Vacuum Sealing and Cooling as Methods to Preserve Surgical Specimens

Kristensen, Thomas Kielsgaard; Engvad, Birte; Nielsen, Ole F.; Pless, Torsten Kjærulf; Walter, Steen; Bak, Martin

doi:10.1097/pai.0b013e318214e523

Cited by 15 publications

(7 citation statements)

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“…From our experience on nucleic acid preservation [7], [9] it seems that it is the prompt cooling that principally influences preservation. This has also been confirmed by independent observations [27] that show that storage at 4°C preserved tissues to a higher degree than storage at room temperature, independently of whether the tissue was subjected to vacuum sealing or not. With this study we prove that VPAC maintains cell viability even after 70 hours, however, as expected, for longer VPAC times, the percentage of cell death increased.…”

Section: Discussionsupporting

confidence: 70%

A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study

et al. 2013

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Primary cultures represent an invaluable tool to set up functional experimental conditions; however, creation of tissue cultures from solid tumors is troublesome and often unproductive. Several features can affect the success rate of primary cultures, including technical issues from pre-analytical procedures employed in surgical theaters and pathology laboratories. We have recently introduced a new method of collection, transfer, and preservation of surgical specimens that requires immediate vacuum sealing of excised specimens at surgical theaters, followed by time-controlled transferring at 4°C to the pathology laboratory. Here we investigate the feasibility and performance of short-term primary cell cultures derived from vacuum packed and cooled (VPAC) preserved tissues. Tissue fragments were sampled from 52 surgical specimens of tumors larger than 2 cm for which surgical and VPAC times (the latter corresponding to cold ischemia time) were recorded. Cell viability was determined by trypan blue dye-exclusion assay and hematoxylin and eosin and immunohistochemical stainings were performed to appreciate morphological and immunophenotypical features of cultured cells. Cell viability showed a range of 84–100% in 44 out of 52 (85%) VPAC preserved tissues. Length of both surgical and VPAC times affected cell viability: the critical surgical time was set around 1 hour and 30 minutes, while cells preserved a good viability when kept for about 24 hours of vacuum at 4°C. Cells were maintained in culture for at least three passages. Immunocytochemistry confirmed the phenotype of distinct populations, that is, expression of cytokeratins in epithelioid cells and of vimentin in spindle cells. Our results suggest that VPAC preserved tissues may represent a reliable source for creation of primary cell cultures and that a careful monitoring of surgical and cold ischemia times fosters a good performance of primary tissue cultures.

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Section: Discussionsupporting

confidence: 70%

A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study

et al. 2013

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“…More extensive exposure of tissues to air, which occurs during conventional autopsies, could also have a negative influence on tissue quality. It is known that vacuum sealing of tissue specimens has a beneficial effect on RNA preservation [33] .…”

Section: Discussionmentioning

confidence: 99%

Post-Mortem Tissue Biopsies Obtained at Minimally Invasive Autopsy: An RNA-Quality Analysis

et al. 2014

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IntroductionBereaved relatives often refuse to give consent for post-mortem investigation of deceased cancer patients, mainly because of the mutilation due to conventional autopsy (CA). Minimally invasive autopsy (MIA) may be a more acceptable alternative and, if implemented in clinical practice, creates an opportunity to more often obtain post-mortem tissue samples of (recurred) primary tumors and metastases for molecular research. As a measure for tissue quality for molecular studies, we hereby present a feasibility study, comparing the RNA quality of MIA and CA samples, and fresh frozen samples as reference.Materials and methodsTissue samples of heart, liver and kidney were prospectively collected from 24 MIAs followed by CA, and compared to corresponding archival fresh frozen tissue. After RNA isolation and RT-qPCR, RNA integrity numbers (RIN) and GAPDH expression (six amplicon sizes ranging from 71 to 530 base pairs) were measured. RIN values and GAPDH Cq values were analyzed and compared between all sample groups and post-mortem intervals (PMI).ResultsRIN values in MIA samples were significantly higher than those in CA samples. GAPDH was expressed significantly higher in MIA samples than in CA samples and 530 bp PCR products could be measured in all cases. GAPDH expression was significantly lower in samples with PMI >15 hours. As expected, the samples of the fresh frozen reference standard performed best in all analyses.ConclusionMIA samples showed better RNA quality than CA samples, probably due to shorter PMI. Both had lower RNA quality and expression levels than fresh frozen tissue, however, remaining GAPDH RNA was still sufficiently intact. Therefore, other highly expressed genes are most likely also detectable. Gene array analysis should be performed to gain insight into the quality of entire post-mortem genomes. Reducing PMI will further improve the feasibility of demanding molecular research on post-mortem tissues, this is most likely more feasible with MIA than CA.

