Bone marrow interpretation

1. BONE MARROW BIOPSY AND ASPIRATION
MODERATOR: Dr. C.J. PRAKASH
PRESENTER: Dr. SPOORTHY

2. INTRODUCTION
Bone Marrow Examination provides a Semi-Quantitative
and Qualitative assessment of the state of Haemopoiesis
Biopsy of Bone Marrow is an indispensable adjunct to the
study of diseases of blood
It may be the only way a correct diagnosis can be made

3. HISTORY
1905 Pianese
Trephine bx in an
infant with
Leishmaniasis.
1909 Pianese
Tibia & femur marrow
aspiration with
attached syringe.
1923 Seyfaith
Surgical trephine to obtain marrow
from ribs & sternum.But there was
excessive bleeding.
1927 Airinkin
Eliminated trephine complication
by using short lumbar needle.
1945 Vandenberg
First obtained marrow
from iliac crest.
Used posterior Iliac

4. STRUCTURE OF BONE
Bone Cortex
Medulla
Cortex:
Strong layer of Compact bone
Gives bone strength
Made of Lamellar bone
contains well organised Haversian
canals

5. MEDULLA
Made up of Cancellous bone or Trabeculae
Trabeculae and inner surface of cortex are lined by
Endosteal cells
Osteoblasts
Osteocytes
Osteoclasts

7. PARENCHYMA:
Haematopoiteic stem cells and Precursors
Mature cells of Erythroid, Myeloid and Megakaryocytic cells
STROMA:
Fat cells, Histiocytes,Fibroblasts,Blood vessels, Intercellular
Matrix

8. BONE MARROW
Normal Bone Marrow
Red Marrow
Yellow Marrow
Red Marrow: Haematopoietic cells
Yellow Marrow : Adipose tissue
Red bone marrow consists of 4% of Total bone marrow
The weight of total bone marrow ranges from- 1600-
3700gm

10. Children: most bones contain Haematopoietic cells
Adults- 1. Skull
2. Sternum
3. Scapulae
4. Ribs
5. Pelvic Bones
6. Proximal ends of long bones

11. PRINCIPLE
The Morphological assessment of aspirated or core biopsy
specimens is based on these principles
1. Bone Marrow has an organised structure
2.In normal health bone marrow cells display distinct
numerical and spatial relationships with each other
3.Individual marrow cells have distinct cytological
appearances
4. This reflects the lineage and stage of maturation
5.Each or all of these may be specifically disordered in a
disease

13. INDICATIONS
ANEMIA
1. MICROCYTIC ANEMIA:
Evaluation of Iron stores and Sideroblasts: allows categorisation of
anaemia
2. MACROCYTIC ANEMIA:
To confirm whether the process is Megaloblastic or not
3.NORMOCYTIC ANEMIA: without an increase in retic count
For quantitative or qualitative abnormalities of Erythropoiesis

14. DIAGNOSIS AND STAGING OF:
Non hodgkin’s lymphoma
Hodgkin’s lymphoma
Malignancy
Metastatic carcinoma
Small round cell tumors of childhood
STROMAL CHANGES:
Fibrosis
Necrosis
Gelatinous marrow transformation

15. • To DIAGNOSE
Aplastic Anaemia
Hypoplastic Myelodysplastic syndrome.
Hypoplastic Leukemia.
• LYMPHOPROLIFERATIVE DISORDERS
Hairy cell leukemia
CLL
• MYELOPROLIFERATIVE DISORDERS

16. NEUTROPENIA,
THROMBOCYTOPENIA,PANCYTOPENIA:
To assess the presence and normality of precursor cells
To assess the probability of decreased production, impaired
maturation or increased destruction
CYTOPENIA
To reveal the presence of leukaemia or another
Haematological neoplasia

17. • Unexplained leukoerythroblastic picture.
• In suspected cases of multiple myeloma and serum paraproteins.
• Pyrexia of unknown origin
• Focal lesions –Metastasis, Granuloma
• Amyloidosis
• Metabolic bone diseases
• To assess the mineralisation front and appositional growth after
tetracycline labelling

