Comparison of histomorphology and DNA preservation produced by fixatives in the veterinary diagnostic laboratory setting

Abstract

Histopathology is the most useful tool for diagnosis of a number of diseases, especially cancer. To be effective, histopathology requires that tissues be fixed prior to processing. Formalin is currently the most common histologic fixative, offering many advantages: it is cheap, readily available, and pathologists are routinely trained to examine tissues fixed in formalin. However, formalin fixation substantially degrades tissue DNA, hindering subsequent use in diagnostics and research. We therefore evaluated three alternative fixatives, TissueTek® Xpress® Molecular Fixative, modified methacarn, and PAXgene®, all of which have been proposed as formalin alternatives, to determine their suitability for routine use in a veterinary diagnostic laboratory. This was accomplished by examining the histomorphology of sections produced from fixed tissues as well as the ability to amplify fragments from extracted DNA. Tissues were sampled from two dogs and four cats, fixed for 24–48 h, and processed routinely. While all fixatives produced acceptable histomorphology, formalin had significantly better morphologic characteristics than the other three fixatives. Alternative fixatives generally had better DNA amplification than formalin, although results varied somewhat depending on the tissue examined. While no fixative is yet ready to replace formalin, the alternative fixatives examined may be useful as adjuncts to formalin in diagnostic practices.

KINFix – A formalin-free non-commercial fixative optimized for histological, immunohistochemical and molecular analyses of neurosurgical tissue specimens

“…Furthermore, alcohol-based fixatives may provide the appropriate material (comparably to fresh or frozen tissue [ 4 , 36 , 37 ]) for biomarker analysis with novel methods requiring high quality (e.g., whole genome amplification [ 25 , 34 ]) or quantity (e.g., methylation arrays [ 25 , 38 ]) of DNA. Although some methods are compatible with FFPE samples (e.g., aCGH or SNP array), a better template, offered by the ethanol-based fixative [ 6 , 10 , 14 , 26 , 27 , 28 , 39 , 40 ], is always beneficial. The formalin-induced artificial mutations might also be mistaken for genuine findings and have several times been incorporated into databases [ 3 , 36 , 41 ].…”

Peer Review #1 of "Determining the utility of veterinary tissue archives for retrospective DNA analysis (v0.2)"

“…While alternative fixatives could be useful in increasing the utility of samples for future molecular diagnostics, these have proved impractical as a routine replacement for formalin in the veterinary diagnostic setting (Craft, Conway & Dark, 2014). Therefore, this study is important to determine how useful a typical veterinary archive is for retrospective studies.…”

“…Each DNA sample was used as a template for PCR amplification, using the primer pairs listed in Table 1 to generate multiple amplicon lengths for the retinol-binding protein 3 (IRBP3) as previously described (Craft, Conway & Dark, 2014) as well as for the succinate dehydrogenase subunit D gene. Briefly, samples were amplified using PCR Master Mix (EmeraldAmp ® GT PCR Master Mix) on a Veriti Thermocycler (Applied Biosystems, Life Technologies Corp., Burlington, Ontario) with the following conditions: 96˚C for 3 min, followed by 35 cycles of 96˚C for 1 min, 60˚C for 1 min, then 72˚C for 1 min.…”

“…A number of previous studies have used these for molecular biology research with varying degrees of success, including polymerase chain reaction (PCR), DNA microarrays, and quantitative reverse-transcriptase PCR (qPCR) (Lewis et al, 2001). Formalin fixation has a profound effect on nucleic acid quality, with a number of parameters altering the ability to recover usable nucleic acids (Craft, Conway & Dark, 2014). In one study using freshly-sampled tissues, formalin-fixed tissues had a mean PCR amplicon size of 300bp, although some samples were able to generate 750bp amplicons (Craft, Conway & Dark, 2014).…”

“…Formalin fixation has a profound effect on nucleic acid quality, with a number of parameters altering the ability to recover usable nucleic acids (Craft, Conway & Dark, 2014). In one study using freshly-sampled tissues, formalin-fixed tissues had a mean PCR amplicon size of 300bp, although some samples were able to generate 750bp amplicons (Craft, Conway & Dark, 2014). Although the degradation of DNA is severe in some samples, altering the size of the target amplicon can help compensate for degradation (Iwamoto et al, 1996, , Lewis et al, 2001, , Taga et al, 2013, von Ahlfen et al, 2007.…”