Considerably simplified, flow cytometry (FCM) counts and analyzes certain aspects of cells using instrumentation similar in principal to many current hematology cell-counting machines. If the cells to be analyzed come from solid tissue, the cells or cell nuclei must first be extracted from the tissue and suspended in fluid. Next, the cell nuclei are stained with a fluorescent dye that stains nucleic acids in order to assay the amount of nuclear deoxyribonucleic acid (DNA); in addition, antibodies with a fluorescent molecule attached can be reacted with cell antigens in order to identify or characterize (phenotype) the cells. The cells or cell nuclei are first suspended in fluid and then forced through an adjustable hole (aperture) whose size permits only one cell at a time to pass. As the cells pass through the aperture each cell also passes through a light beam (usually produced by a laser) that activates (“excites”) the fluorescent molecules and also strikes the cell, resulting in light scatter. A system of mirrors and phototubes detects the pattern of light scatter and the fluorescent wavelengths produced (if a fluorescent dye is used), then records and quantitates the information. The pattern of light scatter can reveal cell size, shape, cytoplasm granules (in complete cells), and other cell characteristics. The electronic equipment can sort and analyze the different pattern. This information is most often collected as a bar-graph histogram, which is then displayed visually as a densitometer tracing of the bar graph; the concentration of cells in each bar appears as a separate peak for each cell category, with a peak height proportional to the number of cells in each bar of the bar graph. If all cells had similar characteristics (according to the parameters set into and recorded by the detection equipment) there would be a single narrow spikelike peak. One great advantage of cell counting by FCM rather than counting manually is that many thousand (typically, 10,000) cells are counted in FCM compared to 100 (possibly 200) in manual counts. Therefore, FCM greatly reduces the statistical error that is a part of manual cell counts.

At present, flow cytometry is most often used for the following functions:

1. Cell identification and phenotyping. Use of fluorescent-tagged antibodies, especially monoclonal antibodies specific for a single antigen, helps to identify various normal and abnormal cells and also subgroups of the same cell type. For example, lymphocytes can be separated into B- and T-cell categories; the T-cells can be further phenotyped as helper/ inducer, suppressor/cytoxic, or natural killer cell types. This technique may also be used to identify subgroups of certain malignancies, most often of hematologic origin (leukemias and lymphomas; see also the discussion of cell lineage studies in Chapter 7).
2. Analysis of nuclear DNA content. Cell nuclear chromatin is stained (usually with a reagent that also contains a fluorescent molecule), and the fluorescence corresponding to total nuclear chromatin content is measured by the flow cytometer. The major determinant of total nuclear chromatin content when cells are in their resting stage is chromosome material, which in turn is related to the number of chromosomes (“ploidy”). Normally, there are 2 sets of chromosomes (“diploid”). As described previously, the FCM produces a visual representation of the DNA content of various cell groups present that in diploid cells from a resting state would be displayed as a single homogeneous spikelike peak appearing in a certain expected location of the FCM densitometer graph. A sufficient number of cell nuclei with more or less DNA than normal (an arbitrary cutoff point most often set at 5 percent deviance from diploid state) is called aneuploidy and often is seen as a separate peak from the usual diploid peak. Another way of demonstrating this is to obtain a DNA index in which the patient DNA content is compared to DNA of a (normal) control cell specimen. Ordinarily, the patient and control DNA patterns would be identical, providing a DNA index of 1.0. A DNA index sufficiently more than or less than 1.0 indicates aneuploidy. Aneuploidy is most often associated with malignancy, and if found in certain tumors may predict a more aggressive behavior. However, it should be noted that aneuploid DNA can be found in some nonneoplastic cells as part of the reaction to or regeneration after inflammation or tissue destruction and has also been reported in some benign tumors.
3. Cell proliferation status. In standard FCM, this is done by determination of the S-phase fraction (percent S-phase; percent S; SPF). A normal cell activity cycle consists of five time periods. The cell normally spends most of its time in the G0 (“gap zero”) resting phase. It may be stimulated to enter the G1 (gap 1) stage in which ribonucleic acid (RNA) and some protein is synthesized. Next comes the S (synthesis) phase when DNA content increases to twice the resting amount in preparation for mitosis. Then comes the G2 (gap 2) period when additional RNA is produced and a very short M (mitosis) period, which is difficult to separate from G2, when mitosis takes place. After this the cell returns to G0. The S-phase (proliferation phase) features doubling of the nuclear DNA content, which is detected on the FCM histogram. The number of cells in the S-phase, if considerably increased, is shown as a small peak or elevation midway between the G0/G1 and the G2M peaks in the FCM histogram. Increase in the S-phase fraction (SPF) area suggests an unusual degree of cell proliferative activity. Since tumor cells tend to replicate more readily than normal cells, increased SPF activity can therefore raise the question of malignancy. In a considerable number of tumors, the degree of SPF activity correlates roughly to the degree of aggressiveness in tumor spread, which has prognostic significance. SPF has become one of the better and more reliable overall tumor prognostic markers (indicators). However, there are certain problems. Not all tumors with increased SPF are malignant; not all malignant tumors with increased SPF metastasize; and not all malignant tumors with relatively small SPF fail to metastasize. In addition, the S-phase peak is usually not large, even when considerable S-activity is occurring. The S-phase peak can be interfered with by cell debris or by poor separation of the G0/G1 and G2/M peaks. SPF can be falsely increased to variable degrees by reactive normal tissue cells (e.g., fibroblasts, endothelial cells, inflammatory cells, or residual areas of nontumor epithelial cells) intermixed with or included with tumor cells.
4. To provide helpful information in patients with cancer. This includes use of information from the previous two categories (analysis of nuclear DNA content and cell proliferation status) to help determine prognosis; to help diagnose cancer in effusions, urine, or other fluids in which cancer cells may be few or mixed with benign cells such as activated histiocytes or mesothelial cells; to detect metastases in lymph nodes or bone marrow when only small numbers of tumor cells are present; or to supplement cytologic examination in fine-needle aspirates.