28-Sep-12

1
Flow Cytometric Findings in
Acute Myeloid Leukemia
Prashant Sharma
Hematology Department,
PGIMER, Chandigarh
FCM in AML
Role: Not just optional for diagnosis
Definitive roles in:
AML-M0
M7 M7
Mixed phenotype acute leukemias
Rapid identification of M3
Emerging role in MRD detection (still
primarily molecular-based)
Aims
To familiarize the participants with the
FCM appearances of the common AML
subtypes.
To introduce them to the core To introduce them to the core
interpretative strategies in FCM analysis of
AML.
To highlight some commonly encountered
issues in the FCM analysis of AML.
Cases
1. AML-M0
2. AML-M2
3. AML-M3
4. AML-M4
5. AML-M7
6. Mixed phenotype acute leukemia
(Bi-lineal, T-myeloid)
Case 1: Background
36 yr/M with fever, pallor
O/E: Splenomegaly 2 cm BCM
Hb 9.7 g/dl; TLC 43,200/l and platelets
2 45 000/l 2,45,000/l
Peripheral smear shows ~96% blasts, negative
for myeloperoxidase
Specimen: EDTA blood
Case 1: CD45-SSC
A prominent
blast cluster and
with marked
depletion of
other cell
populations
Blasts are
CD45
dim
/ SSC
low
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CD45-SSC
P1: 92% of all events
CD45-SSC
P1: 92% of all events
P2: 7% of all events
CD45-SSC
P1: 92% of all events
P2: 7% of all events
P3: 1% of all events
CD34
+
CD117
+ (heterogeneous)
Neg for CD64 & CD14
Co-express CD33 & CD34
Neg for CD13 & CD15
HLA-DR
+
CD13
neg
CD11b
-
CD16
-
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Neg for MPO & CD4/8/TdT CD7
+
CD2
-
CD5
-
CD38
+
CD58
+
CD10
neg
CD20
neg
CD19
neg
Summary of markers
92% blasts expressing:
Myeloid antigens: CD117 (~77%) and CD33 (~96%)
Markers of immaturity: CD34 (~96%), HLA-DR
(~98%), CD58 (~100%) and CD38 (~98%)
Aberrantly express CD7 (~98%)
Blasts are negative for myeloid and monocytic
antigens (cMPO, CD14, CD64, CD11b, CD13,
CD15, CD16), B-cell antigens (cCD79a, CD19,
CD20), T-cell antigens (cCD3, CD5, CD4, CD8,
CD2) and CD10, TdT.
Impression: Acute Myeloid Leukemia with
minimal differentiation and aberrant CD7
expression.
Learning points from case 1
AML with minimal differentiation by FCM
correlates with FAB M0/M1. Distinction
between the 2 requires MPO
+
/Auer rods.
AML M0 requires flow cytometry (or IHC AML-M0 requires flow cytometry (or IHC
or EM) for diagnosis.
CD7 in AML: adverse prognostic marker,
correlates with loss of CEBPA, early
relapse or residual disease.
Rhrs S et al. CD7 in acute myeloid leukemia: correlation with loss of wild-type CEBPA, consequence
of epigenetic regulation. J Hematol Oncol. 2010;3:15.
Morphological correlation: At the very
least, a smear prepared from the flow
sample should always be examined.
Cytochemical (slide) MPO correlates
Learning points from case 1
Cytochemical (slide) MPO correlates
extremely well with the FCM MPO.
(The latter is purported to be more sensitive,
but in borderline +ve cases it may be
difficult to interpret.)
Nakase K et al. Detection of MPO by flow cytometry in acute leukemia. Cytometry. 1998 ;34:198-202.
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Case 2: Background
34-yr/M with bone pain, petechiae x 1m
Hb 9.8 g/dl; TLC 43,000/l and platelets
1,11,000/l.
DLC: Blasts 35%, neutrophils 31%, lymphocytes DLC: Blasts 35%, neutrophils 31%, lymphocytes
24%& monocytes 10%.
Auer rods +
Blasts are MPO +ve.
CD45-SSC
Blast cluster shows continuity with the maturing
granulocytic population.
