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Bone Marrow AspirateExamination

A lecture by:

Maximo B. Axibal,Jr.,

MD, FPSP, MMHA

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HEMATOPOIESIS

A lecture by:

Maximo B. Axibal, Jr.

MD FPSP MMHA

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HEMATOPOIESIS

DEF: Process of ____ of cellularelements of the

blood Production,

Differentiation,

Maturation &

Proliferation Stages: Fetal or

adult?

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HEMATOPOIESIS

Medullary vs

Extramedullary

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Synchronous

Asynchronous

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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

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Introduction

# of circulating blood cells dependson:

Rate of production

Rate of release

Survival

Production & destruction occursimultaneously & constantly

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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

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HEMATOPOIESIS:

Mesenchymal period

Start: 4th wk AOG

End: 2nd mon AOG

Products:

Nucleated megaloblastic RBC's (2

wks AOG) Minor production of MKs

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“Nurse Cell”

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“Nursing Mother”

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HEMATOPOIESIS:

Mesenchymal (Yolk Sac) period

Mesoblastic(19-20d):

ErythropoieticIslands

PNRBCs

Mesodermal (8-12w):

Extraembryonic

3 Embryonic Hgbs

Megakaryopoiesis

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HEMATOPOIESIS:

Hepatic period

Start: End of 11th wk AOG

End: 6th mon AOG

Products:

Extravascular erythroblasts & anucleatedmacrocytic RBC's

Megakaryocytes- 6th wk AOG

Granulocytes- 6th wk AOG Lymphocytes- 4th mon AOG

Monocytes- 5th mon AOG

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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)

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Review of Anatomy/ Histology of the

Bone Marrow

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BM STRUCTURE: Major

compartments

Sinuses(sinusoids)

Hematopoietic

cords Parenchyma

Stroma

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BONE MARROW PARENCHYMA

Erythropoiesis- erythropoieticislands (Histiocyte + Normoblasts)

Granulopoiesis- around reticular cell Megakaryopoiesis- subjacent to

sinus endothelium

Immunocytes

Lymphocytes- lymphocytic nodules atrandom

Plasma cells- around blood vessels

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BONE MARROW STROMA

Histiocyte (Nursecell):perisinusoidal;center of RBC-poiesis; (+) ACP

Reticulum cell:adventitial; center

of WBC-poiesis;(+) Silver stain;(+) ALP

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BONE MARROW STROMA

Fat cell:

Variable amount

0% (NB & infants)

3 y/o discernable fat

40%- 50%- adults

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BONE MARROW STROMA

Endothelial cell:

More visible inhypoplastic marrow

Differentiate fromtumor cells

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BONE MARROW STROMA:

Other cells: Osteo -blast/ -clast

Osteoblasts:

In groups

Resembles plasma

But larger, w/ finechromatin, w/nucleolus & hof

(+) ALP

Osteoblast

Plasma cell

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BONE MARROW STROMA:

Other cells: Osteo -blast/ -clast

Osteoclasts:

Multinucleatedgiant cells

Resembles MKs(lobated nucleus)

But nucleusseparated by

granularcytoplasm

Osteoclast

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BMA Indications

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INDICATIONS: BONE MARROW

STUDIES

Anemias, erthrocytosis,polycythemia

Leucopenia, leucocytosis, immatureor abnormal cells in circulation

Thrombocytopenia, thrombocytosis

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INDICATIONS: BONE MARROW

STUDIES

Malignant tumors Lymphoma, ca & sarcoma mets to BM

Staging & prognosis

Infections ("FUO") Granulomas

Focal necrosis

Histiocytic proliferartion w/ intracytoplasmicorganisms

Dx of hereditary or acquired storage dse Gaucher's

Sea Blue Histiocytosis

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Bone Marrow Examination

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Aspirate Specimen

Cytology:

cellular detail

cellularcomposition

Core BiopsySpecimen

Architecture cellularity

infiltrative lesion-neoplastic orinfectious

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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

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Types of needle:

Aspiration(University of Illinois sternalneedle)

Biopsy (Trephine) Vim Silverman

Westerman-Jensen

Jamshidi 11 G- adults

13 G- children

15 mm length

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Sites of puncture:

Anterior Superior Iliac Crest

Posterior Superior Iliac Crest

(adults) Sternum (most common)

Spinal processes (L3) or vertebralbodies

Proximal end of tibial bone (infants)

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BM Aspiration

BM Biopsy

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Precautions:

Signed informed consent frompatient if adult; parent or guardianif a child w/ matching witness.

Explain indications, risks & benefitsof procedure.

Observe asepsis.

Do procedure in the presence of aRN (assistant) & RMT (propercollection & handling of 1- 1.5 cc).

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Precautions:

Prompt & swift.Avoid clotting(fibrin strips

cytoplasm of cells).

