BONE MARROW INTERPRETATION Made by Dr Mousmi Agrawal

BONE MARROW INTERPRETATION Made by – Dr. Mousmi Agrawal Moderator – Dr. Patil Mam

INTRODUCTION § Bone marrow is the site of hematopoiesis. § Consists of - hematopoietic cells, vascular sinusoids, fibroblasts, fat cells, and macrophages. § No lymphatic channels. § Two types of marrow: red and yellow. 1. Red marrow - active hematopoietic tissue and red coloration is due to developing red cells. 2. Yellow (inactive) marrow - fatty tissue. § The average volume (red and yellow) in an adult is : 3000 -4000 ml. § Red or active marrow constitutes : 1500 ml.

ERYTHROID SERIES 1. PROERYTHROBLAST OR PRONORMOBLAST – large cell, 15 -20 μm in diameter having deeply basophilic cytoplasm and a large central nucleus containing nucleoli. 2. BASOPHILIC (EARLY) ERYTHROBLAST – round cell , 12 -16 μm , large nucleus , more condensed and basophilic cytoplasm. 3. POLYCHROMATIC(INTERMEDIATE) ERYTHROBLAST – diameter 12 -14 μm, nucleus is coarse and deeply basophilic. The cytoplasm is characteristically polychromatic i. e. contains admixture of basophilic RNA and acidophilic haemoglobin. 4. ORTHOCHROMATIC (LATE) ERYTHROBLAST - smaller, 8 -12 μm in diameter, containing a small and pyknotic nucleus with dark nuclear chromatin. 5. RETICULOCYTE - juvenile red cells devoid of nuclei but contain ribosomal RNA so that they are still able to synthesise haemoglobin.

Normal range : • Adults - 0. 5 -2. 5% • Infants - 2 -6% Increased in - haemorrhage, haemolysis and haematopoietic response of anaemia to treatment 6. RED BLOOD CELLS - non-nucleated cells, biconcave disc, 7. 2 μm in diameter, and has a thickness of 2. 4 μm at the periphery and 1 μm in the centre. The lifespan of red cells is 120 +/- 30 days.

MYELOID SERIES 1. MYELOBLAST - 10 -18 μm in diameter, large round to oval nucleus, fine nuclear chromatin, 2 -5 nucleoli. The thin rim of cytoplasm is deeply basophilic and usually devoid of granules. 2. PROMYELOCYTE - 12 -18 μm diameter, round to oval nucleus, fine nuclear chromatin, nucleoli present, cytoplasm contains azurophilic granules. 3. MYELOCYTE - granules present in cytoplasm, nucleus is eccentric, round to oval, coarse nuclear chromatin and no visible nucleoli. 4. METAMYELOCYTE - 10 -18 μm in diameter, indented or horseshoe-shaped nucleus without nucleoli, nuclear chromatin is dense and clumped, cytoplasm contains granules. 5. BAND FORMS - juvenile granulocyte, 10 - 16 μm in diameter, condensation of nuclear chromatin and transformation of nuclear shape into band configuration of uniform thickness. 6. SEGMENTED NEUTROPHILS

MONOCYTE-MACROPHAGE SERIES 1. MONOBLAST- very similar in appearance to myeloblasts except that it has ground-glass cytoplasm with irregular border and therefore, sometimes also called as myelomonoblast. 2. PROMONOCYTE - 20 μm in diameter and possesses a large indented nucleus containing a nucleolus, cytoplasm is basophilic. 3. MONOCYTE - largest mature leucocyte in the peripheral blood measuring 12 -20 μm in diameter. It possesses a large, central notched or indented nucleus which has characteristically fine reticulated chromatin network.

LYMPHOID SERIES 1. LYMPHOBLAST - large cell, 10 -18 μm in diameter, large round to oval nucleus, clumped or stippled nuclear chromatin, few nucleoli. 2. PROLYMPHOCYTE- 9 -18 μm in diameter, round to indented nucleus , stippled or coarse chromatin and may have 0 -1 nucleoli. 3. LYMPHOCYTE – small lymphocytes (9 -12 μm in diameter) and large lymphocytes (12 -16 μm in diameter). Both have round or slightly indented nucleus with coarselyclumped chromatin and scanty basophilic cytoplasm.

