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Principles, Applications and Interpretations.

INTRODUCTION
The concept of flow cytometry has been in existence
for more than five decades.

Flow cytometric immunophenotyping (FCI) first


appeared in clinical laboratories in the 1980s, in the
wake of the AIDS epidemic.

Initially utilized to assess CD4 T-cells, the technique


was soon applied to lymphoid and eventually myeloid
neoplasms.
 Current flow cytometers have the capability of
simultaneously measuring multiple parameters of
individual cells in a cell suspension.

 Thus, a large number of cell specimens can be


processed with a quick turnaround time.

 In addition, flow cytometry is also highly sensitive


and can detect immunophenotype of cells in a
specimen with thousands of cells.
The parameters analyzed by flow cytometry include

 physical properties of cells; the size, cytoplasmic


granularity, and amount of DNA contents; and

 cell antigens/markers (surface, cytoplasmic, and


nuclear) that can be recognized by specific
antibodies.
By using appropriate antibody panels, flow cytometry
can reveal

the cell type (hematopoietic, lymphoid, or


nonhematopoietic),

cell lineage (B- and T cells, natural killer cells,


myeloid/ monocytic cells, neuro/neuroendocrine
cells, and epithelial cells),

cell maturation stage (precursors vs. matured cells)


Flow cytometry involves the analysis of the optical
and fluorescence characteristics of single particle
(e.g. cells, nuclei, chromosomes) during their
passage within a narrow, precisely defined liquid
stream.
For cell analysis, the basic components of a flow
cytometer include:-

OPTICAL ELECTRONIC COMPUTER


FLOW SYSTEM
SYSTEM SYSTEM SYSTEM
SCHEMATIC DIAGRAM OF A FLOW CYTOMETER

PMT-photomultiplier tubes
ADC-analogue-to-digital converter
CONCEPT OF SCATTERING
Physical properties, such as size (represented by
forward angle light scatter) and internal complexity
(represented by right-angle scatter) can resolve certain
cell populations.
 FSC collects light at 180° from the point at which the
laser beam intersects the cells, usually on a linear scale.
It is correlated with cell size, and thus can distinguish
normal lymphocytes (small), monocytes
(intermediate), and neoplastic cells (generally they are
large in size).

 SSC collects right-angle light at 90° and is correlated


with cytoplasmic granularity and nuclear configuration.
The combination of both FSC and SSC can
distinguish normal lymphocytes, granulocytes, and
monocytes.

 The detection of lymphocytes and monocytes


provides a reliable internal control to evaluate the size
of the cells of interest.
IMMUNOPHENOTYPING ANALYSIS
Requires

Antibodies.
Fluorochromes.
ANTIBODY
Highly specific monoclonal antibodies are used that
are produced by cloned antibody secreting cells.
Antibodies are based on cluster of differentiation
(CD)- a protocol used for identification and distinction
of cell surface antigens.
Using CD system we can identify cells by the presence
or absence of particular surface markers for e.g. CD3+
or CD20- etc.
FLUOROCHROMES
 Fluorochromes are substances that can be excited by
certain light source (such as laser) and emit a fluorescent
signal at a single wavelength.
 Fluorescent dyes can directly bind to certain cellular
content, such as DNA and RNA, and allow us to perform
quantitative analysis on individual cells.
 However, in most cases fluorochromes are conjugated with
monoclonal antibodies, which specifically target cellular
antigens/markers.
Characteristics of fluorochromes commonly used in
flow cytometry .
FLUOROCHROMES CONJUGATED TO ANTIBODIES EXCITATION WAVELENGTH(NM) EMISSION WAVELENGTH(NM)

Fluorescein isothiocyanate (FITC) 488 530

Phycoerythrin (PE) 488 580

PE-Texas Red 488 615

PE-Cy5 488 670

Peridinin chlorophyl protein(PerCP) 488 670

Allophycocyanin (APC) 633 670

APC-Cy7 633 767

Interestingly, although some of them can be excited by the same light source, the
different fluorochromes may emit fluorescent signals with different
wavelengths/colors. Thus, multiple fluorochromes can be simultaneously excited by a
light source and detected by their emission fluorescent signals with different
wavelengths, respectively.
IMMUNOPHENOTYPING ANALYSIS
Antibodies conjugated to fluorescent dyes can bind specific
proteins on cell membranes or inside cells. When labeled cells are
passed by a light source, the fluorescent molecules are excited to a
higher energy state. Upon returning to their resting states, the
fluorochromes emit light energy at higher wavelengths. The use of
multiple fluorochromes, each with similar excitation wavelengths
and different emission wavelengths (or “colors”), allows several
cell properties to be measured simultaneously.
Simultaneous detection of multiple cell
antigens/markers.

