High Dimensional Imaging of Postmortem Human Covid-19 Autopsy Samples to Evaluate Pathophysiology and Immunology of SARS-CoV-2

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

As part of a multi-investigator program to study post-mortem samples from COVID-19 patients, we employed highly multiplex imaging (IBEX) to identify and examine the location and state of a wide array of immune cell types (innate and adaptive) in a variety of tissues sites, and to also identify and analyze the parenchymal cells that comprise each of these tissues. We also used antibodies specific for SARS-Cov2 to examine the location of viral antigen and viral replication within these tissues. Because of the harsh fixation conditions necessary to ensure sterility of SARS-Cov2 infected tissue samples, we spent significant time and effort testing various antigen retrieval methods to enable a large set of markers to be properly stained with available antibodies. We have now developed several panels for immune and parenchymal cells in various tissues preserved using 4% PFA for 72 hrs (the standard for material collected by NIH collaborators) and for tissue fixed in formalin for prolonged periods (the method used in Germany). Our experience with post-mortem material from the NIH study indicated significant problems with samples from individuals with long post-mortem intervals prior to tissue removal and fixation during autopsy, with a very limited number of sample showing reasonable tissue preservation. This problem was especially severe for secondary lymphoid tissues such as spleen and lymph node, making a careful assessment of the impact of infection in these critical immune sites impossible. To date, appropriate samples from individuals shorter post-mortem intervals have not been available. In addition, questions have arisen about whether some of the individual actually had a SARS-Cov2 infection prior to death, further complicating the study. Much of the groups efforts have turned to other assays for tissue and infection status and we are awaiting the results of these other assessments before identifying selected tissue samples worth the effort and cost of highly multiplex imaging analysis. Collaborators in Germany have been able to obtain tissue at an earlier time after death and their published data showed generally good tissue architecture, with pathology that seemed related to the infection more so than to post-mortem artifact. These tissues have been very harshly fixed but we have been able to develop antigen retrieval methods that allow nearly all of the extensive list of antibody probes in our multiplex imaging panels to be used effectively with these samples. After some delay in the shipment of samples from Europe, we had to wait for various clearances and collaboration documents to be completed before initiating the planned study and the staining, imaging, and analysis stage of this project has only recently begun, with tissue samples from not only COVID-19 patients, but some from lethal influenza for comparison being examined. The latter parallels our animals studies in AI001292-03. Studies from our European and Boston collaborators suggest that lymph nodes show a vascular proliferation that distorts canonical organization, along with a change in the proportion of lymphocyte subtypes in the node, with possible implications for production of a robust antibody response. We have designed our staining panels to be able to look carefully at this type of pathology, along with the broader features of the tissue to be revealed by the other probes we are using.