Development and implementation of a proficiency testing program for Luminex bead-based cytokine assays

• Published in: Journal of immunological methods Vol. 409; pp. 62 - 71
• Main Authors: Lynch, Heather E., Sanchez, Ana M., D'Souza, M. Patricia, Rountree, Wes, Denny, Thomas N., Kalos, Michael, Sempowski, Gregory D.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2014
• Subjects: Biomarkers - blood; Cooperative Behavior; Cytokines; Cytokines - blood; GCLP; Guideline Adherence - standards; High-Throughput Screening Assays - standards; Humans; International Cooperation; Laboratories - standards; Laboratory Proficiency Testing - standards; Luminex; Monitoring, Immunologic - standards; Multicenter Studies as Topic - standards; Multiplex; Observer Variation; Practice Guidelines as Topic - standards; Predictive Value of Tests; Proficiency; Program Development; Program Evaluation; Quality Control; Quality Improvement - standards; Quality Indicators, Health Care - standards; Reference Standards; Reproducibility of Results; Specimen Handling - standards; Time Factors; Workflow; Proficiency; GCLP; Cytokines; Luminex; Multiplex
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2014.04.011

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.