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Dried blood spot (DBS) sampling for quantitative determination of drugs in blood has entered the bioanalytical arena at a fast pace during the last decade, primarily owing to progress in analytical instrumentation. Despite the many advantages associated with this new sampling strategy, several issues remain, of which the hematocrit issue is undoubtedly the most widely discussed challenge, since strongly deviating hematocrit values may significantly impact DBS-based quantitation. In this review, an overview is given of the different aspects of the 'hematocrit problem' in quantitative DBS analysis. The different strategies that try to cope with this problem are discussed, along with their potential and limitations. Implementation of some of these strategies in practice may help to overcome this important hurdle in DBS assays, further allowing DBS to become an established part of routine quantitative bioanalysis.

The potential of dried blood spot (DBS) sampling as an alternative for classical venous sampling is increasingly recognized, with multiple applications in, e.g., therapeutic drug monitoring and toxicology. Although DBS sampling has many advantages, it is associated with several issues, the hematocrit (Hct) issue being the most widely discussed challenge, given its possible strong impact on DBS-based quantitation. Hitherto, no approaches allow Hct prediction from nonvolumetrically applied DBS. Following a simple and rapid extraction protocol, K(+) levels from 3 mm DBS punches were measured via indirect potentiometry, using the Roche Cobas 8000 routine chemistry analyzer. The extracts' K(+) concentrations were used to calculate the approximate Hct of the blood used to generate DBS. A linear calibration line was established, with a Hct range of 0.19 to 0.63 (lower limit of quantification, LLOQ, to upper limit of quantification, ULOQ). The procedure was fully validated; the bias and imprecision of quality controls (QCs) at three Hct levels and at the LLOQ and ULOQ was less than 5 and 12%, respectively. In addition, the influence of storage (pre- and postextraction), volume spotted, and punch homogeneity was evaluated. Application on DBS from patient samples (n = 111), followed by Bland and Altman, Passing and Bablok, and Deming regression analysis, demonstrated a good correlation between the "predicted Hct" and the "actual Hct". After correcting for the observed bias, limits of agreement of ±0.049 were established. Incurred sample reanalysis demonstrated assay reproducibility. In conclusion, potassium levels in extracts from 3 mm DBS punches can be used to get a good prediction of the Hct, one of the most important "unknowns" in DBS analysis.

Dried blood spot (DBS) sampling and DBS analysis have increasingly received attention during recent years. Furthermore, a substantial number of DBS methods has recently become available in clinical, forensic and occupational toxicology. In this review, we provide an overview of the different DBS-based methods that have been developed for detecting (markers of) abused substances. These include both legal and illegal drugs belonging to different categories, including cannabinoids, cocaine and metabolites, opioids, benzodiazepines and Z-drugs, amphetamines and analogs, gamma-hydroxybutyric acid, ketamine and novel psychoactive substances such as cathinones. Markers of ethanol consumption and tobacco use are also covered in this review. Since the majority of published methods has shown promising results overall, an interesting role for DBS analysis in diverse toxicological applications can be envisaged. For the distinct applications, we discuss the specific potential and benefits of DBS, the associated limitations and challenges, as well as recent developments and future perspectives.

Dried blood spot (DBS) sampling is recognized as a valuable alternative sampling strategy both in research and in clinical routine. Although many advantages are associated with DBS sampling, its more widespread use is hampered by several issues, of which the hematocrit effect on DBS-based quantitation remains undoubtedly the most widely discussed one. Previously, we developed a method to derive the approximate hematocrit from a nonvolumetrically applied DBS based on its potassium content. Although this method yielded good results and was straightforward to perform, it was also destructive and required sample preparation. Therefore, we now developed a nondestructive method which allows to predict the hematocrit of a DBS based on its hemoglobin content, measured via noncontact diffuse reflectance spectroscopy. The developed method was thoroughly validated. A linear calibration curve was established after log/log transformation. The bias, intraday and interday imprecision of quality controls at three hematocrit levels and at the lower and upper limit of quantitation (0.20 and 0.67, respectively) were less than 11%. In addition, the influence of storage and the volume spotted was evaluated, as well as DBS homogeneity. Application of the method to venous DBSs prepared from whole blood patient samples (n = 233) revealed a good correlation between the actual and the predicted hematocrit. Limits of agreement obtained after Bland and Altman analysis were -0.076 and +0.018. Incurred sample reanalysis demonstrated good method reproducibility. In conclusion, mere scanning of a DBS suffices to derive its approximate hematocrit, one of the most important variables in DBS analysis.

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The hematocrit (Hct) effect is one of the most important hurdles currently preventing more widespread implementation of quantitative dried blood spot (DBS) analysis in a routine context. Indeed, the Hct may affect both the accuracy of DBS methods as well as the interpretation of DBS-based results. We previously developed a method to determine the Hct of a DBS based on its hemoglobin content using noncontact diffuse reflectance spectroscopy. Despite the ease with which the analysis can be performed (i.e., mere scanning of the DBS) and the good results that were obtained, the method did require a complicated algorithm to derive the total hemoglobin content from the DBS's reflectance spectrum. As the total hemoglobin was calculated as the sum of oxyhemoglobin, methemoglobin, and hemichrome, the three main hemoglobin derivatives formed in DBS upon aging, the reflectance spectrum needed to be unmixed to determine the quantity of each of these derivatives. We now simplified the method by only using the reflectance at a single wavelength, located at a quasi-isosbestic point in the reflectance curve. At this wavelength, assuming 1-to-1 stoichiometry of the aging reaction, the reflectance is insensitive to the hemoglobin degradation and only scales with the total amount of hemoglobin and, hence, the Hct. This simplified method was successfully validated. At each quality control level as well as at the limits of quantitation (i.e., 0.20 and 0.67) bias, intra- and interday imprecision were within 10%. Method reproducibility was excellent based on incurred sample reanalysis and surpassed the reproducibility of the original method. Furthermore, the influence of the volume spotted, the measurement location within the spot, as well as storage time and temperature were evaluated, showing no relevant impact of these parameters. Application to 233 patient samples revealed a good correlation between the Hct determined on whole blood and the predicted Hct determined on venous DBS. The bias obtained with Bland and Altman analysis was -0.015 and the limits of agreement were -0.061 and 0.031, indicating that the simplified, noncontact Hct prediction method even outperforms the original method. In addition, using caffeine as a model compound, it was demonstrated that this simplified Hct prediction method can effectively be used to implement a Hct-dependent correction factor to DBS-based results to alleviate the Hct bias.

Although dried blood spot (DBS) sampling is increasingly receiving interest as a potential alternative to traditional blood sampling, the impact of hematocrit (Hct) on DBS results is limiting its final breakthrough in routine bioanalysis. To predict the Hct of a given DBS,