This ppt includes detailed bone marrow examination. And each cell lineage described with pictures..

1. BONE MARROW
EXAMINATION

2. Introduction
• Site of hematopoiesis in postnatal life.
• Located within the cavities of the bones
• It consists of hematopoietic cells, vascular sinusoids,
fibroblasts, fat cells, and macrophages.
• There are no lymphatic channels in the bone marrow.
• There are two types of marrow: red and yellow.
• The average volume of bone marrow (red and yellow) in
an adult is 3000-4000 ml.
• Red or active marrow constitutes 1500 ml.

3. ARRANGEMENT OF HEMATOPOETIC
CELLS IN THE BONE MARROW
• The hematopoetic cells are present as cords between the vascular
sinusoids and are supported by a framework of branching processes
of fibroblasts and reticulin fibres.
• The erythroid precursors are present as clusters and closely
associated with centrally placed sinusoids.
• The granulocyte precursors are located close to the bony trabeculae.
• Megakaryocytes are closely apposed to the walls of vascular
sinusoids.
• Lymphocytes and plasma cells are present around capillaries and
arterial vessels.

5. SITES OF HEMATOPOESIS

6. Methods of BM
examination
BONE MARROW ASPIRATION
BONE MARROW BIOPSY

7. Parameter Bone marrow aspiration Bone marrow biopsy
Information
obtained:
Morphology, cytochemistry, iron
stain, immunophenotyping,
culture
Cellularity, architecture,
fibrosis, focal lesions, bone
structure
Main indications: Unexplained cytopenia,
suspected
hematological malignancy
Repeated dry tap, aplastic
anemia, myelofibrosis, focal
lesions, hairy cell leukemia,
staging of lymphoma
Needles
commonly used:
Time for routine
examination
report:
Salah, Klima
Same day
Jamshidi, Islam
Up to 7 days
Studies done: Romanowsky stain, Iron stain
cytochemistry, cytogenetic or
molecular genetic analysis,
immunophenotyping, culture
H and E stain,
immunohistochemistry,
reticulin stain,

8. BONE MARROW ASPIRATION

9. INDICATIONS
Unexplainedcytopenias Myeloproliferativeneoplasm Evaluation of Iron stores
Suspected AcuteLeukemia SuspectedMyelodysplasticsyndrome

10. SuspectedMyeloproliferativedisorders SuspectedPlamacellDyscracias Investigation of PUO
Suspected storage disorders
For Ancillary investigations like
flowcytometry

11. UNEXPLAINED
CYTOPENIAS
• If the cause of cytopenia is not apparent on blood investigations
and clinical evaluation, bone marrow examination is indicated.
• It helps to distinguish among causes of microcytic hypochromic
anemia by evaluation of Iron stores.
• In macrocytic anemia, in absence of vitamin assays and typical
features of megaloblastic anemia, marrow aspiration helps to
distinguish between megaloblastic macrocytosis and
nonmegaloblastic one.
• In leucopenia and thrombocytopenia, it helps in distinguishing
between peripheral destruction and deficient production.

12. SUSPECTED ACUTE
LEUKEMIA
• Bone Marrow examination is carried out in acute leukemia for:
• A) Detection of subleukemic or aleukemic leukemia.
• B) Comparison of baseline marrow smear with follow-up marrow
smears during treatment
• C) Flow cytometry
• D) Cytogenetic analysis
• E) Detection of remission

13. CONTRAINDICATIONS
1. Hemophilia
2. Other coagulation disorders
• However, in these patients it can be performed under
cover of appropriate replacement therapy

14. PROCEDURE OF BONE
MARROW ASPIRATION
• AN INFORMED CONSENT is taken from the patient before the
procedure.
• A sterile tray is prepared which contains:
-Autoclaved Bone marrow aspiration needles
-Sterile disposable syringes with needles
-Local anaesthetic solution
-Clean and dry glass slides, spreader slide, gloves, drape, gauze, and a
skin antiseptic solution.

15. • Salah and Klima
needles are commonly
used
• Salah needle: guard with a side
screw
• Klima needle: guard which
screws along the length of the
needle.
• Guards on these needles are
adjustable to control the depth
of penetration.

16. Jamshidi bone marrow
biopsy needle
• Jamshidi needle is
longer and is used for
both aspiration and
biopsy.

17. SITES
for both aspiration and
biopsy because it has a
large reservoir of marrow, a
large surface area for
maneuvering, easily
accessible and there are no
vital structures close to this
area.
2.Sternum- no longer
preferred because it has risk
of damaging the large blood
vessels and right atrium.
3.Spinous process of
lumber vertebrae
4.Tibia- infants less than
one yr

18. PROCEDURE OF BMA
lie on one side with
back towards the
physician, knees and
hips flexed, and the
knees drawn towards
the chest.
• The site for aspiration
should be selected
and scrubbed with
soap and water.
• After wearing sterile
gloves, antiseptic
solution is applied in
a circular fashion,
moving from center
towards periphery.

