Hematological stains

made from the latest information and dacie and lewis and wintrobes latest edition. A very precise and to the point presentation

1. HEMATOLOGICAL STAINS Presented By: Dr . Akash

2. CONTENTS Introduction Principle Romanowsky Stains Preparations of Solutions Staining Methods Automated Staining Rapid Staining Methods A note on JSB Stain Special Stains- Cytochemical Bibliography

3. INTRODUCTION WHAT IS STAINING? It is a biochemical technique of adding a specific dye to a substrate (DNA, proteins, lipids) to qualify or quantify the presence of a specific compound . Romanowsky stains are universally employed for staining of blood films. It is named after Dmitri Leonovich Romanowsky who invented it in the year 1891. It is a stain made from water soluble eosin and methylene blue (original Romanowsky combination).

4. principle Romanowsky Effect- 2 components: azure B ( trimethylthionine ) and eosinY ( tetrabromofluorescein ). Azure B is bound to anionic molecules, and eosin Y is bound to cationic sites on proteins. Nucleic acids & proteins of the cell nuclei and cytoplasm of primitive cells- stained by azure B The hemoglobin molecule-stained by eosin.

5. Neutrophil granules- weakly stained by the azure complexes. Eosinophilic granules-stains strongly with the acidic component of the dye. Basophilic granules contain heparin which has affinity for the basic component of the dye. A pH of 6.8 is usually recommended for general use. Note: when looking for malarial parasites, a pH of 7.2 is recommended to see Schuffner dots.

6. ROMANOWSKY STAINS These are composed of methylene blue, oxidative products of methylene blue(azures) and eosin dyes. The various stains included under Romanowsky stains are as follows: May- Grunwald Stain Jenner Stain Giemsa Stain Leishman Stain Wright Stain Field Stain Jaswant Singh Bhattacharji (JSB) stain A combination of these stains may also be used, such as Jenner- Giemsa , Wright- Giemsa and azure and May- Grunwald - giemsa,which may give superior results than the individual ones.

7. PREPARATIONS OF SOLUTIONS OF ROMANOWSKY DYES 1. May– Grünwald Stain Weigh out 0.3 g of the powdered dye. Transfer it to a conical flask of 200–250-ml capacity. Add 100 ml of methanol and warm the mixture to 50 °C. Allow the flask to cool to 20 °C and shake several times during the day . After letting it stand for 24 hours, filter the solution. 2. Jenner Stain Prepare a 5 g/l solution in methanol in exactly the same way as described earlier for the May– Grünwald stain.

8. 3. Giemsa Stain 1 g of the powdered dye in 100 ml methanol. At 50 C for 15 min It will improve on standing for a few hours. 4. Leishman Stain 0.2 g of the powdered dye in 100 ml methanol Buffered water Make up 50 ml of 66 mmol /l Sörensen phosphate buffer of the required pH to 1 litre with water at a pH of 6.8. Alternative buffers are available commercially.

9. MANUAL STAINING METHODS 2 types of manual methods are common- Rack and Dip. RACK METHOD: It uses rods overlying a sink or a dish that holds glass slides in a horizontal position during staining. I. Leishman Stain Combined fixative and staining solution. Air-dry the film and flood the top surface of the slide with the stain. The edges of the slides should not touch each other to prevent stain runoff. After 2 min, add double the volume of water and stain the film for 5–7 min. Wash it in a stream of buffered water until it has acquired a pinkish tinge (up to 2 min).

10. DIP METHOD: Several dishes, large enough to contain a portable slide holder and an adequate amount of solution to cover the slides are required. The blood films are fixed for 1-2 min. in a dish with anhydrous, chemically pure methanol. The basket holding the slide should be completely dry to avoid water contamination.

11. INTERPRETATION Nuclei Chromatin Nucleoli Purple Light Blue Cytoplasm Erythroblast Erythrocyte Reticulocyte Lymphocyte Metamyelocyte Monocyte Myelocyte Neutrophil Promyelocyte Basophil Dark blue Dark pink Grey-blue Blue Pink Grey-blue Pink Pink/orange Blue Blue Granules Promyelocyte (primary granules) Basophil Eosinophil Neutrophil Toxic granules Platelet Red or Purple Purple-black Red-orange Purple Dark purple Purple

12. Factors causing variation in staining 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 acidic Film too thick Too Pink Incorrect azure B:eosin Y ratio 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 ( pseudotoxic ) Excess azure B Stain deposit on film Stain solution left in uncovered jar,stain solution not filtered

13. TROUBLESHOOTING STAIN PROBLEMS FIXATION Freshly prepared films should be air dried for atleast 5 mins to prevent drying artifact Glass slides should be clean and clear before use. Methanol should be anhydrous and chemically pure and kept tightly stoppered away from moisture or chemical fumes All staining dishes containing alcohol or alcoholic solutions should be kept covered to prevent evaporation and contamination. Fixation period should be a minimum of 1 min.

14. STAINING: For best results use a staining time atleast twice the fixation period. For the rack method the amount of buffer should be equal to or slightly greater than amount of stain solution. Buffer and stain should be well mixed for uniform staining. RINSE OR WASH: Distilled water or buffered rinse water should be used to wash off the stain. Under washing may result in a blue film precipitate.