No significant monocytic region population.
Residual populations
Blasts (P1) 30%, lymphocytes (P2) 12%, granulocytic cells (P3) 55%
CD34
+
CD33
heterogeneous
CD14
-
CD64
-
CD13
+
, ~12% are CD15
+
CD117
-
CD64
-
HLA-DR
+
CD11b
-
CD16
-
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cMPO
+
cCD79a
-
cCD3
-
TdT
-
Dim CD19
in ~20%
blasts
Negative for
CD2, CD5,
CD8, CD10,
CD20
Summary of markers
~28% blasts expressing CD34 (~92%),
HLA-DR, cMPO (~96%), heterogeneous
CD33 (~61%) and moderate CD13 (~73%)
with aberrant CD19 (20%).
Negative for cCD79a, cCD3, TdT, CD10,
CD20, CD5, CD7, CD2, CD14, CD64, CD
11b and CD16
Impression: AML with maturation (FAB
AML M2) with low level expression of
CD19.
Simply tracking the blast population
(labelled in the appropriate plot) for every
marker yields the diagnosis in
uncomplicated cases
Learning points from case 2
uncomplicated cases.
The aberrant coexpression of CD19 (or
CD22, CD56) is highly associated with
t(8;21), a favorable prognostic indicator.
Ferrara F et al. Immunophenotypic analysis enables the correct prediction of t(8;21) in AML. Br J
Haematol. 1998;102:444-8.
Case 3
19-yr, male in Emergency with altered
sensorium
Bleeding gums, fever x 3 weeks
O/E: Ecchymosis +, No organomegaly. O/E: Ecchymosis , No organomegaly.
Hb 7.1 g%, TLC 2,000/l, Plt 76,000/ l
PT 33 / control 12
Clinical : Pancytopenia, coagulopathy:
? Acute promyelocytic leukemia
? Meningococcal meningitis
Sample for FCM
Blasts 14%, abnormal pro-myelocytes 69%, Auer rods +
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CD45-SSC CD45-SSC: Gated
Markers of immaturity
Heterogeneous
CD13
Negative for CD34 & HLA-DR, heterogeneous (negative to dim +ve) CD13 &
CD64)
Other myeloid markers
Compact CD33
Negative for CD14, heterogeneous (negative to dim +ve) CD117 & compact
moderate CD33)
CD117: 24%
Bright cMPO, low level CD15
(~15%), CD4
+
Negative for TdT, CD16, CD5.
Moderate
CD58
+
, neg
for B- and T-
markers
CD19, CD20, CD19, CD20,
CD10,
cyCD79a,
CD5, CD7,
CD8 and
cyCD3
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Summary of markers
An abnormal blast region cell cluster extending
to granulocytic region on SSC/CD45 plot (~92%)
The blasts express cMPO (~59%), CD33
(~94%), CD15 (~15%), CD13 (~44%), CD38
(~65%) and CD58 (~95%). They lack ( ) ( ) y
expression of CD34 and HLA-DR. and
CD64.aberrant expression of CD4 (~32%).
Negative for CD19, CD10, CD20, CD5, CD7,
CD8, CD2, CD14, CD11b, TdT and CD16
Impression: Consistent with acute
promyelocytic leukemia with aberrant CD4
expression.
Just in case samples: In suspected or
even remotely possible acute leukemia,
keep BM for FCM at the time of aspiration
M3 on FCM: A virtually single major
Learning points from case 3
M3 on FCM: A virtually single major
population with weak to moderate CD45
and wide ranging SSC signals (moderate
to high) is strongly suggestive of AML-M3.
Other corroborating features of AML-M3:
Increased baseline auto-fluorescence even on
unstained / isotype controls
Absence of CD34 & HLA-DR reactivity
Lessons from Case 3
y
Heterogeneous CD13 & compact CD33
clusters
M3 is sometimes equivocal on morphology
alone while RT-PCR takes time. FCM can
corroborate a BMA / PS report.
Mimics of M3
G-CSF therapy: A pitfall in both
morphology and in FCM
Myeloproliferative neoplasms (MPN) and
MDS MPNs: The clinical background MDS-MPNs: The clinical background
helps.
