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

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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.

Introduction

Enzyme-linked immuno-assays have revolutionized our ability to rapidly quantify critical biological mediators such as cytokines, chemokines, hormones, and signal transduction proteins. Traditionally, these assays are performed in 96-well plates using an enzyme-linked monoclonal antibody to cleave a substrate and produce a color change. Although these assays are quite powerful, they have been limited to one dimension — detection of a single analyte per well (50–100 μL sample). With the creation of 100 color-coded fluorescent bead sets by Luminex, each of which can be conjugated with a unique specific reactant (antibody, substrate, etc.), biomarker analysis can now be done with a multiplex approach.

Custom-made or commercially available Luminex bead array assays can be partnered with Luminex bead readers, data analysis software, and instrument validation/calibration kits to generate comprehensive immune monitoring and discovery technology. The power of this system is that up to 100 unique analytes can be quantified in a single well of a 96-well plate (25–50 μL of sample). The automated dual laser or CCD camera flow-based array reader identifies each analyte based on a unique fluorescent bead signature and quantifies fluorescence intensity of the reporter associated with that bead set. This multiplex approach to biomarker analysis has become widely used over the last decade for low- to high-throughput analysis of small volume samples. This assay platform has a broad range of applications, with commercial and lab-developed Luminex-based assays (e.g., mouse, rat, human and non-human primate cytokine panels) being used in basic science discovery, pre-clinical/translational and clinical trial immune monitoring laboratories.

Despite the high-level of integration of Luminex bead-based multiplex assays in pre-clinical and clinical research applications over the past 10 years, there has been no comprehensive analysis of the variables that impact assay performance, nor a national or international proficiency testing program for laboratories performing Luminex-based assays. Efforts to identify inter-laboratory variables which impact assay performance have been a long-standing effort within the Cancer Immunotherapy Consortium (CIC) and NIAID/NIH immune monitoring programs, and have led to implementation of assay harmonization, and optimization across a number of immune assay platforms (van der Burg et al., 2011) as well as development of proficiency testing programs (Jaimes et al., 2011). Luminex assays in particular are complex and subject to variability due to such factors as assay manufacturer and lot number, kit components, antibody pairs/clones, analyte standards, assay execution, instrumentation and data analysis/interpretation (Khan et al., 2004, Djoba Siawaya et al., 2008, Nechansky et al., 2008, Butterfield et al., 2011, Scott et al., 2011). To address these issues, a collaborative effort between the NIH/NIAID and the CIC of the Cancer Research Institute (CRI) was initiated to specifically assess outcome variation of Luminex bead-based cytokine assays performed in immune monitoring laboratories around the world and to develop an external quality assurance (EQA) program to monitor on-going laboratory performance.

The two-pronged mission of this joint NIAID/CIC Luminex proficiency initiative is to 1) identify variables that significantly impact outcomes of Luminex-based assays; and 2) establish a routine proficiency testing program for this assay platform to facilitate assay quality improvement and harmonization among participating laboratory sites. A central oversight laboratory is critical for achieving the later point. The focus of this manuscript is on the development and implementation of an international EQA program for Luminex bead-based cytokine assays.

Section snippets

Program development

As a first-step in developing a Luminex EQA program, the NIH/NIAID-sponsored External Quality Assurance Program Oversight Laboratory (EQAPOL) at Duke University developed and implemented a data gathering survey in December 2010. The survey was developed based on draft questions from the Steering Committee and run electronically through a web survey tool. Survey participants were national and international laboratories identified by NIAID and CIC representatives as performing Luminex assays for

EP1

Twenty-five laboratories participated in EP1. All program participants received a de-identified custom 5-plex Luminex-bead assay kit and two test samples containing known amounts of the five target cytokines (IFNγ, TNFα, IL-6, IL-10 and IL-2). One test sample was culture supernatant from PMA/ionomycin-stimulated human PBMCs, and the other was human AB serum spiked with known concentrations of recombinant cytokines. These samples were created to contain high cytokine concentrations so that

Overall summary

The NIH/NIAID-supported EQAPOL at Duke University has developed and launched the first international EQA/proficiency testing program for Luminex bead-based human cytokine assays. This is the result of a collaborative effort between the EQAPOL, NIH/NIAID and CIC and currently monitors 25 US domestic and international sites with two scored EP panels per year.

Comprehensive scoring based on timeliness, protocol adherence, accuracy, and precision is performed with mixed effects model-based

Acknowledgments

Current and past members of the NIAID/CIC Luminex Steering Committee include: Dr. Patricia D'Souza, representing NIAID; Drs. Michael Kalos and Michael Pride, representing CIC; Dr. Lisa Butterfield, University of Pittsburg and Dr. Gregory Sempowski, Duke University. We are grateful for the guidance and leadership of Dr. Jim Lane (NIAID). The authors are grateful to Jeff Lovingood (EQAPOL Program Management), Ambrosia Garcia, Linda Walker, Sara Brown, Holly Alley and Jennifer Baker (EQAPOL

References (12)

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