Evaluation of Bone Marrow Biopsies and Aspirates ANNA PORWIT DEPARTMENT OF PATHOLOGY, LUND UNIVERSITY

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1 Evaluation of Bone Marrow Biopsies and Aspirates ANNA PORWIT DEPARTMENT OF PATHOLOGY, LUND UNIVERSITY

2 DISCLOSURES NONE

3 Learning objectives To review the rules of BMA evaluation To review the main issues in BMTB evaluation To appreciate the importance of the integrated diagnosis of hematological disorders

4 Integrated Diagnosis of Bone Marrow Samples According to WHO 2016 Cytomorphology (AML, MDS, MPN) PB, BMU Cytogenetics Immunophenotype Mandatory Mandatory Mandatory AML Supportive MDS Histology FISH Molecular Biology Mandatory Recommended in MDS if no cytogenetics Mandatory AML Supportive MDS

5 employees, more than students

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7 Indications for bone marrow examination

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9 Bone marrow aspirate slides 8-10 smears made from the bone marrow particles preferably at beside or collected in EDTA Some smears should be made from crushing the particles in the middle of the slide. Touch preparations should be prepared by repeatedly touching the biopsy specimen to glass slides and exerting a gentle downward pressure. A rotary or smearing motion should be avoided since rupture and destruction of cells will result. If the smears are exposed to formalin vapors, they will be essentially unreadable.

10 Evaluation of bone marrow smears Two air dried smears and one squash slide May-Grünwald-Giemsa or Wright-Giemsa Prussian Blue iron stain Coverslipped Low power: fragments, cellularity, megakaryocytes, abnormal cells High power: in trails to assess cytological details Nucleated differential count: 500 cells if percentage of blasts is necessary, 300 if NDC not necessary for classification, M:E ratio, ring sideroblasts on iron stained smear Squash: cellularity, focal disease

11 Erythropoiesis

12 Normal range Pronormoblast 0 1.5% Basophilic normoblast 0 5% Polychromatophilic normoblast 5 30% Orthochromatic normoblast 5 10%

13 Megaloblastic changes vs dysplasia B12 deficiency MDS

14 SF3B1 mutation

15 Non-clonal causes of dysplasia Vitamin B12 and folic acid deficiency Essential element deficiencies Exposure to heavy metals, particularly lead or arsenic Several commonly used drugs and biologic agents (e.g.bactrim or Mofetil) Chemotherapy Congenital haematological disorders such as congenital dyserythropoietic anaemia Parvovirus B19 infection may cause erythroblastopenia with giant megaloblastoid erythroblasts; Granulocyte colony-stimulating factor Paroxysmal nocturnal haemoglobinuria It is extremely important to be aware of the clinical history including exposure to drugs or chemicals and to consider nonclonal disorders as possible aetiologies

16 CD34+ CD34++ HLA-DR++ CD38++ CD4+/CD13+/CD15+ CD33+/CD36+/ CD64+ HLA-DR+/CD11b+/CD14+/- CD34++ HLA-DR++ CD117+ CD13+ CD33dim MPO- CD13dim CD33+ MPO+ CD65+ CD15+ CD11b+ CD117+/- CD13+ CD33+ MPO+ CD65+ CD15+ CD13+ CD33+ MPO+ CD65+ CD15+ CD11b+ CD35dim CD16+ CD13++ CD33+ MPO+ CD65+ CD15+ CD11b++ CD16++ CD10+ Lin-/CD34+/ CD38/CD123 low / CD45RA+ CD4+/CD13+/CD15+ CD33++/CD36+/ CD64+ HLA-DR+/CD11b++/CD14++ CD34+/CD4+/CD13+/ CD33+/HLADR+/MPO- CD16+/ CD163+ Granulopoiesis

17 Normal ranges Myeloblast 0 2% Promyelocyte 0 4% Myelocytes Neutrophilic 5 20% Eosinophilic 0 3% Basophilic 0 1% Metamyeolocytes and bands: Neutrophilic 5 35% Eosinophilic 0 5% Basophilic 0 1% Segmented neutrophils 5 15%

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19 Dysplasia: Granulopoiesis Bone marrow (500 cells) Small or unusually large size Nuclear hypolobation (pseudo Pelger- Huet) Irregular hypersegmentation Decreased granules Pseudo Chediak-Higashi granules Auer rods Myeloperoxidase negative neutrophils Increased basophils or monocytes