15. DRYING: Air drying is most satisfactory STAIN PRECIPITATE: Filtration will remove the precipitated stain powder if any in the stain solution. Replacement of tubings at regular intervals will alleviate the problem of precipitates within the instrument tubings .

16. II. May– Grünwald – Giemsa Stain Dry the films in the air, fix by immersing in a jar of anhydrous chemically pure acetone free methanol for 5–10 min. For bone marrow films, allow a longer time & then leave it for 15–20 min in the methanol. Films should be fixed as soon as possible after they have dried. Prevent any contact with water before fixation is complete. Methyl alcohol (methanol) is the fixative of choice. Methylated spirits must not be used because it contains water.

17. With 3% water With 1% water

18. With 4% water With 10% water

19. Transfer the fixed films to a staining jar containing May– Grünwald stain freshly diluted with an equal volume (1:1) of buffered water. After 15 min, transfer them without washing to a jar containing Giemsa stain freshly diluted with 9 volumes of buffered water, pH 6.8. After staining for 10–15 min, transfer the slides to a jar containing buffered water, pH 6.8, rapidly wash in three or four changes of water, Allow slides to stand undisturbed in water for a short time (usually 2–5 min) for differentiation to take place.

20. II. Jenner– Giemsa Stain May be substituted for May– Grünwald stain in the technique described above. The results are a little less satisfactory. 4 volumes of buffered water immersed in the Jenner stain for approximately 4 min before being transferred to the Giemsa stain.(7-10 min). Differentiation is carried out as described earlier.

21. AUTOMATED STAINING Enable large batches of slides to be handled. They may be either stand-alone staining machines or a part of a large automated blood counting instrument. Useful for preparing slides for teaching and for quality control purposes. Flat-bed staining Dip- and- Dunk technique Problems increased background staining, inadequate staining of neutrophil granules, degranulation of basophils blue or green rather than pink staining of erythrocytes.

22. RAPID STAINING METHODS The Field Method- introduced to provide a quick method for staining thick films for malaria parasites. The stains are available commercially ready for use, or they can be prepared. Stain A ( Polychromed methylene blue) Methylene blue 1.3 g Disodium hydrogen phosphate (Na2HPO4•12H2O) 12.6 g Potassium dihydrogen phosphate (KH2PO4) 6.25 g Water 500 ml

23. Stain B (Eosin) Eosin 1.3 g Disodium hydrogen phosphate (Na2HPO4•12H2O) 12.6 g Potassium dihydrogen phosphate (KH2PO4) 6.25 g water 500 ml

24. JASWANT SINGH BHATTACHARJI STAIN (JSB ) Rapid staining method for detection of malaria. Useful for the diagnosis of malaria in thick smear samples of blood. Composition: 1 st solution- methylene blue, potassium dichromate, & sulphuric acid diluted in water. 2 nd solution- eosin dissolved in water.

25. CYTOCHEMICAL STAINS Several special stains may be performed on peripheral blood films, bone marrow aspirate smears, bone marrow touch preparations and bone marrow biopsy materials, Provide additional information about the cell lineage. Useful in the diagnosis and classification of acute leukemias (myeloid and lymphoid),basis for subclassification of the acute myeloid leukemias . Widely used in morphological subclassifications , such as French-American-British system(FAB). Use has decreased because of wide availability of flow cytometric and other ancillary tests.

26. 1) MYELOPEROXIDASE Primary granules of neutrophils and secondary granules of eosinophils contain myeloperoxidase . Monocytic lysosomal granules- faintly positive. Lymphocytes and nucleated RBCs lack the enzyme. Staining is caused by oxidation of 3-amino-9-ethylcarbazole or 4-chloro-1-naphthol substrates by myeloperoxidase in the cell - form a brown-colored precipitate. Sensitive to light, smears should be stained immediately or sheltered from light. Enzymatic activity in cells may diminish over time- stain should not be performed in blood or marrow aspirate smears older than 3 weeks. Permount coverslip mounting medium may cause fading of the stain.

27. Also sensitive to heat and methanol treatment. Erythroid cells may stain for peroxidase after methanol treatment - nonenzymatic reaction between the staining reagents and Hb . ( pseudoperoxidase or Lepehne reaction). Antibodies to myeloperoxidase are available for both flow cytometric analysis and immunohistochemical staining in fixed tissue sections.

28. 2) SUDAN BLACK B It stains intracellular lipids and phospholipids. Positive staining of granulocytic cells and eosinophils is seen as the granules are membrane bound &contain phospholipids. Weak monocytic staining. No staining of lymphocytes, although some positivity may be seen in azurophilic granules of lymphoblasts . Sudan Black B has an advantage over myeloperoxidase in that it may be used to stain older blood or bone marrow smears, ad there is little fading of the stain over time.

