Biospecimen Integrity Research Articles

A comprehensive preanalytical protocol for fresh solid tumor biospecimens

Nearly seventy percent of diagnostic lab test errors occur due to variability in preanalytical factors. These are the parameters involved with all aspects of tissue processing, starting from the time tissue is collected from the patient in the operating room, until it is received and tested in the laboratory. While there are several protocols for transporting fixed tissue, organs, and liquid biopsies, such protocols are lacking for transport and handling of live solid tumor tissue specimens. There is a critical need to establish preanalytical protocols to reduce variability in biospecimen integrity and improve diagnostics for personalized medicine. Here, we provide a comprehensive protocol for the standard collection, handling, packaging, cold-chain logistics, and receipt of solid tumor tissue biospecimens to preserve tissue viability.

Delta-S-Cys-Albumin as a Marker of Pediatric Biospecimen Integrity.

Blood plasma storage is a crucial element of pediatric biobanking. Improperly stored or handled specimens (e.g., at > -30°C) can result in altered biomolecular compositions that no longer reflects invivo reality. We report application of a previously developed assay in adults-the ΔS-Cys-Albumin assay, which facilitates estimation of plasma and serum exposure to thawed conditions-to a population of pediatric EDTA plasma samples from patients aged 3-18 years to determine the assay's applicability, estimate its reference range for pediatric samples, and assess the impact of pre-centrifugation delay at 0°C. In addition, the effect of plasma thawed-state exposure to a range of times at 23°C, 4°C, and -20°C on ΔS-Cys-Albumin was evaluated. Using 98 precollected and processed pediatric EDTA plasma specimens, no difference was found in ΔS-Cys-Albumin under conditions of pre-centrifugation delay for up to 10 hours at 0°C. This lack of change allowed us to estimate a pediatric reference range for ΔS-Cys-Albumin of 7.0%-22.5% (mean of 12.8%) with a modest Pearson correlation between ΔS-Cys-Albumin and age (p = 0.0037, R2 = 0.29). ΔS-Cys-Albumin stability in six specimens at 23°C, 4°C, and -20°C was also evaluated. Plateaus in the decay curves were reached by 1 day, 7 days, and 14-28 days at these respective temperatures. The estimated pediatric reference range observed in children was lower than that previously observed in 180 adults of 12.3%-30.6% (mean of 20.0%), and the slope of the age correlation in children was twice as steep as that from adults. ΔS-Cys-Albumin decay curves at 23°C, 4°C, and -20°C were similar to those previously observed in adults. The data reported here support the use of ΔS-Cys-Albumin in evaluating the integrity and overall exposure of pediatric EDTA plasma specimens to thawed conditions. In doing so, they add an important quality control tool to the biobanker's arsenal.

Investigating BioCaRGOS, a Sol-Gel Matrix for the Stability of Heme Proteins under Enzymatic Degradation and Low pH.

There have been significant advances in the development of vaccines for the prevention of various infectious diseases in the last few decades. These vaccines are mainly composed of proteins and nucleic acids. Poor handling and storage, exposure to high temperatures that lead to enzymatic degradation, pH variation, and various other stresses can denature the proteins or nucleic acids present in any vaccine formulation. Therefore, it is necessary to maintain a proper environment to preserve the integrity of biospecimens. To overcome these challenges, we report a practical and user-friendly approach for sol-gels called "BioCaRGOS" that can stabilize heme proteins not only in the presence of degrading enzymes and acidic pH but simultaneously maintain stability at room temperature. Heme proteins, such as myoglobin and cytochrome c, have been used for this study.

Abstract 376: Biospecimen and data resources for cancer research from NCI’s BPV program

