aml- lab diagnosis

8/11/2019 AML- Lab Diagnosis

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PRESENTED BY BHAVYAA BAHL


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Also known as Acute non-lymphocytic leukemiaGroup of clonal hematopoietic stem cell disorders in which,

1. Inhibition of terminal myeloid differentiation2. Over proliferation into STEM CELL compartment

ACCUMULATION OFMYELOBLASTS IN THE

BONE MARROW

NORMAL HEMATOPOIETICPROGENITOR CELL

SUPPRESSED


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NORMAL HEMATOPOIETICPROGENITOR CELL

SUPPRESSION

ANAEMIA

THROMBOCYTOPENIANEUTROPENIA

All responsible for

MAJOR CLINICAL COMPLICATIONS OF AML


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PATIENT SAMPLE COLLECTION

1. Blood Sample2. Bone Marrow Sample

Aspiration Trephine Biopsy

Blood Cell Counting(P. smear)

Routine microscopicexamination

Cytochemistry

Flow cytometry &immunohistochemistry

FISH

PCR

LABORATORY TESTS


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FEATURE ALL AML

Leukemic blasts Lymphoblasts Myeloblasts

Size Smaller, 10-15 m Larger, 12-20 m

N/C Ratio High Low

Chromatin Clumped Spongy

Nucleoli


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FEATURE ALL AML

MPO Negative Positive

Sudan Black B Negative Positive

NSE Negative Positive

TdT Positive Negative

PAS Positive (shows block pattern) Positive in


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CONDITION SPECIFIC MARKERS

ALL

T-cell CD1a, CD2, CD3, CD4, CD5,CD7, CD8, CD11b,CD25,CD45,CD56

B-cell CD10, CD19, CD20,CD21, CD22, CD23,CD79a, Sig, Ig

NK cell CD16, CD56, CD57

AML CD13, CD14, CD15 ,CD33, CD41, CD61, CDw65, CD71,

Glycophorin A, MPO


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CELL MORPHOLOGY

MYELOBLASTS 1. Delicate nuclear chromatin

2. 2-4 nucleoli

3. Azurophilic, peroxidase +ve granules/Auer rods may

be present.

MONOBLASTS 1. Often folded/lobulated nuclei,

2. NO Auer rods

3. PODve, NSE +ve

PROMONOCYTE/

MONOCYTE

Abn nuclear maturation, granulation , loss of basophilia.

ERYTHROID PRECURSORS Normal/varying degrees of dyserythropoiesis

BASOPHILS Rarely in AML/if present show abnormal granule

formation & nuclear maturation.

MEGAKARYOCYTES / . Dysplastic hyperlobated, hypolobated,

multinuclear, small and blastic forms may be present.


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STAIN RESULT

Myeloperoxidase (MPO) Identifies blast cells as myeloid.

Dysplastic neutrophils may be -ve.

Eosinophil granules always +ve.

Monoblasts and promonocytes may be -ve.Sudan Black B Same

Chloroacetate esterase (CAE) Specifically identifies cells of the granulocyte lineage.

Non specific estrase (NSE) Specifically identifies cells of the Monoblast lineage.

Alpha-Naphthyl Acetate

esterase (ANAE)

Stains monocytes and megakaryocytes at all stages of

maturation.

Toluidine blue Stains the granules of basophils and mast cells.

Periodic Acid Schiff (PAS) It is not lineage specific but the pattern of staining

may be helpful


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Diagnosis is confirmed by staining cells for myeloid specific surface markers

CELL MARKERS

Hematopoietic stem cell

ProgenitorCD34, CD117, TdT

Myeloid cell CD11b, CD11c, CD13, CD14, CD15, CD16, CD33, CD34, CD38, CD45,CD71,CD123 , CD163, MPO