1st part of specimen for

smear prep; therest in EDTA tubes

Clotted specimensgood only for

histopath purposes if properly preserved in10% formalin

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BMA Procedure:

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BMA Procedure:

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Types of Smears (BMA):

Direct vs Marrow Particle Smear

Done as in PBS

Ideal for morphologicstudies

Ideal for cell to cellrelationship & cellularity

Done as in PBS butpre- selection of

sample in petri dish Fe staining (Prussian

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Staining of Bone Marrow Smears:

Air dry smears

Fix (Absolute Methanol) for 3- 5mins. ASAP

Stain: 3- 6 mins

Romanowsky type polychromic stains

Wright's

Wright- Giemsa May Grunwald

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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 forclinical staging of lymphomas & Ca)

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BMB Prep for exam:

Touch prep- bone corein between blades of forcep touched severaltimes in 2- 3 clean

slides; no rubbing;done as BM smears

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BM Biopsy Prep for exam:

Histopath section- fixed in 10% formalin/Zenker's/ Carnoys; undergoes standardhistologic processing including

decalcification; 2- 3 u thick; stained w/ H& E or special stains

Most ideal for assessment of cellularity

Advantage: Represents marrow structures inits natural relationship

Disadvantage: Fine cellular details lost; littlevalue in dX of leukemia & refractory anemias

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BM Core Biopsy vs. BM Aspirate Smear

QUANTITATIVE MEASUREMENT

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QUANTITATIVE MEASUREMENT

OF BMA:

EDTA anticoagulated aspirate 1 mltransferred into Wintrobe tube

Centrifuge at 2800 g x 8 min

Layers measured as %age from Wintrobetube

Fat & perivascular cells- Fe content

Plasma

Buffy coat FOR morphology & M:E RBC

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Bone Marrow Aspirate

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w/ particles fresh Wright's stain

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BMA Examination(Wright Stain)

Scan at LPO

Shift to OIO

Evaluate Iron Stores (LPO/ OIO)

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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 formetastatic clumps (black ball)

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BM EXAMINATION at LPO

Area well spread, intact cells, notdiluted by sinusoidal blood

Periphery of BM particles-evaluation of cellularity

# & distribution of MK (3/ LPOadjacent to spicule up to 10/ LPO)

Tumor cells- larger than RBC & WBCprecursors; in clusters

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Marrow cellularity estimate(hematopoietic: fat cells)

Area of examination- between 2uncrushed particles

For diagnosis of aplasia > 1 samplefrom different sites

Correlate w/ M:E (3–

4 : 1)

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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) POSIC60% Sternum

60 y/o 35% - 40%

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BMA Examination (Wright Stain)

Shift to OIO:

Find area where cells are well spread

Determine proportion of differentiatedgranulocytes

Determine M:E (3- 4:1)

Evaluate for orderly granulopoiesis

Evaluate erythropoiesis

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BM EXAMINATION at OIO

Ideal for morphologic studies

500- 1000 cells for diff ct

At birth- granulocytes predominate

1 mo of life- lymphocytes predominate

Limited diagnostic value

Not useful in non- hemopoietic dses

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Evaluate Iron Stores

At LPO, examine Prussian Bluestained smear for iron particles

At OIO, look for ringed sideroblastsif stained iron is increased

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BM EXAMINATION: Fe STORES

Indication: dx of anemia

Refractory

Dyserythropoietic

Use EDTA chelating agts fordecalcification than acid agts topreserve Fe in hemosiderin form

Golden yellow- Unstained Brownish- blue- Wright- Giemsa

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BM EXAMINATION: Fe STORES

Reporting:

Absent

Decreased

Adequate

Moderate

Increased

Markedlyincreased

w/ 2 representingnormal

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Scan at LPO:

Look for marrow spicule Evaluate cellularity Estimate for megakaryocytes Determine presence or absence of

predominance of one cell line(monotony)

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Look for marrow spicule

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Scan at LPO:

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LPO, normal BM, normal cellularity Hypercellular BM, CML

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Hypocellular BM

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Estimate cellularity (2: 1)

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BM Core Biopsy

Bone Marrow Core Biopsy

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(cellularity)

20% cellularity >95% cellularity

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Scan at LPO:

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Estimate MK (2- 5/ LPF)

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Shift to OIO:

Determine proportion of differentiatedgranulocytes

Determine M:E (3- 4:1) Evaluate for orderly granulopoiesis

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Area where cells are well spread

Area for BMA differential

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Area for BMA differential

Good vs Bad

Poor area for BM exam,

appears to be sinusoidal blood

Field of counting

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Field of counting

Good vs Bad

Cells broken, can't count them

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Normal bone marrow differential

Non-diagnostic bone marrow

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Non diagnostic bone marrow

differential, lymphoma patient

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Erythrocytic maturation

Pronormoblast 4%

B. NB 8%

Poly/ & ortho-

chromatophilic NB 18%

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Always count on oil

13 granulocytes

4 metamyelocytes

1 promyelocyte 5 bands

2 myelocytes

6 normoblast:

4 orthochromic NB 2 NB

polychromatophilic

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Always count on oil

2 segs

1 band

1 metamyelocyte

1 myelocyte 1 reticulum cell

2 NBspolychromatophilic

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Shift to OIO:

Determine proportion of differentiated granulocytes

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Determine M:E

Count allgranulocytic & NRBCs in theareas examined

Erythroid hyperplasia, M:E = 1:1 vs.

