids ) to qualify or quantify the presence of a specific compound. The stains most commonly used for staining of blood films are romanowsky stains. Romanowsky stain : it is named after dmitri leonovich romanowsky who invented it in 1891 Definition : a stain made from water soluble eosin,methylene blue and acetone free methanol.

3. VARIOUS STAINS FOR PERIPHERAL BLOOD FILM Romanowsky stains are universally employed for staining of blood films. All Romanowsky combinations have two essential ingredients i.e. methylene blue and eosin . A. Methylene blue: 1)Cationic dye 2)Basic dye 3) Blue-Purple color Structures that stain with methylene blue are termed as Basophilic. B. Eosin: Anionic dye Acid dye Pink red-color Structures that stain with eosin are termed Eosinophilic

4. Methylene blue combines with anionic components of the cell . eg:DNA ,and stain these blue. Eosin combines with cationic components of the cell . Eg:cytoplasm and stain them red . Then there occurs stain –stain interaction. This composition and mode of action allows romanowsky stains to reveal the subtle differences in shades of staining.

5. A well stained –smear with any of romanowsky stains shows following features : 1)Red blood cells : Pink red (or)Deep red color. 2)Polychromatic cells(reticulocytes) : Gray blue color. 3) Neutrophils : Pale , Pink cytoplasm , Purple Granules 4)Eosinophils : Pale , Pink cytoplasm , orange red granules 5) Basophils : Blue cytoplasm , Dark blue-Violet Granules . 6) Monocytes : Gray –Blue cytoplasm. 7)Lymphocytes : Dark blue cytoplasm. 8) Platelets : Purple Color

6. To preserve the morphology of the cells, films must be fixed as soon as possible after they have dried. It is important to prevent contact with water before fixation is complete. Methyl alcohol (methanol) is the choice, although ethyl alcohol ("absolute alcohol") can be used Methylated spirit (95% ethanol) must not be used as it contains water. To fix the films, place them in a covered staining jar or tray containing the alcohol for 2-3 minutes. In humid climates it might be necessary to replace the methanol 2-3 times per day ; the old portions can be used for storing clean slides

7. VARIOUS STAINS INCLUDED UNDER ROMANOWSKY STAINS ARE AS FOLLOWS 1)Leishman stain 2)Giemsa stain 3)Wright stain 4)Field stain 5)Jenner stain 6)JSB stain Among the above mentioned stains ,Jenner is the simplest and Giemsa is the most complex. Leishman stain it occupies intermediate position and it is still widely used in the routine staining of blood films.

8. LEISHMAN STAIN PRINCIPLE: It is used in microscopy for staining blood smears.It provides excellent stain quality . COMPONENTS : It contains methylene blue and eosin in methanol(acetone free)in the ratio of 1.5gm/1 lit. It is generally used to differentiate and identify leucocytes , malaria parasites etc. Preparation Dissolve 0.2 g of powdered Leishman’s dye in 100 ml of acetone-free methyl alcohol in a conical flask. Warm it to 50°C for half an hour with occasional shaking. Procedure for staining 1)Pour Leishman’s stain dropwise (counting the drops) on the slide and wait for 2 minutes. This allows fixation of the PBF in methyl alcohol. 2)Add double the quantity of buffered water dropwise over the slide (i.e. double the number of drops). 3) Mix for 8 minutes 4)Wash in water for 1 to 2 minutes. 5) Dry in air and examine under oil immersion lens of the microscope.

9. GIEMSA STAIN PRINCIPLE: It is used in diagnosis of malaria and other parasites ,also used in cytogenetics. Components for giemsa stain : 1)Methylene blue 2)Azure B 3)Sodium salt of Eosin Y. Preparation Stock solution of Giemsa stain is prepared by mixing 0.15 g of Giemsa powder in 12.5 ml of glycerine and 12.5 ml of methyl alcohol. Before use dissolve one volume of stock solution in nine volumes of buffered water (dilution 1:9). Procedure: 1) Fill staining dish with staining solution 2) Place thin blood film(useful for investigation of anemia ,infections etc ,) and thick blood films(useful for investigation of malaria and microfilaria) into the staining dish 3) Stain blood slides for 45 minutes 4) Wash in water. 5) ) Dry it and examine under oil immersion lens of the microscope

10. WRIGHT'S STAIN PRINCIPLE: Wright’s stain is used to differentiate nuclear and/or cytoplasmic morphology of platelets, RBCs, WBCs, and parasites. Materials: 1)Wright stain powder-1.0gm. 2)methanol(must be water free)-400ml. Preparation: 1)Weigh the wright powder and transfer it to a dry brown bottle. 2)Add a few glass beads (to assist in dissolving the dye) 3)Using a dry cylinder ,measure the methanol and add to this stain. 4)Mix well at intervals until the powder is completely dissolved. 5)Warming the solution in a 37 c water bath will help the dye to dissolve. 6)label the bottle and mark it flammable and toxic.

