Departments of Pathology.
Massachusetts General Hospital Cancer Center, Boston.
Am J Surg Pathol. 2021 Jan;45(1):14-24. doi: 10.1097/PAS.0000000000001563.
Coronavirus disease-19 (COVID-19) is caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although SARS-CoV-2 is visualized on electron microscopy, there is an increasing demand for widely applicable techniques to visualize viral components within tissue specimens. Viral protein and RNA can be detected on formalin-fixed paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. Herein, we evaluate the staining performance of ISH for SARS-CoV-2 and an IHC directed at the SARS-CoV nucleocapsid protein and compare these results to a gold standard, tissue quantitative real-time polymerase chain reaction (qRT-PCR). We evaluated FFPE sections from 8 COVID-19 autopsies, including 19 pulmonary and 39 extrapulmonary samples including the heart, liver, kidney, small intestine, skin, adipose tissue, and bone marrow. We performed RNA-ISH for SARS-CoV-2 on all cases with IHC for SARS-CoV and SARS-CoV-2 qRT-PCR performed on selected cases. Lungs from 37 autopsies performed before the COVID-19 pandemic served as negative controls. The ISH and IHC slides were reviewed by 4 observers to record a consensus opinion. Selected ISH and IHC slides were also reviewed by 4 independent observers. Evidence of SARS-CoV-2 was identified on both the IHC and ISH platforms. Within the postmortem lung, detected viral protein and RNA were often extracellular, predominantly within hyaline membranes in patients with diffuse alveolar damage. Among individual cases, there was regional variation in the amount of detectable virus in lung samples. Intracellular viral RNA and protein was localized to pneumocytes and immune cells. Viral RNA was detected on RNA-ISH in 13 of 19 (68%) pulmonary FFPE blocks from patients with COVID-19. Viral protein was detected on IHC in 8 of 9 (88%) pulmonary FFPE blocks from patients with COVID-19, although in 5 cases the stain was interpreted as equivocal. From the control cohort, FFPE blocks from all 37 patients were negative for SARS-CoV-2 RNA-ISH, whereas 5 of 13 cases were positive on IHC. Collectively, when compared with qRT-PCR on individual tissue blocks, the sensitivity and specificity for ISH was 86.7% and 100%, respectively, while those for IHC were 85.7% and 53.3%, respectively. The interobserver variability for ISH ranged from moderate to almost perfect, whereas that for IHC ranged from slight to moderate. All extrapulmonary samples from COVID-19-positive cases were negative for SARS-CoV-2 by ISH, IHC, and qRT-PCR. SARS-CoV-2 is detectable on both RNA-ISH and nucleocapsid IHC. In the lung, viral RNA and nucleocapsid protein is predominantly extracellular and within hyaline membranes in some cases, while intracellular locations are more prominent in others. The intracellular virus is detected within pneumocytes, bronchial epithelial cells, and possibly immune cells. The ISH platform is more specific, easier to analyze and the interpretation is associated with the improved interobserver agreement. ISH, IHC, and qRT-PCR failed to detect the virus in the heart, liver, and kidney.
新型冠状病毒病（COVID-19）是由一种新发现的冠状病毒，即严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）引起的。尽管电子显微镜可以观察到 SARS-CoV-2，但人们越来越需要广泛适用的技术来观察组织标本中的病毒成分。福尔马林固定石蜡包埋（FFPE）组织中的病毒蛋白和 RNA 可分别通过免疫组织化学（IHC）和原位杂交（ISH）检测到。在此，我们评估了 ISH 检测 SARS-CoV-2 的染色性能，并比较了针对 SARS-CoV 核衣壳蛋白的 IHC，同时将这些结果与金标准，即组织实时定量聚合酶链反应（qRT-PCR）进行比较。我们评估了 8 例 COVID-19 尸检的 FFPE 切片，包括 19 例肺部和 39 例肺外样本，包括心脏、肝脏、肾脏、小肠、皮肤、脂肪组织和骨髓。我们对所有病例进行了 SARS-CoV-2 的 RNA-ISH，对部分病例进行了 SARS-CoV 和 SARS-CoV-2 的 IHC 和 qRT-PCR。COVID-19 大流行前进行的 37 例尸检的肺组织作为阴性对照。4 位观察者对 ISH 和 IHC 切片进行了评估，以记录共识意见。还对选定的 ISH 和 IHC 切片由 4 位独立观察者进行了评估。在 IHC 和 ISH 平台上均发现了 SARS-CoV-2 的证据。在死后的肺中，检测到的病毒蛋白和 RNA 通常是细胞外的，主要位于弥漫性肺泡损伤患者的透明膜内。在个别病例中，肺样本中可检测到的病毒量存在区域性差异。细胞内病毒 RNA 和蛋白定位于肺泡细胞和免疫细胞。在 19 例（68%）COVID-19 患者的肺部 FFPE 块中，有 13 例（68%）通过 RNA-ISH 检测到病毒 RNA。在 9 例（88%）COVID-19 患者的肺部 FFPE 块中，有 8 例（88%）通过 IHC 检测到病毒蛋白，但在 5 例中，该染色被解释为可疑。从对照组来看，所有 37 例患者的 FFPE 块均未检测到 SARS-CoV-2 RNA-ISH，而 13 例中有 5 例呈阳性。与单个组织块的 qRT-PCR 相比，ISH 的灵敏度和特异性分别为 86.7%和 100%，而 IHC 分别为 85.7%和 53.3%。ISH 的观察者间变异性为中度至几乎完美，而 IHC 的观察者间变异性为轻度至中度。COVID-19 阳性病例的所有肺外样本通过 ISH、IHC 和 qRT-PCR 均未检测到 SARS-CoV-2。RNA-ISH 和核衣壳 IHC 均可检测到 SARS-CoV-2。在肺部，病毒 RNA 和核衣壳蛋白主要是细胞外的，在某些情况下位于透明膜内，而在其他情况下则更突出于细胞内。细胞内病毒存在于肺泡细胞、支气管上皮细胞中，可能还存在于免疫细胞中。ISH 平台更具特异性，更易于分析，且其解释与观察者间一致性的提高有关。ISH、IHC 和 qRT-PCR 未能在心脏、肝脏和肾脏中检测到病毒。
