Standardized method to minimize variability in a functional P2X₇ flow cytometric assay for a multi‐center clinical trial

Abstract

Background: Flow cytometric analysis of human P2X 7 pore activity segregates variant from common P2RX7 genotypes 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.Methods: CD14-PE-stained whole blood samples were treated with YO-PRO-1 combined with a P2X 7 agonist (BzATP) or control, followed by the addition of PI after closure of the P2X 7 pore. Recalled instrument settings from previous publications were used to adapt a standardized fluorescent particle-adjusted set-up method. Experiments were performed to compare the two methods while evaluating components of systematic variability and facilitating reliable processing of samples with varied ages.Results: The median YO-PRO-1 fluorescence of BzATP-treated samples had less variability when collected by the bead-adjusted method and was less influenced by the compensation strategy used. The average day-to-day coefficient of variance for assessments of P2X 7 pore activity by this method was 0.11 6 0.04, and the exclusion of nonviable cells was found to accommodate samples aged up to 4 days after phlebotomy. The bead-adjusted set-up method produced measurements differing by only 2.0% 6 1.5% on two analog cytometers, and within similar decades when comparing analog to digital instruments.Conclusions: These results provide a standardized method for quantitative flow cytometric analysis of P2X 7 receptor phenotypes in blood monocytes with minimal intralaboratory variation and potential for interlaboratory comparisons that can greatly facilitate multicenter functional genomic clinical studies. q 2008

Attenuated P2X7Pore Function as a Risk Factor for Virus-induced Loss of Asthma Control

“…Pore activity is reported as the fold change in 39-O-(4-benzoyl)benzoyl adenosine 59-triphosphate (Bz-ATP)-stimulated YO-PRO-1 uptake relative to the buffer control. Using these methods, the present data demonstrate a day-today intraparticipant coefficient of variance of 0.11, which is similar to the reproducibility observed with fluorescent particle-adjusted instrument settings and propidium iodide exclusion of nonviable cells (35). The presence or absence of rhinovirus in nasal lavage samples does not alter the day-to-day variability of this whole blood assay (P 5 0.144).…”

P2X7-Regulated Protection from Exacerbations and Loss of Control Is Independent of Asthma Maintenance Therapy

“…The methods of our pore assay in whole-blood monocytes have been described, including refinements to accommodate loss of viability caused by overnight shipping; nonviable cells were excluded after pore closure by the addition of propidium iodide (23,25,26). The median fluorescence intensity of YO-PRO-1 associated with viable CD14…”

“…At least 10 loss-of-function alleles have been validated in recombinant expression systems (18)(19)(20)(21)(22); however, the frequency of the minor alleles for most of these variants is rare (minor allele frequencies ,3%) and contributes to relatively small blocks of linkage disequilibrium for this gene (23,24). To circumvent these problems, we have developed a functional screening assay for P2X 7 pore activity that detects the presence of five of the most common lossof-function genotypes, and now permits analyses of multicenter studies (25,26).…”

Protection from asthma in a high-risk birth cohort by attenuated P2X7 function

“…37 Briefly, 500 µL room temperature blood was twice with HEPES-buffered saline (HBS; 130 mM NaCl, 5 mM KCl, 20 mM HEPES, 0.1% bovine serum albumin, 10 mM glucose, pH 7.4) and incubated CD14 conjugated to phycoerythrin (CD14-PE, BD Biosciences, San Diego, HBS for 20 minutes. Samples were rinsed twice with potassium glutamate buffer (130 mM potassium glutamate, 5 mM KCl, 20 mM HEPES,0.1% bovine serum albumin, 10 mM glucose, pH 7.4) and incubated with 250 µM 2’(3’)- O -(f-benzoylbenzoyl) adenosine-5’-triphosphate (BzATP; Sigma, St. Louis, MO) and 1 µM YO-PRO-1 (Molecular Probes, Eugene, OR) in potassium glutamate buffer for 20 min before addition of magnesium chloride and HBS washing.…”

“…35 These functional differences allow us to utilize a flow cytometric assay to assess whether an individual’s P2RX7 genotype confers normal or loss-of-function (LOF) potential for cell membrane pore formation. 36,37 Based upon the P2RX7 knockout mouse, 30 we hypothesized that low P2X 7 pore function would confer protection from asthma. We demonstrate that low functioning P2X 7 , as measured in peripheral blood monocytes, is associated with reduced risk of childhood asthma and allergic sensitization.…”