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Common pitfalls in bone marrow biopsy based diagnostic approach
Dr. N. Varma Prof. & Head - HematologyPGIMER, Chandigarh, India
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Bone marrow (BM) examination
• Gold standard investigation for diagnosing and monitoring many hematological diseases
• Useful for investigating various non-hematological conditions
• Combination of bone marrow aspirate and trephine biopsy: fine cytological detail, the organization of BM, and the presence of focal abnormalities
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Good-to-have Information
• Accurate clinical information; context and questions being asked; details of previous investigations
• For neoplastic diseases: ? primary diagnostic investigation/ staging procedure/ re-examination to assess response to treatment (including transplantation)
• The type and timing of previous BM transplantation are also important factors; kinetics of engraftment differ between conditioning regimes and graft types
• Knowledge of the recent therapeutic use of growth factors such as G-CSF; these may transiently have major modifying effects on hemopoiesis that can mask or mimic genuine pathology
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Pitfalls in obtaining and interpreting bone marrow aspirates
• BM aspiration done when not needed• BM aspiration not done when needed• BM aspiration done on the wrong site• The clinical context not adequately assessed and the correct
range of tests is therefore not done on the aspirate• False negative result as a consequence of a sampling error• The aspirate is not interpreted together with the trephine
biopsy sections• The aspirate is misinterpreted
– Problems relating to technical quality– Correct stains not performed– Features present not noted– Misinterpretation of an adequate aspirate
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Limiting factors for interpretation of BMB• Inadequate clinical, hematological (blood and aspirate findings),
genetic and radiological information• Inadequate specimen
– Too small– Too crushed/distorted– Both– Poorly decalcified/processed
• Inadequate sections (thickness, number of levels)• Inadequate stains (poor technical quality, range too limited)• Insufficient experience to avoid common pitfalls (eg, differential
diagnosis of granulomas or fibrosis)• Insufficient confidence to avoid concluding ‘consistent with’• ‘Invisible’ pathology• Forgetting to look at the bone trabeculae and stroma
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Common Ancillary Studies complementary to Bone Marrow Morphologic Examination
• Cytogenetics on BM aspirate or peripheral blood sample• FISH studies on BM aspirate or touch preparations• Molecular studies (PCR or RT-PCR) to detect specific
translocations and/or antigen receptor gene rearrangements
• Flow cytometric Immunophenotyping of BM aspirate or peripheral blood cells
• Immunohistochemistry on paraffin sections• Enzyme cytochemistry on marrow aspirate or peripheral
smear slides
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A systematic approach to diagnosis is required for:
• D/D of hypoplasia/aplasia• D/D of megaloblastic hemopoiesis• Assessing key histological features of myelodysplastic
and myeloproliferative haemopoiesis• D/D of bone marrow fibrosis• Assessing patterns of lymphoid infiltration associated
with various lymphomas, especially small B-cell lymphomas
• D/D of granulomatous pathologies
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1. D/D hypocellular marrow
Normocellular
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• CBC and reticulocyte count• Blood film examination• Bone marrow aspirate and trephine biopsy• HbF% in children • Peripheral blood lymphocyte cultures for clastogens induced
chromosomal breakage studies• Ham’s test and / or flowcytometry for GPI anchored proteins• Urine hemosiderin (if Ham’s test and / or FCM for GPI anchored +)• Vitamin B12 and folate levels• Liver function tests• Renal function tests• Viral markers (hepatitis A, B, C; EBV; CMV; HIV)• Antinuclear antibody and anti ds-DNA• Chest x-ray• Abdominal ultrasound scan
Investigations recommended for suspected AA
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CBS 958
Varma N et al. Multiple constitutional aetiological factors in BMFS patients… Indian J Med Res 2006
Fanconi Anemia associated Aplastic Anemia
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(A-1366/11; Tx-1204/11)
2. Subtle increase of immature cells: in hypocellular marrow ?leukemia/lymphoma
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IHC: Blasts positive for CD34, anti-MPO (A-1366/11; Tx-1204/11) Diagnosis: Hypocellular AML
2. Subtle increase of immature cells in hypocellular marrow
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3. Problem in differentiating AML-M6 and megaloblastic anemia
Megaloblastic anemia ? Megaloblastic anemia
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3. Problem in differentiating AML-M6 and megaloblastic anemia
(A-1305/10; Tx-1056/10) IHC: Blasts positive for anti-MPO, CD34
AML- M6
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Hb TLC Platelet count
Reticulocyte DLC PBF
7 g/dl 7.3 x 109/L
7 x 109/L 5.36% P60L34M4E2 Moderate anisocytosis, microcytes, macrocytes, hypochromia & polychromasia
3. D/D of ‘megaloblastic anemia’ picture
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Bone marrow aspirate (BM A-1404/12)
CellularM:E= 1:1Blast1, Pm2, My 40 ,Mm 1, P44, L8, M1, E2Megakaryocytes: Adequate
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Bone marrow biopsy (T-1226/12)
Mildly hypercellular with relative erythroid hyperplasia with megaloblstic changes. Granulocytes and megakaryocytes are adequate.
