Bioanalysis Zone

Interview with Christophe P. Stove (Ghent University) on DBS in toxicology

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The Laboratory of Toxicology research group at the Faculty of Pharmaceutical Sciences of Ghent University (Ghent, Belgium) have recently published a paper providing an overview of the different DBS applications in toxicology. The strategy of this blood sampling technique and its potential promise are extensively discussed, as well as its relevance and implications for the future in the field of toxicology.

In this interview, Bioanalysis Zone asks Christophe Stove, corresponding author of this review, whether DBS holds just as much of a promising future as it once did, its potential limitations and how they can be overcome, and how bioanalysts, as a community, can help develop this technique to shape the face of drug development in preclinical studies.

In your opinion, what are the most difficult challenges surrounding DBS compared with more traditional based blood-sampling techniques?

The analysis of DBS is associated with several issues, such as contamination, blood volume spotted, blood spot homogeneity, hematocrit and so on. Of these, the hematocrit issue is undoubtedly the most widely discussed challenge, as nicely outlined by Fan and Lee in a recent Editorial in Bioanalysis [1]. Indeed, strongly deviating hematocrit values may significantly impact DBS-based quantitation. First of all, hematocrit strongly influences the spreading of blood on filter paper, with higher hematocrit values leading to smaller, more concentrated spots. Second, the hematocrit may influence parameters such as recovery and matrix effect. Third, when the DBS results are compared with those obtained from plasma, the distribution of an analyte in red blood cells and plasma needs to be examined on a case-by-case basis.

Several strategies have been developed to cope with this ‘hematocrit effect’. The easiest approach is analysis of complete DBS following the volumetric application of DBS, by the use of precision capillaries or other microsampling devices, delivering a fixed amount of blood to filter paper. However, volumetric application requires training and may be less feasible when DBS are obtained by patients at home (e.g., in the context of therapeutic drug monitoring programs). In this case, direct application from a clean fingertip may be the most feasible approach. As this implies nonvolumetric application, DBS punches, rather than complete DBS, should be evaluated, thus necessitating the definition of a hematocrit range and volume range, in which results can still fulfill the acceptance criteria for precision and accuracy. Whereas others have proposed the blood spot diameter as a tool to trace back the hematocrit of volumetrically applied DBS; up until now, it is not possible to trace back one’s hematocrit from a nonvolumetrically applied DBS. 

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To what extent do you believe contamination will affect the future of DBS as a preferred blood sampling technique of choice?

Avoiding and controlling contamination is of the utmost importance in any DBS application. The problem of contamination has primarily been studied in the context of DBS-based analysis of trace elements, where lead contamination is a big issue, especially when DBS are sampled ‘on-field’. In fact, contamination may occur at any time point throughout the procedure from sampling to analysis – the moment of sampling being the most critical point. Therefore, it should be stressed to those responsible for sampling (patients or medical staff) that, in all instances, direct finger contact or contact of any other potentially contaminated object with the paper surface should be avoided. Several options are available to control for potential contamination. These include the analysis of ‘unexposed’ blank paper and ‘exposed’ blank paper near a DBS, as well as the analysis of multiple punches per spot and/or the analysis of multiple spots. The latter option also requires incurred sample reanalysis as part of the validation protocol. For the analysis of trace elements, several specialized techniques are available that may help exclude contamination. However, one cannot completely rule out a scenario in which blank blood, obtained from a contaminated fingertip, gives rise to a positive DBS. This may be particularly relevant when considering the use of DBS in the context of drugs and driving, for example.

As is common practice in forensic hair analysis, demonstrating the presence of metabolites, or determining the ratio of metabolite to the main compound, may provide a potential solution in these cases too.

To what extent do you believe DBS has evolved in the bioanalytical community?

An objective answer on the evolution of DBS can be obtained by performing a simple PubMed abstract query for ‘dried blood spots’ versus ‘blood’. When looking at the number of publications in 2011 versus 2001, one can conclude that during these past 10 years, the increase in publications containing the former keywords, outperforms the increase in publications with ‘blood’ as the keyword by nearly 500%. Especially in the past 5 years, research on DBS is truly thriving. DBS sampling has been ‘rediscovered’ by the bioanalytical community, who holds the primary responsibility in its growth in recent years. Undoubtedly, the main catalysts in DBS progression are the recent improvements in analytical equipment. The more widespread use of LC–MS/MS, for example, has provided bioanalysts with the tools needed to analyze small sample volumes (e.g., a 3-mm DBS punch corresponds to merely 3 µl of blood).

Where do you predict the future of DBS lies within toxicology? And where, by comparison, do you believe the future of DBS lies within drug development?

The emergence of highly advanced inductively-coupled plasma (ICP)-MS configurations and ‘new generation’ MS/MS configurations, for example, will undoubtedly allow the bioanalyst to push the boundaries of DBS applications in toxicology and drug development even further. In toxicology, many avenues still remain unexplored. Possible applications that may become more widespread in the future include: the screening of environmental contaminants in animal or human DBS, the generation of DBS-based ‘metallic profiles’ and the detection of substance-abuse drugs. With respect to the latter, many applications have provided proof of principle, primarily by using DBS, which has been generated by spotting venous blood. However, only very few publications have demonstrated ‘real-life’ applicability, hence this is remaining an under developed area.

The widespread introduction of DBS sampling in clinical studies, conducted by the pharmaceutical industry, is primarily hampered by the lack of formal recognition by regulatory authorities. Here, the largest contribution in the near future may lie in the implementation of DBS sampling – and microsampling in general – in early drug-development phases. In toxicokinetics, for example, the sampling of small animals results in a perfect implementation of the principles of Reduction and Refinement.

What are the advantages of using automation in the field of DBS?

Without a shadow of a doubt, one of the areas currently in full development in DBS research is the automation of DBS analysis, including online extraction, direct desorption or any other approaches that allow high-throughput analysis. Although these techniques have only recently been established, it can be envisaged that their routine implementation may become, in the end, a rapid, easy and economic alternative for the analysis of samples obtained by classical venipuncture, which still requires the ‘classical’ sample-preparation procedure. An additional advantage may be increased analyte stability in DBS – although this requires an analyte-per-analyte evaluation. Having these technologies at hand – and taking into account possible limitations imposed by DBS punches – we feel here, at Ghent University, that DBS will continue to be standing in the spotlight.

[1] Fan L, Lee JA. Managing the effect of hematocrit on DBS analysis in a regulated environment. Bioanalysis 4(4), 345–347 (2012).

Source: Stove CP, Ingels AS, De Kesel PM, Lambert WE. Dried blood spots in toxicology: from the cradle to the grave? Crit. Rev. Toxicol. 42(3), 230–243 (2012).

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