Bone Marrow Aspirate
Examination
A lecture by:
Maximo B. Axibal,Jr.,
MD, FPSP, MMHA
HEMATOPOIESIS
A lecture by:
Maximo B. Axibal, Jr.
MD FPSP MMHA
HEMATOPOIESIS
DEF: Process of
____ of cellular
elements of the
blood
Production,
Differentiation,
Maturation &
Proliferation
Stages: Fetal or
adult?
HEMATOPOIESIS
Medullary vs
Extramedullary
Synchronous
vs
Asynchronous
Introduction
BM largest tissue in the body
Adult: 4 L; Child: 1. 6 L
BM 1° site for blood cell formation
2.5 billion RBCs / kg / day
2.5 billion platelets / kg / day
1 billion granulocytes / kg / day
Introduction
# of circulating blood cells depends
on:
Rate of production
Rate of release
Survival
Production & destruction occur
simultaneously & constantly
INTERRELATIONSHIP OF
BLOOD CELLS: Theories
Monopheletic (Downey, Ferat &
Pappenheim)
Polypheletic (Piney, Naegali,
Schilling, Rosenthal, Osgood, Sabin,
Cunningham & Doan)
Naegali
Modification of Schilling
Sabin & her co-workers
HEMATOPOIESIS:
Mesenchymal period
Start: 4th wk AOG
End: 2nd mon AOG
Products:
Nucleated megaloblastic RBC's (2
wks AOG)
Minor production of MKs
“Nurse Cell”
“Nursing Mother”
HEMATOPOIESIS:
Mesenchymal (Yolk Sac) period
Mesoblastic(19-20 Mesodermal (8-12
d): w):
Erythropoietic Extraembryonic
Islands 3 Embryonic Hgbs
PNRBCs Megakaryopoiesis
HEMATOPOIESIS:
Hepatic period
Start: End of 11th wk AOG
End: 6th mon AOG
Products:
Extravascular erythroblasts & anucleated
macrocytic RBC's
Megakaryocytes- 6th wk AOG
Granulocytes- 6th wk AOG
Lymphocytes- 4th mon AOG
Monocytes- 5th mon AOG
HEMATOPOIESIS:
Medullary period
Start: 10th wk AOG (6th mon AOG-
predominant)
End: Lifetime
Products:
RBC's- 10th wk AOG
Thrombocytes
Granulocytes/ monocytes
Lymphocytes- w/ contributions from
thymus, L.N. & spleen (Ag independent)
HEMATOPOIESIS:
Medullary period
RBC - Alpha 1 & alpha2 beta2
3 wks postpartum – BM (Medullary)
4th y - rate of BM production
exceeds need for blood cells
Review of Anatomy/ Histology of the
Bone Marrow
BM STRUCTURE: Major
compartments
Sinuses (sinusoids)
Hematopoietic
cords
Parenchyma
Stroma
BONE MARROW PARENCHYMA
Erythropoiesis- erythropoietic islands
(Histiocyte + Normoblasts)
Granulopoiesis- around reticular cell
Megakaryopoiesis- subjacent to sinus
endothelium
Immunocytes
Lymphocytes- lymphocytic nodules at
random
Plasma cells- around blood vessels
BONE MARROW STROMA
Histiocyte (Nurse
cell):
perisinusoidal;
center of RBC-
poiesis; (+) ACP
Reticulum cell:
adventitial; center
of WBC-poiesis;
(+) Silver stain;
(+) ALP
BONE MARROW STROMA
Fat cell:
Variable amount
0% (NB & infants)
3 y/o discernable fat
40%- 50%- adults
BONE MARROW STROMA
Endothelial cell:
More visible in
hypoplastic marrow
Differentiate from
tumor cells
