title: Development and implementation of a proficiency testing program for Luminex bead-based cytokine assays.
abstract: Luminex bead array assays are widely used for rapid biomarker quantification due to the ability to measure up to 100 unique analytes in a single well of a 96-well plate. There has been, however, no comprehensive analysis of variables impacting assay performance, nor development of a standardized proficiency testing program for laboratories performing these assays. To meet this need, the NIH/NIAID and the Cancer Immunotherapy Consortium of the Cancer Research Institute collaborated to develop and implement a Luminex assay proficiency testing program as part of the NIH/NIAID-sponsored External Quality Assurance Program Oversight Laboratory (EQAPOL) at Duke University. The program currently monitors 25 domestic and international sites with two external proficiency panels per year. Each panel includes a de-identified commercial Luminex assay kit with standards to quantify human IFNγ, TNFα, IL-6, IL-10 and IL-2, and a series of recombinant cytokine-spiked human serum samples. All aspects of panel development, testing and shipping are performed under GCLP by EQAPOL support teams. Following development testing, a comprehensive site proficiency scoring system comprised of timeliness, protocol adherence, accuracy and precision was implemented. The overall mean proficiency score across three rounds of testing has remained stable (EP3: 76%, EP4: 75%, EP5: 77%); however, a more detailed analysis of site reported results indicates a significant improvement of intra- (within) and inter- (between) site variation, suggesting that training and remediation for poor performing sites may be having a positive impact on proficiency. Through continued proficiency testing, identification of variables affecting Luminex assay outcomes will strengthen efforts to bring standardization to the field.
authors.affiliation: Duke Human Vaccine Institute, Department of Medicine, Duke University Medical Center, Durham, NC 27710, United States.
authors.forename: Heather E
authors.initials: HE
authors.lastname: Lynch
authors.affiliation: Duke Human Vaccine Institute, Department of Medicine, Duke University Medical Center, Durham, NC 27710, United States.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Sanchez
authors.affiliation: Division of AIDS, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892, United States.
authors.forename: M Patricia
authors.initials: MP
authors.lastname: D'Souza
authors.affiliation: Duke Human Vaccine Institute, Department of Medicine, Duke University Medical Center, Durham, NC 27710, United States.
authors.forename: Wes
authors.initials: W
authors.lastname: Rountree
authors.affiliation: Duke Human Vaccine Institute, Department of Medicine, Duke University Medical Center, Durham, NC 27710, United States.
authors.forename: Thomas N
authors.initials: TN
authors.lastname: Denny
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, United States; Cancer Immunotherapy Consortium of the Cancer Research Institute, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Kalos
authors.affiliation: Duke Human Vaccine Institute, Department of Medicine, Duke University Medical Center, Durham, NC 27710, United States. Electronic address: gregory.sempowski@duke.edu.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Sempowski
mesh_terms: D015415:Biomarkers / Q000097:blood; D003299:Cooperative Behavior; D016207:Cytokines / Q000097:blood*; D019983:Guideline Adherence / Q000592:standards; D057166:High-Throughput Screening Assays / Q000592:standards*; D006801:Humans; D007391:International Cooperation; D007753:Laboratories / Q000592:standards*; D059021:Laboratory Proficiency Testing / Q000592:standards*; D015166:Monitoring, Immunologic / Q000592:standards*; D015337:Multicenter Studies as Topic / Q000592:standards*; D015588:Observer Variation; D017410:Practice Guidelines as Topic / Q000592:standards; D011237:Predictive Value of Tests; D016730:Program Development; D015397:Program Evaluation; D011786:Quality Control; D058996:Quality Improvement / Q000592:standards; D019984:Quality Indicators, Health Care / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000592:standards; D013997:Time Factors; D057188:Workflow
chemical_list: D015415:Biomarkers; D016207:Cytokines
keywords: Cytokines; GCLP; Luminex; Multiplex; Proficiency