Bone marrow examination

1. BONE MARROW EXAMINATION
Presentor- Dr. Manju kumari
Moderator- Dr. A.S Nigam

2. BONE MARROW
 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. 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 Vanderberg First obtained marrow from iliac creast.
1952 Bierman Used posterior Iliac creast for biopsy
HISTORY

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

5. INDICATIONS OF BONE MARROW ASPIRATION
• Red cell disorders
• Leucocytic disorders
• Megakaryocyte and platelet disorders
• Myeloproliferative disorders and myelodysblastic
syndrome with BMB
• Paraproteinemias
• Infection
• Storage disorders
• Iron assessment
• Metastasis

6.  Unexplained heptosplenomegaly
 Marrow harvesting for transplant.

7. INDICATIONS OF BONE MARROW BIOPSY
1.To accurately assess marrow cellularity.
2. To diagnose
Aplastic anaemia
Hypoplastic myelodysplastic syndrome.
Hypoplastic leukemia.
3. Lymphoproliferative disorders
Hairy cell leukemia
CLL
4. Myeloproliferative disorders

8. 5. Unexplained leukoerythroblastic picture.
6. In suspected cases of multiple myeloma and serum
paraproteins.
7. Diagnosis and staging of
Non hodgkin’s lymphoma
Hodgkin’s lymphoma
Malignancy
Metastatic carcinoma
Small round cell tumors of childhood
8. Stromal changes
Fibrosis
Necrosis
Gelatinous marrow transformation

9. 9. Pyrexia of unknown origin
10. Focal lesions –Metastasis, Granuloma
11.Amyloidosis
12.Metabolic bone diseases
13.To assess the mineralisation front and appositional
growth after tetracycline labeling

10. CONTRADICATIONS
 Sternal aspirate-osteoporosis and children
 Biopsy in coagulopathies
(For aspiration factor replacement therapy
prior to procedure and observation should be
done for next 24-48 hrs.)

11. COMPLICATIONS
 Hemorrhage
(Risk factors- coagulopathies,
myeloproliferative disorders, aspirin and
warfarin therapy, thrombocytopenia, DIC, liver
disease and disease)
 Pain
 Infection
 Perforation of major vessels
 Risk of general anaesthesia and sedation.

12. BONE MARROW ASPIRATE V/S BIOPSY
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.

13.  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

14. IMPORTANCE OF TOUCH/ IMPRINT
SMEAR
 Gives cytological details when aspirate is not
obtained.
 Shows more neoplastic cells than aspirate.
 Can show marrow infiltration , not seen in
aspirate

15. IMPORTANCE OF CLOT SECTION
 Assessment of bone marrow cellularity.
 For detecting granuloma and tumour infiltrates
complementary to biopsy.
 No decalcification associated nucleic acid or
protein damage.

16. SITE FOR ASPIRATION
1. Posterior superior iliac spine- most preferred.
2. Anterior superior iliac spine. (The iliac spines have the
advantage that if no material is aspirated, a trephine biopsy can be
performed immediately.)
3. Sternum (abt 1cm above the sternomanubrial
angle,to one side of midline). (avoided in babies)
4. Medial aspect of tibia just below tibial tubercle
(small babies).
5. Spinous process of vertebrae

17. 1. Posterior superior iliac spine- most
preferred.
2. Anterior superior iliac spine.
The posterior iliac spine is said to provide samples that are
longer and larger, and the aspiration is less uncomfortable for
the patient.

18. Needles should be stout and made of hard
stainless steel, about 7-8 cm in length, with
a well-fitting stilette, and they must be
provided with an adjustable guard.
1. Jamshidi needle
2. Islam needle
3. Salah needle
4. Klima needle

19.  If larger specimens are needed, trephine
needles that have bores of 4-5 mm may be
used.
 Other needles occasionally used for trephine
biopsy specimens are a 2-mm bore
“microtrephine” needle and a Vim–Silverman
needle.
 However, compared with other needles, these
yield smaller specimens of marrow that are
prone to fracturing.

20.  a: STYLET b : BORE c : PROBE
 The tip gradually widens for next 2cm and then uniform bore the needle.The
marrow biopsy does not get compressed because of narrow cutting edges and
hence cell morhology is well made out.
JAMSHIDI NEEDLE

21. ISLAM
NEEDLEA modified version of the Islam needle has multiple holes in the distal
portion of the shaft in addition to the opening at the tip to overcome
sampling error when the marrow is not uniformly involved in a
pathological lesion.

22. B. Klima needle
These are most common
reusable needles.
A.Salah needle

23. PROCEDURE
Consent- an 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.

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

25.  Either aspirate or biopsy may be performed first.
 But the two should be performed through the
same incision, approx. 0.5-1 cm away from the
other.
 This is done to avoid clotting of aspirate and
hemorrhagic or damaged biopsy.
 It is recommended that the aspirate and biopsy
be obtained using respective needles separately,
and not through a trephine needle.

