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Review Article

The role of Flow Cytometry in the diagnosis of Acute Myeloid Leukaemias

Mervyn Monaheng 212131052

Department of Biomedical Sciences, Cape Peninsula University of Technology

October, 2013



Abstract

The role of Flow Cytometry in the diagnosis of Acute Myeloid Leukaemias is

presented herein. The goal here is to review the use of Immunophenotyping by flow

cytometry in the diagnosis of major haematological disorders falling under Acute

Myeloid Leukaemias with minimal consideration of their subgroups, The discussion

will focus not only on the use of flow cytometry in the differential diagnosis of a

particular disorder, but also link immunophenotypic, molecular, and cytogenetic data

in the description of important subgroups. Information regarding technical aspects of

instrumentation, normal distribution of surface antigens, and intense methodologies

are not extensively discussed.

Flow cytometry is most often used in the clinical laboratory for the purpose of

Immunophenotyping (i.e. using a rather small panel of monoclonal antibodies, which

detect lineage-specific antigens expressed on cells). This immunological

characterization by flow cytometry is routinely used to distinguish between AML and

acute lymphoblastic leukaemia (ALL). The markers used in the immunophenotypical

analysis of AML are the following, CD13, CD15, CD33, CD34, CD36, CD41, CD65,

CD11c (myeloid lineage), cytoplasmic CD3, CD11b, CD13c, CD14, cytoplasmic

CD22, MPO, TdTc, CD4, CD5, CD38, CD56, cytoCD79a, CD117, HLA-DR (not

lineage specific). Expression of each this markers is characterized by the percentage

of cells expressing this marker with the fluorescence level exceeding a background

threshold, determined with an isotype control. Basically Flow cytometry is used to

characterize leukemic blast cells.



Introduction

Hematologic malignancies (HM) are a group of neoplasms that arise through

malignant transformation of cells derived from the bone marrow. There is great

variety in this group of disorders and this reflect how complex normal hemopoiesis

can be. These malignancies are clonal disorders of hemopoietic stem cells whereby

an abnormal clone of cells multiplies autonomously, overwhelming the normal

process of hemopoiesis, and then resulting in tissues and organs infiltration, thereby

disturbing their normal physiological activities. The essential defect is believed to be

due to a genetic abnormality at the level of the hemopoietic stem cell. (Olaniyi, 2011)

All normal cells express a variety of cell surface markers, depending on the specific

type of cell and its degree of maturation. However, abnormal growth such as seen in

haematological malignancies may interfere with the natural expression of these

markers resulting in overexpression of some and under-expression of others. Flow

cytometry can be used to immunophenotype cells and thereby distinguish between

healthy and diseased cells. It is not surprising that today immunophenotyping is one

of the major clinical applications of flow cytometry, and is essentially used to aid the

diagnosis of myelomas, lymphomas and Leukaemias. It can also be used to monitor

the effectiveness of clinical treatments. The differences between the blood profiles of

a healthy individual and one suffering from leukemia, for instance, are very dramatic.

This can be seen from the Forward Scatter (FSC) v Side Scatter (SSC) plots in

Figure 1. In the healthy person the cell types are clearly defined, whereas blood from

a leukaemia patient is abnormal and does not follow the ordinary profile.







The recent WHO classification of AML has placed emphasis on morphological,

immunophenotypic and molecular features, As such, AML is now also classified

according to specific recurrent cytogenetic abnormalities, and these are often directly

associated with specific immunophenotypic profiles, in other words

Immunophenotypic data also correlates reasonably well with the new WHO

classification. In cases of AML, where such genetic abnormalities are not evident,

these are described according to their degree of differentiation and/or differentiation

along monocytic, megakaryocytic or erythroid lineages. As such, the

immunophenotype is indispensable in characterization of the leukaemic cells in the

same way as its utilization in the FAB system. (Ian Chant, 2005)

Despite the fact that no single marker alone allows accurate lineage assignment,

analysis with panels of antibodies allows division of hematologic tumors into various,

very detailed subtypes. (Gorczyca, 2008)

Immunophenotyping by flow cytomctry has been instrumental in recognizing

minimally differentiated AML (AML-MO), acute megakaryoblastic leukaemia (AML-

M7), and AML co-expressing lymphoid-ussociated antigens.It has also been

particularly helpful in distinguishing AML with monocytic differentiation from AML-

MO/M1 or AML subtypes with granulocytic differentiation (i.e. AML-M2/M3) (T.

Benter et al, 2001)

Thanks be to Multiparameter (four- or six-color) flow cytometry analysis which allows

simultaneous evaluation of several markers on a single cell, facilitating accurate

characterization of the analyzed populations (Gorczyca, 2008) for determining the

blast lineage as well as for detecting aberrant antigenic profiles that may prove

useful for disease monitoring, (M. Peters, 2010). This technology provides quick and

detailed determination of antigen expression profiles in acute Leukaemias which, in

union with morphologic assessment, often suggests a conclusive diagnosis or

narrow differential diagnosis.

