Fluorescence intensity calibration for immunophenotyping by flow cytometry

Fluorescence intensity (FI) is the basis far classifying phenotypes by fluo rescence-label Row cytometry. Fl is customarily recorded as an arbitrary re lative value, but with proper calibration it can be expressed in stoichiome tric units called molecules of equivalent soluble fluorochrome (MESF) that reflect the concentrations of the fluorescent conjugates and the receptors they stain. Forthcoming availability of authoritative standards and consens us methods will alleviate many of the difficulties encountered in making va lid MESF measurements. Fl calibration establishes the true Values for the c ritical parameters of the fluorescence measurement, a useful feature for qu ality control. It further allows the establishment of a comparable window o f analysis across different times and laboratories, and it permits numeric assessment of antibody-binding capacity (ABC) values in selected cell popul ations. The relation between ABC values and receptor expression is complica ted by several factors, but careful assessment of the binding chemistry can establish the actual number of receptors on cells stained by fluorescent c onjugates.