Principles and Clinical Applications of Flow Cytometry
Dong Hwan Suh, Byung Woon Min, Keon Boo Yoo
Dept. of Anatomical Pathology, Chonnam University Hospital
- Abstract
- Flow cytometry is a technique of single cell analysis, based on the scattering of light and emlsslon of fluorescence by laser. Flow cytometry offers an rapid, objective and quantitative measured results for analysis and sorting of cells in suspension condition. The fundamental concept of flow cytometry is simple : cells interact indiviually with a laser beam as they pass by single file in suspension. The interaction with the laser is measured as laser scatter, commonly measured at “ forward" and “ 90℃angles" to the incident light source. Other parameters measured include fluorescence emission(FL, and FL2). In a flow sorter, single cell can be passed in charged droplets based on a compos ite of these parameters, and physically separated from the larger cell populations. Analysis and sorting of other particular material is also possible. The microscope has been the primary tool of pathologists and medical researchers since its invention in the early 1600s. The major disadvantages of light microscopy are its subjective, mechanical nature and based on the training and experince of the observer and is limited to the inherent resolution of the human eye. In addition, light microscopy is laborious and not suitable for the rapid examination of large numbers of cells. Flow cytometry is examined at the single-cell level and quantitative measurements of physical or biological properties of the cells are accurately made. Furthermore, multiparameters of the cells can be simultaneously measured and cell populations can be separated(sorting function) on the bases of one or several parameters. Modern flow cytometry resulted from the application of knowledge and techniques which were developed independently in the fields of laser technology, monoclonal antibody production, immunohistochemical staining, fluorochrome chemistry, optical system, fluidic system, electronic system and computer science. The identification and classifica tion of cells by the presence of. surface antigen began with the revolution in immunology brought about by the discovery of T and B lymphocytes and has now expanded to the analysis of other cells, such as monocytes, macrophages, myeloid stem cells, tumor cells. The currently utilization of flow cytometer is widely increase in the clinical assessment of immunological, hematological, oncological and neoplastic diseases and has introduced a new era into the pathological laboratory. Routinely used tests in clinical laboratory are lymphocyte immunophenotyping, cell cycle analysis and leukemia immunophenotyping. In DNA ploidy analysis, single-cell analysis has been used to study tumor kinetics and metastasis. Quantitative measurements of nuclear DNA content at the single-cell level have great impact on clinical oncology and pathology. In general, flow cytometric DNA analysis in the clinical laboratory is bases on the following three principles : (1) cells in different stages of the cell cycle have different but predictable amount of DNA, (2)fluorochromes are available that stoichiometrically bind to DNA and (3)normal cells are euploidy(diploidy) and have a known amount of DNA , whereas malignant cells tend to be aneuploidy, malignant cells often have abnormal DNA content. DNA aneuploidy is believed to reflect the numerical or structural chromosome aberrations which are frequently present in neoplastic cells , and cell cycle activities is a fundamental parameter related to the rate of tumor growth We explained the principles and clinical applications of flow cytometry in view of Korea clinical and anatomical pathological laboratory.
- Keywords : flowcytometry, laser, fluidic system, FITC, PE, Iymphocyte immunophenotyping, leukemia immunophenotyping, cell cycle analysis, diploidy, aneuloidy
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