Flow Cytometry – What It Is & How It Works – Basic Information

Nov 23, 2011
Donald Reinhardt

Technician Expert Evaluates Cell Sorter Data - NIAID NIH

Flow cytometry is a molecular or cellular way to separate, different cells or particles and determine the unique and characteristic chemistry and features.

Dynamic View of Fluorescent Cell Sorter Activity

A good place to start is to watch a short animation of a fluorescent activated cell sorter FACS such as this animation produced by the Sandia Laboratory. Watch the animation twice and then read aspects of its functions below.

Flow Cytometry Significance and Importance

Scientists are always interested in similarities and differences. In what ways is this cell similar to that cell is important, but also of significance is the question of why this cell is different from that cell. An important example of this concept becomes very important when we consider normal cells and cancer cells. What makes a cancer cell different from a normal cell? What makes a leukemia cell different from a normal white blood cell? When normal cells are made abnormal by chemical or physical treatment what changes can we see and how can we detect them? Flow cytometry helps scientists to separate different types of white blood cells such as lymphocytes from monocytes or neutrophils from basophils or eosinophils. How are these cell separations and collections done and made possible?

Flow Cytometry Features and Concepts

Flow cytometry (FCM) first relies upon the use of special stains or fluorescent markers to specifically label the different types of cells or particles to be analyzed. After appropriate-effective labeling, large numbers of these cells or particles in suspension can then be differentiated and detected by light scatter as they pass and flow rapidly through a sensing-detection region that detects forward scatter (FS) of light or fluorescence and another detector for side scatter (SS). Once detected each cell is then quickly identified based on size and fluorescence characteristics and separated and passed into selected, collection tubes.

Effective flow cytometry involves certain unique and distinctive features or attributes of the cells or particles (e.g., size, morphology, enzymes, specific structural chemicals or biochemicals) which can be quantified simultaneously on a single-particle basis. FCM enables thousands of cells, cell fractions or particles to be examined per second.

Flow Cytometry Works to Separate and Differentiate White Blood Cells

Faucher et al. were able to use 6 different markers and 5 colors as referenced in their paper in "Sources" to separate leukocytes and tell them apart. Here are the essentials of this experimental work:

379 subjects were involved
11 of 12 different circulating cell types were identified and quantified
the quantitative data obtained by FCM and conventional staining and differentiation were similar for whole leucocytes, neutrophils, eosinophils, basophils, monocytes, or lymphocytes
FCM was better than standard cytology for detection and quantification of circulating blast cells or immature granulocytes
All cases of lymphocytosis were detected by FCM
FCM identified a group of patients with excess of CD16+ monocytes as an inflammatory syndrome
FCM is superior to conventional cytology in that FCM can detect and characterize the lineage orientation of blast cells or lymphocytes and specific markers of interest such as CD16 on inflammatory monocytes.

Flow cytometry will continue to be a useful research, discovery and clinical tool of medicine and science throughout the 21st century and beyond.