Bone marrow pathology

1. BONE MARROW EXAMINATION

3. When?
Indications for BM examination
• Haematological abnormalities that cannot be explained by available
clinical and laboratory data.
• 3 main indications :
– Diagnostic purposes
– Staging for malignant diseases
– Monitoring
• A good indication is essential for an accurate diagnosis.

4. Indication of marrow examination:
• Investigation of unexplained peripheral blood abnormalities
• Diagnosis of suspected primary hematopoietic neoplasm
• Infectious disease work-up if other systemic investigations non-contributory
• Evaluation of suspected constitutional disorder
• Assess for storage disorder
• Assess for involvement by metastatic neoplasm
• Staging of lymphoma
• Ongoing monitoring after therapy

5. What
specimens to
collect ?

7. Importance of touch/ Imprint smear
• Gives cytological details when aspirate is not
obtained.(fibrotic or inaspirable)
• Shows more neoplastic cells than aspirate.
• Can show marrow infiltration , not seen in
aspirate
• Important for potential ancillary techniques
(e.g., cytochemistry, FISH, iron stains)
• Particularly if BM aspirate is hemodilute/dry tap
• ≥ 3 air-dried imprints
Be gentle, be delicate !
Do NOT CRUSH !

8. Importance of Clot Section
• Assessment of bone marrow cellularity.
• For detecting granuloma and tumor infiltrates
complementary to biopsy.
• No decalcification associated nucleic acid or
protein damage.
• Immunostains may also be performed.

9. BM Aspirate or Trephine Biopsy

10. BM aspirate or trephine biopsy

11. How to collect these specimens ?
• Anatomic sites
• Collection procedure
• Adequacy

12. Anatomic sites
Posterior iliac crest aspiration is
suitable for children, infants and
many neonates.
Tibial aspiration is suitable for very
small babies but has no advantages
over iliac crest aspiration in older
infants.

13. BM Aspirate Adequacy
• Obtain at least 3 particles per slide
• Obtain at least 4 slides
– 2 for routine staining
– 1 for iron (as needed)
– 1 or more for potential ancillary tests (e.g.,cytochemistry, FISH)
• Not crushed/Not too thick/Not clotted
• Allow to dry quickly
• Dry tapes represent 2-7% of the cases
• If an adequate aspirate has not been possible, considerations should be
given to preparing touch imprints of the core biopsy prior to placing it in
fixative.

14. BM Trephine Biopsy
• 11-gauge needle AT LEAST
• If osteopenic, a 8-gauge needle allows the collection of an intact core
biopsy with minimal crush artifact
• 13-gauge biopsy needle for pediatric patients
Adequate core biopsy:
• At least 1.6 cm to 2 cm long
• Exclusive of cortical bone, cartilage, or periosteum
• Free of crush artifact or interstitial hemorrhage or fragmentation

15. BM Evaluation
• Clinical history
• Clinical impression
• Laboratory data
• Morphology
• Immunophenotype
• Genetic features

18. • Erythrocytes remain macrocytic from the first 11 weeks of
gestation until day 5 of postnatal life.
Peripheral blood film for a
normal newborn
demonstrating a normal
lymphocyte, macrocytes,
polychromasia, and one
nucleated red blood cell
(×1000).

19. • Orthochromic normoblasts frequently are observed in the full-term
infant on the first day of life but disappear within postnatal days 3-5.
• NRBCs may persist longer than a week in immature infants.
• The average number of NRBCs ranges from 3 to 10 per 100 WBCs in a
normal full-term infant to 25 NRBCs per 100 WBCs in a premature
infant.
• The presence of NRBCs for more than 5 days suggests hemolysis,
hypoxic stress, or acute infection.

21. Bone Marrow Aspiration

22. Evaluating Aspirate Smears
Screen all slides at low power (4 x)
• Evaluate cellularity, look for abnormal infiltrate
• Find good areas of slides for counting
• Evaluate number of megakaryocytes
Count cells from several areas (100 x)
• If spicules are lacking, accurate counts may be obtained at edges of smears
• If cells are mainly neutrophils and lymphocytes, smears may represent peripeheral
blood rather than marrow
The preparation can be considered satisfactory only when marrow
particles and free marrow cells can be seen in stained films.

