Why every step before analysis matters

Variations in the way biological samples are collected, processed and stored can greatly impact downstream research results. To avoid this and ensure sample quality, IBBL invests in internal biospecimen research. Two flagship projects in 2013 focused on investigating the impact of these variations on blood sample analysis.

High-throughput analyses that facilitate or enable the simultaneous study of thousands of genes, proteins or metabolites, have substantially advanced biomedical research in recent years. Importantly, they have led to the discovery of a number of biomarkers for diagnosis, prognosis or drug response. However, new technologies also bring along new challenges. The large scale of these technologies means that samples from many different patients need to be processed in exactly the same fashion before they are analysed to ensure that observed differences in genes or proteins reflect differences between patients rather than differences in the pre-analytical process. This is especially crucial in biomarker research. Variations in the way in which samples are collected or processed can lead to molecules being misidentified as biomarkers. Similarly, scientists may fail to identify biomarkers if small differences between patients are masked by the variations due to processing.

Tools to assess sample quality

As one of a few biobanks to carry out their own research, IBBL, specifically the team around its Chief Scientific Officer, Dr Fay Betsou, focuses on studying the impact of these pre-analytical variations on the down-stream results. One of the team’s main aims is to discover and validate markers for biospecimen quality control (QC). These QC markers are molecules (DNA, RNA, protein, metabolites) that are sensitive to the method of sample collection, processing or storage, allowing researchers to detect when samples have not been handled correctly.  QC markers are especially important for biobanks, which need to ensure that their samples are fit-for-purpose, meaning that they have a level of quality suitable for the specific type of analysis they are intended for. Indeed, the parameters that need to be controlled during processing depend strongly on the type of downstream analysis and the type of sample. In 2013, IBBL’s biospecimen research team focused on two main research projects to determine the best ways to handle and process blood samples.

Camille Bellora completed a project as part of her BSc studies at IBBL, supervised and supported by Dr Olga Kofanova, IBBL’s Biospecimen Research Scientist. The project aimed to investigate what impact pre-analytical variations have on the levels of gene expression (activity) in white blood cells. When a gene is active it produces gene-specific RNA. So gene expression is generally assessed by measuring the amount of specific RNA extracted from the cells. IBBL’s scientists decided to focus on 4 genes and evaluate how different anti-coagulants, temperatures and times before processing affect their expression in white blood cells. Their results show that the expression of these 4 genes starts changing as early as 3 hours after the time blood is taken from the patient. As the time increases, so does the change in gene expression. One gene in particular was strongly activated the longer the processing was delayed, making it a candidate pre-analytical biomarker. In general, the expression of these 4 genes was altered by differences in all of the tested parameters (anti-coagulants, temperatures and delay), underlining the importance of controlling all variables during collection and processing. Dr Kofanova and Ms Bellora are currently validating their initial results in additional samples to determine if that particular gene can indeed be used as a QC marker to identify samples with compromised pre-analytical conditions.

Time and temperature are critical

IBBL’s second big pre-analytics project in 2013 is a collaboration with the Metabolomics research group at the Luxembourg Centre for Systems Biomedicine (LCSB). For his joint PhD at IBBL and the LCSB, Jean-Pierre Trezzi studies the effect of pre-analytical variations on blood plasma metabolomics. Metabolomics is a fairly new but exciting field that systematically studies the output of all biochemical reactions in cells by measuring their end-products, so-called metabolites. Being downstream of genomics, transcriptomics and proteomics, metabolomics analyses the final phenotype of a cell. Since metabolites can tell us more precisely what is happening within cells, they are excellent candidate biomarkers. However, they are also less robust and more time-sensitive than genes or proteins, thus making the control of pre-analytical variation even more important.  For the collaborative project, blood samples from volunteers were collected and processed at IBBL and the metabolites were analysed at the LCSB by gas chromatography–mass spectrometry (GC-MS). Jean-Pierre Trezzi’s results suggest that, if researchers intend to study metabolomics on plasma, the blood should be stored on ice immediately after blood collection, in tubes with EDTA as anti-coagulant and be processed within 90 minutes to avoid any detrimental effect on the end results. Indeed, the concentration of 28 metabolites, most of which play a crucial role in the body’s energy metabolism cycle,, was altered when samples were stored at room temperature, even for a short time. On the other hand, almost all metabolites levels were stable for up to 90 minutes when stored on ice.

Both of these projects underline the importance of tightly controlling every step a biological sample goes through; from the patient, to the biobank, to the researcher. By implementing the results from biospecimen research into efficient Standard Operating Procedures (SOPs), IBBL can ensure all samples are of high quality and fit-for-purpose.