Multiple cell antigens ( Ag ) are recognized by fluorochromeconjugated specific antibodies (


Ab ). Because different fluorochromes have different emission wavelengths/colors, they can
be simultaneously detected by a flow cytometer.
FITC fluorescein isothiocyanate; PE phycoerythrin; PerCP peridinin chlorophyll protein; PE-T Red PE-Texas
Red .
Abnormal/ aberrant antigenic expression can be
grouped into four basic categories:
• Abnormally increased or decreased levels of antigenic
expression (aberrant expression)

• Gain of antigens not normally expressed in the cell type

• Expression of antigens not synchronized with normal


development and maturation stage of the cell type or
lineage

• Homogeneous expression of antigen(s) by a cell


population that normally show more heterogeneous
expression
SPECIMENS SUITABLE FOR FLOW
CYTOMETRY
Theoretically, any specimens from which a single
cell suspension can be generated are suitable for flow
cytometry analysis.

However, a lack of distinct antigens or markers in


the cells of interest or tissues limits the diagnostic
value of flow cytometry.
Common specimens suitable for flow
cytometry analysis include
Peripheral blood,

Bone marrow,

Body fluids,

Cerebrospinal fluid,

Urine,

Lymph node (cells or fresh tissues),

Any fine-needle aspirates,

Fresh tissues suspicious for hematopoietic and lymphoid disorders.


SPECIMEN STORAGE
For blood and bone marrow specimens, anticoagulants
such as EDTA, heparin, or acid citrate dextrose are needed.

Fresh tissue specimens are best transported and stored


in sterile tissue culture medium.

Although specimens may be stored at room


temperature, refrigeration is preferred, particularly when
there is a delay for flow cytometric analysis.

For flow cytometry analysis, single-cell suspensions of


the fresh tissues can be achieved by mechanical
dissociation.
General Notes On Cell Preparation
1.Single cell suspensions are required for optimal
staining of samples for flow cytometry.

2. The narrow bores of the sample injection needle and


tubing on a flow cytometer will be easily clogged by
aggregated cells and debris.

3. Preparation of single cell suspensions from solid


tissue requires mechanical dissociation and/or
enzymatic digestion for optimal recovery of cells from
the tissue.
PROCESSING OF SOLID TISSUES
1) Tissue is weighed and mechanically and enzymatically
disaggregated into a single cell suspension.

2) Collagenase is the most commonly used enzyme, followed by


dispase and trypsin.

3) Enzymatic digestion is performed in an incubator or a


shaking water bath.

4) Mechanical disaggregation can be accomplished with paired


scalpels or scissors.

5) The process often requires centrifugation, harvest of single


cells and redigestion of tissue fragments.
6) The sample should be visually inspected at all phases of
tissue digestion.

7) In the final stage, cell suspensions are passed through a 70


to 200 micron filter to remove aggregates.

8) Cell suspensions are then counted and viability is


determined by a dye exclusion assay, such as trypan blue.

9) Sample is finally incubated with required antibodies


attached with fluorochrome in optimal temperature and pH
and analyzed by flow cytometer.
DNA content Analysis
The measurement of cellular DNA content by flow
cytometry uses fluorescent dyes, such as propidium
iodide, that intercalate into the DNA helical structure.

The fluorescent signal is directly proportional to the


amount of DNA in the nucleus and can identify gross
gains or losses in DNA.

Abnormal DNA content, also known as “DNA content


aneuploidy”, can be determined in a tumor cell
population.

DNA aneuploidy generally is associated with malignancy


DNA analysis by flow cytometer
Erythrocyte analysis
Detection and quantification of fetal red cells in
maternal blood. The use of flow cytometry for the
detection of fetal cells is much more objective,
reproducible, and sensitive than the Kleihauer-Betke
test .
Diagnosis of PNH
Conventional laboratory tests for the diagnosis of PNH
include the sugar water test and the Ham’s acid hemolysis test .
Antibodies to CD55 and CD59 are specific for decay-
accelerating factor and membrane-inhibitor of reactive lysis,
respectively, and can be analyzed by flow cytometry to make a
definitive diagnosis of PNH.
Reticulocyte analysis
Reticulocyte counts are based on identification of residual
ribosomes and RNA in immature nonnucleated red blood cells
by using supravital stain. The flow cytometric enumeration
of reticulocytes uses fluorescent dyes that bind the residual
RNA, such as thiazole orange .

