Acceptability of dried blood spot collection by primary caregivers of Filipino patients with maple syrup urine disease (MSUD) and phenylketonuria (PKU).

MSUD and PKU require lifetime management hence, regular monitoring of amino acid levels is needed to achieve good metabolic control. Ideally, plasma amino acid analysis (PLAA) is used to monitor concentrations but is expensive and not widely available in local laboratories. The newborn screening program in the Philippines uses dried blood spot (DBS) analysis as an alternative where only trained healthcare providers are allowed to perform the collection at selected facilities. With the increasing number of patients, DBS monitoring has been noted to be delayed due to multiple factors. This issue became even more evident during the COVID-19 pandemic where high-risk patients need to travel outside for blood collection. The study used a cross-sectional study design to determine the primary caregivers' perspective on DBS self-sampling for patients with MSUD and PKU and the acceptability of the samples collected. This was done through a series of collection training, pre-/post- surveys, and 10-item questionnaire, and an in-depth 1-on-1 interview for thematic analysis. The acceptability of samples was processed and evaluated by the newborn screening laboratory. At-home DBS collection by primary caregivers was found to be acceptable. The provision of knowledge and routine collection training by the medical team aids in the increase of sample acceptability as well as a source of empowerment in being equipped to take care of their child. It is highly recommended that DBS samples collected by caregivers be considered acceptable for more time and cost-saving monitoring of the patients' metabolites. This practice also promotes timely and appropriate management which can lead to better patient health outcomes.

Utility of an Archival Dried Blood Spot (DBS) Collection from HIV-Infected Individuals with and without Cancer in a Resource-Limited Setting.

The frequency of virus-associated cancers is growing worldwide, especially in resource-limited settings. One of the biggest challenges in cancer research among people living with HIV (PLWH) has been understanding how infection with both HIV and Kaposi sarcoma-associated herpesvirus (KSHV) promotes the pathogenesis of Kaposi sarcoma (KS), the most common cancer among PLWH worldwide and a significant public health problem in regions with high prevalence of HIV such as Sub-Saharan Africa (SSA). The AIDS and Cancer Specimen Resource (ACSR) provides samples for research, including dried blood spots (DBS) that were collected from large clinical epidemiology studies of KSHV and KS in PLWH conducted more than a decade ago in SSA. Here, we validated the quality of DNA derived from DBS samples from SSA studies and provided evidence of quantitative recovery of inflammatory cytokines using these DBS samples through comparison with paired frozen plasma. Significant differences in DNA, protein yields, and inflammatory biomarker levels were also observed between PLWH with/without KS. Establishing the fitness of DBS samples for studies of KS pathogenesis extends the number of projects that can be supported by these ACSR special collections and provides evidence that DBS collection for future KS research is a practical option in resource-limited settings.

Evaluating the detection of barbiturates in dried blood spots: A comparative analysis using gas chromatography-mass spectrometry, gas chromatography-tandem mass spectrometry, and liquid chromatography-tandem mass spectrometry with different extraction methods.

Rapid and accurate characterization and quantitation of blood barbiturates and their combination drugs are very important for the clinical treatment of acute barbiturate poisoning. A comparison of dried blood spot (DBS) and traditional liquid-liquid extraction (LLE) in the pre-treatment stage, as well as a comparison of gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS/MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) as instrumental analysis methods, revealed differences in the analysis results of barbiturates and their combination drugs under different conditions. Based on these findings, we introduce a DBS-GC-MS/MS method. The developed and validated method showed good selectivity, sensitivity (LOD: 0.1 µg mL-1, LOQ: 0.2 µg mL-1), linearity (R2>0.9992), trueness (<15 %, except for carbamazepine, at 29.4 %), and precision (<15 %). Recovery was also good for most target compounds, but significant matrix effects were evident. Compared with the LLE method, the DBS method has the benefits of easy sample collection, storage, and transport, as well as simple pre-treatment and reduced reagent and energy consumption. Compared to LC-MS/MS, GC-MS/MS requires no switching between positive and negative ion modes and uses the MRM detection mode, meaning that more information about the sample compounds can be obtained in less analysis time. Using actual sample analysis, we have demonstrated the advantages of the DBS-GC-MS/MS method for the qualitative and quantitative analysis of barbiturates and poisoning events due to combinations of these drugs. Comparison of the three instruments and the two treatment methods revealed their analysis characteristics. From the perspective of practical application, the broad practical value and advantages of DBS should be embraced in more applications, and future analytical laboratory development should continue to recognize GC-MS/MS as a useful supplement to LC-MS/MS.

