Lessons Learned During Three Decades of Operations of Two Prospective Bioresources.

Prospective collection is a model through which biospecimens are provided for research. Using this model, biospecimens are collected based on real-time requests from the research community instead of being collected based on the prediction of future requests. We describe the lessons learned by two bioresources that have operated successfully using a prospective model for over three decades. Our goal is to improve other bioresources by increasing utilization of biospecimens that honor consented donors who provide biospecimens to the research community; this provides strong evidence of stewardship of the public trust. The operation of these sites requires flexibility, close communication, and cooperation with the investigator in developing a standard operating procedure (protocol) based on the investigator's needs described in their initial request. If practicable, almost any preparation can be provided, including fresh (nonfrozen) biospecimens and tissue blots. A quality management system includes rigorous quality control of the specific biospecimens provided to an investigator. The informatics approach focuses on the investigator, the investigator's request, and the biospecimens collected for the investigator; the informatics focus of classic biobanks is on the biospecimens collected to match expected future requests. These lessons have been incorporated into our current operations. Standard investigator agreements (e.g., indemnification and no unapproved biospecimen transfers to third parties) replace material transfer agreements. We have operated under the prospective model of the Cooperative Human Tissue Network (CHTN), which has been successful and has provided over 1.2 million biospecimens since it began in 1987. These tissues have supported over 4300 peer-reviewed scientific articles. Since 2012, about 1000 publications have indicated support by CHTN tissues; their average citation rate is 31 with an H factor of 61. Also, during this period, 114 patents cited the CHTN. We also describe disadvantages of prospective bioresources (e.g., inadequate distribution of rare tissues, biospecimens not immediately available, and delayed clinical outcomes).

Protein extraction from methanol fixed paraffin embedded tissue blocks: A new possibility using cell blocks.

BACKGROUND: Methanol fixed and paraffin embedded (MFPE) cellblocks are an essential cytology preparation. However, MFPE cellblocks often contain limited material and their relatively small size has caused them to be overlooked in biomarker discovery. Advances in the field of molecular biotechnology have made it possible to extract proteins from formalin fixed and paraffin embedded (FFPE) tissue blocks. In contrast, there are no established methods for extracting proteins from MFPE cellblocks. We investigated commonly available CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) buffer, as well as two commercially available Qiagen(®) kits and compared their effectiveness on MFPE tissue for protein yields. MATERIALS AND METHODS: MFPE blocks were made by Cellient™ automated system using human tissue specimens from normal and malignant specimens collected in ThinPrep™ Vials. Protein was extracted from Cellient-methanol fixed and paraffin embedded blocks with CHAPS buffer method as well as FFPE and Mammalian Qiagen(®) kits. RESULTS: Comparison of protein yields demonstrated the effectiveness of various protein extraction methods on MFPE cellblocks. CONCLUSION: In the current era of minimally invasive techniques to obtain minimal amount of tissue for diagnostic and prognostic purposes, the use of commercial and lab made buffer on low weight MFPE scrapings obtained by Cellient(®) processor opens new possibilities for protein biomarker research.

Archived formalin-fixed paraffin-embedded (FFPE) blocks: A valuable underexploited resource for extraction of DNA, RNA, and protein.

Formalin-fixed paraffin-embedded (FFPE) material presents a readily available resource in the study of various biomarkers. There has been interest in whether the storage period has significant effect on the extracted macromolecules. Thus, in this study, we investigated if the storage period had an effect on the quantity/quality of the extracted nucleic acids and proteins. We systematically examined the quality/quantity of genomic DNA, total RNA, and total protein in the FFPE blocks of malignant tumors of lung, thyroid, and salivary gland that had been stored over several years. We show that there is no significant difference between macromolecules extracted from blocks stored over 11-12 years, 5-7 years, or 1-2 years in comparison to the current year blocks.

Effect of thaw temperatures in reducing enzyme activity in human thyroid tissues.

An identified impediment to the advancement of science in the field of proteomics is the deterioration of proteins in tissue upon removal of the tissue from its natural state. To reduce this degradation, human tissues are frozen and stored in either liquid nitrogen or -80°C environments. It is believed that frozen tissue in ultralow temperatures preserves proteins against enzyme degradation. Various molecular, biophysical, and biochemical analytical studies require that frozen tissues be thawed before being used for analyses. Depending on downstream analyses, tissues are thawed at different temperatures (37°C, room temperature or 4°C). However, there is very little literature that describes the effects of different thaw temperatures on enzymatic inactivation in tissue lysates. We investigated the effects of preprocessing variable thaw temperature on postprocessed lysates using tyrosine phosphatase and phosphatase and tensin homolog activity assays. In our study we examined the thawing of frozen human thyroid tissues at the traditional temperatures of 4°C (on ice), 37°C (in an oven), and the novel temperature of 95°C (using Stabilizor T1™). The tissue lysates were processed without the addition of enzymatic inhibitors. Our results showed that in benign, malignant, and diseased tissues, high temperature thawing is effective in reducing enzymatic activity. In normal tissue, the reduction is dependent on individual enzymes. This suggests that if tissue lysates are to be obtained from frozen tissues without the addition of inhibitors, high temperature thawing might have marked improvement in downstream non-enzymatic analyses of diseased and neoplastic tissues.