special and routine stains in haematology 1

SHAHID

1. SPECIAL AND ROUTINE STAINS IN HAEMATOLOGY PRESENTOR-Dr SHAHID

2. OUTLINE Introduction Routine Stain Special stains Summary

3. INTRODUCTION Differentiation of the peripheral blood is an important procedure in the diagnosis of hematologic disorders. The slide must be absolutely clean to avoid introducing artifacts . Slides are cleaned most effectively by degreasing in alcohol for 24 h, drying with a lint-free cloth,

4. Blood films are usually examined to investigate hematological problems (disorders of the blood) and Occasionally, to look for parasites within the blood such as malaria and filaria . Examination of thin blood films is important in the investigation and management of anaemia , infections, and other conditions which produce changes in the appearance of blood cells and differential white cell count.

5. Three basic steps to make blood film: Preparation of blood smear. Fixation of blood smear. Staining of blood smear.

6. The peripheral blood film (PBF) is of two types: Thin blood film Thick blood film

7. THIN BLOOD FILM O btained by venepuncutre or from free flowing finger prick blood by any of the following three techniques : Slide method Cover glass method Spin method

8. Preparation of blood smears Blood collection for thick or thin blood smears Capillary blood obtained by fingerstick :

9. 1. Label pre-cleaned slides (preferably frosted-end) with patient’s name (or other identifier), date and time of collection. 2. Wear gloves. 3. Clean slides with 70 to 90% alcohol and allow to dry. Do not touch the surface of the slide where the blood smear will be made.

10. 4. Select the finger to puncture, usually the middle or ring finger. In infants, puncture the heel. 5. Clean the area to be punctured with 70% alcohol; allow to dry 6. Puncture the ball of the finger, or in infants puncture the heel.

11. 7. Wipe away the first drop of blood with clean gauze. 8. Touch the next drop of blood with a clean slide. Repeat with several slides (at least two thick and two thin smears should be made). If blood does not well up, gently squeeze the finger.

12. For venous blood obtained by venipuncture : 1. Label collection tubes and pre-cleaned slides (preferably frosted-end) with the patient’s name (or other identifier), date and time of collection. 2. Clean the site for blood collection well using 70% alcohol; allow to dry.

13. 3. Collect the venous blood in a vacuum tube containing anticoagulant (preferably EDTA); alternatively, collect the blood in a syringe and transfer it to a tube with anticoagulant; mix well. 4. Prepare at least two thick smears and two thin smears as soon as possible after collection.

14. Making thick and thin blood smears 1. Whenever possible, use separate slides for thick and thin smears. 2. Thin film (a): Bring a clean spreader slide, held at a 45° angle, toward the drop of blood on the specimen slide. 3. Thin film (b): Wait until the blood spreads along the entire width of the spreader slide.

15. 4. Thin film (c): While holding the spreader slide at the same angle, push it forward rapidly and smoothly. 5. Thick film: Using the corner of a clean slide, spread the drop of blood in a circle the size of a dime (diameter 1-2 cm). Do not make the smear too thick or it will fall off the slide. (You should be able to read newsprint through it.)

16. 6. Wait until the thin and thick films are completely dry before staining. Fix the thin film with methanol (100% or absolute) and let it dry completely before staining. The thick film should not be fixed. 7. If both thin and thick films need to be made on the same slide, fix only the thin film with methanol. The thick film should not be fixed.

17. Cover Glass Method Procedure Take a clean cover glass. Touch it on to the drop of a blood. Place it on another similar cover glass in crosswise direction with side containing drop of blood facing down. Pull the cover glass quickly. Dry it and stain it. Mount it with a mountant , film side down on a clean glass slide .

18. Spin Method This is an automated method Procedure Place a drop of blood in the centre of a glass slide. Spin at a high speed in a special centrifuge, cytospin . Blood spreads uniformly. Dry it and stain it.

19. THICK BLOOD FILM Procedure Place a large drop of blood in the centre of a clean glass slide. Spread it in a circular area of 1.5 cm with the help of a stick or end of another glass slide. Dry it Stain

20. Parts of a Thin Blood Film

21. The shape of blood film

22. Examples of unacceptable smears

23. Fixation of blood smear

24. 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.

25. 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

26. 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 or azure .

27. Methylene blue is the basic dye Has affinity for acidic component of the cell (i.e. nucleus) Eosin/azure is the acidic dye and has affinity for basic component of cell (i.e. cytoplasm). Most Romanowsky stains are prepared in methyl alcohol so that they combine fixation and staining .

