Kinetic Analysis of Intracellular ionized Calcium Level from Human Peripheral Blood Lymphocytes Using Flow Cytometry / 대한임상병리학회지

ABSTRACT

BACKGROUND: Intracellular ionized calcium plays a central role in the transduction of external stimuli as a critical second messenger. The spectral properties of fluo-3 allows the analysis of intracellular ionized calcium level by flow cytometers. The aim of this study is to assess the performance of flow cytometer for measuring intracellular ionized calcium level using fluo-3 and to define the reference interval of intracellular ionized calcium level of lymphocytes from healthy people, and to find out the clinical implications according to various disorders. METHODS: For the analytical performance of flow cytometer on determining the concentration of intracellular ionized calcium, precision study, lowest limit of detection, analytical range, and the loading stability of fluo-3 were per foamed. Fifty-four cases of healthy people, 52 cases of renal transplant patients, and 20 cases of diabetes mellitus patients were included in this study. RESULTS: Loading effect of fluo-3 at room temperature was stable upto 5 hours. Lowest limit of detection of ionized calcium concentration was 4.34 nM at in-situ calibration procedure. Within-run and among-day intraindividual CVs of in-situ calibration procedure were 6.67% and 13.99% respectively, and of optical calibration procedure were 13.86% and 16.12% respectively. The reference interval of cytosolic free calcium level for healthy people ranged 73.54 - 155.09 nM without sexual differences. The level of intracellular ionized calcium was lowered by 36.9% on renal transplant group in comparison with healthy control group. But, level of cytosolic free calcium was Increased upto 276.0% on acute rejection group and 159.1% on diabetes mellitus group compared to control group. CONCLUSIONS: These results reveal that in-situ calibration method for intra cellular ionized calcium using flow cytometry with flue-3 can be regarded as an accurate and standardized method. Quantitation of intracellular ionized calcium level might be used as the monitoring test for early detection of acute rejection after renal transplantation.