Coronavirus disease 2019 (COVID-19) is the highly contagious and potentially debilitating disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is associated with increased levels of pulmonary thrombosis (1–3). The COVID-19 pandemic has resulted in massive global suffering (4) including job losses, travel restrictions (5), mental and physical illness (6, 7), and mortality (7). Several treatments for COVID-19 have been given emergency use authorization by the FDA, but these treatments are less than optimal. Remdesivir, for example, improves time to recovery in hospitalized patients with moderate disease (NIAID ACTT-1 trial), but has not been shown to improve survival (8, 9). A major limitation of Remdesivir is its requirement for intra-venous delivery. Another example, Dexamethasone, reduces mortality in persons with severe COVID-19 (RECOVERY trial) (10), but corticosteroid treatment in patients with SARS-CoV, MERS, and influenza can result in increased mortality, incidence of secondary infections, and impaired viral clearance. Convalescent plasma is another treatment granted emergency use authorization by the FDA for hospitalized COVID-19 patients, but a mortality benefit of this treatment has not yet been shown (11). The FDA has also granted emergency use authorization to monoclonal antibodies, for the treatment of non-hospitalized mild to moderate COVID-19. These treatments have importantly shown a reduction in hospitalization in those at high risk for disease progression (BLAZE-1 trial) (12, 13). However, monoclonal antibodies are not approved for hospitalized or critically ill individuals requiring mechanical ventilation The REMAP-CAP and RECOVERY trials are also assessing the application of Tocilizumab and Sarilumab, which are interleukin 6 receptor antagonists, in critically ill COVID-19 patients (14). Another immunomodulator, Baricitinib, which inhibits januse kinase 1 and 2, has been shown to improve survival in COVID-19 patients receiving Baricitinib plus Remdesivir versus Remdesivir alone (15). Regarding the use of Ivermectin, current evidence is inconclusive (16, 17), but additional randomized controlled trials are underway.

While clinical studies of COVID-19 treatments crucial are ongoing, including tests of anti-coagulant therapies (18, 19), an improved understanding of the pathogenesis and pathological characteristics of COVID-19 may lead to the development of novel treatment options. To this end, several studies have used established or emerging histological techniques to characterize the pathological features of lung injury in COVID-19 patients, including widespread pulmonary thrombosis. Such techniques can also be useful for disease diagnosis or retrospective tissue analysis. For example, a recent study has demonstrated the potential for high-resolution cleared-tissue microscopy in the 3-dimensional (3D) analysis of thrombosis in COVID-19 lungs (20). Although this proof-of-concept study was limited to a single patient, such emerging technological advances enable for the analysis of thrombosis in COVID-19 lungs in cubic millimeter volumes and provide novel insights into COVID-19 disease pathogenesis. While the organ-specific pathological characteristics of COVID-19 have been reviewed elsewhere (21–26), this mini-review describes the 2D and 3D histological studies of COVID-19 lungs, with a focus on thrombosis and vascular abnormalities. To this end, we searched PubMed and Google Scholar for articles that included the following terms: “coagulation or thrombosis or thrombus” and “COVID-19 or COVID19” and “histological or histology” and “lung or pulmonary”.

While both 2D and 3D histological techniques remain temporally limited by the analysis of one timepoint and spatially limited by the amount of tissue that can be imaged, such techniques can provide valuable insights into COVID-19 disease pathology. Often, 2D techniques are quicker and cheaper to perform compared with 3D techniques, but the 3D techniques yield more information and provide improved visualization of disease features. In the manuscript by Li et al. (20), for instance, 3D renderings from a 7.8 mm × 5.9 mm × 0.9 mm formalin-fixed block of lung tissue from the left peripheral upper lobe were generated, which enabled the visualization of lung features from millimeter dimension vessels to single cell nuclei. However, 3D histology from serial sectioning and whole slide scanning with image reconstruction is expensive and time consuming. In future, it is likely that established 2D histological techniques will be complemented with emerging 3D virtual histology. While the histological studies of COVID-19 patients are often limited in sample size, the key findings from studies of COVID-19 lung that have used 2D and 3D techniques appear to be congruent so far (Table 1). However, it remains to be seen what (if any) additional information will be uniquely generated from each type of histological technique. Furthermore, it is important to note that the findings from histological studies are observational and are usually limited to the demonstration of associations between pathological features and death in diseased patients.

Analysis of 2D lung tissue sections remains the gold-standard for histological examination, but 2D tissue sectioning can create cutting artifacts that confuse interpretation. Pathological interpretation may be improved in future studies by 3D analysis of tissue microstructure (49). Emerging histological techniques have used fluorescence contrast to simulate H&E staining (50, 51) and optical tissue clearing and high throughput sectioning microscopes to acquire 3D virtual histology of different tissue types (49, 52–54). Such 3D virtual imaging techniques could be improved by: (i) increasing resolution with higher aperture waveguide optics, enhanced pixel detector technology, and improvements in holographic reconstruction; (ii) including more than two zoom levels; and (iii) developing cell-specific markers coupled to radiocontrast agents (28). In both 2D and 3D histology, the consistency and reliability of novel and emerging histological techniques should be an area of focus in future studies. It would also be beneficial if 2D and 3D histological techniques could be upscaled and carried out in multiple different patient sub-groups and at various stages of disease severity. Furthermore, it could be useful if histological imaging studies in future could be performed in an automated manner, perhaps eventually in living patients. Methodological factors pertaining to both 2D and 3D histology should be carefully considered when designing and carrying out histological studies of COVID-19 lungs (55, 56). Nevertheless, histological studies will likely continue to improve understanding of COVID-19 pathogeneses and progression, which will add to understanding of COVID-19 from other pre-clinical and clinical observations, and hopefully lead to the development of novel therapies and treatment strategies.

Novel treatments for COVID-19 patients and an improved understanding of COVID-19 pathogenesis may arise from in-depth analyses of the histological features of COVID-19 lungs, which include enhanced levels of thrombosis. Such analyses will likely be facilitated by novel histological techniques for high-resolution and large-volume imaging of lung structure and pulmonary thrombosis at the microvascular level.

CE and AS wrote and edited the manuscript. All authors contributed to the article and approved the submitted version.

CE is supported in part by an American Heart Association Career Development Award (19CDA34500000).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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