26. • Operator should wear Surgical gloves
• Skin at the site should be cleaned with 70%
alcohol or 0.5% chlorhexidine.
• Infiltrate the skin, s/c tissue and periosteum
over the site with 2-5ml of lignocaine.
• Children are either sedated or given general
anaesthesia.

27.  With boring movement pass the needle
perpendicularly at center of PSIS.
 When bone has been penetrated, remove the
stilette, attach 30 ml syringe and suck up
marrow contents (0.3ml) for making smears
immediately.

28.  If large sample is needed for cytogenetics and
immunophenotyping attach another 5 or 10 ml
syringe and aspirate.
(should be kept in preservative free heparin than in EDTA)
• Fix the slides in absolute methanol (20 min) as
soon as they are thoroughly dry for subsequent
staining by a Romanowsky method or Perls' stain
for iron.

29.  The preparation can be considered satisfactory
only when marrow particles and free marrow
cells can be seen in stained films.
 These films are also suitable for cytochemical
staining.

30.  Some material (clots) can be preserved in
fixative rather than anticoagulant for preparation
of histological sections.
(clot section).
 Clot is processed same as bone marrow biopsy
but without decalcification
• Peripheral blood sample is also taken along with
it.

31. • Dry tap- Failure to aspirate marrow (suggests
bone marrow fibrosis or infiltration.)
• If there has been a “dry tap,” insert the
stilette into the needle and push any material
in the lumen of the needle onto a slide; in
lymphomas and carcinomas, especially,
sufficient material can be obtained to make a
diagnosis.
• CT–guided marrow sampling-obese, in whom
it is difficult to locate the iliac spine.

32. Look for
marrow
fragments
while making
the smears

33. EXAMINATION OF ASPIRATED BONE
MARROW
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)

34. High power (40x,100x oil immersion)
Identify all stages of maturation of myeloid
and erythroid cells
Maturation abnormalities are noted
Determine the myeloid:erythroid ratio
Perform a differential count using the
categories erythroid, myeloid, lymphoid, plasma
cell, and “others,” simultaneously noting any
morphological abnormalities.
Look for areas of bone marrow necrosis
Assess the iron content

35. Area for myelogram near fragment

36. Cellurarity

37. Normocellular bone marrow on aspirate

38. High cellularity of 95-100%

39. Hypercellular for adult and normocellular for
child

40. Cellularity of 40-50%

41. Hypocellular bone marrow (10-20%)

42. 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)

43. Myeloid series of cells

44. Erythroid cells

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

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

47. Plasma cells present in pericappilary region

48. 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.

49. 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.

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

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

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

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

54. 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

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

56. 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

57. Perl’s stain
sideroblast

58. Macrophage with iron
Perl’s stain

59. Ringed sideroblast

60.  Example of
report form for
bone marrow films

61. BIOPSY (Procedure)
• The trephine specimen is obtained by inserting
the biopsy needle into the bone and using a to-
and-fro rotation to obtain a core of tissue.
• If an aspirate has been performed first the
needle should be inserted through the same
incision but the needle should be advanced at a
slightly different angle.

62. • The bony core is gently dabbed or rolled
across the slide to form imprint smears, which
is then fixed and stained as for bone marrow
aspiration smears.
This allows immediate examination of cells that fall out of the
specimen onto the slide and may provide a diagnosis several
days before the trephine biopsy specimen has been
processedaspirate. and also useful in dry
• Bilateral trephine biopsies may be performed
to increase the yield of detecting focal lesions.

63.  Length at least 1.5 cm
 At least 10 partialy preserved trabecular
spaces seen
 Sections of 3-4 micron in thickness cut at a
distance of 50 micron each.
(WHO Classification of tumours of
haematopoietic and lymphoid tissue 2008)

64. FIXATION:
 Biopsy is fixed in 10% neutral buffered
formalin for 6 hrs.(ICSH preferred)
 Other fixatives: Zinc formaldehyde
Acetic acid formalin
Isotonic buffered formalin
 B5: not used due to presence of mercuric
chloride (poisonous)
 Bouin’s fixative: not used in some countries,
due to presence of picric acid.

65.  It is important to ensure that the formol -
saline is not left for long periods at ambient or
high temperature before being used because
formic acid and formalin pigment may be
produced.
 The reactivity of antibodies used for
immunohistochemical staining may also be
affected by the choice of fixative (use of
Bouin’s solution is particularly limiting) and
Zenker’s fixative can destroy chloroacetate
esterase activity.

66.  B5 fixative- if fixation lasts for more than 6
hours hardening of the tissue can make it
difficult to cut sections.

67. Decalcifier agent volume to tissue volume should
be 20:1
1. 10-15% EDTA( 1-3 days) preserves morphology,
enzymes and immunological epitopes. ICSH
recommended
2. 10% Formic acid( 2-3 days) causes more tissue
distortion.

68. 3. 5-10% nitric acid(3-6 hrs) used for urgent
processing. (nitric acid can cause
megakaryocytes to give positive reactions
with antibodies to CD34 and affect IHC.)
4. In our lab-EDTA.

69. DECALCIFICATION :PITFALLS
 It chelates storage iron.
 Affects the morphology and cytological details.
 Affects ability to perform immunohistochemistry
and to retrieve material for molecular analysis.