BONE MARROW STROMA:
Other cells: Osteo -blast/ -clast
Osteoblasts:
In groups
Resembles plasma
cells Osteoblast
But larger, w/ fine
chromatin, w/
nucleolus & hof
(+) ALP
Plasma cell
BONE MARROW STROMA:
Other cells: Osteo -blast/ -clast
Osteoclasts:
Multinucleated
giant cells
Resembles MKs Osteoclast
(lobated nucleus)
But nucleus
separated by
granular
cytoplasm
MKs
BMA Indications
INDICATIONS: BONE MARROW
STUDIES
Anemias, erthrocytosis,
polycythemia
Leucopenia, leucocytosis, immature
or abnormal cells in circulation
Thrombocytopenia, thrombocytosis
INDICATIONS: BONE MARROW
STUDIES
Malignant tumors
Lymphoma, ca & sarcoma mets to BM
Staging & prognosis
Infections ("FUO")
Granulomas
Focal necrosis
Histiocytic proliferartion w/ intracytoplasmic
organisms
Dx of hereditary or acquired storage dse
Gaucher's
Sea Blue Histiocytosis
Bone Marrow Examination
Bone Marrow Examination
Aspirate Specimen
Cytology:
cellular detail
cellular
composition
Core Biopsy
Specimen
Architecture
cellularity
infiltrative lesion-
neoplastic or
infectious
Specimen Collection &
Handling
Types of needle
Sites of puncture
Precautions
Types of Smears (BM ASPIRATES)
Staining of Bone Marrow Smears
BONE BIOPSY
QUANTITATIVE MEASUREMENT OF BMA
Types of needle:
Aspiration
(University of
Illinois sternal
needle)
Biopsy (Trephine)
Vim Silverman
Westerman- Jensen
Jamshidi
11 G- adults
13 G- children
15 mm length
Sites of puncture:
Anterior Superior Iliac Crest
Posterior Superior Iliac Crest
(adults)
Sternum (most common)
Spinal processes (L3) or vertebral
bodies
Proximal end of tibial bone (infants)
ASIC
PoSIC
BM Biopsy
BM Aspiration
Precautions:
Signed informed consent from
patient if adult; parent or guardian if
a child w/ matching witness.
Explain indications, risks & benefits
of procedure.
Observe asepsis.
Do procedure in the presence of a
RN (assistant) & RMT (proper
collection & handling of 1- 1.5 cc).
Precautions:
Prompt & swift.
Avoid clotting
(fibrin strips
cytoplasm of
cells).
1st part of Clotted specimens
specimen for good only for
smear prep; the histopath purposes if
rest in EDTA tubes properly preserved in
10% formalin
BMA Procedure:
BMA Procedure:
Types of Smears (BMA):
Direct vs Marrow Particle Smear
Done as in PBS
Ideal for morphologic
studies
Ideal for cell to cell
relationship &
cellularity
Done as in PBS but
pre- selection of
sample in petri dish
Fe staining (Prussian
Blue)
Staining of Bone Marrow Smears:
Air dry smears
Fix (Absolute Methanol) for 3- 5
mins. ASAP
Stain: 3- 6 mins
Romanowsky type polychromic stains
Wright's
Wright- Giemsa
May Grunwald
BMB
BONE MARROW BIOPSY:
Most reliable for assessment of
cellularity
Indications:
Done routinely w/ aspirates.
Dry tap aspirates.
