01 bm
TRANSCRIPT
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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
vs
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
cells
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
MKs
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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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Bone Marrow Examination
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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ASIC
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PoSIC
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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
Blue)
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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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BMB
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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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BMA:
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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BM EXAMINATION at LPO
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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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) 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
0- 5
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)
Scan for abnormal cells; Look formetastatic clumps (black ball)
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Look for marrow spicule
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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)
Scan for abnormal cells; Look formetastatic clumps (black ball)
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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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Bone Marrow Core Biopsy
(cellularity)
20% cellularity >95% cellularity
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Bone Marrow Core Biopsy
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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)
Scan for abnormal cells; Look formetastatic clumps (black ball)
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Estimate MK (2- 5/ LPF)
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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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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
1 seg
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:
Find area where cells are well spread
Determine proportion of differentiated granulocytes
Determine M:E (3- 4:1)
Evaluate for orderly granulopoiesis
Evaluate erythropoiesis
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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:
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
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p ,
them
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Are RBC precursors normal in #?
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Erythropoeisis type?
Is there orderly granulocytic
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y g y
maturation?
… or is there a preponderance of early
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p p y
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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y y (
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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Iron content examination
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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Iron content examination
When iron ispresent, look forringed sideroblasts
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BONE MARROW REPORT
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
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 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)
Bone Marrow Examination
(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
Determine M:E (3- 4:1)
Evaluate for orderly granulopoiesis
Evaluate erythropoiesis
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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