Special stains in Bone marrow examination

Special stains in Bone marrow examination

1. SPECIAL STAINS IN BONE MARROW EXAMINATION

2. Bennett" has divided cytochemical stains into three groups: ( i ) cytochemical stains of diagnostic significance - peroxidase , naphthol AS-D acetate esterase without and with fluoride, leucocyte alkaline phosphatase ; (it) specialized cytochemical stains - leucocyte acid phosphatase , naphthol AS-D chloro -acetate esterase, toluidine blue (iii) cytochemical stains of limited value - ,B- glucuronidase . -acetyl-,B- glucosaminidase , periodic acid-Schiff, Oil red 0, Sudan black B. The Present Status of Cytochemistry in the Diagnosis of Haematological Malignancy, W. G. STAPLES, E. P.GETAZ, S. Afr. med. l., 51, 838 (1977).

3. " Perls ' Prussian blue Prussian blue - Berlin in the early 1700s The original stain formula, known historically (1867) as " Perls ' Prussian blue" after its inventor, German pathologist Max Perls Separate solutions of potassium ferrocyanide and acid to stain tissue (these are now used combined, just before staining). The formula is also known as Perls Prussian blue and (incorrectly) as Perl's Prussian blue.

4. ( Perls's Prussian Blue reaction) The majority of non- haem iron is stored as haemosiderin (a ferric iron-protein complex), a small amount as ferritin and the remainder is contained in myoglobin and transferrin . Ferritin - normally in all cells in the body, Haemosiderin - macrophages in the bone marrow, liver ( Kupffer cells) and spleen. Most intracellular iron is in macrophages, a small amount in erythroblasts ( sideroblasts ) .

5. A Perls ’ or Prussian blue stain demonstrates haemosiderin in bone marrow macrophages and within erythroblasts. Assessment of both the amount of iron in reticulo - endothelial stores and the availability of iron to developing erythroblasts. Detection of increased or decreased proportion of sideroblasts and abnormal sideroblasts .

6. Principle Ferric iron in haemosiderin is released from protein by treatment with dilute hydrochloric acid . The free iron reacts with a dilute solution of potassium ferrocyanide to produce an insoluble blue compound, ferric ferrocyanide (Prussian Blue). By contrast, ferritin , which is a water-soluble non- haem compound of iron with the protein apoferritin , is not detectable by Perls ’ reaction. Any contrasting, non-acidic counterstain may be used but Neutral Red, safranin or eosin are the most popular.

7. MATERIALS Fixative Methanol as for MGG 4% HCL 4ml HCL + 96% distilled water 4% potassium ferrocyanide 4g potassium ferrocyanide + 1000ml distilled water Nuclear fast red Store at room temperature

8. PROCEDURE Prepare working solution by adding two parts of 4% HCL and 4% Potassium ferrocyanide 2-3ml a slide Flood the slide with working solution and allow to stand for 15-20 mins Wash well with distilled water for 5 mins Counter stain with nuclear fast red in a coplin jar for 5-7mins Wash in tap water Air dry and observe marrow fragment and erythroid precursor

9. Positive- bright green/bright blue Cytoplasm of stain pink A bone marrow film or squash preparation - both intracellular and extracellular iron, the latter being derived from crushed macrophages. Assessment of iron stores mainly on intracellular iron

10. INTERPRETATION A BM smear with increased iron stores - positive control. Low power to assess storage iron 40 or × 50 objective to detect abnormally prominent siderotic granulation 100 objective to assess whether siderotic granulation is reduced, normal or increased

11. Gale E, Torrance J, Bothwell T. The quantitative estimation of total iron stores in human bone marrow. J Clin Invest 1963;42:1076-82.

12. Intensive histological grading method Intensive histological grading method—the fragments, macrophages and erythroblasts. Iron assessed in the fragments and macrophages - iron stores Iron in the erythroblast - utilisable iron. 100 erythroblasts were examined and the percentage containing iron granules in their cytoplasm ( ie , sideroblasts ) were enumerated. Improved method for assessing iron stores in the bone marrow, K S Phiri et al, J Clin Pathol . 2009 August; 62(8): 685–689.