Diagnosis of neoplastic/ granulomatous
diseases (bilateral POSiC biopsy for
clinical staging of lymphomas & Ca)
BMB Prep for exam:
Touch prep- bone core
in between blades of
forcep touched several
times in 2- 3 clean
slides; no rubbing;
done as BM smears
BM Biopsy Prep for exam:
Histopath section- fixed in 10% formalin/
Zenker's/ Carnoys; undergoes standard
histologic processing including
decalcification; 2- 3 u thick; stained w/ H
& E or special stains
Most ideal for assessment of cellularity
Advantage: Represents marrow structures in
its natural relationship
Disadvantage: Fine cellular details lost; little
value in dX of leukemia & refractory anemias
BM Core Biopsy vs. BM Aspirate
Smear
QUANTITATIVE MEASUREMENT
OF BMA:
EDTA anticoagulated aspirate 1 ml
transferred into Wintrobe tube
Centrifuge at 2800 g x 8 min
Layers measured as %age from Wintrobe
tube
Fat & perivascular cells- Fe content
Plasma
Buffy coat FOR morphology & M:E
RBC
Bone Marrow Aspirate
BMA:
w/ particles fresh Wright's stain
BMA Examination
(Wright Stain)
Scan at LPO
Shift to OIO
Evaluate Iron Stores (LPO/ OIO)
BMA Examination (Wright Stain)
Scan at LPO:
Look for marrow spicule
Evaluate cellularity
Estimate for megakaryocytes
Determine presence or absence of
predominance of one cell line (monotony)
Scan for abnormal cells; Look for
metastatic clumps (black ball)
BM EXAMINATION at LPO
Area well spread, intact cells, not
diluted by sinusoidal blood
Periphery of BM particles-
evaluation of cellularity
# & distribution of MK (3/ LPO
adjacent to spicule up to 10/ LPO)
Tumor cells- larger than RBC & WBC
precursors; in clusters
BM EXAMINATION at LPO
Marrow cellularity estimate
(hematopoietic: fat cells)
Area of examination- between 2
uncrushed particles
For diagnosis of aplasia > 1 sample
from different sites
Correlate w/ M:E (3 – 4 : 1)
BM EXAMINATION at LPO
Infants - 100%
2- 3 y/o - start of fat formation
Theory: Centripetal due to
Variation in body temperature
Poor vascularity
Inherited factors
20 y/o 50% (+ or -10) POSIC
60% Sternum
60 y/o 35% - 40%
BMA Examination (Wright Stain)
Shift to OIO:
Find area where cells are well spread
Determine proportion of differentiated
granulocytes
Determine M:E (3- 4:1)
Evaluate for orderly granulopoiesis
Evaluate erythropoiesis
BM EXAMINATION at OIO
Ideal for morphologic studies
500- 1000 cells for diff ct
Atbirth- granulocytes predominate
1 mo of life- lymphocytes predominate
Limited diagnostic value
Not useful in non- hemopoietic dses
Evaluate Iron Stores
At LPO, examine Prussian Blue
stained smear for iron particles
At OIO, look for ringed sideroblasts
if stained iron is increased
BM EXAMINATION: Fe STORES
Indication: dx of anemia
Refractory
Dyserythropoietic
Use EDTA chelating agts for
decalcification than acid agts to
preserve Fe in hemosiderin form
Golden yellow- Unstained
Brownish- blue- Wright- Giemsa
BM EXAMINATION: Fe STORES
Reporting: 0- 5
Absent w/ 2 representing
Decreased normal
Adequate
Moderate
Increased
Markedly
increased
Scan at LPO:
Look for marrow spicule
Evaluate cellularity
Estimate for megakaryocytes
Determine presence or absence of
predominance of one cell line
(monotony)
Scan for abnormal cells; Look for
metastatic clumps (black ball)
Look for marrow spicule
Scan at LPO:
Look for marrow spicule
Evaluate cellularity
Estimate for megakaryocytes
Determine presence or absence of
predominance of one cell line
(monotony)
Scan for abnormal cells; Look for
metastatic clumps (black ball)
LPO, normal BM, normal cellularity Hypercellular BM, CML
Hypocellular BM
Estimate cellularity (2: 1)
BM Core Biopsy
Bone Marrow Core Biopsy
(cellularity)
>95% cellularity 20% cellularity
Bone Marrow Core Biopsy
Scan at LPO:
Look for marrow spicule
Evaluate cellularity
Estimate for megakaryocytes
Determine presence or absence of
predominance of one cell line
(monotony)
Scan for abnormal cells; Look for
metastatic clumps (black ball)
Estimate MK (2- 5/ LPF)
Shift to OIO:
Find area where cells are well spread
Determine proportion of differentiated
granulocytes
Determine M:E (3- 4:1)
Evaluate for orderly granulopoiesis
Evaluate erythropoiesis
Area where cells are well spread
Area for BMA differential
Good vs Bad
Poor area for BM exam,
appears to be sinusoidal blood
Field of counting
Good vs Bad
Cells broken, can't count them
Normal bone marrow differential
Non-diagnostic bone marrow
differential, lymphoma patient
Granulocytic maturation
Myeloblast 2%
Promyelocytes 5%
Myelocytes
12%
Metamyelocytes 22%
Bands & PMNs 20%
Erythrocytic maturation
Pronormoblast 4%
B. NB
8%
Poly/ & ortho-
chromatophilic NB
18%
Always count on oil
13 granulocytes
1 seg
4 metamyelocytes
1 promyelocyte
5 bands
2 myelocytes
6 normoblast:
4 orthochromic NB
2 NB
polychromatophilic
Always count on oil
2 segs
1 band
1 metamyelocyte
1 myelocyte
1 reticulum cell
2 NBs
polychromatophilic
Shift to OIO:
Find area where cells are well spread
Determine proportion of
differentiated granulocytes
Determine M:E (3- 4:1)
Evaluate for orderly granulopoiesis
Evaluate erythropoiesis
Determine M:E
Count all
granulocytic &
NRBCs in the areas
examined
Erythroid hyperplasia, M:E = 1:1 vs.
Granulocytic hyperplasia, M:E inc
What is the M:E?
Non-diagnostic BM, M:E normal
Shift to OIO:
Find area where cells are well spread
Determine proportion of
differentiated granulocytes
Determine M:E (3- 4:1)
Evaluate for orderly granulopoiesis
Evaluate erythropoiesis
If cells w/ round nuclei predominate, ID
them
Are RBC precursors normal in #?
Erythropoeisis type?
Is there orderly granulocytic
maturation?
… or is there a preponderance of early
cells?
Other Cells:
Tissue basophil (mast cell)
Foamy histiocytes, liver dse (similar to
Nieman-Pick)
Gaucher cell
Basophilic MK (MK1)
Granular MK (MK2)
Platelet producing MK (MK3)
MK w/ cells overlying cytoplasm
Plasma cells
Reticulum cell vs. Phagocytic histiocyte
Evaluate Iron Stores
At LPO, examine Prussian Blue
stained smear for iron particles
At OIO, look for ringed sideroblasts
if stained iron is increased
Iron content examination
Determine
whether or not
there is a normal
amt of
hemosiderin
Iron content examination
If normal amount of iron is present,
patient is not iron def
If iron is absent, patient is iron def
Iron content examination
When iron is
present, look for
ringed sideroblasts
BONE MARROW REPORT
BONE MARROW REPORT
Patient's addressograph data, age &
relevant clinical history
Description of material received
CBC, WBC, Differential, Platelet &
reticulocyte counts w/ PBS taken on
the day of the marrow sampling
B.M. Differential count
Cellularity
M:E
Bone Marrow Biopsy Report
Sample of actual Report
Stanford Hospital and Clinics 300 Pasteur Drive, Stanford, CA 94305
Name: XXXXXXXXXX (CROSSED OUT)
CLINICAL HISTORY: 32-year-old male with a history of APL (SHS-01-29962). A recent biopsy showed no morphologic evidence of residual
disease (SHS 01-37736).
ORDER DOCTOR: MARTIN, BETH
OPERATION: Bone marrow biopsy
GROSS DESCRIPTION: The specimen "left PSIC bone marrow biopsy" is received in Bouin's solution and consists of one elongated
cylindrical tan-brown fragment of bony material that measures 1.5 x 0.2 x 0.2 cm. The specimen is submitted entirely between sponges in a
single cassette following decalcification (MARROW HEME tag). Estrada for Morgan/pal
LABORATORY DATA:
WBC: 6.4 K/uL
HGB: 11.0 g/dL
HCT: 34.0 %
MCV: 74.2 fL
PLT: 358 K/uL
RDW: 14.5%
ABS NEUT: 3.92 K/uL
ABS LYM: 1.70 K/uL
PERIPHERAL BLOOD SMEAR: Red blood cells are normochromic and range from microcytic to normocytic. There is occasional polychromasia.
A few scattered teardrop forms are identified. Platelets are normal in number with occasional large and hypogranular forms. -White blood
cells are normal in number, with a normal absolute number of neutrophils. White blood cells are predominantly normal appearing
segmented and band neutrophils, with fewer numbers of lymphocytes and monocytes.
