Laboratories that measure emissions from fluorescent particles in a wide range of applications from clinical chemistry to biodefense research to pharmaceutical development now can have more confidence in their results thanks to new theory, standards and methods developed by the National Institute of Standards and Technology (NIST).
The culmination of five years of research, NIST's newest reference material (RM 8640) is a set of calibrated microspheres coated with fluorescent dye. The tiny spheres, each one about one-tenth the width of a human hair, are used to calibrate flow cytometry instruments that measure fluorescence intensity.
Fluorescent markers often are used to "tag" antibodies, cancer cells, specific genes or other biomolecules. For example, the brightness of the signal from a sample can indicate whether a disease is getting worse or is in remission. Until now, the intensity of the signal, reflecting numbers of target molecules, has been judged visually or with benchmarks developed by individual manufacturers. Previous research has shown that measurements of the same samples can vary by more than 100 percent depending on the instrument used and a variety of experimental conditions.
The new reference material, combined with previously developed NIST standards and measurement procedures, now will provide an authoritative national fluorescence measurement scale. Each kit contains six vials of microspheres that emit fluorescent light at six different intensity levels from zero to very bright. A flow cytometer is used to analyze the contents of all the vials and an unknown sample. By calibrating the cytometer to match the intensity values provided by NIST for the reference vials, the intensity of the unknown sample can be measured more accurately.
NIST's work has been supported in part by the Centers for Disease Control and Prevention, with supplies provided by the Food and Drug Administration, Becton Dickinson Biosciences, Molecular Probes, Inc., and Bangs Laboratory.
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