bone marrow ppt.pptx

bone marrow

1. PA 17.2 BONE MARROW ASPIRATION AND BIOPSY

2. INTRODUCTION Bone Marrow Examination provides a Semi-Quantitative and Qualitative assessment of the state of Haemopoiesis Biopsy of Bone Marrow is an indispensable adjunct to the study of diseases of blood It may be the only way a correct diagnosis can be made

3. HISTORY 1905 Pianese Trephine bx in an infant with Leishmaniasis. 1909 P i anese Tibia & femur marrow aspiration with attached syringe. 1923 Seyfaith Surgical trephine to obtain marrow from ribs & sternum.But there was excessive bleeding. 1927 A i rinkin Eliminated trephine complication by using short lumbar needle. 1945 Vandenberg First obtained marrow from iliac crest. Used posterior Iliac

4. STRUCTURE OF BONE Bone Cortex Medulla Cortex: Strong layer of Compact bone Gives bone strength Made of Lamellar bone contains well organised Haversian canals

5. MEDULLA Made up of Cancellous bone or Trabeculae Trabeculae and inner surface of cortex are lined by Endosteal cells Osteoblasts Osteocytes Osteoclasts

6. PARENCHYMA: Haematopoiteic stem cells and Precursors Mature cells of Erythroid, Myeloid and Megakaryocytic cells STROMA: Fat cells, Histiocytes,Fibroblasts,Blood vessels, Intercellular Matrix

7. BONE MARROW Normal Bone Marrow Red Marrow Yellow Marrow Red Marrow: Haematopoietic 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

8. Children: most bones contain Haematopoietic cells Adults- 1. Skull Sternum Scapulae Ribs Pelvic Bones Proximal ends of long bones

9. PRINCIPLE The Morphological assessment of aspirated or core biopsy specimens is based on these principles Bone Marrow has an organised structure In normal health bone marrow cells display distinct numerical and spatial relationships with each other Individual marrow cells have distinct cytological appearances This reflects the lineage and stage of maturation Each or all of these may be specifically disordered in a disease

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

11. 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

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

13. NEUTROPENIA, TH R OMBO C YTOPENIA,PANCYT O PENIA: 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

14. 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

15. CONTRAINDICATIONS  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

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

17. STER N UM Usual sites: Manubrium 1st and 2nd parts of Body of Sternum Precaution: Appropriate Guard is supposed to be used Danger of perforating the Inner cortical layer and Damaging the underlying large blood vessels Right atrium

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

19. PROCEDURE Consent: A written consent should be taken from patient. An appropriate clinical history should accompany the bone marrow, as they relate to possible findings within the bone marrow examination.

20.  It is useful to know relevant laboratory data such as Iron studies, Folate or Vitamin B-12 studies, transfusion therapy, or history of chemotherapy.   The physician’s clinical impression should be included on the form.  Lignocaine sensitivity test should be done.

21. COMPLICATIONS  Haemorrhage   Pain  Infection  Perforation of major vessels  Risk of general anaesthesia and sedation.

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

23. 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

24. 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

25. CENTRIFUGATION Centrifugation can be used to concentrate the Marrow cells To assess the relative proportion of Marrow cells, Peripheral blood and Fat in Aspirated material Useful : 1. Poorly cellular sample 2. Abnormal cells are present in small numbers

26. DIRE C T F ILMS 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

27. CRUSH 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

28. IMPRINTS 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

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

30. INTERPRETATION

31. STAINING 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

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

33. Periosteal Connective Tissue Bony Trabecu l ae

34. TB- NORMAL BONE SECTION

35. CELLULARITY 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

36. NORMAL CELLULARITY Hyper cellular Hypo cellular

37. 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

38. 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

39. 95% RANGE MEAN [ 12 ] MEAN [ 11 ] Myeloblasts 0– 3 1. 4 0. 4 7. 8 13.7 [ * ] 7. 6 – P ro m y e l oc y t e s Myelocytes (N) Me t a m y e l oc y t e s 3–12 2–13 2–6 4. 1 – 32.1 M ; 37.4 W 35. 5 1. 3 1. 6 2. 2 1. 7 0. 1 0. 2 13. 1 16. 1 1. 3 2. 5 Neutrophils Myelocytes (E) Eosinophils Basophils L y m phoc y t e s Monocytes Plasma cells 22–46 0–3 0.3–4 0–0.5 5–20 0–3 0–3.5 0. 6 1. 9 28.1 M ; 22.5 W 23. 5 0. 5 Erythroblasts [ † ] Megakaryocytes Macrophages 5–35 0–2 0–2 0. 4 2. Normal ranges for differential counts on aspirated bone marrow (500 cells should be counted)

40. ERYTHROID CELLS MYELOID CELLS Para Trabecular Mature cells MEGAKA R YOCYTES Centre around Sinusoids Centre in Colonies STROMA Fat cells Reticulin Fibres Fibroblasts Macrophages

41. ASPIRATION- ERYTHROID ISLAND

42. H&E- ERYTHROID ISLAND

43. Maturation sequence of RBC

44. Maturation sequence- Neutrophils

45. Myelocyte Pro M y elo c yte Myeloblast

46. EOSINOPHILIC PRECURSORS

47. MATURATION SEQUENCE OF MEGAKARYOCYTES

48. ME G A K AR Y OC Y T E

49. BUDDING MEGAKARYOCYTES

50. OSTEOB L ASTS Extruding nuclei 1-4 Nucleoli Regular Chromatin Position of Golgi Zone

51. OSTEO C LASTS 1. Highly Granular Cytoplasm 2 . Multiple Nuclei (2-100) 3. Single Nucleoli

52. PLASMA CELLS

53. BONE MARROW-NECROSIS

54. ASSESSMENT OF IRON STORES

55. PERL’S STAIN Also called Prussian blue stain Demonstrates Haemosiderin in Bone marrow Macrophages and Erythroblasts Reticulo-Endothelial cells Hence it allows the assessment of Iron Developing Erythroblasts

56. REQUIREMENTS 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

57. ASSESSMENT 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

58. 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

59. NORMAL IRON STORES NO STAINABLE IRON

60. GEIMSA STAIN PERL’S STAIN

61. ASSESSMENT OF RETICULIN

62. 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

63. 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 Diffuse fibre network with 3 scattered thick coarse fibres but no mature collagen Diffuse often coarse fibre 4 network with areas of collagenization

64. 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

65. GRADE-1 GRADE-2

66. GRADE-3 GRADE-4

67. 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

68. BONE MARROW- INFECTIONS

69. PARVO B19 INFECTION Giant Pro Erythroblasts

70. HIV- Histoplasma HIV-Cryptoccocus

71. HIV- CHANGES Increased Cellularity Increased fibrosis Lymphoid aggregates

72. LEISHMANIASIS

73. TB- GRANULOMA ACID FAST BACILLI

74. GAUCHERS DISEASE Tissue like crumpled cytoplasm

75. IRON DEFICIENCY ANEMIA

76. MEGALOBLASTIC ANEMIA BONE MARROW SMEAR OF MEGALOBLASTIC ANEMIA

77. AML BBBOMM TREPHINE BIOPSY BM ASPIRATION SMEAR ACUTE MYELOID LEUKEMIA

78. MULTIPLE MYELOMA

79. THANK YOU