Recent developments in quantitative fluorescence calibration for analyzing cells and microarrays

Over the last two decades, cellular immunophenotyping by fluorescence-labeled flow cytometry has been the principal technology behind efforts to quantify fluorescence measurements by constructing a calibration curve based on mass units (1, 2, 3, 4). These efforts have finally resulted in several coordinated activities that will soon reach fruition. In 1998, a U.S. federal interagency workgroup for fluorescence calibration was formed as a partnership between the National Institute for Standards and Technology (NIST) and agencies of the U.S. Public Health Service: the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA), and the National Institutes of Health (NIH). NIST then established a Fluorescence Intensity Standards program (5) which will soon release the first standard reference material (SRM) for fluorescence intensity: a fluorescein solution of known concentration packaged in sealed ampules. This fluorescein SRM will serve as a model to be applied to other fluorochromes. NIST is also developing fluorescein microbead standards calibrated in molecules of equivalent soluble fluorochrome (MESF). To complement the availability of authoritative fluorochrome standards, NCCLS has convened a subcommittee to propose consensus guidelines for quantitative fluorescence calibration (QFC) to be submitted next year (3). The NCCLS subcommittee recently held its second meeting in conjunction with the European Union Task Force on Antigen Quantitation, providing an opportunity to develop guidelines that best reflect international consensus. The impetus for comprehensive QFC started with flow cytometry, but it is even more important to the emerging technology of microarrays. Microarrays are orderly high-density arrangements of microscopic dots, each about the size of a cell, that serve as the targets for fluorochrome-labeled probes. They provide a highly flexible system for analyzing an immense number of probe-target interactions. Microarrays 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 as diagnosis is complemented by individual patient profiling. The fluorescence measurements on these microdot arrays share many features with fluorescence measurements on flow, image and laser scanning cytometers. Microarray technology was prominent at the 20th Congress of the International Society for Analytical Cytology: a tutorial, several workshops and full sessions were devoted to the topic. NIST convened a meeting in June devoted to quantitative fluorescence measurements for microarrays, and a follow-up meeting sponsored by the entire federal interagency workgroup is planned for next year. The need for QFC in microarray technology was evident from these meetings. Current estimates suggest that microarray readers should be able to detect around one fluorochrome per square micron, but this estimate has been questioned. The final results from microarray assays of differential expression involve a number of variables that can be best characterized using QFC. Calibrating fluorescence intensity across the various microarray platforms is a challenging task which will be the topic of a forthcoming review. Fortunately, the basic principles have already been elucidated in developing QFC for flow cytometers. As microarray research begins to impact clinical practice and public health, the clinical cytometry community can appreciate, anticipate and assist in resolving the challenges of this powerful technology.