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1. Cell Identification in b one marrow

2. Normal b one marrow Normal Bone Marrow Red Marrow: Hematopoietic cells Yellow Marrow : Adipose tissue Red bone marrow consists of 4% of Total bone marrow The weight of total bone marrow ranges from- 1600-3700gm

3. PARENCHYMA: Hematopoietic stem cells and Precursors Mature cells of Erythroid, Myeloid and Megakaryocytic cells STROMA: Fat cells, Histiocytes , Fibroblasts, Blood vessels, Intercellular Matrix STRUCTURE OF BONE MARROW

4. Children: most bones contain Haematopoietic cells Adults- 1. Skull 2. Sternum 3. Scapulae 4. Ribs 5. Pelvic Bones 6. Proximal ends of long bones

5. INDICATIONS ANEMIA 1. MICROCYTIC ANEMIA: Evaluation of Iron stores and Sideroblasts: allows categorisation of anaemia 2. MACROCYTIC ANEMIA: To confirm whether the process is Megaloblastic or not 3. NORMOCYTIC ANEMIA: without an increase in retic count For quantitative or qualitative abnormalities of Erythropoiesis

6. DIAGNOSIS AND STAGING OF: Non hodgkin’s lymphoma Hodgkin’s lymphoma Malignancy Metastatic carcinoma Small round cell tumors of childhood STROMAL CHANGES: Fibrosis Necrosis Gelatinous marrow transformation

7. To DIAGNOSE Aplastic Anaemia Hypoplastic Myelodysplastic syndrome. Hypoplastic Leukemia. LYMPHOPROLIFERATIVE DISORDERS Hairy cell leukemia CLL MYELOPROLIFERATIVE DISORDERS

8. NEUTROPENIA, THROMBOCYTOPENIA,PANCYTOPENIA: To assess the presence and normality of precursor cells To assess the probability of decreased production, impaired maturation or increased destruction CYTOPENIA To reveal the presence of leukaemia or another Haematological neoplasia

9. Unexplained leukoerythroblastic picture. In suspected cases of multiple myeloma and serum paraproteins. Pyrexia of unknown origin Focal lesions –Metastasis, Granuloma Amyloidosis Metabolic bone diseases To assess the mineralisation front and appositional growth after tetracycline labelling

10. C ontraindications BIOPSY IN COAGULOPATHIES (For aspiration : factor replacement therapy prior to procedure and observation should be done for next 24-48 hrs.) STERNAL ASPIRATE - OSTEOPOROSIS AND CHILDREN

11. SITES 1. Sternum 2. Anterior Iliac spine 3. Posterior Iliac spine: 1.overlies a large marrow space 2. Larger samples can be obtained 4.Upper end of Tibia- Children < 1year old

12. Klima Sternal Needle Salah Bone Marrow Aspiration Watherfield Iliac Crest Bone Aspiration Modern Jamshidi Needle

13. ASPIRATION BIOPSY Better cytological detail Topographical details, cellularity and infiltration More range for Cytochemical stains, Flow cytometry. IHC Less Range Ideal for Cytogenics and Molecular Genetics Can be used for both Dry tap in fibrosis Essential for diagnosis in Dry tap Less painful More painful

14. ASPIRATE Smears should be made without delay at the bedside Remaining material should be delivered into a bottle containing EDTA Preservative free Heparin should be used if Immunophenotyping or Cytogenetic studies are needed. Some material can be fixed in Fixative rather than anticoagulant for preparing histological sections Some films should be fixed in Absolute Methanol for subsequent staining by Romanowsky Method or Perl’s stain

15. Appropriate amounts of anticoagulant for the volume of marrow to be anticoagulated are used Gross excess of anticoagulant: masses of pink-staining amorphous material may be seen Clumping of some erythroblasts and reticulocytes may be seen

16. Marrow Stim Device PLASMA Nucleated cells RBC

17. D irect films A drop of marrow is placed on a slide a short distance away from one end A film 3-5cm is made with a spreader, not wider than 2cm Dragging the particles behind them but not squashing them. A trail of cells is left behind each particle

