key: cord-0690981-z6qj5aob authors: Garcia-Beltran, W. F.; Miller, T. E.; Kirkpatrick, G.; Nixon, A.; Astudillo, M. G.; Yang, D.; Mahanta, L. M.; Murali, M.; Dighe, A.; Lennerz, J.; Thierauf, J.; Naranbhai, V.; Iafrate, A. J. title: Remote fingerstick blood collection for SARS-CoV-2 antibody testing date: 2020-11-01 journal: nan DOI: 10.1101/2020.10.27.20221028 sha: 0d44e177cbcac68710abdd6eff2a71f9c4fb033d doc_id: 690981 cord_uid: z6qj5aob The rapid worldwide spread of SARS-CoV-2 infection has propelled the rapid development of serological tests that can detect anti-SARS-CoV-2 antibodies. These have been used for studying the prevalence and spread of infection in different populations, helping establish a diagnosis of COVID-19, and will likely be used to confirm humoral immunity after infection or vaccination. However, nearly all lab-based high-throughput SARS-CoV-2 serological assays require a serum sample from venous blood draw, limiting their applications and scalability. Here, we present a method that enables large scale SARS-CoV-2 serological studies by combining self or office collection of fingerprick blood with an FDA-approved dried blood spot collection device (Neoteryx Mitra(R)) with a high-throughput electrochemiluminescence-based SARS-CoV-2 total antibody assay (Roche Elecsys(R)) that is EUA approved for use on serum samples and widely used by clinical laboratories around the world. We found that the Roche Elecsys(R) assay has a high dynamic range that allows for accurate detection of SARS-CoV-2 antibodies in serum samples diluted 1:20 as well as contrived dried blood extracts. Extracts of dried blood from Neoteryx Mitra(R) devices acquired in a community seroprevalence study showed near identical sensitivity and specificity in detection of SARS-CoV-2 antibodies as compared to neat sera using predefined thresholds for each specimen type. Overall, this study affirms the use of Neoteryx Mitra(R) dried blood collection device with the Roche Elecsys(R) SARS-CoV-2 total antibody assay for remote or at-home testing as well as large-scale community seroprevalence studies. The ongoing pandemic of coronavirus disease 2019 , caused by infection with severe acute respiratory system coronavirus 2 (SARS-CoV-2), has continued to ignite the rapid development of diagnostic tests that can detect active or past infection. To date, molecular tests that detect viral RNA are the gold standard for diagnosing and reporting active infection. However, detection of antibodies to SARS-CoV-2 via serological assays is necessary to identify recently infected individuals that have cleared the virus, which is critical for studies that measure the prevalence and spread of infection and is likely to be used to confirm the development of humoral immunity after infection or vaccination. Currently, there are over 30 emergency use authorization (EUA)-approved serological tests and there currently exists no wide-spread or gold-standard platform 1 . Several studies have compared the performance of these various platforms, including: Euroimmun anti-spike IgG enzyme-linked immunosorbent assay (ELISA), Bio-Rad Platelia anti-nucleocapsid total antibodies ELISA, Wantai anti-spike total antibodies ELISA, Diasorin Liaison anti-spike IgG chemiluminescence immunoassay (CLIA), Abbott Alinity anti-nucleocapsid IgG chemiluminescent microparticle-based immunoassay (CMIA), Siemens anti-spike IgG CMIA, and Roche Elecsys® anti-nucleocapsid total antibodies electrochemiluminescence-based immunoassay (ECLIA) [2] [3] [4] . Based on our previous work, we chose at our institution to deploy the Roche Elecsys® SARS-CoV-2 total antibody ECLIA that detects IgG, IgM, and/or IgA antibodies to SARS-CoV-2 nucleocapsid 2 , which many other centers around the world have also done 5 . While this has provided great benefit to diagnosing recent SARS-CoV-2 infection for inpatients and outpatients, this test requires collection of serum from whole blood via phlebotomy, as do the other EUA-approved serological tests mentioned above. This exposes clinical staff to potentially infectious patients and limits the scalability of SARS-CoV-2 antibody testing due to phlebotomy requirements. Consequently, many investigators have searched for a strategy that involves remote collection or self-collection of serum. Several methods of remote blood collection have long existed and have been used to detect SARS-CoV-2 antibodies, including dried blood spot (DBS) cards 6, 7 , yet extraction of serum from these specimens is cumbersome and low throughput. However, a novel device that uses a filter tip that collects a standardized volume of finger prick blood was developed, called Neoteryx Mitra®. This FDA-approved medical device allows for remote fingerprick blood self-collection that can be mailed back to a central facility and processed through a