Monitoring SARS-CoV-2 IgA, IgM and IgG antibodies in dried blood and saliva samples using antibody proximity extension assays (AbPEA).

Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from ø1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

Development and validation of UPLC-MS/MS method for 5-Fluorouracil quantification in dried blood spot.

5-Fluorouracil is an antimetabolite drug indicated for cancer treatment. Therapeutic drug monitoring of 5-Fluorouracil is necessary because 5-Fluorouracil has narrow therapeutic window and its concentration in blood is affected by individual conditions, like gene polymorphisms. Dried Blood Spot (DBS) is one of the biosampling methods used for therapeutic drug monitoring. Asides from reducing patients' discomfort, the use of DBS can increase 5-Fluorouracil stability by stopping the enzymes activity in blood. Therefore, this research developed a method to monitor 5-Fluorouracil levels in DBS using ultra-high performance liquid chromatography-tandem mass spectrometry. Sample preparation was carried out by extracting DBS using 2-Propanol: ethyl acetate (16:84). Reconstituted samples were analyzed using ultra high performance liquid chromatography equipped with Acquity® UPLC BEH C18 column (2.1 × 100 mm; 1.7 µm). The ionization process was carried out in negative electrospray ionization mode. Multiple Reaction Monitoring (MRM) values were set at m/z 128.97 > 41.82 for 5-Fluorouracil and 168.97 > 57.88 for propylthiouracil as the internal standard. Optimum analytical conditions were obtained with acetonitrile-ammonium acetate 1 mM (95:5) as mobile phase, flow rate of 0.15 mL/min, and column temperature of 40 °C. The lowest level of quantification obtained from this method was 0.1 µg/mL with a calibration curve range of 0.1 µg/mL-60 µg/mL. This method was proven to be valid according to the requirements set by the US Food and Drug Administration and the European Medicines Agency.

Prevalence and predictors of residual antibiotics in children's blood in community settings in Tanzania.

OBJECTIVES: Children account for a significant proportion of antibiotic consumption in low- and middle-income countries, with overuse occurring in formal and informal health sectors. This study assessed the prevalence and predictors of residual antibiotics in the blood of children in the Mbeya and Morogoro regions of Tanzania. METHODS: The cross-sectional community-based survey used two-stage cluster sampling to include children aged under 15 years. For each child, information on recent illness, healthcare-seeking behaviour, and use of antibiotics, as well as a dried blood spot sample, were collected. The samples underwent tandem mass spectrometry analysis to quantify the concentrations of 15 common antibiotics. Associations between survey variables and the presence of residual antibiotics were assessed using mixed-effects logistic regression. RESULTS: In total, 1742 children were surveyed, and 1699 analysed. The overall prevalence of residual antibiotics in the blood samples was 17.4% (296/1699), the highest among children under the age of 5 years. The most frequently detected antibiotics were trimethoprim (144/1699; 8.5%), sulfamethoxazole (102/1699; 6.0%), metronidazole (61/1699; 3.6%), and amoxicillin (43/1699; 2.5%). The strongest predictors of residual antibiotics in the blood were observed presence of antibiotics at home (adjusted odds ratio [aOR] = 2.9; 95% CI, 2.0-4.1) and reported consumption of antibiotics in the last 2 weeks (aOR = 2.5; 95% CI, 1.6-3.9). However, half (145/296) of the children who had residual antibiotics in their blood, some with multiple antibiotics, had no reported history of illness or antibiotic consumption in the last 2 weeks, and antibiotics were not found at home. DISCUSSION: This study demonstrated a high prevalence of antibiotic exposure among children in Tanzanian communities, albeit likely underestimated, especially for compounds with short half-lives. A significant proportion of antibiotic exposure was unexplained and may have been due to unreported self-medication or environmental pathways. Incorporating biomonitoring into surveillance strategies can help better understand exposure patterns and design antibiotic stewardship interventions.

A Systematic Literature Review on the Use of Dried Biofluid Microsampling in Patients With Kidney Disease.

BACKGROUND: Kidney disease is fairly unique due to the lack of symptoms associated with disease activity, and it is therefore dependent on biological monitoring. Dried biofluids, particularly dried capillary blood spots, are an accessible, easy-to-use technology that have seen increased utility in basic science research over the past decade. However, their use is yet to reach the kidney patient population clinically or in large-scale discovery science initiatives. The aim of this study was to systematically evaluate the existing literature surrounding the use of dried biofluids in kidney research. METHODS: A systematic literature review was conducted using three search engines and a predefined search term strategy. Results were summarised according to the collection method, type of biofluid, application to kidney disease, cost, sample stability and patient acceptability. RESULTS: In total, 404 studies were identified and 67 were eligible. In total, 34,739 patients were recruited to these studies with a skew towards male participants (> 73%). The majority of samples were blood, which was used either for monitoring anti-rejection immunosuppressive drug concentrations or for kidney function. Dried biofluids offered significant cost savings to the patient and healthcare service. The majority of patients preferred home microsampling when compared to conventional monitoring. CONCLUSION: There is an unmet need in bringing dried microsampling technology to advance kidney disease despite its advantages. This technology provides an opportunity to upscale patient recruitment and longitudinal sampling, enhance vein preservation and overcome participation bias in research.