19. PROCEDURE CONTINUED
• Skin and periosteum are infiltrated ith a local anaesthetic, first inject
beneath the skin surface and advancing the needle further, a larger
amount is injected into the periosteal surface.
• After waiting for 5 minutes for anaesthsia to take effect, bone marrow
aspiration needle is inserted along with the fitted stylet. When the
bone is reached, the needle is rotated clockwise and anticlockwise
and slowly advanced into the bone, maintaining steady and firm
pressure.
• When the marrow is reached, a slight “give” will be noted.
• The needle is advanced for 1-2 mm into the marrow and stylet is
removed.

20. PROCEDURE CONTINUED
• A 5 or 10 ml syringe is attached to the needle and a small amount of
marrow is aspirated by quickly pulling the plunger of the syringe.
Aspiration is associated with sharp pain.
• If no material is aspirated, stylet is replaced, needle is redirected, and
aspiration attempted again.
• The smears should be made promptly, before clotting occurs, by
putting one drop of the aspirated material near one end of a glass slide
and spreading it similar to a blood film.
• Before making smears, any excess blood on the slide should be sucked
away by Pasteur pipette, leaving behind marrow particles.
• After completion of aspiration, the stylet should be reinserted into the
needle and the needle is removed. Sterile gauze is placed over the site
and light pressure is applied till bleeding ceases.

21. PROCESSING OF MARROW SPECIMENS
Bone marrow clots faster than
peripheral blood. So smears should be
made at the bedside without delay.
Rest of the aspirated marrow can be
stored in a bottle containing EDTA.
If there are insufficient marrow
fragments, they can be concentrated.
At least 5-6 smears are made.
After thorough drying, fix the smears
in methanol for 20 minutes.
Stain with any of
theRomanowskydyes (atleast3
smears)

22. One of the smears
stained by Perl’s
method (Prussian
Blue reaction) to
assess iron stores.
Rest of the smears
may be used
forimmunophenotypin
g/
cytogenetic analysis.

23. • Bone marrow aspiration provides following information:
o • Assessment of morphology of bone marrow cells.
o • Assessment of nature of hematopoiesis (normal,
dyshematopoiesis)
o • Cytogenetic analysis
o • Immunophenotyping of abnormal cells in leukemias.
o • Cytochemistry for typing of leukemia
o • Iron stain for assessing iron stores and sideroblasts
o • Microbial culture, e.g. for tuberculosis
With marrow aspiration, marrow architecture and cellular relations
cannot be studied(biopsy is better).

24. BONE MARROW TREPHINE
BIOPSY

25. Indications
BM failure States
Aplastic anaemia
Hypoplastic Acute Leukemia
Hypoplastic Myelodysplastic
syndrome
Marrow fibrosis
Idiopathic Myelofibrosis
Hairy Cell Leukemia
AML –M 7
Multiple Myeloma (with fibrosis)
Chronic Myeloproliferative
disorders – CML , PV , ET
Systemic Mastocytosis

26. Focal involvement of marrow
Granulomatous lesions – tuberculosis ,sarcoidosis ,
histoplasmosis , cryptococcosis , mycobacterium
avium intracellulare disease.
Hodgkin’s disease
Metastatic deposits
Non-Hodgkin’s lymphoma
Miscellaneous
Bone marrow necrosis
Pyrexia of unknown origin
Post bone marrow transplantation
Osteopathies
Storage Disorders

27. PROCEDURE
• Percutaneous trephine biopsy of bone marrow is commonly obtained
from PSIS. Jamshidi or Islam needles are commonly used.
• For adults- 4-inch, 11 gauge needle used.
• For children- 2or 4-inch, 13 gauge needle used.
• Often aspiration and biopsy are performed together; aspiration is
carried out first followed by biopsy. After aspiration, needle is
advanced a little further (1-3 cm) into the bone. Or its withdrawn and
placed about 1 cm away from the previous site.
• For biopsy, needle should be advanced through the bone by rotating
it atleast 10 times. The needle should be removed by anticlockwise
rotation.
• For adequate assessment, biopsy should measure at least 1 cm in
length.
• Biopsy should be placed in 10% neutral buffered formalin for at least
18-24 hours.

