The National Cancer Institute’s Biorepositories and Biospecimen Research Branch (BBRB) Biospecimen Pre-analytical Variables Program rigorously investigated variables affecting the integrity of biospecimens prior to their analysis. The ultimate goal was to develop evidence-based protocols and best practices for the collection, processing and storage of biospecimens, thereby enhancing the field’s standards and adding confidence to research results.
A large number of highly annotated biospecimens were collected following well-defined protocols, including tumor tissue and blood from kidney, ovary, lung and colon cancer patients, which were used to assess the impact of various pre-analytical factors on the molecular integrity of biospecimens. Pre-analytical factors examined included delay to fixation (DTF) and time in fixative (TIF) for FFPE tissues (see table 1). Additionally, freezing methods and/or storage temperature variations were studied for tissue and plasma.
As the Comprehensive Biospecimen Resource for this program, Van Andel Research Institute’s Biorepository compiled and shipped collection kits to sites across the country. Upon return to the Biorepository, these samples were collected, processed, inventoried and stored. The specimens also were assessed for histological quality and were analyzed by next-gen sequencing, miRNA assay, immunohistochemistry and mass spectrometry (see tables 2 and 3).
Although collection has ended, analysis is ongoing. A large number of specimens remain available for research use through a collaboration agreement with the NCI BBRB. Through this collaboration, the Core continues to store, maintain and distribute these biospecimens.
Table 1: Tissue Module Description
Table 2: Analysis Platforms
Table 3: Immunohistochemical Staining
Along with the samples, matched blood was collected as well as normal adjacent tissue, when possible.
Following surgical removal, tumors were dissected into six blocks—one was frozen for use in future research, a second block was formalin-fixed and paraffin-embedded for quality control and an H&E slide was generated, and the remaining four were randomly assigned to an experimental protocol.
The H&E section taken from the QC block was reviewed by a pathologist for confirmation of diagnosis and to ensure the requirement of at least 50% tumor cells by surface area and less than 20% necrosis was met. The samples were used to evaluate the effects of pre-analytical factors on downstream molecular analysis results, such as:
Tissue microarrays (TMAs) also are available for each tissue type collected. All TMAs were created using duplicate 1.0 mm cores randomly placed in each TMA block. Three control tissues were included in each TMA. The TMAs represent modules I and II. Except for kidney, the modules are randomized throughout the TMAs. Each TMA was created in duplicate. A total of 293 patients are represented across these 21 TMAs. Available TMAs include the following:
Colon TMAs: 4 Colon TMAs (plus 4 duplicates), 56 patients
Kidney TMAs: 11 Kidney TMAs (plus 11 duplicates), 165 patients
Lung TMAs: 2 Lung TMAs (plus 2 duplicates), 20 patients
Ovary TMAs: 4 Ovary TMAs (plus 4 duplicates), 52 patients
To obtain access to this collection, please download the BPV application. All applications must be submitted to email@example.com. Access requests will be accepted on a continuing basis. NCI BPV program staff will review each request for completeness and follow up as necessary until all the required data is received. All applicants will be expected to pay for cost recovery fees such as cost for retrieving, processing and shipping the samples.
The criteria used to approve each request will include the following: