Comparison of In Situ Hybridization, Immunohistochemistry, and Reverse Transcription-Droplet Digital Polymerase Chain Reaction for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Testing in Tissue

* Context.--Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in formalin-fixed, paraffin-embedded tissue using reverse transcription-polymerase chain reaction, immunohistochemistry (IHC), and/or RNA in situ hybridization (RNAish). Objective.--To compare droplet digital polymerase chain reaction, IHC, and RNAish to detect SARS-CoV-2 in formalin-fixed, paraffin-embedded tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients. Design.--Droplet digital polymerase chain reaction and RNAish used commercially available probes; IHC used clone 1A9. Twenty-six autopsies of COVID-19 patients with formalin-fixed, paraffin-embedded tissue blocks of 62 lung specimens, 22 heart specimens, 2 brain specimens, and 1 liver, and 1 umbilical cord were included. Control cases included 9 autopsy lungs from patients with other infections/inflammation and virus-infected tissue or cell lines. Results.--Droplet digital polymerase chain reaction had the highest sensitivity for SARS-CoV-2 (96%) when compared with IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between droplet digital polymerase chain reaction and IHC or RNAish was fair ([kappa] = 0.23 and [kappa] = 0.35, respectively). Agreement between IHC and in situ hybridization was substantial (k = 0.75). Interobserver reliability was almost perfect for IHC ([kappa] = 0.91) and fair to moderate for RNAish ([kappa] = 0.380.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by droplet digital polymerase chain reaction (P =.03, Pearson correlation = -0.65) and were more likely to be positive by RNAish (P =.02) than lungs from patients who died later. We identified SARS-CoV-2 in hyaline membranes, in pneumocytes, and rarely in respiratory epithelium. Droplet digital polymerase chain reaction showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative. Conclusions.--Droplet digital polymerase chain reaction was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients. (Arch Pathol Lab Med. 2021;145:785-796; doi: 10.5858/arpa.2021-0008-SA)
Coronavirus disease 2019 (COVID-19), the disease that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, can be life-threatening, largely because of development of acute respiratory distress syndrome. [...]