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Clinical applications of flow cytometry.

Flow cytometry is coming of age. Its roles in establishing or confirming difficult diagnoses and in therapeutic drug monitoring are recognized worldwide. Nevertheless, the clinical relevance of any new technology must be accepted by the practicing physician. Unfortunately, the medical community's ability to assimilate new methodologies of flow cytometry has lagged behind developments in the field.

Flow cytometry has three major functions. It is an excellent diagnostic adjunct for pathologists; it can help the physician determine a patient's prognosis; and it enables the clinician to track a patient's progress and make immediate therapeutic adjustments.

Development. Several emerging technologies have contributed to the evolution of flow cytometry. First and probably foremost is the engineering of the instruments. Several basic areas of physics are involved: optics, hydrodynamics, and electronics, encompassing data handling or computer science. The optical system includes a source of illumination. This originally used lasers, although more recent light sources include halogen or arc lamps.

Other parts of the optical system are a monochromator to provide a specific wavelength of light, lenses to focus the light beam, an optical bench that allows accurate aligning of the light source, optical filters to eliminate unwanted fluorescence, and detectors to detect and measure the intensity of fluorescent and/or scattered light.

The electronics component provides the electronic acquisition, display, analysis, and recording of the acquired data pulses. Hydrodynamic principles control the flow system, which keeps the cells in single file as they travel through the instrument in a stream. This is accomplished by introducing cells, suspended in a fluid, through a narrow injector into a stable flowing cylinder of sheath fluid. When properly controlled, the pressures of specimen and sheath fluid keep the cells moving single file in a straight path through the flow chamber. The flow paths of both cylinder and specimen must be balanced to keep the cells in a straight line.

Not only instruments but also reagents had to be developed. With the advent of hybridoma technology in 1975, immunologists could prepare almost unlimited quantities of antibodies. (These chemically, physically, and immunologically homogeneous antibodies are specific for certain epitopes. There is little or no cross-reactivity.)

Hybridoma technology was combined with the fluorescein isothiocyanate (FITC) fluorescent antibody technique developed in the late 1950s. More recent fluorochrome technologies gave immunologists an excellent method for identifying specific antigens in and on cells. Subsequent developments included a large number of fluorochromes with excitation wavelengths ranging from 360 to 660 nm and emission wavelengths from 440 to 675 nm.

Preparation. Since flow cytometers analyze individual cells, cellular preparation is an important step. While many body fluids consist of suspensions of individual cells, solid tissue must be broken down into single cells. The tissues are minced in a buffer-a citrate buffer, perhaps-and treated with a proteolytic enzyme such as trypsin.

The pathologist must give the technologist a proper specimen. Tissues from paraffin block must be fixed for a longer period of time than is typically done for staining, for example. Delivery of fresh or frozen tissue must be prompt as well.

We have achieved fine results by disrupting fresh or frozen tissues between the frosted ends of two microscope slides. Cellular debris and particles are removed by centrifugation and filtered through 37-Km nylon mesh. The resulting suspensions are checked for viability and stained with a fluorochrome or a fluorochrome labeled antibody.

Applications. Flow cytometry has both clinical and research applications. he size and refractility differential of cellular populations have been analyzed to classify cell types. The concentration of different types of cells is important in various areas of cell biology. Cell viability, cell structure, and cell cycle are among the many parameters studied by flow cytometry. The technology has also been used to examine DNA, PNA, protein content, intracellular pH, and cell membrane potential.

For genetic disorders, flow cytometry may be helpful in chromosome analysis and karyotyping. Immunologic studies, including the determination of the types and density of epitopes and the identification of cell surface receptors, have been used in the analysis of lymphocyte subsets.

Two standouts among the many clinical assays adapted for flow cytometry are surface marker analysis and DNA analysis. The development of monocional antibodies labeled with fluorochromes, which fluoresce at different wavelengths such as those of red and green, opened the door for multiparameter analysis.

