Unexpected False-Positive Rates in Pediatric SARS-CoV-2 Serology Using the EUROIMMUN Anti-SARS-CoV-2 ELISA IgG Assay

Daniel Geisler, MD; Megan Culler Freeman, MD, PhD; Glenn J. Rapsinski, MD, PhD; Sarah E. Wheeler, PhD


Am J Clin Pathol. 2021;155(6):773-775. 

In This Article

Abstract and Introduction


Objectives: Serologic assay performance studies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in pediatric populations are lacking, and few seroprevalence studies have routinely incorporated orthogonal testing to improve accuracy.

Methods: Remnant serum samples for routine bloodwork from 2,338 pediatric patients at UPMC Children's Hospital of Pittsburgh were assessed using the EUROIMMUN Anti-SARS-CoV-2 ELISA IgG (EuroIGG) assay. Reactive cases with sufficient volume were also tested using 3 additional commercial assays.

Results: Eighty-five specimens were reactive according to the EuroIGG, yielding 3.64% (95% confidence interval [CI], 2.91%-4.48%) seropositivity, of which 73 specimens had sufficient remaining volume for confirmation by orthogonal testing. Overall, 19.18% (95% CI, 10.18%-28.18%) of samples were positive on a second and/or third orthogonal assay. This 80.82% false positivity rate is disproportionate to the expected false positivity rate of 50% given our pediatric population prevalence and assay performance.

Conclusions: In pediatric populations, false-positive SARS-CoV-2 serology may be more common than assay and prevalence parameters would predict, and further studies are needed to establish the performance of SARS-CoV-2 serology in children.


Serologic assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific antibodies provide several important applications in monitoring and responding to the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV-2–specific antibody testing assists in surveillance, complex diagnoses, convalescent plasma donation, and confirmation of appropriate vaccine response.[1] Characterization of the humoral response to SARS-CoV-2 and specific, robust detection methods are essential for meeting these needs. Prior validation studies across multiple testing platforms have demonstrated sufficient specificity (>99%) in the detection of anti–SARS-CoV-2 immunoglobulin G (IgG) antibodies, which included patients with prior common endemic coronavirus infections detected by nucleic acid testing.[2] Large-scale seroprevalence surveys are underway, however, and minor cross-reactivities can lead to significant inaccuracies in prevalence estimates because of low pretest probabilities. To minimize false-positive tests, the current Centers for Disease Control and Prevention (CDC) interim clinical testing guidelines for SARS-CoV-2 antibody testing recommend orthogonal testing, which employs 2 sequential independent tests when the initial test yields positive results.[3] Seroprevalence studies in the pediatric population are especially lacking, and neither adult nor pediatric studies have routinely incorporated orthogonal testing. Between February 12, 2020, and April 20, 2020, only 1.7% of reported cases in the United States were in children younger than 18 years of age.[4] Children generally develop milder disease, raising the possibility that seroprevalence studies are underestimating true seroprevalence. SARS-CoV-2 serologic assay validation in the pediatric population is lacking. We present a subset of cases from our pediatric SARS-CoV-2 seroprevalence study using an orthogonal testing strategy to assess false-positive SARS-CoV-2 antibody detection rates in our low-prevalence population and maximize analytic specificity.