Formalization of the MESF unit of fluorescence intensity

Quantitative flow cytometry of ZAP‐70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome

“…Taking advantage of this powerful feature, we performed quantitation of expression levels in (CD19 þ /CD5 þ ) CLL cells and (CD19 þ ) B cells from healthy volunteers using calibration beads, MESF. Such an approach allows exact quantitation of ZAP-70 molecules in B cells and enables uniform standardization of the assay in any laboratory, because of platform-to-platform and lab-to-lab reproducibility (18,24). This quantitative assay demonstrates that a sample labeled with a fluorochrome has the same fluorescence intensity as an equivalent number of molecules of the fluorochrome, which is free in a solution under the same environmental conditions.…”

“…In the study reported here, we provide the first clinically relevant data obtained using a highly sensitive and reproducible, computerized quantitative analysis of the molecules of equivalent soluble fluorochrome (MESF) of ZAP-70 in CLL cells. MESF is a relatively new type of analysis utilized in the field of flow cytometry to quantitate soluble cellular proteins (16)(17)(18). The MESF concept designates that a sample labeled with a fluorochrome has the same fluorescence intensity as an equivalent number of molecules of the fluorochrome free in a solution under the same environmental conditions (19,20).…”

How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards

“…If not properly considered, these differences lead to considerable errors in fluorophore quantification. As an approach to overcome these problems, the quantification of the fluorescence intensity of samples is often performed in units of molecules of equivalent soluble fluorochrome (MESF) [56,57]. This type of reference system, that circumvents differences in the molar absorption coefficients and fluorescence quantum yields of the standard and the sample, however, relies on matching fluorescence spectra and the sample and is not designed to derive the absolute number of fluorophores in the sample.…”

“…This is the reason why for fluorescence techniques using integral measurements of relative fluorescence intensities for quantification purposes like e.g. flow cytometry, spectra matching is recommended [32,56]. Due to the instrument specifity of spectra matching of a sample-standard pair, its dependence on dye microenvironment, and the ever increasing variety of fluorescent labels and fluorophorecontaining systems, general solutions to quantitative fluorescence analysis with small uncertainty are not very realistic.…”

Recommendations for the development and validation of flow cytometry‐based receptor occupancy assays

“…For each measurement type, assay results may be expressed in terms of relative frequency (percent positive, absolute cell count) or antigen expression quantitated using antigen quantitation beads or cell reference controls (3)(4)(5)(6). Free Receptor can be measured either in a competitive assay, provided that monoclonal antibodies (mAb) against competitive binding sites are available, or with directly conjugated drug (7).…”