| Autor: |
KorpiSteiner, N. L., Sheerar, D., Puffer, E. B., Urben, C., Boyd, J., Guadarrama, A., Schell, K., Denlinger, L. C. |
| Zdroj: |
Cytometry Part B: Clinical Cytometry; September 2008, Vol. 74 Issue: 5 p319-329, 11p |
| Abstrakt: |
BackgroundFlow cytometric analysis of human P2X7pore activity segregates variant from common P2RX7genotypes and may serve as a biomarker for cancer, pain, inflammation, and immune responses to infection. Standardization is needed to accommodate variable sample age and instrumentation differences in a multicenter clinical trial.MethodsCD14PEstained whole blood samples were treated with YOPRO1 combined with a P2X7agonist BzATP or control, followed by the addition of PI after closure of the P2X7pore. Recalled instrument settings from previous publications were used to adapt a standardized fluorescent particleadjusted setup method. Experiments were performed to compare the two methods while evaluating components of systematic variability and facilitating reliable processing of samples with varied ages.ResultsThe median YOPRO1 fluorescence of BzATPtreated samples had less variability when collected by the beadadjusted method and was less influenced by the compensation strategy used. The average daytoday coefficient of variance for assessments of P2X7pore activity by this method was 0.11 ± 0.04, and the exclusion of nonviable cells was found to accommodate samples aged up to 4 days after phlebotomy. The beadadjusted setup method produced measurements differing by only 2.0 ± 1.5 on two analog cytometers, and within similar decades when comparing analog to digital instruments.ConclusionsThese results provide a standardized method for quantitative flow cytometric analysis of P2X7receptor phenotypes in blood monocytes with minimal intralaboratory variation and potential for interlaboratory comparisons that can greatly facilitate multicenter functional genomic clinical studies. © 2008 Clinical Cytometry Society |
| Databáze: |
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