PRESENTED BY BHAVYAA BAHL

Also known as Acute non-lymphocytic leukemia

Group of clonal hematopoietic stem cell disorders in which,

1. Inhibition of terminal myeloid differentiation
2. Over proliferation into STEM CELL compartment
ACCUMULATION OF
MYELOBLASTS IN THE
BONE MARROW
NORMAL HEMATOPOIETIC
PROGENITOR CELL
SUPPRESSED
NORMAL HEMATOPOIETIC
PROGENITOR CELL
SUPPRESSION
ANAEMIA
THROMBOCYTOPENIA NEUTROPENIA
All responsible for
MAJOR CLINICAL COMPLICATIONS OF AML
PATIENT SAMPLE COLLECTION

1. Blood Sample
2. Bone Marrow Sample
Aspiration
Trephine Biopsy
Blood Cell Counting
(P. smear)
Routine microscopic
examination
Cytochemistry
Flow cytometry &
immunohistochemistry
FISH
PCR
LABORATORY TESTS
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 <2; indistinct 2-5, distinct
Nuclear membrane Irregular, convoluted Regular
Auer Rods Not present Present in 10-20%
FEATURE ALL AML
MPO Negative Positive
Sudan Black B Negative Positive
NSE Negative Positive
TdT Positive Negative
PAS Positive (shows block pattern) Positive in <25% of cells
Lysozyme Negative Positive
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
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. POD ve, 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.
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
Diagnosis is confirmed by staining cells for myeloid specific surface markers
CELL MARKERS
Hematopoietic stem cell
Progenitor
CD34, 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 myeloid
linage, not biphenotypic differentiation

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 megakaryocytic
leukemia
1%
MORPHOLOGY
CYTOCHEMISTRY
IMMUNOPHENOTYPING
1. CLASS 1. CRITERIA
M0 1. Myeloid blasts >20% of nucleated Bone marrow cells
2. MPO +ve on ultrastructural cytochemistry
3. <3% blasts +ve for Sudan Black B
4. CD 13, 33, 117
M1 1. Myeloid blasts >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. Promonocytes <10% of nonerythroid marrow cells
4. >3% blasts +ve for Sudan Black B
5. CD13, 33, 117, MPO
M2 1. Blasts 20-89% of bone marrow nonerythroid cells
2. Maturing granulocytic cells (i.e promyelocytes to polymorphonuclear cells)
>10% of nonerythroid cells
3. Monocytic cells (monoblasts to monocytes) <20% of nonerythroid cells & not
meeting other criteria for M4
M3 1. Promyelocytes (hypergranular) >20% of BM nucleated cells
M3 variant 1. Promyelocytes (hypogranular) >20% of BM nucleated cells
1. CLASS 1. CRITERIA
M4 1. Blasts >20% of BM nucleated cells
2. Monocytic cells,precursors and neutrophils ,precursors are each more than
20%.
3. MPO +ve >3% blasts.
4. NSE +ve in cells of monocytic lineage
5. 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, 11c
M6 1. More than 20% of non-erythroid cells are myeloblasts and more than 50%
of all nucleated cells are erythroblasts Or
2. More than 80% of marrow cells are erythroblasts with no significant
Myeloblastic component
3. PAS stain gives a diffuse or block positivity in erythroblasts.
4. MPO +ve in myeloblasts
5. Erythroblasts react with monoclonal antibody against glycophorin A
M7

1. Megakaryoblasts are 20% or more in the marrow
2. Marked bone marrow fibrosis.
3. Blasts are platelet peroxidase, CD41 (glycoprotein IIb/IIIA) & CD61
(glycoprotein III a) +ve
MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE
Blasts >20% of nucleated
BM cells
No evidence of maturation
CYTOPLASM :
Scant
Grey to light blue in color
No granules
No Auer rods
Lack definitive
cytological &
cytochemical markers
of myeloblasts .

NEGATIVE FOR ALL
(<3% +ve for MPO,SB)
CD 13
CD33
CD34
CD38
CD117
HLA-DR

*T-cell marker CD7 -
frequently positive e.g of
biphenotypic, mixed
lineage, or hybrid acute
leukemias.
M0
















Bone marrow
aspirate smear,
Wright-Giemsa
stain
M0 M0, MPO +ve <3%
MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE
1. Blasts vary in size
2. Nuclei is round to oval/
irregular
3. Scant, Agranular usually, blue-
grey cytoplasm
4. Auer rods few
5. In this setting, if Auer rods are
seen, the diagnosis of M1
AML is established.

*By morphology alone, M1 blasts
cannot be distinguished from
M0, agranular M2 blasts or L2
blasts.

MPO,SB PAS NS
E
Bu CD 13
CD33
CD34
CD19
CD117
HLA-DR

>3% - - -
M1










Bone marrow
aspirate smear,
Wright-Giemsa
stain
M1, MPO+ve M1
MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE
Maturation down the granulocytic
line
Cells beyond Promyelocyte
Differentiated neutrophils, some
eosinophils , and rarely basophils
Maturing cells are dysplastic
(mostly)
Nuclear chromatin coarser,
clumped
Type II blasts significant in number
Unlike normal promyelocytes and
early 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 13
CD33
CD34
CD99
CD117
HLA-DR

CD56
CD19
>3% - - +
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
M2
AUER ROD
M2















Bone marrow
aspirate smear,
Wright-Giemsa
stain
M2 AML with
eosinophilia
(M2Eo)










Bone marrow
aspirate smear,
Wright-Giemsa stain
Sudan black B +ve blasts, stronger in dysplastic
metamyelocytes.
Inset: Chloroacetate in maturing granulocytes.
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 wing like nucleus
The nuclear chromatin -coarse,
clumped
Nucleoli may /may not be visible.