13. s Multiple transfusions Anemia of chronic disease Hemolytic anemia Haemoglobinopathy Aplastic anemia

14. SIDEROBLASTS Kaplan and others (1954) introduced the term ' sideroblast ' to indicate a normal red cell precursor with visible iron-containing granules, A proportion of normal erythroblasts - few (one to five) fi ne iron - containing granules randomly distributed in the cytoplasm These granules are seen in Ramanowsky -stained blood or bone marrow smears as basophilic granules ( Pappenheimer bodies) The Sideroblastic Anaemias , J. J. Morrow and A. Goldberg, Postgrad Med J 1965 41: 740-747

15. INTRACELLULAR IRON The total number of sideroblasts (normal, reduced or increased) should be reported and the frequency and location ( cytoplasmic or perinuclear ) of siderotic granules (Bowman, 1961; Cartwright & Deiss , 1975) should be noted, At least 100 erythroblasts should be evaluated for the percentage of ring sideroblasts , if present ICSH guidelines for the standardization of bone marrow specimens and reports S.-H. LEE*, W. N. ERBER†, A. PORWIT‡, M. TOMONAGA§, L. C. PETERSON– FOR THE INTERNATIONAL COUNCIL FOR STANDARDIZATION IN HEMATOLOGY INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

16. The percentage of sideroblasts is increased - haemolytic anaemias , megaloblastic anaemias and in haemochromatosis and haemosiderosis , in proportion to the degree of saturation of transferrin (i.e. to the amount of iron available). A disproportionate increase in the percentage of sideroblasts - synthesis of haemoglobin is impaired , in which case the siderotic granules are both more numerous and larger than normal

17. RINGED SIDEROBLASTS Iron deposits are visualized with iron stain as blue granules (10 or more granules which encircles >1/3 of the nucleus) May Resemble: Sideroblast is similar on iron stain but with less than 5 granules

18. Ring sideroblasts - Refractory anaemia with ring sideroblasts and refractory cytopenia with multilineage dysplasia and ring sideroblasts , Other categories of MDS. Primary myelofibrosis Acute myeloid leukaemia (AML), particularly erythroleukaemia WHO categories of therapy related AML and AML with multilineage myelodysplasia . States of iron overload such as hemochromatosis (sometimes ) Zinc toxicity induced copper deficiency

19. Plasma cells contain haemosiderin inclusions, which are irregular in shape and relatively large. MGG stain - greenish black Iron overload (for example in haemochromatosis and transfusional siderosis ) and in chronic alcoholism

20. VARIANTS Films that have previously been stained by Romanowsky dyes, even after years of storage. Stand in methanol overnight to remove most of the Romanowsky stain. The film should be checked before carrying out Perls ’ reaction to ensure that there is no residual blue staining that could obscure Prussian-blue staining. Rapid method- demonstrating siderotic granules by staining with 1% bromochlorphenol blue for 1 min. Iron-containing granules stain dark purple

21. Sundberg and Bromann - films were stained first by a Romanowsky dye (Wright’s stain) and then overstained by the acid- ferrocyanide method. Hayhoe and Quaglino described a method for combined periodic acid–Schiff (PAS) and iron staining. This may be helpful in the investigation of abnormal erythropoiesis in which the erythroblasts give a positive PAS reaction

22. Aspirate films are more sensitive than trephine biopsy sections for the detection of haemosiderin when the biopsy specimens are decalcified in formic acid. Decalcification leads to an unquantifiable loss of iron. Decalcification - ethylenediaminetetraacetic acid, which is a less powerful decalcifying agent.

23. Distributed irregularly within bone marrow macrophages - minimum of seven fragments before concluding that storage iron is absent or reduced. If less than seven particles are present and no iron is seen, the sample should be reported as lacking stainable iron, but the assessment should be stated to be unreliable How should stainable iron in bone marrow films be assessed? D A Hughes, S E Stuart-Smith, B J Bain J Clin Pathol 2004;57:1038–1040. doi : 10.1136/jcp.2003.015834

24. LEUCOCYTE CYTOCHEMISTRY To identify diagnostically useful enzymes or other substances in the cytoplasm of haemopoietic cells. To characterize the blast cells in acute leukaemia as myeloid (leading to a diagnosis of AML unless there is also evidence of lymphoid differentiation) To diagnosis a mixed-phenotype acute leukaemia,

25. To identify granulocytic and monocytic components in AML To identify unusual lineages occasionally involved in clonal myeloid disorders (e.g. basophils and mast cells) To detect cytoplasmic abnormalities and enzyme deficiencies in myeloid disorders (e.g. MPO neutrophils in myelodysplasia or acute leukaemia, NAP -deficient neutrophils in CML) To identify Auer rods in MDS

26. Periodic Acid – Schiff [PAS] Reaction Periodic acid oxidizes the 1-2 glycol groups in  sugars , to produce dialdehydes The oxidation condition has to be sufficiently regulated so as to not oxidize the aldehydes further. Aldehydes +  Schiff reagent = purple-magenta color .