70. Factors affecting decalcification
 Conc. of decalcifying agent.
 Temperature
 Agitation
 Age of patient
 Type of bone
 Size of spicules

71. Decalcification endpoint
 Specimen radiography (FAXITRON,most
reliable)
 Chemical method
Calcium oxalate test
(5ml of used decalcifying agent+Ca(OH)+ 5
mlsat. ammonium oxalate)
 Physical method
 Bubble test

72. :
Causes shrinkage and loss of some cellular
detail but better for IHC.
:
Glycol methacrylate or methyl methacrylate
used
 Finer sections with better cellular details.
 No decalcification required so used for
metabolic bone diseases.

73. SECTIONING
 At least 6 sections should be cut at 3 levels
into cross sectional diameter of core-
25%
50%
75%
Additional sections needed for IHC or
histochemical stains

74. 1. H&E
2. GIEMSA-helpful for plasma cells,mast
cells,lymphoid cells,eosinophils,myeloblasts and
proerythroblasts.
3. RETICULIN(silver impregnation)
4. TRICHROME
5. PERL STAIN
6. OTHERS: toluidine blue, congo red,ziehl
neelson,PAS

75. EVALUATION OF BONE MARROW BIOPSY
4x or 10x
 Adequacy
 Cellularity
 Pattern
 Presence of focal lesions
 Megakaryocyte number
 Abnormal cell clusters and location
 Bone structure
 Osteoclastic and osteoblastic activity.

76. 40x
 Assess haemopoietic cells(erythroid, myeloid,
megakaryocytes, lymphoid cells, plasma cells
and macrophages) and cytological details.
Oil immersion
 For finer cytological details (eg.intracellular
granules, organisms.)

77. Normal topography
 Myeloid cells
 Paratrabecular
 Mature cells towards centre
 Erythroid cells
 Centre in colonies
 Megakaryocytes
 Centre around sinusoids

78.  Monocyte precursors:
Not recognizable.
Some can be seen randomly distributed.
 Lymphoid precursors:
Seen in periarteriolar region.
 Stroma
Fat cells, fibroblasts, reticulin fibres

79.  Osteocytes:
Seen in bony
lacunae.
 Osteoblasts:
Seen lining the
trabeculae.
 Osteoclasts:
Seen in howship’s
lacunae
.

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

81. Cellularity:

82. Normal bone marrow cellularity
In adults

83. Hypocellular bone marrow
Aplastic anaemia

84. Hypercellular bone marrow
In AML WITH MATURATION

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

86. Erythroid cells in the centre

87. Myeloblast and promyelocytes
paratrabecular

88. Megakayocyte in bone marrow
parasinusoidal

89. Mast cells in immunoperoxidase stain
Usually seen in giemsa . Present irregularly in medullary cavity

90. Quantification of bone marrow reticulin
and collagen
 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

91. grade 0 grade 1
Fine fibre network throughout
most of the section; no
Occasional fine individual
fibres and foci of fine fibre
network
grade 2,

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

93. Marrow fibrosis
Reticulin (silver impregnation method)

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

95. Bone marrow biopsy report (ICSH guidelines
2008):
 Name of institution
 Unique specimen identifier
 Details of patient- name,age,gender, contact details
 Name of responsible physician
 Name of requesting doctor
 Date of procedure
 Clinical history, examination,therapy
 Indication of bone marrow examination
 Procedure performed
 Site of procedure

96. Signature and date of report
 Length of biopsy core
 Adequacy and appearance of the core
 Percentage and pattern of cellularity
 Bone architechture
 Location, number,morphology and pattern of differentiation
of
1. Erythroid
2. Myeloid
3. Megakaryocytic
4. Lymphoid
5. Plasma cells
6. Macrophages
 Abnormal cells
 Reticulin stain
 IHC
 Other Ix

97. Turn around time
 Varies from lab to lab
 24 hrs in case of microwave decalcification and as
long as 1 week if EDTA based decalcifying agent
is used.
 5 days for less urgent cases
 Additional 1-2 days if IHC is required
 In our lab aspiration is reported within 24hrs and
biopsy within 4-5 days.

98. Storage
 No guideline
 BM slides should be stored for 20yrs or
indefinitely if possible.

99. Quality assurance
 North America and Europe have well trenched
external quality assessment for histopathology
which includes bone marrow biopsy.
 Situation in India is less advanced due to
 vast unorganised structure of private labs
and
 highly variable training, technological facilities
and reporting styles.

100. TAKE HOME MESSAGE
 Biopsy of the bone marrow 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.
 A minimum definition of an adequate bone
marrow biopsy specimen stipulates a length of
atleast 1.5 cm, 5-6 marrow spaces and no
aspiration, crush or processing artifacts.

101. • Not to assess histology in isolation, ideally reported
along with bone marrow aspirate and peripheral
smear and preferrably reported by same pathologist.
• There should be an integrated approach in reporting
which includes
 Clinical history
 Laboratory investigations,
 Imaging information
 IHC wherever applicable.