28. Various stains included under Romanowsky stain are as under 1. Leishman stain 2. Giemsa stain 3. Wright stain 4. Field stain 5. Jenner stain 6. JSB stain

29. Principle And Interpretation Leishman stain, is used in microscopy for staining blood smears. It provides excellent stain quality. It is generally used to differentiate and identify leucocytes, malaria parasites, and trypanosomas . It is based on a methanolic mixture of " polychromed " methylene blue. (i.e. demethylated into various azures) and eosin.The methanolic stock solution is stable and also serves the purpose of directly fixing the smear eliminating a prefixing step .

30. Quality Control Appearance Blue coloured solution Clarity Clear without any particles Microscopic Examination Blood staining is carried out and staining characteristic is observed under microscope by using oil immersion lens

31. Leishman Stain Preparation Mix and dissolve 0.15 g of Eosin- Methylene blue in 100 ml Methanol dried p.A. at 56°C. When the stain is dissolved completely remove the solution from the heater. (Alternatively, dissolve at RT over night. Cover container with Parafilm in order to prevent contamination by moisture). After the solution reached RT, clear the solution by using a dry Whatman paper filter. Collect filtered solution in a clean and dry brown glass bottle. Age the solution at least 2-3 days before using it the first time.

32. Procedure for staining 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. Add double the quantity of buffered water drop wise over the slide (i.e. double the number of drops). Mix by rocking for 8 minutes. Wash in water for 1 to 2 minutes. D r y in air and e x ami n e unde r oi l imme r si o n lens o f the microscope.

33. Giemsa Stain PROCEDURE Giemsa May- Grünwald Dilute Giemsa Stain 1:20 with deionized water. For bluer coloration, water buffered at pH 7.2 may be used in place of deionized water. 2. Place slides in May- Grünwald Stain for 5 minutes. 3. Place slides in Working Phosphate Buffer or Trizma ® Buffer (20-70 mmol /L). pH 7.2, for 1.5 minutes.

34. 4. Place slides in dilute Giemsa solution from step 1 for 15-20 minutes. 5. Rinse slides BRIEFLY in DEIONIZED water. 6. Air dry and evaluate.

35. 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).

36. .

37. Caution Thick films need careful rinsing! (since they are not fixed before staining) Mistake : Thick films are not Rinsed properly! Blood is lost

38. Wright Staining FIXATION Streak thin smears across a sterile slide by means of a second slide or cover glass placed at an angle. Air-dry completely. Make sure all slides are clean prior to making the blood or bone marrow smear. If slides are not completely clean the stain will not absorb into the smear.

39. Procedure 1. Place 1.0 ml of the Wright Stain Solution upon the smear approximately 1-3 minutes. 2. Add 2.0 ml of distilled water or Cat. Wright Stain Phosphate Buffer pH 6.8 and let the slide stand twice as long as in step 1.

40. 3. Rinse off the slide with water or Cat. Wright Stain Phosphate Buffer pH 6.8 until a faint pinkish red appears on the edges. If a Giemsa appearance is desired, stain 10 minutes in one volume of Wright Stain Solution and 4 volumes of Wright Stain Phosphate Buffer pH 6.8. 4. Dry thoroughly using bibulous paper to blot dry. Allow the slide to dry at room temperature before examination to determine if adjustments are needed in the dilution or timing.

41. Jenner stain The Jenner stain Solution is a mixture of several thiazin dyes in a methanol solvent. Ionic and non-ionic forces are involved in the binding of these dyes The staining solution has anionic and cationic properties

42. The negatively charged phosphoric acid groups of DNA attract the purple polychromatic cationic dyes to the nuclei. Th e blu e b a sophi lic g r anules a r e s t ai n ed b y the polychromatic cationic dyes.

43. Immersion Staining Protocol Thoroughly dry blood or bone marrow smears Fix smears in absolute methanol for 15 seconds to 5 minutes Stain smears in Jenners Stain Solution for 2 minutes Stain in mixture of 50ml of Jenners Stain Solution, 75ml of PH 6.6 Phosphate Buffer Solution and 175ml deionized water for 5 minute

44. Rinse in standing deionized water for 1.5 minutes or rinse briefly in running deionized water Air dry smears Examine smears under a microscope

45. Staining Protocol Place slid e with tho r ou g hl y dri ed fil m in a hori z o n t a l staining rack Flood smear with absolute methanol for 15-30 seconds and then drain Flood smear with 1ml Jenners Stain Solution and let stand for 3 minute

46. Add 1mL of pH 6.6 Phosphate Buffer solution and 1 ml deionized water to smear and let stand for 45 seconds. Rinse briefly with running deionized water Air dry and examine under a microscope Perform immunochemical staining procedure according to manufacturer.