27. PAS A variety of intracellular compounds react with the PAS reagents, but in the blood and marrow cells glycogen is the most abundant compound Intensity of colour - number of aldehyde groups liberated For blood smears, the recommended fixative is  methanol.  Glutaraldehyde - not recommended - free aldehyde groups may be available to react with the Schiff reagent, which may result in  false positive staining .

28. Fixative- Methanol 1% periodic acid 10g HIO4.2H2O+ 1000ml Distilled water Schiff’s reagent Counter stain Aqeous haematoxylin

29. PROCEDURE Fix films 20 min in methanol Rinse gently under tap water If required expose to diastase Flood slides with 1% PAS , allow to stand for 5-7min Rinse under running tap water, 5-7min Immerse in Schiff’s reagent for 15 min Rinse in running tap water Counter stain- Aqeous haematoxylin for 30 seconds QC control- high neutrophil count

30. PAS stain can be used in old but well-preserved smear and smear stained with Wright’s stain. Smear stained with Wright’s stain should be decolorized with ethanol before performing PAS staining . Microscopic examination should be performed immediately after PAS staining, since positive reaction will turn weaker after one week.

31. Neutrophil precursors contain many mitochondria , respiration is the main source of metabolic energy. As they mature, mitochondria decrease in number Glycolysis is now the predominant mechanism for energy production and the neutrophil can be sustained anaerobically to operate in hypoxic infected tissues and exudates Glycogen particles accumulate in the metamyelocyte and in band and mature neutrophils which represent the non-dividing non- secretory stages.'·

32. Granulocyte Myeloblasts and promyelocyte diffuse faint positive Myelocyte - some coarse and some fine granules Neutrophils - numerous granules packed tightly Eosinophils - diffuse intergranular cytoplasm with large unstained granules

33. Normal erythroid precursors- variable Normoblasts may be positive in sideroblastic anemia, iron deficiency, thallassemia , and severe hemolytic anemias ‑

34. It was found that megakaryocytes are rich in glycogen which is not only confined to the intensely PAS-positive granules and inclusion bodies, but also makes up a good part of the diffuse cytoplasmic staining. Glycogen content and PAS staining pattern of human megakaryocytesDr . F. V. Shammas * , A. Engeset Scandinavian Journal of Haematology , September 1986

35. Monocytes - weakly positive reaction . Monocytes have active aerobic glycolysis Macrophages - strong positive reaction. Lymphocytes- smaller content Ewings sarcoma and alveolar rhabdomyosarcoma cells are PAS positive

36. AML Usually negative, but may show a weak diffuse reaction with superimposed fine granular positivity. AML M1 and M2, Auer rods- weak positive a cytoplasmic ‘blush ’ – a fine diffuse or dust-like positivity-AML M3 Negative to focal or granular positivity, to strongly positive staining.- AML M7 Acute basophilc lekemia - positive

37. PAS IN AML Positive in acute erythroid leukemia Pronormoblasts and other stages of normoblasts positive Nucleated RBCs PAS positive in MDS

38. PAS IN ALL 95% of cases show positive blocks or granules of bright red PAS-positive material. This may be present in very few blasts (<1%) or the majority. The critical difference from granular or block positivity in other leukaemic cells is the glass-clear background cytoplasm in lymphoblasts . Myeloblasts , monoblasts , leukaemic erythroblasts and megakaryoblasts all show some degree of diffuse cytoplasmic positivity and occasionally block positivity is seen.

