A Buffered Alcohol-Based Fixative for Histomorphologic and Molecular Applications

Abstract

Formalin-fixed paraffin-embedded (FFPE) tissue is the predominant preparation for diagnostic histopathological evaluation and increasingly the biospecimen on which molecular diagnostics are performed. However, formalin is carcinogenic and results in cross-linking of proteins and nicking and alterations of nucleic acids. Alternative fixatives, including 70% ethanol, improved biomolecular integrity; however, they have yet to replace neutral-buffered formalin (NBF). Herein, we describe the phosphate-buffered ethanol 70% (BE70) fixative. The histomorphology of BE70-fixed tissue is very similar to that of NBF; however, it is a non-cross-linking fixative and lacks the carcinogenic profile of formaldehyde-based fixatives. RNA isolated from tissue fixed in BE70 was of substantially higher quality and quantity than that was recovered from formalin-fixed tissue. Furthermore, the BE70 fixative showed excellent RNA and DNA integrity compared with that of NBF fixative based on real-time polymerase chain reaction analysis results. Immunohistochemical staining was similar for the antigen tested. In conclusion, BE70 is a non-cross-linking fixative that is superior to NBF and 70% ethanol with reference to biomolecule recovery and quality from paraffin-embedded tissue. Additional studies to compare the histomorphologic and immunohistochemical performance and utility in a clinical setting are required.

Keywords: RNA integrity; alcohol; fixation; formalin; histomorphology; paraffin embedded; real-time RT-PCR; tissue.

Figure 1.

Histomorphological assessment of different fixatives on mouse kidney. Mouse kidney tissue section stained with hematoxylin and eosin (H&E) after buffered ethanol 70% (BE70) (A), neutral-buffered formalin (NBF) (B), 70% ethanol (E) (C), 70% ethanol + 0.5× PBS (EP) (D), 70% ethanol + 1% glycerol + 0.5× PBS (EGP) (E), or 70% ethanol + 0.5% glacial acetic acid + 0.5× PBS (EAP) (F) fixation. Scale bar, 20 µm. Abbreviation: PBS, phosphate-buffered saline.

Figure 2.

Comparison between buffered ethanol 70% (BE70) and neutral-buffered formalin (NBF) fixation. Tissue sections were stained with hematoxylin and eosin (H&E) after BE70 or NBF fixation. H&E images of liver (A), pancreas (B), spleen (C), duodenum (D), heart (E), lung (F), brain (G), and skin (H). Scale bar, 50 µm.

Figure 3.

Immunohistochemical staining between buffered ethanol 70% (BE70) and neutral-buffered formalin (NBF) fixation. Immunohistochemistry staining with anti-Ki-67 in the mouse spleen (A and B), anti-aquaporin 1 (AQP1) in the mouse kidney (C and D), anti-CD31 in the mouse brain (E and F), rabbit immunoglobulin G (IgG) isotype control (G), and no primary antibody control (H). Scale bar, 50 µm.

Figure 4.

Protein quantity and quality of buffered ethanol 70% (BE70) fixative. (A) Amount of protein extracted from each condition was measured using the bicinchoninic acid (BCA) Protein Assay Kit. The protein extraction yield was expressed as the mean of three replicated samples (mean ± SD). (B) Protein integrity of different fixative solutions was assessed by western blotting. Proteins extracted from different fixative solutions were separated by 4% to 12% reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted to nitrocellulose membrane, and probed with anti-aquaporin 1 (AQP1; 1:1000). (C) Western blotting by anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. Relative GAPDH signal of each entity was normalized to neutral-buffered formalin (NBF). Abbreviations: E, 70% ethanol.

Figure 5.

RNA quantity and quality from tissue fixed in different fixative solutions. (A) RNA quantity extracted from each specimen was measured by the Nanodrop spectrophotometer. RNA extraction yield is expressed as the mean of three replicates (mean ± standard deviation [SD]). (B) Representative data are presented as an electropherogram. To compare the quality of RNA extracted under each condition, we used an electropherogram to overlay all seven different conditions. (C) RNA integrity is presented as paraffin-embedded RNA metric (PERM). Abbreviations: E, 70% ethanol; BE70, buffered ethanol 70%; NBF, neutral-buffered formalin; FF, fresh frozen mouse kidney (positive control).

Figure 6.

RNA integrity profile of RNA samples derived from experimental mouse kidney tissues. Gene expression profile of mouse kidney tissue was assessed by multiplex reverse transcription–polymerase reaction (RT-PCR). Tumor necrosis factor signaling genes profiling was measured using the Multiplex Polymerase Chain Reaction (MPCR) kit (Maxim Biotech). One microliter of the PCR reaction was run on the Agilent 2100 Bioanalyzer using the DNA 1000 chip. Representative data are presented as a gel-like image (A) and an electropherogram (B). The fixative conditions of each sample are indicated on the gel-like image above each lane: 1, E (70% ethanol); 2, EP (70% ethanol + 0.5× PBS); 3, EGP (70% ethanol + 1% glycerol + 0.5× PBS); 4, EAP (70% ethanol + 0.5% glacial acetic acid + 0.5× PBS); 5, BE70 (buffered ethanol 70%); 6, NBF (neutral buffered formalin); F, fresh frozen mouse kidney; P, positive control; N, negative control (water). To compare the quality of PCR amplicon, we used an electropherogram to overlay the BE70 and NBF fixative conditions. Each symbol represents the difference between BE70 and NBF. (C) The mean cycle threshold (Ct) value of the toll-like receptor-4 (TLR-4) was determined in kidney tissue under different fixative conditions. Gene expression levels are shown as a box plots. The values are the average quantitative real-time RT-PCR cycle threshold numbers (Ct-values). (D) The mean Ct-value of the housekeeping gene (18S rRNA) was determined in kidney tissue under different fixative conditions. Bars indicate standard deviation. Fresh frozen (FF) mouse kidney is used as a positive control. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7.

DNA quantity and quality from tissue fixed in different fixative solutions. (A) Quantity of RNA extracted from each specimen was measured with the Nanodrop spectrophotometer. DNA extraction yield is expressed as the mean of three replicates (mean ± SD). (B) DNA quality was assessed using the BioScore™ Screening and Amplification kit. We amplified approximately 10 µg DNA using a 100-ng DNA template from buffered ethanol 70% (BE70). (C) Mean cycle threshold (Ct) values of the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) were determined in kidney under different fixative conditions. Gene expression levels are shown as box plots. The values are the mean polymerase chain reaction Ct-values. Bars indicate standard deviations. Abbreviations: E, 70% ethanol; NBF, neutral-buffered formalin; FF, fresh frozen mouse kidney (positive control). *p < 0.05, **p < 0.01, ***p < 0.001.