PubMed · 8777359
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C T Nebe. 1996. [Flow cytometry and data analysis].. https://pubmed.ncbi.nlm.nih.gov/8777359/
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UNLABELLED: BACKGROUND Three flow cytometric methods for anti-D quantification have been published. All use different cell sensitization and antibody detection conditions that may lead to varied results. Therefore, a direct comparison of the three methods is timely. STUDY DESIGN AND METHODS: The published flow cytometric methods and two new in-house modifications were compared. Ten serum samples containing anti-D at levels between 11.6 and 915 IU per mL were selected for analysis, and each was tested a minimum of three times. Anti-D bound to cells was detected with fluorescence-labeled anti-human IgG reagents. RESULTS The interassay CV of the standard curves for each of the five methods was less than 10 percent. The intra-assay CV was consistently <10 percent with four out of the five methods, but, by the fifth method, it was >20 percent in more than one-third of the tests. In 72 percent of the sample and method combinations, the interassay CV was <25 percent. Plotting of the mean anti-D value for each sample as a percentage of the value determined by an automated technique (AutoAnalyzer) revealed wide variability between the methods. CONCLUSION: Anti-D quantification by flow cytometry is influenced by the serum antibody characteristics and the method used. The differences between the flow cytometric and AutoAnalyzer techniques indicate that further validation of the flow cytometric method is required before routine use.
BACKGROUND: The Kolmogorov-Smirnov test is a valid statistical test for comparing distributions that has been recommended for flow cytometric histogram analysis. However, this test is frequently found to be too sensitive for flow cytometric histogram comparisons. Here, a parametric alternative to the Kolmogorov-Smirnov test is proposed that is based on fitting suitable models to flow cytometric data. METHODS: Several flow cytometric histograms derived from cell surface immunophenotyping for intercellular adhesion molecule-1 (ICAM-1) on K562 cells were analyzed using numerical modeling. The prediction intervals derived from the modeling were used for decision making. RESULTS: The residuals after peak fitting flow cytometric data are normally distributed and this permits the use of the prediction limit methodology. The usefulness of the approach for analyzing flow cytometry histograms is examined and the method is shown to avoid the "sensitivity" disadvantages associated with the Kolmogorov-Smirnov test. CONCLUSIONS: The prediction limit method is a viable alternative to the Kolmogorov-Smirnov method.
BACKGROUND: We previously reported a new optical configuration, in which both the side scatter and the fluorescence are collected using the index-guided, total internal reflection of a flow stream in air (the flow-stream waveguide). METHODS: Using a mixture of 0.202-microm and 0.093-microm diameter polystyrene beads, we have characterized the side scatter (SSC) sensitivity of a custom-built flow cytometer (miniFlo) which incorporates a flow-stream waveguide. RESULTS: The SSC-triggered SSC signal of 0.093-microm polystyrene beads in water was almost baseline resolved from the background. We also measured the SSC-triggered SSC signal of the same beads in water on our FACScan, which is a commercial unit with the conventional optical arrangement that uses a custom imaging objective to collect light from a sheath flow cuvette in perpendicular direction-the signal from 0.093-microm beads was not resolved from the background. CONCLUSIONS: The SSC sensitivity of miniFlo is one of the best reported in the literature. Cytometry 37:160-163, 1999. Published 1999 Wiley-Liss, Inc.