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 d): Erythropoietic Islands PNRBCs

Mesodermal (8-12 w): Extraembryonic 3 Embryonic Hgbs 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 Granulocytes6th wk AOG Lymphocytes4th mon AOG Monocytes5th mon AOG

HEMATOPOIESIS: Medullary period

Start: 10th wk AOG (6th mon AOGpredominant) 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 RBCpoiesis; (+) 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 But larger, w/ fine chromatin, w/ nucleolus & hof (+) ALP

Osteoblast

Plasma cell

BONE MARROW STROMA: Other cells: Osteo -blast/ -clast

Osteoclasts: Multinucleated giant cells Resembles MKs (lobated nucleus) But nucleus separated by granular cytoplasm

Osteoclast

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 Granulomas Focal necrosis Histiocytic proliferartion w/ intracytoplasmic organisms Gaucher's Sea Blue Histiocytosis

Infections ("FUO")

Dx of hereditary or acquired storage dse

Bone Marrow Examination

Bone Marrow Examination

Aspirate Specimen

Cytology: cellular detail cellular composition

Core Biopsy Specimen

Architecture cellularity infiltrative lesionneoplastic 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 WestermanJensen 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 specimen for smear prep; the rest in EDTA tubes

Clotted specimens good only for histopath purposes if 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 particlesevaluation 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

At birth- 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: Absent Decreased Adequate Moderate Increased Markedly increased

0- 5 w/ 2 representing normal

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 Promyelocytes Myelocytes Metamyelocytes Bands & PMNs

2% 5% 12% 22% 20%

Erythrocytic maturation

Pronormoblast B. NB Poly/ & orthochromatophilic NB

4% 8% 18%

Always count on oil

13 granulocytes

1 4 1 5 2

seg metamyelocytes promyelocyte bands 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: OPERATION: MARTIN, BETH

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: HGB: HCT: MCV: PLT: RDW: ABS NEUT: ABS LYM:

6.4 K/uL 11.0 g/dL 34.0 % 74.2 fL 358 K/uL 14.5 % 3.92 K/uL 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 leftshifted. 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 LEFTSHIFTED 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