Flow cytometry analysis

Definition

Flow cytometry analysis is the classification of cell populations based upon the analysis of light scattering and fluorescence facilitated by a laser. Cells are counted and analyzed as they pass singly through the counting area created by a liquid sheath that flows past the laser. Cells scatter the light from the laser; forward and right-angle scatter are measured to determine size and granularity. This initial light scattergraph (dot plot) is used to select a specific cell population for testing using specific antibodies covalently bound to fluorescent dyes. The laser excites the fluorochrome causing it to emit visible light, so that the cells bound to the dye can be detected.

Purpose

Principles of flow cytometry are incorporated into some automated hematology analyzers to determine the reticulocyte (stage preceding a mature red cell) count and the percentage of each type of white blood cell (automated differential count). Flow cytometers are specialized instruments that can measure specific cell subpopulations in blood, bone marrow aspirates, body fluids and tissues. Flow cytometry has many applications including:

• Counting of lymphocyte subpopulations to evaluate immunological function and immunodeficiency states. The number of B, T and NK lymphocytes can be counted by flow cytometry to evaluate a person's cellular immune status. T helper and suppressor cells can be counted to assist in the diagnosis and staging of persons with HIV disease.
• Counting of immature white blood cells (blasts) to determine the cell lineage. Cell lineage must be defined to properly classify acute and chronic leukemias and non-Hodgkins lymphomas. Flow cytometry tests for surface markers on early white blood cells to determine the cell lineage (lymphoid vs myeloid), and to determine the stage of cell maturation.
• Determining the DNA content of cells. Malignant cells often possess an abnormally high DNA content. Determination of DNA content, called ploidy analysis, is used to investigate tumor cell populations. Cells from solid tissues (for example, breast tissue) can be made into a suspension and analyzed.
• Physically sorting cell subpopulations by applying an electrostatic charge to the cells and using a fluid collecting device to harvest them from droplets passing through the flow chamber.
• Evaluation of autoimmune thrombocytopenia, transplant rejection, and autoimmune diseases.

Precautions

Universal precautions for the prevention of transmission of bloodborne pathogens is observed when collecting and processing blood, bone marrow, body fluids and tissues for flow cytometry analysis. Blood or bone marrow aspirate specimens may be submitted in sodium heparin (green top tube), EDTA (lavender top tube), or ACD (yellow top tube). Of these, the preferred anticoagulant is sodium heparin. Lithium heparin and other anti-coagulants are not used.

Lymph node or other tissue specimens are not placed in fixative. They should be submitted fresh, in isotonic saline or transport medium. Specimens should be kept at room temperature if the analysis is done within 24 hours. Otherwise the specimen should be refrigerated, but not frozen.

Description

A flow cytometer consists of a laser light source, flow measurement chamber, and an optical system consisting of lenses, filters, and light detectors. Two photo-multiplier tubes (light detectors), one at 180 degrees and one at 90 degrees to the laser, are used to measure forward and right-angle scatter, respectively. Three fluorescence detectors, each consisting of a filter and photomultiplier tube, are used to detect fluorescence. The three detectors sense green, orange, and red fluorescence. Cells are identified by sort logic applied to all five of the detector signals using a computer.

A typical analysis of blood is performed by first measuring the right-angle and forward light scatter of the cells. The resulting scattergraph is used to identify the counting gate, a set of parameters used to select a subpopulation of cells for further study. The gated area of the scattergraph is the portion in which the cells of interest are found. The gate parameters are selected so that only this cell subpopulation is reported in subsequent fluorescence studies.

Portions of the specimen are treated with two monoclonal antibodies, each specific for a cell surface antigen. Each monoclonal antibody is covalently bound to a diferent

KEY TERMS

Aneuploidy —An abnormal number of chromosomes within a cell.

Antigen —A molecule, usually a protein, that elicits the production of a specific antibody. In flow cytometry studies, an antigen is a cell surface marker that is recognized by a specific antibody and referred to by its cluster of differentiation (CD) number.

CD marker —A monoclonal antibody for a specific CD antigen.

Gating —The selection of cells that fit a specific set of parameters for further analysis. Only those cells belonging to the gated cell subpopulation are measured.

Immunophenotyping —Identification of antigens on the surface of cells using fluorescent-labeled antibodies. The phenotype profile is used to classify the cell.

Immune system —The body's system of defenses against infectious diseases, which includes both cellular and humoral (antibody) responses.

fluorescent dye. There are approximately two-dozen fluorochromes in use with flow cytometry, but the most commonly used labels are fluorescein isothiocyanate (FITC) which produces apple-green light and phycoerythrin (PE) which produces orange-red light when excited by the laser. For DNA analysis of cells the label most often used is propidium iodide (PI), which binds to DNA of nuclei and emits orange-red light when excited. The specific surface antigen to which an anti-body binds is defined by the cluster of differentiation (CD) number. Each CD number represents a specific antibody combining site on the white cell surface. When the white cell surface marker is recognized by a fluorescent-labeled monoclonal antibody, the antibody binds to the cell membrane. When the cell passes through the flow chamber, the laser will excite the fluorochrome and it will emit light stimulating one of the detectors. This process is interpreted by the computer as an event. An event is a cell that meets a set of criteria required to register a dot on the scattergraph.

