10 years of IBBL publications (4/4) – Evaluating and optimising sample processing methods

IBBL recently celebrated 10 years of service to the national and international scientific community through the publication of its 100th scientific paper, sharing its knowledge and expertise in biobanking and biospecimen research to promote the standardisation of biobanking practices and ultimately advance biomedical research. To this end, IBBL’s Biorefinery Department carries out studies to validate and optimise sample preanalytics. Some of these include the optimisation of fecal sample collection and DNA extraction, as well as the evaluation of the PAXgene Tissue Fixation System.

The optimisation and validation of biospecimen processing methods is crucial in order to guarantee the quality and fitness-for-purpose of biological samples, ensuring that the results of downstream biomedical analyses are reliable, accurate and reproducible. Indeed, method optimisation and validation is key in the context of standardising biobanking practices, and is particularly relevant for biobanks seeking accreditation according to international standards and for biomarker development.

The collection of stool samples, and the subsequent extraction and sequencing of the bacterial DNA present therein, allows the characterisation of the resident bacterial population. This gives researchers an insight into the gastrointestinal microbiome and enables the study of its impact on a variety of diseases. The choice of stool collection container type and DNA extraction method has been shown to affect the quality of the extracted DNA. For this reason, IBBL’s Biorefinery Department carried out a study to identify the optimal combination of stool collection container and DNA extraction procedure, in collaboration with the Luxembourg Centre for Systems Biomedicine (LCSB). Specifically, the team compared three stool DNA extraction methods and analysed the fitness-for-purpose of the resulting DNA for human and microbiome analysis by assessing its integrity, concentration and quality with different techniques. Moreover, the impact of six different containers was also analysed against these parameters for each extraction method. The team found that extraction using a Genotek stabiliser and the Chemagic Magnetic Separation Module provided a very good DNA yield, quality and purity when compared to the other two methods tested, particularly in combination with Sarstedt tubes and Genotek tubes. This validated stool collection and processing method was considered optimal also due to its simple collection and shipment logistics, as well as for its automation potential. The full publication can be accessed here.

When it comes to tissue samples, these need to be prepared, preserved and be made amenable to further research through fixation or stabilisation. Tissues are often fixed using formalin and are 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 IBBL Biorefinery Department therefore carried out a critical and independent evaluation of an alternative non-formalin tissue fixative, the PAXgene tissue fixation system. Dr William Mathieson, Scientist at IBBL and leader of the study, and his team therefore compared PAXgene-fixed paraffin-embedded (PFPE) tissues with FFPE tissues and fresh-frozen (FF) blocks in terms of morphologic preservation, as well as yield, quality and integrity of extracted RNA, DNA and proteins. The team observed that, in terms of overall histology, the morphology of PFPE and FFPE tissues was similar. Conversely, RNA yields were lower when RNA was extracted from PFPE tissues compared to FFPE. However, in terms of integrity, RNA extracted from PFPE sections was significantly less degraded than that from FFPE sections, although the best results were obtained from FF blocks. Similarly, DNA from FFPE sections was much more degraded compared to PFPE blocks, while no significant differences between the two fixatives were found in terms of yield. Regarding protein expression levels, the study found that FFPE-extracted proteins more closely represent the FF gold standard compared to those extracted from PFPE tissues. The team therefore concluded that the PAXgene fixation system is a suitable alternative to formalin in a research setting due to the well-preserved tissue morphology and better integrity of nucleic acids, particularly for specific molecular diagnostics projects entailing downstream DNA and RNA analyses or when the sample is too small to obtain both a frozen and an FFPE block. The full publication can be accessed here.