Assessing and improving the validity of COVID-19 autopsy studies - a multi center approach to establish essential standards for immunohistochemical and ultrastructural analyses

ABSTRACT

BackgroundAutopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, e.g. by an overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. MethodsWe assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 spike protein and nucleocapsid, dsRNA, and non-structural protein Nsp3 in cultured cell lines and COVID-19 autopsy tissues. In a multicenter study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 nucleocapsid staining. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. FindingsPublications show high variability in the detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. In our study, we show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (r=-0.83, p-value <0.0001). For EM, we refined criteria for virus identification and also provide recommendations for optimized sampling and analysis. 116 of 122 publications misinterpret cellular structures as virus using EM or show only insufficient data. We provide publicly accessible digitized EM and IHC sections as a reference and for training purposes. InterpretationSince detection of SARS-CoV-2 in human autopsy tissues by IHC and EM is difficult and frequently incorrect, we propose criteria for a re-evaluation of available data and guidance for further investigations of direct organ effects by SARS-CoV-2. Key messagesO_LIDetection of SARS-CoV-2 proteins by IHC in autopsy tissues is less sensitive in comparison to SARS-CoV-2 RNA detection by RT-qPCR. C_LIO_LIFor determination of SARS-CoV-2 protein positive cells by IHC in autopsy tissues, detection of spike protein is less sensitive than nucleocapsid protein. C_LIO_LICorrect identification of SARS-CoV-2 particles in human samples by EM is limited to the respiratory system. C_LIO_LIInterpretation of IHC and EM should follow substantiated consensus criteria to enhance accuracy. C_LIO_LIExisting datasets describing SARS-CoV-2 presence in human autopsy tissues need to be critically re-evaluated. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/22269205v1_ufig1.gif" ALT="Figure 1"> View larger version (44K) org.highwire.dtl.DTLVardef@eafd97org.highwire.dtl.DTLVardef@1aed770org.highwire.dtl.DTLVardef@1c21ab9org.highwire.dtl.DTLVardef@68a101_HPS_FORMAT_FIGEXP M_FIG C_FIG