Alternative tissue fixation for combined histopathological and molecular analysis in a clinically representative setting

Abstract

Formalin is the principal tissue fixative used worldwide for clinical and research purposes. Despite optimal preservation of morphology, its preservation of DNA and RNA is poor. As clinical diagnostics increasingly incorporates molecular-based analysis, the requirement for maintaining nucleic acid quality is of increasing importance. Here we assess an alternative non-formalin-based tissue fixation method, PAXgene Tissue system, with the aim of better preserving nucleic acids, while maintaining the quality of the tissue to be used for vital existing diagnostic techniques. In this study, these criteria are assessed in a clinically representative setting. In total, 203 paired PAXgene Tissue and formalin-fixed samples were obtained. Blind-scored haematoxylin and eosin (H&E) sections showed comparable and acceptable staining. Immunohistochemistry (IHC) staining was suboptimal using existing protocols but improved with minor method adjustment and optimisation. Quality of DNA and RNA was significantly improved by PAXgene tissue fixation [RIN 2.8 versus 3.8 (p < 0.01), DIN 5.68 versus 6.77 (p < 0.001)], which translated into improved performance on qPCR assay. These results demonstrate the potential of PAXgene Tissue to be used routinely in place of formalin, maintaining adequate histological staining and significantly improving the preservation of biological molecules in the genomic era.

Keywords: DNA; FFPE; Fixative; Formalin; PAXgene; RNA.

Fig. 1

Representative H&E-stained sections from formalin- and PAXgene-fixed tissues. Images presented are from a range of tissue types and demonstrate the hypereosinophilia observed in the PAXgene-fixed tissues. Scoring of the quality of these sections can be found in Table 2. Scale bars represent 100 µm

Fig. 2

IHC staining. Representative images of IHC staining performed on paired FFPE and PFPE samples using MLH1, MSH6, MSH2, PMS2, CD3, CD20, TIF-1, CK7, CK5, p53 and HMWCK (34BE12) antibodies. Staining was performed using pre-optimised conditions for FFPE tissues. Scale bars represent 50 µm

Fig. 3

Representative images of FISH signals from EGFR probe. EGFR represented in red and centrometric enumeration probe (CEP) in green. Left PFPE, right FFPE

Fig. 4

DNA and RNA assessment of quality. DNA and RNA obtained from paired formalin- or PAXgene-fixed samples were analysed using a number of methods to assess quality. No differences were observed by tissue type. Following exclusion of unquantifiable samples, the numbers of paired cases analysed were as follows: DNA yield (n = 56), DNA purity (n = 66), DIN (n = 44), DNA fragment length (n = 58), dCt (n = 39), RNA yield (n = 55), RIN (n = 55), RNA purity (n = 63), GUSB dCt (n = 19). Data represent mean score + SD. A paired Student’s t-test was performed to determine significance (*p < 0.05, **p < 0.01, ***p < 0.001). Details on how values were explained can be found in “Materials and methods”

Fig. 5

NGS quality metrics. NGS was performed on n = 4 triplets of DNA samples extracted from PFPE, FFPE or fresh frozen (FF) tissue. Data represent mean score + SD. Statistical significance was determined by Friedman test (*p < 0.05)