T-cell CD1a, CD2, CD3, CD4, CD5,CD7, CD11b, CD25,CD45, CD56, HLA-DR

B-cell CD10, CD19, CD20,CD21, CD22, CD23,CD79a, SIg, HLA-DR,Ig

Megakaryocyte CD41, CD61

Erythrocytes CD 71, CD235a, Glycophorin A

Neutrophil CD11c, CD15, CDw65, MPO

Monocytes/Macrophages CD4,CD33, CD64, CD 163, HLA-DR, MPO

cCD79a-In AML, presence usually represents aberrant B cell antigen in leukemias of distinct myeloidlinage, not biphenotypic differentiation


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Revised FAB classification of AML

S.NO CLASS INCIDENCE

1. M0 Minimally differentiated AML 2-3%

2. M1 AML without differentiation 20%

3. M2 AML with maturation 30-40%

4. M3 Acute promyelocytic leukemia 5-10%

5. M4 Acute myelomonocytic

leukemia

15-20%

6. M5 Acute monocytic leukemia 10%

7. M6 Acute erythroleukemia 5%

8. M7 Acute megakaryocyticleukemia 1%


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MORPHOLOGYCYTOCHEMISTRYIMMUNOPHENOTYPING


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1. CLASS 1. CRITERIA

M0 1. Myeloid blasts >20% of nucleated Bone marrow cells

2. MPO +ve on ultrastructural cytochemistry

3. 90% of BM nonerythroid cells (i.e also excluding

lymphocytes, plasma cells, mast cells & macrophages from the count)

2. Maturing granulocytic cells (i.e promyelocytes to polymorphonuclear cells)10% of nonerythroid cells

3. Monocytic cells (monoblasts to monocytes) 20% of BM nucleated cells

M3 variant 1. Promyelocytes (hypogranular) >20% of BM nucleated cells


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1. CLASS 1. CRITERIA

M4 1. Blasts >20% of BM nucleated cells2. Monocytic cells,precursors and neutrophils ,precursors are each more than

20%.3. MPO +ve >3% blasts.

4. NSE +ve in cells of monocytic lineage5. CD13, 33 (myeloid); CD14, LYSOZYME (monocytic)

M5 1. >80% cells in the bone marrow are monocytic (monoblasts, promonocytes& monocytes).

2. There is intense NSE +vity

3. CD 14, 36, 64, 11cM6 1. More than 20% of non-erythroid cells are myeloblasts and more than 50%

of all nucleated cells are erythroblasts Or2. More than 80% of marrow cells are erythroblasts with no significant

Myeloblastic component3. PAS stain gives a diffuse or block positivity in erythroblasts.

4. MPO +ve in myeloblasts5. Erythroblasts react with monoclonal antibody against glycophorin A

M7 1. Megakaryoblasts are 20% or more in the marrow2. Marked bone marrow fibrosis.3. Blasts are platelet peroxidase, CD41 (glycoprotein IIb/IIIA) & CD61

(glycoprotein III a) +ve


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MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE

Blasts >20% of nucleatedBM cellsNo evidence of maturation

CYTOPLASM : Scant

Grey to light blue in color No granules No Auer rods

Lack definitivecytological &cytochemical markers

of myeloblasts .

NEGATIVE FOR ALL(


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M0

Bone marrowaspirate smear,Wright-Giemsa

stain


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M0 M0,MPO +ve


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1. Blasts vary in size

2. Nuclei is round to oval/

irregular

3. Scant, Agranular usually, blue-

grey cytoplasm4. Auer rods few

5. In this setting, if Auer rods are

seen, the diagnosis of M1

AML is established.

*By morphology alone, M1 blastscannot be distinguished from

M0, agranular M2 blasts or L2

blasts.

MPO,SB PAS NSE

Bu CD 13CD33CD34

CD19CD117HLA-DR

>3% - - -


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M1

Bone marrowaspirate smear,Wright-Giemsa

stain


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M1, MPO+ve M1


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Maturation down the granulocytic

line

Cells beyond Promyelocyte

Differentiated neutrophils, some

eosinophils,and rarely basophilsMaturing cells are dysplastic

(mostly)

Nuclear chromatin coarser,

clumped

Type II blasts significant in number

Unlike normal promyelocytes andearly myelocytes, the Golgi

apparatus is poorly developed.

