Making clinical samples fit for research (1/3) – Optimising fixation during sample preparation

Clinical biospecimens are the most accurate sample-type for research purposes, but are often of poor quality given their susceptibility to uncontrolled and unrecorded preanalytical variables. IBBL’s Biorefinery Department carries out biospecimen research to guide researchers in their sample preparation, selection and processing decisions. Recently, Dr. William Mathieson and his colleagues investigated the suitability of the non-formalin PAXgene tissue fixative, with the aim of providing better insight into this aspect of sample preparation prior to processing.

Tissue samples to be used for diagnosis or research need to be preserved and be made amenable to further analyses such as immunohistochemistry and molecular biology, either by fixation or stabilisation. Most tissue biospecimens are fixed using formalin then embedded in paraffin, resulting in a formalin-fixed paraffin-embedded (FFPE) tissue block. Despite the many advantages of this method, nucleic acids extracted from FFPE tissue blocks are often fragmented and chemically modified. The PAXgene tissue fixative was therefore developed as an alternative to formalin to address these shortcomings. However, it was not known how resistant PAXgene-fixed paraffin-embedded (PFPE) tissue blocks are to storage.

The IBBL research team, together with Imperial College London and the Wales Cancer Bank, tackled this question by comparing the integrity of RNA and microRNA (miRNA)[1] extracted from PFPE blocks immediately after fixation with that extracted from the same blocks seven years later. They found that RNA degraded in the PFPE blocks during storage but the miRNA was much more stable. However, no degradation occurred during the seven-year period when RNA was extracted from the PFPE blocks immediately upon collection and stored at -80 °C.

“Our work supports researchers in choosing the most suitable fixative according to the type of downstream analyses to be carried out, and on the specific logistics of sample collection and processing”, explains Dr. Mathieson. “The degradation during long-term storage of tissues as PFPE blocks makes them unsuitable for certain molecular biology analyses, so it is advisable to collect enough material to have both a fresh-frozen tissue block and an FFPE block. However, PFPE fixation is a viable alternative when it is not possible to freeze the tissue or when the sample is too small to obtain both a frozen and an FFPE block”, he adds.

The detailed results of the study can be found in the full paper here.

The study provides insight on the impact of pre-analytical variables related to the choice of tissue fixatives and suggests ways to deal with such issues. However, pre-analytical variables linked to sample collection methods also represent a risk for the sample’s fitness-for-purpose, and therefore need to be understood and addressed. The next article in this series will focus specifically on how the impact of cold ischemic time can be assessed. Stay tuned!

[1] miRNAs: small noncoding RNAs involved in the regulation of gene expression by degrading their target mRNAs and/or inhibiting their translation, and therefore have diagnostic and therapeutic value for several diseases.