Role of Flowcyometry in

Acute Leukemia
• Leukaemia is a disease resulting from the neoplastic
proliferation of haemopoietic or lymphoid cells

• Classically leukaemic blast cells have been considered to

represent the neoplastic counterpart of normal
immature cells blocked at a specific differentiation stage

• Historical classification systems relied predominantly on

morphologic and cytochemical features
• Mutation in a single stem cell, progeny of which form
a clone of leukaemic cells

• Often there is a series of genetic alterations rather

than a single event

• Leukaemic cells – not perfect replica of normal cells,

asynchrony of gene expression
• Antigenic expressions have been found to be frequently
aberrant
– presence of cross-lineage antigens
– presence of abnormal expression of normal antigens

• Anomalous antigen expression

– not directly related to abnormal differentiation pattern
– reflects underlying abnormal genetic pattern
• 2008 WHO Classification of Tumours of Haematopoietic
and Lymphoid Tissues

– Recent advances in immunophenotypic, cytogenetic,

and molecular aspects

– Categorization into the most clinically relevant entities

possible facilitating management of patients

– Criteria for prior entities have been revised and new

entities introduced based primarily on recurrent
molecular and genetic lesions
 Primary modalities for
diagnosis

Clinical Molecular
Information + Morphology +Immunophenotyping+ Cytogenetics + genetics
 MOLECULAR
STUDIES
Currently impractical to perform
a comprehensive, undirected
molecular analysis owing to the
myriad different genes involved
Require a high level of
technical expertise, available
only in specialized reference
laboratories, necessitating
prolonged specimen processing
and analysis times, which may
be incompatible with timely
clinical management of acute
leukemia
MORPHOLOGY &
IMMUNOPHENOTYPING
Initial rapid interpretation of
morphologic and
immunophenotypic data can
efficiently direct a narrow and
specific search for molecular
lesions
Analysis can be completed
within a matter of
hours on virtually any case
and is often sufficient to
arrive at a narrow differential
or even definitive diagnosis
 Immunophenotyping
• Identification of antigens within (cytoplasmic & nuclear)
or on the surface of leukaemic cells

• Using labelled antibodies that recognize specific

epitopes of cellular antigens

• Analysis of heterogeneous populations

– identification of pathologic cells
– their phenotypic characterization
 Immunophenotyping can be performed by:

•Immunoenzymatic techniques
•Using Peroxidase or ALP
•Utilise cytospin/ paraffin
embedded/ fresh frozen tissue
• Flow cytometry - preferred method for the lineage
assignment and maturational analysis of hematological
malignancies

The results can be stored in list-mode files and interpreted by

other observers to facilitate objective interpretation
 Flowcytometry
Flow ~ motion, Cyto ~ cell, Metry ~ measure
Measuring properties of cells while in a fluid stream

• Measurement and analysis

of physical and chemical
properties while the cells
pass, in single file, through
the apparatus in a fluid
stream through a beam of
light
 Using multiple Abs labelled with
fluorochromes expression of antigens
can be evaluated and correlated

Forward light scatter (proportional

to cell size) and sideways light
scatter (determined by cell
structure, including granularity)
can be analysed and related to
antigen expression
 Type of specimen
Bone marrow aspirate –
preferred, especially in low
blast counts

Peripheral blood – high

blast counts (>80%)

Malignant effusions, e.g. ascites or pleural effusions

Biopsies from solid organs and bone marrow can also be used after
manual disaggregation and filtration of large particles
• Volume – 2 - 5 ml

• Anticoagulant
– EDTA - reduces leukocytes and platelets adhesivity and
aggregation

– Heparin - may interfere with morphology, labelling,

fixation, permeabilization
 RBC Lysis
a) Ammonium chloride buffer
b) Hypotonic sodium chloride solutions
c) Commercial reagents
Density-gradient centrifugation with either Percoll or
Ficoll is an acceptable alternative for this purpose

Remaining leukocytes suspended in cell

culture medium and subjected to cell count
so that controlled no. of cells can be
submitted for staining with abs

Aliquots of cells are then

added to tubes for staining
with panels of antibodies
 RBC Lysis
a) Ammonium chloride buffer
b) Hypotonic sodium chloride solutions
c) Commercial reagents

Remaining leukocytes suspended in cell

culture medium and subjected to cell count
so that controlled no. of cells can be
submitted for staining with abs

Aliquots of cells are then

added to tubes for staining
with panels of antibodies
Forward scatter
• Forward Angle Light Scatter
(FSC)

• Large objects will scatter

more light in the forward
direction than small objects

• Voltage Signals received by

detectors are directly
proportional to cell size
Side Scatter

• Side Scatter near 90° (SSC)