On FCM: CD11b & CD16 expression favor
non-APML disorders.
Case 4: Background
54/M, fatigue and bleeding gums
Hepatosplenomegaly, skin rash
Hb 8.8 g/dl; platelets 39,000/ml, TLC
4 300/ l 4,300/ml
BM: 23% blasts, 30% promonocytes (total
53%), MPO negative, NSE intense
positive.
Case 4: Morphology
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CD45-SSC
A large cluster is seen shifted upward and to the right (almost merging with the
sizeable monocytic region).
Gating monocytic AMLs
The neoplastic cluster displays bimodal CD45 with merging of cellular events from
blast region and monocytic regions.
Ensure that the tumor is not actually composed of 2 separate populations.
Red: Blasts; Blue: Monos
Blasts: CD34
+
, CD117
+
, CD64
+
(25%) CD14
-
, Monos: CD34
-
, CD117
-
, CD64
+
CD14
+
Red: Blasts; Blue: Monos
Blasts: CD33
+
, CD15
+
, CD64
+
(25%) CD14
-
,
Monos: CD33
bright
, CD15
-
, CD64
+
CD14
+
(admixed granulocytic population is ve for CD14, CD64, dimmer CD33)
Red: Blasts; Blue: Monos
Blasts: CD13
+
, CD11b
-
, HLA-DR
+/-
, CD4
-
, Monos: CD13
+
, CD11b
+
, CD16
+
, CD4
+
Red: Blasts; Blue: Monos
Blasts: TdT
+
, CD79a
-
, CD3
-
, CD10
-
, CD20
-
, CD2-, CD8-
Monos: TdT
-
, CD79a
-
, CD3
-
, CD10
-
, CD20
-
, CD2
-
, CD8
-
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Summary of findings
A prominent CD45
dim
cluster (blast region,
~31%) merging with the mono region (12%,
CD45
moderately bright
, higher SSC).
Blasts: CD34 (89%), CD13 (94%), CD33 (84%),
CD117 (50%), CD38 (85%) and TdT (60%). ( ) ( ) ( )
Maturing monocytes: CD16, CD64, CD11b,
CD4, CD13, CD33.
Blasts neg: cMPO, cCD79a, cCD3, CD19,
CD10, CD20, CD5, CD8, CD2, CD14, CD64,
CD11b, CD16.
Impression: AML with monocytic
differentiation {c/w BMA AML-M5b}.
Distinction between M4 / M5a / M5b by
FCM alone is difficult. Require correlation
with morphology.
Look for the monocytic patterns
Learning points from case 4
Look for the monocytic patterns.
Identify and track all discrete cell
populations separately to provide
information about differentiation patterns
of blasts.
Case 5
3 yr, F
Downs syndrome, now with fever and
pallor x2 weeks
N li l LN No liver, spleen, LN
PS: 14% abnormal cells
BMA: 40% abnormal cells (?blasts)
negative for MPO.
FSC-SSC, CD45-SSC
Multiple populations: lymphocytes, blasts, blasts merging with mono, nRBC/debris
Gating on CD45-SSC
P1 (red): Hematogones, P3 (blue): negative for myeloid, B- & T- markers. P2
(green): showed myeloid antigens. Was this the neoplastic clone?
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CD34 back-gating
On back gating from CD34, the violet cells (~30%) localized to P2, thus confirming
their immature nature.
Tracking P2 (CD34
+
, blast region)
Expressed CD34, CD33, CD14, CD11b, CD15, HLA-DR
CD7
+
, cMPO
neg
Add-on testing: CD41, CD61
Summary of markers
Cell cluster in the blast region extending to
monocytic region (~40%)
Express CD41, dim CD61, CD33, CD13,
CD11b, CD15, HLA-DR, CD34, aberrant CD7
(~93%). ( )
Negative for cMPO, cCD3, CD19, CD117,
CD10, CD2, CD5, TdT, CD14 & CD 64.
Impression: AML-M7 in Down syndrome
Transient abnormal myelopoiesis unlikely:
Patients age
CD13, CD11b +ve blasts .
