In 2018, IBBL’s Biorefinery Department took another step forward in advancing biospecimen research. The team developed an assay to evaluate the quality and fitness for purpose of PBMC samples, thus adding to IBBL’s portfolio of discoveries to the benefit of biomedical research.
Biomedical research and precision medicine rely on precision preanalytics. When preanalytical variables – factors 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 peripheral blood mononuclear cells (PBMCs) – white blood cells containing one nucleus, such as lymphocytes and macrophages – some of the most critical parameters include the type of anticoagulant used for blood collection, as well as pre-centrifugation conditions, namely the temperature and time during which the blood cells have been in contact with plasma prior to being centrifuged. Long pre-centrifugation times have been linked to a series of metabolic alterations within the sample, with repercussions on its quality attributes.
IBBL’s Biorefinery Department, led by Dr. Fay Betsou, has been actively investigating ways to retrospectively assess the impact of pre-centrifugation times on the quality and fitness for purpose of biospecimens of undocumented preanalytical history for clinical or experimental immunology. Dr. Olga Kofanova, Biospecimen Quality Team Leader at IBBL and project coordinator, and her team developed an assay that can ‘diagnose’ prolonged pre-centrifugation times, with the aim of guaranteeing the accuracy of downstream applications such as downstream gene expression or functional analyses. The team isolated PBMCs from blood samples from healthy subjects and patients with inflammatory and infectious conditions. Subsequently, the combined expression of two target genes – specifically IL8 and EDEM3, which are known to be upregulated and downregulated in response to extended blood pre-centrifugation delays, respectively – was assessed for its ‘diagnostic performance’ in identifying PBMC samples altered by significant pre-centrifugation delays. The team thus established a ‘PBMC preanalytical score’, i.e. an assay based on the ratio of the expression of the two genes, which can differentiate RNA samples extracted from PBMCs with long (≥24 hour or ≥48 hours) pre-centrifugation times, from RNA samples extracted from PBMCs with short (≤3 hours) pre-centrifugation times.
“Our PBMC preanalytical score will allow researchers to objectively qualify PBMC samples for downstream assays that are sensitive to blood pre-centrifugation delays, such as gene expression analyses and antigen-specific response assays, with a very high sensitivity and specificity”, explains Dr. Kofanova. “This improved qualification will in turn result in new and more accurate insights into disease mechanisms and experimental immunology”, she concludes.
The PBMC preanalytical score adds to a previous assay developed by the team in 2017 for the qualification of serum and plasma samples with long pre-centrifugation delays.
The study was conducted in cooperation with the ISBER Biospecimen Science Working Group, and specifically with Rocio Aguilar Quesada and Sonia Panadero-Fajardo (SSPA Biobank), Alexandre Bulla and Pierre Lescuyer (Hôpitaux Universitaires de Genève), Kathi Shea (Precision for Medicine), Marc Keipes (Zitha Gesondheets Zentrum) and Mars Stone (Blood Systems Research Institute), who collected, processed and provided samples for the validation of the assay.
The full scientific publication is available here.