A region has been drawn on the red cells in the scatter plot. The other
major cluster in the scatter plot are the platelets.
The histogram was gated on the red cells and the regions on it delineate
cells with high (H), medium (M) and low (L) fluorescence corresponding
to increasing reticulocyte maturity. N marks nucleated red cells.
In the blood bank, flow cytometry can be used as a
complementary or replacement test for red cell
immunology, including RBC-bound
immunoglobulins and red cell antigens. Flow
cytometry has been used to accurately identify and
phenotype the recipient’s red cells.

Flow cytometry is being used increasingly in the


blood bank to assess leukocyte contamination in
leukocyte-reduced blood products .
Leukocyte Analysis
Perhaps the best example of simultaneous analysis of
multiple characteristics by flow cytometry involves the
immunophenotyping of leukemias and
lymphomas.

WHO classification has divided non-Hodgkin


lymphoma into B-cell and T/NK cell subtypes, which
are further subclassified into precursor and peripheral
lymphomas.

Immunophenotyping by flow cytometry (FCM) is an


essential aid for accurately diagnosing and
prognosticating leukemia and lymphoma.
The ability to analyze multiple cellular characteristics,
along with new antibodies and gating strategies, has
substantially enhanced the utility of flow cytometry in
the diagnosis of leukemias and lymphomas.

Different leukemias and lymphomas often have subtle


differences in their antigen profiles that make them
ideal for analysis by flow cytometry.

B cell: CD5, CD10, CD19, CD20, CD45, Kappa, Lambda;


T cell: CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD56;
Myelomonocytic: CD7, CD11b, CD13, CD14, CD15, CD16, CD33, CD34, CD45,
CD56, CD117, HLA-DR;
Plasma cell: CD19, CD38, CD45, CD56, CD138
Diagnosis of B-Cell Lymphomas by using
specific antibodies in flow cytometer
Identification of lineage specific antigens for perfect
diagnosis
Immunologic monitoring of HIV-infected
patients is a mainstay of the clinical flow cytometry
and provides the best possible way for enumeration of
CD4+ T lymphocytes and HIV viral load.
Flow cytometry can be used for lymphoma
phenotyping of fine needle aspirates, and is a
powerful adjunct to cytologic diagnosis.

Neutropenia may be immune or nonimmune in


nature. Immune neutropenia may result from
granulocytespecific autoantibodies, granulocyte-
specific alloantibodies, or transfusion-related anti-
HLA antibodies. Flow cytometry can readily identify
anti-neutrophil antibodies that are either bound to
granulocytes or free in plasma and confirm the origin
of neutropenia, possibly eliminating the need for a
bone marrow procedure.
Functional deficiencies of leukocytes can be
assessed by flow cytometry. Assays for oxidative burst,
phagocytosis, opsonization, adhesion, and structure
are available.
One of the clinical example is LAD type I is caused by
a genetic deficiency of β2 -integrins, which are
heterodimers of CD11 and CD18. This deficiency leads
to a loss of neutrophil and monocyte migration.
The high sensitivity and capacity for simultaneous
analysis of multiple characteristics make flow
cytometry useful for the detection of minimal
residual disease, especially if abnormal patterns of
antigen expression are present.
Platelet analysis
Flow cytometry is an excellent method for direct
analysis of platelet-bound antibodies, and it has
also been shown to be of benefit in detection of free
plasma antibodies in ITP.

The reticulated platelet count can be quantified by


flow cytometry in order to assess the rate of
thrombopoiesis. This measurement can separate
unexplained thrombocytopenias into those with
increased destruction and those with defects in
platelet production.
The pathogenesis and molecular defects of many
primary thrombocytopathies are well known and
relate to defects in structural or functional glycoproteins,
such as the abnormal expression of gpIIb/IIIa in
Glanzmann thrombasthenia and gpIb in Bernard-
Soulier disease.
Flow cytometry is a rapid and useful method of
obtaining a diagnosis.
Other applications
Flow cytometry is indicated in the evaluation of serous effusions
and CSF, including aqueous or vitreous humor of patients
with a history of hematolymphoid neoplasia.

Flow cytometry assists in the differential diagnosis between


plasma cell myeloma and monoclonal gammopathies of
undetermined significance by determining the percentage of
aberrant or clonal plasma cells of all bone marrow plasma cells.

Flow cytometry is useful in diagnostic evaluation of unexplained


marrow plasmacytosis by assessing phenotypically aberrant or
clonal plasma cells and its ability to detect other underlying
monoclonal B-cell process.
Tissue-based lymphoid neoplasias commonly affect
lymph nodes, spleen, mucosa-associated lymphoid
tissue, skin, or nonlymphoid solid organs resulting in
masses or organomegaly.