39. PAS IN ALL ALL –varied staining pattern Lymphoblasts - coarse block, fine diffuse or both or negative Negative staining does not rule out ALL Burkitt’s lymphome – negative

40. Some authors suggest that strong PAS-positivity indicates a good prognosis (Laurie, 1968;Vowels and Willoughby, 1973; Feldges et al., 1974; Ascari et al., 1975) but others claim that it has no such significance (Bennett and Henderson 1969; Berrebi et al., 1973; Humphrey et al., 1974; Shaw et al., 1977). There are varying reports of the relation of P.A.S. positivity, to the duration of first remission and survival. Kurz , R. and Haas, H.: Value of the com­bined cytological and cytochemical classi­fication in the management of acute child­hood leukaemia. Acta . Haematol . 52: 1-7, 1974 Feldges , A. J., Aur , R. J. A., Verzosa , M. S. and Daniel, S.: Periodic acid Schiff re­action-a useful index of duration of com­plete remission in acute childhood lym­phocytic leukaemia. Acta . Haematol . 52: 8-13, 1974. 

41. Myeloperoxidase (MPO) Primary and secondary granules of neutrophils and their precursors Eosinophil granules Azurophilic granules of monocytes . The MPO in eosinophil granules is cyanide resistant , whereas that in neutrophils and monocytes is cyanide sensitive. MPO has a  heme pigment, which causes its green color in secretions rich in neutrophils , such as pus and some forms of mucus

42. Peroxidase stain : Principle: In the presence of peroxidase , H2O2 is split liberating O2 that oxidizes benzidine or 4-chlor-1-naphthol

43. Myeloperoxidase (MPO) Bluish-black granules red brown precipitate The reaction product is blue/ black/ red brown.

44. Dissolve 1,4 chloro 1 naphthol in 15ml ethanol Add 45ml distilled water 10 drops of tris ( hydromethylaminomethane )- Hcl buffer and H2O2 each Fix in leucognost for 1 min Wash and place in staining solution for 10 min Wash and stain with Harris Haematoxylin Wash under tap water and dry in air Staining can be enhanced by immersing the slides in copper sulphate or nitrate, but this is generally not required in normal diagnostic practice

45. SUBSTRATES The most common substrates used are benzidine derivatives (Graham, 1918; Goodpasture , 1919). Benzidine dihydrochloride (BDC) according to Kaplow's (1965) technique. Alternative non- benzidine -based techniques use 4-chloro-1-naphthol (4CN) or 3-amino-9-ethylcarbazole. The former gives very crisp staining but is soluble in some mounting media and immersion oil The latter shows some diffusibility and does not stain as strongly as DAB.

46. MPO is not inhibited by heparin, oxalate or EDTA anticoagulants. Films should be made within 12 h of blood collection. Staining is satisfactory on slides kept at room temperature for at least a week. QC control slide- High neutrophil count

47. NORMAL MARROW The most primitive myeloblasts are negative- granular positivity appearing progressively as they mature toward the promyelocyte stage- positivity may be localized to the Golgi region. Promyelocytes and myelocytes are the most strongly . Metamyelocytes and neutrophils - fewer positive (secondary) granules. Neutrophils and eosinophils - primary granules Eosinophils – also secondary granules.

48. Monocyte lineage- peroxidase activity detectable at the promonocyte stage, fewer scattered granules than neutrophils and their precursors. MPO activity - basophil granules- not demonstrable in mature basophils by the DAB reaction described earlier. Erythroid positive when treated with methanol-staining reagent- Hb - pseudoperoxidase / Lepehne reaction

49. Some individuals have congenital deficiency of neutrophil MPO. All stages of the neutrophil lineage, from the myeloblast onward, are negative. In these individuals, the eosinophils stain normally.

50. MPO in AML AML with MDS- MPO may be aberrant, because patients may develop an acquired MPO deficiency as part of the dysplastic process. AML 1, 2- >3% positive Auer rods- MPO + AML M3- MPO- strongly positive, reaction product covering cytoplasm and nucleus Monoblasts - MPO negative, Promonocytes -scattered positivity AML M7- Consistently negative for MPO and SBB

51. s AML without maturation AML with maturation

52. Pox type 1- upto 5% Peroxidase positive blasts- AML without maturation Pox type 2- 5-65%- AML without maturation/ Acute myelomonocytic leukemia Pox type 3- >65% - AML with maturation, Acute promyelocytic leukemia

53. Percentage of MPO-positive blast cells has an impact on the clinical outcome of AML . MPO-negative M0 subtype with immature features showed a disappointing clinical outcome, higher resistance rate in patients MPO-positive rate of blast cells is associated with drug-resistance mechanisms. The percentage of myeloperoxidase -positive blast cells is a strong independent prognostic factor in acute myeloid leukemia , even in the patients with normal karyotype T Matsuo 1 , K Kuriyama 1 , Y Miyazaki 1 , S Yoshida 1 , M Tomonaga 1 , N Emi 3 , T Kobayashi 4 , S Miyawaki 5 , T Matsushima 6 , K Shinagawa 7 , S Honda 2  and R Ohno 8 for the Japan Adult Leukemia Study Group Leukemia (2003)  17,  1538–1543. doi:10.1038/sj.leu.2403010

54. SUDAN BLACK More sensitive than MPO in early precursors More reliable and stable Principle : Sudan black B dye is fat soluble, then it stains fat particles ( Steroles , phospholipids and neutral fats) in the primary and secondery granules of myelocytic and lysosomes in monocytic cells.

