3d virtual pathohistology of lung tissue from COVID-19 patients based on phase contrast x-ray tomography

  1. Marina Eckermann
  2. Jasper Frohn
  3. Marius Reichardt
  4. Markus Osterhoff
  5. Michael Sprung
  6. Fabian Westermeier
  7. Alexandar Tzankov
  8. Mark Kühnel
  9. Danny Jonigk  Is a corresponding author
  10. Tim Salditt  Is a corresponding author
  1. Georg-August-Universität Göttingen, Germany
  2. Deutsches Elektronen-Synchrotron (DESY), Germany
  3. Universitätsspital Basel, Switzerland
  4. Medizinische Hochschule Hannover (MHH), Germany

Abstract

We present a three-dimensional (3d) approach for virtual histology and histopathology based on multi-scale phase contrast x-ray tomography, and use this to investigate the parenchymal architecture of unstained lung tissue from patients who succumbed to Covid-19. Based on this first proof-of-concept study, we propose multi-scale phase contrast x-ray tomography as a tool to unravel the pathophysiology of Covid-19, extending conventional histology by a third dimension and allowing for full quantification of tissue remodeling. By combining parallel and cone beam geometry, autopsy samples with a maximum cross section of 4mm are scanned and reconstructed at a resolution and image quality which allows for the segmentation of individual cells. Using the zoom capability of the cone beam geometry, regions-of-interest are reconstructed with a minimum voxel size of 167 nm. We exemplify the capability of this approach by 3d visualisation of the DAD with its prominent hyaline membrane formation, by mapping the 3d distribution and density of lymphocytes infiltrating the tissue, and by providing histograms of characteristic distances from tissue interior to the closest air compartment.

Data availability

All datasets were uploaded to zenodo: 10.5281/zenodo.3892637

The following data sets were generated

Article and author information

Author details

  1. Marina Eckermann

    Institute for x-ray physics, Georg-August-Universität Göttingen, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Jasper Frohn

    Institute for x-ray physics, Georg-August-Universität Göttingen, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Marius Reichardt

    Institute for x-ray physics, Georg-August-Universität Göttingen, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Markus Osterhoff

    Institute for x-ray physics, Georg-August-Universität Göttingen, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Michael Sprung

    Petra III, P10, Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Fabian Westermeier

    Petra III, P10, Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Alexandar Tzankov

    Institut für Medizinische Genetik und Pathologie, Universitätsspital Basel, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  8. Mark Kühnel

    Pathology, Medizinische Hochschule Hannover (MHH), Hannover, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Danny Jonigk

    Pathology, Medizinische Hochschule Hannover (MHH), Hannover, Germany
    For correspondence
    Jonigk.Danny@mh-hannover.de
    Competing interests
    The authors declare that no competing interests exist.
  10. Tim Salditt

    Institute for x-ray physics, Georg-August-Universität Göttingen, Göttingen, Germany
    For correspondence
    tsaldit@gwdg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4636-0813

Funding

Bundesministerium für Bildung und Forschung (05K19MG2)

  • Tim Salditt

H2020 European Research Council (771883)

  • Danny Jonigk

Max-Planck School (Matter to Life)

  • Marius Reichardt
  • Tim Salditt

Deutsche Forschungsgemeinschaft (-EXC 2067/1-390729940)

  • Tim Salditt

Botnar Research Center of Child Health (BRCCH)

  • Alexandar Tzankov

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States

Ethics

Human subjects: The study was approved by and conducted according to requirements of the ethics committees at the Hannover Medical School (vote Nr. 9022 BO K 2020).

Version history

  1. Received: June 25, 2020
  2. Accepted: August 17, 2020
  3. Accepted Manuscript published: August 20, 2020 (version 1)
  4. Accepted Manuscript updated: August 25, 2020 (version 2)
  5. Version of Record published: September 4, 2020 (version 3)

Copyright

© 2020, Eckermann et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Marina Eckermann
  2. Jasper Frohn
  3. Marius Reichardt
  4. Markus Osterhoff
  5. Michael Sprung
  6. Fabian Westermeier
  7. Alexandar Tzankov
  8. Mark Kühnel
  9. Danny Jonigk
  10. Tim Salditt
(2020)
3d virtual pathohistology of lung tissue from COVID-19 patients based on phase contrast x-ray tomography
eLife 9:e60408.
https://doi.org/10.7554/eLife.60408

Share this article

https://doi.org/10.7554/eLife.60408

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    Background:

    Prinflammatory extracellular chromatin from neutrophil extracellular traps (NETs) and other cellular sources is found in COVID-19 patients and may promote pathology. We determined whether pulmonary administration of the endonuclease dornase alfa reduced systemic inflammation by clearing extracellular chromatin.

    Methods:

    Eligible patients were randomized (3:1) to the best available care including dexamethasone (R-BAC) or to BAC with twice-daily nebulized dornase alfa (R-BAC + DA) for seven days or until discharge. A 2:1 ratio of matched contemporary controls (CC-BAC) provided additional comparators. The primary endpoint was the improvement in C-reactive protein (CRP) over time, analyzed using a repeated-measures mixed model, adjusted for baseline factors.

    Results:

    We recruited 39 evaluable participants: 30 randomized to dornase alfa (R-BAC +DA), 9 randomized to BAC (R-BAC), and included 60 CC-BAC participants. Dornase alfa was well tolerated and reduced CRP by 33% compared to the combined BAC groups (T-BAC). Least squares (LS) mean post-dexamethasone CRP fell from 101.9 mg/L to 23.23 mg/L in R-BAC +DA participants versus a 99.5 mg/L to 34.82 mg/L reduction in the T-BAC group at 7 days; p=0.01. The anti-inflammatory effect of dornase alfa was further confirmed with subgroup and sensitivity analyses on randomised participants only, mitigating potential biases associated with the use of CC-BAC participants. Dornase alfa increased live discharge rates by 63% (HR 1.63, 95% CI 1.01–2.61, p=0.03), increased lymphocyte counts (LS mean: 1.08 vs 0.87, p=0.02) and reduced circulating cf-DNA and the coagulopathy marker D-dimer (LS mean: 570.78 vs 1656.96 μg/mL, p=0.004).

    Conclusions:

    Dornase alfa reduces pathogenic inflammation in COVID-19 pneumonia, demonstrating the benefit of cost-effective therapies that target extracellular chromatin.

    Funding:

    LifeArc, Breathing Matters, The Francis Crick Institute (CRUK, Medical Research Council, Wellcome Trust).

    Clinical trial number:

    NCT04359654.