Evaluation of a single Eporatio® micro-dose in urine and dried blood spots.

Recombinant human erythropoietin (rhEPO) has been abused as a performance enhancer in sports for several years, but with advancements in detection methods, even micro-doses can be detected in dried blood spot (DBS) samples. Here, we present the results from an Eporatio® (epoetin theta) micro-dose administration study to detect rhEPO in DBS samples. Five healthy male volunteers received a 15 IU/kg subcutaneous dose of Eporatio®. Urine and DBS samples (Mitra® VAMS and Capitainer® B50) were collected 1, 10, 24, 36, 48 and 72 h after drug administration. After 1 h, all urine samples were negative for rhEPO, whereas 40% of DBS samples were considered suspicious. All samples between 10 and 48 h were suspicious for the presence of Eporatio®, except one urine sample that was negative at 48 h. After 72 h, 40% of urine samples and 60% of DBS samples were suspicious and would have proceeded to a confirmation analysis. DBS is an efficient complementary matrix to urine for detection of rhEPO micro-doses.

Revolutionizing HIV-1 Viral Load Monitoring in India: The Potential of Dried Blood Spot Analysis for Expanding Access and Improving Care.

India continues to grapple with a significant burden of HIV infections. Despite notable progress in prevention and treatment efforts, multiple challenges, such as high-risk populations, inadequate testing facilities, and limited access to healthcare in remote areas, persist. Though the Government of India offers HIV-1 plasma viral load testing at various medical centers, aiding treatment decisions and monitoring antiretroviral therapy effectiveness, enhancing care for individuals living with HIV under the National AIDS Control Program (NACP), the nation's large population and diverse demographics further complicate its outreach and response. Hence, strategic interventions and alternative methods of testing remain crucial to curbing HIV transmission and improving the quality of life for those affected. Dried blood spot (DBS) sampling has emerged as a convenient and cost-effective alternative for HIV-1 viral load testing, revolutionizing the landscape of diagnostic and monitoring strategies for HIV infection. Though the plasma-based viral load remains the gold standard for monitoring HIV-1, DBS-based HIV-1 viral load testing holds immense promise for improving access to care, particularly in resource-limited settings where traditional plasma-based methods may be logistically challenging. DBS entails the collection of a small volume of blood onto filter paper, followed by drying and storage. This approach offers numerous advantages, including simplified sample collection, transportation, and storage, reducing the need for cold-chain logistics. Recent studies have demonstrated the feasibility and accuracy of DBS-based HIV-1 viral load testing, revealing a strong correlation between DBS and plasma measurements. Its implementation can enhance the early detection of treatment failure, guide therapeutic decisions, and ultimately contribute to better clinical outcomes for HIV-infected individuals. Hence, this review explores the principles, advancements, feasibility, and implications of DBS-based HIV-1 viral load testing.

A comparative study of plasma and dried blood spot metabolomics and its application to diabetes mellitus.

Metabolomics has become a promising method for understanding pathological mechanisms. Plasma (PLS) is the most common sample type used for metabolomics studies, and dried blood spot (DBS) sampling has been regarded as a good strategy due to its unique characteristics. However, how results obtained from DBS can be correlated to results obtained from PLS remains unclear. To bridge the results and to investigate the feasibility of using DBS to study metabolomics, we performed a comparative study using 64 paired PLS and DBS samples. The number of features extracted from the two different sample types was investigated. The concentration correlations of the identified metabolites between the DBS and PLS were individually studied. Approximately 47 % showed a strong correlation, 19 % showed a moderate correlation, and 34 % showed a low or even negligible correlation. Finally, we applied both PLS- and DBS-based metabolomics to explore the dysregulated metabolites in diabetes mellitus (DM) patients. Thirty-two non-DM subjects and 32 DM patients were enrolled, and 2 significant metabolites were found in both PLS and DBS samples. In summary, detailed correlation information between PLS and DBS metabolites was first explored in this study, and it is anticipated that these results could facilitate future applications in DBS-based metabolomics.