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Granulocytes
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Granulocytes
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Monocytes
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RBCs
Final diagnosis: Classical PNH
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RCMD-RS
3. D/D of ‘megaloblastic anemia’ picture: characterization of ‘MDS’ like pathology.45 M, bicytopenia
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Bone marrow infiltration in a case of Hepatosplenic lymphoma (A-408/10; Tx-323/10)
4. Pattern of bone marrow infiltration by NHL
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Bone marrow intravascular infiltration in a case of Hepatosplenic lymphoma. IHC for CD34 and CD3 (A-408/10; Tx-323/10)
CD34 CD3
4. Pattern of bone marrow infiltration by NHL
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Plasma cells in a case of Multiple myeloma (A-1441/08; Tx-1161/08)
5. Differentiation between reactive and malignant plasma cells
IHC: Kappa light chain
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Reactive plasmacytosis + LD bodies(A-1535/11; Tx-1356/11)
5. Differentiation between reactive and malignant plasma cells
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Reactive increase in plasma cells in a case of Tubercular Granuloma (A-1198/12; Tx-1042/12)
5. Differentiation between reactive and malignant plasma cells
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BM: GranulomaAFB stain
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Renal Osteodystrophy (A-391/12; Tx-339/12)
6. Identification of etiology in fibrosis
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6. Identification of etiology in fibrosis
Acute panmyelosis with myelofibrosis (A-185/13; Tx-167/13)
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ALL with fibrosis(A-1476/12; Tx-1288/12)
7. Identification of subtle infiltration of leukemia/lymphoma in fibrotic marrow
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IHC for CD34
7. Identification of subtle infiltration of leukemia/lymphoma in fibrotic marrow; IHC is required
IHC for TdT
IHC for CD20
ALL with fibrosis(A-1476/12; Tx-1288/12)
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Amyloid deposition in vessel wall(A-1554/12; Tx-1356/12)
Congo Red stain
8. Problem in cases with subtle Amyloid deposition- need to be confirmed by special staining by Congo Red
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9. Problem in assigning myelodysplasia as reactive or primary
RCMD- predominantly dysplastic megakaryocytes (A-803/12; Tx-690/12)
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9. Problem in assigning myelodysplasia as reactive or primary
Case with sepsis- dysplastic megakaryocytes(A-55/12; Tx-51/12)
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Metastatic carcinoma of GIT (A-105/13; Tx-93/13)
10. Problem in assessment of focal lesions- like metastasis may be missed if sample is inadequate, and also in identification of primary site. These non-hematologic malignancies may mimic hematological malignancies also.
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Metastatic carcinoma- Prostate (A-983/09; Tx-763/09)
10. Problem in assessment of focal lesions- like metastasis may be missed if sample is inadequate, and also in identification of primary site. These non-hematologic malignancies may mimic hematological malignancies also.
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Granuloma- TB (A-1198/12; Tx-1042/12)
11. Problem in assessment of focal lesions- like granuloma may be missed if inadequate sample and also in differentiation of granuloma etiology
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Granuloma- Hodgkin’s Lymphoma(A-1252/12; Tx-1091/12)
11. Problem in assessment of focal lesions- like granuloma may be missed if inadequate sample and also in differentiation of granuloma etiology
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12. Problem in cases with only necrosis- where etiology can not be assessed
BM Necrosis- (A-330/11; Tx-286/11)
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13. Problem in identification of lymphocytosis, esp in NK/ T-cell infiltration as reactive increase or malignant
NK leukemia/lymphoma (A-444/12; Tx-314/12)
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Bone Marrow Trephine Biopsy 314/12
Splenectomy section (S-12985/12) of this case.IHC for CD56 highlighting NK cell increase in spleen; case with increased lymphocytes on bone marrow.