20 Pseudo Pelger-Huet cells Hypersegmented granulocytes Del 17p

21 Pesudo Chediak-Higashi granula

22 Dysplasia: Hypogranular granulopoiesis

23 Dysgranulopoiesis

24 Megakaryocytes Normal range: mm Multilobated nuclei 8-32 lobes No nucleoli Coarse chromatin

25 Dysmegakaryopoiesis Micromegakaryocytes: 15-30mm Nonlobulated, N/C asynchrony, eosinophilic cytoplasm Nuclear hypolobation Multinucleation (normal are uninucleate with lobated nuclei) Less specific Numerous megakaryoblasts och promegakaryocytes Large dysplastic megakaryocytes Cytoplasmic vacuoles Apoptotic nuclei megakaryocytes should be evaluated

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27 Dysmegakaryopoiesis

28 WHO: The contribution of adequate BM biopsy sections in the diagnosis of myeloid neoplasms cannot be overstated Information on: Overall cellularity Topography Proportions of haematopoietic lineages Maturation of haematopoietic cells Bone Marrow stroma (fibrosis) Immunohistochemical studies combine information on morphology and immunophenotype of cells

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30 Normal CD34 Normal CD117 CD34 in MDS

31 CD34 Enumeration in BM Bx (QBEND10): CD34/IHC counts on BMTB aid in diagnosis and classification of MDS and other hematological malignancies, especially in presence of fibrosis

32 B-cell lymphoma CD20 Diffuse pattern

33 Paratrabecular pattern follicular lymphoma

34 B-cell lymphoma CD20 Nodular pattern

35 CD20 Splenic marginal zone lymphoma Hepatosplenic T-cell lymphoma Intrasinusoidal pattern

36 Interstitial pattern CD20

37 Significance of integrated approach: Case 1 70 years old female admitted Sept.2006 due to bleeding from stomach ulcus Hb 85g/L with signs of iron deficiency WBC 30x10 9 /L with 12% ly, 82% neutrophils, 1% eosinophils, 4% basophils, no blasts, no precursors Platelets 2200x10 9 /L

38 Anna Porwit 38

39 Anna Porwit 39

40 Anna Porwit 40

41 Interactive question 1: Your diagnosis 1. Reactive thrombocytosis 2. Essential thrombocytemia 3. Primary myelofibrosis 4. Polycythemia vera with post bleeding anemia 5. Chronic myeloid leukemia

42 Follow up Clinical diagnosis of Polycythemia vera with secondary bleeding was made Started immediately on Hydrea treatment and later with Anagrelid July 2007 JAK2 analysis was performed and was negative Therefore new BM was taken including chromosomal analysis, which showed t(9;22)(q34;q11). Hb was 112, Plt 764x10 9 /L WBC was 26x10 9 /L, still no precurors but basophilia 9% Revised diagnosis: BCR-ABL1+ CML Things are not always what they seem to be

43 Significance of integrated approach: Case 2 89 years old male admitted due to anemia and leukocytosis Hb 94 g/l WBC 48x10 9 /L Differential count showed presence of all stages of granulopoiesis Plt 444x10 9 /L Spleen slightly enlarged Clinically suspect CML

44 44 Bone marrow smears hemodiluted due to fibrosis Blood smears showed all stages of granulopoiesis and some erythropoietic precursors

45 Anna Porwit 45

46 Interactive question 2: Your diagnosis 1. Reactive leukocytosis 2. Primary myelofibrosis 3. Polycythemia vera with post bleeding anemia 4. Chronic myeloid leukemia 5. Chronic myelomonocytic leukemia

47 Genetic studies BCR-ABL1 negative JAK2 V617F mutation positive in peripheral blood DIAGNOSIS: PRIMARY MYELOFIBROSIS IN FIBROTIC STAGE

48 Case 3 67 year old woman Fatigue, nos signs of infection Hb 86 g/l WBC 0.8x10 9 /L Differential count showed neutropenia Plt 60x10 9 /L No lymphadenopathy Spleen borderline Bone marrow smears showed mostly blood (bad quality)

49 Bone marrow biopsy

50 Interactive question 3 Your diagnosis 1. Aplastic anemia 2. Hemophagocytosis 3. Leishmania infection 4. Large granular lymphocyte leukemia 5. Hairy cell leukemia

51 Immunohistochemistry CD20

52 Flow cytometry: Hairy cell leukemia

53 Summary Standardization is required in diagnostics to be able to compare treatment results of patients in various countries and in various clinical trials Follow the guidelines in BM evaluation and reporting

54 Summary Multidisciplinary approach and integrating all available diagnostic studies, including cytology, morphology, immunophenotyping and genetic/molecular studies is required for adequate diagnostics and classification of hematological malignancies

55 Questions?