Nuclear maturation lags behind

the cytoplasmic maturation.

Auer rods are frequently visible.

MPO,SB PAS NSE CAE CD 13CD33CD34

CD99CD117HLA-DR

CD56CD19

>3% - - +


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A variant associated with eosinophilia

The eosinophils may show mild

atypia, particularly in the more

immatures ones, characterized by the

appearance of coarse cytoplasmic

granules ranging in color from deeply

basophilic, resembling primary or

basophil granules to those that have a

salmon-like color.

The more mature eosinophils usually

are not atypical.

A very rare subtype, is also an

example of AML with differentation-

in this case down the basophil lineage.

The leukemic blasts are type II and the

cytoplasmic granules are coarse and

basophilic, resembling those in

normal mature basophils, and the

cytoplasm is basophilic in color and

may contain vacuoles.

Some of the more mature forms often

are dysplastic


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M2AUER ROD


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M2

Bone marrowaspirate smear,Wright-Giemsastain


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M2 AML with

eosinophilia

(M2Eo)

Bone marrowaspirate smear,

Wright-Giemsa stain


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Sudan black B +ve blasts, stronger in dysplasticmetamyelocytes.Inset: Chloroacetate in maturing granulocytes.


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Arrest at the promyelocyte -

late myelocyte stage

Cell size & shape vary

Poorly developed Golgi

apparatus

Variable nuclear:cytoplasmic

ratio

Cytoplasmic color - light blue to

pink, variable number of granules

that may have a pink, red, or dark

purple color. The granules may

obscure the nucleus.Auer rods & Faggot cells seen

Round, oval, indented, reniform,

Angel winglike nucleus

The nuclear chromatin -coarse,

clumped

Nucleoli may /may not be visible.

MPO,SB

CAE PAS NSE Bu CD2CD4CD11cCD13

CD33CD34CD45CD56CD64CD117

+ + - - -


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HYPERGRANULAR HYPOGRANULAR / MICROGRANULAR

Most common subtype, 80% of cases

Cytoplasmic granulation prominentLarge (giant) granules may be seenBlasts vary in sizeAuer rods are common

Granulation sparce, in some cells

granules are not seen on a Wright-Giemsa stainDistinct morphological features such asbilobed nucleusAuer rods less frequent(confused with monoblast)


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M3 AMLwith

Auer rods



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M3



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M3 Angel wingnucleus


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M3HYPOGRANULAR

Bone marrowaspirate smear,

Wright-Giemsa stain


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Sudan black B +veTypical heavy cytoplasmic

positivity

Promyelocyte CAE +ve (blue)Inset: atypical ANAE +ve 1%of M3

cases.


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Appear as typical myeloblasts ormonoblasts/promonocytes, & cellsthat are difficult to categorize asbelonging to either lineage .Depending upon which lineage they

appear like the following varies,1. Size & shape2. The cytoplasm-

Scant- Myeloblasts likeAbundant- Monocytic like

3. N:C ratio4. Nucleus variable in size and shape

5. Nucleoli prominent in those withmonoblastic and promonocyticfeatures.

In the more differentiated cells,cytoplasmic granules that resemblepromonocytes (fine granulation with a"salt and pepper" appearance)Auer rodsmay be seen

MPO,SB

CAE PAS NSE Bu Myeloid lineageCD13+CD33

Monocyte lineageCD4CD14+CD116CD11c

Lysozyme

+ + - + +


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1. May represent up to 10% of all adult AMLs2. Usually occurs at a younger age3. Variable number of differentiating eosinophils present

4. Eosinophils are atypical (dysplastic). More matureeosinophils are less atypical5. Coarse basophilic granules seen with a variable number

of eosinophilic granules.6. The morphologic picture is diagnostic and is associated

with the inv(16) cytogenetic abnormality.


