Approch to bone marrow examination-PATHOLOGY

1. APPROCH TO BONE
MARROW EXAMINATION
Dr Shashi Bansal
Consultant Hemato-Pathology
BMCHRC, Jaipur

2.  Bone marrow examination is an
indispensable adjunct to the study of
diseases of the blood and may be the
only way in which a correct diagnosis can
be made.

3.  HISTORY
 G/P/E
 LN-….,S…..,L……
 CBC ,PBF,R/C
 BMA,IRON
 IMPRINT,SPECIAL STAIN
 BIOPSY,RETIC
 IHC
 FLOW
 CYTOGENETIC
 FISH
 RT-PCR
 NGS

4. 1. Bone marrow
aspiration
2. Clot section
3. Bone marrow biopsy
4. Biopsy imprint
smears

5. Aspiration biopsy and trephine biopsy are
complementary to each other.
Better cytological detail.
More range for
cytochemical stains,
flowcytometry and IHC.
Ideal for cytogenetics
and molecular genetics.
Topographical details,
cellularity and
infiltration.
Less range.
Can be used for both.

6. Dry tap in fibrosis
Can be performed alone in
iron deficiency anemia,
anemia of chronic
disease , megaloblastic
anemia and acute
leukaemia.
Less painful
Essential for diagnosis
in dry tap.
Helpful for aplastic
hypoplastic anaemia,
lymphoma, metastatic
carcinoma,
myeloproliferative
neoplasms and
diseases of the bones.
More painful

7. Gives cytological details when aspirate is not
obtained.
Shows more neoplastic cells than aspirate.
Can show marrow infiltration , not seen in
aspirate

8. Assessment of bone marrow cellularity.
For detecting granuloma and tumour infiltrates
complementary to biopsy.
No decalcification associated nucleic acid or
protein damage.

11. Low power (10x)
Determine cellularity
Identify megakaryocytes and note
morphology and maturation sequence
(higher power may be needed for smaller immature megakaryocytes and
micromegakaryocytes).
Look for clumps of abnormal cells
(higher power needed to examine content and morphology of clumps)
Identify macrophages
(higher power for evidence of haemophagocytosis, malaria pigment, and
bacterial or fungal infections that may be present in the cytoplasm)

12. High power (40x
Identify all stages of maturation of myeloid
and erythroid cells
Look for areas of bone marrow necrosis
Assess the iron content

13. High power (100x oil immersion)
Perform a differential count using the
categories erythroid, myeloid, lymphoid, plasma
cell, and “others,” simultaneously noting any
morphological abnormalities.
Maturation abnormalities are noted
Determine the myeloid:erythroid ratio

14. Area for myelogram near fragment

17. Cellularity of fragments is of more importance than the
cellularity
of trails,
Fragments should be examined not only to assess cellularity but
also
to determine if any cells have been preferentially retained in the
fragments, e.g. mast cells or myeloma cells

18. If an aspirate is partly clotted, small bone marrow
clots may be mistaken for bone marrow particles,
leading to a mistaken attempt to assess cellularity
or the presence or absence of storage iron in the
clot.
The presence of fibrin strands and the lack of
any organized structure of the apparent particle is
a clue to its true nature.
In patients with essential thrombocythaemia, solid
clumps of large numbers of platelets can also be
mistaken for bone marrow fragments.

19. Hypercellular for adult and normocellular for
child

20. Hypocellular bone marrow (10-20%)

21. The cell count should be performed in
the trails behind fragments so that the
cells counted represent cells that have
come from fragments rather than
contaminating peripheral blood cells.

22. Haemopoietic cells

23. Erythroid cells

24. Erythroid island on aspirate. Macrophage is surrounded by
developing normoblast, hemosiderin form of iron is stored in it.

26. (i) in normal bone marrow they(Erythroid island)
occur in distinctive erythroblastic islands
containing several generations of cells of varying
size and
maturity
(ii) erythroblasts adhere tightly to one another
(iii) their nuclei are round
(iv) in late erythroblasts the chromatin is
condensed in a regular manner whereas nuclei of
small lymphocytes show coarse clumping.

27. Myelocyte
Pro
Myelocyte

28. Bone marrow (BM) aspirate fi lm from a patient with
severe infection showing heavy toxic granulation and
vacuolation of neutrophil precursors.

29. Mature megakaryocyte having loblated nucleus and pink granular
cytoplasm..platelets are formed by budding of the cytoplasm which are
shed in the circulation.

30. It should be noted that whether or not
megakaryocytes appear to be producing platelets
shows little correlation with the number of
platelets being produced

31. Aspirate of non - infiltrated BM from a patient with Hodgkin lymphoma: a mature
megakaryocyte exhibiting emperipolesis. MGG× 100.

33. When haemopoiesis is normal, megakaryocytes do not
form clusters of more than two or three cells.
Larger clusters of megakaryocytes are seen in
regenerating marrow, following chemotherapy and bone
marrow transplantation, and also in variouspathological
states; this feature is diagnostically useful.

35. Plasma cell has an eccentric nucleus,cartwheel nuclear chromatin,basophilic cytoplasm
with perinuclear hoff.
Present in pericapillary location.

36. Plasma cells present in pericappilary region

37. 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.

38. Section of BM from a patient with Fanconi anaemia: the trabecula is
lined by osteoblasts; note the distinct Golgi zones which do not abut
on the nuclear
membrane. Resin - embedded, H & E × 40.

39. An osteoclast; note the highly granular cytoplasm and the multiple nuclei (2-
100)which are uniform in size and have indistinct, medium - sized, single nucleoli.
MGG × 100.