It is now possible to separate and purify monodispersed cell suspensions in the form of either citrated blood or as prepared from lymph nodes or other tissues. This is done with density centrifugation over Ficoll Hypaque. Red blood cells may be lysed with ammonium chloride. The cells are washed to remove nonspecific antibody and then reacted with fluorochrome-labeled specific monoclonal antibodies.

For DNA analysis, monodispersed cell suspensions of fixed or unfixed cells are stained with fluorochromes-propidium iodide, ethidium bromide, acridine orange, or diamidine phenylindole-which bind stoichiometrically to DNA. The cells are analyzed on the flow cytometer. The percentages of the cells in the three stages of the cycle are calculated from the histogram derived from the flow cytometric analysis. These stages consist of diploid (2N) resting cells" in GO/Gl, actively DNA-synthesizing S-phase cells 2N-4N), and cells undergoing mitosis in the G2/M stage (4N). Cells with aneuploid concentrations of DNA are readily differentiated from euploid cells by means of a diplold control, such as human lymphocytes, or chicken or trout RBCS.

Software is available to calculate these percentages. If an aneuploid population is observed, its DNA index (DI) is determined by dividing the peak channel number of the aneuploid population GO/ GI phase by the peak channel number of the diplold population GO/G1 phase. (The DI of a normal diploid population is, by definition, I.O.) Once the DI has been determined, it is possible to calculate the percentage of the S-phase cells in the aneuploid population.

The most frequently studied solid tumors include breast, colorectal, and lung masses. Many investigators have studied melanomas, mesotheliomas, salivary gland tumors, and other masses, or used specimens to identify cervical or bladder cancer.

Researchers at the University of Texas Health Science Center at San Antonio evaluated the risk of recurrence of breast tumors at five years.' They found an approximate risk of 10 per cent for recurrence of diplold breast tumors with low <7 per cent) S-phase; 26 per cent for ancuploid breast tumors, regardless of S-phase; and 30 per cent for diploid breast tumors with high S-phase. This ability to predict possible recurrence gives clinicians a means of determining a patient's prognosis.

Italian scientists have divided the DNA index of lung tumors into the following ranges: Dl< 1, hypodiploid (aneuploid); DI = 1, diploid; DI ranging from 1. I to 2, aneuploid; DI = 2, tetraploid; and DI >2, hypertetraploid aneuploid).2

They noted that the occurrence of hyperdiploidy and/or hypertetraploidy is associated with tumors that have relatively high growth rates. So too did an Australian study comparing lymph node status and ploidy with survival.

The results of this comparison are shown in Table 1. The Australian investigators observed that all patients with two positive nodes had aneuploid tumors and most had a shorter survival rate than those with diploid masses.

Several researchers have analyzed colorectal malignancies. On the basis of DNA content, 70 to 80 per cent of Duke's stage B colon cancers are aneuploid. Patients with such tumors have significantly shorter survival times than those with diploid tumors; tumors recurred in 38 per cent of patients with aneuploid tumors. Sixty-eight per cent of colon cancers have an S-phase of <20 per cent. These patients have a better prognosis than those whose tumors are in a higher proliferative state. 6The investigators who reported the data shown in Table 11 provided laboratorians with an important reference range.

Applications of flow cytometry in urine cytology are developing rapidly. Researchers at Memorial Sloan-Kettering Cancer Center, whose investigators are widely recognized as the diagnostic trailblazers in bladder cancer, have determined that bladder cancer can be diagnosed by flow cytometry. They base this determination on the technology's ability to uncover any exfoliated epithelial cells with true aneuploidy or abnormal hyperdiploid (S + G2M) DNA values > 15 per cent).

A specimen is considered negative for cancer if fewer than I I per cent of the cells measured are hyperdiploid and no aneuploid stemline is detected. A specimen is deemed suspicious when 11 to 15.9 per cent of measured cells are hyperdiploid and no aneuploid stemline is detected. For detecting malignant cells in urine, the Sloan-Kettering group found, bladder wash flow cytometry is more sensitive than irrigation cytology, which is more sensitive than voided urine cytology.

