Flow Cytometry is a modern method of studying cells individually in population to determine their multiple physical and biological properties. We have two types of instruments, analyzers which are useful mostly in the clinical diagnostic set up and the second category is sorters which can collect the cells after analysis for further use.
Sample Preparation and Analysis
Cell suspensions are required for flow cytometric analysis. These are easily prepared from the blood and other body fluids. Solid tissue can be minced and passed through a graded series of nylon meshes to prepare a cell suspension, which can be subsequently analyzed by the flow cytometry. Fixed, embedded tissues also can be made into suspension and analyzed by flow cytometer.
After isolation and purification of the cells and the preparation of a cell suspension, fluorescent-labeled monoclonal antibodies or other labels are applied to the cells. The monoclonal antibodies which are highly specific to the surface antigens or some internal cellular substances of interest such as DNA/ RNA or Cytokines. The choice of probes in each case is dependent upon the clinical questions to be answered.
In the flow Cytometer , the labeled cells are pumped under slight pressure into a detection zone where they are illuminated by precisely focused laser beam. The cells may scatter light from the laser due to their size and internal granularity or the energy of the laser may excite the fluorochrome attached to the cells through monoclonal antibodies. In either case, light intensity is measured photodiodes or Photomultiplier tubes ( PMTs) and the resulting electrical signals are amplified , digitized through Analaogue to Digital conversion and are sent to the computer for further processing and analysis. This process is accomplished at a very rapid rate (typically 5000 cells / sec).
Clinical Utility
Quantitative cell analysis, at the single cell level allows flows cytomtery to be used for the clinical assessment of immunological, hematological and neoplastic diseases. Cellular parameters such as DNA content , RNA content or enzyme activity can be rapidly and accurately determined. Cell populations and subpopulations in a body fluid or tissue can be identified by the number and type of cell surface antigens, and by their light scatter properties. In addition to proving an objective confirmation of morphological diagnosis, the data has independent diagnostic and prognostic significance in many circumstances.
In addition, flow cytometry allows the rapid examination of large number of cells, far beyond the capability of traditional cytopath methods. By this technique, small numbers of neoplastic (cancer) cells can be detected among a population of normal cells.
Immunophenotypic Analysis
Monoclonal antibodies have been developed against antigenic determinants on different types and subtypes of blood cells, and against blood cells at different stages of differentiation. Their utilization in the characterization and quantitation of blood (immunophenotypic analysis) has been very useful in the diagnosis of leukemias and lymphomas, inherited and acquired immuno deficiency diseases, autoimmune diseases, cell ploidy analysis for solid tumors, stem cell enumeration, apoptosis (programmed cell death). In addition, chemotherapy is being increasingly used based on the antigenic characteristics of the malignant cells, rather than the histological features alone.
The existence of unique phenotypic markers, present on the immature leukemic cells, but not on normal white blood cells, has allowed the detection of very small numbers of neoplastic cells (say 1:100,000) in blood or other body fluids. By the use of these markers, early recurrences of hematological malignancies can often be detected. Recently a precise technique of detecting small number of circulating abnormal B lymphocytes, called clonal cells, has been developed for the diagnosis of B cell neoplasms.
Immunophenotyping is performed by labeling cells with fluorescence tagged (FITC / PE/ECD/PC5) monoclonal antibodies, passing these cells through the flow cytometer and measuring the number and percentage of positive cells. In most cases, a panel of monoclonal antibodies is necessary for he exact classification of cell populations.
DNA Analysis
Early studies, using microphotometric and audioradiographic techniques, showed that cancer cells often demonstrate abnormal DNA content (DNA Aneuploidy) when compared to normal cells, which have a " diploid" (2N) or "Euploid" DNA content. Aneuploidy is believed to reflect the high incidence of numerical or structural chromosomal aberrations present in neoplastic cells. In contrast to early methods of DNA analysis, flow cytometry offers the advantage of high-speed analysis of large number of cells with smaller coefficient of variation (CV). Small number of aneuploid cells can be detected which may go unnoticed by other techniques.
The capacity of rapid cell division is the feature of neoplastic cells, which is ultimately responsible for most of the clinical manifestations of cancer. A direct relationship between patient survival and proliferative activity of the tumor cells has been demonstrated for most neoplastic diseases. Although histological grading provides a rough estimate of proliferative capacity, precise measurements can only be made by flow cytometry.
DNA analysis is performed by staining cells or nuclei with one of the several available fluorescent, intercalating dyes. The fluorescence intensity of the dye in each cell is directly proportional to the mount of nuclear DNA.
By flow cytometric analysis the precise number of cells in each phase of the cell cycle (G0/G1, S, and G2/M) can be determined and the proliferative activity can be calculated. One of the advantages of DNA analysis is that it can be performed on fixed cells, embedded tissue, essentially without any loss of data. This has allowed retrospective studies of DNA content in a diversity of neoplasms and has contributed greatly to the practical utilization of DNA analysis.
The list of clinical applications in which flow cytometry data unequivocally aids in diagnosis and prognosis are:
· Lymphocyte sub population analysis
· Leukemia / Lymphoma diagnosis
· Detection and quantitation of neoplastic cells
· DNA/RNA Analysis
· Bone Marrow indices
· Chemotherapeutic monitoring
· Platelet quantification and analysis
· Reticulocyte analysis
· Immune complex detection /quantitation
· Estrogen receptor analysis
· Cell function analysis
· Apoptosis
Some of the applications have been discussed in detail in the article.
Beckman Coulter is one of the leading manufacturers of the FlowCytometers in the world and in India, these instruments are supplied and supported by Wipro Biomed, a division of Wipro Healthcare and Life Science. One of their latest models, FC-500 is shown in the picture which can do 5-color analysis with 1 or 2 lasers.
-- The author is marketing manager, Wipro Healthcare and Life Sceince, Sarjapur Road, Bangalore