Principles, Applications and Interpretations.

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

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.

COMPUTER SYSTEM

ELECTRONIC SYSTEM

OPTICAL SYSTEM

FLOW SYSTEM

For cell analysis, the basic components of a flow cytometer include:-

PMT-photomultiplier tubes ADC-analogue-to-digital converter

SCHEMATIC DIAGRAM OF A FLOW CYTOMETER

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

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.

ANTIBODYHighly 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.

FLUOROCHROMESFluorochromes 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.

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

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.

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

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

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.

Common specimens suitable for flow cytometry analysis include

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

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 TISSUES1) 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 AnalysisThe measurement of cellular DNA content by flow cytometry uses fluorescent dyes, such as propidiumiodide, 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 analysisDetection 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-Betketest .

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

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

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 AnalysisPerhaps 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 analysisFlow 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 applicationsFlow cytometry is indicated in the evaluation of serous effusions

and CSF, including aqueous or vitreous humor of patientswith 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 & SSCAs 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 briefGating 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.

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

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

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

Gating the T-Lymphocytes

Quadrant regions showing the percentage of cells in each sub-population

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

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

CONCLUSIONFlow 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.