Flowcytometry 1

FLOW CYTOMETRY
.SUNIL KUMAR.P
Department of Clinical Pathology
St.John’s Medical College
Bangalore
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SUNIL KUMAR. P ST.JOHN'S MEDICAL
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INTRODUCTION
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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.
INTRODUCTION
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 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.
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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.
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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)
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PRINCIPLES OF CYTOMETRY
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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.
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COMPUTER
SYSTEM
ELECTRONIC
SYSTEM
OPTICAL SYSTEMFLOW SYSTEM
For cell analysis, the basic components of a flow
cytometer include:-
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SCHEMATIC DIAGRAM OF A FLOW CYTOMETER
PMT-photomultiplier tubes
ADC-analogue-to-digital converter
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Physical properties, such as size (represented by forward
angle light scatter) and internal complexity (represented
by right-angle scatter) can resolve certain cell populations.
CONCEPT OF SCATTERING
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 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.
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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.
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IMMUNOPHENOTYPING ANALYSIS
Requires
• Antibodies.
• Fluorochromes.
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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.
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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.
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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.
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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.
IMMUNOPHENOTYPING ANALYSIS
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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 .
Simultaneous detection of multiple cell antigens/markers.
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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 heterogeneous10/13/2018 22
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PROCESSING OF SPECIMEN FOR FLOW
CYTOMETRY
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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.
SPECIMENS SUITABLE FOR FLOW
CYTOMETRY
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Peripheral blood,
Bone marrow,
Body fluids,
Cerebrospinal fluid,
Lymph node (cells or fresh tissues),
Any fine-needle aspirates,
Fresh tissues suspicious for hematopoietic and lymphoid disorders.
Common specimens suitable for flow
cytometry analysis include
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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.
SPECIMEN STORAGE
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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.
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APPLICATIONS OF
FLOW CYTOMETRY
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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
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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 .
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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.
Diagnosis of PNH
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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.
Reticulocyte analysis
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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 .
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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.
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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
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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.
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Flow cytometry can be used for lymphoma phenotyping
of fine needle aspirates, and is a powerful adjunct to
cytologic diagnosis.
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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.
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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.
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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.
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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.
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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.
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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.
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FLOW CYTOMETRY
INTERPRETATION
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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.
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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
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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
Types of Gating
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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
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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 Monocytes
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Gating the T-Lymphocytes
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Quadrant regions showing the percentage of cells in each sub-
population
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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.
QUALITY CONTROL IN FLOW CYTOMETER
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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
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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.
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REFERENCE
1.Text book of haematology Wintrobes 10th edition
2.Textbook of haematology Williams.
3.Manual of Flowcytometry BD FACS calibaur.
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THANK YOU
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Flowcytometry 1

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