47. J.S.B. Stain ( Jaswant –Singh– Bhattacharji   stain) Materials and reagents required : 1. Eosin yellow (water soluble) 2. Methylene blue 3. Potassium dichromate 4. Di-sodium hydrogen phosphate ( dihydrate ) 5. 1% sulphuric Acid. 6. Round bottom flask (2 lit.) 7. Healing mantle 8. Distilled water 9. Staining jars .

48. Preparation of the smear Use universal precautions while preparing the smears for malarial parasites – use gloves; use only disposable needles/lancets; wash hands; handle and dispose the sharp instruments and other materials contaminated with blood carefully to avoid injury.

49. Hold the third finger of the left hand and wipe its tip with spirit/ Savlon swab; allow to dry Prick the finger with disposable needle/lancet; allow the blood to ooze out Take a clean glass slide. Take 3 drops of blood 1 cm from the edge of the slide, take another drop of blood one cm from the first drop of blood

50. Take another clean slide with smooth edges and use it as a spreader and make thick and thin smears. Allow it to dry Slide number can be marked on the thin smear with a lead pencil

51. Staining technique Prepare thin and thick smears from malaria cases on micro slides De- haemoglobinise the thick smear Fix the thin smear in methanol for few minutes

52. Take 3 staining jars for J.S.B. I, J.S.B.II and tap water Dip the smears in J.S.B. II for few seconds and immediately wash in water Drain the slides free of excess water

53. Dip the smears in J.S.B.I for 30-40 seconds Wash well in water and dry Examine the smears under oil immersion

54. Staining of Thick Smear It can be stained with any of the Romanowsky stains listed above except that before staining, the smear is dehaemoglobinised by putting it in distilled water for 10 minute

55. Jaswant Singh Battacharya (JSB) Stain for thick and thin films : This is the standard method used by the laboratories under the National Malaria Eradication Programme in India

56. Special Stains In Hematology

57. Periodic assay Schiff (PAS) Perl’s Prussian Blue Reaction Leucocyte alkaline phosphatase (LAP) Myeloperoxidase Sudan Black B Toluidine Blue Specific esterases Non- Specific esterases Tartarate resistant acid phosphatase Pappenheim’s Stain (Panoptic Stain) Mayer’s Acid Hemalum Nuclear Stain Heilmeyer’s Reticulocyte Stain Heinz Body Test of Beutler1 Nile Blue Sulfate Stain

58. 15. Kleihauer - Betke Stain for Demonstrating Fetal Hemoglobin in Red Blood Cells 16. Kleihauer - Betke Stain for Demonstrating Methemoglobin - Containing Cells in Blood Smears 17. Berlin Blue Iron Stain 18. Cytochemical Determination of Peroxidase

59. Periodic Acid Schiff Reaction

60. Principle Periodic Assay oxidizes 1-2 glycol groups to produce stable dialdehydes which give a red reaction product when exposed to Schiff’s reagent ( leucobasic fuchsin ) Positive reaction occurs with carbohydrates, principally glycogen

61. Reagents Fixative : Methanol 1% Periodic acid Schiff’s Reagent: 5g basic fushcin in 500ml of hot distilled water and then saturated with SO 2 for 1-12 hr. Shake vigorously with 2g activated charcoal for 1 min. Counterstain : Aqueous Haematoxylin.

62. M e thod Fix films for 15 min in methanol Rinse with tap water Flood slides with 1%periodic acid for 10 min Immerse in Schiff reagent for 30 min Rinse in running tap water for 10 min Counterstain

63. Results and Interpretation PAS-positive material in the cytoplasm may produce a diffuse red stain Or may appear as pink to burgundy-red granules, flakes, or clumps of varying size that may occupy large areas of the cytoplasm. Some plasma cells, macrophages, and osteoblasts may also show a positive PAS reaction Megakaryocytes are strongly positive.

