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![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 32.1M; 37.4W 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 28.1M; 22.5W 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)](https://image.slidesharecdn.com/bonemarrow-4-180106181202/75/Bone-marrow-biopsy-and-interpretation-55-2048.jpg)
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Bone marrow biopsy and interpretation
- 1. BONE MARROW BIOPSYAND ASPIRATION MODERATOR: Dr. C.J. PRAKASH PRESENTER: Dr. SPOORTHY
- 2. INTRODUCTION Bone Marrow Examinationprovides 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 bxin an infant with Leishmaniasis. 1909 Pianese Tibia & femur marrow aspiration with attached syringe. 1923 Seyfaith Surgical trephine to obtain marrow from ribs & sternum.But there was excessive bleeding. 1927 Airinkin 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: Stronglayer of Compact bone Gives bone strength Made of Lamellar bone contains well organised Haversian canals Cortex Medulla
- 5. MEDULLA Made up ofCancellous bone or Trabeculae Trabeculae and inner surface of cortex are lined by Endosteal cells Osteoblasts Osteocytes Osteoclasts
- 7. PARENCHYMA: Haematopoiteic stem cellsand Precursors Mature cells of Erythroid, Myeloid and Megakaryocytic cells STROMA: Fat cells, Histiocytes,Fibroblasts,Blood vessels, Intercellular Matrix
- 8. BONE MARROW Normal BoneMarrow 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 Red Marrow Yellow Marrow
- 10. Children: most bonescontain Haematopoietic cells Adults- 1. Skull 2. Sternum 3. Scapulae 4. Ribs 5. Pelvic Bones 6. Proximal ends of long bones
- 11. PRINCIPLE The Morphological assessmentof aspirated or core biopsy specimens is based on these principles 1. Bone Marrow has an organised structure 2. In normal health bone marrow cells display distinct numerical and spatial relationships with each other 3. Individual marrow cells have distinct cytological appearances 4. This reflects the lineage and stage of maturation 5. Each or all of these may be specifically disordered in a disease
- 13. INDICATIONS ANEMIA 1. MICROCYTIC ANEMIA: Evaluationof 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
- 14. DIAGNOSIS AND STAGINGOF: Non hodgkin’s lymphoma Hodgkin’s lymphoma Malignancy Metastatic carcinoma Small round cell tumors of childhood STROMAL CHANGES: Fibrosis Necrosis Gelatinous marrow transformation
- 15. • To DIAGNOSE AplasticAnaemia Hypoplastic Myelodysplastic syndrome. Hypoplastic Leukemia. • LYMPHOPROLIFERATIVE DISORDERS Hairy cell leukemia CLL • MYELOPROLIFERATIVE DISORDERS
- 16. NEUTROPENIA, THROMBOCYTOPENIA,PANCYTOPENIA: To assess thepresence 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
- 17. • Unexplained leukoerythroblasticpicture. • 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
- 18. CONTRAINDICATIONS BIOPSY IN COAGULOPATHIES (Foraspiration : factor replacement therapy prior to procedure and observation should be done for next 24-48 hrs.) STERNAL ASPIRATE - OSTEOPOROSIS AND CHILDREN
- 19. SITES 1. Sternum 2. AnteriorIliac 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
- 21. STERNUM Usual sites: 1. Manubrium 2.1st 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
- 22. Klima Sternal NeedleSalah Bone Marrow Aspiration Watherfield Iliac Crest Bone Aspiration Modern Jamshidi Needle
- 25. PROCEDURE Consent: A writtenconsent 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.
- 26. It is usefulto 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.
- 28. COMPLICATIONS Haemorrhage Pain Infection Perforation of majorvessels Risk of general anaesthesia and sedation.
- 29. ASPIRATION BIOPSY Better cytologicaldetail 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
- 30. ASPIRATE Smears should bemade 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
- 31. • Appropriate amountsof 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
- 32. CENTRIFUGATION Centrifugation can beused 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
- 33. Marrow Stim Device PLASMA Nucleatedcells RBC
- 34. DIRECT FILMS A dropof 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
- 37. CRUSH PREPARATIONS Marrow particlesin 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
- 38. IMPRINTS Marrow particles canalso 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
- 40. ADEQUACY OF BIOPSY Length-1.6cm( 1.5- 2.5cm) 25% shrinkage during processing 5-6 Trabecular spaces Good quality Staining
- 44.
- 45. STAINING OF SECTIONS Bonemarrow 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
- 46. Other stains thatare usually done are: 1. PERL’S STAIN - IRON 2. SILVER IMPREGNATION METHOD - RETICULIN Both Plastic and Paraffin embedded specimens can be used for IHC
- 47.
- 48. TB- NORMAL BONESECTION
- 49. CELLULARITY Expressed as aratio 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
- 50. Cellularity varies withthe 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
- 51. NORMAL CELLULARITY Hyper cellularHypo cellular
- 52. SYSTEMATIC SCHEME FOREXAMINATION 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
- 54. HIGHER POWER: Identify allstages 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
- 55. 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 32.1M; 37.4W 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 28.1M; 22.5W 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)
- 56. MYELOID CELLS MEGAKARYOCYTES ERYTHROIDCELLS STROMA Para Trabecular Mature cells Centre around Sinusoids Centre in Colonies Fat cells Reticulin Fibres Fibroblasts Macrophages
- 57.
- 58.
- 59.
- 60.
- 61.
- 62.
- 63. MATURATION SEQUENCE OFMEGAKARYOCYTES
- 64.
- 65.
- 67. OSTEOBLASTS 1. Extruding nuclei 2.1-4 Nucleoli 3. Regular Chromatin 4. Position of Golgi Zone
- 68. OSTEOCLASTS 1. Highly GranularCytoplasm 2 . Multiple Nuclei (2-100) 3. Single Nucleoli
- 69.
- 70.
- 71. ASSESSMENT OF IRONSTORES
- 72. PERL’S STAIN Also calledPrussian blue stain Demonstrates Haemosiderin in Bone marrow Macrophages and Erythroblasts Hence it allows the assessment of Iron Reticulo-Endothelial cells Developing Erythroblasts
- 73. REQUIREMENTS Assessment of storageiron 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
- 74. ASSESSMENT Iron stores maybe 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
- 75. GRADING OF IRONSTORES 0 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
- 76. NORMAL IRON STORES NOSTAINABLE IRON
- 77.
- 78.
- 79. RETUCULIN STAIN Histological sectionscan 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
- 80. BAUERMEISTER GRADING 0 No ReticulinFibres 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
- 81. Many normal subjectsmay 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
- 82.
- 83.
- 84. OTHER STAINS USED Chloroacetateesterase 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
- 85.
- 86. PARVO B19 INFECTION GiantPro Erythroblasts
- 87.
- 88. HIV- CHANGES Increased Cellularity Increasedfibrosis Lymphoid aggregates
- 89.
- 90. TB- GRANULOMA ACID FASTBACILLI
- 91.
- 92. SUPPLEMENTARY INVESTIGATIONS 1. Immunohistochemistry- fordemonstration 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
- 93. REFERENCES 1. Barbara Bain-BoneMarrow 2. Dacie and Lewis 3. Wintrobes 4.Mckenzie 5.Henry Laboratory Methods
- 94.