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“…Each step in this process is critical: this shed light on the role that is played by an organized team and a correct triage evaluation of the sample in the Pathology Room. The VPAC system, already proven to preserve specimens integrity, was adopted as gold standard procedure in order to obtain viable tissues [ 6 – 8 ]. However, while in breast cancer tissues RNA, DNA and proteins may be optimally preserved for >24h [ 7 , 13 ], we here showed that lung tissues are much more sensible to this time.…”

Section: Discussionmentioning

confidence: 99%

“…The specimens were preserved and transferred to the pathology lab using the vacuum packing and cooling (VPAC) procedure [ 6 , 7 ]. Briefly, in the OR, explanted surgical specimens were immediately placed into beta-ray sterilized plastic bags and vacuum-sealed using the TissueSAFE machine (Mod.…”

Section: Methodsmentioning

confidence: 99%

The Influence of Tissue Ischemia Time on RNA Integrity and Patient-Derived Xenografts (PDX) Engraftment Rate in a Non-Small Cell Lung Cancer (NSCLC) Biobank

et al. 2016

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IntroductionBio-repositories are invaluable resources to implement translational cancer research and clinical programs. They represent one of the most powerful tools for biomolecular studies of clinically annotated cohorts, but high quality samples are required to generate reliable molecular readouts and functional studies. The objective of our study was to define the impact of cancer tissue ischemia time on RNA and DNA quality, and for the generation of Patient-Derived Xenografts (PDXs).MethodsOne-hundred thirty-five lung cancer specimens were selected among our Institutional BioBank samples. Associations between different warm (surgical) and cold (ex-vivo) ischemia time ranges and RNA quality or PDXs engraftment rates were assessed. RNA quality was determined by RNA integrity number (RINs) values. Fresh viable tissue fragments were implanted subcutaneously in NSG mice and serially transplanted.ResultsRNAs with a RIN>7 were detected in 51% of the sample (70/135), with values of RIN significantly lower (OR 0.08, P = 0.01) in samples preserved for more than 3 hours before cryopreservation. Higher quality DNA samples had a concomitant high RIN. Sixty-three primary tumors (41 adenocarcinoma) were implanted with an overall engraftment rate of 33%. Both prolonged warm (>2 hours) and ex-vivo ischemia time (>10 hours) were associated to a lower engraftment rate (OR 0.09 P = 0.01 and OR 0.04 P = 0.008, respectively).ConclusionRNA quality and PDXs engraftment rate were adversely affected by prolonged ischemia times. Proper tissue collection and processing reduce failure rate. Overall, NSCLC BioBanking represents an innovative modality, which can be successfully executed in routine clinical settings, when stringent Standard Operating Procedures are adopted.

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Vacuum Sealing and Cooling as Methods to Preserve Surgical Specimens

Cited by 15 publications

References 0 publications

A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study

A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study

Post-Mortem Tissue Biopsies Obtained at Minimally Invasive Autopsy: An RNA-Quality Analysis

The Influence of Tissue Ischemia Time on RNA Integrity and Patient-Derived Xenografts (PDX) Engraftment Rate in a Non-Small Cell Lung Cancer (NSCLC) Biobank

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