Common pitfalls in bone marrow biopsy based diagnostic approach

Dr. N. Varma Prof. & Head - HematologyPGIMER, Chandigarh, India

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

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

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

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

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

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

1. D/D hypocellular marrow

Normocellular

• 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

CBS 958

Varma N et al. Multiple constitutional aetiological factors in BMFS patients… Indian J Med Res 2006

Fanconi Anemia associated Aplastic Anemia

(A-1366/11; Tx-1204/11)

2. Subtle increase of immature cells: in hypocellular marrow ?leukemia/lymphoma

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

3. Problem in differentiating AML-M6 and megaloblastic anemia

Megaloblastic anemia ? Megaloblastic anemia

3. Problem in differentiating AML-M6 and megaloblastic anemia

(A-1305/10; Tx-1056/10) IHC: Blasts positive for anti-MPO, CD34

AML- M6

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

Bone marrow aspirate (BM A-1404/12)

CellularM:E= 1:1Blast1, Pm2, My 40 ,Mm 1, P44, L8, M1, E2Megakaryocytes: Adequate

Bone marrow biopsy (T-1226/12)

Mildly hypercellular with relative erythroid hyperplasia with megaloblstic changes. Granulocytes and megakaryocytes are adequate.

Granulocytes

Granulocytes

Monocytes

RBCs

Final diagnosis: Classical PNH

RCMD-RS

3. D/D of ‘megaloblastic anemia’ picture: characterization of ‘MDS’ like pathology.45 M, bicytopenia

Bone marrow infiltration in a case of Hepatosplenic lymphoma (A-408/10; Tx-323/10)

4. Pattern of bone marrow infiltration by NHL

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

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

Reactive plasmacytosis + LD bodies(A-1535/11; Tx-1356/11)

5. Differentiation between reactive and malignant plasma cells

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

BM: GranulomaAFB stain

Renal Osteodystrophy (A-391/12; Tx-339/12)

6. Identification of etiology in fibrosis

6. Identification of etiology in fibrosis

Acute panmyelosis with myelofibrosis (A-185/13; Tx-167/13)

ALL with fibrosis(A-1476/12; Tx-1288/12)

7. Identification of subtle infiltration of leukemia/lymphoma in fibrotic marrow

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)

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

9. Problem in assigning myelodysplasia as reactive or primary

RCMD- predominantly dysplastic megakaryocytes (A-803/12; Tx-690/12)

9. Problem in assigning myelodysplasia as reactive or primary

Case with sepsis- dysplastic megakaryocytes(A-55/12; Tx-51/12)

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.

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.

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

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

12. Problem in cases with only necrosis- where etiology can not be assessed

BM Necrosis- (A-330/11; Tx-286/11)

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)

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

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)

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)

15. Problem in identification T-cell rich B-cell lymphoma

IHC for CD3 IHC for CD20

16. There can be technical artefacts leading to inconclusive findings

Washed off marrow spaces

Washed off marrow spaces, hemorrhage and cartilage

16. Procedural artefacts leading to inconclusive findings

16. Technical artefacts leading to inconclusive findings

Crushed marrow spaces

17. 15yrs/M, TLC 32x109/L, Blasts 95%Scanty aspirate smears

Tx BxPB smear

MPO +

Positive Markers: CD13, CD33, Anti MPO, CD19, CD10, CD34, CD45, CD123, HLADRNegative Markers: T lineage

FCM-IP Diagnosis: Mixed Phenotype Acute Leukemia (B/ Myeloid)

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.

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

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

19. 5 ys. FCh; AML with increased mast cells/ basophils

MPO cytochemistry

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)]

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

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.

Thank You

Common pifalls in BMB interpretation can be avoided