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M4 Someblasts withmyeloblastic

features andsome withmonoblastic

Bone marrowaspirate smear,Wright-Giemsastain,


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M4Eo AMLwithatypical

youngeosinophils


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M4E0


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M4 AML

MPO +ve

M4 AML

Sudan Black B +ve


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M4, NSE +ve


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CATEGORY M5aMONOBLASTIC,Undifferentiated

M5bMONOCYTIC, Differentiated

1. Predominant cell type2. Size3. Cytoplasm

4. Vacuole5. Auer rods6. Nucleus7. Chromatin8. Nucleoli

9. Erythrophagocytosis

Monoblast1-1.5 times neutrophil sizeAbundant, color ranges froma medium to dark blue

PresentAbVaries in shape n sizeFine1 or


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TYPE CYTOCHEMISTRY IMMUNOPHENOTYPE

M5a

Vacuoles are PAS +veMPO,SB ANAE PAS NSE Bu Stem cell/hematopoietic

progenitor cell

CD34-

HLA-DR -

Monocytic lineage

CD4,

CD14

CD11c

CD64

CD68

CD116lysozyme

Myeloid lineage

CD33

CD13

CD117

- + - + +

M5b

Both nuclear & cytoplasmic

differentiation

+/- + - + +


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M5a


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M5 AMLNon-specific esterase (NSE) stain


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M5b

M6a : Proerythroblasts mixed with myeloblasts


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Dysplastic proerythroblasts

predominate Varied-size and shape Presence of bizarre, giant

forms Megaloblastic & dysplastic

nuclear features prominent,including multilobated nucleiwith Howell-Jolly bodies

Marked lag in nuclearmaturation wrt cytoplasmic

Vacuoles prominent On Fe stain, ring sideroblasts A myeloblastic component

may be present, tendency totransform to either an M1, M2,or M4 subtype with time

MPO,SB CAE PAS NSE GlyA Stemcell/hematopoietic

progenitor cell

CD34-

HLA-DR-Erythroid lineage

Glycophorin A +ve

Myeloid lineage

CD13+

CD33+

CD117+Monocytic lineage

CD4+

CD14+

CD11b+

CD11a+

CD64+

+/-

(M6a)-

(M6b)

+/-

-

+

+

+/-

(M6a)-

(M6b)

+

+

y y

M6b : Proerythroblasts


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M6a AML withmyeloblasts &erythroblasts

Bone marrow aspiratesmear, Wright-Giemsa stain


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M6b AML withmultiple Howell-Jollybodies-

M6a AML - myeloblasts& erythroblasts andwith many cytoplasmicvacuoles

M6


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M6a

PAS+ve


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M6b


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Giant multinucleate late

normoblasts

PAS +veGranular in proerythroblasts

Homogeneous in normoblasts


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Variable

Microblasts like lymphoblasts

a. Irregular cytoplasmic blebs

b. Membrane projections

c. Fine cytoplasmic granules(micromegakaryoblastic subtype)

or very heterogenous population

of blasts including,

a. Large megakaryoblasts

b. Blue cytoplasm

c. With/without nuclear lobation& cytoplasmic granules

On a biopsy specimen, fibrosis,

ranging from reticulin to

collagenous seen.

MPO,SB PAS NSE AP Stem cell/hematopoieticprogenitor cell

CD34-

HLA-DR-

*CD45-Myeloid lineage(may be +ve)

CD13

CD33

Megakaryocyte lineage

CD41 (glycoprotein IIb/IIIa)

CD61 (glycoprotein IIIA)CD36 (glycoprotein IIIb)

Platelet Peroxidase +ve

- + + +

*Common Leukocyte Antigen CD45 is NEGATIVE

M AML


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M7 AMLMicromegak

-aryoblasticsubtype

Bone marrow aspiratesmear, Wright-Giemsastain


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M7


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M7 AML with large,immature andbilobedmegakaryoblast

Peripheral bloodsmear, Wright-Giemsa stain


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aml- lab diagnosis

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