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Erythroid hyperplasia, M:E 1:1 vs.

Granulocytic hyperplasia, M:E inc

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What is the M:E?

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Non-diagnostic BM, M:E normal

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Shift to OIO:

Determine proportion of differentiated granulocytes

If cells w/ round nuclei predominate, ID

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Are RBC precursors normal in #?

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Erythropoeisis type?

Is there orderly granulocytic

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maturation?

… or is there a preponderance of early

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cells?

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Other Cells:

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Tissue basophil (mast cell)

Foamy histiocytes, liver dse (similar to

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Nieman-Pick)

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Gaucher cell

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Basophilic MK (MK1)

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Granular MK (MK2)

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Platelet producing MK (MK3)

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MK w/ cells overlying cytoplasm

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Plasma cells

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Reticulum cell vs. Phagocytic histiocyte

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Evaluate Iron Stores

At LPO, examine Prussian Blue

stained smear for iron particles

At OIO, look for ringed sideroblasts

if stained iron is increased

I t t i ti

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Iron content examination

Determinewhether or notthere is a normalamt of hemosiderin

I t t i ti

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If normal amount of iron is present,patient is not iron def

If iron is absent, patient is iron def

I t t i ti

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When iron ispresent, look forringed sideroblasts

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BONE MARROW REPORT

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BONE MARROW REPORT

Patient's addressograph data, age & relevant clinical history

Description of material received

CBC, WBC, Differential, Platelet & reticulocyte counts w/ PBS taken onthe day of the marrow sampling

B.M. Differential count

Cellularity

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 ld l ith hi t f APL (SHS 01 29962) A t bi h d h l i id f id l

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CLINICAL HISTORY: 32-year-old male with a history of APL (SHS-01-29962). A recent biopsy showed no morphologic evidence of residualdisease (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 ina 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 hypogranularforms. -White blood cells are normal in number, with a normal absolute number of neutrophils. White blood cells are predominantly normalappearing 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 thenumber of promyelocytes or blasts. Erythroid precursors are present in normal numbers and are also mildly left-shifted . Iron stores arepresent within histiocytes, no ringed sideroblasts are identified.

BONE MARROW BIOPSY: The bone marrow biopsy is mildly hypercellular for age, approximately 70%. Megakaryocytes are present innormal numbers and morphology. The M:E ratio is 3:1. Myeloid and erythroid precursors are normal in number and mature fully. Themyeloid precursors are mildly left-shifted. There are no excess blasts. Notable, there is an area of fibrosis, which appears to be associatedwith 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, correlationwith 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

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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

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PERIPHERAL BLOOD SMEAR:

Red blood cells are normochromic andrange from microcytic to normocytic.There is occasional polychromasia. A fewscattered teardrop forms are identified.Platelets are normal in number withoccasional large and hypogranular forms.White blood cells are normal in number,with a normal absolute number of neutrophils. White blood cells arepredominantly normal appearing

segmented and band neutrophils, withfewer numbers of lymphocytes andmonocytes.

BMA Report:

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BMA Report:

The bone marrow aspirate is adequate.Megakaryocytes are present in normalnumbers with normal morphology. TheM:E ratio is 2-3:1. Myeloid precursors are

normal in number and are left-shiftedwithout a relative increase in the numberof promyelocytes or blasts. Erythroidprecursors are present in normal numbersand are also mildly left-shifted. Iron

stores are present within histiocytes, noringed sideroblasts are identified.

BONE MARROW BIOPSY:

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BONE MARROW BIOPSY:

The bone marrow biopsy is mildlyhypercellular for age, approximately 70%.Megakaryocytes are present in normalnumbers and morphology. The M:E ratio

is 3:1. Myeloid and erythroid precursorsare 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 subcorticalbone.

COMMENT: The bone marrow aspirate andbiopsy reveal left-shifted myelogenesis and

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p y y gerythrogenesis. There is no relative increase in

the number of promyelocytes or blasts. Sincemorphology is limited in distinguishing normalmyelogenesis from residual disease, correlationwith cytogenetic and/or molecular studies isrecommended.

DIAGNOSIS: BONE MARROW ASPIRATE,BIOPSY, AND PERIPHERAL BLOOD SMEARMILDLY HYPERCELLULAR MARROW WITH LEFT-SHIFTED MYELOID AND ERYTHROIDMATURATION (SEE COMMENT)

(Ancillary Techniques)

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(Ancillary Techniques)

Special stains

Flow cytometry

Cytogenetics Molecular

Diagnostics

SUMMARY

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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

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SUMMARY

Scan under OIO

SUMMARY

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SUMMARY

Examine iron stain

Determine amount of hemosiderin

Look for ringed sideroblast (OIO) if

stainable iron is increased

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01 bm

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