23. Normocellular bone marrow on aspirate High cellularity oaf 95-100%
Cellularity of 40-50% Hypocellular bone marrow (10-20%)

24. Epidemiology
• Dramatic age-related variations in BM cellularity
-Highest cellularity in neonates: ~ 80-100%
-Gradual decline in cellularity with age
• BM cellularity in elderly patients: ~ 20-30%

25. Mean values (observed range) for bone marrow
cells in healthy infants and children

26. • The bone marrow at birth has major erythroid and myeloid components with
few lymphocytes and very few plasma cells.
• The percentage of erythroid cells declines steeply in the first weeks.
• The percentage of lymphocytes increases during the first month and remains
at a high level until 18 months of age (up to 40-50% in young children)
• Numbers decline during childhood and in adults they do not generally
comprise more than 15–25% of nucleated cells, unless the marrow aspirate
has been considerably diluted with peripheral blood.
Evaluating Aspirate Smears

27. • Megakaryocytes of haematologically normal neonates and infants, up to
the age of 10 months, are smaller and more homogeneous in size than
those of older children and adults.
• In children above the age of 2 years, the proportions of different cell types
do not differ greatly from those in normal adult bone marrow.
• Osteoblasts are uncommon in bone marrow aspirates of healthy adults but,
when present, often appear in small clumps. They are much more
numerous in the bone marrow of children and adolescents.
Evaluating Aspirate Smears

28. Osteoclast: note the highly
granular cytoplasm and the
multiple nuclei (2- 100)which
are uniform in size and have
indistinct, medium - sized, single
nucleoli.
OSTEOBLAST: have basophillic
cytoplasm,extruding nucleus and regular
chromatin with 1-4 nucleoli. Can be
distinguished from plasma cells by their
larger size and the position of the Golgi
zone, which is not immediately adjacent
to the nucleus.
Mature megakaryocyte having
loblated nucleus and pink granular
cytoplasm.platelets are formed by
budding of the cytoplasm which are
shed in the circulation.

29. 95% Range Mean[12] Mean[11]
Myeloblasts 0–3 1.4 0.4
Promyelocytes 3–12 7.8 13.7[*]
Myelocytes (neutrophil) 2–13 7.6 –
Metamyelocytes 2–6 4.1 –
Neutrophils 22–46 32.1M; 37.4W 35.5
Myelocytes (eosinophil) 0–3 1.3 1.6
Eosinophils 0.3–4 2.2 1.7
Basophils 0–0.5 0.1 0.2
Lymphocytes 5–20 13.1 16.1
Monocytes 0–3 1.3 2.5
Plasma cells 0–3.5 0.6 1.9
Erythroblasts[†] 5–35 28.1M; 22.5W 23.5
Megakaryocytes 0–2 0.5
Macrophages 0–2 0.4 2.0
Normal ranges for differential counts on aspirated bone marrow (500 cells should be counted)

30. BM Evaluation
Bone Marrow Biopsy

31. How To Examine A Trephine Section
10X Evaluation
•Evaluate bone trabeculae
•Look for gross abnormalities
•Evaluate marrow cellularity
•Screen slide for adequacy of sample
20X Evaluation
•Marrow architecture
40X Evaluation
•Morphology of erythroid, myeloid elements
and Megakaryocytes
Have an organized approach!
1. Assessment of architecture and
cellularity
2. Assessment of accessory structures
3. Distribution of cellular elements
(topography)
4. Assessment of cell morphology

32. Adequacy:
Quality of marrow more important than length of core
-Adequate sampling: 1.5 cm (1.6-2cm)
-H&E (at least 2 levels) and reticulin stains
•Avoid
 Crushed areas
 Subcortical fatty marrow
 Intertrabecular hemorrhage
This BM core biopsy is suboptimal for
evaluation with cartilage on both ends
of the biopsy flanking minimal
subcortical BM. Subcortical marrow is
often not representative of the marrow
as a whole, and a statement to this
effect should be issued in the final
report.

