tetano
Editor, Senior Moderator
Arch Pathol Lab Med
. 2021 Mar 15.
doi: 10.5858/arpa.2021-0008-SA. Online ahead of print.
Comparison of In Situ Hybridization, Immunohistochemistry and Reverse Transcription-Droplet Digital Polymerase Chain Reaction for Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV-2)-Testing in Tissue
Anja C Roden[SUP] 1 [/SUP], Julie A Vrana[SUP] 1 [/SUP], Justin W Koepplin[SUP] 1 [/SUP], Angela E Hudson[SUP] 1 [/SUP], Andrew P Norgan[SUP] 1 [/SUP], Garrett Jenkinson[SUP] 1 [/SUP], Satoko Yamaoka[SUP] 1 [/SUP], Hideki Ebihara[SUP] 1 [/SUP], Robert Monroe[SUP] 1 [/SUP], Matthias J Szabolcs[SUP] 1 [/SUP], Ramanath Majumdar[SUP] 1 [/SUP], Ann M Moyer[SUP] 1 [/SUP], Joaqu?n J Garc?a[SUP] 1 [/SUP], Benjamin R Kipp[SUP] 1 [/SUP]
Affiliations
Abstract
Context: Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-detection in formalin-fixed paraffin-embedded (FFPE) tissue using reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and/or RNA-in situ hybridization (RNAish).
Objective: To compare droplet digital PCR (ddPCR), IHC, and RNAish to detect SARS-CoV-2 in FFPE tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients.
Design: ddPCR and RNAish used commercially available probes; IHC utilized clone 1A9. Twenty-six autopsies of COVID-19 patients with FFPE 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: ddPCR had the highest sensitivity for SARS-CoV-2 (96%) when compared to IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between ddPCR and IHC or RNAish was fair (?=0.23, ?=0.35, respectively). Agreement between IHC and ISH was substantial (?=0.75). Interobserver reliability was almost perfect for IHC (?=0.91) and fair to moderate for RNAish (?=0.38-0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by ddPCR (P=.03, pearson corr = -0.65) and were more likely to be positive by RNAish (P=.02) than lungs from patients who died later. SARS-CoV-2 was identified in hyaline membranes, pneumocytes, and rarely in respiratory epithelium. ddPCR showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative.
Conclusions: ddPCR was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.
. 2021 Mar 15.
doi: 10.5858/arpa.2021-0008-SA. Online ahead of print.
Comparison of In Situ Hybridization, Immunohistochemistry and Reverse Transcription-Droplet Digital Polymerase Chain Reaction for Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV-2)-Testing in Tissue
Anja C Roden[SUP] 1 [/SUP], Julie A Vrana[SUP] 1 [/SUP], Justin W Koepplin[SUP] 1 [/SUP], Angela E Hudson[SUP] 1 [/SUP], Andrew P Norgan[SUP] 1 [/SUP], Garrett Jenkinson[SUP] 1 [/SUP], Satoko Yamaoka[SUP] 1 [/SUP], Hideki Ebihara[SUP] 1 [/SUP], Robert Monroe[SUP] 1 [/SUP], Matthias J Szabolcs[SUP] 1 [/SUP], Ramanath Majumdar[SUP] 1 [/SUP], Ann M Moyer[SUP] 1 [/SUP], Joaqu?n J Garc?a[SUP] 1 [/SUP], Benjamin R Kipp[SUP] 1 [/SUP]
Affiliations
- PMID: 33720333
- DOI: 10.5858/arpa.2021-0008-SA
Abstract
Context: Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-detection in formalin-fixed paraffin-embedded (FFPE) tissue using reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and/or RNA-in situ hybridization (RNAish).
Objective: To compare droplet digital PCR (ddPCR), IHC, and RNAish to detect SARS-CoV-2 in FFPE tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients.
Design: ddPCR and RNAish used commercially available probes; IHC utilized clone 1A9. Twenty-six autopsies of COVID-19 patients with FFPE 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: ddPCR had the highest sensitivity for SARS-CoV-2 (96%) when compared to IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between ddPCR and IHC or RNAish was fair (?=0.23, ?=0.35, respectively). Agreement between IHC and ISH was substantial (?=0.75). Interobserver reliability was almost perfect for IHC (?=0.91) and fair to moderate for RNAish (?=0.38-0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by ddPCR (P=.03, pearson corr = -0.65) and were more likely to be positive by RNAish (P=.02) than lungs from patients who died later. SARS-CoV-2 was identified in hyaline membranes, pneumocytes, and rarely in respiratory epithelium. ddPCR showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative.
Conclusions: ddPCR was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.