Abstract The Biospecimen Preanalytical Variables (BPV) Program was initiated by the National Cancer Institute’s Biorepositories and Biospecimen Research Branch to evaluate the impact of preanalytical factors on the molecular integrity of biospecimens. Selected preanalytical factors including cold ischemic time (delay to formalin fixation (DTF)), time in formalin (TIF), freezing methods, and storage temperatures and durations were examined for their potential effects on molecular profiles from surgical resection tissues and matched blood from four cancer types (kidney, ovary, colon and lung). The BPV program has collected tumor specimens from 364 cancer patients. Each specimen was annotated with 300+ data elements that cover steps in the collection, handling, and processing procedures, pathology review, and clinical information. NCI conducted multiple studies using these specimens to evaluate the preanalytical impacts on different analytical platforms including gene expression profiling, copy number variation, proteomics and metabolomics profiling. The program invites interested organizations to work with NCI through collaboration to further evaluate preanalytical effects on molecular analyses (https://techtransfer.cancer.gov/availabletechnologies/e-000-2013). The remaining specimens are available to support relevant research focusing on biospecimen science and/or clinical biomarker assay development (https://specimens.cancer.gov/search/). The IT infrastructure that was developed to support BPV biospecimen collection and management has been further developed into open source products (https://github.com/NCIP/CDR and https://github.com/NCIP/CDR-Lite). The controlled vocabulary that records the terms and definitions used in describing the overall biospecimen collection efforts has been refined and published at publicly accessible CDE repositories: NCI’s caDSR (https://cdebrowser.nci.nih.gov/CDEBrowser/ ) and NIH’s CDE portal (https://cde.nlm.nih.gov/cde/search ). An ongoing collaboration with an academic ontology consortium will map the CDEs to existing biobanking ontology frameworks and make them publicly available. The BPV program has generated a wide range of ~omics data. We are preparing a BPV data compendium to be submitted to dbGaP at NCBI. These data will be used as the experimental evidence to develop evidence-based best practices for fit-for-purpose collection, processing, and storage of biospecimens for cancer research. Citation Format: Ping Guan, Helen M. Moore. Biospecimen and data resources for cancer research from NCI’s BPV program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 376. doi:10.1158/1538-7445.AM2017-376

Abstract 1377: Impact of biospecimen pre-analytical factors on molecular analysis

Abstract The Biospecimen Pre-analytical Variables (BPV) Program of the Biorepositories and Biospecimen Research Branch (BBRB) at the National Cancer Institute (NCI) is designed to systematically investigate the effects of preanalytical factors on the molecular integrity of biospecimens. Specific parameters related to formalin fixation and paraffin embedding (FFPE) of cancer tissues were examined, including the effects of cold ischemic time (delay to fixation (DTF)) and time in fixative (TIF). Additional preanalytical studies focus on snap freezing method for tissue specimens (dry Ice vs. LN2 vapor), storage temperature (-80°C vs. LN2 vapor), and duration of storage for frozen specimens. Biospecimens for the BPV program were collected at four medical centers, within a tightly regulated infrastructure and strict adherence to SOPs, to enable consistent collection and handling across all sites. Biospecimens were collected from research participants undergoing surgical treatment for renal cell carcinoma; ovarian, fallopian tube, and peritoneal carcinoma; lung adenocarcinoma and squamous cell carcinoma; and colorectal adenocarcinoma. Biospecimens were subjected to specific experimental protocols to systematically vary the preanalytical factors of interest. Extensive annotation was performed with 300+ data elements that include steps in the collection, handling, and processing of the biospecimens, pathological evaluation of the tumor, and clinical information collected from donors. Biospecimen collections concluded in April 2015 with a total of 364 tumor tissue cases collected. The BPV program has conducted multiple, simultaneous molecular analyses to understand the impact of FFPE preanalytical factors on the expression and detection of various molecular analytes. Initial efforts were focused on evaluating the impact of DTF and TIF on the quality of DNA and RNA from FFPE samples using multiple methods and approaches such as NanoDrop 8000 UV-Vis spectrophotometer, Qubit 2.0 Fluorometer, Agilent Bioanalyzer and KAPA Human Genomic DNA Quantification. The QC data from both RIN and Kappa assays showed that FFPE samples have a significant drop in RNA and DNA quality compared with matched frozen samples. 72 hr TIF samples showed a significant drop in both RNA and DNA quality compared to shorter time points (6, 12, or 23 hr TIF), as measured by DV200 and Kappa assays. No significant differences were observed in RNA/DNA quality between the shorter TIF time points or between the DTF time points (1, 2, 3, 12 hr DTF). Further studies are now underway to evaluate additional preanalytical factors. The data from BPV studies will be widely shared with the research community through publication and deposition at a public data repository. The results from these studies will be used to develop evidence-based protocols and best practices for fit-for-purpose collection, processing, and storage of biospecimens. This project is funded by NCI Contract No. HHSN261200800001E. Citation Format: Rachana Agarwal, Ping Guan, Mary Barcus, Jasmin Bavarva, Robin Burges, Philip Branton, Latarsha Carithers, Corinne Camalier, Biswajit Das, Jason Lih, Hana Odeh, Nancy Roche, Dan Rohrer, Michael Sachs, Leslie Sobin, Jewell Scott, Anna Smith, Conrado Soria, Kimberly Valentino, Dana Valley, Mickey Williams, Helen Moore. Impact of biospecimen pre-analytical factors on molecular analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1377.

