A simultaneous three-color T cell subsets analysis with single laser flow cytometers using T cell gating protocol. Comparison with conventional two-color immunophenotyping method.

We describe a method for simultaneous analysis of CD3, CD4, and CD8 positive cells from whole blood utilizing single laser flow cytometers. All three T cell values are attained from a single test tube. CD4 and CD8 positive cells are identified only if they are CD3 positive. Thus the values obtained by this method for T helper/inducer and T cytotoxic/suppressor cells can be reported directly as a percentage of T lymphocytes. Analysis for CD4 and CD8 positive cells is accomplished, by first gating on CD3 positive T lymphocytes, hence the approach is referred to as a T gating method. As the third dye, conjugated to anti-CD3 monoclonal antibodies (MAbs), we utilized peridinin chlorophyll protein (PerCP), a new red fluorochrome. The proposed method may prove to be practical for monitoring disease progression in AIDS, where longitudinal T helper/inducer and T cytotoxic/suppressor cell enumeration must be performed unambiguously by a simple, reproducible, and fast method.

Overview and application of suspension array technology.

A cell-based solid-phase immunoassay usually is called immunophenotyping when performed on a flow cytometer. Using the same principles, there is a new flow cytometric application available with the suspension array technology. The significant difference is that the immunologic reaction does not occur on the surface of leukocytes, but rather on the surface of plastic fluorospheres. This is a multiplexed solid-phase flow cytometry-based technology with some clinically relevant potential.

Suspension array technology: new tools for gene and protein analysis.

Flow cytometry has long been a key tool in the analysis of lymphocytes and other cells, owing to its ability to make quantitative, homogeneous, multiparameter measurements of particles. New developments in illumination sources, digital signal processing and microsphere chemistry are driving the development of flow cytometry in new areas of biomedical research. In particular. the maturation of approaches to perform highly parallel analyses using suspension arrays of microspheres with different morphospectral features is making flow cytometry an important tool in protein and genetic analysis. In this paper, we review the development of suspension array technology (SAT), current applications in protein and genomic analysis, and the prospects for this platform in a variety of large scale screening applications.

Principles of flow cytometry.

Clinical flow cytometry is a relatively new and rapidly growing medical technology. According to estimates, there were less than 1000 instruments in operation globally prior to 1985. Most of these instruments were used exclusively for research, and required dedicated facilities and operators with extensive backgrounds in electronics. In the mid-1980s, with the availability of benchtop clinical models, the number of flow cytometers jumped dramatically, surpassing 4000 by 1990. Most of the instruments that have been sold in the past decade are equipped with low power, air-cooled argon ion lasers with a fixed emission light wavelength at 488 nm. They are capable of multi-color immunophenotyping, and are usually connected to powerful personal computers for data analysis. By 1992, there were an estimated 7000 flow cytometers in operation worldwide. Today, the three general fields where this technology is well established are clinical immunology, laboratory hematology, and medical oncology. The most prominent uses of flow cytometers are for immunological characterization of lymphomas and leukemias, crossmatching tissues for organ transplants, and counting lymphocyte subpopulations in the peripheral blood of HIV-infected individuals. In this review, a brief historical introduction will be followed by a general description of some of the salient features of clinical flow cytometers.

Evolution of leukocyte immunophenotyping as influenced by the HIV/AIDS pandemic: a short history of the development of gating strategies for CD4+ T-cell enumeration.

This brief review of the development of gating strategies for CD4+ T-cell enumeration is really the story of contemporary clinical flow cytometry. It is the chronicle of its birth, and its slow invasion into the clinical immunology laboratory over the past 15 years. The driving force behind the technological evolution of leukocyte immunophenotyping was, and still is, the impact of the HIV/AIDS pandemic on the discipline of immunology. While, at times, this review of analytical flow cytometry makes metaphorical excursions into the literary world, the objective is not to trivialize technology. Rather, it attempts to illustrate how, in his drive to solve the practical problems of modern medicine, man's essentially eclectic nature excels in an environment of diverse knowledge. This review will examine how technological advancements, such as the introduction of clinical instruments with multi-color capabilities, and the evolution of leukocyte gating strategies, has influenced the evolution of leukocyte immunophenotyping.