high-throughput extraction pipeline in 96-well plate format. Accordingly, the National Institute of Health (NIH) has partnered with the developers of this device and have validated its use with an in-house-developed serological assay that measures IgG and IgM antibodies to SARS-CoV-2 spike protein 8 , and have an ongoing clinical trial assessing the sero-prevalence of SARS-CoV-2 infection in different areas of the country 9 . In this study, we assess the performance of the Neoteryx Mitra® device pipelined into the Roche Elecsys® SARS-CoV-2 antibody assay to expand our SARS-CoV-2 testing capability to at-home testing and large-scale community seroprevalence studies. Contrived whole blood was generated by mixing serum with an equal volume of type O-negative packed red blood cells (hematocrit of approximately 80%). Whole blood was obtained from discarded segments (tubing) from whole blood donations. Fingerprick blood expressed using a spring-loaded lancet (Becton Dickinson). 20-μL Neoteryx Mitra® devices were used to collect the aforementioned specimens, followed by dry storage for at least 24 h. Neoteryx Mitra® devices containing dried blood samples were placed in a deep (2-mL) 96-well plate with a fitted holder (Neoteryx) that allows for the devices to be slightly suspended. The deep 96-well plates contained 200 μL/well of extraction buffer, which consisted of 1% BSA, 50 mM Tris (pH 8.0), 140 mM NaCl, and 0.05% Tween-20. Our extraction buffer contained BSA to help stabilize antibodies as well as 0.02% Tween-20 to help facilitate extraction and inactivate any potentially infectious virus 13 . Plates were then placed on a shaker for 2 h at 600 RPM. Devices were subsequently removed, leaving 200 μL of dried blood extract in each well. Of note, extracts exhibited significant hemolysis (dark red in color). We used the electrochemiluminescence-based Roche Elecsys® Anti-SARS-CoV-2 immunoassay, which detects IgG, IgM, and/or IgA antibodies to the SARS-CoV-2 nucleocapsid protein. The assay was run on a Roche Cobas® 8000 e801 Immunoassay Analyzer, which also measured hemolysis and lipemia indices. A minimum volume of approximately 200 μL of sample per run was required when using a tube insert. Use of patient samples for the development and validation of SARS-CoV-2 diagnostic tests was approved by Partners Institutional Review Board (protocol 2020P000895). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 1, 2020. ; In order to determine the feasibility of running dried blood extracts from 20-μL Neoteryx Mitra® devices on the Roche Elecsys® SARS-CoV-2 total antibody assay, we first tested serum samples from COVID-19 patients (confirmed by nasopharyngeal swab PCR) diluted 1:20 in extraction buffer. A dilution of 1:20 was chosen based on a conservative estimate that assumes that of the 20 μL of whole blood that dries onto 20-μL Neoteryx Mitra® device, there will be at Having demonstrated that the Neoteryx-Roche assay can accurately detect SARS-CoV-2 antibodies in diluted serum samples, we proceeded to generate extracts from dried blood collected on Neoteryx Mitra® devices using contrived whole blood with known serostatus. For this, we mixed serum samples from 48 COVID-19 patients and 48 pre-pandemic individuals with an equal volume of O-negative donor packed red blood cells to mimic whole blood, which was subsequently collected onto 20-μL Neoteryx Mitra® devices. After 24 hours of dry storage, samples were extracted and run on the Roche Elecsys® assay. Quality control measurements made by the Roche Cobas® instrument of hemolysis and lipemia were included, and each extracted sample, which was dark red in color, unsurprisingly contained high hemolysis and lipemia indices due to the nature of the sample (data not shown). Nevertheless, this did not affect the SARS-CoV-2 antibody assay results, and using a threshold of 1.0 for neat serum and 0.15 for extracted samples, a concordance of 98% (94/96) was achieved, with the only two discordant specimens being COVID-19 patient samples ( Figure 1B ). Of the two discrepant COVID-19 patient samples, one was positive by the neat serum specimen, but just below the threshold by the dried blood specimen, and the other was positive by the dried blood specimen but just below the threshold by the neat serum specimen. In addition, a false positive pre-pandemic specimen, which was selected as one of two pre-pandemic specimens that were false positive in the Roche assay in a cohort of 1,200 pre-pandemic specimens, was still All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 1, 2020. ; detected in the extract sample, indicating that false positive signals indeed dilute with sample dilution, and could reflect truly cross-reactive antibodies. We also generated duplicate Neoteryx Mitra® collections devices from contrived whole blood and performed extractions