• Cell with more granularity

scatter more side light
Light emission

• Flowcytometer can detect

light emission from single
cell that binds fluorescently
conjugated mAb

• Multiparameter FCM - Can

detect 3 or more colours,
(currently upto 10)
fluorochrome conjugated
mAb that emit light at
different wavelength
• Data so obtained can be analysed for:

1. Gain of antigens not normally expressed by cell type or lineage

(linage infidelity markers)

2. Abnormally increased or decreased levels of expression (intensities)

of antigens normally expressed by cell type or lineage, including the
complete loss of normal antigens in some instances

3. Asynchronous antigen expression (i.e., expression of antigens

normally expressed by cell type or lineage), but at an inappropriate
time during maturation

4. Abnormal scatter patterns

 Scope of FCM in Acute Leukemias

• Assignment of cellular lineage of the malignant cell

• Analysis of cellular maturation

• Aberrant features of the malignant cell populations

• Detection of minimal residual disease(MRD)

 Indications for immunophenotyping
• ALL – B/ T - Lineage

• Leukaemic blasts are morphologically undifferentiated

and negative for the cytochemical reactions
characteristic of myeloid cells

• In these cases immunophenotypic analysis may clarify

whether the case corresponds to one of the subtypes of
ALL or to poorly differentiated acute myeloid leukaemias
(AML-MO), early erythroleukaemias, or
megakaryoblastic leukaemias.
• Identification of hematopoietic neoplasia by IP relies on the
patterns of antigen expression

• Antigen expression in normal cells is a tightly regulated

process, resulting in a characteristic pattern of antigen
acquisition and loss with maturation that is cell lineage
specific
 Stem cell
TdT, CD 34
Immature megakaryoblast Myelomonocytic stem cell
CD 33, 34, Gp IIIa CD 13, 14, 15, 33, 34

Proerythroblast
Myeloblast Monoblast
Gp A
Megakaryoblast CD 13, 15, 33, 34 CD 13, 15, 33, (11c)
Gp IIIa (CD 61),
Gp Iib (CD 41) Erythroblast
Gp A Promyelocyte Promonocyte
CD 13, 15, 33,(11c) CD 13, 14, 33, 11c
Megakaryocyte
Gp IIIa, Gp IIb Myelocyte Monocyte
CD 13, 15, 33, 11c CD 13, 14, 33, 11c

Granulocyte Macrophage
Thrombocyte Erythrocyte
CD 13, 15, 11c CD 13, 14, 33, 11c, 1
Gp IIIa, Gp IIb Gp A

NORMAL PATTERNS OF ANTIGEN EXPRESSION

 Antibodies should be combined in an efficient way for use
in the identification of abnormal hematopoietic populations
 Immunophenotyping in Acute Leukemia

(Lineage
assignment)

(Definite phenotype,
Maturation)
• B cell – CD 19
• T cell – CD 3, CD 7
• Myeloid – CD 13, CD 33, CD 117
• Monocytic lineage – CD 14
• Megakaryocytic – CD 61
• CALLA – CD 10
The CD45/Side Scatter “Blast Gate”

Borowitz et al., 1993

Acute Myeloid Leukemia With t(8;21)(q22;q22);
RUNX1-RUNX1T1

G
T
B

HLA –DR +,
CD 34+,
MPO +, CD
13 +, weak
CD 33
Acute Promyelocytic Leukemia With
t(15;17)(q22;q12); PML-RARA
 Acute Promyelocytic Leukemia With
t(15;17)(q22;q12); PML-RARA

• Hypogranular variant –
– Frequent expression of CD34, CD2, at least on a
fraction of cells
Immunophenotyping in AML, NOS
AML M0
AML, M1 HLA DR- +, CD 34+
(70%) , CD 13+, CD
117 +, CD 33 +,
MPO + (fraction)

CD 11b, CD 15,
CD 14, CD 64 -
NEG
AML, M1
AML, M2
AML, M4
CD 33 (bright), HLA –DR (intermediate), CD 117 (intermediate)
CD 34 neg
CD 13 (intermediate),
CD 15 (intermediate)
CD11b (low to intermediate), CD64 (bright) and CD36 (intermediate to
bright), CD14 (low)
AML, M5
This CD45 vs SSC plot corresponds
to the above acute monocytic
leukemia case. Abnormal monocytic
population is green. Note the higher
SSC. AML M5 b
This CD45 vs SSC plot corrponds to
the above acute monoblastic
leukemia case. Abnormal monocytic
population is green. Note the lower
SSC. AML M5a
AML, M5
AML, M6
AML, M6
AML, M6
AML, M7
Immunophenotyping in ALL
Precursor B cell lymphoblastic leukemia
Precursor B cell lymphoblastic leukemia
T-lymphoblastic leukemia
T-lymphoblastic leukemia
 Additionally, some nonhematolymphoid tumors such as small cell
carcinoma or pediatric small round cell tumors can resemble leukemic
blasts on smears but are readily distinguished from leukemic blasts by flow
cytometry, where they appear as CD45-negative cells lacking specific
markers of B cell, T cell, or myeloid lineage, often with higher side scatter
than might be expected for a cell with so little cytoplasm (Chang, 2003)
Biphenotypic, bilineal, ambiguous or mixed lineage: strange
leukemias!
Requirements for assigning more than one lineage to a single
blast population

