Olga Mizrahi1, Eliran Ish Shalom1, Michal Baniyash1,2, Yair Klieger1. 1. ImProDia LTD, Herzliya Pituah, 46723, Israel. 2. Lautenberg Center for General and Tumor Immunology, Israel-Canada Medical Research Institute, Faculty of Medicine, Hebrew University, Jerusalem, 91120, Israel.
Abstract
BACKGROUND: Quantitative flow cytometry (QFCM) can be an important element within the developing toolbox for clinical diagnostics which relies on precise and rapid tests that provide a conclusive answer for physicians. The FC technology combines all of these features. Until recently, this imperative discipline was based on qualitative assessments of cell populations. However, due to the enormous advancement in FC technology, which allows the quantification of a number of antigens on cell surface and within the cells by units of median fluorescence intensity (MFI), this method becomes meaningful and fits the clinical needs. METHODS: On the basis of our experience in the field of quantitative FC, we wish to highlight some of the key concerns related to this methodology and suggest possible solutions for achieving uniform and standardized QFCM tests based on MFI. RESULTS: Several parameters are responsible for inter and intra laboratory variations. The standardization of quantitative FC relies on three major components; Samples and reagents handling, FC maintenance and data analysis. The use of specialized beads as a part of the routine calibration process lowers inter-test variability between different operators and different FC instruments. Similarly, the use of agreed biological controls contributes significantly to lowering test variability. CONCLUSIONS: The field of QFCM displays a significant part in the diagnostic clinical toolbox. We believe that the recommendations described herein can improve significantly the stability and accuracy of this method, thus assuring a more standardized cell analyses.
BACKGROUND: Quantitative flow cytometry (QFCM) can be an important element within the developing toolbox for clinical diagnostics which relies on precise and rapid tests that provide a conclusive answer for physicians. The FC technology combines all of these features. Until recently, this imperative discipline was based on qualitative assessments of cell populations. However, due to the enormous advancement in FC technology, which allows the quantification of a number of antigens on cell surface and within the cells by units of median fluorescence intensity (MFI), this method becomes meaningful and fits the clinical needs. METHODS: On the basis of our experience in the field of quantitative FC, we wish to highlight some of the key concerns related to this methodology and suggest possible solutions for achieving uniform and standardized QFCM tests based on MFI. RESULTS: Several parameters are responsible for inter and intra laboratory variations. The standardization of quantitative FC relies on three major components; Samples and reagents handling, FC maintenance and data analysis. The use of specialized beads as a part of the routine calibration process lowers inter-test variability between different operators and different FC instruments. Similarly, the use of agreed biological controls contributes significantly to lowering test variability. CONCLUSIONS: The field of QFCM displays a significant part in the diagnostic clinical toolbox. We believe that the recommendations described herein can improve significantly the stability and accuracy of this method, thus assuring a more standardized cell analyses.
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