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 +/- 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% +/- 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.
BACKGROUND: Flow cytometric analysis of humanP2X(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 +/- 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% +/- 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.
Authors: Pandurangan Vijayanand; Grégory Seumois; Chris Pickard; Robert M Powell; Gilbert Angco; David Sammut; Stephan D Gadola; Peter S Friedmann; Ratko Djukanovic Journal: N Engl J Med Date: 2007-04-05 Impact factor: 91.245
Authors: Wilfried H B M Levering; Frank W M B Preijers; Wessel N van Wieringen; Jaco Kraan; Wil A M van Beers; Kees Sintnicolaas; Dick J van Rhenen; Jan W Gratama Journal: Cytometry B Clin Cytom Date: 2007-05 Impact factor: 3.058
Authors: F Di Virgilio; P Chiozzi; D Ferrari; S Falzoni; J M Sanz; A Morelli; M Torboli; G Bolognesi; O R Baricordi Journal: Blood Date: 2001-02-01 Impact factor: 22.113
Authors: R Coutinho-Silva; J L Perfettini; P M Persechini; A Dautry-Varsat; D M Ojcius Journal: Am J Physiol Cell Physiol Date: 2001-01 Impact factor: 4.249
Authors: Claudia Jursik; Ronald Sluyter; Jennifer G Georgiou; Stephen J Fuller; James S Wiley; Ben J Gu Journal: J Immunol Methods Date: 2007-06-26 Impact factor: 2.303
Authors: G Malerba; M C Lauciello; T Scherpbier; E Trabetti; R Galavotti; V Cusin; L Pescollderungg; G Zanoni; L C Martinati; A L Boner; R C Levitt; P F Pignatti Journal: Am J Respir Crit Care Med Date: 2000-10 Impact factor: 21.405
Authors: J Xu; D S Postma; T D Howard; G H Koppelman; S L Zheng; O C Stine; E R Bleecker; D A Meyers Journal: Am J Hum Genet Date: 2000-10-06 Impact factor: 11.043
Authors: David M Manthei; Daniel J Jackson; Michael D Evans; Ronald E Gangnon; Christopher J Tisler; James E Gern; Robert F Lemanske; Loren C Denlinger Journal: J Allergy Clin Immunol Date: 2012-06-27 Impact factor: 10.793
Authors: Loren C Denlinger; David M Manthei; Max A Seibold; Kwangmi Ahn; Eugene Bleecker; Homer A Boushey; William J Calhoun; Mario Castro; Vernon M Chinchili; John V Fahy; Greg A Hawkins; Nicolina Icitovic; Elliot Israel; Nizar N Jarjour; Tonya King; Monica Kraft; Stephen C Lazarus; Erik Lehman; Richard J Martin; Deborah A Meyers; Stephen P Peters; Dagna Sheerar; Lei Shi; E Rand Sutherland; Stanley J Szefler; Michael E Wechsler; Christine A Sorkness; Robert F Lemanske Journal: Am J Respir Crit Care Med Date: 2012-11-09 Impact factor: 21.405
Authors: Xiang Ding; Nancy A Wilson; Robert R Redfield; Sarah E Panzer; Bret Verhoven; Shannon R Reese; Weixiong Zhong; Lei Shi; William J Burlingham; Loren C Denlinger; Arjang Djamali Journal: Kidney360 Date: 2020-02-03
Authors: Loren C Denlinger; Lei Shi; Arturo Guadarrama; Kathy Schell; Dawn Green; Alison Morrin; Kirk Hogan; Ronald L Sorkness; William W Busse; James E Gern Journal: Am J Respir Crit Care Med Date: 2008-11-21 Impact factor: 21.405