Preanalytical variables affecting the integrity of human biospecimens in biobanking.

Most errors in a clinical chemistry laboratory are due to preanalytical errors. Preanalytical variability of biospecimens can have significant effects on downstream analyses, and controlling such variables is therefore fundamental for the future use of biospecimens in personalized medicine for diagnostic or prognostic purposes. The focus of this review is to examine the preanalytical variables that affect human biospecimen integrity in biobanking, with a special focus on blood, saliva, and urine. Cost efficiency is discussed in relation to these issues. The quality of a study will depend on the integrity of the biospecimens. Preanalytical preparations should be planned with consideration of the effect on downstream analyses. Currently such preanalytical variables are not routinely documented in the biospecimen research literature. Future studies using biobanked biospecimens should describe in detail the preanalytical handling of biospecimens and analyze and interpret the results with regard to the effects of these variables.

Abstract 2493: Molecular biomarkers for cancer biospecimen integrity

Abstract Successful implementation of personalized health care requires the availability of biospecimens of sufficient quality to ensure accurate downstream test results, which will in turn determine optimal patient management. Cancer biospecimens of known quality with appropriate clinical annotation are also critical to the process of biomarker discovery and validation that lead to the development of clinical tests. Due to widely varying methods of biospecimen collection, processing, and storage, numerous pre-analytical factors can affect human biospecimen integrity for biomarker research. Such pre-analytical variability can affect the integrity of research biospecimens including plasma/serum and fixed cancer tissues used for biomarker analysis. The National Cancer Institute (NCI) Biorepositories and Biospecimen Research Branch (BBRB) was established to address the scientific and policy issues that affect the procurement of and access to high quality specimens for research. The NCI Biospecimen Research Network (BRN) was established to sponsor and conduct research on biospecimen pre-analytical factors and their affect on molecular integrity, to support the development and adoption of evidence-based standardized biospecimen handling procedures. In this presentation we will summarize BRN-supported studies to identify biomarkers that may be used as sentinel markers of plasma stability using mass spectroscopy proteomics and analysis of circulating miRNA, and the development of intrinsic controls for FFPE tissues. In one of the projects, identification of protein biomarkers using mass-spectrometry and Illumina arrays in plasma led to the development of a biomarker panel for serum/plasma integrity; these biomarkers are currently being validated. A second project studied the effects of pre-analytical variables on circulating miRNA and identified and validated new and improved housekeeping miRNA, as well as novel biomarkers associated with breast cancer. In another study, a series of biomarkers have been validated by construction of tissue microarrays from 93 breast cancer specimens with known time to fixation delays. A tissue quality index (TQI) model was generated to predict the time to fixation and tissue quality by measuring a subset of biomarker proteins in breast cancer tissues using AQUA technology. The progressive efforts taken by BRN investigator-led projects to identify and validate biomarkers for sample integrity will be incorporated into efforts to develop new biospecimen evidence-based practices for clinical biomarker research. Citation Format: Lokesh Agrawal, Jim Vaught, Helen Moore. Molecular biomarkers for cancer biospecimen integrity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2493. doi:10.1158/1538-7445.AM2014-2493

Veterinary and Human Biobanking Practices

Animal models have historically informed veterinary and human pathophysiology. Next-generation genomic sequencing and molecular analyses using analytes derived from tissue require integrative approaches to determine macroanalyte integrity as well as morphology for imaging algorithms that can extend translational applications. The field of biospecimen science and biobanking will play critical roles in tissue sample collection and processing to ensure the integrity of macromolecules, aid experimental design, and provide more accurate and reproducible downstream genomic data. Herein, we employ animal experiments to combine protein expression analysis by microscopy with RNA integrity number and quantitative measures of morphologic changes of autolysis. These analyses can be used to predict the effect of preanalytic variables and provide the basis for standardized methods in tissue sample collection and processing. We also discuss the application of digital imaging with quantitative RNA and tissue-based protein measurements to show that genomic methods augment traditional in vivo imaging to support biospecimen science. To make these observations, we have established a time course experiment of murine kidney tissues that predicts conventional measures of RNA integrity by RIN analysis and provides reliable and accurate measures of biospecimen integrity and fitness, in particular for time points less than 3 hours post-tissue resection.

MC13-0047 Serum and tissue biomarkers for cancer biospecimen integrity

MC13-0047 Serum and tissue biomarkers for cancer biospecimen integrity

Abstract 5149: Translating biospecimen science research results to improved biospecimen practices.

