Flow Cytometry Analysis Health Article

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