Dynex M² Multiplexed Automated Platform Performance and reliability of Dynex M² Multiplexed Assay System for immunity assessment for the Democratic Republic of the Congo –Demographic Health and Survey #85 Patrick Mukadi1, Stephen Higgins2,  Anis Karmali2, Melanie Poncheri2, Andrew Fusellier2, José Ngamboli1, Nathalie Kavira1, Reena H. Doshi3, Nicole A. Hoff3, Sue Gerber5,  Emile Okitolonda-Wemakoy 4, Jean-Jacques Muyembe-Tamfum1, Anne W. Rimoin3     1 Institut National de Recherche Biomedicale Kinshasa DRC; 2 Dynex Technologies Inc. Chantilly VA; 3 UCLA Fielding School of Public Health Los Angeles CA; 4 Kinshasa School of Public Health, Kinshasa DRC; 5 The Bill and Melinda Gates Foundation, Seattle, WA  Abstract Childhood vaccine-preventable diseases continue to be a major public health problem in terms of global morbidity and mortality. In support of the 2013 Demographic and Health Survey in the Democratic Republic of Congo, our lab completed an immunity assessment using dried blood spots. We used a multiplex panel capable of testing for antibodies against measles, mumps, rubella, tetanus, and chickenpox. In total, 423 DBS were collected as part of a pilot study from children visiting Kinshasa health centers and 9906 DBS collected during the principal DRC-DHS survey. Including optimization runs, data from 60 plates comprising 32,400 data points have been collected. This system in conjunction with DBS processing offers a very cost-effective reliable multiplexed immunoassay processing system for use in country-wide assessments of immunity status in challenging environments. Results 423 DBS were collected as a pilot study from children visiting Kinshasa health centers and 9906 DBS during the principal DRC-DHS survey. Including optimization runs data from 60 plates comprising 32,400 data points have been collected, which included 423 pilot DBS and 1150 of the total DHS samples. During the initial optimization of the M² testing platform all samples were tested in replicate and gave excellent concordance of clinical calls regardless of processing variables used. Sensitivity and specificity of the M² system in Kinshasa was shown to be equivalent to that at Dynex based on extraction of the 32-member reference set as well as to fresh dilutions of the control sera. Extraction of the 7-point DBS calibration series in the DRC shows an equivalent assay response to the same set extracted in the Dynex labs. Samples tested in replicate during optimization runs in Kinshasa gave 94.5% concordance of clinical calls regardless of processing variables used, with discrepant results found within the indeterminate range. Assay response histograms reveal very few naïve samples for measles, mumps and rubella, and distinct low- and high response populations. Varicella response indicate a substantial naïve population as well as low- and high responders. In contrast, tetanus response indicate a naïve population and a single responder distribution. Introduction Childhood vaccine- preventable diseases continue to be a major public health problem in terms of global morbidity and mortality. In endemic regions the only available tools of diagnosis are based on clinical and serological criteria. Serosurveys measuring specific antibodies are a direct and accurate method to assess population susceptibility and can provide critical insight into ongoing immunity gaps and operational program efficiency, however they are logistically challenging and expensive to undertake. As a result, they are generally limited to small geographic areas or convenience samples that may produce biased results and cannot be interpreted on a national scale. In support of the 2013 Democratic Republic of Congo -Demographic Health Survey (DRC-DHS) and in collaboration with University of California, Los Angeles, Fielding School of Public Health (UCLA-FSPH) an immunity assessment is being undertaken using dried blood spots (DBS). Based on this experience, a standard set of materials for add-on serosurveys are being developed that can be used in future DHS surveys and/or adapted for use with any existing data collection effort in DRC and other countries. This project is also developing local capacity to analyze and interpret these data alongside existing immunization program indicators and validate questionnaire-based surveys of coverage. Additionally, data on population immunity to measles, mumps, rubella varicella, and tetanus (MMRVT) has been obtained which will provide baseline for future elimination and eradication programs. Methods The Dynex Technologies M² ®multiplex immunoassay platform with a Measles, Mumps, Rubella, Varicella and Tetanus (MMRVT) panel is being used in which 10 polystyrene beads have been coated and immobilized within 54-well M² plates: separate assay beads with MMRVT antigens; positive controls with horseradish peroxidase, total human IgG, goat anti-human IgG; negative controls with MRC-5 and E6 cell lysate. Positive control DBS are 5-donor normal defibrinated serum and negative control DBS are pooled normal IgG-stripped serum. DBS are extracted into 1ml PBS, 0.5% tween20, 5.0% dried milk and processed on a modified Dynex DS2® automated ELISA system. DBS from 32 reference sera and 7-point dilutions of pooled sera were independently processed in DRC and USA. Assay cutoffs were set by reference to 13 singleplex and 1 multiplex FDA-approved ELISA kits. Dynex M² Multiplexed Automated Platform MMRVT Assay Layout Discussion Having been deployed to a substantially resource-limited environment the Dynex M² multiplex immunoassay system has proven to be a very robust assay platform with excellent sensitivity and specificity. This system in conjunction with DBS processing offers a very cost-effective reliable multiplexed immunoassay processing system for use in country-wide assessment of immunity status in challenging environments. Acknowledgements: We wish to thank the DRC Ministry of Health, National Institute for Biomedical Research (INRB), the Bill and Melinda Gates Foundation, and the Faucett Family Foundation for their support. Contact Information Patrick Mukadi, MD Institut National de Recherche Biomedicale UCLA-DRC Research Program Patrickmukadi@gmail.com