Biobanking and Biospecimen Quality - Inseparable Entities

It is critical to determine the best method for storing tissue. When researchers request tissue from our biobank, we must ensure that we are providing them with the highest quality tissue possible. Therefore, the biobank at Windber Research Institute (WRI) is constantly designing and performing experiments to determine how the tissue collected from the many donors is impacted by pre-analytical variables and how these “uncontrollable” factors could impact the derivatives of the tissue such as DNA, RNA and Protein.

Pre analytical variables may be represented by the following 1) Physiology of the human research participant 2) Specimen collection practices 3) Specimen handling practices before downstream testing (NCI Best practices, 2014; http://biospecimens.cancer.gov/bestpractices/to/bcpsrd.asp).  There are other variables that could potentially affect tissue quality, for example,  it is believed that “minor” aspects such as sample storage vessels (snap cap and screw lid, non-sterile, sterile or RNase/DNase free tubes) could impact tissue quality (Pfaendner 2006). Additionally, tubes can be made out of different plastics which can result in adherence of biomolecules to the vessel walls or leaching of plastic components into the sample (McDonald et al., 2008). This makes it important to ensure that samples are stored in appropriate tubes that will maintain tissue quality over time and at specific storage conditions (Smith 2011). Additionally, the storage temperature of tissue and nucleic acids is very critical (Andreasson et al. 2013). Normally, samples intended for long term storage are stored in liquid nitrogen vapor tanks at -190 degrees Celsius and samples intended for short term storage are stored in -80 degree Celsius freezers (Karisson and Toner 1996). However, not all biobanks have the ability to store in liquid nitrogen vapor and as such utilize mechanical freezers at -80 degree Celsius.  It is essential for biobankers to know which types of samples can be stored at which temperature and for what length of time (Hubel, Spindler and Skubitz 2014). These are the many areas awaiting evidence based data to support biobank storage protocols. Biospecimen Science Research is the umbrella whereby these issues are being addressed and in the future, this science should provide more evidence based data to allow biobanks fully understand all that impacts tissue quality.  Since biobanks have their individual interest in what they store, what these tissue samples are used for and for how long they will be stored, these evidence based studies will have to meet a variety of needs.

Interestingly, studies have shown that the concentration of the RNA stored could have an impact on the integrity of the RNA over time (Olivieri et al. 2014). In their study Olivieri et. al, showed the effects of storing RNA at high concentrations (250ng/µL) and low concentration (25ng/µL). They found that after 8 months of storage, RNA stored at high concentrations preserved its integrity while RNA stored at low concentrations showed significant degradation. Therefore, while low concentrations of RNA may be acceptable for immediate analysis, storage for a few months may not be suitable. These kinds of results are a clear indication of the complex issues that biobanks face when working to maintain tissue integrity and provide quality DNA, RNA and Protein for downstream analysis.

Proper storage of tissue and nucleic acids can greatly affect experiments and their outcome (Yang and Chang 2012). If tissue is not stored correctly, valuable proteins can be degraded (Auer et al. 2014). Additionally, degradation of RNA and/or DNA can affect critical analyses such as gene expression (Shane, Kohlmeyer & Hunter 2010). Biobanks need to be on top of these issues either by designing internal experiments that provide data for the design of evidence based Standard Operating Procedures (SOPs) or keeping abreast with research results in the area of Biospecimen Science as the source of new information/findings that will drive biobanking SOPs. The International Society for Biological and Environmental Repositories (ISBER  http://www.isber.org/) and the Biorepository and Biospecimen Research Branch of the NCI (BBRB  http://biospecimens.cancer.gov/default.asp) remain great resources for biobanks to tap into for current ideas and information that will enable them maintain the desired standards that will drive biobanking for quality tissue. 

