Abstract
Simple SummarySpecimen collection and preservation can be challenging, especially when suboptimal conditions occur. A relevant amount of time could be required from sampling to the tissue analysis, and therefore a high-quality conservation technique is vitally important for diagnostic purposes. The aim of this study was the identification of a reliable and economic method for tissue preservation to be used in complex “in-field” situations, suitable for both histological and molecular analysis. Punch biopsies were collected from six cattle livers. Comparison among methods of preservation using RNAlater, silica beads, or under-vacuum was carried out using different times and temperatures. Three days were assumed as a maximum time interval from sampling to laboratory and 4 °C and 24 °C chosen as references for refrigeration temperature and room temperature, respectively. Histological and biomolecular analyses were performed. RNAlater and silica beads poorly preserved the histological parameters evaluated; conversely, vacuum-sealed samples showed a good grade of preservation for 48 h. DNA quality was acceptable for each sample. RNA integrity showed promising results for samples preserved with silica beads.A high quality of samples is crucial for the success of the analysis and diagnostic purposes, and therefore the right method of conservation is vitally important for an optimal preservation of tissues. Indeed, the time to deliver the sample to the laboratory could be remarkably long, especially under suboptimal conditions, and the use of specific fixatives or cold storage may not be possible. Moreover, the portability and cost of storage equipment, their toxicity, and their ease of use play a central role when choosing the correct preservation method. The aim of this study was the identification of a reliable and economic method for tissue preservation, to be used in “in-field” sampling, suitable for both histological and molecular analysis. Punch biopsies were collected from six cattle livers. Comparisons among methods of preservation using RNAlater, silica beads, and under-vacuum was carried out. These methods were tested through considering different times and temperatures, assuming three days as a maximum time interval from sampling to laboratory and choosing 4 °C and 24 °C as references for refrigeration temperature and room temperature, respectively. Histologically, the integrity of nucleus, cytoplasm, preservation of liver structure, and easiness of recognition of inflammatory infiltrate were evaluated. The integrity of the extracted DNA and RNA was evaluated through PCR and by means of an automated electrophoresis station, respectively. RNAlater and silica beads poorly preserved the histological parameters evaluated, independently from the temperature. Conversely, the vacuum-sealed samples showed a good grade of preservation until 48 h. DNA quality was acceptable for each sample. RNA integrity showed promising results only for samples preserved with silica beads.
Highlights
Samples’ conservation, from collection to laboratory, is critical for the success of the analyses and diagnostic purposes
All the samples in silica beads and RNAlater showed a statistically significant worsening when compared with the control samples at any time
The nuclei of the samples stored in silica beads at 24 ◦ C showed a statistically significant worsening of the scores over time, precisely between T4 and T24 (p < 0.01)
Summary
Samples’ conservation, from collection to laboratory, is critical for the success of the analyses and diagnostic purposes. Sometimes, when cold storage may not be possible, for instance, sample preservation can be challenging. This is true in forensic science and in “in-field” critical situations, such as mass disasters, when successful DNA sampling is fundamental for the victim’s identification. Multiple factors should be analyzed to reach a full understanding of this issue, especially when evaluating tissue preservation protocols in sub-optimal conditions. The efficiency of the method and toxicity, portability of storage equipment or materials, cost, and ease of use play a central role in guiding the correct choice [1]. Fixatives are fundamental in order to obtain reliable slides for histological analyses
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