Multiplexed detection of SARS-CoV-2 genomic and subgenomic RNA using in situ hybridization.

The widespread Coronavirus Disease 2019 (COVID-19) is caused by infection with the novel coronavirus SARS-CoV-2. Currently, we have a limited toolset available for visualizing SARS-CoV-2 in cells and tissues, particularly in tissues from patients who died from COVID-19. Generally, single-molecule RNA FISH techniques have shown mixed results in formalin fixed paraffin embedded tissues such as those preserved from human autopsies. Here, we present a platform for preparing autopsy tissue for visualizing SARS-CoV-2 RNA using RNA FISH with amplification by hybridization chain reaction (HCR). We developed probe sets that target different regions of SARS-CoV-2 (including ORF1a and N) as well as probe sets that specifically target SARS-CoV-2 subgenomic mRNAs. We validated these probe sets in cell culture and tissues (lung, lymph node, and placenta) from infected patients. Using this technology, we observe distinct subcellular localization patterns of the ORF1a and N regions, with the ORF1a concentrated around the nucleus and the N showing a diffuse distribution across the cytoplasm. In human lung tissue, we performed multiplexed RNA FISH HCR for SARS-CoV-2 and cell-type specific marker genes. We found viral RNA in cells containing the alveolar type 2 (AT2) cell marker gene (SFTPC) and the alveolar macrophage marker gene (MARCO), but did not identify viral RNA in cells containing the alveolar type 1 (AT1) cell marker gene (AGER). Moreover, we observed distinct subcellular localization patterns of viral RNA in AT2 cells and alveolar macrophages, consistent with phagocytosis of infected cells. In sum, we demonstrate the use of RNA FISH HCR for visualizing different RNA species from SARS-CoV-2 in cell lines and FFPE autopsy specimens. Furthermore, we multiplex this assay with probes for cellular genes to determine what cell-types are infected within the lung. We anticipate that this platform could be broadly useful for studying SARS-CoV-2 pathology in tissues as well as extended for other applications including investigating the viral life cycle, viral diagnostics, and drug screening.

The Implementation of an Introductory Surgical Pathology Didactic Series to Transition First Year Residents and Facilitate Upper Level Resident Teaching.

The increasing complexity of the practice of pathology and health care in general requires that pathology residents acquire a vast number of skills during their training. This has been reflected by the broad range of skills addressed in the Accreditation Council for Graduate Medical Education (ACGME) milestones. In order to address some of these milestones, our residency program instituted an introductory didactic series in surgical pathology that focused on 2 objectives. First, the didactics provided basic grossing and histology training to first year residents transitioning from medical school. Second, the sessions allowed upper level residents to refine their teaching and communication skills at the microscope and therefore served as an important career development tool. Surveys of both first year residents and the upper level residents that led these sessions confirm the utility of these didactics and the use of upper level residents to teach junior trainees. In addition, these sessions led to a dramatic increase in RISE scores among first year trainees. An introductory series with upper level residents leading slide sessions could easily be replicated at other institutions and provide similar benefits.