An evaluation of a FluoroSpot assay as a diagnostic tool to determine SARS-CoV-2 specific T cell responses

PLoS One. 2021 Sep 30;16(9):e0258041. doi: 10.1371/journal.pone.0258041. eCollection 2021.


Numerous assays evaluating serological and cellular responses have been developed to characterize immune responses against SARS-CoV-2. Serological assays are both cost- and time-effective compared to cellular assays, but cellular immune responses may provide a diagnostic value to determine previous SARS-CoV-2 infection in seronegative individuals. However, potential cross-reactive T cell responses stemming from prior encounters with human coronaviruses (HCoVs) may affect assay specificity. In this study, we evaluated the specificity and sensitivity of a SARS-CoV-2 IFN-γ Release Assay (IGRA) based on the FluoroSpot method employing commercially available SARS-CoV-2-specific peptide pools, as well as an in-house designed SARS-CoV-2 peptide pool restricted to 5 amino acid stretches or less aligning with endemic HCoVs. Blood samples were obtained from healthcare workers (HCW) 5-6 months post SARS-CoV-2 spike (S) IgG and nucleocapsid (N) IgG dual seroconversion (n = 187) and HCW who had been S IgG and N IgG dual seronegative at repeated occasions, including the current sampling time point (n = 102). In addition, samples were obtained 4 to 5 months post infection from 55 polymerase chain reaction (PCR)-confirmed COVID-19 patients. Assay specificity and sensitivity were calculated with serology as a reference standard for HCW. The in-house generated peptide pool displayed a specificity of 96.1%, while the commercially available peptide pools displayed specificities of 80.4% and 85.3%, respectively. Sensitivity was higher in a cohort of previously hospitalized COVID-19 patients (96.4% and 84.0% for the commercially available peptide pools and 92.7% for the in-house generated peptide pool) compared to the HCW cohort (92.0% and 66.8% for the commercially available peptide pools and 76.0% for the in-house generated peptide pool). Based on these findings, the individual diagnostic value of T cell immune responses against SARS-CoV-2 currently appears to be limited but remain an important research tool ahead.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Viral / blood
  • Antibodies, Viral / immunology
  • COVID-19 / blood
  • COVID-19 / diagnosis
  • COVID-19 / immunology*
  • COVID-19 Testing / methods*
  • Health Personnel
  • Humans
  • Immunity, Cellular*
  • Interferon-gamma / blood
  • Interferon-gamma / immunology
  • SARS-CoV-2 / immunology*
  • Sensitivity and Specificity
  • Seroconversion
  • T-Lymphocytes / immunology*


  • Antibodies, Viral
  • Interferon-gamma

Grants and funding

This research was supported by the Swedish Research council to JK (2020-05782), a fellowship grant from the Science for Life Laboratory to GC, Knut and Alice Wallenberg to MP, CT, SH, and SM, the Swedish Research Council to MP (2020-05880, 2018-02552), the Heart and Lung foundation (20190468) to MP, Leif Lundblad Family Foundation to CT, Region Stockholm and Erling Persson Family Foundation to SH, and Svenska Sällskapet för Medicinsk Forskning to SM. SciCross AB provided support in the form of salaries for author PD. The specific roles of these authors are articulated in the ‘author contributions’ section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.