Finding better ways

While everyone agrees that biospecimens need to be collected and processed in a standardised way to eliminate bias introduced during pre-analytical steps, in many instances the best way to handle these biospecimens is simply not known. In reality, most researchers do not have the time to optimise each step of their processing and analysis protocols or develop alternative methods. IBBL sees one of its roles as a biobank, in investigating these kinds of questions and has over the years become an international leader in the field of biospecimen research. Other organisations that are interested in optimizing the way biospecimens are processed and analysed are equipment, consumable and assay suppliers. IBBL works closely with these companies to optimize and evaluate their products and workflows. In 2015, IBBL’s biospecimen researchers carried out two independent evaluations of new methods to improve the utilisation of tissue biospecimens.

An alternative to formalin

The first project evaluated the use of the PAXgene tissue fixative system as an alternative to formalin fixation, which is currently used, in combination with paraffin embedding, as the gold standard for tissue preservation. While formalin fixation is routine in pathology laboratories around the world, it has one major shortfall, which is the poor quality of molecules (DNA, RNA, protein, etc.) extracted from the tissue. For this reason molecular biologists favour frozen tissue over formalin-fixed paraffinembedded tissue (FFPE). Unfortunately, though, frozen tissue preserves the morphology less well than fixed tissue. The PAXgene tissue fixation system was developed to address this dilemma.

Collaborating with the Imperial College London, the University of York and the Wales Cancer Bank, IBBL’s researchers performed a comprehensive independent evaluation of the system. They compared the morphological preservation, the performance of immunohistochemistry and the quality of moleculesextracted from frozen, FFPE and PAXgene-fixed paraffin-embedded tissue (PFPE) tissue. They found that the PAXgene system preserved nucleic acids better than FFPE preservation, but not as well as cryopreservation. Thus, while the PAXgene system is unlikely to replace formalin in the routine clinical setting, because of the cost associated with switching to a new system, it is a valuable alternative for molecular diagnostics.

Enhancing the value of biospecimens

IBBL’s second tissue biospecimen research project of 2015 evaluated the CryoXtract CXT350 Frozen Sample Aliquotter, which can be used not only to take cores of frozen tissue, but also liquid biospecimens, like stool, urine or blood. Unlike a cryostat, which slices frozen tissue, coring allows researchers to target a specific section of the tissue, thus enabling the separate analysis of molecules from different parts of a biospecimen (e.g. tumoral and adjacent normal tissue). IBBL’s researchers used a total of 614 cores from different types of tissue in their evaluation of the CryoXtract equipment. They found that the technology was easy to use and integrate into the workflow of a tissue biobank. It provided reproducible cores without nuclease- or crosscontamination. The quality of RNA extracted from cores was marginally poorer than that of RNA extracted from tissue slices, thus not representing a barrier for the effective utilisation of the technology. Overall, the researchers concluded that the CryoXtract system enhances the value of frozen tissue biospecimens, because one can get a lot more data out of each biospecimen.

Ideally, researchers and biobanks would have a frozen specimen for coring with the CXT350 for molecular analysis and a mirrored FFPE tissue block for histological analysis. “While IBBL has applied this principle to its routine processing of tissues, this may not always be feasible”, comments Dr William Mathieson, the scientist at IBBL who led both projects. “Particularly when dealing with rare tumours or remote collection locations, the PAXgene system is a viable alternative.”

Sharing knowledge

While Dr Mathieson and his team focused primarily on tissue biospecimen research, other researchers within IBBL’s Biorefinery department investigated aspects relating to the quality of other types of biospecimens. In 2015, IBBL’s researchers evaluated the impact of different collection tubes on biospecimen quality, compared RNA quality assessment methods, investigated different aspects of RNA extraction protocols, developed new quality control assays and validated a number of processing and analysis methods, including 16S rRNA gene sequencing and the nucleic acid extraction from whole blood. These various projects led to 10 scientific publications in peer-reviewed journals. These publications are not only a reward for the researchers who worked for months on their projects, but also a crucial tool for IBBL to share its knowledge with the biobanking and the biomedical research community.

References:

A critical evaluation of the PAXgene tissue fixation system. W Mathieson, N Marcon, L Antunes, DA Ashford, F Betsou, SG Frasquilho, OA Kofanova, SC McKay, S Pericleous, C Smith, KM Unger, C Zeller, GA Thomas. American Journal of Clinical Pathology. 2016;146(1):25-40.

An Independent Evaluation of the CryoXtract Instruments’ CXT350 Frozen Sample Aliquotter Using Tissue and Fecal Biospecimens W. Mathieson, I. Sanchez, K. Mommaerts, S. Frasquilho, F. Betsou Biopreservation and Biobanking, Volume 14, Issue 1, Page: 2-8

Photo (c) Shutterstock