11. Procedure: Thin blood films (only) – Dip Method 1. Dip air dried blood film in undiluted stain for 15 to 30 seconds (double the staining time for bone marrow smears). 2.Decolorize the stained smears by immersion in distilled or deionized water and air dry. 3. Let air dry in a vertical position. Thin Blood films (only)-Rack Method. 1) Lay air dried slides on staining rack and flood with stain , stain for 10 to 15 seconds(double the staining time for bone marrow smears). 2)Add an equal volume of deionized/distilled water and stain for 10 seconds. 3)Rinse the slide by dipping in deionized/distilled water for 30 seconds. 4. Allow the film to air dry. 5.Dip air dried blood film in undiluted stain for 15 to 30 seconds (double the staining time for bone marrow smears). 6.Decolorize the stained smears by immersion in distilled or deionized water and air dry 7.Let air dry in a vertical position.

12. THICK BLOOD FILMS (ONLY): Allow film to air dry thoroughly for several hours or overnight. Do not dry films in an incubator or by heat, because this will fix the blood and interfere with the lysing of the RBCs. Note: If a rapid diagnosis of malaria is needed, thick films can be made slightly thinner than usual, allowed to dry for 1 h, and then stained

13. FIELD STAIN : (THIN FILM) Materials: 1) Methanol (absolute) 2) Field’s stain A and B 3) Tube with water 4) Staining dishes 5)Filter paper Procedure: A. Fix thin film with methanol for 1 min. B. Dry microscopic slide on filter paper C. Immerse slide in Field’s stain B (Eosin) for 5 seconds D. Immediately wash with water E. Immerse slide in Field‘s stain A (Methylene blue) for 10 secs. F. Immediately wash with water G. Dry thin films

14. FIELD STAIN: (THICK FILM) Materials: 1)Field‘s stain A and B 2) Tube with water 3) Filter paper Procedure: Immerse thick film in Field‘s stain A (Methylene blue) for 3 secs Do not forget: Thick films need to be hemolysed and are therefore not fixed with methanol. Rinse immediately in tap water Immerse thick film in Field’s stain B (Eosin) for 3 seconds Then rinse immediately with tap water E. Let the slide carefully dr

15. JENNER STAIN: PRINCIPLE: It is similar to wright stain ,used for staining blood smears. The Jenner stain Solution is a mixture of several thiazin dyes in a methanol solvent. Ionic and nonionic forces are involved in the binding of these dyes. The staining solution has anionic and cationic properties. The negatively charged phosphoric acid groups of DNA attract the purple polychromatic cationic dyes to the nuclei. The blue basophilic granules are stained by the polychromatic cationic dyes.

16. IMMERSION STAINING PROTOCOL 1.Thoroughly dry blood or bone marrow smears. 2.Fix smears in absolute methanol for 15 seconds to 5 minutes. 3.Stain smears in Jenners Stain Solution for 2 minutes. 4.Stain in mixture of 50ml of Jenners Stain Solution, 75ml of pH6.6 Phosphate Buffer Solution and 175ml deionized water for 5 minutes. 5.Rinse in standing deionized water for 1.5 minutes or rinse briefly in running deionized water. 6.Air dry smears. 7.Examine smears under a microscope.

17. HORIZONTAL STAINING PROTOCOL: 1)Place slide with thoroughly dried film in a horizontal staining rack. 2) Flood smear with absolute methanol for 15-30 seconds and then drain. 3) Flood smear with 1ml Jenners Stain Solution and let stand for 3 minutes. 4) Add 1mL of pH 6.6 Phosphate Buffer solution and 1 ml deionized water to smear and let stand for 45 seconds. 5) Rinse briefly with running deionized water. 6) Air dry and examine under a microscope.

18. J.S.B. STAIN Materials and reagents required: 1)Eosin yellow (water soluble) 2)Methylene blue 3)Potassium dichromate 4)hydrogen phosphate (dihydrate) 5)1% sulphuric Acid. 6)Round bottom flask (2 lit.) 7)Heating mantle 8)Distilled water 9)Staining jars.

19. STAINING TECHNIQUE: 1)Prepare thin and thick smears from malaria cases on micro slides. 2) De- haemoglobinise the thick smear. 3) Fix the thin smear in methanol for few minutes. 4) Take 3 staining jars for J.S.B. I, J.S.B.II and tap water. 5)Dip the smears in J.S.B. II for few seconds and immediately wash in water. 6)Drain the slides free of excess water. 7)Dip the smears in J.S.B.I for 30-40 seconds. 8)Wash well in water and dry. 9)Examine the smears under oil immersion lens of microscope

20. STAINING OF THICK SMEAR It can be stained with any of the Romanowsky stains except that before staining, the smear is de- hemoglobinised by putting it in distilled water for 10 minutes. Auto- stainer currently, automatic staining machines are available which enable a large batch of slides to be stained with a uniform quality. Jaswant Singh Battacharya (JSB) Stain for thick and thin films: • This is the standard method used by the laboratories under the National Malaria Eradication Program in India

21. PRECAUTIONS IN STAINING OF PERIPHERAL BLOOD FILM 1)Dark blue blood film: It can be due to overstaining, inadequate washing or improper pH of the buffer. In this RBCs are blue, nuclear chromatin is black , granules of the neutrophils are overstained and granules of the eosinophils are blue or grey. 2)Light pink blood film: In this RBCs are bright red, the nuclear chromatin is pale blue and granules of the eosinophils are dark red. It can be due to under staining , prolonged washing, mounting the film before drying and improper pH of the buffer. 3)Precipitate on the blood film: This could be due to inadequate filtration of the stain, dust on the slide, drying during staining and inadequate washing. REFERENCES: DACIE AND LEWIS BANCROFT Lynch’s Medical laboratory

22. Thank you …