Fluorescence Intensity Calibration for Immunophenotyping by Flow Cytometry

Fluorescence intensity (FI) is the basis for classifying phenotypes by fluorescence-label flow cytometry. FI is customarily recorded as an arbitrary relative value, but with proper calibration it can be expressed in stoichiometric 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 consensus methods will alleviate many of the difficulties encountered in making valid MESF measurements. FI calibration establishes the true values for the critical parameters of the fluorescence measurement, a useful feature for quality control. It further allows the establishment of a comparable window of analysis across different times and laboratories, and it permits numeric assessment of antibody-binding capacity (ABC) values in selected cell populations. The relation between ABC values and receptor expression is complicated by several factors, but careful assessment of the binding chemistry can establish the actual number of receptors on cells stained by fluorescent conjugates.