Quantitative flow cytometry in the clinical laboratory

Flow cytometry is most often used in the clinical laboratory for the purpose of immunophenotyping. Here, fluorescently labeled antibodies are bound to cell surface receptors, and their presence on the cell is most often defined in bivariate terms of positive or negative, with a cutoff set relative to a nonstaining control population. It has long been recognized that the intensity of the fluorescent signal is proportional to the amount of antibody bound per cell and therefore related to the number of antigen sites expressed. This relationship makes flow cytometers, at least theoretically, capable of quantifying antigen expression in terms of molecules per cell. There were numerous obstacles to the development of such methods and clinical utilization of fluorescence intensity measures by flow cytometry has in the past been largely overlooked. The first widespread recognition of the clinical utility for fluorescence intensity measures came from laboratories where malignant phenotypes were defined by aberrant intensity of staining due to over or under expression of various cellular proteins. These semiquantitative measures were relative in nature and described staining as bright or dim compared to that normally seen in healthy individuals. Recent advances within the past decade have resulted in the development of flow cytometric methods and materials that now permit one to conduct measures of quantitative fluorescence with improved levels of control and interlaboratory precision. With these advances have come increasing interest in quantitative flow cytometry as a method to quantify the expression and activities of a variety of proteins and enzymes for diagnostic, prognostic, and therapeutic purposes. This article discusses the background and theoretical and practical considerations, as well as the current use of quantitative flow cytometry measures in the clinical laboratory.