64. Monocytes and their precursors shows diffuse PAS positivity in AML M5 Normal erythroid precursors and RBCs are negative but dysplastic erythroblasts are positive- AML M6-Coarse granules are seen in cytoplasm Megakaryocytes and Platelets are strongly positive -AML M7-fine cytoplasmic granularity and blebs are positive Lymphocytes shows PAS blocks or granules ie block positivity ALL-L1,ALL-L2.

65. Perl’s Prussian Blue Reaction Principle The reaction occurs with the treatment of tissue sections with acid ferrocyanide solution. Any ferric ion (Fe3+) in the tissue combines with ferrocyanide and results in the formaion of a bright blue pigment called “ prussian blue ” or ferric ferrocyanide .

66. Fixation Avoid the use of acid fixatives. Chromates will also interfare with the preservation of iron

67. Staining solution 1% aqueous potassium ferrocyanide = 20 ml 2% aqueous hydrochloric acid = 20 ml Mix both

68. Procedure Deparaffinize and bring the sections to water. Treat the sections with freshly prepared acid ferrocyanide solution for 10-30 minutes Wash well in distilled water.

69. Lightly stain the nuclei with 0.5% aqueous neutral red or 0.1% nuclear fast red. Wash rapidly in distilled water. Dehydrate, clear and mount

70. Result and Interpretation Ferric ion = blue Nuclei = red Background = pink

71. Differential diagnosis by iron stain in the bone marrow

72. Sideroblasts Iron storing reticulum cells, sideromacrophages Special features Normal bone marrow 20–60 % finely granular, 1– 4 granules Isolated, mostly finely granular deposits Siderocytes in peripheral blood 0-0.3% Iron deficiency < 15 % finely granular None None Serum Fe decreases – Lead poisoning > 90 % coarsely granular; ringed sideroblasts Greatly increased, many diffuse or coarsely granular deposits Serum Fe increase siderocytes may be increased Thalassemia > 90 % coarsely granular; ringed sideroblasts Greatly increased, many diffuse or coarsely granular deposits Serum Fe increses siderocytes may be increased Hemolytic anemias <80 % finely granular Increased finely granular or (rarely) coarsely granular deposits

73. Hemochromatosis 80 % finely granular Increased Plasma cells contain iron Bone marrow is not useful for diagnosis except positive plasma cells

74. Leucocyte Alkaline Phosphatase

75. PRINCIPLE Naphthol AS phosphate in the presence of LAP gets hydrolysed then in the presence of Coupling Azo -dye: Fast blue RR salt gives insoluble ppt at the site of enzyme activity

76. REAGENTS Fixative: 4% formalin methanol Substrate : Naphthol AS phosphate Buffer: Tris Buffer (pH 9.0) Stock Substrate solution 30mg naphthol AS phosphate in 0.5ml N,N dimethyl formamide . Add 100ml Tris buffer Coupling Azo -dye: Fast blueRR salt Counter stain: Neutral red, 0.02% aqueous solution

77. M e thod Fix air dried smears for 30 s in 4% formalin methanol. Rinse with tap water. Prepare working solution by adding 24mg Fast blue BB salt in 40ml stock substrate solution. Incubate slides in working solution for 15 min Rinse with running tap water Counterstain for 3 min

78. Results and interpretation Reaction product is blue and granular. LAP score is determined by evaluation of the staining intensity (ranging from to 4+) of 100 counted neutrophils or bands. Normal LAP scores range from 15 to 130

79. USE Useful as a screening test to differentiate chronic myelogenous leukemia from leukemoid reactions and other myeloproliferative disorders

80. POSITIVE LAP REACTION NEGATIV LAP REACTION

81. Score Interpretation Negative, no granules 1 Occasional granules scattered in cytoplasm 2 Moderate number of granules 3 Numerous granules 4 Heavy positivity with numerous coarse granules, frequently overlying the nucleus

82. Low LAP score (<15) High LAP score (>130) CML Infections Paroxysmal nocturnal hemoglobinuria Growth factor therapy Myelodysplastic syndromes Inflammatory disorders Pregnancy, oral contraceptives Stress

83. Myeloperoxidase

84. Principle of Procedure Antigen detection in tissues and cells is a multi-step immunohistochemical process. The initial step binds the primary antibody to its specific epitope . A secondary antibody may be applied to bind the primary antibody, followed by an enzyme labeled polymer; or an enzyme labeled polymer may be applied directly to bind the primary antibody. The detection of the bound primary antibody is evidenced by an enzyme-mediated colorimetric reaction