In Down syndrome, AML blasts are express myeloid associated antigens CD13 and CD 11b as
compared to blasts in most cases of TMD; Am J Clin Pathol 2001;116:204-210.
CD34 back-gate in cases with multiple
populations (also CD117, HLA-DR back
gate).
A primary acute leukemia panel with add-
Learning points from case 5
p y p
on testing as indicate clinically or by the
initial results.
TAM vs. AML-M7 in DS
Hematogones may lie in the blast region
esp. in children.
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Case 6: Background
4-year-old boy
Pallor, hepatosplenomegaly, pleural
effusion, blasts in PS. Clinical : ALL.
Hb 8 1 /dl l t l t 106 000/ l d Hb: 8.1 g/dl; platelets: 106,000/l and
TLC: 21,400/l (Neutrophils 15%,
lymphocytes 49%, monocytes 13%,
myelocytes 02%, metamyelocytes 02%
and blasts 21%).
BM
BMA- Diluted
BM touch showed 70% blasts of variable
morphology (small lymphoid with cleavage
and larger undifferentiated to myeloid) and larger undifferentiated to myeloid)
The larger blasts showed ~10% MPO
positivity.
FCM on peripheral blood.
Morphology
CD45-SSC
Multiple populations in the blast window.
CD45-SSC
P1: CD45 dim, higher SSC, P2: CD45 brighter, lower SSC
Establishing the nature of the 2
populations
P1: CD34
+
, CD33
+
, CD64
-
, CD14
-
, P2: CD34
+
, CD33
-
, CD14
-
, CD64
-
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Myeloid & mono markers
P1: CD13
+
, CD15
+
, CD11b
-
, CD16
-
, P2: CD13-, CD15
-
, CD11b
-
, CD16
-
P1: HLA-DR
+
, cCD3
-
, CD11b
-
, CD11b
-
, P2: HLA-DR-, CD11b
-
, cCD3
+
, TdT
-
P1: CD7-, CD4-, cMPO
+
, CD79a
-
, P2: CD7+, CD4+, cMPO
-
, aCD79a
-
T-cell markers
P1: CD8-, CD4-/+, CD5-, CD2-, P2: CD8-, CD4+, CD2+, CD5+
Summary of markers
P1: ~18% of all acquired events. Dimmer CD45 with
higher side scatter. Express CD117, CD33, CD13, HLA-
DR, CD38 and CD34. MPO is expressed
immunophenotypically by ~7% blasts.
P2: ~14% of all cells. Moderate CD45, lower SSC. Co-
CD3 (81%) CD2 CD4 CD5 CD7 CD10 express cCD3 (81%), sCD2, CD4, CD5, CD7, CD10
and CD34 along with CD1a. CD8, CD10 and TdT are
negative in this population.
Both the types of blasts are negative for CD19,
cyCD79a, CD20, CD11b, CD16, CD14 and CD64.
Diagnosis: Mixed Phenotype Acute Leukemia (bi-
lineal, T/Myeloid).
FCM is mandatory for the diagnosis of
mixed phenotype acute leukemia.
Pattern-recognition and tracing the
immuno phenotype of each and every
Learning points from case 6
immuno-phenotype of each and every
population in the blast region and nearby
can yield significant information.
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Take home message: Basics
Use all the information available: clinical,
blood counts, morphology, cytochemistry.
Apply a consistent, logical approach to
analysis analysis.
Know the biological basics.
Know the artifacts: sample-related,
processing-related, machine-related.
Get familiar with the software.
Take home message: Analysis
Apply a variety of gating strategies to the
same population of cells to obtain the
maximum information.
Use consistent gating strategies to avoid Use consistent gating strategies to avoid
missing subtle alterations / populations.
Use cell numbers / percentages as
adjunctive information, not as the sole
means to evaluate a population.
Take home message:
Troubleshooting
If faced with a pattern one does not
understand:
Re-check the technique, the reagents and the
machine machine,
Call the clinician for more information,
Read, search the internet, email the *.fcs files
to someone else to take a look.
sharma.prashant@pgimer.edu.in