Interlaboratory study of cellular fluorescence intensity measurements with fluorescein‐labeled microbead standards
- 1 January 1991
- Vol. 12 (6), 525-536
- https://doi.org/10.1002/cyto.990120609
Abstract
To determine the precision of cellular fluorescence intensity (FI) measurements derived from labeled microbead standards, FI results were compared from 43 different flow cytometers in 34 laboratories. All laboratories analyzed prepared aliquots of fluoresceinated calf thymocyte nuclei (Fluorotrol), human lymphocytes stained with fluoresceinated anti‐CD4 antibody, and fluoresceinated microbeads used as both internal and external standards. Measurements were conducted by most laboratories on the third and fourth days after sample preparation. Results for percent of events within the gates and the histograms returned by participants indicated that the samples had remained stable and that gated populations had been properly identified. All standard curves showed strong linearity, and the pooled results from all standards produced a best‐fit curve that was in close agreement with the assigned values. Nonetheless, results for cellular FI were highly variable, with CVs of 20–34%. Agreement within lab/instrument was much better, with CVs ranging from 3.0 to 9.9%. The overall variability was not obviously attributable to differences in the types of cytometer, nor could it be explained by attributes of the standard curves or any other single variable examined. However, the application of a corrective factor based on FI results for Fluorotrol allowed a two‐fold improvement in the precision of FI measurements on CD4‐stained lymphocytes, with an overall CV of 11%. Uncharacterized differences in the operating conditions of flow cytometers can influence cellular FI measurements, but consistent results can be obtained if a stained cellular calibrator is analyzed in addition to the proper microbead standards.Keywords
This publication has 8 references indexed in Scilit:
- Model system evaluating fluorescein‐labeled microbeads as internal standards to calibrate fluorescence intensity on flow cytometersCytometry, 1989
- Calibration of flow cytometric fluorescence standards using the isoparametric analysis of ligand bindingCytometry, 1987
- Fluorescence Intensity as a Quality Control Parameter in Clinical Flow CytometryAmerican Journal of Clinical Pathology, 1987
- Relationship between T200 antigen expression and stages of B cell differentiation in resurgent hyperplasia of bone marrowBlood, 1987
- Controls for Flow Cytometers in Hematology and Cellular ImmunologyAnnals of the New York Academy of Sciences, 1986
- Flow-cytometric determination of fluorescence ratios between differently stained particles is dependent on excitation intensity.Journal of Histochemistry & Cytochemistry, 1985
- Expression of human B cell-associated antigens on leukemias and lymphomas: a model of human B cell differentiationBlood, 1984
- Determination of linear fluorescence intensities from flow cytometric data accumulated with logarithmic amplifiersCytometry, 1983