The process of determining the specific cell type from a panel of antibody-conjugated fluorescent stains is called immunophenotyping. For example, CD45 is a marker common to all white blood cells. CD2, CD3, CD5, and CD7 are markers for T lymphocytes. CD4 is the site that defines a T-helper lymphocyte and CD8 is the marker or surface antigen that defines a suppressor or cytotoxic T lymphocyte. Therefore, a cell subpopulation that tests positive (i.e., produces a significant number of events) with antibodies to CD45, CD2, CD3, CD5, CD7 and CD4 is defined as a T-helper cell. Normally, a panel of antibodies is selected for use depending upon the characteristics of the gated population. For example, if the gated (selected) population is located in the region of the scattergraph where lymphocytes normally are seen, then lymphocyte markers are used. Two fluorescent-labeled antibodies are mixed with a small portion of the sample and measured simultaneously. One will be labeled with FITC and the other with PE. The events are shown as a plot of colored dots. The most commonly used plot consists of a square divided into four quadrants. The position of a dot (event) on the plot depends upon whether the cell is positive for one marker, both, or neither. For example, a positive staining reaction with FITC but not PE causes a dot in the lower right quadrant of the square. The percentage of events that fall into each quadrant is reported by the computer, and this report correlates with the density of the dots in the respective quadrant. A typical immunophenotyping for lymphocytes consists of the markers mentioned above and CD19 and CD20, which recognize B cells; HLA-DR, which recognizes B cells, T cells, monocytes and precursor cells; and anti-lamda and anti-kappa, which recognize the light chains of surface immunoglobulin molecules. The corresponding profile of positive results will identify the type of lymphocyte and its stage of maturation.

Preparation

If possible, a person should avoid eating a heavy meal within hours of the test or engaging in strenuous exercise for the 24 hours preceding the blood test.

Aftercare

The puncture site or biopsy site should be observed for excessive bleeding or infection .

Complications

In rare cases, the puncture site or biopsy site may show excessive bleeding or become infected.

Results

Interpretation of immunophenotyping requires the careful evaluation of known control cells to insure that the signals measured are not the result of background or nonspecific fluorescence. When performing the test to determine the lineage of a cell line as in leukemia, the percentage of cells that are positive for each marker is reported. These results are evaluated along with the mor phology of the cells in blood and bone marrow, and the use of cytochemical stains to determine the type of pre cursor cell (lymphocytic, monocytic, granulocytic) and its maturation stage.

When performing immunophenotyping of lympho cytes for the evaluation of immunological function, such as the staging of HIV disease, the percentage of cells pos itive for the defining marker is multiplied by the absolute lymphocyte count (i.e., number of lymphocytes per microliter). The absolute lymphocyte count is measured by the automated hematology analyzer used for the complete blood count . For example, to quantify the number of T-helper cells, the percentage of CD4-positive gated cells is multiplied by the lymphocyte count.

DNA content is measured by comparing the cells inthe G0/G1 phase (resting or presynthesis of DNA) of the cell cycle to the G0/G1 phase of a normal diploid control. The ratio of G0/G1 DNA peaks is called the DNA index (DI), and is normally 1.00. Values grater than 1.00 indicate that an increased amount of DNA is present in the sample. A DI of 2.0 indicates that the cells are tetraploid (i.e, have twofold the normal number of chromosomes). Benign tissues do not display aneuploidy (an abnormal number of chromosomes), so the finding of aneuploidy points to a malignant state. Some malignant cells do not display aneuploidy, so a normal finding cannot rule out malignancy. In general DNA aneuploidy is not well cor related with prognosis, since the course of malignant disease is dependent upon the stage (progression), histological type of the tumor, and the tissue of origin. However, in some malignancies, the cancer is associated with a greater chance of recurrence or decreased survival when aneupoloidy is present

Health care team roles

The physician will order the specific type of flow cytometry study, and if a biopsy is needed, will obtain a sample of the tissue. If blood is needed, the nurse or phlebotomist will draw the blood and transport the specimen to the laboratory. A clinical laboratory scientist/medical technologist with special training in flow cytometry will perform the analysis. Results are interpreted by a clinical pathologist who issues an interpretive report of the cell subpopulation(s) studied.

Resources

BOOKS

American Society of Clinical Pathologists. Practical Diagnosis of Hematologic Disorders, 3rd ed., edited by Carl Kjeldsberg et al. Chicago, IL: ASCP Press, 2000.

Corbett, Jane Vincent. Laboratory Tests & Diagnostic Procedures with Nursing Diagnoses, 4th ed. Stamford, CT: Appleton & Lange, 1996.

Harmening, Denise M. Clinical Hematology and Fundamentals of Hemostasis, 3rd ed. Philadelphia, PA: F.A. Davis Company, 1997.

Henry, John B. Clinical Diagnosis and Management by Laboratory Methods, 20th ed. Philadelphia, PA: W. B. Saunders Company, 2001.

Owens, Marilyn A. and Michael R. Loken. Flow Cytometry Principles for the Clinical Laboratory Practice. New York: Wiley-Liss, Inc., 1995.

Shapiro, Howard M. Practical Flow Cytometry, 3rd. ed. New York: Wiley-Liss, Inc., 1995.

Turgeon, Mary Louise. Immunology & Serology in Laboratory Medicine. St. Louis, MO: Mosby-Year Book, Inc., 1996.

Mark A. Best