28. DRY TAP
• Failure to aspirate marrow i.e. a dry tap – suggests bone
marrow fibrosis or infiltration.
• Most common causes of marrow fibrosis :
Metastatic tumor
Lymphoma
• Bone marrow trephine biopsy is indicated in these cases.

29. IMPORTANT ADD NOTE
• Before Bone Marrow Aspiration, peripheral venous blood
should be withdrawn and collected in EDTA
anticoagulant. Smears should be made for cell counts.

30. ADEQUATE BMB
• 1.5 cm to 2 cm in length,
>5 inter trabecular
spaces.
• 10 intertrabecular space
s (WHO)
• C/L BMB if the first one
is <1.6 cm
• Small BMB is adequate
if +ve for
granuloma/metastasis/fu
ngus/lymphoma deposit.

31. EXAMINATION OF MARROW
SPECIMENS
(normoblasti
c,
micronormob
lastic,
megaloblasti

32. EXAMINATION OF BONE
MARROW SPECIMENS CONTD.
malaria
parasites,
microfilaria,
Leishmania

33. EVALUATION OF BMA
1. Detailed clinical history .
2. PBF taken at time of bone marrow aspirate /biopsy should be
stained and evaluated followed by marrow examination.
3. Choose the smears with bone marrow particle and cell trails of the
particles.
4. Select the area close to the particles.
5. Selected area should be well preserved and cell should just touch
each other.
6. Naked nuclei not included in differential count
7. 500 cells should be counted for differential on the BM.

35. ASSESSMENT OF
CELLULARITY
• CELLULARITY refers to the proportion of hematopoetic cells as
compared to the fat cells in the marrow particle.
• It can be expressed either as percentage or stating whether
marrow particles are normocellular, hypercellular or hypocellular
for age.
• Megakaryocytes are found in the tail of smear or near the
marrow particles. About 1-3 megakaryocytes are seen normally
per low power field.
• A rough estimate of normal cellularity is obtained by deducting 1
for each year of age from 100 and expressing it as a percentage;
expected normal cellularity is +/- 10% of this value.
• The cellularity for first decade of life is about 80%
• In adults, it is about 50%
• In older age, it decreases to about 30%

36. CAUSES OF DILUTED
MARROW
• Causes-
• 1 Procedural
• 2 Disease process affecting BM eg-fibrosis of the marrow
• 3 Aplasia of marrow
• BMB Indicated

38. DIFFERENTIAL COUNT IN BONE MARROW IN
ADULTS
CELLS COUNTS
MYELOBLASTS 0-3%
PROMYELOCYTES 2-5%
NEUTROPHIL MYELOCYTES 8-15%
METAMYELOCYTES 9-24%
NEUTROPHILS AND BAND FORMS 14-26%
ERYTHROBLASTS 15-36%
LYMPHOCYTES 5-20%
PLASMA CELLS 0-3%

39. MYELOID:ERYTHROID (M:E) RATIO
• Normal M:E ratio ranges from 2:1 to 4:1. It is the ratio of all
granulocytes and their precursors to all erythroid precursors. On an
average, there are 3 myeloid precursors for every erythroid
precursors.
• Increased M:E ratio is observed when:
a) myeloid series is hyperplastic as in infections and
myeloproliferative disorders (CML or AML)
b) erythroid series is suppressed as in aplastic crisis of hemolytic
anemia.
• Reduced M:E ratio is observed when:
a) myeloid series is depressed
b) erythroid hyperplasia (e.g. hemolytic anemia).

40. ERYTHROPOEISIS
• Erythroblasts are examined for reaction- normoblastic,
megaloblastic or micronormoblastic,
• The maturation of erythroid cells and
• The proportion of early, intermediate & late erythroblasts

41. Proerythroblasts/pronormoblast -
earliest precursors of RBCs
Size-15 to 20 microns
Basophilic cytoplasm
Fine, uniform chromatin
One or more nucleoli
Early normoblasts(Baophilic
Normoblasts)
Size-12-16 microns
Do not yet contain hemoglobin.
basophilic cytoplasm that
sometimes shows pseudopodia.
Coarse chromatin
Inconspicuous nucleoli

42. Intermediate
normoblast(Polychromatic
normoblast)
Size-12-15 microns
Polychromatic cytoplasm because
of presence of both blue RNA and
pink Hb
Clumped chromatin
Late normoblast( Orthochromatic
normoblast)
Size is 8-10 microns
Orthochromatic cytoplasm as it
resembles that of mature RBCs
Nucleus is pyknotic

43. RETICULOCYTE
• Anucleated
• Remnants of ribosomal
RNA are present in the
cytoplasm
• Reticulocyte mature into
RBCs within 1-2 days in
the peripheral blood.

