Tag Archives: Olga Kofanova

DNA extracted from Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks is increasingly being used for applications such as Next Generation Sequencing (NGS) both in oncology research and clinical diagnostics. However, the accuracy and reproducibility of this analysis, as well as its resilience to variations in the quality of the samples used, remain to be ascertained. IBBL recently released its 100th publication, reviewing specifically the preanalytical factors affecting the quality of DNA extracted from fixed tissue. Dr. Olga Kofanova, Biospecimen Quality Team Leader at IBBL, explains the main findings.

Interview by Federica Amato (FA)

FA: In this publication you focus on DNA extraction from fixed tissues. Can you explain what fixation entails and why it is an important factor to consider when evaluating the quality of the extracted DNA?

OK: Before tissue samples can be used for diagnostics or research purposes, they need to be preserved and be made amenable to further analyses. This is done either chemically (by fixation or stabilisation) or cryogenically (by freezing). Typically, most tissue biospecimens are fixed using formalin and are then embedded in paraffin, which results in what is known as a formalin-fixed paraffin-embedded (FFPE) tissue block. Despite the many advantages of this method, the process of formalin fixation often induces the fragmentation of the extracted DNA, as well as chemical modifications and crosslinking, which can negatively affect the efficiency of the extraction and of the amplification of the extracted DNA. Non-formalin alternatives such as the PAXgene tissue fixative were developed to address these shortcomings, resulting in so-called PAXgene-fixed paraffin-embedded (PFPE) tissue blocks.

FA: In addition to the type of fixative, what are the other critical parameters that can affect the quality and quantity of the DNA extracted from FFPE tissues?

OK: Considerable work has been done by the international community, including the National Cancer Institute (NCI) and the European Committee for Standardization (CEN), on this subject. The most critical preanalytical variables to be considered are tissue fixation time, storage conditions of the FFPE blocks and the choice of DNA extraction method. Indeed, the goal is to have enough DNA of sufficient integrity after extraction, if we want to obtain reliable results when performing downstream NGS analyses. This is why, prior to NGS, it is important to carry out Quality Control of the extracted DNA to assess its yield and integrity. The former is typically done by techniques such as spectrofluorometry or quantitative PCR, while DNA integrity is evaluated by the DNA Integrity Number (DIN). In this paper, we specifically assessed the impact of different types of fixatives – namely FFPE and PFPE – and DNA extraction methods on the quality of the extracted DNA.

FA: Can you briefly describe the experimental setup?

OK: The idea behind the study design is simple. We took 15 FFPE tissue blocks and 15 PFPE tissue blocks, extracted DNA using four different DNA extraction methods and then performed a series of analyses to assess whether the type of fixative and extraction technique affected various quality parameters in the extracted DNA. Specifically, we measured DNA concentration and purity by spectrophotometry and spectrofluorometry, we assessed DNA integrity and the extent of crosslinking by PCR and Whole Genome Amplification (WGA) and carried out NGS followed by bioinformatics analysis to estimate any damage in the DNA samples which would result in incorrect sequencing data. We also compared the results with 15 paired “gold standard” tissue samples, collected in a nucleic acid stabiliser.

FA: What did you find out?

OK: In terms of DNA yield and purity, we found that FFPE and PFPE processing resulted in lower quantities of extracted DNA compared to the reference samples. When looking specifically at DNA extracted from FFPE samples, higher yields were observed when using the QIAamp DNA FFPE extraction method, compared to the other extraction techniques. Conversely, we found the yield of DNA extracted from PFPE tissue to be higher when using the PAXgene Tissue DNA and Chemagic DNA extraction methods. With regard to the purity of the extracted DNA, the best quality DNA from fixed tissue was obtained with the PAXgene Tissue DNA kit from PFPE tissue and with the QIAamp DNA FFPE kit from FFPE tissue. As for DNA integrity, the best quality DNA from fixed material was DNA extracted with the Chemagic DNA Tissue kit from either PFPE or FFPE tissue. Essentially, we showed that the combination of fixative and DNA extraction method affect the yield, purity and integrity of the resulting DNA. In particular, the use of non-formalin fixatives such as PAXgene coupled with the Chemagic extraction method has shown better results in terms of DNA yield and integrity, but also accuracy of NGS analyses.

FA: And what are the implications for NGS and Quality Control?

OK: Once we have measured parameters such as the ones discussed above, we can use these values to detect the DNA samples of higher quality for the purpose of NGS analysis. Namely, the best ‘diagnostic’ performance in identifying the DNA samples that would be best suited for NGS was obtained with the combination of double stranded (ds) DNA proportion and DNA integrity. Specifically, we found that the DNA samples most amenable to NGS should contain at least 25% of double-stranded DNA (dsDNA), with DNA fragments having a length of at least 200 base pairs – as measured by multiplex PCR – and at least an ’intermediate’ integrity score – as measured by WGA. Researchers can use this finding as a useful combinatorial Quality Control (QC) assay to assess whether their extracted DNA samples will give reliable results when being subjected to NGS.

FA: This paper is a particularly special one for IBBL! 

