What is flow cytometry?
Flow cytometry is a laboratory method used to detect, identify, and count specific cells. This method can also identify particular components within cells. This information is based on physical characteristics and/or markers called antigens on the cell surface or within cells that are unique to that cell type. This method may be used to evaluate cells from blood, bone marrow, body fluids such as cerebrospinal fluid (CSF), or tumors.
How is it performed?
Flow cytometry involves several steps:
- A sample of cells is suspended in a fluid.
- Prior to testing and depending on the cells being analyzed, the sample may be treated with special dyes to further define cell sub-types. The dyes (fluorochromes) that are used are attached to monoclonal antibodies that bind to particular cells or key components of cells.
- The sample containing the cells passes through an instrument called a flow cytometer.
- In the instrument, the fluid in which the cells are suspended passes through very narrow channels so that the cells are organized in a single file as they pass the detector(s). This is accomplished at a high rate of speed (hundreds to thousands of cells per second.)
- The flow cytometer contains one or more lasers and a series of photo detectors that are able to identify certain characteristics unique to various cell types. The single-cell suspension creates unique light-scattering events that occur when each cell passes through the laser light. These initial events are characteristic of the size and shape of the cell, as well as the intensity of the signal that is generated by the specific dyes, thus creating patterns that reflect cell type.
- The signals from the detectors are amplified and sent to a computer. They are converted to digital read-outs displayed on a computer screen or in a printout.
- The data are usually displayed as graphs.
This analysis allows evaluation of the types and numbers of cells in the sample. The flow cytometer is sensitive enough to analyze cells or particles as small as one micron in diameter (about the size of 1/75th of a human hair) and can be performed on relatively small sample sizes. Thousands of cells can be counted and analyzed in a few minutes providing a highly accurate picture of any tissue or body fluid’s cellular composition.
One additional function of a flow cytometer is the ability to physically separate unique cell types based on the characteristics mentioned above. Once a sample has passed through the laser light and photo detectors, an electric charge can be applied to the cells of interest. This occurs when a fluid sample is broken into droplets that are positively or negatively electrically charged and then deflected by oppositely- charged deflection plates. The cells of interest can then be physically collected into separate vessels for further testing.
How is it used?
Flow cytometry has been available for several decades and been adapted for use in many areas of clinical testing. Below are just a few examples of tests described on this site that use flow cytometry:
- Reticulocyte counts
- CD4 count
- HLA typing
- Sperm analysis
- Platelet function tests
- Bone marrow aspiration and biopsy
- Lymph node biopsy
Flow cytometry has been applied in identifying various cell types unique to certain diseases. One of the most common is in the diagnosis of blood-related cancers such as leukemia and lymphoma. Very specific monoclonal antibodies that have been treated with a fluorochrome are utilized to detect the presence or absence of various cellular components that are commonly seen in certain types of cancers. This information is used in the diagnosis, prognosis, and treatment of these diseases. This is especially useful in the early stages of a malignant disease where there may be only a few cancer cells present in the sample and these could go undetected by ordinary examination under a microscope.
Brown M, Wittwer C, Flow Cytometry: Principles and Clinical Applications in Hematology, Clinical Chemistry 46:8(B) 1221–1229 (2000). Available online: http://clinchem.aaccjnls.org/content/46/8/1221
Craig F E and Foon KA (2008) Flow cytometric immunophenotyping for hematologic neoplasms. Blood 111:3941-3967.
Kern W., et.al, (2008) Monitoring of minimal residual disease in acute myeloid leukemia. Cancer 112:4-16.
Martz, E. (© 2000). Introduction to Flow Cytometry for Microbiology 542, Immunology Laboratory [30 paragraphs]. University of Massachusetts [On-line information]. Accessed August 2018 Available FTP: http://www.bio.umass.edu/micro/immunology/facs542/facsprin.htm
Basic Information on Flow Cytometry [17 paragraphs] Flow Cytometry Facility, University of California, Berkeley [On-line information]. Accessed August 2018 Available FTP: http://flowcytometry.berkeley.edu/pdfs/Basic%20Flow%20Cytometry.pdf
Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. McPherson R, Pincus M, eds. Philadelphia, PA: Saunders Elsevier: 2011. 48-49, 656-660.
Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Nader Rifai, ed; senior editors, Andrea R. Horvath, Carl T. Wittwer., St. Louis, Missouri: Elsevier Saunders; 2018. Chap 25.