TITLE:
Flow Cytometer Performance Characterization, Standardization and Calibration against CD4 on T Lymphocytes Enables Quantification of Biomarker Expressions for Immunological Applications
AUTHORS:
Heba Degheidy, Steven Bauer, Gerald Marti, Lili Wang
KEYWORDS:
Flow Cytometry, Biomarker Quantification, Instrument Standardization, Target Median Fluorescence Intensity (MFI)
JOURNAL NAME:
Journal of Biomedical Science and Engineering,
Vol.7 No.9,
July
28,
2014
ABSTRACT: There is an urgent need for developing a procedure for biomarker standardization and relative quantificationin clinical laboratories. Measuring the expression levels of cell antigens is critical for the diagnosis of many diseases, e.g. leukemia, lymphoma and immunodeficiency diseases. One of the most significant challenges in flow cytometry is obtaining inter-laboratory and intra-laboratory consistent and reproducible results across multiple cytometer platforms and locations longitudinally over time. To obtain measurement consistency, the target flow cytometer voltages should be optimized to segregate the negative population from the electronic noise, and to keep the brightest positive population within the dynamic range of each detector. Then target values should be determined and transferred to selected cytometers. In this study, we optimized a procedure for instrument standardization across three different flow cytometer platforms from the same vendor and in two different locations. The biomarker quantification was implemented on standardized instruments using CD4 expression on T lymphocytes with a known amount of antibody bound per cell as a quantification standard. Our results on blood cell subset typing and CD19 quantification demonstrated that consistent and reliable results could be accomplished between instruments using the developed procedure. Quantitating the expression levels of certain cell biomarkers relative to a known reference marker before, during, and after therapy would provide important information for monitoring antibody-based therapy and could be potentially used to adjust dosing. Presently, we are implementing this protocol to quantify critical disease biomarkers, and making necessary modifications to the procedure to include instruments from different instrument manufacturers.