OK: Indeed it is! It is our 100th publication, a considerable milestone! From a scientific standpoint, the paper reviews critical aspects pertaining to the preanalytics of DNA extraction and proposes a combinatorial QC metric that guides researchers in the qualification of DNA samples and in the assessment of their fitness for purpose in terms of NGS. In addition, the publication is the result of fruitful collaborations with partners from the Greater Region and from the Luxembourg biomedical research environment, specifically the Luxembourg Institute of Health (LIH), which assisted us with bioinformatics analyses. Moreover, the publication is part of a collection of articles published in the context of the European project SPIDIA4P.

FA: Thank you very much Olga for taking the time to answer my questions!

The detailed results of the study can be found in the full paper here.

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.

In a new study published in the Biopreservation & Biobanking journal IBBL scientists show that there is still no validated urine biomarker for pre-analytical variations.

High-throughput analysis in genomics and proteomics, which facilitate or enable the simultaneous study of thousands of genes or proteins, have substantially advanced biomedical research in recent years. Importantly, they have led to the discovery and subsequent use of a number of biomarkers for diagnosis, prognosis or drug response. However, new technologies always bring along new challenges. The large scale of these technologies means that samples from different patients need to be treated in exactly the same fashion before analysis to ensure that observed differences in proteins or genes are not due to variations in the pre-analytical process.

As one of a few biobanks to carry out their own research, IBBL, specifically the team around Chief Scientific Officer, Dr Betsou, has focussed on studying the impact of such pre-analytical variations on the down-stream results, in an effort to discover and validate markers for biospecimen quality control (QC). These QC markers are molecules, for example proteins, that are sensitive to the method of collection, processing or storage, allowing researchers to detect when samples have not been handled correctly.

In a recently published study Dr Betsou and Dr Kofanova, Biospecimen Research Scientist at IBBL, evaluated the usefulness of lithostatine as a quality control tool for urine sample processing in routine biobanking. A previous guideline suggested that the concentration of this protein could be a useful pre-analytical biomarker, but IBBL’s researchers were unable to confirm this finding. Using a routine protein assay, they showed that lithostatine concentration in urine does not change significantly in response to common pre-analytical variations, such as a delay in sample processing or repeated freeze-thaw cycles. Thus, lithostatine concentration cannot be considered a useful tool to assess the integrity of urine samples and the urgent need for a urine quality control marker remains.

The full article is available here 

Given the importance of low temperature storage in biobanks and biorepositories, IBBL’s biospecimen researchers have published a concept paper to guide the implementation and standardisation of fundamental cryobiological principles in biobanks.

Choosing the right storage and processing methods is key in biobanking and biopreservation, because they are determining factors for the quality of biospecimens. Since many specimens and samples are stored at low temperature, cryobiology (from the Greek: “cryo” = cold) is a fundamental branch of science applied in biobanks. However, there seems to be a certain lack of understanding and knowledge of low temperature storage principles within the biobanking sector. On the other hand, the objective in biobanks is not always the preservation of cell viability but increasingly includes the maintenance of genetic stability, meaning that traditional cryobiological principles do not always apply.

In order to improve understanding and communication, scientists at IBBL have published their strategy, based on five core concepts, to help translate cryobiological knowledge from scientific publications into practices applicable in biobanks, where quality assurance, risk management and Standard Operating Procedure (SOPs) govern daily operations. The concept paper was written in collaboration with experts in both biorepositories and cryobiology and through close interactions with the three main societies in these fields; ISBER (International Society for Biological and Environmental Repositories), SLTB (Society for Low Temperature Biology) and SfC (Society for Cryobiology).

“Although distinct sectors, we share many common interests.” says Dr Olga Kofanova, Biospecimen Research Scientist at IBBL. ”This concept paper is a first step to achieving consensus across these different disciplines. It identifies critical factors as a stepping stone for the implementation of low temperature storage and processing standards within the biobanking sector.”

The full article is available here

IBBL’s Biospecimen Research team has published new data which suggests that heat stabilisation of tissues is an alternative processing method for the analysis of certain phosphoproteins.

Phosphoproteins are crucial in the regulation and execution of a variety of processes within human cells and have been linked to multiple diseases. Their potential as biomarkers means that they are being extensively studied in the field of personalised medicine. The quality and amount of phosphoproteins that are extracted from human tissues is of paramount importance in this type of research. Unfortunately this type of protein is particularly susceptible to degradation and modifications during processing, which can distort research results, especially if there is variation in the collection or processing methods.

In collaboration with the NorLux laboratory of Neuro-Oncology of the CRP-Santé, IBBL scientists carried out a study to compare 2 very different stabilisation methods that aim to preserve tissues, and thus phosphoproteins, in their native state. Their data, published in the Biopreservation and Biobanking journal, shows that heat stabilisation, which uses instant 95˚C heating and pressure, preserved certain phosphoproteins significantly better than snap-freezing, where samples are rapidly frozen to below -80˚C. In addition, the team around Dr Fay Betsou reported that the stability of these proteins was affected by the method of extraction from tissues.

IBBL’s Biospecimen Research Scientist, Dr Olga Kofanova, who carried out the study, comments: “Our results show that heat stabilisation may be a good alternative for the specific analysis of phosphoproteins and that the phosphoprotein assay may also be a useful tool for quality control. It also underlines the importance of biospecimen research in continuously improving our techniques to offer the highest quality samples to our collaborators and clients”.

The full article is available here