BONE MARROW ASPIRATE: The bone marrow aspirate is adequate. Megakaryocytes are present in normal numbers with normal
morphology . The M:E ratio is 2-3:1. Myeloid precursors are normal in number and are left-shifted without a relative increase in the number
of promyelocytes or blasts. Erythroid precursors are present in normal numbers and are also mildly left-shifted . Iron stores are present
within histiocytes, no ringed sideroblasts are identified.
BONE MARROW BIOPSY: The bone marrow biopsy is mildly hypercellular for age, approximately 70%. Megakaryocytes are present in
normal numbers and morphology. The M:E ratio is 3:1. Myeloid and erythroid precursors are normal in number and mature fully. The
myeloid precursors are mildly left-shifted. There are no excess blasts. Notable, there is an area of fibrosis, which appears to be associated
with subcortical bone.
COMMENT: The bone marrow aspirate and biopsy reveal left-shifted myelogenesis and erythrogenesis. There is no relative increase in the
number of promyelocytes or blasts. Since morphology is limited in distinguishing normal myelogenesis from residual disease, correlation
with cytogenetic and/or molecular studies is recommended.
DIAGNOSIS: BONE MARROW ASPIRATE, BIOPSY, AND PERIPHERAL BLOOD SMEAR MILDLY HYPERCELLULAR MARROW WITH LEFT-SHIFTED
MYELOID AND ERYTHROID MATURATION (SEE COMMENT)
LABORATORY DATA:
WBC: 6.4 K/uL
HGB: 11.0 g/dL
HCT: 34.0 %
MCV: 74.2 fL
PLT: 358 K/uL
RDW: 14.5 %
ABS NEUT: 3.92 K/uL
ABS LYM: 1.70 K/uL
PERIPHERAL BLOOD SMEAR:
Red blood cells are normochromic and
range from microcytic to normocytic.
There is occasional polychromasia. A few
scattered teardrop forms are identified.
Platelets are normal in number with
occasional large and hypogranular forms.
White blood cells are normal in number,
with a normal absolute number of
neutrophils. White blood cells are
predominantly normal appearing
segmented and band neutrophils, with
fewer numbers of lymphocytes and
monocytes.
BMA Report:
The bone marrow aspirate is adequate.
Megakaryocytes are present in normal
numbers with normal morphology. The
M:E ratio is 2-3:1. Myeloid precursors are
normal in number and are left-shifted
without a relative increase in the number
of promyelocytes or blasts. Erythroid
precursors are present in normal numbers
and are also mildly left-shifted. Iron stores
are present within histiocytes, no ringed
sideroblasts are identified.
BONE MARROW BIOPSY:
The bone marrow biopsy is mildly
hypercellular for age, approximately 70%.
Megakaryocytes are present in normal
numbers and morphology. The M:E ratio is
3:1. Myeloid and erythroid precursors are
normal in number and mature fully. The
myeloid precursors are mildly left-shifted.
There are no excess blasts. Notable, there
is an area of fibrosis, which appears to be
associated with subcortical bone.
COMMENT: The bone marrow aspirate and
biopsy reveal left-shifted myelogenesis and
erythrogenesis. There is no relative increase in
the number of promyelocytes or blasts. Since
morphology is limited in distinguishing normal
myelogenesis from residual disease, correlation
with cytogenetic and/or molecular studies is
recommended.
DIAGNOSIS: BONE MARROW ASPIRATE, BIOPSY,
AND PERIPHERAL BLOOD SMEAR MILDLY
HYPERCELLULAR MARROW WITH LEFT-SHIFTED
MYELOID AND ERYTHROID MATURATION (SEE
COMMENT)
Bone Marrow Examination
(Ancillary Techniques)
Special stains
Flow cytometry
Cytogenetics
Molecular
Diagnostics
SUMMARY
Scan at LPO
Find spicule
Evaluate cellularity
Look for metastatic clumps (black ball)
Estimate for megakaryocytes
Determine presence or absence of
predominance of one cell line (monotony)
Scan for abnormal cells
SUMMARY
Scan under OIO
Determine M:E (3- 4:1)
Evaluate for orderly granulopoiesis
Evaluate erythropoiesis
SUMMARY
Examine iron stain
Determine amount of hemosiderin
Look for ringed sideroblast (OIO) if
stainable iron is increased