13. Problem in identification of lymphocytosis, esp in NK/ T-cell infiltration as reactive increase or malignant
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14. Problem in differentiation of syntitial variant of Hodgkin’s lymphoma and ALCL
Reported as Anaplastic large cell lymphoma (A-1255/08; Tx-1020/08)
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IHC for CD30
IHC for CD15
14. Problem in differentiation of syntitial variant of Hodgkin’s lymphoma and ALCL- IHC required for differentiation
Reported as Anaplastic large cell lymphoma (A-1255/08; Tx-1020/08)
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15. Problem in identification T-cell rich B-cell lymphoma
IHC for CD3 IHC for CD20
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16. There can be technical artefacts leading to inconclusive findings
Washed off marrow spaces
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Washed off marrow spaces, hemorrhage and cartilage
16. Procedural artefacts leading to inconclusive findings
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16. Technical artefacts leading to inconclusive findings
Crushed marrow spaces
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17. 15yrs/M, TLC 32x109/L, Blasts 95%Scanty aspirate smears
Tx BxPB smear
MPO +
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Positive Markers: CD13, CD33, Anti MPO, CD19, CD10, CD34, CD45, CD123, HLADRNegative Markers: T lineage
FCM-IP Diagnosis: Mixed Phenotype Acute Leukemia (B/ Myeloid)
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1444 bp
943 bp
754 bp
585 bp
458 bp
341 bp258 bp
NC P1 P2 P3 P4 P5 P6 M
bcr-abl transcripts in 1 MPAL (P1) and 5 different CML (P2-6) patients
b3a2 – 385 bp b2a2 – 310 bp
Bhatia P, Binota J, Varma N, Bansal D, Trehan A, Marwaha RK, Malhotra P, Varma S. A Study on the Expression of BCR-ABL Transcript in Mixed Phenotype Acute Leukemia (MPAL) Cases Using the Reverse Transcriptase Polymerase Reaction Assay (RT-PCR) and its Correlation with Hematological Remission Status Post Initial Induction Therapy. Mediterr J Hematol Infect Dis. 2012;4(1):e2012024.
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18. 22M, DOA: 19.11.06; DOD: 26.11.06C/O fever (↑↑-↑↑↑) 4 weeks PUO: ? Disseminated TB / lymphoreticular malignancyCBC: Hb 8.7 g/dl, Retics 1.6-2.6%, TLC 1800-700/ml, platelets 27000-12000/ml; PTI 86%Bilirubin 4.2/2.8, 4.9/3.6; Serum ferritin 7557.8 mg/dl; Fibrinogen 2.85 g/l; TG 410
BM no. A-1083/06
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EBV Zebra LMP 1 EBNA
HPS / HLH•Screen for underlying genetic, autoimmune, infectious and malignant diseases•Uncontrolled hypercytokinemia & many triggers•Early diagnosis and Rx
PM no. 21703
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19. 5 ys. FCh; AML with increased mast cells/ basophils
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MPO cytochemistry
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P 30 / 07: CD 117 (APAAP)
Varma N, Varma S, Wilkins B. Br J Haematol 2000;111:991.
Mast cell tryptase
AML with mastocytosis [Systemic mastocytosis with associated clonal hematological non-mast cell disease (SM-AHNMD)]
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Few representative examples• Assessment of focal lesions• Differentiation between reactive lymphoid infiltrate and NHL• Differentiation between reactive and malignant plasma cells• Identification of malignancies with associated fibrosis• Effect of growth factors• Differentiation between hematogones and blasts• Differentiation between megaloblastic anemia and acute leukemia• Differentiation between aplastic bone marrow and hypoplastic
myelodysplastic syndrome or hypoplastic acute leukemia• Identification of lymphomas having a tendency for intravascular infiltration
in the BM• Subtle amyloid deposition• Differentiation of macrophage infiltrates and other pathologies that
resemble granulomatous infiltration• Procedure related artefacts
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Take home message• Integration of clinical, laboratory and imaging information • Not to assess histology in isolation; uni- / bilateral bx; dry aspirate• Components of an integrated approach to interpretation are:
– Adequate size of trephine core, with minimal disruption by trauma caused during collection.
– Access to detailed clinical information and results of additional tests (specially, peripheral blood cell counts, blood and BM aspirate cytomorphology, flow cytometry, cytogenetic analysis and radiological imaging).
– Systematic assessment of all BM components, including trabecular bone and interstitial stroma.
– Awareness of pathologies that may be ‘invisible’ in trephine specimens without immunostaining.
– Use of preselected antibody panels for immunostaining and familiarity with the expected results, including controls.
– Experience in interpreting additional molecular studies, such as clonality PCR and fluorescence in-situ hybridization.
– Familiarity with the major patterns of bone marrow involvement by reactive and neoplastic conditions and their differential diagnosis.
– A collaborative approach to working with diverse clinical and laboratory colleagues.– Ideally, hematopathologists should report BM Bx along with BM aspirate.
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Thank You
Common pifalls in BMB interpretation can be avoided