Download - 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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MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE
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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MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE
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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MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE
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
Bone marrowaspirate smear,Wright-Giemsastain
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M3
Bone marrowaspirate smear,Wright-Giemsastain
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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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MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE
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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TYPE CYTOCHEMISTRY IMMUNOPHENOTYPE
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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TYPE CYTOCHEMISTRY IMMUNOPHENOTYPE
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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