32.7: Flow Cytometry
The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
In 1967, Kamentsky and Melamed achieved the diversion of cells away from the flow, allowing cell sorting. But it was not until 1973 that a refined electrostatic sorter, known as a fluorescence assisted cell sorter or FACS, was developed. Typically, the cell suspension passes through a laser beam, and the light scatter, or fluorescence, is detected. The changes in scatter patterns or fluorescence determine how the cells should be sorted.
Applications of Flow Cytometry
Apart from cell counting and sorting, flow cytometry is also used to estimate the DNA and RNA content of cells. When a cell suspension is incubated with fluorochromes, viable cells take up the stains to different degrees based on cell type and growth phase. These differences are detected by the FACS software, which then estimates the relative proportions of the cell types in different growth phases. Since flow cytometry estimates cell parameters such as shape, size, and granularity, it can also be used to differentiate necrotic and apoptotic cells. For example, apoptotic cells are typically more granular and therefore tend to have a higher side scatter of light than the less granular necrotic cells.
Limitations of Flow Cytometry
An obvious limitation of flow cytometry is that it cannot analyze cells that are not suspended in culture, such as those growing on an adherent medium or organized in a tissue. All samples must be dissociated into cell suspensions to generate single-cell droplets; therefore, cell-cell interactions cannot be analyzed. Another constraint stems from the limit on the number of simultaneous detectors. Despite many fluorophores being available, a flow cytometer usually does not have more than 12 detectors to efficiently and consistently detect the different cell subpopulations. While flow cytometry is a powerful technique, it can also generate an overwhelming quantity of data that requires a human expert to analyze and process. And finally, given the high sensitivity of this technique, standard protocols for sample preparation, data recording, and analysis are essentia. However, a lack of standardization of these aspects makes it difficult to compare results from different studies.
