The Differing Pathophysiologies That Underlie COVID-19-Associated Perniosis and Thrombotic Retiform Purpura

A Case Series

C.M. Magro; J.J. Mulvey; J. Laurence; S. Sanders; A.N. Crowson; M. Grossman; J. Harp; G. Nuovo


The British Journal of Dermatology. 2021;184(1):141-150. 

In This Article

Materials and Methods

Samples of COVID-19-associated perniosis were sectioned at a thickness of 5 μm and stained on positively charged glass slides stored at 4 °C within 3 days after sectioning. Deparaffinization, rehydration and antigen retrieval were performed on the BOND-III Leica automated slide stainer (Leica Biosystems, Newcastle upon Tyne, UK) following the methodologies that have been previously described.[14,15] Specimens were incubated and variably stained with CD3 (LN10), CD4 (4B12), CD8 (4B11), CD7 (LP15), CD11c (5D11), CD20, myeloperoxidase, and granzyme (11F1) from Leica Biosystems; lysozyme (Rabbit Poly) from Agilent (Santa Clara, CA, USA); and myxovirus virus resistance protein A (MXA, C-1) from Santa Cruz Biotechnology (Santa Cruz, CA, USA) at room temperature, followed by visualization with the Leica BOND detection kit for 20 min at room temperature.[16] The specimens were then counterstained with haematoxylin and coverslipped.[14,15] For the cases of thrombotic retiform purpura, immunohistochemical staining of C3d (ready-to-use polyclonal antibody; Cell Marque, Rocklin, CA, USA), C4d (1: 50, monoclonal antibody; Quidel, San Diego, CA, USA), mannan-binding lectin serine peptidase (MASP)-2 (Sigma, St Louis, MO, USA; HPA029313) and C5b-9 (1: 250, clone aE11; Dako, Glostrup, Denmark) was accomplished using the BOND-III autostainer.

Further viral studies were performed on the cases of COVID-19-associated perniosis and COVID-19 thrombotic retiform purpura and three cases of perniosis unrelated to COVID-19. Positive and negative controls were used with every run, including autopsy lung tissue from patients with COVID-19 pneumonia and normal lung tissue obtained prior to 2019, respectively. Antibodies specific for the envelope protein (1: 300) and spike protein (1: 7000) of COVID-19 (ProSci, Poway, CA, USA) were optimized with the lung tissues. Optimal pretreatment conditions included ethylenediaminetetraacetic acid antigen retrieval solution (pH 9·0) for 30 min. The analyses were done on the automated Leica BOND platform, with the modification that the Enzo Life Sciences (Farmingdale, CA, USA) peroxidase antimouse/rabbit conjugate (catalogue #ADI-950-113-0100) was used in place of the equivalent Leica conjugate as this reduced background.

Detection of SARS-CoV-2 RNA was done using the ACD RNAscope probe (catalogue #848561-C3; ACD, Newark, CA, USA) as per a previously published protocol in which only the viral RNA probe is changed. The SARS-CoV-2 probe targets the target region nt21631–23303 derived from the genomic sequence of the virus (NC_045512·2). In brief, pretreatment in the ACD RNA retrieval solution and protease digestion is followed by overnight hybridization at 37 °C and detection using the ACD 2·5 High Definition diaminobenzidine detection kit.