Highlights

A retrospective cohort of liver biopsies collected from over 20 healthcare centres has been assembled.

The cohort is characterized on the basis of collagen staining used for liver fibrosis assessment.

A computational pipeline for the quantification of collagen from liver histology slides has been developed and applied to the described cohorts.

Uncertainty estimation is evaluated as a method to build trust in deep-learning based collagen predictions.

The introduction of digital pathology has revolutionised the way in which histology-based measurements can support large, multi-centre studies. How-ever, pooling data from various centres often reveals significant differences in specimen quality, particularly regarding histological staining protocols. These variations present challenges in reliably quantifying features from stained tissue sections using image analysis. In this study, we investigate the statistical variation of measuring fibrosis across a liver cohort composed of four individual studies from 20 clinical sites across Europe and North America.

In a first step, we apply colour consistency measurements to analyse staining variability across this diverse cohort. Subsequently, a learnt segmentation model is used to quantify the collagen proportionate area (CPA) and employed uncertainty mapping to evaluate the quality of the segmentations. Our analysis highlights a lack of standardisation in PicroSirius Red (PSR) staining practices, revealing significant variability in staining protocols across institutions. The deconvolution of the staining of the digitised slides identified the different numbers and types of counterstains used, leading to potentially incomparable results.

Our analysis highlights the need for standardised staining protocols to ensure reliable collagen quantification in liver biopsies. The tools and methodologies presented here can be applied to perform slide colour quality control in digital pathology studies, thus enhancing the comparability and reproducibility of fibrosis assessment in the liver and other tissues.

The authors have declared no competing interest.

No external funding has been received.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study involved secondary analysis of anonymised digital histopathology slides from previously approved clinical studies. No new patient data were collected specifically for this analysis. Ethical approvals for the original studies were obtained as follows: - CALM study: Approved by the West Midlands - Black Country Research Ethics Committee (REC reference: 14/WM/0010; IRAS reference: 140543), United Kingdom. - HepaT1ca study: Approved by the South East Scotland Research Ethics Committee 02 (REC reference: 17/SS/0049; IRAS reference: 223180), United Kingdom. - PREV study: Approved by the Institutional Review Board at Brooke Army Medical Center, United States. The study was sponsored by The Geneva Foundation and is registered on ClinicalTrials.gov (Identifier: NCT03142867). - UK-AIH study: Approved by the London - Camden & Kings Cross Research Ethics Committee (REC reference: 14/LO/0303; IRAS reference: 144806), United Kingdom. In all cases, ethical approval covered the use of liver biopsy samples and associated clinical data for research purposes.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Data produced in the present study are available upon reasonable request to the authors.