Hematocrit-related issues remain a major barrier for (regulatory) acceptance of the classical dried blood spot (DBS) analysis in the bioanalytical and clinical field. Lately, many attempts to cope with these issues have been made. Throughout this review, an overview is provided on new strategies that try to cope with this hematocrit effect (going from avoiding to minimizing), on methods estimating a DBS volume, and on methods estimating or measuring the hematocrit of a DBS. Although many successful strategies have been put forward, a combination of different technologies still provides the most complete solution. Therefore, further efforts and the availability of a straightforward guideline for analytical and clinical method validation should help to overcome the hurdles still associated with DBS sampling.

Dried blood spots (DBS) have been used in newborn screening programs for several years. More recently, there has been growing interest in using DBS as a home sampling tool for the quantitative determination of analytes. However, this presents challenges, mainly because of the well-known hematocrit effect and other DBS-specific parameters, including spotted volume and punch site, which could add to the method uncertainty. Therefore, new microsampling devices that quantitatively collect capillary dried blood are continuously being developed. In this review, we provided an overview of devices that are commercially available or under development that allow the quantitative (volumetric) collection of dried blood (-based) microsamples and are meant to be used for home or remote sampling. Considering the field of therapeutic drug monitoring (TDM), we examined different aspects that are important for a device to be implemented in clinical practice, including ease of patient use, technical performance, and ease of integration in the workflow of a clinical laboratory. Costs related to microsampling devices are briefly discussed, because this additionally plays an important role in the decision-making process. Although the added value of home sampling for TDM and the willingness of patients to perform home sampling have been demonstrated in some studies, real clinical implementation is progressing at a slower pace. More extensive evaluation of these newly developed devices, not only analytically but also clinically, is needed to demonstrate their reallife applicability, which is a prerequisite for their use in the field of TDM.

In the field of bioanalysis, dried matrix spot sampling is increasingly receiving interest, as this alternative sampling strategy offers many potential benefits over traditional sampling, including matrix volume sparing properties. By using a microsampling strategy, e.g. volumetric absorptive microsampling (VAMS), the number of samples that can be collected from a patient can be increased, as a result of the limited sample volume that is required per sample. To date, no VAMS-based methods have been developed for the quantification of analytes in cerebrospinal fluid (CSF). The objective of this study was to develop and validate two LC-MS/MS methods for the quantification of paracetamol in dried blood and dried CSF, with both matrices sampled using VAMS. Both methods were fully validated based on internationally accepted guidelines. Paracetamol was chromatographically separated from its glucuronide and sulfate metabolites and no carry-over or unacceptable interferences were detected. The total precision (%RSD) was below 15% for all QC levels and accuracy (%bias) was below 7% (17% for the LLOQ of aqueous VAMS). The influence of the hematocrit on the recovery of blood VAMS samples appeared to be limited within the hematocrit range of 0.21 to 0.62.The blood VAMS samples were stable for 1 week if stored at 50 °C, and for at least 8 months when stored between -80 °C and room temperature. The aqueous VAMS samples were stable for at least 9 months when stored between -80 °C and 4 °C, and for 1 month when stored at room temperature.Application of the methods on external quality control material and analysis of patient samples demonstrated the validity and utility of the methods and provided a proof of concept for the analysis of CSF microsamples obtained via VAMS devices.

Nodding syndrome is a highly debilitating, generalized seizure disorder, affecting children in subregions of sub-Saharan Africa. Despite many efforts towards finding its etiology, the exact cause of the syndrome still remains obscure. Therefore, to date, patients only receive a symptomatic care, including the administration of first-generation anti-epileptic drugs (AEDs) for seizure control. Since information about medication effectiveness within this population is completely lacking, the aim of this study was to perform therapeutic drug monitoring (TDM) to seek whether an answer could be provided to the question why for some patients the symptoms decrease, whilst in others the epileptic seizures remain poorly controlled. Seeing the challenging context in which sampling needed to take place (remote areas, devoid of electricity, running water, etc.), dried blood matrices (i.e. dried blood spots (DBS) and volumetric absorptive microsampling (VAMS) devices) were considered fit-for-purpose.Seeing the similarities between the syndrome and other forms of epilepsy, also samples originating from patients suffering from (onchocerciasis-associated) epilepsy were included.In total, 68 patients with Nodding syndrome from Uganda, 58 Ugandan patients with epilepsy and 137 patients with onchocerciasis-associated epilepsy from the Democratic Republic of the Congo (DRC) were included in this study. VAMS samples and DBS were analyzed using fully validated methods, involving manual extraction or fully automated extraction, respectively, prior to quantification using liquid chromatography coupled to tandem mass spectrometry.Analysis revealed that serum concentrations (calculated from DBS) within the respective reference ranges were attained for only 52.9% of the 68 Nodding syndrome patients treated with valproic acid, for 21.4% of the 56 Ugandan epilepsy patients treated with carbamazepine, and for 65.7% of the 137 onchocerciasis-associated epilepsy patients from the DRC treated with phenobarbital. In all other instances, concentrations were subtherapeutic. Furthermore, when comparing DBS to VAMS concentrations, an inexplicable overestimation was observed in the latter. Finally, no obvious link could be observed between the obtained drug concentrations and the amount of seizures experienced during the last month before sampling, disclosing the fact that the level of improvement of some patients cannot simply linked to reaching therapeutic concentrations.

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