• Myeloid lineage – MPO (FCM, IHC, CC) OR

Monocytic – ( at least 2 of the following:
NSE, CD 11c, CD14, CD64, Lysozyme)

• T-lineage - cCD3, surface CD3

• B-lineage – Strong CD19 with any one CD79a, cCD22, CD10

Weak CD19 with 2 of the above
MPAL,
B/myeloid
T/MYELOID leukaemia with two separate populations of T
(blue) & myeloid (green) . (Bilineage leukemia).
T/MYELOID leukaemia with single population of blasts
(red) . (Biphenotypic leukemia).
T/MYELOID leukaemia in which both co-expression (red) &
separate populations of T(blue) & myeloid (green) blasts
can be seen.
 Immunophenotyping and Acute
Undifferentiated Leukemia

• Acute leukaemia, NOS, Stem cell leukaemia

• No clear evidence of myeloid or lymphoid differentiation

even after IP with a comprehensive panel of abs

• Often express CD34, CD38 and HLA-DR and may express

TdT
Aberrant Expressions - LAIP
 Minimal Residual Disease
• Sensitivity - 1 abnormal cell per 104 to 105

• Tailor postinduction therapy

• LAIP:
– Absence of expected ags
– Overexpression of ags
– Asynchronous expression
– Lineage infidelity
– Aberrant light scatter property
Minimal Residual Disease
• Drawbacks
– lack of antigen specificity for malignant cells as these
cells represent the counterparts of normal cells with, in
many cases, identical or similar antigen profiles

– existence of several subpopulations, some of them as

minor clones, that are difficult to identify

– inability to identify phenotypic switch, a phenomenon

that may occur at relapse, albeit at a low rate
Prognosis
CD1O-negative phenotype –
worse prognosis
Therapeutic Targets

B- ALL - anti-CD22
(epratuzamab) and
CD52 (alemtuzamab)
 CD 13, CD 33 + Blasts

- MPO CD 19, CD 22, CD10 +

+ cCD3, CD 7 +

CD 41, CD 61 T-ALL B-ALL

AML, M7
HLA DR+, CD 34+
AML, M4
CD 71, GPA CD14 +, CD 64+
AML, M6

HLA DR-, CD 34- AML, M3

HLA DR +, CD 34+
AML, M0
HLA DR+, CD 34+
AML, M1
CD 15 -, CD 14-
CD11b, CD 64, CD 14 +
AML, M5 HLA DR+, CD 34+
AML, M2
CD 15 +, CD 14-
 Conclusion
• Immunophenotypic analysis &
morphology remain as the foundation for
initial evaluation as well as establishing
the diagnosis in leukemias

• Provide a rapid assessment for directing

specific molecular genetic tests

• The unique combination of antigens may

be utilised to identify malignant cells in
fluids or for MRD
 References
• WHO Classification of tumours of haematopoietic and lymphoid tissue, 2008 4th edition
• Wintrobe’s Hematology 12th edition
• Henry’s Diagnosis by laboratiory methods. Chapter 34
• Hoffbrand’s Post Garduate Hematology
• Multiparameter Flow Cytometry in Acute Leukemia—Peters & Ansari Arch Pathol Lab Med—
Vol 135, January 2011
• Leukemia Diagnosis. Barbara J. Bain – Third edition
• Alexandra M. Harrington. A Dissection of the CD45/Side Scatter “Blast Gate”. Am J Clin
Pathol 2012;137:800-804
• Fiona E. Craig and Kenneth A. Foon - Flow cytometric immunophenotyping for hematologic
neoplasms. Blood, 15 April 2008 Volume 111, number 8
• Brent Wood. Multicolor Immunophenotyping: Human Immune System Hematopoiesis.
METHODS IN CELL BIOLOGY, VOL. 75
• C. Darrell Jennings and Kenneth A. Foon. Recent Advances in Flow Cytometry: Application to
the Diagnosis of Hematologic Malignancy. Blood, Vol 90, No 8 (October 15), 1997: pp 2863-
2892
• Konstantin U. Slobodnyuk. Detection of minimal residual disease in patients with acute
myeloid leukemia with multicolor fl ow cytometry. Cellular Therapy and Transplantation
(CTT), Vol. 3, No. 10
• Dario Campana. Detection of Minimal Residual Disease in Acute Cytometry
(Communications in Clinical Cytometry) 38:139–152 (1999)
THANK YOU
AML, M5