55. Fix air dried smears in formalin vapor Wash in running water and air dry Immerse in working stain solution ( 60 ml of 0.3 Sudan Black B in 100 ml absolute alcohol+ 40 ml of crystalline phenol in absolute alcohol and disodium hydrogen phosphate) Transfer to staining rack- dip in 70% alcohol and rinse Counter stain with nuclear fast red Wash in running water

56. SBB stains the granules of neutrophils (both the primary and the specific granules) , specific granules of eosinophils The staining of eosinophil granules may be peripheral with the central core remaining unstained . Promyelocytes - few sudanophilic granules, mature polymorphonuclear neutrophils - large numbers of sudanophilic granules.

57. Monoblasts - either negative or few small SBB positive granules. Promonocytes and monocytes - variable number of fine positively staining granules. Hereditary neutrophil , eosinophil and monocyte peroxidase deficiencies- granules of cells of the deficient lineages are SBB-negative. Basophils - generally not positive - may show bright red/purple metachromatic staining of the granules.

58. ALL Rare cases (1–2%) of acute lymphoblastic leukaemia (ALL) show non-granular smudgy positivity not seen with MPO staining. Very rarely a stronger reaction - lymphoblasts of ALL or in lymphoma cells of T or B lineage

59. AML: Acute Myeloid Leukemia (M1, M2 and M3) Auer rods AMML: Acute Myelomonocytic Leukemia Erythroleukemia : pos in myeloblasts , neg in normoblasts

60. ESTERASES Leucocyte esterases - enzymes that hydrolyse acyl or chloroacyl esters of a- naphthol or naphthol AS. Li et al Nine isenzymes of esterases in leucocytes - Napthol AS-D Chloroacetate (1,2,7,9) - Alpha- naphtyhl acetate - Alpha- naphthyl butyrate (3,4,5,6) Specific- myelocyte Non specific- other cells Debris digesting action of the neutrophil .

61. SPECIFIC ESTERASE A specific esterase (capable of liberating naphthol from a naphthol AS-D chloroacetate substrate) present in the non specific granules - granulocytes and mast cells Chloracetate esterase has an optimum pH between 7.0 and 7.6 and is insensitive to fluoride inhibition

62. Specific esterase or chloroacetate Principle: Interpretation: Myeloid cells (+ ve ) Mast cells Monocyte and basophile (– ve ) to weak (+ ve ) Other cells {lymph – plasma –megakaryocyte – nrbc } (- ve ) A naphthol compound is released which combines with a diazonium salt to produce a brightly coloured compound at the site of enzyme activity

63. The incubation time is important- most haemopoietic cells show some scattered granular staining if the incubation is prolonged. The substrate used is naphthol AS- D chloro -acetate and the coupler is garnet GBC diazo salt Haematoxylin is used as a counterstain . Positivity is represented by a reddish coloration. The reaction is resistant to sodium fluoride inhibition . Aminocaproate esterase is found in mast cells but cytochemically has no practical value.

64. CAE in ACUTE MYELOID LEUKEMIA CAE - more often positive in M2 than in M1 AML and reactions are stronger. Auer rods- usually weak or negative except in M2 AML associated with t(8;21) and AML M3 in which Auer rods are often positive for CAE

65. Non Specific Esterase: { with fluoride inhibition} Specificity - sodium fluoride as an inhibitor (NASDA-F). Sodium fluoride, inhibits the activity in monocytes , megakaryocytes , platelets, and plasma cells but not that in lymphocytes

66. Nonspecific esterases Monocytes and precursors macrophages, megakaryocytes and platelets Mature T cells and T-all ( cytoplasmic dot) Normal granulocytes are negative- MDS or AML may give positive reactions of varying intensity.  Carcinomas, megaloblastic erythrocytes, cytoplasm focally in AML-M7

