Standardized method to minimize variability in a functional P2X(7) flow cytometric assay for a multi-center clinical trial.
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.
Korpi-Steiner
N L
NL
Department of Medicine-Allergy, Pulmonary & Critical Care, University of Wisconsin-Madison, Madison, WI 53792, USA.
Sheerar
D
D
Puffer
E B
EB
Urben
C
C
Boyd
J
J
Guadarrama
A
A
Schell
K
K
Denlinger
L C
LC
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000375:Aging / Q000187:drug effects; D001249:Asthma / Q000175:diagnosis; D001583:Benzoxazoles; D002470:Cell Survival / Q000187:drug effects; D002986:Clinical Trials as Topic; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000187:drug effects; D015337:Multicenter Studies as Topic; D018962:Phlebotomy; D011806:Quinolinium Compounds; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7
D001583:Benzoxazoles; D018950:Lipopolysaccharide Receptors; C546468:P2RX7 protein, human; D011806:Quinolinium Compounds; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C089813:YO-PRO 1; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate