Novel tissue preservative and tissue fixative for comparative pathology and animal research

doi:10.1016/j.jeas.2006.09.002

Journal of Experimental Animal Science

Volume 43, Issue 4, 26 February 2007, Pages 271-281

https://doi.org/10.1016/j.jeas.2006.09.002 Get rights and content

Abstract

Aim

To determine whether universal molecular fixative (UMFIX), a novel human tissue fixative, could also be used as an animal tissue fixative for histomorphology and a preservative of RNA at subtropical temperatures. Cat, dog, mouse, pigeon, rabbit and rat tissue, as well as ant, beetle, earthworm and lizard were collected. Tissue was fixed in UMFIX for up to a week at room or ambient temperatures, processed and paraffin embedded. Histomorphology and RNA quality were evaluated and compared to formalin-fixed and fresh frozen tissue.

Results

Animal tissue fixed in UMFIX at room temperature or high ambient temperature (30–34 °C) provides similar histomorphology. Comparable to other alcohol-based fixatives, UMFIX produces a histomorphology similar but not identical to formalin. All minor histopathological differences, however, in no way interfere with establishing the correct diagnostic conclusion. Whereas RNA extracted from animal tissue fixed in formalin was completely degraded, tissue fixed up to 1 week in UMFIX at high ambient temperatures rendered completely intact RNA.

Conclusions

UMFIX represents a new class of preservative/fixative that protects RNA and provides acceptable histomorphology. Tissue fixation and preservation of RNA can be achieved at high ambient temperatures. This allows collection of animal tissue in field research without the need for immediate freezing of tissue and also provides histomorphology comparable to formalin-fixed tissue. Furthermore, as RNA is also preserved in UMFIX preserved paraffin-embedded tissue, if amount of tissue is limited the same tissue that is used to determine histomorphology can be used to extract RNA.

Introduction

Procurement of animal and human tissue is of great importance for biomedical research. Histomorphological evaluation of formalin-fixed, paraffin-embedded tissue stained with hematoxylin and eosin represents common practice in pathology. At the same time, the tissue provides a source of DNA, RNA and proteins (macromolecules) necessary for molecular analysis. Unfortunately, formalin, the most commonly used tissue fixative, does not protect macromolecules (Srinivasan et al., 2002). It degrades RNA and modifies DNA and proteins. Therefore, macromolecules are most commonly extracted from fresh or frozen tissue. However, immediate extraction of macromolecules, particularly during field studies, is impossible in most situations and the storage and transportation of frozen tissue represents a logistical challenge. Furthermore, fresh or frozen tissue has limited value for the assessment of histomorphology or for the performance of ancillary tests. As formalin fixation is of limited value in preserving macromolecules and is also not environmentally friendly, alternative tissue fixatives were tried but with partial success. Recently we have developed a novel tissue fixative and preservative (Vincek et al., 2003). The universal molecular fixative (UMFIX), a mixture of methanol (serving as coagulative fixative) and polyethylene glycol (PEG) (used to decrease excessive nuclear shrinkage), was shown to preserve macromolecules and histomorphology in human tissue at room temperature. It is currently introduced to human clinical practice and is replacing formalin. In this study we tested whether UMFIX can also be used for histomorphological evaluation of animal tissue and whether it preserves RNA at high ambient temperatures so that it could be used as a tissue preservative of macromolecules for animal field studies.

Section snippets

Tissue collection

Mouse, cat, dog, pigeon, rabbit and rat tissue was obtained as residual animal tissue through the approved program for the use of residual animal tissue by the Animal Research Committee at University of Miami. Ant (Camponotus floriadanus), beetle (Phylophaga), earthworm (Lumbricus terrestris) and lizard (Anolis sagrei sagrei) were collected during a field trip.

Tissue fixation

Tissue was fixed in neutral-buffered 10% formalin or UMFIX (Sakura Finetek USA, Inc.) for a period of time ranging from 24 h to 7 days.

Results

To assess histomorphology of animal tissue fixed in our novel fixative we have fixed all major organs of cat, dog, mouse, pigeon, rabbit and rat for 24 h or 7 days in UMFIX at room temperature or environmental temperature (30–34 °C). We have also fixed the same tissue in formalin for 24 h or 7 days at room temperature for comparison. We found that UMFIX preserved tissue, fixed for 1 day or 1 week, shows similar histomorphology regardless of the type of the tissue processor used. Fixation in UMFIX

Discussion

The time-honored processes of formalin fixation and routine overnight tissue processing form the cornerstones of human and veterinary pathology but are of limited value in preserving intact RNA, protein and even DNA. It has, with minor modifications, been used for over 100 years (Blum, 1893). From the beginning, histologists pointed to various problems with formalin. It causes various tissue artifacts and some regarded formalin unsuitable for visualization of nuclear structures, microscopy of

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Citation Excerpt :
Unfortunately, RNAlater alone resulted in uneven immunohistochemical staining and preserved noticeably less of the finer structural details compared to formalin (Paska et al., 2004). This could be improved by post-fixing with formalin, but formalin is known to damage RNA (Gugic et al., 2007; Masuda et al., 1999). Alcohol-based molecular fixatives have been introduced that aim to combine the best attributes of formalin and freezing (Delfour et al., 2006; Ergin et al., 2010; Stanta et al., 2006; van Hemel and Suurmeijer, 2013; Viertler et al., 2012; Vincek et al., 2003).
Formalin-fixed tissue has been a mainstay of clinical pathology laboratories, but formalin alters many biomolecules, including nucleic acids and proteins. Meanwhile, frozen tissues contain better-preserved biomolecules, but tissue morphology is affected, limiting their diagnostic utility. Molecular fixatives promise to bridge this gap by simultaneously preserving morphology and biomolecules, enabling clinical diagnosis and molecular analyses on the same specimen. While previous reports have broadly evaluated the use of molecular fixative in various human tissues, we present here the first detailed assessment of the applicability of molecular fixative to both routine histopathological diagnosis and molecular analysis of cervical tissues. Ten specimens excised via the loop electrosurgical excision procedure, which removes conical tissue samples from the cervix, were cut into alternating pieces preserved in either formalin or molecular fixative. Cervical specimens preserved in molecular fixative were easily interpretable, despite featuring more eosinophilic cytoplasm and more recognizable chromatin texture than formalin-fixed specimens. Immunohistochemical staining patterns of p16 and Ki-67 were similar between fixatives, although Ki-67 staining was stronger in the molecular fixative specimens. The RNA of molecular fixative specimens from seven cases representing various dysplasia grades was assessed for utility in expression microarray analysis. Cluster analysis and scatter plots of duplicate samples suggest that data of sufficient quality can be obtained from as little as 50 ng of RNA from molecular fixative samples. Taken together, our results show that molecular fixative may be a more versatile substitute for formalin, simultaneously preserving tissue morphology for clinical diagnosis and biomolecules for immunohistochemistry and gene expression analysis.
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We assessed the effect of DNA extraction and sample preservation methods on the DNA yield and quality obtained from earthworm (Aporrectodea caliginosa Savigny) gut samples and on the results obtained by bacterial and fungal automated ribosomal intergenic spacer analysis (ARISA) of DNA extracts. Methods based on a hexadecyltrimethylammonium bromide dithiotreitol (CTAB-DTT) extraction buffer yielded more favourable results than those based on a sodium dodecyl sulphate (SDS) buffer. For both of these buffers, incorporation of a bead-beating during the lysis step increased the ARISA-derived bacterial ribotype numbers and diversity estimates, as determined for gut wall samples (P < 0.01). Although spectrophotometric analysis indicated that DNA extracted by the CTAB-DTT and SDS-based methods were of comparable quality (P ≥ 0.05), the former method yielded > 1.5 times more DNA from both gut contents and gut walls of earthworms than the latter method (both incorporating the bead beating step) (P < 0.01). ARISA analysis detected more reproducible ribotypes and more microbial diversity in DNA extracted by the CTAB-DTT- as compared to the SDS-based method (P < 0.01). Significant difference between bacterial communities of gut contents and gut walls were detected within DNA extracted by the CTAB-DTT (but not by the SDS-based) method (Global R = 0.76, P < 0.001, analysis of similarity). Using the CTAB-DTT-based method, we showed that earthworm preservation in ethanol yielded higher quality DNA from gut contents than preservation in either chloroform or liquid N, as determined by spectrophotometry, PCR inhibition analysis and bacterial and fungal ARISA (P < 0.05). Bacterial or fungal communities in the gut contents of fresh and ethanol-preserved earthworms were more similar and were significantly different from those of earthworms preserved in chloroform or liquid N (Global R = 0.79 and 0.83 for bacteria and fungi, respectively; P < 0.001, analysis of similarity). We propose that ethanol preservation and the CTAB-DTT-based DNA extraction method described herein are also suitable for the analysis of gut-associated microbiota in other soil and sediment feeding invertebrates.
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View full text

Novel tissue preservative and tissue fixative for comparative pathology and animal research

Abstract

Aim

Results

Conclusions

Introduction

Section snippets

Tissue collection

Tissue fixation

Results

Discussion

Path. Res. Pract.

Kidney Int.

Mod. Pathol.

Am. J. Pathol.

J. Mol. Diagn.

Am. J. Pathol.

Methods

Pathol. Res. Pract.

Am. J. Pathol.

Am. J. Pathol.

Lab. Invest.

Lab. Invest.

Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications

Proteomics

American Academy of Clinical Toxicology practice guidelines on the treatment of methanol poisoning

J. Toxicol. Clin. Toxicol.

Der formaldehyd als hartungsmittel

Z. Wiss. Microsc.

Establishment of the formalin-free surgical pathology laboratory. Utility of an alcohol-base fixative

Arch. Pathol. Lab. Med.