References

Andreasson, A., Kiss, NB., Juhlin, CC and Höög, A (2013). Long-Term Storage of Endocrine Tissues at -80^oC Does Not Adversely Affect RNA Quality or Overall Histomorphology. Biopres. & Biobanking, 10.1089/bio.2013.0038

Auer, H., Mobley, J., Ayers, L., Bowen, J., Chuaqui, R., Johnson, L., … Ramirez, N. (2014). The effects of frozen tissue storage conditions on the integrity of RNA and protein. Biotechnic & Histochemistry : Official Publication of the Biological Stain Commission, 89(7), 518–528. http://doi.org/10.3109/10520295.2014.904927

Hubel, A., Spindler, A and Skubitz, A (2014). Storage of Human Biospecimens: Selection of the Optimal Storage Temperature. Biopres. & Biobanking, 10.1089/bio.2013.0084

Karlsson, JOM and Toner, M (1996). Long-term storage of tissues by cryopreservation: critical issues. Biomaterials, 17(3): 243-256.

McDonald, GR et al., (2008). Bioactive contaminants leach from disposable laboratory plasticware.  Science, 322(5903):917

Olivieri, EHR., Franco, LA., Pereira, RG., Mota, LDC., Campos, AH and Carraro, DM (2014). Biobanking Practice: RNA Storage at Low Concentrations Affects Integrity. Biopres. & Biobanking, 10.1089/bio.2013.0056

Pfaendner, R (2006). How will additives shape the future of plastics? Polymer Degrad. Stabil. 91:2249-2256.

Shane, M. L., Kohlmeyer, M., Hunter, T., and Tighe, S. (2010). RT-qPCR Analysis of Degraded RNA using Five Different Pre-Amplification Methods. Journal of Biomolecular Techniques : JBT, 21(3 Suppl), S45.

Smith, C (2011). Selecting the Right Tubes or Plates for Your Research. Life Science Articles, http://www.biocompare.com

Yang, T-H and Chang, P-L (2012). Determination of RNA degradation by capillary electrophoresis with cyan light-emitted diode-influences fluorescence. J. of Chromatogr. A, http://dx.doi.org/10.1016/j.chroma.2012.03.070

THE BIOREPOSITORY AT WINDBER RESEARCH INSTITUTE (WRI) RECEIVES CAP ACCREDITATION

Biobanking is the fastest growing component in translational research and the Windber Research Institute established a biorepository in 2001 with the mission of “Providing high quality biospecimens to enhance the scope and reliability of molecular research.”

We are happy to announce that the Accreditation Committee of the College of American pathologists (CAP) has awarded accreditation to the WRI Biorepository based on the results of an onsite inspection on 4/7/2015.

The Senior Director of the Biorepository, Dr. Stella Somiari, PhD, was advised of this national recognition on 5/14/2015 and congratulated for the excellence of the services being provided. The repository at WRI is one of the 39 CAP accredited biorepositories to date and one of the 2 CAP accredited biorepositories in Pennsylvania!

The accreditation program for biorepositories by CAP was started in 2012 and is the first of such programs offered for biorepositories.  It is a three-year accreditation cycle which includes on-site inspection, desk review, and optional education modules and gap assessment. As part of the on-site inspection, the CAP uses Accreditation Checklists to provide a comprehensive and up-to-date blueprint of quality practices to assist biorepositories in improving their operations and ensure quality. Further, a desk review offers a remote review of a biorepository’s quality plan, certain procedures and select quality and process statistics. The program is designed to ensure that standards are maintained and there is uniformity in all operational procedures which should result in the management and distribution of the highest quality biospecimens.   

The recent CAP accreditation status for the WRI puts the official “stamp” of quality to all its operations and with the added oversight by CAP users of our biorepository resources will have increased confidence in the quality of the biospecimens which we provide to support high impact molecular research.  We are proud to continue to serve the John P. Murtha Cancer Center  of Walter Reed Bethesda in the many capacities that we do, even more so, as an accreditaed biorepository.

About the WRI Biorepository

The biorepository at WRI was established in 2001 to manage specimen procurement, processing, storage, and distribution to the Department of Defense funded Clinical Breast Care Project (CBCP). To date, its activities have expanded to include biobanking activities for the John P. Murtha Cancer Center of Walter Reed, Bethesda, MD.  The repository has established and maintained extensive Quality Management Systems (QMS) to cover all areas of its operations and participates in regular Proficiency/Quality Testing activities in areas that include DNA/RNA Extraction and Quantification, RNA Integrity assessment, and Tissue Histology. The repository has expertise in the establishment and management of multiple collection sites. It has experience working with regional, urban, and community hospitals and has managed collections from international sites and organ procurement organizations. Tissue processing is performed in a variety of formats and new processing protocols are developed as needed. Services provided include:

General

Acquisition, processing and distribution of human biospecimens for research

Current inventory available for research includes serum, plasma, DNA, RNA, PAXgene Blood (for RNA extraction), assorted tissue types (breast, heart, kidney, lung, colon, and lymph node, etc)

Laser Microdissection (LMD) of Tissue for Research

General Histology Services

Long or Short Term Biospecimen Storage in ultra-low or liquid nitrogen freezers

DNA and RNA Extraction

Nucleic Acid Extraction from a variety of biological materials

Nucleic Acid Quality Assessment using a variety of technologies

Consulting Services

Customized Biorepository Design and Setup.

Development of Standard Operating Procedures (SOP) for biorepositories.

Design and Review of Biospecimen Research Study Protocols.

In recent years, Biospecimen Science Research has become an integral part of the biorepository’s operations. Current biospecimen research activities include elucidation of the effects of pre- analytical variables on biospecimen quality, effects of storage time and condition of storage on biospecimen quality, evaluation of new collection methods that will not only conserve scare tissue but also allow the banking of minimal tissue from surgical procedures which would normally have not been available for research and banking.  The repository is also conducting cutting-edge research aimed at developing non-destructive methods for tissue quality assessment. 

My Experience as an Intern at Windber Research Institute by Hunter Bomba

            My passion has always cemented itself in mathematics and science. Before I ventured off to college, I sought to gain experience in a laboratory setting; Windber Research Institute (WRI) offered me just that opportunity. During my internship at WRI, I worked with the Tissue Bank and Cell Biology groups and assisted the Laboratory Manager.

            The Tissue Bank at WRI gathers normal and diseased tissue samples from donors for various research studies in the institute. In the Tissue Bank department, I mastered a variety of skills including: Hematoxylin and Eosin (H&E) staining, cover slipping techniques for histological slides, and the filing of slides. I also processed blood donated to the Clinical Breast Care Project (CBCP). In addition to my time in the Tissue Banking lab, I assisted with data entry of clinical pathological information of the CBCP donors. Working in the Tissue Bank department was unique, as it allowed me not only to see the exciting lab work but also the necessary formalities associated with Human Subject’s Research.

            In addition to working in the Tissue Bank, I spent significant time in the Cell Biology laboratory. The Cell Biology department not only studies the advancement of breast cancer, but also works to identify new biomarkers for breast cancer. Apart from learning basic laboratory skills like adjusting the pH of buffers and solutions, I assisted in maintaining about ten different cell lines during the period of my internship. This included changing the media in the dishes where the cells grew, splitting the cells once they were confluent, and freeze/thawing cell lines when necessary. Additionally, it was my responsibility to check the liquid nitrogen level in the Dewar flask where the cell lines were stored, record temperatures of the shaker and incubator, and clean the incubator every week.

I also had the opportunity to run an acrylamide gel while working with the Cell Biology group. Running my own gel helped me to better understand the associated processes/terminologies such as electrophoresis, transfer, blocking, primary and secondary anti-bodies, and it was a great learning experience. Discussing the results with my coworkers was rewarding as well.

In addition to mammalian cell lines, I worked with DH5alpha bacteria. In the lab, I grew bacteria in flasks using LB Broth and then proceeded to follow a protocol in order to isolate the plasmids. Next, I measured the concentration of the plasmids obtained using the NanoDrop spectrophotometer. The plasmids acquired were then used to transfect mammalian cells. Through transfection, we were then able to study how a gene affected breast cancer progression. Through my time in Cell Biology, I learned a vast amount about cells, bacteria, and lab skills.

            Lastly, my work with the Laboratory Manger encompassed the challenge of updating the Material Safety Data Sheets (MSDS) binders to meet the new Occupational Safety & Health Administration’s (OSHA) Safety Data Sheet (SDS) standards. The SDS documents provide safety and health facts that help keep employees informed and vigilant about the products they are working with. I alphabetized nearly 700 chemical documents, gathered electronic copies of the documents that met the new SDS criteria, and created a new master spreadsheet of the chemicals in the building. While the task was tedious, I learned the importance of keeping accurate records and maintaining safety information in the laboratory.