THROMBOPOIESIS 1. MEGAKARYOBLAST - The earliest precursor of platelets in the bone marrow is megakaryoblast. 2. PROMEGAKARYOCYTE - A megakaryoblast undergoes endo-reduplication of nuclear chromatin. 3. MEGAKARYOCYTE – large cell, 30 -90 μm in diameter, and contains 4 -16 nuclear lobes, coarsely clumped chromatin, cytoplasm is abundant, light blue in colour and contains purple granules. Each megakaryocyte may form up to 4000 platelets. 4. PLATELETS - formed from pseudopods of megakaryocyte cytoplasm, small (1 -4 μm in diameter) and discoid shape.

NORMAL DISTRIBUTION OF CELLS IN BONE MARROW • The hematopoietic cells are present as cords between vascular sinusoids and are supported by a framework of branching processes of fibroblasts and reticulin fibers. • Erythroid precursors are present as clusters (islands) and are closely associated with centrally placed sinusoids. • An erythroid island consists of a centrally placed macrophage around which erythroid precursors are concentrically arranged. • Early granulocyte precursors are located close to the bony trabecule • Mature granulocytes are located more centrally, between adjacent trabeculae. • Megakaryocytes, the largest of the hematopoietic cells, are closely apposed to the walls of the sinusoids.

• Lymphocytes and plasma cells are mostly present around capillaries and arterial vessels. • The proportion of fat cells increases with advancing age with corresponding decrease in hematopoietic tissue. Fats cells occupy § children and adolescents -20 -30% § adults - 50% § elderly - 70%

TECHNIQUES FOR SAMPLING OF BONE MARROW: ASPIRATION ü Bone marrow fluid is obtained by a needle and syringe. ü Smears from this material are prepared on glass slides. ü Stained. ü Examined under the microscope. BONE MARROW TREPHINE BIOPSY ü Core biopsy. ü A small tissue piece of bone marrow is removed with needle. ü Processed to obtain histological sections. ü Examined.

INDICATIONS FOR BONE MARROW ASPIRATION 1. Unexplained cytopenia. 2. Suspected acute leukemia : Comparison of baseline marrow smear with follow-up marrow aspiration smears during treatment, detection of remission. 3. Suspected myeloproliferative disorders : chronic myeloid leukemia, polycythemia vera, essential thrombocythemia and myelofibrosis. 4. Suspected plasma cell dyscrasia: multiple myeloma. 5. Suspected chronic lymphoid leukemias like chronic lymphocytic leukemia, hairy cell leukemia. 6. Investigation of pyrexia of unknown origin. 7. Suspected storage disorder like Gaucher’s disease.

INDICATIONS FOR BONE MARROW BIOPSY 1. Repeated failure of aspiration (dry tap) – myelofibrosis, hairy cell leukemia. 2. Suspected aplastic anaemia. 3. Suspected focal lesions like granuloma, metastatic deposit, or infiltrate of lymphoma. 4. Suspected bone disorder, e. g. osteopetrosis.

CONTRAINDICATIONS 1. Infection at the site of aspiration. 2. Hemophilia and un - corrected coagulation disorders.

SITES FOR BONE MARROW ASPIRATION OR BIOPSY 1. Posterior iliac crest (posterior superior iliac spine) : This site has a large reservoir of marrow, is located just beneath the skin easily accessible, no large blood vessels or nerves close to this area, and as the patient’s back is towards the physician, patient’s apprehension is less. 2. Anterior superior iliac spine : obese 3. Spinous processes of lumbar vertebra. 4. Tibia: less than 1 year of age, marrow can be aspirated from the medial aspect of upper end of tibia just beneath tibial tuberosity. 5. Sternum : manubrium or the first part of the body of the sternum.

METHOD : BONE MARROW ASPIRATION 1. An informed consent should be obtained before the procedure. 2. Bone marrow aspiration needles : Salah and Klima needles. 3. For both aspiration and biopsy from iliac crest : Jamshidi needle. 4. Aspiration is associated with sharp pain (suction pain).

