IBBL carries out biospecimen research for the benefit of the international biomedical science community, providing researchers with fit-for-purpose biospecimens and with valuable insight into the factors that can affect sample quality. IBBL’s Biorefinery Department recently published a series of 3 review articles in the journal Current Pathobiology Reports, summarising the key preanalytical aspects to be considered when preparing, processing and storing cfDNA, PBMCs and FFPE tissue biospecimens.
The first article — published in April 2019 in Current Pathobiology Reports as part of the Topical Collection on Biospecimens Science and Evidence-Based Standards for Precision Medicine — focuses specifically on the preanalytical factors affecting the quality and quantity of cell free DNA (cfDNA). This analyte is frequently used as a biomarker for the monitoring of cancers or pregnancy status through a variety of downstream genetic analyses by next-generation sequencing (NGS) or digital PCR. Given its role in diagnostics, it is important that cfDNA samples be of good enough quality to ensure the accuracy of the results of the genetic analyses. Uncontrolled preanalytical variables can compromise the quality and quantity of the analyte and subsequently undermine its utility. The article presents the main factors that need to be addressed to avoid irreproducible results. “The yield, size range, and integrity of the recovered cfDNA can be impacted by in-vivo pre-analytical factors in the donor blood sample, such as physical/psychosocial stress, the presence of viral infections and donor age. Similarly, ex-vivo factors related to the handling of the cfDNA samples prior to downstream analysis — such as blood collection tube type, the time and temperature between blood collection and plasma isolation, as well as cfDNA extraction kit type — have been shown to affect sample fitness-for-purpose”, explains Wim Ammerlaan, corresponding author of the review. “Standardisation of the cfDNA pre-analytical phase is therefore key for the accuracy of genetic analyses and the reproducibility of research results”, he says.
The second review — published in May 2019 in the same topical collection — deals with the preanalytical factors affecting the quality of Peripheral Blood Mononuclear Cells (PBMCs) isolated from blood samples. PBMCs are used in preclinical and clinical research as a source of biomarkers in infectious and chronic diseases, but also in epidemiological studies, vaccine trials and clinical trials. The quality and value of isolated PBMCs can be affected by a variety of preanalytical parameters, including all steps and operations occurring between blood collection and the moment PBMCs are delivered to the analytical laboratory. “The most critical preanalytical factors to take into account during blood collection are the blood collection tube type and anticoagulant, any stabilisers used and the blood storage temperature and time prior to PBMC isolation”, explains Dr. Fay Betsou, Chief Scientific Officer at IBBL and corresponding author. A variety of other parameters during PBMC isolation – such as the isolation method and centrifuge type – cryopreservation and subsequent thawing process – such as the cryomedium type, the long-term storage temperature the thawing procedure used – also need to be closely monitored. “To avoid biased results in PBMC assays, it is important that these critical preanalytical factors be standardised and documented, always taking into account the specific goals of the study and the functional applications that are being investigated. Participation of PBMC processing laboratories in external quality assurance programmes is also recommended, since it allows to benchmark the performance of the laboratory”, she adds.
The third review article — published in June 2019 under the same topical collection — deals with the issue of extracting DNA from Formalin-fixed paraffin-embedded (FFPE) tissue biospecimens for NGS analysis. Despite fixing clinical biospecimens in formalin and embedding them in paraffin being the preferred method of preparing biospecimens for diagnosis, nucleic acids extracted from FFPE tissue blocks are fragmented and low in quantity, containing an increased number of modifications and mutations compared to those extracted from fresh-frozen biospecimens. Preanalytical factors such as tissue degradation due to enzymatic activity, the efficiency of transport of the resected specimen from the operating theatre to the pathology department, the fixation method, fixation time and the chosen DNA extraction protocol are shown to affect the quality and yield of the extracted DNA. “To minimise the impact of these parameters on the quality and quantity of DNA extracted from FFPE tissues, labs can optimise their DNA extraction protocols by testing different reagents or kits or by participating in external quality assurance schemes to compare the performance of their protocols with those of other laboratories, thereby identifying the best performing protocols”, explains Dr. William Mathieson, corresponding author. “By continuously applying and strengthening quality control assays, we will be able to reduce the technical issues associated with FFPE blocks and increase the percentage of FFPE biospecimens that are fit-for-purpose and amenable to be sequenced through NGS”, he concludes.
The Topical Collection on Biospecimens Science and Evidence-Based Standards for Precision Medicine, edited by Dr. Fay Betsou, also includes other review articles focusing on exosome isolation and miRNA extraction and quantification.