flow cytometry; fluorescence-activated cell sorting (FACS)

Indications

Flow cytometry is used for:

• complete blood count (CBC) with white blood cell (WBC) differential
• immunophenotyping (fluorochrome-conjugated monoclonal Ab)
  • lymphoma
  • leukemia
  • immune status in HIV (CD4/CD8)
• cell cycle analysis
  • propidium iodide labeling of DNA

Principle

Measures properties of cells moving through a fluid medium.

As cells pass single file through a sensing point, they are intercepted by laser beam (generally argon laser). The transmitted light (including forward scatter, right angle scatter & fluorescent light) is focused onto photomultiplier tubes. The analogue signal from the photomultiplier tubes are converted into a digital signal that the computer can use for quantitation.

Forward light scatter is proportional to cell size. Right angle scatter is related to cell granularity & density. If the cells are labeled with appropriate fluorochromes, fluorescent signals can be measured.

Simultaneous measure of forward & right angle scatter allows for separation of granulocytes, monocytes & lymphocytes based upon their size & granularity.

Flow cytometers may be designed as cell sorters. Cells of interest are identified with electronic gating & are given an electrical charge. The electrically charged cells are deflected by an electric field into suitable containers for further analysis. Other cells remain uncharged & pass through the electric field undeflected.

Modern high-speed flow cytometers can handle 70,000 events/ second. Analyses include number of cells, cell size & presence of surface & cytoplasmic markers. Fluorochromes conjugated with monoclonal antibodies

More general terms

• detection technique
• separation technique