85. R ea g e n ts Fixative : buffered formal acetone. Substrate : 3,3’diaminobenzidine (DAB). Buffer : Sorenson’s phosphate buffer pH7.3. Hydrogen peroxide. Counterstain : hematoxylin . Working substrate solution: 30mg DAB in 60ml buffer, add 120  l H 2 O 2 and mix well

86. Method Fix air dried smears in buffered formal acetone for 30s. Rinse thoroughly in running water. Incubate for 10 min in working substrate solution. Counterstain

87. Reactions and interpretations Reaction product is brown and granular. Red cells and erythroid precursors show diffuse brown cytoplasmic staining. Most primitive myeloblasts are negative with granular positivity appearing progressively as they mature toward promyelocyte stage. Promyelocytes and myelocytes are strongly staining cells in granulocyte series

88. Metamyelocytes and neutrophils have fewer positive granules. Eosinophil granules stain strongly and the large specific eosinophil granules are easily distinguished from neutrophil granules. Monoblasts and monocytes have variable positive reaction

89. Use To differentiate a myelogenous or monocytic leukaemia from acute lymphocytic leukaemia . Auer rods are better visualized with MPO than Romanowsky stains.

90. Pathological variants Some individuals have congenital deficiency of neutrophil MPO. All stages of neutrophil lineage from myelocyte onwards are negative, although the eosinophils stain normally. Some individuals may have eosinophil MPO or Monocyte MPO deficiency

91. Red brown precipitate

92. References 1. Toth B, et al. Immunophenotyping of acute lymphoblastic leukaemia in routinely processed bone marrow biopsy specimens. J Clin Pathol . 1999 Sep;52(9):688-92. 2. Pinkus GS, Pinkus JL. Myeloperoxidase : a specific marker for myeloid cells in paraffin sections. Mod Pathol . 1991 Nov;4(6)733-41. 3. Center for Disease Control Manual. Guide: Safety Management, NO. CDC-22, Atlanta, GA. April 30, 1976 "Decontamination of Laboratory Sink Drains to Remove Azide Salts.“ 4. Clinical and Laboratory Standards Institute (CLSI). Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline-Fourth Edition CLSI document M29-A4 Wayne, PA 2014.

93. Sudan Black B

94. PRINCIPLE PRINCIPLE :  Sudan Black  is slightly basic dye and will combine with acidic groups in compound lipids, thus  staining  phospholipids also. An alternative  stain to the  Sudan Black B stain . CONTROL: Use a positive control of a fat smeared slide, and a negative control slide of a paraffin processed tissue, such as lung.

95. PRINCIPLE Sudan Black B is a lipophilic dye . That binds irreversibly to an undefined granule component in granulocytes, eosinophils and some monocytes .

96. M e thod Fix air dried smears in formalin vapors. Immerse the slides in working stain solution for 1 hr. in a coplin jar with lid on. Transfer the slides to a staining rack and flood wth 70% alcohol. After 30s, tip off and repeat three times in total Rinse in gently running tap water Counterstain with May- Grunwald - Giemsa or Lieshman stain

97. REAGENTS 1. Dye 60 ml 2. Buffer 40 ml ADDITIONAL REAGENTS : Fixative : Available on request . - Formaldehyde Fixative . Or - Formaline – ethanol fixative Counterstain : Methyl green or Giemsa

98. STABILITY Reagents are stable up to the expiration date given when stored at 4 °C

99. PROCEDURE Fixation: Fix air – dried films of blood or bone marrow for 10 min in formalin vapour , formaldehyde fixative or formaline – ethanol fixative . 2. Wash gently in water for 5 – 10 min ;

100. Staining Working Reagent: Mix Reagent R1 and Reagent R2 in ratio of 1. 5 : 1, Filter before use. 1. Incubate the fixed film in 20 drops working reagent for 10 min. in closed area to avoid evaporation.

101. 2. Wash in 70 % ethanol by waving the slides in the alcohol for 3 - 5 min . 3. Wash with water for 2 min, dry . 4. Counterstain for 5 min and mount .

102. Sudan Black B staining Positive stain in a patient of AML Black stained cytoplasm in myeloblasts .

103. RESULT The reaction product in the cytoplasm is black; the nuclei stain blue or red depending on the counterstain used. ( Giemsa , safranin or methyl green.)