18. C rush preparations Marrow particles in a small drop of aspirate is placed on a slide at one end Another slide is placed on the first Slight pressure is exerted to crush the particles and slides are separated by pulling them apart in a direction parallel to their surfaces

19. I mprints Marrow particles can also be used for preparation of imprints One or more particles are picked up capillary pipet Transferred immediately to a slide and made to stick to it by a gentle smearing motion. The slide is air dried rapidly by waving, then it is stained

20. ADEQUACY OF BIOPSY Length-1.6cm ( 1.5- 2.5cm) 25% shrinkage during processing 5-6 Trabecular spaces Good quality Staining

21. INTERPRETATION

22. S taining of sections Bone marrow sections are routinely stained with Haematoxylin and Eosin It is excellent for demonstrating the cellularity and pattern of the Marrow. This reveals the pathological changes such as presence of Granuloma or Carcinoma cells Haematopoietic cells may be more easily identified in a Romanowsky stained preparation

23. Other stains that are usually done are: 1. PERL’S STAIN - IRON 2. SILVER IMPREGNATION METHOD - RETICULIN Both Plastic and Paraffin embedded specimens can be used for IHC

24. Periosteal Connective Tissue Bony Trabeculae

25. TB- NORMAL BONE SECTION

26. C ellularity Expressed as a ratio of volume of Hematopoietic cells to the total volume of marrow spaces It is best judged by Histological sections of biopsy or aspirated particles Can also be estimated from the particles present in the Marrow films Done my comparing the areas occupied by Fat spaces and By Nucleated cells Also by the density of Nucleated cells in the Tail or fallout of particles

27. Cellularity varies with the age of the subject and the site 50 years of age : Vertebrae : 75% Sternum : 60% Iliac crest : 50% Rib : 30% If the percentage is increased for patients age: Hypercellular If the percentage is decreased for patients age : Hypocellular

28. NORMAL CELLULARITY Hyper cellular Hypo cellular

29. SYSTEMATIC SCHEME FOR EXAMINATION OF BONE MARROW ASPIRATE LOW POWER: Determine Cellularity Identify Megakaryocytes Note Morphology and Maturation Sequence Look for clumps of abnormal cells: METASTATIC TUMOUR Identify Macrophages

30. HIGHER POWER: Identify all stages of maturation of Myeloid and Erythroid cells Determine the Myeloid:Erythroid ratio Perform Differential count: Erythroid, Myeloid,Lymphoid,Plasma cells and Others Look for areas of bone marrow Necrosis Assess the Iron content of the Macrophages Look for Iron granules in Erythroid cells: Perl’s stain

31.   95% Range Mean [ 12 ] Mean [ 11 ] Myeloblasts 0–3 1.4 0.4 Promyelocytes 3–12 7.8 13.7 [ * ] Myelocytes (N) 2–13 7.6 – Metamyelocytes 2–6 4.1 – Neutrophils 22–46 32.1 M ; 37.4 W 35.5 Myelocytes (E) 0–3 1.3 1.6 Eosinophils 0.3–4 2.2 1.7 Basophils 0–0.5 0.1 0.2 Lymphocytes 5–20 13.1 16.1 Monocytes 0–3 1.3 2.5 Plasma cells 0–3.5 0.6 1.9 Erythroblasts [ † ] 5–35 28.1 M ; 22.5 W 23.5 Megakaryocytes 0–2 0.5   Macrophages 0–2 0.4 2.0 Normal ranges for differential counts on aspirated bone marrow (500 cells should be counted)

32. ASPIRATION- ERYTHROID ISLAND

33. H&E- ERYTHROID ISLAND

34. Maturation sequence- Neutrophils

35. Myelocyte Pro Myelocyte Myeloblast

36. EOSINOPHILIC PRECURSORS

37. MATURATION SEQUENCE OF MEGAKARYOCYTES

38. MEGAKARYOCYTE

39. BUDDING MEGAKARYOCYTES

40. OSTEOBLASTS 1. Extruding nuclei 2. 1-4 Nucleoli 3. Regular Chromatin 4. Position of Golgi Zone

41. OSTEOCLASTS 1. Highly Granular Cytoplasm 2 . Multiple Nuclei (2-100) 3. Single Nucleoli

42. PLASMA CELLS

43. BONE MARROW-NECROSIS

44. ASSESSMENT OF IRON STORES

45. PERL’S STAIN Also called Prussian blue stain Demonstrates Haemosiderin in Bone marrow Macrophages and Erythroblasts Hence it allows the assessment of Iron