33. • A biopsy specimen containing at least five or six intertrabecular
spaces is desirable, not only for an adequate assessment of cellularity
but also to give a reasonable probability of detecting focal bone
marrow lesions.
• Ideally this requires a core of 2–3 cm in length. A core length of at
least 0.5 cm has been advised in children but one study found 1.0 cm
was necessary to avoid a high rate of non‐interpretable specimens.
Adequacy:

34. • A section of a trephine biopsy specimen of adequate
size from a patient with Hodgkin lymphoma showing
only a small area of infiltration at one end of the
specimen, illustrating how a small biopsy may miss
focal lesions. at least 16 mm
Adequacy:

35. Common artifacts in marrow trephine biopsy
• Washed off of marrow space- technical artifact leading to inconclusive findings
• Aspiration artifact
• Crushed artifact

36. 1. ASSESMENT OF ARCHITECTURE AND CELLULARITY
HOW TO EXAMINE A TREPHINE SECTION

37. • Cellularity varies according to location and age
• Hypocellular in subcortical marrow
(three intertrabecular space)
Evaluation of cellularity

38. Evaluation of cellularity
• Average cellularity in the bone marrow of children, assessed on core
biopsy or clot sections:
• 80% at 2 years
• 69% at 2–4 years
• 59% at 5–9 years
• around 60% thereafter
• 20-40% in elderly

40. Architecture
Myeloid cells
Paratrabecular
Immature myeloid elements
are adjacent to bone
trabeculae
Mature cells towards centre
Erythroid cells
Centre in colonies
Megakaryocytes
Centre around sinusoids
are singletons (<3 together)

42. 2. ASSESMENT OF ACCESSORY STRUCTURES
HOW TO EXAMINE A TREPHINE SECTION

43. Accessory structures
• Both osteoclasts and osteoblasts normally evident along bony
trabeculae in specimens from pediatric patients.
• Incomplete ossification typical in infants/young children
• Evidence of bony remodeling normal in children, abnormal in middle-
aged to elderly adults.

44. Trabeculae consists of lamellar bone Osteoblasts along endosteal surface
Ossifying cartilage. Osteocytes within
lacuna (arrow head).
Osteoclast

45. BM trephine biopsy section from a child
showing endochondrial ossification; a bony
spicule with a core of cartilage is lined by
osteoblasts. Cartilage cells are
dispersed singly or in small groups
and are aligned into columns.
BM trephine biopsy section
from an adult showing cartilage
adjacent to the cortex. By contrast
with childhood appearances, a
well‐defined layer of cortical bone
separates this cartilage from the
bone marrow.

46. HOW TO EXAMINE A TREPHINE SECTION
3. DISTRIBUTION OF CELLULAR ELEMENTS (TOPOGRAPHY)
4. ASSESMENT OF CELL MORPHOLOGY

47. Diagrammaticrepresentation of the topography of normal bonemarrow.
Osteoclasts, osteoblasts, myeloblasts, and promyelocytes are adjacent to the spicule of bone.
Deeper in the intertrabecular space are maturing cells of neutrophil lineage, erythroid islands with a central macrophage, and interstitial lymphocytes.
Eosinophils and their precursorsare apparently randomly scattered, plasma cells are interstitial or form a sheath around capillaries, and
megakaryocytes abut on a sinusoidat one extremity of the cell.

48. Marrow elements
1-Hematopoietic
component
• Erythroid series
• Myeloid Series
• Megakaryocyticseries
• Lymphoid cells
t
2-Stromal componen
• Macrophage
• Fat cells
• Fibroblast
• Delicate fiber network
• Blood vessels(mostly sinusoid)
• Nerve bundles
• Trabecular bone
• Extracellular matrix
• Osteoblast and Osteoclast
Stromal component facilitate maintenance of HSC and support differentiation and maturation of the progenitors
These two components not only coexist but
closely interact with each other

49. Special Stains on Marrow
-Giemsa(routine)
• Differentiates cytoplasm of erythroid elements and myeloid elements
• Plasma cells, mast cells more obvious
-Reticulin (special)
• Evaluates bone marrow fibrosis
• clues: crush artifact, dry tap, enlarged vascular spaces
-PAS stains highlighting megakaryocytes, fungal organism in institutions with large populations of
immunosuppressed patients
-Silver stain for assessment of reticulin fibrosisIncases of myeloproliferative neoplasms
-Not recommend the routine Perl stain of the core biopsyif a satisfactory marrow aspirate cannot be
obtained, iron stains of the clot or biopsysections>>>potential false-negative results.