104. REFERENCE Sheehan , H .L . and Story , G . W . ( 1977 ) J . Path . and Bacter . 59 , 336

105. Toluidine Blue Stain

106. Principle Toluidine blue staining is useful for the enumeration of basophils and mast cells. It binds strongly to the granules in these cells

107. M e thod Place air dried smears on a staining rack and flood with toluidine blue solution Incubate for 5-10min. Rinse briefly in gently running tap water

108. RESULTS The granules of basophils and mast cells stain a bright red/purple and are discrete and distinct. Nuclei stain blue and cells with abundant RNA may show a blue tint to the cytoplasm

109. Toluidine blue staining showing strongly positive basophils

110. Specific Esterases

111. Use The specific ( naphthol AS-D chloroacetate ) esterase stain, also called the Leder stain, is used to identify cells of the granulocytic series

112. M e thod Fix smears in cold buffered formal acetone. Rinse in gently running tap water. Immerse slides in working solution for 10min. Rinse in tap water. Counterstain for 1 min with aqueous hematoxylin

113. RESULT Reaction product is bright red. It is confined to cells of neutrophil series and mast cells. Myeloblasts stain rarely. Promyelocytes and myelocytes show strong positvity

114. Non specific esterases

115. Use To identify monocytic cells but do not stain granulocytes or eosinophils Include α- naphthyl butyrate and α- naphthyl acetate

116. α- Naphthyl butyrate Reagents Fixative: Buffered formal acetone. Buffer: phosphate buffer (pH8.0). Substrate stock solution α- Naphthyl butyrate 100  l in 5ml acetone stored at -20 o C Coupling reagent : Fast Garnet GBC 15mg. Counterstain : aqueous hematoxylin

117. M e thod Fix air dried smears in buffered formal acetone for 30 sec and then rinse in tap water . Add Fast Garnet GBC to 50ml buffer and mix . Add 0.5 ml of substrate solution . Pour incubation medium in coplin jar containing fixed slides and incubate for 20-40min . Air dry and counterstain for 1 min aqueous hematoxylin

118. Results and interpretation Reaction product is brown and granular. Majority of monocytes stain strongly. More specific for identifying monocytic component in AML

119. α-naphthyl acetate

120. Results and interpretation Reaction product is brown and granular. Majority of monocytes stain strongly. More specific for identifying monocytic component in AML

121. M e thod Fix air dried smears in buffered formal acetone for 30s . Rinse in running tap water . Immerse slides for 30-60min in incubation medium . Rinse in running tap water . Counterstain for 2-5min

122. Results and interpretation Reaction product is red/brown in color . Normal and leukemic monocytes stain strongly. Normal granulocytes are negative but in MDS or AML may give varying positive reaction. Megakaryocytes stain strongly

123. Tartrate Resistant Acid Phosphatase

124. Principle The tartrate-resistant acid phosphatase (TRAP) is an isoenzyme of acid phosphatase that is found in high levels in the cells of hairy cell leukemia and osteoclasts .

125. M e thod Air dry films for at least 24hr . Fix for 10 min in methanol . Incubate for 1hr at 37 o C in working solution A . Rinse in tap water and air dry . Counter stain with 1% aqueous methyl green or hematoxylin for 5 min . Rinse in tap water and mount

126. Results and interpretation Reaction product is red with a mixture of granular and diffuse positivity. Almost all T-lineage leukemias show string activity In Hairy cell leukaemia, majority of leukemic cells react equally positively.

127. Tartrate-resistant Acid Phosphatase (TRAP) Activity Of Lymphocytes

128. Pappenheim’s Stain (Panoptic Stain ) It is based on a combination of the Jenner-May-Gru¨nwald stain and Giemsa stain . Used to differentiate basophilic granules of erythrocytes and nuclear fragments Uses-

129. Heinz Body Test of Beutler1 This test is used to detect defects of red cell metabolism that do not allow glutathione , maintained in a reduced state . The defect may result from a glucose-6-phosphate dehydrogenase deficiency , a glutathione reductase deficiency,diseases In which there is“unstable hemoglobin ,” or an “ idiopathic” Heinz body anemia . The test involves the oxidative denaturation of hemoglobin to intraerythrocytic“Heinz bodies” following incubation of the red cells with acetylphenylhydrazine.

130. INTERPRETATION - The percentage of erythrocytes that contain more than four Heinz bodies is determined . Normal values range from 0 % to 30 %.