MPO,S
B
CAE PAS NSE Bu CD2
CD4
CD11c
CD13
CD33
CD34
CD45
CD56
CD64
CD117
+ + - - -
HYPERGRANULAR HYPOGRANULAR / MICROGRANULAR
Most common subtype, 80% of cases
Cytoplasmic granulation prominent
Large (giant) granules may be seen
Blasts vary in size
Auer rods are common
Granulation sparce, in some cells
granules are not seen on a Wright-
Giemsa stain
Distinct morphological features such as
bilobed nucleus
Auer rods less frequent
(confused with monoblast)
M3 AML
with
Auer rods













Bone marrow
aspirate smear,
Wright-Giemsa
stain
M3















Bone marrow
aspirate smear,
Wright-Giemsa
stain
M3
Angel wing
nucleus
M3
HYPOGRANULAR
















Bone marrow
aspirate smear,
Wright-Giemsa stain
Sudan black B +ve
Typical heavy cytoplasmic
positivity
Promyelocyte CAE +ve (blue)
Inset: atypical ANAE +ve 1% of M3
cases.
MORPHOLOGY CYTOCHEMISTRY IMMUNOPHENOTYPE
Appear as typical myeloblasts or
monoblasts/promonocytes, & cells
that are difficult to categorize as
belonging to either lineage .
Depending upon which lineage they
appear like the following varies,
1. Size & shape
2. The cytoplasm-
Scant- Myeloblasts like
Abundant- Monocytic like
3. N:C ratio
4. Nucleus variable in size and shape
5. Nucleoli prominent in those with
monoblastic and promonocytic
features.
In the more differentiated cells,
cytoplasmic granules that resemble
promonocytes (fine granulation with a
"salt and pepper" appearance)
Auer rods may be seen

MPO,S
B
CAE PAS NSE Bu Myeloid lineage
CD13+
CD33

Monocyte lineage
CD4
CD14+
CD116
CD11c
Lysozyme
+ + - + +
1. May represent up to 10% of all adult AMLs
2. Usually occurs at a younger age
3. Variable number of differentiating eosinophils present
4. Eosinophils are atypical (dysplastic). More mature
eosinophils are less atypical
5. 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.

M4 Some
blasts with
myeloblastic
features and
some with
monoblastic










Bone marrow
aspirate smear,
Wright-Giemsa
stain,
M4Eo AML
with
atypical
young
eosinophils
M4E0
M4 AML
MPO +ve
M4 AML
Sudan Black B +ve
M4, NSE +ve
CATEGORY M5a
MONOBLASTIC,
Undifferentiated
M5b
MONOCYTIC, Differentiated
1. Predominant cell type
2. Size
3. Cytoplasm



4. Vacuole
5. Auer rods
6. Nucleus
7. Chromatin
8. Nucleoli
9. Erythrophagocytosis
Monoblast
1-1.5 times neutrophil size
Abundant, color ranges from
a medium to dark blue


Present
Ab
Varies in shape n size
Fine
1 or <, Prominent
Ab
Promonocyte
More variable
Abundant, light blue to blue-
grey, fine granules "salt and
pepper appearance

More Prominent
Ab
Varies, Convoluted
Clumped
1 or <, Less Prominent
Present
TYPE CYTOCHEMISTRY IMMUNOPHENOTYPE
M5a
Vacuoles are PAS +ve
MPO,SB ANAE PAS NSE Bu
Stem cell/hematopoietic
progenitor cell
CD34-
HLA-DR -
Monocytic lineage
CD4,
CD14
CD11c
CD64
CD68
CD116
lysozyme
Myeloid lineage
CD33
CD13
CD117
- + - + +
M5b
Both nuclear & cytoplasmic
differentiation
+/- + - + +
M5a
M5 AML
Non-specific esterase (NSE) stain
M5b
TYPE CYTOCHEMISTRY IMMUNOPHENOTYPE
Dysplastic proerythroblasts
predominate
Varied-size and shape
Presence of bizarre, giant
forms
Megaloblastic & dysplastic
nuclear features prominent,
including multilobated nuclei
with Howell-Jolly bodies
Marked lag in nuclear
maturation wrt cytoplasmic
Vacuoles prominent
On Fe stain, ring sideroblasts
A myeloblastic component
may be present, tendency to
transform to either an M1, M2,
or M4 subtype with time
MPO,SB CAE PAS NSE GlyA
Stem
cell/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)
+

+
M6a : Proerythroblasts mixed with myeloblasts
M6b : Proerythroblasts
M6a AML with
myeloblasts &
erythroblasts















Bone marrow aspirate
smear, Wright-
Giemsa stain
M6b AML with
multiple Howell-Jolly
bodies-
M6a AML - myeloblasts
& erythroblasts and
with many cytoplasmic
vacuoles
M6a
PAS+ve
M6b
Giant multinucleate late
normoblasts
PAS +ve
Granular in proerythroblasts
Homogeneous in normoblasts
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/hematopoietic
progenitor 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
M7 AML
Micromegak
-aryoblastic
subtype












Bone marrow aspirate
smear, Wright-Giemsa
stain
M7
M7 AML with large,
immature and
bilobed
megakaryoblast















Peripheral blood
smear, Wright-
Giemsa stain