29. 3) SPECIFIC(NAPHTHOL AS-D CHLOROACETATE) ESTERASE Used to identify cells of the granulocytic series. Does not stain lymphocytes and monocytes . Extremely useful for identifying granulocytes and mast cells in tissue sections Helpful in the diagnosis of extramedullary myeloid tumours (granulocytic sarcoma and chloroma ) of blasts found in tissues. The cellular esterase enzyme hydrolyzes the naphthol AS-D chloroacetate substrate. This reaction product is then coupled to a diazo salt to form a bright red-pink reaction product at the site of enzymatic activity. The enzyme activity is inhibited by the presence of mercury, acid solutions, heat, and iodine that may give rise to false-negative staining results.

30. 4) NON-SPECIFIC (ALPHA-NAPHTHYL BUTYRATE OR ALPHA-NAPHTHYL ACETATE) ESTERASE Used to identify monocytic cells, Do not stain granulocytes or eosinophils . Mature T-lymphocytes stain with a characteristic focal, dot-like pattern. NSE is positive for monocytoid series(M4, M5A, and M5B). The stain also reacts with macrophages, histiocytes , megakaryocytes , and some carcinomas. The alpha- naphthyl butyrate is considered to be more specific, slightly less sensitive than the alpha- naphthyl acetate stain. Differential staining with different esterases is seen in megakaryoblasts , which do not stain with the alpha- naphthyl butyrate but stain with alpha- naphthyl acetate.

31. 5) TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE ( TdT ) An intranuclear enzyme that catalyzes the addition of deoxynucleotide triphosphates to the 3’-hydroxyl ends of oligonucleotides or polydeoxynucleotides without need for a template strand. Found normally in the nucleus of thymocytes and immature lymphoid cells within the bone marrow Not found in mature lymphocytes. Useful marker in identifying acute lymphoblastic leukemias and lymphomas as well as in a small subset of acute myeloid leukemias . TdT levels may be measured by cytochemical staining with an immunofluorescent detection technique, by flow cytometry or by immunohistochemical methods.

32. 6) LEUKOCYTE ALKALINE PHOSPHATASE(LAP) Alkaline phosphatase activity is found in the cytoplasm of neutrohil , osteoblasts , vascular endothelial cells and some lymphocytes. The alkaline phosphatase level of peripheral blood neutrophils is quantitated by the LAP score. Useful as a screening test to differentiate CML from leukemoid reactions and MPD. The LAP score is usually performed using the Kaplow procedure. Determined by evaluation of the staining intensity(ranging from 0 to +4) of 100 counted neutrophils or bands. Normal LAP scores range from 15 to 130. Many different disease states may have elevation or depression of LAP score:

33. Low LAP scores(usually between 0 and 13)- CML, PNH, few MDS. Elevated LAP score- Leukemoid reactions in response to infection, other myeloproliferative neoplasms (Primary Myelofibrosis & Polycythemia Vera). Rapid loss of alkaline phosphatase activity in samples drawn in EDTA anticoagulant. Optimally performed on fresh capillary blood fingerstick smears or on blood anticoagulated with heparin Should be performed within 48 hours after collection of the sample. The blood smears may be held in the freezer for 2 to 3 weeks with little loss of activity.

34. 7) ACID PHOSPHATASE It is found in all hematopoietic cells, highest levels are found in macrophages & osteoclasts . Localized dot-like pattern is seen in many T- lymphoblasts . Tartarate -resistant acid phosphatase (TRAP) is an isoenzyme of acid phosphatase that is found in high levels in the cells of hairy cell leukemia and osteoclast . Positive TRAP staining may also be seen in some activated T-lymphocytes, macrophages, some histiocytes (such as Gaucher cells), mast cells and some marginal zone lymphomas. TRAP stain may also be detected by immunohistochemical methods in fixed tissue sections.

35. 8) PERIODIC ACID-SCHIFF(PAS) The PAS stain detects intracellular glycogen and neutral mucopolysaccharides , which are found in variable quantities in most hematopoietic cells. Seen in blasts of both ALL and AML, although there is great variability between cases. Erythroleukemias demonstrate an intense diffuse cytoplasmic positivity with PAS, which may be helpful in diagnosis. Very useful in demonstrating the abnormal glucocerebrosidase accumulation in Gaucher disease.

36. 9) IRON Cellular iron is found as either ferritin or hemosiderin . Identified in cells by the Perls or Prussian blue reaction, in which ionic iron reacts with acid ferrocyanide to impart a blue colour . Used to identify iron in nucleated RBCs ( sideroblastic iron) and histiocytes ( reticuloendothelial iron) or to identify Pappenheimer bodies in erythrocytes. When increased number of these granules surround atleast two-thirds of the nucleus of red cell precursor, the cell is called a ringed sideroblast . Best used on bone marrow aspirate smears but can also be used on blood films and aspirate clot tissue sections. Decalcification of the bone marrow core biopsy may lead to loss of iron from the cells, leading to a false impression of low iron.

37. 10) TOLUIDINE BLUE It specifically marks basophils and mast cells by reacting with the acid mucopolysaccharides in the cell granules to form metachromatic complexes. Malignant mast cells or basophils may have low levels of acid mucopolysaccharides and may not react with this stain. Specific immunohistochemical markers such as staining for mast cell tryptase may be more specific in identification of mast cells than toluidine blue staining.

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