NIST/ISAC standardization study: variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads
A procedure is described for assigning the number of equivalent reference fluorophores (ERF) values to microspheres labeled with a fluorophore designed to produce a fluorescence response in a given fluorescence channel of a multicolor flow cytometer. The ERF value was assigned using a fluorimeter which was calibrated by a series of solutions of a reference fluorophore. The fluorimeter was used to obtain the microsphere fluorescence intensity, and a multicolor flow cytometer was used to obtain the microsphere concentration. The microsphere fluorescence intensity and the concentration were used to obtain the value of ERF for each surface labeled microsphere reference standard. The procedure is described for microsphere reference standards for four fluorescence channels, fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC) and Pacific Blue (PB) using four different reference fluorophores, fluorescein SRM 1932, Nile Red, APC and Coumarin 30, respectively. The four surface labeled microsphere standards provided a one point calibration for the four channels of a flow cytometer and were used in the 2nd part of the standardization study, which assigned ERF values to hard dyed microspheres through cross calibration.