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flow cytometry; fluorescence-activated cell sorting (FACS)
Indications:
Flow cytometry is used for:
1) complete blood count (CBC) with white blood cell (WBC) differential
2) immunophenotyping (fluorochrome-conjugated monoclonal Ab)
a) lymphoma
b) leukemia
c) immune status in HIV (CD4/CD8)
3) 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
General
detection technique
separation technique
References
- Clinical Diagnosis & Management by Laboratory Methods,
19th edition, J.B. Henry (ed), W.B. Saunders Co.,
Philadelphia, PA. 1996, pg 62-63
- ARUP Consult:
Leukemia/Lymphoma Phenotyping Evaluation by Flow Cytometry
https://arupconsult.com/ati/leukemia-lymphoma-phenotyping-evaluation