Abstract Cancer tissues and blood are collected, processed and stored in different ways, across the country and across the world. Biospecimen Science is the study of how different methods of biospecimen collection, processing, and storage affect downstream analysis. The NCI Biospecimen Research Network (BRN) program sponsors, conducts, and collaborates on Biospecimen Science research to better understand how biospecimen pre-analytical factors influence the molecular integrity of biospecimens, and in turn, how such variation affects the reproducibility of research results. New data on how pre-analytical variation affects molecular integrity will support best practice Standard Operating Procedures for research and clinical trials biospecimens. This presentation will focus on results from the first phase of the BRN program and current efforts to translate research results to evidence-based biospecimen practices. A summary of research results will be presented from several research studies which have performed systematic perturbations of blood and tissue collection and processing procedures, with subsequent molecular analysis. We will also present an overview of the Biospecimen Research Database (BRD; https://brd.nci.nih.gov), developed to improve access to the literature in Biospecimen Science. The BRD is a free and publicly accessible web-based database that currently contains over 1500 review and research articles covering a broad range of topics spanning the biospecimen lifecycle. Articles are meticulously categorized and annotated by a team of Ph.D. level scientists according to the type of biospecimen and technology platform used, the experimental variables investigated, and many other parameters. Progress will be described on a new approach to perform meta-analyses of the literature evidence related to different tissue preservation methodologies, such as freezing and temperature storage methods and formalin fixation and paraffin embedding. In addition, we will describe current progress on the development of evidence-based biospecimen practices based on BRN research results, literature evidence, and expert input. Citation Format: Helen M. Moore, Kelly Engel, Sarah Greytak, B. Paige Bass, Jim Vaught. Translating biospecimen science research results to improved biospecimen practices. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5149. doi:10.1158/1538-7445.AM2013-5149

Abstract 973: The NCI Biospecimen Research Network

Abstract The National Cancer Institute's (NCI) Office of Biorepositories and Biospecimen Research (OBBR) initiated the Biospecimen Research Network (BRN) in early 2006 to coordinate and support systematic investigation into how collection, processing, and storage of human biospecimens affect subsequent analysis. To support this goal, the BRN sponsors, conducts and collaborates on studies to evaluate the effects of biospecimen pre-analytical variables on the outcomes of molecular assays for cancer diagnosis and research. BRN supported findings will contribute to the development of evidence-based best practices for the collection, processing, storage and transport of biospecimens; building on the “NCI Best Practices for Biospecimen Resources.” Currently the BRN sponsors a variety of initiatives aimed to develop and support the research efforts of the biospecimen science community. Through its newly launched extramural research program the BRN supports a portfolio of projects addressing biospecimen pre-analytical variables and biospecimen integrity. Furthermore, the BRN aims to significantly improve biospecimen-based diagnostic and research capabilities by communicating the results of such research to the scientific and clinical communities. The Biospecimen Research Database and the annual BRN symposium, Advancing Cancer Research Through Biospecimen Science are forums established for effective exchange of biospecimen research results. The BRN also supports new research and innovation through its collaborations with other NCI programs such as NCI Innovative Molecular Analysis Technologies (IMAT), the NCI Small Business Innovation Research Development Center (SBIR), the Clinical Proteomics Technologies for Cancer (CPTC) and the Cancer Genome Atlas (TCGA). Through these partnerships, the BRN serves as a resource for program awardees and presents new opportunities for research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 973.

Methodologic Data: Important Foundation for Molecular and Biomarker Studies

In this issue of Cancer Epidemiology, Biomarkers & Prevention (CEBP) , we have included a Focus section featuring a series of papers with the themes of Biomarkers, -Omics & Systems Biology and Biospecimens & Biorepositories to highlight the importance and/or emergence of these topics in cancer

Cryovial with partial membrane sealing can prevent liquid nitrogen penetration in submerged storage

Cryopreservation is one of the fundamental techniques in life science. To preserve the viability of cells and tissues, many researchers use plastic cryogenic vials and immerse them into liquid nitrogen for long-term storage. However, the non-sterile liquid nitrogen usually infiltrates into the vials and may cause a high rate of microbial contamination, and even some explosive incidents upon retrieval. To prevent these drawbacks while retaining the benefit of constant ultra-low temperature in submerged liquid nitrogen, we used a heat-sealable membrane to cover the upper portion of vials. After heat-sealing, the vials were completely free of liquid nitrogen penetration in the submerging test. Moreover, the sealing process did not affect the cell viability. This modified protocol provides an easy and efficient tool to ensure the integrity of biospecimens in long-term storage without interfering with existing cryobox storage systems.