A Comparison Between Conventional Formalin and Novel PAXgene Tissue Fixation: the European Spidia Morphology Ring Trials Experience

Abstract

Introduction and Aim: Molecular pathology is an emerging discipline requiring high quality tissue samples which allow simultaneous molecular and histopathological analysis. Within the European FP7 project (grant agreement no. 222916) \u201cStandardisation and improvement of generic pre-analytical tools and procedures for in-vitro diagnostics\u201d (SPIDIA) different morphology ring trials were implemented. The aim of these ring trials was to evaluate the quality of histomorphology and suitability of PAXgene-fixed and paraffin-embedded (PFPE) samples for routine diagnostics, with special emphasis on the tumor grading, in comparison to the current state-of-the-art technique for routine morphological diagnostics formaldehyde-fixed and paraffin-embedded (FFPE) samples in breast and colon cancer tissues. Methods: Each cancer tissue sample was divided into two mirrored samples for PFPE and FFPE. Haematoxylin and eosin and Periodic-Acid-Schiff stained sections were scanned and evaluated in a blinded, randomized ring trial by pathologists from Europe and the US using virtual microscopy. Each participant evaluated only one of the two virtual slides obtained from each case according to a randomized scheme implying the subdivision of both, cases and participants, in two groups. Links to the randomized virtual slides were distributed by email to the participants together with the study protocol, the electronic evaluation form and all instructions and timeline for the ring trial implementation. The reproducibility between fixation methods (PFPE vs. FFPE) was assessed by computing the modal category within each method for each case (Modal Scenario, MS). By starting from these values, the jackknifed estimate of the weighted kappa statistic (Kwj)1 was computed together with the relative 95% Confidence Interval (CI)2. In addition the inter-observer reproducibility was assessed by computing the Kwj and the kappa category-specific statistics (Kcs) and their weighted average (Cohen\u2019s kappa statistic, Kc) were estimated by jointly considering all participant\u2019s data1,3. Results: Tissue samples of 18 breast and 15 colon cancer cases were evaluated by 23 and 20 pathologists, respectively. By using the MS, a high and quite satisfactory level of reproducibility between PFPE and FFPE samples in evaluating the histological grading score was observed in breast (kwj=0.92, 95% CI: 0.76-1.00) and colon (kwj=0.73, 95% CI: 0.41-0.94) cancer, respectively. Moreover the inter-observer reproducibility for grading was not completely satisfactory in both ring trials, with G2 as the most critical category, but showed a similar performance level in each of the two considered groups of pathologists. Conclusions: Our findings suggest that the methodological approach we developed is suitable for evaluating the reproducibility between alternative fixation methods in the ring trial setting implemented here and that histomorphology is excellently preserved in PFPE tissues. References: 1. Fleiss JL. 2nd Ed. New York: Wiley and Sons 1981. 2. Corletto V, Verderio P, Giardini R et al. Anal Cell Pathol. 1998;16:83-93. 3. Holman CD. Am J Epidemiol. 1984;120:154-60. 4. Landis JR, Koch GG. Biometrics. 1977;33:159-74