67. Air dried smears in Leucognost fixing mixture for 1-3 mins Wash with distilled water for 1 min Place in staining solution(60 ml of phosphate buffer in distilled water+1 naphthyl acetate in acetone+pararosaniline Hcl + nitrite solution)and incubate in dark for 1-2 hr Wash in distilled water for 10 sec Stain with haematoxylin – 2min Wash in tap water – 2 min Air dry and examine QC Control-slide with high platelet count

68. a- Naphthyl Acetate Esterase (ANAE) The reaction product is diffuse red/brown in colour Sensitive but not specific ( picks up T-lymphocytes ( punctate staining pattern) as well as monocytes (diffuse staining pattern) The T lymphocytic pattern is resistant to NaF , staining in monocytes is NaF sensitive and favoured at an alkaline pH   The reaction product is brown and granular More specific, although slightly less sensitive Differential staining with the different esterases - megakaryoblasts , which do not stain with the α- naphthyl butyrate, but stain with the α- naphthyl acetate substrate α- Naphthyl Butyrate Esterases

69. Peroxidase type- < 20% EST- positive blasts- AML Pox EST mixed type- 25-50% pos blasts- AML M5 Esterase type- > 50% EST positive blasts- AML M5

70. USES OF NON SPECIFIC ESTERASES Helps diagnosis in AML of M5 and AML M5 ALL of T-ALL and in CLL T-CLL (POS) from B-CLL (NEG) Erythroblasts are usually ANAE NEG, however they may react in megaloblastic anaemia and erythroleukaemia AML M7- They are alpha naphthyl butyrate esterase negative Variable alpha naphythyl acetate esterase activity - scattered clumps or granules inhibited by flouride ( vs diffuse cytoplasmic positivity)

71. Monocytes , monoblasts and promonocytes - NSE positive

72. Alpha- naphthyl acetate esterase as substrate and fast blue B as coupler- histiocytes in lymph node imprints in true histiocytic lymphoma. Histiocytic lymphoma is extremely rare and can only be identified positively by this stain.' By this means we can differentiate between the histiocyte and the transformed lymphocyte or immunoblast

73.   Double esterase- Sequential Combined Esterase Stain Using ANAE and CAE  Double esterase combines two substrates: naphthol AS-D chloracetate that reacts with primary granules in the neutrophilic series and  - naphthyl butyrate that stains material in the endoplasmic reticulum of cells in the monocytic lineage. The combined methods have the advantage of demonstrating pathological double staining of individual cells. Identification of monocytic and granulocytic component in AML M4

74. Step 1 - Fix in buffered formalin acetone Add 10 ml of non specific buffer+ 0.5 ml butrate 50 microl pararosaniline + 50 microl sodium nitate Stand for 1 min 50microl from 3 to 2 Add5 to slide and incubate in dark room for 45 min Step 2 - 10 ml specific esterase buffer to 0.5ml chloracetate substrate+ 5 ml of fast blue BB salt Wash and air dry Counter and counter stain with haematoxyline

75. The ANAE - brown reaction product CAE - granular bright blue product Staining patterns are identical to those seen with the two stains used separately. Avoids the need to compare results from separate slides and reveals aberrant staining patterns.

76. In myelomonocytic leukaemias – cells staining with both esterases may be present. In MDS and AML with dysplastic granulocytes, double staining of individual cells Non- clonal dysplastic states such as megaloblastic anaemia

77. Acid phosphatase ( with tartrate resistance) Principle: The acid phosphatase hydrolyzes naphthol AS‑BI phosphoric acid. The hydrolyzed substrate + dye - colored complex is insoluble, it precipitates out at the site of enzyme activity. Tartaric acid when added to the incubation mixture, will not inhibit the enzyme fraction found in hairy cell leukemia .  