METHOD : BONE MARROW TREPHINE BIOPSY 1. Preparation of the patient and local anesthesia are similar to aspiration. 2. Often, bone marrow aspiration and biopsy are combined together; aspiration is carried out first followed by biopsy. 3. After aspiration, either (i) the needle is advanced a little further (1 -3 cm) into the bone, or (ii) the needle is withdrawn and reinserted through the same skin incision but placed at a different site in the bone (about 1 cm away). 4. For biopsy, the needle should be advanced through the bone rotating it clockwise 10 times. 5. The needle should be removed by anticlockwise rotation. 6. For adequate assessment, biopsy length = 1. 6 cm OR 5 -6 trabecular spaces. 7. Fixed in 10 % formalin or Helly’s fluid. 8. Dressing applied.

COMPLICATIONS OF BONE MARROW ASPIRATION AND/OR BIOPSY 1. Local infection: neutropenic patients. 2. Hemorrhage: (i) Marrow biopsy is done without adequate replacement cover in coagulation disorders (ii) Great vessels or heart is injured during sternal aspiration. 3. Cardiac tamponade or mediastinitis: if posterior sternal plate is penetrated during sternal aspiration.

PREPARATION OF SMEARS 1. Wedge-spread films - Put one drop of the aspirated material near one end of a glass slide and spread it similar to a blood film. 3– 5 cm in length and no more than 2 cm in width. 2. Squash preparation - A drop of aspirated marrow is placed in the centre of a slide. A second slide is then placed on top of the first and the fragments are crushed by rotating one slide on the other. The preparation can be considered satisfactory only when marrow particles and free marrow cells can be seen in stained films.

PROCESSING OF MARROW SPECIMENS BONE MARROW ASPIRATION : § Staining : - Romanowsky stains § On smears, marrow particles are dragged towards the tail end of the smear and after staining appear as dark blue-violet irregular granules. § Rest of the film stains even pink. § For assessment of iron storage : - Perl’s stain § Imprint smears should be prepared by gently rolling the marrow biopsy specimen on a glass slide. § In acute leukemia - cytochemical stains.

CYTOCHEMICAL STAINS CELLULAR ELEMENT STAINED COLOUR PRODUCED POSITIVE RESULT 1. Myeloperoxidase (MPO) Granules of neutrophils Brown Promyelocyte and myelocyte 2. Sudan black B Phopspholipids Black Myeloblasts 3. Periodic acid schiff Glycogen Pink to bright Pronormoblast, red lymphoblast 4. Specific esterase Cellular enzyme Bright red Promyelocyte 5. Non specific esterase Cellular enzyme Brown Monoblast 6. Toludine blue Granules Bright red / purple Basophils and mast cells

BONE MARROW TREPHINE BIOPSY : 1. The specimen should be fixed in 10% formal saline, buffered to p. H 7. 0, for 12– 48 h. 2. Dehydrating and embedding in paraffin wax. 3. Less than 4 μm thick sections are cut and stained with hematoxylin and eosin stain and reticulin. 4. It is recommended to mount 5 stepwise serial sections on one slide to increase the chance of detecting small focal lesions. 5. Giemsa stain - easier differentiation between blood and marrow cells and mast cells.

Parameter Bone marrow aspiration Bone marrow biopsy 1. Site Iliac spine, sternum, tibia, spinous process of vertebra Iliac spine (posterior superior) 2. Information obtained Morphology, cytochemistry, iron Cellularity, architecture, stain, immunophenotyping, culture fibrosis, focal lesions, bone structure 3. Main indications Unexplained cytopenia, suspected hematological malignancy Repeated dry tap, aplastic anemia, myelofibrosis, focal lesions, hairy cell leukemia, staging of lymphoma 4. Needle used commonly Salah, Klima Jamshidi 5. Studies done Romanowsky stain, Iron stain, cytochemistry, cytogenetic or molecular genetic analysis, immunophenotyping, culture H and E stain, reticulin stain, immunohistochemistry 6. Time for report Same day Up to 7 days

EXAMINATION OF MARROW SPECIMENS Bone Marrow Aspiration Smears • Cellularity • Differential count • Myeloid: erythroid ratio • Erythroid series: maturation sequence, type of maturation (normoblastic, micronormoblastic, megaloblastic), cytologic abnormalities. • Myeloid series: maturation sequence, cytologic abnormalities. • Megakaryocyte series: number, abnormal forms. • Lymphocyte series • Plasma cell series • Abnormal cells: blasts, carcinoma cells, and necrotic cells. • Parasites: malaria parasites, microfilaria, Leishmania donovani, and Histoplasma. • Iron content of marrow (on iron stain).