            Overall, I appreciated my time at the WRI. Working in multiple departments gave me a broader view of a research setting and taught me the value of teamwork, professionalism, and communication. I am thankful for the opportunity and would like to thank WRI and all of the employees, for welcoming me and providing me with such an amazing experience. I am anxious to incorporate the knowledge I gained here into my college work and future career.

Record Keeping……..Never Too Much: A Lesson Learned in Laser Micro Dissection (LMD)

A major research activity that we perform for our scientists is Laser Micro Dissection (LMD). Over the years we have performed LMD on many of our donated tissue samples.  We have microdissected breast tissue and lymph node tissue including both normal and tumor cell types. A good part of this is performed on OCT embedded tissue.  The first step usually is to prepare H&E section(s) and review the section/area of interest with the pathologist.  Often times we draw lines on the coverslip to mark the areas of interest and use as a reference while performing LMD.  This slide will be saved in our file for future reference (see Figure 1).

The next step is cutting tissue sections with the laser microdissecting microscope. In our establishment, we use Leica AS LMD (Leica microsystems). Cut tissue sections are then mounted on PEN foil slides (W. Nuhsbaum Inc.), stained and LMD performed to cut out the areas of interest. 

 Many times, both cell types of interest are represented in the same OCT block (as shown in Figure 1 and 2 above) and often one of the cell types is much smaller compared to the other.  When genomic data for example, is generated from these cells, there could be potential questions raised about cell contamination. In order to understand if this was a possibility or not, an image of the LMD slide and written record of how LMD was performed (that is, both cell types were obtained from the same slide, or each cell type was obtained from independent slides during LMD) will provide clear answers about any potential contamination of cells during the downstream processing into nucleic acids (DNA/RNA). 

Record keeping is therefore very essential here and you can never have too much detail maintained during LMD processing.  We have always recorded how many sections we cut from each block and used for LMD, how the cells were captured within each slide and images of the LMD slide.  For example, if we are studying tumor and adjacent normal tissue from the same donor, we document if we captured both normal and tumor from separate slides or from the same slide.  However, even detailed notes cannot beat the usefulness of an image of the slide. It is beneficial to capture the H&E image of the slide and an image of the LMD slide before and after performing LMD.  If an imaging system is unavailable, another option would be to save the PEN foil slide and the original H&E slide.  Both of these options will tell you exactly what areas of the slide were laser microdissected. This exercise may seem tedious and you may need a lot of storage space in the months or years ahead after performing multiple LMD, but when you are faced with a research scientist who has questions regarding the tissue morphology, and who is trying to eliminate potential cell contamination in the interpretation of research results, you will be glad you kept all of these different pieces of information!

Using the Smallest Amount Possible

The success of translating bench research to the clinic depends on the availability of good quality biological speciemens. As the demand for well characterized biospecimens increases with increased interest in personalized medicine, Biobanks will face the problem of meeting biospecimen needs for research. The observation today is that sample sizes, for example, from surgical procedures, are decreasing. Factors contributing to this include new advances in diagnostic procedures resulting in much earlier detection of tumors, for example, in cancer. Thus the excised tumor is very small and available only for pathological diagnosis. Also, new techniques such as minimally invasive surgical procedures result in less “left over” material for research.  

These advances are great for medical science. However research activities need to continue so that we can improve and advance our knowledge of disease development and progression. Biobanks therefore will have to design new routine collection methods to feed research needs.  Some of these new methods will include tissue touch imprint preparations, tissue scrapings, and bronchial washes/gastric lavage collections.   These could become sources for isolating DNA and RNA for research. Biobanks will have to assess the potential of incorporating these methods into their routine collection protocols. Specific research/technological platforms may benefit from some or all of these methods while others may not. It will be left to Biobanks to determine what new sample collection methods can be incorporated into their collection protocols when biospecimens from surgical procedures is not an option, and what body fluids can be collected and utilized for nucleic acid extraction other than blood/blood products. These collection methods will not jeopardize patient treatment and “scarce and “small” surgical specimens will now provide biospecimens for research.