18. CONTRAINDICATIONS
🞪BIOPSY IN COAGULOPATHIES
(For aspiration : factor replacement therapy prior to procedure and
observation should be done for next 24-48 hrs.)
🞪STERNAL ASPIRATE - OSTEOPOROSIS AND CHILDREN

19. SITES
1. Sternum
2. Anterior Iliac spine
3. Posterior Iliac spine: 1.overlies a large marrow space
2. Larger samples can be obtained
4.Upper end of Tibia- Children < 1year old

21. STERNUM
Usual sites:
1. Manubrium
2. 1st and 2nd parts of Body of Sternum
Precaution: Appropriate Guard is supposed to be used
Danger of perforating the Inner cortical layer and Damaging
the underlying large blood vessels
Right atrium

22. Klima Sternal Needle Salah Bone Marrow Aspiration
Watherfield Iliac Crest Bone Aspiration
Modern Jamshidi Needle

25. PROCEDURE
Consent: A written consent should be taken from
patient.
• An appropriate clinical history should accompany the
bone marrow, as they relate to possible findings within
the bone marrow examination.

26. 🞪Itis useful to know relevant laboratory data such as
Iron studies, Folate or Vitamin B-12 studies,
transfusion therapy, or history of chemotherapy.
🞪
🞪The physician’s clinical impression should be included
on the form.
🞪Lignocaine sensitivity test should be done.

28. COMPLICATIONS
🞪Haemorrhage
🞪
🞪Pain
🞪Infection
🞪Perforation of major vessels
🞪Risk of general anaesthesia and sedation.

29. ASPIRATION BIOPSY
Better cytological detail
Topographical details, cellularity
and infiltration
More range for Cytochemical
stains, Flow cytometry. IHC
Ideal for Cytogenics and Molecular
Genetics
Dry tap in fibrosis
Less painful
Less Range
Can be used for both
Essential for diagnosis in Dry tap
More painful

30. ASPIRATE
Smears should be made without delay at the bedside
Remaining material should be delivered into a bottle
containing EDTA
Preservative free Heparin should be used if
Immunophenotyping or Cytogenetic studies are needed.
Some material can be fixed in Fixative rather than
anticoagulant for preparing histological sections
Some films should be fixed in Absolute Methanol for
subsequent staining by Romanowsky Method or Perl’s stain

31. • Appropriate amounts of
anticoagulant for the volume
of marrow to be
anticoagulated are used
• Gross excess of
anticoagulant: masses of
pink-staining amorphous
material may be seen
• Clumping of some
erythroblasts and
reticulocytes may be seen

32. CENTRIFUGATION
Centrifugation can be used to concentrate the Marrow cells
To assess the relative proportion of Marrow cells,
Peripheral blood and Fat in Aspirated material
Useful : 1. Poorly cellular sample
2. Abnormal cells are present in small numbers

33. Marrow Stim Device
PLASMA
Nucleated cells
RBC

34. DIRECT FILMS
A drop of marrow is placed on a slide a short distance away
from one end
A film 3-5cm is made with a spreader, not wider than 2cm
Dragging the particles behind them but not squashing them.
A trail of cells is left behind each particle

37. CRUSH PREPARATIONS
Marrow particles in a small drop
of aspirate is placed on a slide at
one end
Another slide is placed on the first
Slight pressure is exerted to crush
the particles and slides are
separated by pulling them apart in
a direction parallel to their
surfaces

38. IMPRINTS
Marrow particles can also be used for preparation of imprints
One or more particles are picked up capillary pipet
Transferred immediately to a slide and made to stick to it by a
gentle smearing motion.
The slide is air dried rapidly by waving, then it is stained

40. ADEQUACY OF BIOPSY
Length-1.6cm ( 1.5- 2.5cm)
25% shrinkage during processing
5-6 Trabecular spaces
Good quality Staining