44. MICRONORMOBLASTIC
REACTION
• MICRONORMOBLAST
IC REACTION
Late normoblast show
fraying of cytoplasmic
borders and persistent
basophilia

45. MEGALOBLASTIC REACTION
MEGALOBLASTIC
REACTION
Erythroid precursors larger
in size ,early and
intermediate erythroblast
have stippled
chromatin(sieve like)
.

46. MYELOPOEISIS
• Myeloid precursors are examined for
hypogranulation/hypergranulation,
• Blast cell number and their maturation,
• Proportion of blast cells, promyelocyte, myelocyte,
metamyelocyte, band form including neutrophils.

47. MYELOBLASTS
Size -12 to 20 microns.
The cytoplasmic rim is basophilic
The cytoplasm is agranular
The nucleus shows a very fine, dense chromatin structure, many
nucleoli, which generally are distinct and pale blue.

48. MYELOBLASTS

49. PROMYELOCYTE
• Size:15-20 microns,
• Abundant primary or azurophilic granules,
• Have a golgi zone( pale area adjacent to the nucleus)
• Nucleoli +nt

50. PROMYELOCYTE

51. MYELOCYTE
smaller than promyelocytes,
10-20 microns in size,
secondary granules appear,
nucleolus is no longer visible,
focal perinuclear hof is visible.

52. MYELOCYTE

53. METAMYELOCYTE AND
BAND FORMS
• Metamyelocytes- 10-12 microns, differ from myelocyte
in having some indentation/reniform shape of nucleus.
• Band or juvenile neutrophils- smaller cells of neutrophil
lineage with ribbon shaped non segmented nuclei

54. METAMYELOCYTE AND BAND FORMS

55. MACROPHAGE MORPHOLOGY
• Macrophage with nuclei, erythrocytes and platelets in the
cytoplasm (nucleus at lower right)
• Two macrophages with cellular residues
• Macrophage showing high acid phosphatase activity (red) and
nuclear residues (bluish gray)
Binucleated macrophage with predominantly fine-grained hemosiderin
in the cytoplasm (yellow-
• gold)

56. MEGAKARYOCYTE MORPHOLOGY
MEGAKARYOBLASTS: 10-15 micro
meter in size, blue nongranular
cytoplasm,large irregular single nucleus
with many nucleoli. The diploid forms
develop into polyploid forms.
PROMEGAKARYOCYTE- size-15-25 um
,lower N:C ratio,scant dark blue
cytoplasm,dense non lobulated and partly
lobulated nucleus with heavy
chromatin.Azurophilic granules start
appearing.
MATURE MEGAKARYOCYTE- size
ranging from 40-60 um in size ,single large
multilobed nucleus,cytoplasm varies from
blue to pink. Granular platelets found in
pseudopods like structure about the
periphery of cells.

57. PLASMA CELLS
• Typical mature
plasma cell in the
bone marrow with an
eccentric nucleus and
perinuclear halo
• Group of mature
plasma cells with a
spoked wheel nuclear
structure
• Binucleated mature
plasma cell

58. LEISHMANIA DONOVANI BODIES

59. HISTOPLASMOSIS

60. IRON STORES IN BONE MARROW
Section of BM showing
haemosiderin within
macrophages in a patient
with iron overload
• Sections are stained by
Perls method and graded
0-4
• Marrow iron is leached
during fixation with
formalin and therefore
exact iron status cant be
assesed unless biopsy has
been fixed in Zenker
fluid.
• Coarse iron granules
lying free or in
macrophage suggests
increased iron stores.

61. IRON STAINING: Iron stain on bone marrow aspiration
smear.
Left figure shows blue stained iron granules. Right
figure shows no stainable iron

62. PERL’S STAIN

63. • On marrow smears, stainable iron can be demonstrated in
siderocytes, sideroblasts, and macrophages of
reticuloendothelial system.
• Siderocytes are mature, non-nucleated red cells, which
contain 1-2 granules of non-heme iron. (On Romanowsky-
stained smears, these are called as Pappenheimer bodies).

64. • Erythroblasts containing aggregates of iron are called as
sideroblasts.
• three types—I, II, and III.
• In type I sideroblasts (seen normally), iron granules are small
and few (1-4) in number.
• In Type II sideroblasts,iron granules are large and
numerous(hemolytic anemia and iron overload).
• In type III sideroblasts, iron granules are distributed in the form
of a ring around the nucleus(‘ringed’ sideroblasts are seen in
sideroblastic anemias).

65. • In iron deficiency anemia, there is complete lack of
stainable iron in the erythroblasts and macrophages.
• Lack of stainable iron in erythroblasts and increased
amount of iron in macrophages is a feature of anemia of
chronic disease.
• Marked increase of storage iron in macrophages occurs in
thalassemias and iron overload.

