Recent developments in quantitative fluorescence calibration for analyzing cells and microarrays.

Abstract

Over the last two decades, cellular immunophenotypingby fluorescence-labeled flow cytometry has been the princi-pal technology behind efforts to quantify fluorescence mea-surements by constructing a calibration curve based on massunits (1,2,3,4). These efforts have finally resulted in severalcoordinated activities that will soon reach fruition. In 1998,a U.S. federal interagency workgroup for fluorescence cali-bration was formed as a partnership between the NationalInstitute for Standards and Technology (NIST) and agenciesof the U.S. Public Health Service: the Centers for DiseaseControl and Prevention (CDC), the Food and Drug Adminis-tration (FDA), and the National Institutes of Health (NIH).NIST then established a Fluorescence Intensity Standardsprogram (5) which will soon release the first standard refer-ence material (SRM) for fluorescence intensity: a fluoresceinsolution of known concentration packaged in sealed am-pules. This fluorescein SRM will serve as a model to be appliedto other fluorochromes. NIST is also developing fluoresceinmicrobead standards calibrated in molecules of equivalentsoluble fluorochrome (MESF). To complement the availabil-ity of authoritative fluorochrome standards, NCCLS has con-vened a subcommittee to propose consensus guidelines forquantitative fluorescence calibration (QFC) to be submittednext year (3). The NCCLS subcommittee recently held itssecond meeting in conjunction with the European Union TaskForce on Antigen Quantitation, providing an opportunity todevelop guidelines that best reflect international consensus.The impetus for comprehensive QFC started with flowcytometry, but it is even more important to the emergingtechnology of microarrays. Microarrays are orderly high-den-sity arrangements of microscopic dots, each about the size ofa cell, that serve as the targets for fluorochrome-labeledprobes. They provide a highly flexible system for analyzingan immense number of probe-target interactions. Microar-rays have proven remarkably useful in screening for disease-related genes through assays of mRNA differential expression(6,7,8,9), and they will soon have profound clinical impact asdiagnosis is complemented by individual patient profiling.The fluorescence measurements on these microdot ar-rays share many features with fluorescence measurementson flow, image and laser scanning cytometers. Microarraytechnology was prominent at the 20th Congress of theInternational Society for Analytical Cytology: a tutorial,several workshops and full sessions were devoted to thetopic. NIST convened a meeting in June devoted to quan-titative fluorescence measurements for microarrays, and afollow-up meeting sponsored by the entire federal inter-agency workgroup is planned for next year.The need for QFC in microarray technology was evidentfrom these meetings. Current estimates suggest that microar-ray readers should be able to detect around one fluoro-chrome per square micron, but this estimate has been ques-tioned. The final results from microarray assays of differentialexpression involve a number of variables that can be bestcharacterized using QFC. Calibrating fluorescence intensityacross the various microarray platforms is a challenging taskwhich will be the topic of a forthcoming review. Fortunately,the basic principles have already been elucidated in devel-oping QFC for flow cytometers. As microarray research be-gins to impact clinical practice and public health, the clinicalcytometry community can appreciate, anticipate and assist inresolving the challenges of this powerful technology.