Clinical Heterogeneity in MT-ATP6 Pathogenic Variants: Same Genotype-Different Onset.

Human mitochondrial disease exhibits large variation of clinical phenotypes, even in patients with the same causative gene defect. We illustrate this heterogeneity by confronting clinical and biochemical data of two patients with the uncommon pathogenic homoplasmic NC_012920.1(MT-ATP6):m.9035T>C variant in MT-ATP6. Patient 1 presented as a toddler with severe motor and speech delay and spastic ataxia without extra-neurologic involvement. Patient 2 presented in adolescence with ataxia and ophthalmoplegia without cognitive or motor impairment. Respiratory chain complex activities were normal in cultured skin fibroblasts from both patients when calculated as ratios over citrate synthase activity. Native gels found presence of subcomplexes of complex V in fibroblast and/or skeletal muscle. Bioenergetic measurements in fibroblasts from both patients detected reduced spare respiratory capacities and altered extracellular acidification rates, revealing a switch from mitochondrial respiration to glycolysis to uphold ATP production. Thus, in contrast to the differing disease presentation, biochemical evidence of mitochondrial deficiency turned out quite similar. We conclude that biochemical analysis remains a valuable tool to confirm the genetic diagnosis of mitochondrial disease, especially in patients with new gene variants or atypical clinical presentation.

A challenging diagnosis of a nonsecretor plasma cell dyscrasia with pleomorphic plasmablastic morphology.

This report highlights the importance of integrating clinical, radiological, genetic, and pathological laboratory findings to make a correct diagnosis especially with challenging and rare entities.

Hematocrit prediction in volumetric absorptive microsamples.

Recently, volumetric absorptive microsampling (VAMS) has been suggested as an alternative to DBS sampling. With VAMS, a fixed volume of blood (approximately 10 µL) is wicked up by the absorbent tip of a collection device, independent of the hematocrit (HT) of the blood sample. This way, VAMS effectively avoids the HT bias which occurs in partial-punch DBS analysis. Nonetheless, the HT remains an important variable in VAMS analysis, particularly if VAMS-based blood results need to be converted to serum or plasma values to allow comparison with e.g. plasma-based therapeutic intervals. Indeed, an analyte's plasma to whole blood ratio may be HT-dependent. Therefore, we developed two straightforward methods to derive the HT value from a VAMS sample based on its potassium content. One of these methods uses an aqueous extraction procedure, whereas the other one requires an organic extraction. Both methods have the potential to be seamlessly integrated with most existing VAMS analyses, allowing both target analyte quantitation and potassium analysis on a single VAMS extract.

Development, validation and application of an inductively coupled plasma - mass spectrometry method to determine cobalt in metal-on-metal prosthesis patients using volumetric absorptive microsampling.

Metal-on-metal (MoM) prostheses, in which the bearing surfaces are made of a metal alloy, may release metal ions upon wear and corrosion, potentially inducing both local and systemic toxicity. As the systemic cobalt concentration increases with the degree of implant wear, this concentration needs to be monitored as a means of assessing implant function and the risk of adverse effects. Here, we report on the development, validation and application of a method to quantitatively assess these Co concentrations in whole blood, based on the combination of volumetric absorptive microsampling (VAMS) and inductively coupled plasma - mass spectrometry (ICP-MS). This method could allow patients to collect the required samples at home, as VAMS samples are easy to collect and can be transported to the laboratory via regular mail. The extraction procedure utilized an alkaline extraction mixture with yttrium as internal standard and proved to be independent of the hematocrit and age of the VAMS samples. The Co concentrations in the VAMS extracts were measured using quadrupole-based ICP-MS. The analytical method covers a range of 2-300 µg/L and displays excellent accuracy (bias ≤4%) and imprecision (relative standard deviation ≤ 5% and ≤15% at the lower limit of quantitation (LLOQ)). The method was applied to venous VAMS samples of MoM prosthesis patients (n = 78), yielding promising results. The comparison of these results with those obtained on the corresponding liquid whole blood samples, showed a correlation coefficient of 0.99 and 87% of the data fulfilled the criteria proposed by the Royal College of Pathologists of Australasia (RCPA).

Official International Association for Therapeutic Drug Monitoring and Clinical Toxicology Guideline: Development and Validation of Dried Blood Spot-Based Methods for Therapeutic Drug Monitoring.

Dried blood spot (DBS) analysis has been introduced more and more into clinical practice to facilitate Therapeutic Drug Monitoring (TDM). To assure the quality of bioanalytical methods, the design, development and validation needs to fit the intended use. Current validation requirements, described in guidelines for traditional matrices (blood, plasma, serum), do not cover all necessary aspects of method development, analytical- and clinical validation of DBS assays for TDM. Therefore, this guideline provides parameters required for the validation of quantitative determination of small molecule drugs in DBS using chromatographic methods, and to provide advice on how these can be assessed. In addition, guidance is given on the application of validated methods in a routine context. First, considerations for the method development stage are described covering sample collection procedure, type of filter paper and punch size, sample volume, drying and storage, internal standard incorporation, type of blood used, sample preparation and prevalidation. Second, common parameters regarding analytical validation are described in context of DBS analysis with the addition of DBS-specific parameters, such as volume-, volcano- and hematocrit effects. Third, clinical validation studies are described, including number of clinical samples and patients, comparison of DBS with venous blood, statistical methods and interpretation, spot quality, sampling procedure, duplicates, outliers, automated analysis methods and quality control programs. Lastly, cross-validation is discussed, covering changes made to existing sampling- and analysis methods. This guideline of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology on the development, validation and evaluation of DBS-based methods for the purpose of TDM aims to contribute to high-quality micro sampling methods used in clinical practice.

Correction for the Hematocrit Bias in Dried Blood Spot Analysis Using a Nondestructive, Single-Wavelength Reflectance-Based Hematocrit Prediction Method.

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.

A Novel, Nondestructive, Dried Blood Spot-Based Hematocrit Prediction Method Using Noncontact Diffuse Reflectance Spectroscopy.

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.

Spot them in the spot: analysis of abused substances using dried blood spots.

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.

Potassium-based algorithm allows correction for the hematocrit bias in quantitative analysis of caffeine and its major metabolite in dried blood spots.

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, potassium (K(+)) proved to be a reliable marker. The aim of this study was to evaluate whether application of an algorithm, based upon predicted Hct or K(+) concentrations as such, allowed correction for the Hct bias. Using validated LC-MS/MS methods, caffeine, chosen as a model compound, was determined in whole blood and corresponding DBS samples with a broad Hct range (0.18-0.47). A reference subset (n = 50) was used to generate an algorithm based on K(+) concentrations in DBS. Application of the developed algorithm on an independent test set (n = 50) alleviated the assay bias, especially at lower Hct values. Before correction, differences between DBS and whole blood concentrations ranged from -29.1 to 21.1%. The mean difference, as obtained by Bland-Altman comparison, was -6.6% (95% confidence interval (CI), -9.7 to -3.4%). After application of the algorithm, differences between corrected and whole blood concentrations lay between -19.9 and 13.9% with a mean difference of -2.1% (95% CI, -4.5 to 0.3%). The same algorithm was applied to a separate compound, paraxanthine, which was determined in 103 samples (Hct range, 0.17-0.47), yielding similar results. In conclusion, a K(+)-based algorithm allows correction for the Hct bias in the quantitative analysis of caffeine and its metabolite paraxanthine.

Hemato-critical issues in quantitative analysis of dried blood spots: challenges and solutions.

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.

Prediction of the hematocrit of dried blood spots via potassium measurement on a routine clinical chemistry analyzer.

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.