Now, at least four to six cellular antigens can be measured simultaneously in

combination with two intrinsic parameters, such as cytoplasmic complexity and cell

size by leukaemic blast light scatter properties (i.e. forward and side scatter

characteristics, FSC and SSC) Immunophenotyping by multiparameter flowcytometry has emerged as the best method for immunodiagnosis of haematopoietic



malignancies, largely replacing immunocytochemical microscopic analysis.

(T.Benter, 2001)

Methodology

Flow cytometry analysis requires fresh (unfixed) material. Types of specimen

suitable for flow cytometry include blood, bone marrow aspirates (and fresh core

biopsy), fresh tissue samples (excisional or core biopsies), fine needle aspirates, and

body fluids, (Gorczyca, 2008) although in general, the best results are achieved with

fluid-based specimens. (M. Peters, 2010) In the flow cytometry protocol, the sample

containing leukocyte population suspended in cell culture medium is incubated with

antibodies conjugated to a variety of fluorochromes, preceded by red blood cell lysis with ammonium chloride, washing, fixation in paraformaldehyde, and flow cytometry

analysis. Whole blood lysis is the most commonly used technique for sample

preparation. (Gorczyca, 2008)

Finally, the samples are scanned by the flow cytometry instrument, and the resultant

data histograms saved for evaluation. (M. Peters, 2010)

Monoclonal antibodies used in flow cytometry are conjugated with fluorochromes,

which are excited or stimulated by laser(s). The commonest fluorochromes excited at

488 nm (argon laser) include fluorescein isothiocyanate (FITC), phycoerythrin (PE),

propidium iodide (PI), 7-amino-actinomcyin D (7-AAD), peridin-chlorophyll-A-protein

(PerCP), and dimers of thiazole orange (TOT-1).

fluorescently labeled antibodies are bound to cell surface receptors, and their

presence on the cell is most often defined in bivariate terms of positive or negative,

with a cutoff set relative to a nonstaining control population

Immunologists from the far corners of the world who have produced monoclonal

antibodies directed to surface molecules on cells, these are usually functional

molecules reflecting the state of cellular differentiation. Every so often they meet to

compare the specificities of their reagents in international workshops, these has

resulted in discovery of a cluster of monoclonals are found to react with the same

polypeptide, they clearly represent a series of reagents defining a given marker and

they label it with a CD (cluster of differentiation) number. Up to date there are nowover 250 CD specificities assigned, With the impressive number of monoclonal



antibodies to hand, highly detailed phenotypic analysis of single-cell populations is

now a practical proposition.( A. Rabson, 2005)

Cluster of Differentiation (CD) molecules are markers on the cell surface, as

recognized by specific sets of antibodies, used to identify the cell type, stage of

differentiation and activity state of a cell (G. Ciuperc,2005) here is an example:

There are three main components to the flow cytometer :

1 The Fluidics System

Presentation of the sample to the laser.

2 The Optical System

Gathering information from the scattered light of the analysis.

3 The Computer/Electronic System

Conversion of optical to digital signals for display.



Light scatter

As a cell passes through the interrogation point, light from the laser beam is

scattered in forward and 90º angles. The amount of light scattered is dependent on

the physical properties of the cell, such as, cell size, nuclear complexity and

cytoplasmic granularity. These light scattering signals are gathered by specific

detectors, converted to digital signals and finally displayed as dot plots for analysis.

Light diffracted at narrow angles to the laser beam is called forward scattered light

(FSC) or forward angle light scatter (FALS). The amount of FSC is proportional to

the surface area or size of the cell. The forward scattered light is collected by a

detector placed in line with the laser beam on the opposite side of the sample



stream. Some light will pass through the cell membrane and is refracted and

reflected by cytoplasmic organelles or nucleus of the cell. This light is collected by a

photodiode positioned at approximately 90º to the laser beam and is known as side

scattered light (SSC). Side scattered light is proportional to the granularity or internal

complexity of the cell (Figure 2.4).

Together, FSC and SSC signals provide information on the physical properties of the

cells allowing differentiation of cells within a heterogeneous population, for example

the differentiation of white blood cells (Figure 2.5).





The gold standard for classifying acute myeloid leukaemia (AML) has been based on

morphological, cytochemical, and iinmunophenotypic criteria as defined by the

French—

American—

British (FAB) system 11-4]. Eight subgroups of AML (AML MO-

AML M7) have now been identified by this classification and by using lineage

commitment and the degree of blast cell differentiation. ( T.Benter, 2001)

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