46. R equirements Assessment of storage iron requires that an adequate number of fragments are obtained A Minimum of 7 Bone marrow fragments in one or more bone marrow films are needed to be examined. To state that the bone marrow Iron is reasonably absent A Bone marrow film or Crush preparation will contain both Intracellular and Extra cellular Iron It is basic to count only Intra cellular iron

47. A ssessmen t Iron stores may be assessed as: NORMAL, DECREASED, INCREASED May be graded as +1 - +6 Where +1 - +3 is regarded as Normal A Proportion of Normal Erythroblasts have few(1-5) fine iron containing granules randomly distributed in the cytoplasm These are called SIDEROBLASTS

48. GRADING OF IRON STORES NO STAINABLE IRON 1+ SMALL IRON PARTICLES JUST VISIBLE IN RETICULUM CELLS UNDER OIL IMMERSION 2+ SMALL IRON PARTICLES VISIBLE IN RETICULUM CELLS UNDER LOW POWER 3+ NUMEROUS SMALL PARTICLES IN RETICULUM CELLS 4+ LARGER PARTICLES WITH A TENDENCY TO AGGREGATE INTO CLUMPS 5+ DENSE LARGER CLUMPS 6+ VERY LARGE CLUMPS AND EXTRA CELLULAR IRON

49. NORMAL IRON STORES NO STAINABLE IRON

50. GEIMSA STAIN PERL’S STAIN

51. As es s ment of reticulin

52. RETUCULIN STAIN Histological sections can be stained by Silver Impregnation Method for Reticulin For Collagen- Trichrome stain Reticulin is closely concentrated more around the blood vessels and bony trabeculae Hence these areas should be disregarded while grading

53. bauermeister grading No Reticulin Fibres Demonstrable 1 Occasional fine Individual fibres and foci of a fine fibre network 2 Fine fibre network throughout. No coarse fibres 3 Diffuse fibre network with scattered thick coarse fibres but no mature collagen 4 Diffuse often coarse fibre network with areas of collagenization

54. Many normal subjects may have Reticulin grade 0- +1 Some may even have a grade of +2 There is a tendency of reticulin to be deposited more in Iliac crest or than in the Sternum

55. GRADE-1 GRADE-2

56. GRADE-3 GRADE-4

57. OTHER STAINS USED Chloroacetate esterase Identification of Granulocyte differentiation and Mast cells PAS Staining of complex carbohydrates, identification of fungi Toulidine blue Identification of Mast cells Congo Red Identification of Amyloid Z-N stain Identification of Mycobacteria

58. BONE MARROW- INFECTIONS

59. PARVO B19 INFECTION Giant Pro Erythroblasts

60. HIV- Histoplasma HIV-Cryptoccocus

61. HIV- CHANGES Increased Cellularity Increased fibrosis Lymphoid aggregates

62. LEISHMANIASIS

63. TB- GRANULOMA ACID FAST BACILLI

64. GAUCHERS DISEASE Tissue like crumpled cytoplasm

65. SUPPLEMENTARY INVESTIGATIONS 1. Immunohistochemistry- for demonstration of antigens in biopsy Ex.- CD 34, CD 45, Lysozyme, MPO, CD 68, CEA etc. 2. Cytogenetic analysis- for chromosomal rearrangements 3. Molecular genetics- by PCR, RTPCR 4. FISH

66. R eferences 1. Barbara Bain-Bone Marrow Pathology 2. Dacie and Lewis – Practical hematology 3. Wintrobe ’ s hematology 4.Mckenzie 5.Henry Laboratory Methods