131. Nile Blue Sulfate Stain This stain is used for the visualization of Heinz inclusion bodies . The Heinz bodies appear as small,dark blue bodies situated at the margin of the yellow to bluish erythrocytes.

132. Kleihauer-Betke Stain for Demonstrating Fetal Hemoglobin in Red Blood Cells Principle Normal adult hemoglobin ( HbA ) is dissolved out of the red cells by incubating air-dried and fixed blood smears in citric acid phosphate buffer (of McIlvain ), pH 3.3, at 37 8C . Fetal haemoglobin( HbF ) is left undissolved in the red cells and can be made visible by staining

133. Method Prepare thin blood smears, air dry, and fix in 80 % ethyl alcohol for 5 min. Wash in water and dry. If further processing is delayed, the slides may be stored in a refrigerator for 4–5 days. For elution , place the slides upright in a beaker containing the buffer in a 37 C water bath for 3 min,moving the slides up and down after 1 and 2 min to keep the buffer mixed. Then wash in running water .

134. Staining Stain in Ehrlich hematoxylin for 3 min,then post stain in 0.1 % aqueous erythrosin solution for 3 min . Examine at 40 using dry or oil immersion magnification

135. Interpretation . Erythrocytes that contain HbA appear as unstained “shadows,” while cells that contain HbF will stain a bright red color . The method can be used for the diagnosis of thalassemia major and for the detection of fetal erythrocytes that have entered the maternal circulation .

136. Kleihauer-Betke Stain for Demonstrating Methemoglobin -Containing Cells in Blood Smears Principle . Methemoglobin combines with KCN to for cyanmethemoglobin , while oxyhemoglobin does not react with cyanides. Oxyhemoglobin functions as a peroxidase, whereas cyanmethemoglobin has very low peroxidase activity .

137. Interpretation Oxy- Hb -containing cells stain a bright red . Cells that contain met- Hb ( converted to cyanmet-Hb ) are eluted and appear as shadows

138. Cytochemical Determination of Acid Phosphatase Used in the cases of plasmacytomas , the abnormal plasma cells tend to show stronger activity than normal plasma cells or plasma cells affected by reactive changes . A dot like staining pattern is seen in T-lymphocytes, while the blasts of T-ALL usually show a circumscribed (focal) paranuclear acid phosphatase reaction.

139. SUMMARY

140. Stain Structure stained Use Periodic assay Schiff (PAS) Carbohydrates, principally glycogen In AML and MDS to identify abnormal erythroblasts and dysplastic megakaryocytes To confirm the diagnosis of acute promyelocytic leukaemia Perl’s Prussian Blue Reaction Iron For demonstration of ring sideroblasts and Pappenheimer bodies Leucocyte alkaline phosphatase (LAP) Neutrophil alkaline phosphatase Differentiate chronic myelogenous leukemia from leukemoid reactions and other myeloproliferative disorders

141. Stain Structure stained Use Myeloperoxidase Myeloperoxidase in neutrophils, monocytes and eosinophils To differentiate a myelogenous or monocytic leukemia from acute lymphocytic leukemia . To visualize auer rods Sudan Black B Intracellular lipid Similar to MPO Toluidine Blue Basophils and mast cells To identify dysplastic basophils in myeloproliferative diseases Specific esterases Neutrophil series and mast cells Marker of cytoplasmic maturation in myeloid leukemias Non- Specific esterases Monocytes Monocytic component in AML Tartarate resistant acid phosphatase T-cells and granulocytes Diagnosis of T-cell ALL and hairy cell leukemia

142. References- Bacus , JW. Erythrocyte morphology and “spinner” blood film preparations. J Histochem Cytochem . 1974;22:506–516 . Crossref | PubMed | Bentley, SA. Quality control and the differential leukocyte count. Clin Lab Haematol . 1990;12:101–109. PubMed Beutler , E, Lichtman , MA, Coller , BS et al, Williams Hematology . ed 5. McGraw Chanarin , I. Laboratory Haematology. Churchill Livingston, Edinburgh; 1989 . Dacie , JV, Lewis, SM. Practical Haematology. ed 5. Churchill Livingston, Edinburgh; 1975 . Devices for collection of skin puncture blood specimens. NCCLS Approved Guideline H14-A2. 1990 ;. Gleeson, RM. An improved method for thick film preparation using saponin as a lysing agent. Clin Lab Haematol . 1997;19:249–251 .