Two-color analysis. This is one of the most frequently used immunologic procedures for determining the presence of lymphocyte subsets in immunodeficiencies. Monitoring the number of populations of T cells, B cells, T helper inducer) cells, T suppressor (cytotoxic) cells, suppressor-inducer cells, and helper-inducer cells, and calculating the helper: suppressor (CD4:CD8) ratio aid in the periodic evaluation of a patient's immune status. The technique has been especially helpful in AIDS and ARC cases, particularly in following patients with T4 cell counts less than 475/mm (sup 3)and T4/T8 ratios less than 0.75.

Surface marker analysis. This assists in the diagnosis of lymphomas. In Hodgkin's lymphoma, viable cancer cells-mostly lymphocytes-are scarce in the preparations studied by flow cytometry. These are mostly T cells, and the T4/T8 ratio is usually high. Analysis of B cell surface markers reveals polyclonal B cells. DNA analysis of Hodgkin's lymphoma cells may show a small aneuploid peak. In many cases, however, the peak may be obscured because the cancer cells are diluted by the abundant normal cells.

?Malignancies. Most of the malignancies in non-Hodgkin's lymphoma are B cell tumors, which express B cell antigens, and surface immunoglobulins (sIG) in a manner similar to normal cells. Most B cell neoplasms, however, have been found to be clones-they arise from a single cell that has become malignant and whose progeny will perpetuate its characteristics. Non-Hodgkin's lymphoma cells demonstrate light chain restriction, primarily expressing a single light chain (either kappa or lambda) on their surfaces. The normal kappa/lambda ratio is 1.5 to 1. If the kappa/ lambda ratio is -3:1, many researchers view the resulting proliferation as a kappa clone. If the lambda/kappa ratio is >/=2:1, the proliferation is termed a lambda tumor.

Lymphoma cells. Researchers have determined that small lymphocyte lymphoma cells express sIG very faintly, whereas follicular lymphoma cells express sIG intensely. Some B cell tumors express antigens that are not normally found on B cells. This is true with CD5 (TI, OKTI, Leul), which, although usually found on T cells, is also found on most small lymphoma B cells. The CALLA (CDIO) antigen, found on follicular lymphoma cells, is sometimes seen on normal follicular cells as well. Although T cell lymphomas occur infrequently, they express early T cell differentiation antigens and are less often aneuploid than are B cell tumors.

Aneuploidy, highly variable among the lymphomas, is more likely to be seen in aggressive tumors than in less aggressive ones. A direct relationship between histologic grade, patient survival, and the percentage of S-phase cells in the lymphomas has been demonstrated. *Cost. Establishing a flow cytometry laboratory is an expensive undertaking. The outlay for instruments alone ranges from 85,000 for a clinical analyzer to $500,000 for research equipment with dual lasers and cell-sorting capabilities.

What's more, flow cytometry and computer expenses are only one portion of setup and operating costs. A satisfactory swinging bucket centrifuge and assorted smaller instruments would add another $10,000. Monoclonal antibodies for surface marker analysis cost $3.50 to $4.00 per monoclonal per test; the typical lymphoma panel requires 12 to 15, including controls. Depending on the number of specimens processed, a flow cytometry lab may take a year to break even, much less show a profit. 9 Staff. Technologists must be trained for several months and assigned exclusively to the flow cytometry laboratory. Financial compensation must be sufficient to retain skilled operators and reduce or eliminate turnover. Flow cytometry requires a solid knowledge and integration-of the basic principles of biology, chemistry, and physics, involving more than pushing buttons in the proper sequence. The technologist must know what is going on in each step to insure satisfactory quality.

Invaluable adjunct. Flow cytometry is quickly becoming an invaluable adjunct to clinical laboratory medicine. Already, for example, it can help predict the likelihood of post-transplant rejection. Such knowledge enables physicians to adjust patients' immunosuppressive medication. As researchers continue to accumulate and analyze data, flow cytometry will become an even more important tool in the laboratory. n
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Author:Scholes, Vernon E.
Publication:Medical Laboratory Observer
Date:Jul 1, 1990
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