Use of dried blood spots for monitoring inflammatory and nutritional biomarkers in the elderly.

OBJECTIVES: Blood microsampling, particularly dried blood spots (DBSs), is an attractive minimally-invasive approach that is well suited for home sampling and predictive medicine associated with longitudinal follow-up of the elderly. However, in vitro diagnostic quantification of biomarkers from DBS poses a major challenge. Clinical mass spectrometry can reliably quantify blood proteins in various research projects. Our goal here was to use mass spectrometry of DBS in a real-world clinical setting and compared it to the standard immunoassay method. We also sought to correlate DBS mass spectrometry measurements with clinical indices. METHODS: A clinical trial of diagnostic equivalence was conducted to compare conventional venous samples quantified by immunoassay and DBSs quantified by mass spectrometry in an elderly population. We assayed three protein biomarkers of nutritional and inflammatory status: prealbumin (transthyretin), C-reactive protein, and transferrin. RESULTS: The analysis of DBSs showed satisfactory variability and low detection limits. Statistical analysis confirmed that the two methods give comparable results at clinical levels of accuracy. In conclusion, we demonstrated, in a real-life setting, that DBSs can be used to measure prealbumin, CRP and transferrin, which are commonly used markers of nutritional status and inflammation in the elderly. However, there was no correlation with patient frailty for these proteins. CONCLUSIONS: Early detection and regular monitoring of nutritional and inflammatory problems using DBS appear to be clinically feasible. This could help resolve major public health challenges in the elderly for whom frailty leads to serious risks of health complications.

Validating blood microsampling for per- and polyfluoroalkyl substances quantification in whole blood.

Microsampling allows the collection of blood samples using a method which is inexpensive, simple and minimally-invasive, without the need for specially-trained medical staff. Analysis of whole blood provides a more holistic understanding of per- and polyfluoroalkyl substances (PFAS) body burden. Capillary action microsamplers (Trajan hemaPEN®) allow the controlled collection of whole blood as dried blood spots (DBS) (four 2.74 µL ± 5 %). The quantification of 75 PFAS from DBS was evaluated by comparing five common extraction techniques. Spiked blood (5 ng/mL PFAS) was extracted by protein precipitation (centrifuged; filtered), acid-base liquid-liquid extraction, trypsin protease digestion, and weak anion exchange (WAX) solid-phase extraction with analysis by high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Filtered protein precipitation was the most effective extraction method, recovering 72 of the 75 PFAS within 70 to 130 % with method reporting limit (MRL) for PFOS of 0.17 ng/L and ranging between 0.05 ng/mL and 0.34 ng/mL for all other PFAS. The optimised method was applied to human blood samples to examine Inter- (n = 7) and intra-day (n = 5) PFAS blood levels in one individual. Sixteen PFAS were detected with an overall Σ16PFAS mean = 6.3 (range = 5.7-7.0) ng/mL and perfluorooctane sulfonate (branched and linear isomers, ΣPFOS) = 3.3 (2.8-3.7) ng/mL being the dominant PFAS present. To the authors knowledge, this minimally invasive self-sampling protocol is the most extensive method for PFAS in blood reported and could be a useful tool for large scale human biomonitoring studies.

Monitoring Exposure to Five Chemical Warfare Agents Using the Dried Urine Spot Technique and Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-In Vivo Determination of Sarin Metabolite in Mice.

Dried urine spot (DUS) is a micro-sample collection technique, known for its advantages in handling, storage and shipping. It also uses only a small volume of urine, an essential consideration in working with small animals, or in acute medical situations. Alkyl-phosphonic acids are the direct and indicative metabolites of organophosphorus chemical warfare agents (OP-CWAs) and are present in blood and urine shortly after exposure. They are therefore crucially important for monitoring casualties in war and terror scenarios. We report here a new approach for the determination of the metabolites of five CWAs in urine using DUS. The method is based on a simple and rapid sample preparation, using only 50 µL of urine, spotted and dried on DBS paper, extracted using 300 µL methanol/water and analyzed via targeted LC-MS/MS. The detection limits for the five CWAs, sarin (GB), soman (GD), cyclosarin (GF), VX and RVX in human urine were from 0.5 to 5 ng/mL. Recoveries of (40-80%) were obtained in the range of 10-300 ng/mL, with a linear response (R2 > 0.964, R > 0.982). The method is highly stable, even with DUS samples stored up to 5 months at room temperature before analysis. It was implemented in a sarin in vivo exposure experiment on mice, applied for the time course determination of isopropyl methylphosphonic acid (IMPA, sarin hydrolysis product) in mice urine. IMPA was detectable even with samples drawn 60 h after the mice's (IN) exposure to 1 LD50 sarin. This method was also evaluated in a non-targeted screening for multiple potential CWA analogs (LC-Orbitrap HRMS analysis followed by automatic peak detection and library searches). The method developed here is applicable for rapid CWA casualty monitoring.

