ISAC Task Force on Fluorescence Calibration

The ISAC Standards Committee is commissioning a Task Force to evaluate a new approach to fluorescence intensity calibration in flow cytometry. We invite participation from instrument and reagent manufacturers, as well as from users interested in fluorescence intensity calibration. Interested persons should send their contact info and any other relevant information to ISAC Executive Directors Todd Philbrick (toddp@isac-net.org).
 

Summary

For the calibration of fluorescence measurements, and flow cytometry measurements especially, the use of the Mean Equivalent Soluble Fluorophoes (MESF) as an intensity measurement standard has facilitated the reporting of absolute intensity measurements. Reference particles whose intensities are calibrated in MESF units are available for a few of the most frequently used fluorophores (for example, fluorescein and phycoerythrin), but extension of this approach to highly multiparameter measurements is limited by the availability of reference materials.
 

An alternative approach is to use a calibration particle with a broad emission spectrum and referenced to a single reference fluorophore to calibrate the response of multiple fluorescence channels over defined spectral ranges. We refer to this as the Equivalent Reference Fluorophore (ERF) unit. The ERF unit has practical advantages with respect to the MESF unit, most notably the possibility to use a single reference particle for calibration across multiple fluorescence channels. The ERF unit will complement the MESF unit by providing practical and usefully accurate calibration of absolute intensities for many fluorescence channels.
 

Calibration in ERF units is not restricted to multifluorophore beads. Fluorochrome specific beads may also be calibrated in ERF intensity units to reference ERF standards. Cross calibration of fluorochrome-specific beads (e.g. stained with fluorescein or phycoerythrin) to multifluorophore beads will be practical using ERF units.
 

Reference

L Wang, AK Gaigalas, G Marti, F Abbasi, and RA Hoffman. Toward Quantitative Fluorescence Measurements with Multicolored Flow Cytometry. Cytometry 73A: 279-288 (2008).