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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