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.

Keywords: Cytokines; GCLP; Luminex; Multiplex; Proficiency.

Figure 1

EQAPOL administrative structure and support teams. Highlighted along the bottom are the key groups involved in executing the international Luminex EQA.

Figure 2

Location of sites participating in EQAPOL Luminex EQA. A) International. B) Domestic, United States of America.

Figure 3

EP1 reported concentration of IFNγ in serum (A) and activated PBMC culture supernatant (B). Solid lines indicate the upper and lower bounds for Site Reported data. Dashed lines indicate upper and lower bounds for data from Central Analysis.

Figure 4

Site performance across three scored EPs. A) Individual site results. Performance ranges: Excellent (91–100), Good (75–90), Fair (66–74), and Poor (0–65). B) Between (inter-) site variation. C) Within (intra-) site variation. Box and whisker plots represent the 25^th and 75^th percentile, the line represents the median and the end of the whiskers are drawn to the most extreme values. The dot represents the mean. Note: Two sites were excluded from this analysis, as they received zeros for failure to complete a shipped EP. (LN = natural log-transformed)