The Impact of Freeze-Thaw Cycles on the Integrity of SARS-COV-2 Viral Culture Fluids and Clinical Remnant Samples in Antigen or Nucleic Acid Testing

Preservation at ultra-low temperatures has been a gold standard for long-term storage of many types of clinical specimens including the SARS-CoV-2 virus. The frozen specimens can be easily transported and tested later. In addition, de-identified frozen remnant samples are resources for many preclinical or clinical studies. It is therefore crucial to understand whether freeze and thaw cycles (FTCs) can adversely affect SARS-CoV-2 test performance when frozen samples are tested. Some early studies suggest that the FTCs increased the cycles threshold (Ct) of RT-PCR indicating the potential degradation of the SARS-CoV-2 nucleic acid after FTCs, while the others did not report any significant changes in the SARS-CoV-2 nucleic acids after the FTCs. Moreover, the impact of FTCs on the performance of the SARS-CoV-2 antigen test is scarcely reported. In this study, we performed paired nucleic acid and rapid antigen tests on the same samples to investigate and directly compare how FTCs affect the performance of two types of tests. Both inactivated viral culture fluid samples and clinical remnant samples were studied. Our results showed that FTCs had minimal negative effects on the performance of the rapid SARS-CoV-2 antigen test, and the test results remained largely consistent throughout the FTCs, whereas the Ct values of RT-PCR increased with the increase of the FTC numbers. In addition, our data also demonstrated that the SARS-CoV-2 is preserved better in VTM than PBS during FTCs in regard to nucleic acid testing.