10 years of IBBL publications (3/4) – Quality Control assays: assessing the quality of liquid and solid samples

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 fill the remaining gaps in terms of biospecimen qualification, IBBL’s Biorefinery Department developed a series of quality control (QC) assays to evaluate the quality and fitness for purpose of both fluid – specifically serum, plasma and PBMCs – and solid samples – namely FFPE tissues.

Successful biomedical research and precision medicine go hand in hand with precision preanalytics. To minimise the negative impact of unknown and uncontrolled preanalytical variables and ensure the accuracy and reproducibility of downstream analytical results, Quality Control (QC) assays need to be carried out to assess biospecimen fitness-for-purpose. However, only a few reliable and evidence-based QC assays currently exist. IBBL’s Biorefinery Department has therefore been investigating ways to retrospectively assess the impact of various preanalytical factors that may affect the quality of liquid and solid samples.

Some of the most critical preanalytical variables to monitor in serum, plasma and peripheral blood mononuclear cells (PBMCs)[1] include the type of anticoagulant used for blood collection, as well as pre-centrifugation conditions, the delay between collection and processing and the temperature. Long pre-centrifugation times have been associated with a series of metabolic alterations within the sample, with repercussions on its quality attributes. In order to help the scientific community identify serum and plasma samples that have been subject to precentrifugation times in the order of magnitude of 24 hours, Dr. Olga Kofanova, Biospecimen Quality Team Leader at IBBL, and her team developed an easy and accurate QC tool, in collaboration with members of the ISBER Biospecimen Science Working Group. Namely, the team validated two cytokines[2], interleukin 8 (IL-8) and interleukin 16 (IL-16), in serum and plasma samples that showed significant changes in concentration when subjected to extended pre-centrifugation delays at room temperature. These were validated for their ‘diagnostic ability’ to detect serum or plasma samples altered by this preanalytical factor, providing a reliable QC assay for serum and plasma samples. The full paper can be accessed here.

In parallel, Dr. Kofanova and her team have been working on a related assay focusing specifically on the assessment of the quality of PBMC samples of undocumented preanalytical history for clinical or experimental immunology. In collaboration with the ISBER Biospecimen Science Working Group, they looked at the combined expression of two target genes, namely IL-8 and EDEM3, as an indicator of pre-centrifugation delays and established a ‘PBMC preanalytical score’, i.e. an assay based on the ratio of the expression of the two genes. This QC tool 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, allowing researchers to objectively qualify PBMC samples for downstream assays, such as gene expression analyses. The full publication is available here.

Similarly, formalin-fixed paraffin-embedded (FFPE) tissue blocks are often of poor quality given their susceptibility to uncontrolled and unrecorded preanalytical variables. For this reason, IBBL’s Biorefinery Department developed two assays based on markers that denote two of the most important preanalytical factors for FFPE tissues: cold ischemic (CI) time[3] and fixation time. CI time is not usually recorded upon collection, and therefore researchers do not know the extent of its impact on their results. Dr. William Mathieson and his team devised an assay based on the expression of three genes, namely PRKACA, FOS, and EGR1, which were found to be upregulated in response to CI. Using a gene (POLR2A) which remains unvaried as a baseline, the combined expression of the three genes gives a Cold Ischemia (CI) Score that accurately differentiates between samples with CI times shorter than 3 hours and those with 12-hours. The full publication can be accessed here.

Finally, IBBL’s Biorefinery Department tackled the issue of fixation time in FFPE tissues. Indeed, the longer the fixation time, the higher the degree of cross-linking and the lower the quality of nucleic acids that can be extracted from the tissue for sequencing and other molecular analyses. There are currently no assays that allow the identification of formalin overfixed tissues. A study led by Wim Ammerlaan, Technological Platforms Team Leader at IBBL, sought to find at least one biomarker – a small RNA target – that is sensitive to overfixation and that could be used as a QC tool to assess overfixed tissue samples, particularly in legacy collections of unknown preanalytics. The team identified a set of 24 micro RNAs (miRNA[4]) and small nuclear RNAs (snRNAs[5]) as the best determinants of fixation time and developed an assay generating a simple ‘snoRNA score’. This score accurately detects FFPE tissue samples with a fixation time of 72 hours or more and can therefore be used to assess the fitness-for-purpose of FFPE samples for DNA or RNA-based research or clinical assays, which are known to be particularly sensitive to formalin overfixation. The scientific publication can be found here.

Despite the availability of QC assays such as the ones described above, their use is still not sufficiently widespread in the research community, also due to the lack of tools guiding researchers in the choice of the most appropriate assay in the context of a specific downstream application. IBBL therefore launched Findmyassay.com, a free web-based application that helps the user identify and select the most relevant QC assay based on the type of sample, through a series of questions and answers. Findmyassay.com can be accessed through the dedicated webpage and is expected to be updated in 2020.

[1] White blood cells containing one nucleus, such as lymphocytes and macrophages.

[2] Cell signaling proteins that regulate several biological functions including immunity and inflammation.

[3] The time between tissue collection and fixation, during which the tissue is deprived of oxygen and is below body temperature.

[4] A class of small noncoding RNAs which are involved in the regulation of gene expression at the posttranscriptional level by degrading their target mRNAs and/or inhibiting their translation.

[5]  A class of small RNA that assist in chemical modifications of other RNAs such as ribosomal RNAs.