组织中用于严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 检测的原位杂交、免疫组织化学和逆转录-液滴数字聚合酶链反应的比较。

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.

机构信息

From the Department of Laboratory Medicine and Pathology (Roden, Vrana, Koepplin, Hudson, Norgan, Majumdar, Moyer, García, Kipp), Mayo Clinic Rochester, Minnesota.

Department of Health Sciences Research (Jenkinson), Mayo Clinic Rochester, Minnesota.

出版信息

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

DOI:10.5858/arpa.2021-0008-SA

PMID:33720333

Abstract

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.

摘要

背景

已有小样本病例系列研究评估了使用逆转录聚合酶链反应（RT-PCR）、免疫组织化学（IHC）和/或 RNA 原位杂交（RNAish）对福尔马林固定、石蜡包埋组织中严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的检测。

目的

通过对来自 2019 年冠状病毒病（COVID-19）患者的大量肺标本的福尔马林固定、石蜡包埋组织，比较液滴数字 PCR、IHC 和 RNAish 检测 SARS-CoV-2 的方法。

设计

液滴数字 PCR 和 RNAish 使用了商业化的探针；IHC 使用了克隆 1A9。纳入了 26 例 COVID-19 患者的尸检，包括 62 个肺标本、22 个心脏标本、2 个脑标本和 1 个肝标本以及 1 个脐带组织的福尔马林固定、石蜡包埋组织块。对照病例包括 9 例因其他感染/炎症以及病毒感染组织或细胞系而行尸检的患者的肺组织。

结果

与 IHC（31%）和 RNAish（36%）相比，液滴数字 PCR 对 SARS-CoV-2 的检测灵敏度最高（96%）。所有 3 种检测方法的特异性均为 100%。液滴数字 PCR 与 IHC 或 RNAish 的一致性为中等（κ=0.23 和 κ=0.35）。IHC 与原位杂交的一致性较高（κ=0.75）。IHC 的观察者间可靠性几乎为完美（κ=0.91），RNAish 的观察者间可靠性为中等至较好（κ=0.38-0.59）。症状发作后更早死亡的患者的肺组织通过液滴数字 PCR 检测到的拷贝数更高（P=0.03，Pearson 相关系数=-0.65），且更有可能通过 RNAish 检测到阳性（P=0.02）。我们在透明膜、肺泡细胞中发现了 SARS-CoV-2，在呼吸道上皮细胞中罕见。来自 ProteoGenex Inc 的 7 例尸检心脏的液滴数字 PCR 显示低拷贝数。所有其他肺外组织均为阴性。