66. Grading of iron stores on BMA
• 0 -- No iron granules seen
• 1 -- Small granules in reticulum cells only under oil immersion
• 2 -- Few small granules visible with low power lens
• 3 -- Numerous small granules in the marrow particle
• 4 -- Large granules in small clumps
• 5 -- Dense large clumps of granules
• 6 -- very large deposits obscuring the marrow cells
• Interpretation :-
• Grade 0 : IDA
• Grade 1, 2 ,3 : Normal iron stores
• Grade 4, 5, 6 : Increased iron stores

67. Marrow failure states
• Hypoplasia /aplasia refers to replacement of hemopoietic tissue
of marrow by fat.
• Pancytopenia with reticulocytopenia without organomegaly is
suggestive of hypoplasia.
• Hypoplasia is observed in
- Aplastic anemia
- Paroxysmal nocturnal hemoglobinuria
- Hypoplastic MDS / Acute Leukemia

68. Marrow failure states
• Trephine biopsy assess both cellularity and cytological
features of residual cells .
• In hypoplastic cases marrow demonstrates a cellularity of
less than 25 percent with depletion of erythroid, myeloid
and megakaryocytic precursors.
• Lymphocytes if present as tiny nodules suggest poorer
prognosis.

69. Marrow fibrosis
• Increase in reticulin or collagenisation of the marrow is
the main pathology.
• Fibrosis may be diffuse or patchy.
• Diffuse fibrosis is seen in Idiopathic Myelofibrosis
(IMF) or Chronic Myeloproliferative Disorders (CMPD)
• Focal fibrosis is seen in metastatic deposit, Hodgkin’s
infiltration or granuloma.

70. Bone marrow reticulin
grading(modified
Bauermeister)
Grade 0 No reticulin fiber demonstrable
Grade 1 Occasional fine individual fibres and foci of the fine fibre
network
Grade 2 Fine fibre network throughout most of the marrow section;
no coarse fibre
Grade 3 Diffuse fibre network with scattered thick coarse fibres but
no mature collagen
Grade 4 Diffuse; offen coarse fibre network with area of
collagenisation (positive Masson”s trichome staining)

71. European consensus
grading of bone marrow
fibrosis(MF)
MF-- 0 Scattered linear reticulin with no intersections
corresponding to normal bone marrow
MF-- 1 Loose network of reticulin throughout most of the section
MF-- 2 Diffuse and dense increase in reticulin with extensive
intersections with focal collagen bundles or focal
osteosclerosis
MF-- 3 Diffuse and dense increase in reticulin with extensive
intersections and collagen bundles of collagen often
associated with significant osteosclerosis

72. Bone marrow necrosis
• BMN is the necrosis of myeloid tissue or necrosis of the
neoplastic tissue that has replaced the normal
hemopoietic elements of the bone marrow.
• Associated necrosis of the medullary stroma of the
marrow or necrosis of the bone characterized by loss of
osteocytes from the lacunae. There is disruption of
normal bone marrow architecture with considerable loss
of fat spaces.
• Cytokines like TNF play a role in BMN.
• BMA is serosanguinous or amorphous-pink staining with
necrotic and smudged cells.
• In BMB necrotic cells appear as ghost cells /amorphous
material with nuclear karyorrhexis.

73. Bone marrow necrosis
• BMN is suspected when patient develops sudden bone
pain and fever. Sudden anaemia, thrombocytopenia or
leucoerythroblastic blood picture.
• LDH and Alkaline phosphatase are elevated in 50%
cases.
• BMN is a poor prognostic factor.
• BMN grading : - MILD : < 20% of marrow biopsy
specimen is necrotic
MODERATE : 20-50% necrotic
SEVERE : >50% necrotic

74. Bone marrow necrosis
A) Hematologic
malignancies
B) Metastatic tumor
deposits
C) Non malignant
conditions
ALL Sickle cell disease
AML HIV infections
CML Parvovirus B-19
infections
NHL Typhoid fever
Hodgkin’s disease Q fever
Multiple myeloma

75. COMPLICATIONS OF
BMA/BMB
1. Local infection: occur in neutropenic patients, can be
prevented if strict aseptic precautions are observed.
2. Hemorrhage: Serious hemorrhage can occur if
(i)marrow biopsy is done without adequate replacement
cover in coagulation disorders,
(ii) great vessels or heart is injured during sternal
aspiration.
3. Cardiac tamponade or mediastinitis: if posterior
sternal plate is penetrated during sternal aspiration

76. THANK YOU