78. ACID PHOSPHATASE Enzymes which hyrolysze phosphate esters at acid Ph Seven isoenzymes (0,1,2,3,3b,4 and 5) All haematopoetic cells Most intense - macrophages and osteoclasts Moderate staining - plasma cells, megakaryocytes , and monocytes . Weak reactions- neutrophils , bands, metamyelocytes , myelocytes , and promyelocytes . Main diagnostic use - diagnosis of T-cell ALL and hairy cell leukaemia

79. ISOENZYMES POSITIVE CELLS 2 AND 4 NEUTROPHILS AND PROSTATE EPITHELIAL CELLS 4 MONOCYTES 3 3b LYMPHOCYTES AND PLATELETS PRIMITIVE BLASTS 5 HCL EPITHELIOID CELLS GAUCHER CELLS OSTEOCLASTS

80. Fixative- Formalin vapour for 10 mins Bromathine fast quarnet GBC salt Substrate- 10mg Naphthol AS-BI Counter stain- Haematoxylin Tartaric acid Working solution A- 40ml stock substrate+ 20 mg Bromathine fast quartnet GBC salt. B- 266 mg crystalline tartaric acid+40 ml stock solution A

81. Air dry for several hours Fix in formalin vapour Incubate in working solutions A and B FOR 1 HOUR Rinse in tap water, air dry Counter stain with haematoxylin Rinse in tap water and mount slide QC control- high neutrophil count

82.  Increased acid phosphatase activity - lymphocytes from patients with macroglobulinemia , atypical lymphocytes from infectious mononucleosis(<40 granules) Lymphoblasts from patients with T-cell leukemia .( golgi region) Tartarate -resistant acid phosphatase reaction diffusely prominent in the cytoplasm of neoplastic cells is highly characteristic of hairy-cell leukemia .

83. Not all Hairy Cells are Tartrate resistant and not all other cells are Tartrate sensitive  a. Infectious mononucleosis has increased acid phosphatase activity with some of the atypical lymphs Tartrate resistant.  b. Gaucher cells are acid phosphatase positive, Tartrate resistant. c. Sezary cells are acid phosphatase positive, Tartrate resistant. d. Occasionally, some CLL clones are Tartrate resistant.

84. REACTION The naphthol --ASBI phosphoric acid-fast garnet GBC method is sensitive, cytochemical demonstration of acid phosphatase and TRAP activity in cytologic preparations. The naphthol --ASBI phosphoric acid-- pararosaniline method is highly specific - histochemical demonstration of acid phosphatase TRAP in tissue sections. The reaction product is red with a mixture of granular and diffuse positivity The test is considered positive when two or more cells are found with 4+ activity.

85. Positive staining - disease Osteoclastic giant cells in tumors Acute basophilic leukemia ,   T-ALL  (focal paranuclear ),  T cell CLL AML-M6b ,  T cell large granular lymphocytic leukemia ,  T cell prolymphocytic leukemia

86. Toluidine blue Basic thiazine metachromatic dye Metachromatic property Red violet – positive It is a member of the thiazine group and is partially soluble in both water and alcohol.

87. Toluidine blue Enumeration of basophils and mast cells . It binds strongly to the granules in these cells Particularly useful in pathological states in which the cells may not be easily identifiable on Romanowsky stains. In AML and in CML and other myeloproliferative neoplasms , basophils may be dysplastic and poorly granular, as may the mast cells in systemic mastocytosis .

88. Nuclei stain blue and cells with abundant RNA may show a blue tint to the cytoplasm. Although toluidine blue is said to be specific for these granules, with >10 min incubation, the primary granules of promyelocytes are stained red/purple. However, these are smaller and finer than the mast cell or basophil granules and easily distinguished

89. Acute basophilic leukemia CML in blast crisis Acute mast cell leukemia

90. Leukocyte Alkaline phosphatase (LAP): Purpose: Distinguishing the cells of leukemoid reactions with increase activity from these of ( CML) with decreased activity. Secondary granules of neutrophils . The substrate naphthol AS-BI phosphate is hydrolyzed by the enzyme at an alkaline pH. This hydrolyzed substrate in combination with a dye such as fast garnet produces a colored precipitate at site of the enzyme activity

91. Leukocyte Alkaline phosphatase (LAP): Mature neutrophils , but not eosinophils , have alkaline phosphatase in specific cytoplasmic organelles - secretory vesicles or phosphosomes . The cytochemical reaction - within 8 hours of obtaining the blood specimen Films can be fixed and stored, in the dark, at room temperature. EDTA)- anticoagulated blood is not ideal as enzyme activity is inhibited Capillary blood preferred

92. The films should be made within 10–20 minutes of obtaining the blood, but even then there is some loss of activity N,N- dimethylformamide may dissolve some types of plastic; therefore a glass tube should be used to dissolve the substrate. . Once spread, the blood film should be stained within 6 h.