50. Reticulin fibrosis
 Reticulin is normally present in BM
 ‘Normal’ reticulin
• Around blood vessels
• Short wisps
 • Day-to-day variation in silver
• Underdone reticulin stain
• - No staining around blood vessels -> repeat
• Overdone reticulin stain
• - Staining of cell membranes, cell nuceli stained darkly - >
repeat or discount
•
Collagen fibrosis
Most notably seen in association with
myeloproliferative neoplasms
Detected with trichrome stain
Not normally present in BM
Unlikely to be reversible
2 types of BM fibrosis:
- Reticulin fibrosis - Collagen fibrosis

52. grade 0 grade 1 grade 2,
grade 3 grade 4

53. Iron Assessment
• Typically assess 2 types of iron stores
• Sideroblastic iron (a.k.a. erythroid iron)
• Macrophage iron (a.k.a. storage iron)
• Assessment of storage iron requires that an adequate number of
fragments are obtained
• A minimum of seven fragments in one or more bone marrow films
need to be examined in order to state with reasonably reliability that
bone marrow iron is absent .

54. Erythroid iron
Preferred specimen: Spicular, unstained,
air-dried BM aspirate smear/touch
preparation containing adequate red
blood cell precursors
Normal: 20-50% of red blood cell
precursors demonstrate 1-3 small
cytoplasmic granules
Abnormal
– Abnormal size (large), shape (chunky),
or location (perinuclear, ring) of granules
– Ring sideroblast is defined by WHO as
1/3 of red blood cell nucleus tightly
surrounded by 5 or more iron granules.
Storage iron
Preferred specimen: Spicular, unstained, air-dried BMA
smear
Grading may be semiquantitatively assessed (normal,
none, increased, decreased) or assessed by grading scale
(0-6)
In routine practice, grading as absent, scanty, reduced,
normal or increased is also a practical approach.
Need adequate positive control for accuracy
May have heterogeneous iron deposition in BMA
Formalin fixation/histologic processing/decalcification
may interfere with staining of iron stores (ferritin)

55. Grading of bone marrow storage iron
0 No stainable iron
1+ Small iron particles just visible in
reticulum cells using an oil objective
2+ Small, sparse iron particles in reticulum
cells, visible at lower power
3+ Numerous small particles in reticulum cells
4+ Larger particles with a
tendency to aggregate into clumps
5+ Dense, large clumps
6+ Very large clumps and extracellular iron

56. Perl’s stain
sideroblast
Perl’s stain
Macrophage with iron
Ringed sideroblast

57. ,CD41

58. Majorpatternsof theIHCstainingin formalin-fixed andparaffin-embedded tissuesections

59. MPO showing distribution of normal myeloid cells
CD79a showing distribution of normalB cells
CD20
CD3 showing distribution of normalT cells
CD34 showing positivity of endothelial cells
CD34showing very occasional positive cells

60. Pathologic Key Features Of Pediatric Bone
Marrow Interpretation
• Interpretation of pediatric bone marrow samples requires that the
pathologist integrate clinical, morphologic, and ancillary study results
to arrive at the correct diagnosis.
• The morphology of normal pediatric bone marrow overlaps
significantly with that of adults.

61. • The evaluation of pediatric bone marrow poses specific challenges
when compared with the general adult population.
• These challenges stem in part from the higher likelihood of congenital
disorders with hematopoietic manifestations, some of which may give
rise to hematologic malignancies.
Pathologic Key Features Of Pediatric Bone
Marrow Interpretation

62. • Congenital syndromes and florid reactive processes must always be
considered in the differential diagnosis of hematologic malignancies
in the pediatric population
• Recognition of the general pathologic pattern (hypercellular,
hypocellular, or single-lineage defect) aids in narrowing the
differential diagnosis when interpreting bone marrow samples from
pediatric patients.
Pathologic Key Features Of Pediatric Bone
Marrow Interpretation

63. • Two features are significantly different and may affect bone marrow
interpretation: the overall cellularity and the presence of benign
lymphoid precursors (hematogones), which may be markedly
increased in some patients.
• Also it is important to realize that a large proportion of the benign
and malignant disorders diagnosed during childhood are likely to be
related to underlying genetic abnormalities, sometimes as the first
manifestation of these defects
Pathologic Key Features Of Pediatric Bone
Marrow Interpretation

64. • Finally, certain types of malignant neoplasms with partially
overlapping blastic morphologic features (so called “small blue-cell
tumors”), but with vastly different biology requiring distinct
therapeutic approaches, occur relatively frequently in young patients.
Pathologic Key Features Of Pediatric Bone
Marrow Interpretation

65. Pitfalls Of Pediatric Bone Marrow
Interpretation
• Benign B-cell precursors (hematogones) can occur in significant
numbers in the bone marrow of children with cytopenias of
nonneoplastic causes and may be mistaken for involvement by B-
lymphoblastic leukemia.

