1. Common pitfalls in bone marrow
biopsy based diagnostic approach
Dr. N. Varma
Prof. & Head - Hematology
PGIMER, Chandigarh, India

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 1. D/D hypocellular marrow
Normocellular

9. • 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

10. CBS 958
Varma N et al. Multiple constitutional aetiological factors in BMFS patients… Indian J Med Res 2006
Fanconi Anemia associated Aplastic Anemia

11. (A-1366/11; Tx-1204/11)
2. Subtle increase of immature cells: in hypocellular marrow ?leukemia/lymphoma

12. 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

13. 3. Problem in differentiating AML-M6 and megaloblastic anemia
Megaloblastic anemia ? Megaloblastic anemia

14. 3. Problem in differentiating AML-M6 and megaloblastic anemia
(A-1305/10; Tx-1056/10) IHC: Blasts positive for anti-MPO, CD34
AML- M6

15. 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

16. Bone marrow aspirate
(BM A-1404/12)
Cellular
M:E= 1:1
Blast1, Pm2, My 40 ,Mm 1, P44, L8, M1, E2
Megakaryocytes: Adequate

17. Bone marrow
biopsy
(T-1226/12)
Mildly hypercellular with relative erythroid hyperplasia with
megaloblstic changes. Granulocytes and megakaryocytes are adequate.

19. Granulocytes

20. Granulocytes

21. Monocytes

22. RBCs
Final diagnosis: Classical PNH

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

24. Bone marrow infiltration in a case of Hepatosplenic lymphoma
(A-408/10; Tx-323/10)
4. Pattern of bone marrow infiltration by NHL

25. 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

26. 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

27. Reactive plasmacytosis + LD bodies
(A-1535/11; Tx-1356/11)
5. Differentiation between reactive and malignant plasma cells

28. 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

29. BM: GranulomaAFB stain

30. Renal Osteodystrophy
(A-391/12; Tx-339/12)
6. Identification of etiology in fibrosis

31. 6. Identification of etiology in fibrosis
Acute panmyelosis with myelofibrosis
(A-185/13; Tx-167/13)

32. ALL with fibrosis
(A-1476/12; Tx-1288/12)
7. Identification of subtle infiltration of leukemia/lymphoma in fibrotic marrow

33. 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)

34. 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

35. 9. Problem in assigning myelodysplasia as reactive or primary
RCMD- predominantly dysplastic megakaryocytes (A-803/12; Tx-690/12)

36. 9. Problem in assigning myelodysplasia as reactive or primary
Case with sepsis- dysplastic megakaryocytes
(A-55/12; Tx-51/12)

37. 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.

38. 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.

39. 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

40. 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

41. 12. Problem in cases with only necrosis- where etiology can not be assessed
BM Necrosis- (A-330/11; Tx-286/11)

42. 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)

43. 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

44. 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)

45. 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)

46. 15. Problem in identification T-cell rich B-cell lymphoma
IHC for CD3 IHC for CD20

47. 16. There can be technical artefacts leading to inconclusive findings
Washed off marrow spaces

48. Washed off marrow spaces, hemorrhage and cartilage
16. Procedural artefacts leading to inconclusive findings

49. 16. Technical artefacts leading to inconclusive findings
Crushed marrow spaces

50. 17. 15yrs/M,
TLC 32x109/L, Blasts 95%
Scanty aspirate smears
Tx BxPB smear
MPO +

51. Positive Markers: CD13, CD33, Anti
MPO, CD19, CD10, CD34, CD45, CD123, H
LADR
Negative Markers: T lineage
FCM-IP Diagnosis: Mixed Phenotype Acute Leukemia (B/ Myeloid)

52. 1444 bp
943 bp
754 bp
585 bp
458 bp
341 bp
258 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.

53. 18. 22M, DOA: 19.11.06; DOD: 26.11.06
C/O fever (↑↑-↑↑↑) 4 weeks
PUO: ? Disseminated TB / lymphoreticular malignancy
CBC: Hb 8.7 g/dl, Retics 1.6-2.6%, TLC 1800-700/ l, platelets 27000-12000/ l; 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

54. 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

55. 19. 5 ys. FCh; AML with increased mast cells/ basophils

56. MPO cytochemistry

57. 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)]

58. 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

59. 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.

60. Thank You
Common pifalls in BMB interpretation can be avoided