93. CONTROLS Low, normal and high controls should be stained in parallel with the patient’s sample. A low control - patient with chronic granulocytic leukaemia (CGL), or can be prepared by immersing an appropriately fixed film of normal blood in boiling water for 1 minute. A high control - be obtained from a patient with infection or from a pregnant or postpartum woman or from a woman taking oral contraceptives.

94. Fixative- con formalin methanol Fix and refrigerate for 90 seconds Wash with distilled water and air dry Fix the substrate solution(10mg naphthol acid phosphate+10mg fast garnet+10ml working propanediol solution) onto the slides Allow to stand for 15 min at 20 degrees Rinse and wash with distilled water Counter stain with nuclear fast red

95. LAP Intracellular metabolic activity Ruby red/ blue purple colour 100 consecutive neutrophils Colourless 1Diffuse positivity- occasional granules 2 Diffuse positive moderate amouns of granules 3 Strong- numerous granules 4 Very strong- dark confluent granules

96. The reaction is scored from 0 to 4 depending on the number of stained granules and the intensity of the stain. The number of cells is multiplied by the score and added up with a normal range being from 40 to 100

97. Leukocyte Alkaline phosphatase (LAP) Positive LAP reaction Negative LAP reaction

98. LAP decreased in: LAP elevated in: CML. Leukemoid reaction. Paroxymal Nocturnal Hemoglobinuria. Pregnancy Sickle cell anemia. Polycythemia vera. Hypophosphatasia. Aplastic anemia. Multiuple myeloma Obstructive juindice. Hodgkins` disease. Myelofibrosis Blast crisis-CML

99. Normal- 20-100 CML <13 Leukemoid reaction>100 Polycythemia Vera- 100- 200 Secondary polycythemia : 10- 100 Cortisol and stress Osteoblasts and endothelial cells

100. Stimulated by granulocyte colony-stimulating factor (G-CSF) Inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL3) and interferon. Neonates - very high NAP scores, usually exceeding 200. A fall to levels more typical of childhood occurs between 5 and 10 months of age Premature and low birth-weight babies - lower scores than full-term babies. Children have higher NAP scores than adults with a gradual fall to adult levels occurring before puberty

101. Patients with CGL - normal or elevated NAP during pregnancy, postoperatively (particularly following splenectomy ), during bacterial infection, blast crisis, when the bone marrow is rendered hypoplastic by chemotherapy, and following the onset of transformation.. In multiple myeloma, the increased NAP score correlates with disease activity.

102. TdT TdT catalyses the addition of dinucleoside triphosphate onto the free end of ssDNA without a template Not in normal, mature lymphocytes but is present in 65% of the total thymic population of lymphocytes 1-3% of all normal BM cells are POS T-ALL are TdT POS but B-ALL are NEG Minority of patients with acute nonlymphocytic leukemia (ANLL) Red to brown staining/lime green flouresence

103. N-acetyl-,B-glucosaminidase - lysosomal enzyme that hydrolyses ,B- glycosidic bonds and releases N-acetyl-,B-glucosamine. Naphthol AS- Bl -N-acetyl-,B- glycosaminide - substrate Fast garnet GBe - coupler Monocytes and Neutrophils

104. B- Glucuronidase - lysosomal enzyme. Naphthol -AS-BI-B-D glucuronide as substrate and hexazonium pararosanilin as coupler Granulocytes,T lymphocytes More sensitive than the PAS reaction in lymphoblastic leukaemia. B- glucuronidase activity is low in ,B-cell chronic lymphatic leukaemia and high in the T-cell variety. There is a close correlation between acid phosphatase and , Bglucuronidase activity.

105. Oil red 0, a diazo dye, selectively stain neutral or simple fats - lipid substance, showing up as round orange-pink areas. Both lymphoid precursors and histiocytes exhibit positivity. Positive in ALL L3/ Burkitt’s lymphoma

106. Atlas of Hematology  By Shauna Christine Anderson, Keila Poulsen Dacie and Lewis Practical Haematology Barbara Bain The diagnosis of leukemia Cytochemical Markers of Differentiation in Acute Leukemia1D aniel Catovsky, Luigi de Salvo Cardullo, Maureen O'Brien, Ricardo Morilla, Christine Costello, David Galton, Kanagabasai Ganeshaguru , and Victor Hoffbrand Standard operating protocol- Dept of clinical pathology, CMC Vellore

107. THANK YOU