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

Arch Pathol Lab Med. 2021 Jul 1;145(7):785-796. doi: 10.5858/arpa.2021-0008-SA.


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 (κ = 0.23 and κ = 0.35, respectively). Agreement between IHC and in situ hybridization was substantial (κ = 0.75). Interobserver reliability was almost perfect for IHC (κ = 0.91) and fair to moderate for RNAish (κ = 0.38-0.59). 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.

Publication types

  • Comparative Study

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • COVID-19 / diagnosis*
  • COVID-19 / virology
  • COVID-19 Testing / methods*
  • Female
  • Humans
  • Immunohistochemistry*
  • In Situ Hybridization / methods*
  • Lung / virology*
  • Male
  • Middle Aged
  • Observer Variation
  • Prospective Studies
  • RNA, Viral / isolation & purification
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • SARS-CoV-2 / genetics
  • SARS-CoV-2 / isolation & purification*
  • Sensitivity and Specificity


  • RNA, Viral