bone marrow pdf
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R.S.Riley,M.D.,Ph.D.
BoneMarrowPathology
Part1
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Bone marrow basicsRed cell diseases
White cell diseases
Other diseases
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Bone marrow basics
HematopoiesisBone marrow structure
Obtaining bone marrow
Interpreting bone marrow
Red cell diseases
White cell diseases
Other diseases
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Bone marrow basics
HematopoiesisBone marrow structure
Obtaining bone marrow
Interpreting bone marrow
Red cell diseasesWhite cell diseases
Other diseases
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1950 1975
LightMicroscopy Cytochemical
StainsKaryotypicAnalysis
FlowCytometryIPO Stains
MolecularAssays
1925 2000
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Review patient history, laboratory data, previous specimens
Examine peripheral blood smear
Examine bone marrow aspirate
Examine at least two films
10x - Cellularity, megakaryocyte #, infiltrates
40-50x - Examine each cell line, search for nonhematopoietic
cells
100x - Fine cytologic detail, representative differential countin fragment trails
Prussian blue stain for iron content and abnormal
sideroblasts
Examine bone marrow biopsy
Examine slides at three levels or more
4x - Evaluate cellularity, megakaryocyte #, bone structure,
focal lesions
10x - Evaluate each cell line, bony structure, focal lesions
Evaluate flow cytometry, immunohistochemical stains etc.
Assign final diagnosis
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SpecimensPeripheral blood smear
Bone marrow aspirate
Bone marrow biopsyLymph nodes
Body fluids
Other tissues
StainsWright-Giemsa
Hematoxylin and eosin (H&E)
Perls Prussian blue
Gordon-Sweet reticulin
Cytochemical stains
Immunohistochemical stains
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Bone Marrow Aspirate
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Bone Marrow Biopsy
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Aspirated fragments or biopsyspecimens
Determined by patient age,specimen site, and technicalfactors
Methods for assessment
Subjective estimate
Computerized image analysis orhistomorphology
Adequate biopsy required (20-30mm)
Aspirate only, evaluate fragmentsrather than trails
CELLULARITY
OF 25-75% IS
USUALLY
NORMAL INPATIENTS 20-70
YEARS OF AGE
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0
20
40
60
80
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Bone
Marrow
Cellularity
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1
12
2
22
23
8
14
15
21Myeloblasts
Promyelocytes
Myelocytes
Metamyelocytes
Band Neutrophils
Segmented Neutrophils
Eosinophils
Erythroid Precursors
Lymphocytes
Plasma Cells
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M:E Ratio =
Best determined in bone marrowaspirate
Normal ratio = 2:1 to 4:1
Increased M:E ratio in myeloid
hyperplasia or erythroid hypoplasia
Decreased M:E ratio in myeloidhypoplasia and erythroid hyperplasia
Cells in myeloid series
Erythroblasts
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Glycophorin-A Stain
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Perls Prussian blue
Best performed on bonemarrow aspirate smears
Intracellular stores should be
evaluated, extracellular storescan be confused with artifact
Most intracellular iron is inmacrophages, a small amountin erythroblasts (sideroblasts)
Normally 20-50% oferythroblasts are sideroblasts
Ringed sideroblasts areatypical, with iron inmitochondria forming a ringaround nucleus
Grading Iron Stores
0 - No stainable iron
1+ - Small intracellular iron
stores using oil objective2+ - Small, sparse intracellular
iron particles at low power
3+ - Numerous small intracellular
iron particles
4+ - Larger particles with a
tendency to aggregate into
clumps
5+ - Dense, large clumps
6+ - Very large clumps and
extracellular iron
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Perls Prussian Blue
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Reticular fibers formed byfibroblasts
Normally few, primarily
perivascular and periendostealIncreased in many conditions,may be associated withcollagen
Cause dry tap aspirate
Evaluated by Gordon-Sweetand trichrome stain
Interpretation must avoid areasof crush artifact andperivascular regions
Grading Reticulin Content
0 - No reticulin fibers
1+ - Occasional fine individual
fibers2+ - Fine fiber network
throughout section, no
coarse fibers
3+ - Diffuse fiber network with
scattered thick coarse fibers,
no collagen
4+ - Diffuse often coarse fiber
network with areas of
collagenization
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Biopsy of Aspiration Tract
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ImunohistochemicalStains
Flow Cytometry
Cytospin or
Tissue SectionSingle-Cell
Suspension
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Cells are incubated withfluorochrome labeled MoAbs
Cells are passed in single filethrough highly focused laser
beam
Different fluorochromes emitlight at different wavelengths
Emitted light analyzed bycomputer and plotted on ahistogram
Data analysis shows number andimmunophenotypiccharacteristics of the cellpopulation
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International Workshops on HumanLeukocyte Differentiation Antigens
Sponsored by World Health Organization
Hybridoma technology, antibodies shared,common reactivity identified, antigensdefined
8th Workshop - Adelaide, Australia, 2004
CD1 - CD247
General conclusions
Complex interrelationships
Few lineage-specific antigens
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CD34
TdT
T-Lymphoblast
CD3
CD7
CD34
B-Lymphoblast
CD10
CD19TdT
CD3
T
CD4/8
T-Lymphocyte
B CD19
CD20
CD7
B-Lymphocyte
MYO
CD34
CD13
CD33 MYO
CD13
CD33
CD45
CD14
Myeloblast Myeloid Cells
MonocytesAll Leukocytes
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Cytogenetic analysis
GTG banding
Spectral karyotyic analysis(SKY)
Molecular techniques
Fluorescent in situhybridization
Polymerase chain reaction
Restriction FragmentLength Polymorphisms(RFLPs)
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Avaunt! and quit my sight!Let the earth hide thee! Thy
bones are marrowless, thy
blood is cold ; Thou hast no
speculation in those eyeswhich thou dost glare with!
William ShakespeareMacBeth
Act 3 Scene 4