44. INTERPRETATION

45. STAINING OF SECTIONS
Bone marrow sections are routinely stained with
Haematoxylin and Eosin
It is excellent for demonstrating the cellularity and pattern of
the Marrow.
This reveals the pathological changes such as presence of
Granuloma or Carcinoma cells
Haematopoietic cells may be more easily identified in a
Romanowsky stained preparation

46. Other stains that are usually done are:
1. PERL’S STAIN - IRON
2.SILVER IMPREGNATION METHOD -
RETICULIN
Both Plastic and Paraffin embedded specimens
can be used for IHC

47. Periosteal
Connective
Tissue
Bony
Trabeculae

48. TB- NORMAL BONE SECTION

49. CELLULARITY
Expressed as a ratio of volume of Hematopoietic cells to the
total volume of marrow spaces
It is best judged by Histological sections of biopsy or aspirated
particles
Can also be estimated from the particles present in the Marrow
films
Done my comparing the areas occupied by Fat spaces and By
Nucleated cells
Also by the density of Nucleated cells in the Tail or fallout of
particles

50. Cellularity varies with the age of the subject and the site
50 years of age : Vertebrae : 75%
Sternum : 60%
Iliac crest : 50%
Rib : 30%
If the percentage is increased for patients age: Hypercellular
If the percentage is decreased for patients age : Hypocellular

51. NORMAL CELLULARITY
Hyper cellular Hypo cellular

52. SYSTEMATIC SCHEME FOR EXAMINATION OF BONE MARROW ASPIRATE
LOW POWER:
Determine Cellularity
Identify Megakaryocytes
Note Morphology and Maturation Sequence
Look for clumps of abnormal cells: METASTATIC TUMOUR
Identify Macrophages

54. HIGHER POWER:
Identify all stages of maturation of Myeloid and Erythroid
cells
Determine the Myeloid:Erythroid ratio
Perform Differential count: Erythroid,
Myeloid,Lymphoid,Plasma cells and Others
Look for areas of bone marrow Necrosis
Assess the Iron content of the Macrophages
Look for Iron granules in Erythroid cells: Perl’s stain

55. 95% RANGE MEAN[12] MEAN[11]
Myeloblasts 0–3 1.4 0.4
7.8 13.7[*]
7.6 –
Promyelocytes
Myelocytes (N)
Metamyelocytes
3–12
2–13
2–6 4.1 –
32.1M; 37.4W 35.5
1.3 1.6
2.2 1.7
0.1 0.2
13.1 16.1
1.3 2.5
Neutrophils
Myelocytes (E)
Eosinophils
Basophils
Lymphocytes
Monocytes
Plasma cells
22–46
0–3
0.3–4
0–0.5
5–20
0–3
0–3.5 0.6 1.9
28.1M; 22.5W 23.5
0.5
Erythroblasts[†]
Megakaryocytes
Macrophages
5–35
0–2
0–2 0.4 2.0
Normal ranges for differential counts on aspirated bone
marrow (500 cells should be counted)

56. ERYTHROID CELLS
MYELOID CELLS
Para Trabecular
Mature cells
MEGAKARYOCYTES
Centre around Sinusoids
Centre in Colonies
STROMA
Fat cells
Reticulin Fibres
Fibroblasts
Macrophages

57. ASPIRATION- ERYTHROID ISLAND

58. H&E- ERYTHROID ISLAND

59. Maturation sequence of RBC

60. Maturation sequence- Neutrophils

61. Myelocyte
Pro
Myelocyte
Myeloblast

62. EOSINOPHILIC PRECURSORS

63. MATURATION SEQUENCE OF MEGAKARYOCYTES

64. MEGAKARYOCYT
E

65. BUDDING MEGAKARYOCYTES

67. OSTEOBLASTS
1. Extruding nuclei
2. 1-4 Nucleoli
3. Regular Chromatin
4. Position of Golgi Zone

68. OSTEOCLASTS
1. Highly Granular Cytoplasm
2 . Multiple Nuclei (2-100)
3. Single Nucleoli

69. PLASMA CELLS

70. BONE MARROW-NECROSIS

71. ASSESSMENT OF IRON STORES

72. PERL’S STAIN
Also called Prussian blue stain
Demonstrates Haemosiderin in Bone marrow Macrophages
and Erythroblasts
Reticulo-Endothelial cells
Hence it allows the assessment of Iron
Developing Erythroblasts