  

The image here shows a tissue touch imprint preparation which involves the transfer by direct contact of the superficial cellular content of a fresh tissue (excised from surgery) onto filter paper or a glass slide. Cells from the tissue will adhere to the paper or glass slide. Specific protocols will then be utilized to process the paper or slide for nucleic acids. Touch imprint preparations are widely used by histologists and cytologists.

Safety in the Biorepository

The biorepository is a very valuable asset to Windber Research Institute (WRI), and because of the importance of our samples we take every measure possible to insure safety of samples as well as our personnel. WRI has an active safety committee whose purpose is to bring workers and management together in a cooperative effort to promote safety and health in the workplace.  It is important for each department to be represented and we have several members of the tissue bank on WRI’s safety committee. The committee is formed by lab staff and managers representing at least one member from each department.  The safety committee’s job is to ensure proper lab and workplace safety is being practiced. As members of the committee, we participate in safety inspections of the building and check all areas for safety violations. These inspections are very important and help us to identify existing and potential hazards and recommend corrective action. We check lab equipment, emergency supplies and evacuation plans.

Because operation of a biorepository can potentially expose employees to hazards, it is important for everyone to be involved in training and education activities.  Our biorepository receives annual safety training and participates in periodic educational activities for things such as the safe handling and use of liquid nitrogen, proper personal protective equipment, etc.

Members of the Tissue Bank participate in training for the safe packaging and transport of dangerous goods. The packaging and shipment training helps keep us up to date according to national and international regulations.

Not only is the safety of our staff important, it is critical that we take measures to protect the biospecimens. We have comprehensive standard operating procedures covering everything from physical security and backup power, to monitoring and maintenance of storage equipment and troubleshooting.  We constantly strive to put procedures into place and ensure they are followed with the ultimate goal of protecting our most valuable resource – the biospecimens! 

Our contribution to the TCGA findings of a Comprehensive Breast Cancer Analysis

The Windber Research Institute is proud to be part of the just published online article in the journal Nature http://www.nature.com/nature/journal/vaop/ncurrent/full/nature11412.html  (September 23^rd, 2012) by The Cancer Genome Atlas (TCGA) Network entitled “Comprehensive molecular portraits of human tumors.” Together with our military collaborators, the Walter Reed National Military Medical Center (WRNMMC), we provided breast tissue specimens, DNA and relevant donor data for this ground breaking research. A total of fifteen Tissue Source Sites were involved in this national scientific project sponsored by the National Institutes of Health (NIH). Our journey to such a success story was as expected not very easy. Our efforts through the years to acquire and maintain the very best quality tissue specimens paid off when we qualified to become tissue providers for TCGA. The strict qualification criteria of this project kept us on our toes. Our qualification rates were usually between 85% and 100%. We were highly commended by the Biospecimen Core Resource group (Nationwide Children’s Hospital Biospecimen Core) as they compared our performance with that of the other tissue source sites. All this attests to the hard work and dedication of our staff. Their constant and daily efforts to provide the highest level of efficiency and perfection made it possible for us to be part of this major scientific project.  As a small not for profit research institute established twelve years ago, we are proud to be in the same playing field with institutions such as Duke University, Mayo Clinic, MD Anderson Cancer Center, University of Pittsburgh and University of North Carolina at Chapel Hill to mention just a few of the other Tissue Source Sites for this project. 

In this project, 800 breast tumors were genetically characterized using multiple technological platforms. The study was able to characterize four major sub-types of breast cancer (HER-2 enriched, Luminal A, Luminal B, and Basal-like) as previously described and provided a more comprehensive understanding of the mechanism behind each type of breast cancer. Furthermore, the study identified molecular similarities between one sub-type of breast cancer and ovarian cancer. Charles Perou, PhD, the corresponding author says “This study has now provided a near complete framework for the genetic causes of breast cancer which will significantly impact clinical medicine in the coming years as these genetic markers are evaluated as possible markers of therapeutic responsiveness.” Dr. Perou is the May Goldman Shaw Distinguished professor of Molecular Oncology at the University of North Carolina at Chapel Hill and a member of UNC Lineberger Comprehensive Cancer Center.

The tissue bank at Windber will continue to engage in ground breaking research activities in the coming years. This will encourage our donors and help with new therapeutic targets for treatment to alleviate the sufferings of the many breast cancer patients out there. We are working for you!

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature11412.html