in two independent facilities and found high precision ( Figure 1C , R 2 = 0.97), highlighting the reproducibility of this testing strategy. To test the performance of the combined Neoteryx-Roche assay on low-and high-seroprevalence cohorts during the pandemic, we collected specimens from pre-screened blood donors at the Massachusetts General Hospital Blood Donor Center as well as in a community seroprevalence study conducted in Chelsea, Massachusetts, the densest and most affected area for SARS-CoV-2 infection 11 . For blood donors, we collected serum and dried venous blood on Neoteryx Mitra® devices, and found that the assay showed a seroprevalence of 1% (1/143), with a 100% concordance ( Figure 2A ). For community samples, we collected dried fingerprick blood on Neoteryx Mitra® devices and serum from antecubital vein phlebotomy in 402 participants. Dried fingerprick blood extracts and sera were run on the Roche Cobas® instrument, and the results revealed a seroprevalence of 24% (98/402) in serum samples, 24% in extracted sample (97/402), and a concordance of 99% (397/402) ( Figure 2B ). Interestingly, we saw a threshold effect in neat serum samples, with extracted samples that showed the highest COI demonstrating slightly decreased COI in the neat serum, most likely representing a 'hook effect' ( Figure 2C ). This effect, however, did not decrease COI sufficiently to approximate the COI threshold, and thus is unlikely to affect Roche results as a qualitative read-out. The need for remote and large-scale testing for SARS-CoV-2 antibodies is increasing as the population of convalescent individuals continues to increase and vaccines are soon to be implemented. In this study, we validate the use of an FDA-approved dried blood collection device (Neoteryx Mitra®) on a widely used, EUA-approved SARS-CoV-2 antibody test (Roche Elecsys®). Currently, the Roche Elecsys® test detects IgG, IgM, and/or IgA antibodies to SARS-CoV-2 nucleocapsid protein, an antigen that is not present in most SARS-CoV-2 vaccines, but a Roche Elecsys® assay that detects antibodies to SARS-CoV-2 spike is currently being developed 12 and will be useful to detect immune responses to vaccines, all of which contain all or a portion of the spike antigen. Both tests together can even help discriminate individuals who were infected with circulating virus (i.e. positive for both nucleocapsid and spike antibodies) or administered one of the vaccines being developed (i.e. negative for nucleocapsid antibodies but positive for spike antibodies). As such, this protocol and collection method, which All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 1, 2020. ; is readily adaptable to the upcoming version of Roche Elecsys® assays, can enable remote confirmation of vaccination responses. Currently, the NIH is using this collection method in conjunction with an in-house developed serological assay, but our study validates its use in facilities already deploying the Roche Elecsys® SARS-CoV-2 total antibody assay. In addition, although performance may not be guaranteed due to potential interferences from extracted dried blood, this study provides a straightforward framework for validating other SARS-CoV-2 serological testing platforms, such as from Euroimmun, Bio-Rad, Diasorin, Abbott, and Siemens. Overall, we believe these findings to be of significance for clinical laboratories around the world as we combat this pandemic. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 1, 2020. ; All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 1, 2020. Evaluation of Three Commercial SARS-CoV-2 Serologic Assays and their Performance in Two-Test Algorithms Comparison of different serological assays for SARS-CoV-2 in real life Performance characteristics of five immunoassays for SARS-CoV-2: a head-to-head benchmark comparison Roche to launch laboratory SARS-CoV-2 antigen test to support high-volume testing of suspected COVID-19 patients Sensitive Detection of SARS-CoV-2-Specific Antibodies in Dried Blood Spot Samples High seroprevalence for SARS-CoV-2 among household members of essential workers detected using a dried blood spot assay Standardization of enzyme-linked immunosorbent assays for serosurveys of the SARS-CoV-2 pandemic using clinical and at-home blood sampling. medRxiv NIH Begins Study to Quantify Undetected Cases of Coronavirus Infection Clinical Performance of the Elecsys Electrochemiluminescent Immunoassay for the Detection of SARS-CoV-2 Total Antibodies High seroprevalence of anti-SARS-CoV-2 antibodies in Chelsea Inactivation of West Nile virus during serologic testing and transport We wish to thank Dr. Robert S. Makar for providing whole blood samples from blood donors. We also thank Dr. Edward T. Ryan for providing pre-pandemic serum samples. A.J.I. is supported by the Lambertus Family Foundation.