73. REQUIREMENTS
Assessment of storage iron requires that an adequate
number of fragments are obtained
A Minimum of 7 Bone marrow fragments in one or more
bone marrow films are needed to be examined.
To state that the bone marrow Iron is reasonably absent
A Bone marrow film or Crush preparation will contain both
Intracellular and Extra cellular Iron
It is basic to count only Intra cellular iron

74. ASSESSMENT
Iron stores may be assessed as: NORMAL, DECREASED,
INCREASED
May be graded as +1 - +6
Where +1 - +3 is regarded as Normal
A Proportion of Normal Erythroblasts have few(1-5) fine
iron containing granules randomly distributed in the
cytoplasm
These are called SIDEROBLASTS

75. GRADING OF IRON STORES
0 NO STAINABLE IRON
1+
SMALL IRON PARTICLES JUST VISIBLE IN
RETICULUM CELLS UNDER OIL IMMERSION
2+
SMALL IRON PARTICLES VISIBLE IN
RETICULUM CELLS UNDER LOW POWER
3+
NUMEROUS SMALL PARTICLES IN
RETICULUM CELLS
4+
LARGER PARTICLES WITH A TENDENCY TO
AGGREGATE INTO CLUMPS
5+ DENSE LARGER CLUMPS
6+
VERY LARGE CLUMPS AND EXTRA
CELLULAR IRON

76. NORMAL IRON STORES
NO STAINABLE IRON

77. GEIMSA STAIN
PERL’S STAIN

78. ASSESSMENT OF
RETICULIN

79. RETUCULIN STAIN
Histological sections can be stained by Silver Impregnation
Method for Reticulin
For Collagen- Trichrome stain
Reticulin is closely concentrated more around the blood
vessels and bony trabeculae
Hence these areas should be disregarded while grading

80. BAUERMEISTER GRADING
0
No Reticulin Fibres
Demonstrable
1
Occasional fine Individual fibres
and foci of a fine fibre network
2
Fine fibre network throughout.
No coarse fibres
Diffuse fibre network with
3 scattered thick coarse fibres but
no mature collagen
Diffuse often coarse fibre
4 network with areas of
collagenization

81. Many normal subjects may have Reticulin grade 0-
+1
Some may even have a grade of +2
There is a tendency of reticulin to be deposited
more in Iliac crest or than in the Sternum

82. GRADE-1
GRADE-2

83. GRADE-3
GRADE-4

84. OTHER STAINS USED
Chloroacetate esterase
Identification of Granulocyte
differentiation and Mast cells
PAS
Staining of complex
carbohydrates, identification of
fungi
Toulidine blue Identification of Mast cells
Congo Red Identification of Amyloid
Z-N stain Identification of Mycobacteria

85. BONE MARROW- INFECTIONS

86. PARVO B19 INFECTION
Giant Pro Erythroblasts

87. HIV- Histoplasma
HIV-Cryptoccocus

88. HIV- CHANGES
Increased Cellularity
Increased fibrosis
Lymphoid aggregates

89. LEISHMANIASIS

90. TB- GRANULOMA
ACID FAST BACILLI

91. GAUCHERS DISEASE
Tissue like
crumpled
cytoplasm

92. SUPPLEMENTARY
INVESTIGATIONS
1.Immunohistochemistry- for demonstration of antigens in
biopsy
Ex.- CD 34, CD 45, Lysozyme, MPO, CD 68, CEA etc.
2. Cytogenetic analysis- for chromosomal rearrangements
3. Molecular genetics- by PCR, RTPCR
4. FISH

93. REFERENCES
1. Barbara Bain-Bone Marrow
2. Dacie and Lewis
3.Wintrobes
4.Mckenzie
5.Henry Laboratory Methods

94. THANK YOU