66. Pitfalls Of Pediatric Bone Marrow
Interpretation
• Benign B-cell precursors (hematogones) often represent a significant
proportion of the cells in hypocellular marrow aspirates from acute
megakaryoblastic leukemia (with marrow fibrosis) potentially leading
to a misdiagnosis of B lymphoblastic leukemia.

67. Pitfalls Of Pediatric Bone Marrow
Interpretation
• B-lymphoblastic leukemias with a CD45-,CD20-, CD99+
immunophenotype may be mistaken for Ewing sarcoma, especially in
small bone or bone marrow biopsies

68. Pitfalls Of Pediatric Bone Marrow
Interpretation
• Hemophagocytic syndromes may be associated with peripheral T-cell
lymphomas; diagnostic lymphoma cells are sometimes present in
small numbers in the bone marrow aspirates and may be missed.

69. Pitfalls Of Pediatric Bone Marrow
Interpretation
• Variably prominent populations of polyclonal CD8+ T lymphocytes
with immunophenotypic aberrancies may be present in familial
hemophagocytic histiocytosis; they may be mistaken for peripheral T-
cell lymphoma.

70. Pitfalls Of Pediatric Bone Marrow
Interpretation
• Parvovirus B19 infections occurring in patients with constitutional red
cell disorders may cause erythroid hyperplasia and significant
dyserythropoiesis, resembling congenital dyserythropoietic anemia.

71. • Acute lymphoblastic leukaemia can be confused with small cell
tumors of childhood, e.g. neuroblastoma, Ewing’s tumour and other
primitive neuroectodermal tumours (PNET), rhabdomyosarcoma,
medulloblastoma and retinoblastoma.
• It should be noted that leukaemic lymphoblasts may fail to express
CD45, that neuroendocrine tumors can express PAX5 and that blast
cells in a large proportion of ALL patients express CD99, an antigen
commonly expressed in Ewing’s tumour/PNET.
Pitfalls Of Pediatric Bone Marrow
Interpretation

72. Pitfalls Of Pediatric Bone Marrow
Interpretation
• Terminal deoxynucleotidyl transferase (TDT) in addition to ALL,
positive in up to 15% of AML cases.
• It has been reported to be positive in a proportion of cases of
medulloblastoma and occasionally in other small cell tumors of
childhood

73. • Small cell tumors of childhood, e.g. those with heavily vacuolated
cells such as alveolar rhabdomyosarcoma, have been misdiagnosed
and even treated as Burkitt lymphoma
Pitfalls Of Pediatric Bone Marrow
Interpretation

74. Pitfalls Of Pediatric Bone Marrow
Interpretation
• It should be noted that, in children, apparent aplastic anemia may
represent an aplastic presentation of ALL.

75. Pitfalls Of Pediatric Bone Marrow
Interpretation
• Acute megakaryoblastic leukemia (AMKL), which may yield clusters of
cohesive cells mimicking metastatic solid tumors in some cases
• Some lymphoblastic leukemias with near-tetraploid DNA content may
also show very large blasts reminiscent of solid tumor cells.

76. Pitfalls Of Pediatric Bone Marrow
Interpretation
• Some leukemias (especially hyperdiploid B-ALL and AMKL) may be
accompanied by significant marrow fibrosis, leading to a paucity of
neoplastic cells available for ancillary studies in the aspirate samples

85. CONCLUSION
• Integration of clinical, morphologic, immunophenotypic, genetic, and
other biologic features is mandatory to define specific disease entities
• The relative contribution of each feature varies, depending on the
case.
• Make your cytologists/pathologists/geneticians good ! by
providing them relevant clinical information's and optimal samples.