Flow cytometry is extremely useful in the diagnosis and


subclassification of tissue-based lymphoid neoplasias,,
organomegaly and tissue infiltrates.
Basic parameters and Windows of
cell population
Forward light scatter (FSC) and side light scatter (SSC) . FSC collects
light at 180° from the point at which the laser beam intersects the cells .It is
correlated with cell size.and thus can distinguish normal lymphocytes
(small), monocytes (intermediate), and neoplastic cells (generally they are
large in size).

SSC collects right-angle light at 90° and is correlated with cytoplasmic


granularity and nuclear configuration.

The combination of both FSC and SSC can distinguish normal lymphocytes,
granulocytes, and monocytes.

The detection of lymphocytes and monocytes provides a reliable internal


control to evaluate the size of the cells of interest.
CD45 & SSC
As the first step, it is most important to determine whether the cells of
interest are hematopoietic.

Generally speaking, all hematopoietic/lymphoid cells express CD45


antigens (CD45+).

Thus, a histogram of CD45 on a logarithmic scale vs. SSC on a linear


scale is indispensable as a starting point of flow cytometry analysis.

Based on antigen expression, cells are divided into CD45+ and CD45–
groups.

Among the CD45+ group, the cells can further separated into
subgroups (windows in the histogram) based on expression levels of
CD45 and intensity of cytoplasmic granularity.
CD45 negativity is seen in non-
haemopoietic cells, erythroid
precursors and abnormal plasma cells.
Weak positive in blasts.

More CD45 positivity more the


brightness
Concept of Gating in brief
Gating is the most important first step in
immunophenotyping analysis.

It is critical particularly in a specimen that contains


mixed cell populations, such as bone marrow aspirate.

Gating sets upper and lower limits on the type and


amount of material that passes through.

It is used to separate a sub-population from


heterogeneous population.

It permits very specific questions to be asked about a


particular population.
Types of Gating
1. By cell distribution in the CD45 vs. SSC. This is most useful
in a specimen containing mixed cell populations . The grouped
cells in individual windows represent different cell lineages.

2. By cell size: In FSC vs. SSC histograms, neoplastic cells


(usually large in size) can be gated by using lymphocytes (small)
and monocytes (intermediate) as an internal size control . Once
the cells of interest are gated, further analysis of cell lineage can
be performed.

3. By cell lineage-specific antigens (immunophenotype): If


cells are CD45+ but do not fit into particular windows in the
CD45 vs. SSC histogram, identification of lineage-specific
antigen expression is needed
Gating the Lymphocytes

A region, R1, has been drawn around the lymphocytes (A).

In B, the lymphocytes are coloured red.

In C a gate has been set to show only the cells in R1


Gating the Monocytes

A region, R2, has been drawn around the monocytes (A).

In B, the monocytes are coloured blue.

In C a gate has been set to show only the cells in R2


Gating the T-Lymphocytes
Quadrant regions showing the percentage of cells in each
sub-population
QUALITY CONTROL IN FLOW CYTOMETER
There are several kinds of quality controls.

First the flow cytometer itself must be evaluated for proper


function.This is usually accomplished with standardized
fluorescent beads. These give very precise, reproducible
patterns, which quickly assess instrument function.

A second quality control material is used to set up the


appropriate instrument settings for the type of staining used.
These can be beads or antibody-stained cells.

The third level of quality control is a control substance that


mimics actual specimens. These controls are available
commercially and usually consist of stabilized blood,
sometimes with added tissue-culture cells that mimic a specific
cancer cell.
Comparison of immunophenotypic techniques .
FLOW CYTOMETRY IMMUNOHISTOCHEMISTRY

Shorter turnaround time (minutes to hours) Longer turnaround time (hours to days)

Less subjective result interpretation Subjective result interpretation

Quantitative results Semiquantitative results

Multiple antibodies/fluorochromes per test Usually limited to a single antibody per slide

Greater antibody selection Fewer antibodies available

Data/results can be electronically transferred Slides can be shipped by mail or courier service

Need fresh cells or tissue Can use fixed/archived tissue

Limited morphologic correlation Architectural and cytologiccorrelation

Cannot assess nonviable cells Can assess nonviable “ghost” cells


CONCLUSION
Flow cytometry is a powerful technique for correlating
multiple characteristics on single cells. This qualitative
and quantitative technique has made the transition
from a research tool to standard clinical testing.

Smaller, less expensive instruments and an increasing


number of clinically useful antibodies are creating
more opportunities for routine clinical laboratories to
use flow cytometry in the diagnosis and management
of disease

And last but not the least, keeping pace with scientific
and clinical advancements is the need of hour.