41. Mast cells on bone marrow.
With PAS stain they display magenta coloured granules

42. Aspirate of normal BM: a macrophage containing granular and refractile debris
and several normoblast nuclei. MGG × 100.

43. Fat Cells

44. ERYTHROID HYPERPLASIA
The marrow reveals greatly increased numbers of maturing
erythroid progenitors (normoblasts)

45. The bone marrow imprint smear on low power showing
optimum cellularity

46. Gelatinous Transformation

47. Bone Marrow Necrosis

49. 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

50. Grade 1 Grade 2 GRADE 3
Grading for iron on bone marrow aspirate
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

51. Iron grading
4+ Larger particles with a tendency to aggregate into clumps
5+ Dense, large clumps
6+ Very large clumps and extracellular iron
GRADE 4 GRADE 5 GRADE 6

52. Perl’s stain
sideroblast

53. Macrophage with iron
Perl’s stain

54. Ringed sideroblast

55. In certain pathological conditions, plasma cells
contain haemosiderin inclusions, which are
irregular in shape and relatively large. With an
MGG stain they are greenish black
Their nature is confirmed by a Perls ’ stain .
Haemosiderin inclusions in plasma cells are
observed mainly in iron overload (for example in
haemochromatosis and transfusional siderosis)
and in chronic alcoholism

56. Plasma Cells - with Prussian Blue
Stain

57. Length at least 1.5 cm
At least 10 preserved trabecular spaces seen
Sections of 3-4 micron in thickness cut at a
distance of 50 micron each.

58. 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

59. 4x or 10x
Adequacy
Cellularity
Pattern
Presence of focal lesions
Megakaryocyte number
Abnormal cell clusters and location
Bone structure
Osteoclastic and osteoblastic activity.

60. 40x
Oil immersion
For finer cytological details (eg.intracellular
granules, organisms.)

61. Myeloid cells
Paratrabecular
Mature cells towards centre
Erythroid cells
Centre in colonies
Megakaryocytes
Centre around sinusoids

62. Usually not seen.
Some can be seen randomly distributed.
Lymphoid precursors:
Seen in periarteriolar region.
Stroma
Fat cells, fibroblasts, reticulin fibres

63. Topography of cells
Bone marrow is highly organised structure with haemopoietic
elements maturing in different micro-anatomical sites.

64. Normal bone marrow cellularity
In adults

65. If the pelvis has previously been irradiated,
biopsies will show bone marrow hypoplasia or
aplasia which isnot indicative of the appearance
of the bone marrow at other sites.
Specimen includes only subcortical
bone, which is often markedly hypocellular.
This can create a mistaken impression of aplastic
anaemia.

66. Trephine biopsy specimens from children may
contain cartilage as well as bone, and endochondrial
bone formation may be observed

67. Hypocellular bone marrow

68. Hypercellular bone marrow
In AML WITH MATURATION

69. Erythroid cells in the centre

70. Myeloblast and promyelocytes
paratrabecular

71. Megakayocyte in bone marrow
parasinusoidal

72. 0 No reticulin fibres demonstrable
1 Occasional fine individual fibres and foci of
fine fibre network
2 Fine fibre network throughout most of the
section; no coarse fibres
3 Diffuse fibre network with scattered thick
coarse fibres but no mature collagen
4 Diffuse often coarse fibre network with areas
of collagenization

73. grade 0
Fine fibre network
throughout most of
the section; no
coarse fibres
Occasional fine individual
Ffibre
grade 2,

74. Diffuse often coarse fibre network withgrade 4,
areas
grade 3 Diffuse fibre network with
scattered thick coarse fibres but
no mature collagen
,

76. If slides are fixed before they have dried adequately there is an
appearance suggesting that nuclear contents are leaking into the
cytoplasm and cellular outline is indistinct.
Delayed fixation and staining of archival bone marrow slides
usually leads to a strong blue or turquoise tint to the film

77. Crystals of glove powder in a BM aspirate.
MGG

78. BM trephine biopsy section, crushed bone.
Paraffin -embedded, H & E × 20

79. BM trephine biopsy section from a patient with
chronic
lymphocytic leukaemia showing torsion artefact.
Paraffin - embedded, H & E × 20.

80. BM trephine biopsy section showing megakaryocytes
surrounded by an empty space as a consequence of
shrinkage artefact.
Paraffin - embedded, H & E × 40.

81. Tissue from other biopsy specimens can
contaminate the water bath in which sections are
floated prior to mounting on glass slides.
Examination of reticulin stains can be helpful if there
is doubt as to whether or not abnormal tissue is an
intrinsic part of the biopsy specimen
Floater

82. Washed off marrowspaces

83. BM trephine biopsy specimen showing an
artefact caused by using a blunt knife.
Paraffin -embedded, H & E × 40.

84. BM trephine biopsy showing a piece of epidermis
which has been driven into the biopsyspecimen.
Paraffin - embedded, H & E× 50

85. A hair follicle and ducts of a sweat gland which
have been driven into a trephine biopsy specimen.
Paraffin - embedded, H & E× 10.

86. BM trephine biopsy specimen showing a needle
track from a bone marrow aspiration performed
immediately before the trephine biopsy.
Paraffin - embedded,H & E × 4

87. BM trephine biopsy specimen showing a linear
scarresulting from damage by a previous biopsy
at the same site. Paraffin -embedded, H & E

88. • D/D of hypoplasia/aplasia
• D/D of megaloblastichemopoiesis
• Assessingkey histological features of myelodysplastic
and myeloproliferative haemopoiesis
• D/D of bone marrowfibrosis
• Assessingpatterns of lymphoid infiltration associated
with various lymphomas, especially small B-cell
lymphomas
• D/D of granulomatouspathologies