UNDER LOW POWER OBJECTIVE (× 10) ASSESS : • Cellularity of marrow particles : Cellularity refers to the proportion of hematopoietic cells as compared to the fat cells in a marrow particle. Expressed as normocellular, hypercellular, or hypocellular for age. • Number of megakaryocytes - Megakaryocytes are found in the tail of the smear or near the marrow particles. About 1 -3 megakaryocytes are seen normally per low power field. • Focal metastatic deposits. • Cell distribution and selection of suitable area for detailed cytologic examination.

UNDER HIGH POWER (× 40) AND OIL IMMERSION OBJECTIVE (× 100) ASSESS : § Differential count – 200 - 500 cells should be counted in cellular trails of particles. § Myeloid: erythroid (M: E) ratio - 2: 1 to 4: 1. a. Increased M: E ratio : chronic or acute myeloid leukemia, aplastic crisis of hemolytic anemia. b. Reduced M: E ratio : hemolytic anemia, on treatment of anaemia. § Nature of erythroid maturation : normoblastic, micronormoblastic or megaloblastic.

Normal Differential Count in Bone Marrow in Adults : • • • • Myeloblasts: 0 -3% Promelocytes: 3 -12% Neutrophil myelocytes: 2 -13% Metamyelocytes: 2 -6% Neutrophils (including band forms): 22 -46% Lymphocytes: 5 -20% Monocyte : 0 -3% Eosinophils : 0 – 4% Basophils : 0 – 1% Erythroblasts: 5 -35% Plasma cells: 0 -3% Megakaryocyte : 0 -2% Macrophages : 0 -2% Myeloid: erythroid (M: E) ratio: 2: 1 to 4: 1

IRON STAINING OF BONE MARROW ASPIRATION SMEARS § Perls’ Prussian blue reaction. § Principle : ionic iron reacts with acid ferrrocyanide solution to form blue-colored ferric ferrocyanide. Iron appears as bright blue granular aggregates. § Perls’ stain does not detect heme iron of hemoglobin. § Detection of iron deficiency. § At least seven particles should be examined to optimally assess a bone marrow aspirate for iron stores. § On marrow smears, stainable iron can be demonstrated in siderocytes, sideroblasts, and macrophages of reticuloendothelial system.

BONE MARROW TREPHINE BIOPSY § Low power examination - cellularity, to identify focal lesions like granuloma, lymphoma infiltrates and clumps of metastatic malignancy, and to assess bone structure and number of megakaryocytes. § High power examination - proportion of myeloid and erythroid cells, location of hematopoietic precursors, pattern of infiltration of lymphoma cells, morphology of abnormal cells, and parasites.

§ Silver impregnation technique - reticulin fibers. § Reticulin fibers are increased in : ü ü ü Myelofibrosis acute leukemias chronic myeloid leukemia polycythemia vera hairy cell leukemia metastatic carcinoma § Special stains - Ziehl-Neelsen stain, Giemsa stain, Congo red stain.

IRON DEFICIENCY ANAEMIA SIDEROBLASTIC ANAEMIA PERLS’ STAIN

MEGALOBLASTIC ANAEMIA IDIOPATHIC THROMBOCYTOPENIC PURPURA (ITP)

CHRONIC MYELOID LEUKAEMIA (CML) ACUTE MYELOID LEUKAEMIA (AML)

ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL) CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL)

HAIRY CELL LEUKAEMIA BIOPSY MULTIPLE MYELOMA

BONE MARROW NECROSIS KALA - AZAR

Bone marrow aspirate from a patiant of fever with splenomegaly of two months duration showing marrow macrophages loaded with brown nodule of malarial pigment. One P. falciparum gametocyte is also seen.

TUBERCULOSIS

REFRENCES 1. Dacie and Lewis , Practical Haematology – 12 th edition. 2. Tejindar Singh, Atlas and Text of Hematology – 4 th edition. 3. Barbara J. Bain, David M. Cark, Bone Marrow Pathology – 3 rd edition. 4. Shirlyn B. Mc. Kenzie, Clinical Laboratory Hematology – 2 nd edition. 5. Robbins and Cotran, Pathologic Basis of Disease – 9 th edition.

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