Accuracy of whole slide image based image analysis is adversely affected by preanalytical factors such as stained tissue slide and paraffin block age.

Background: Personalized medicine and accurate quantification of tumor and biomarker expression have become the cornerstone of cancer diagnostics. This requires Quality Control (QC) of research tissue samples to confirm adequate targeted tumor tissue sampling. Digitalization of stained tissue slides offer a precious way to archive, preserve, and retrieve necessary information when needed. This study is aimed to assess the most significant pre-analytic and analytic factors that might contribute to the efficacy of obtaining accurate whole slide images (WSIs) interpretation. Various studies are needed to identifysuch factors to allow for appropriate AI application and adequate tumor area/percentage quantification. Methods: Hematoxylene and Eosine (H&E) satined WSIs collected from tissue specimens provided by the Cooperative Human Tissue Network (CHTN) Midwestern Division (CHTNMWD) were analyzed. Tissue specimens were processed, fixed, stained, and scanned contemporaneously (within 1 month). Two cohorts of malignant, colorectal cancer, 20X WSI (ScanscopeXT, Leica Biosystems, Illinois), were assembled. The study identified a "recent cohort" that included 76 WSIs created on 2018 or later. "Aged cohort" included 73 WSIs from specimens procured in the period of (2012-2014). Twenty recent WSIs of adenocarcinoma cases were used to construct WSIs analysis algorithms (VIS, Visiopharm A/S, Denmark) using machine learning to produce morphometric maps and calculate tissue and tumor areas. Results: Algorithmic analysis of 69 WSIs from rescanned aged slides vs. that of contemporaneous WSIs concluded 18 (28%) similar finding in tumor areas (within 10%), 56 (82%) had identicaltissue areas, and 54 (79%) had similar tumor percentages. Conclusion: WSIs of aged H&E slides and stained paraffin block re-cuts produce different tumor quantification compared to those of original scanned sslides most likely due to pre-analytical factors. The difference in tumor area detected between original and rescanned WSIs trended upward in the period between 2012 and 2014. Less tumor area was detected as the slides age. Recut and H&E-stained tissues from stored paraffin blocks may detect more tumor due to excess eosinophilia. These results highlights the value of documenting archives of H&E WSIs collected at the procurement time. Such images provide a superior archive over glass slides and Formalin-Fixed Paraffin-Embedded (FFPE) blocks and contribute betterg to WSIs analysis application.