Acid-free glyoxal as a substitute of formalin for structural and molecular preservation in tissue samples

<div><p>Tissue fixation in phosphate buffered formalin (PBF) remains the standard procedure in histopathology, since it results in an optimal structural, antigenic and molecular preservation that justifies the pivotal role presently played by diagnoses on PBF-fixed tissues in precision medicine. However, toxicity of formaldehyde causes an environmental concern and may demand substitution of this reagent. Having observed that the reported drawbacks of commercially available glyoxal substitutes of PBF (Prefer, Glyo-fix, Histo-Fix, Histo-CHOICE, and Safe-Fix II) are likely related to their acidity, we have devised a neutral fixative, obtained by removing acids from the dialdehyde glyoxal with an ion-exchange resin. The resulting glyoxal acid-free (GAF) fixative has been tested in a cohort of 30 specimens including colon (N = 25) and stomach (N = 5) cancers. Our results show that GAF fixation produces a tissue and cellular preservation similar to that produced by PBF. Comparable immuno-histochemical and molecular (DNA and RNA) analytical data were obtained. We observed a significant enrichment of longer DNA fragment size in GAF-fixed compared to PBF-fixed samples. Adoption of GAF as a non-toxic histological fixative of choice would require a process of validation, but the present data suggest that it represents a reliable candidate.</p></div

Representative sample pair showing a better DNA preservation in GAF-fixed <i>versus</i> PBF-fixed tissues.

<p>Representative fragmentation analysis and sequencing results from different methods of two DNA specimens, GAF- and PBF- fixed, respectively (sample pair 4–12). At Bioanalyzer analysis a higher degree of DNA fragmentation was observed in the specimen fixed in PBF (B) compared to the corresponding parallel GAF-fixed sample (A). In addition, the allele frequency of the <i>KRAS</i> p.G12D mutation was considerably higher in GAF fixed specimen with all the employed molecular techniques: panel C shows results of the GAF-fixed sample (Direct sequencing, Pyrosequencing, Mass Spectrometry, from left to right), whereas panel D shows corresponding results in the PBF fixed sample (Direct sequencing, Pyrosequencing, Mass Spectrometry, from left to right). Of note, by NGS this mutation showed a mutant allele frequency of 32% in the GAF-fixed sample <i>versus</i> 7% in the PBF-fixed sample.</p

Antibodies and antigen retrieval methods used for immunohistochemical reactions.

<p>Ab: antibody; CC1/CC2: cell conditioning Ventana Apparatus; CEA: carcinoembryonic antigen; CGA: chromogranin; CK: cytokeratin; F: formalin fixation; G: glyoxal fixation; SMA: smooth muscle actin; CAD-E: E-cadherin; PHH3: phospho-histone H3; TTF1: thyroid transcription factor 1.</p

Preservation of antigens in tissues fixed either in GAF or in PBF.

<p>(A) CDX2 expression in a GAF-fixed colorectal adenocarcinoma; (B) CDX2 expression in a PBF-fixed sample of the same colorectal adenocarcinoma illustrated in A; (C) HER2 overexpression in a GAF-fixed gastric adenocarcinoma; (D) HER2 overexpression in a PBF-fixed gastric sample of the same adenocarcinoma illustrated in C; (E) proliferation index assessed by Ki67 in a GAF-fixed colorectal adenocarcinoma; (F) proliferation index assessed by Ki67 in a PBF-fixed sample of the same colorectal adenocarcinoma depicted in E.</p

DNA FISH for <i>HER2</i> and CEP17 in parallel tissue samples fixed in GAF and in PBF.

<p>Representative fields of a breast gastric carcinoma where we performed <i>HER2</i>/CEP17 FISH analysis. (A) FISH analysis in a GAF -fixed specimen. (B) The same sample as in A in which sections were treated with a short wash in Tris-HCl 0.01 M at pH 8.5 leading to disappearance of the mild fluorescent background observed in A. (C) Sampling of the same specimen parallel to (A), but fixed in PBF.</p

Preservation of nucleic acids in GAF-fixed tissues.

<p>Bioanalyzer results of DNA samples extracted from 8 GAF-fixed and matching PFB-fixed colorectal carcinoma specimens. (A) Fragment size distribution of the analyzed DNA samples (threshold for longer fragments: 5000 bp). GAF samples were significantly enriched for less fragmented DNA compared with PBF specimens (Chi-square test). (B) Fragmentation analysis example of two DNA specimens. In both cases the difference of the fragment size average between GAF and PBF samples was statistically significant. (C) RNA quality, determined by Agilent Bioanalyzer traces, was satisfactory in both GAF- and PBF-fixed samples.</p

Histology (H&E staining) of tissues fixed either in GAF or in PBF.

<p>A colorectal adenocarcinoma fixed in GAF (A, 20X) or in PBF (B, 20X), where the similarity in preservation of structural and cell components can be appreciated. A detailed examination revealed preservation of erythrocytes and no signs of lysis or loss of staining of eosinophils, as shown here in a GAF-fixed colorectal adenocarcinoma (C, 20x and inset, where an area enriched of eosinophils is captured at a higher magnification).</p