DIAGNOSING SAMPLE ‘HEALTH’

In 2017, IBBL’s Biorefinery Department took yet another step forward in advancing biospecimen research. The team published the results of a study that identified and validated two cytokine biomarkers to be used as an assay to evaluate the quality and fitness for purpose of serum and plasma samples. This discovery bears important implications for the accuracy and reliability of downstream biomedical research.

“We will now be able to determine whether the quality of samples with undocumented preanalytics, such as historical collections, is good enough to allow their use in downstream research applications.”

Fay Betsou

PhD, HDR, Chief Scientific Officer

No precision preanalytics, no precision medicine

It is nowadays universally acknowledged that the success of biomedical research and precision medicine requires precision preanalytics. When preanalytical variables – parameters that influence the sample before it is processed – are unknown and uncontrolled, they can negatively affect the accuracy and reproducibility of downstream analytical results. When it comes to serum, plasma and peripheral blood mononuclear cells (PBMCs) – white blood cells containing one nucleus, such as lymphocytes and macrophages – some of the most critical variables include pre-centrifugation conditions, namely the temperature and time during which the blood cells have been in contact with plasma prior to being centrifuged. Indeed, prolonged pre-centrifugation times, particularly at room temperature, mean greater chances of protein degradation, extended metabolic activity in the blood cells and ensuing oxygen depletion, in turn leading to a series of alterations within the sample, with repercussions on its quality attributes. IBBL’s Biorefinery Department, led by Dr. Fay Betsou, has therefore been actively investigating ways to assess the pre-centrifugation times of biospecimens of undocumented preanalytical history. In 2017, the team made a significant discovery that will enable the objective qualification of biological samples.

“We can now offer a robust and accurate quality control tool to diagnose the ‘preanalytical health’ of serum and plasma samples”

Olga Kofanova

PhD, Biospecimen Quality Team Leader

Qualifying serum and plasma

The challenge that the IBBL Biorefinery team embarked on was to identify an easy and reliable way to assess whether serum and plasma samples had been subject to long pre-centrifugation times, in the order of magnitude of 24 hours. Such delays have an impact on the downstream quantification of several proteins and metabolites in the serum and plasma. By leveraging on simple and readily available ELISA kits, Dr. Olga Kofanova, Biospecimen Quality Team Leader at IBBL and project coordinator, together with Estelle Henry, successfully devised an accurate method to address this research question. They screened twenty cytokines – cell signaling proteins that regulate several biological functions including immunity and inflammation – in serum and plasma samples for variations in their concentration as a result of extended pre-centrifugation delays at room temperature.

Two cytokines, namely interleukin 8 (IL-8) and interleukin 16 (IL-16), showed the largest changes in concentration. These were subsequently validated for their ‘diagnostic ability’ to detect serum or plasma samples altered by significant pre-centrifugation delays. The validation was performed on 7 independent sample collections from both healthy and diseased donors, in close collaboration with members of the ISBER Biospecimen Science Working Group. “Our work allowed us to identify the two most promising cytokine markers to be used in the characterisation of pre-centrifugation delays of over 24 and 48 hours. We can now offer a robust and accurate quality control tool to diagnose the ‘preanalytical health’ of serum and plasma samples”, explains Dr. Kofanova. “We will now be able to determine whether the quality of samples with undocumented preanalytics, such as historical collections, is good enough to allow their use in downstream research applications” adds Dr. Betsou.

In parallel, Dr. Kofanova and Camille Bellora have been working on a related assay focusing specifically on the assessment of the quality of PBMC samples. The research looked at the ratio of the expression of two target genes, namely IL-8 and EDEM3, as an indicator of pre-centrifugation delays. The results of the study will be published in 2018 and are set to revolutionise PBMC sample qualification for downstream applications such as gene expression and functional assays, in turn enabling new and more accurate insights into disease mechanisms and experimental immunology.

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