Dried blood spots for erythropoietin analysis: Detection of micro-doses, EPO c.577del variant and comparison with in-competition matching urine samples.

The abuse of recombinant erythropoietin (rEPO) and other erythropoietin (EPO) receptor agonists (ERAs) in sports prompted the need for sensitive detection methods of these substances. Dried blood spot (DBS) samples offer an easy solution for simultaneous collection of blood and urine during a doping control, but sensitivity issues are often presented as a challenge for routine EPO analysis from DBS. Its potential use for detecting rEPO micro-doses and the EPO gene c.577del variant thus needed further demonstration. Here, capillary blood collected from the arm skin of 111 athletes with Tasso-M20 (17.5 µL/spot), collected during professional triathlon competitions, were analysed. Also, venous blood samples from healthy volunteers were used to prepare several spots of 20 µL on Mitra VAMS (from an rEPO micro-dose study) and Whatman filter paper (from an EPO gene variant study). Immunopurification of 2 spots with MAIIA EPO Purification Gel Kit and analysis with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE)/Western blot resulted in sensitive detection of (1) micro-doses of rEPO from Mitra VAMS, (2) endogenous EPO from Tasso-M20 in all in-competition subjects, and (3) the EPO c.577del variant from Whatman filter paper. Additionally, in-competition endogenous EPO was detected in DBS even when matching urine samples had undetectable EPO. In conclusion, this work demonstrated that DBS can be a useful complementary matrix to urine samples for EPO detection.

Rapid LC-MS/MS detection of 25-hydroxyvitamin D in dried blood spots.

BACKGROUND: The detection of 25-hydroxyvitamin D (25OHD) from dried blood spots (DBS) has been widely studied. However, the existing pretreatment methods suffer from limitations in terms of throughput (usually exceeding 2 h), complexity (involving liquid-liquid extraction or solid-phase extraction), and contamination (including multiple steps of organic solvent evaporation). RESULTS: We first released 25OHD from DBS samples by 50% acetonitrile solution through ultrasonication. Subsequently, the cold-induced phase separation technique was introduced for in-situ concentration and purification. Afterward, the PTAD derivatization of 25OHD was performed directly, profiting from the high acetonitrile content in the concentrated solution. In the end, the resulting solution was submitted to LC-MS/MS for quantification. The established LC-MS/MS methodology possessed favorable analytical performance, possessing lower limit of quantification of 1 ng/mL pointing to plasma, accuracy of 86.8-110.1% and imprecision of 5.4-16.8%. Method comparison with plasma samples demonstrated that over 93% of the detections met the acceptance limit for cross-validation of ±20%. SIGNIFICANCE AND NOVELTY: The novel sample preparation can be finished within 15 min and eliminated the traditional steps of extraction and organic solvent evaporation. Based on this high-throughput, reliable and applicable LC-MS/MS method, the detection of 25OHD in DBS samples can be better achieved for clinical patients and researchers with relevant demands.

Portuguese Neonatal Screening Programme: A Retrospective Cohort Study of 18 Years of MS/MS.

INTRODUCTION: The Portuguese Neonatal Screening Programme (PNSP) identifies patients with rare diseases through nationwide screening. Currently, 27 diseases are diagnosed, amongst which are 24 Inborn Errors of Metabolism (IEM), covering approximately 100% of neonates (1). In 2004, the national laboratory implemented a new screening method, tandem mass spectrometry (MS/MS) to test for amino acids and acylcarnitines. This new protocol revolutionized the PNSP and allowed for the analysis of an increased number of IEM, with clear improvements in treatment timings and clinical outcomes (2). METHODS: From 2004 to 2022, 1 764 830 neonates were screened with MS/MS technology. Those who displayed biochemical profiles indicating an IEM were subjected to molecular characterization via genomic DNA extraction, PCR amplification, and direct Sanger sequencing method of dried blood spot samples. RESULTS/CASE REPORT: A cohort of 681 newborns were diagnosed with an IEM. MCAD deficiency is the most frequent, with 233 confirmed diagnoses, showing predominantly c.985A>G (p.K329E) mutation of the ACADM gene in homozygosity. Approximately 1/3 of the 33 confirmed cases of Glutaric Aciduria type I present homozygous for the c.1204C>T (p.Arg402Trp) mutation in GCDH. Around 60% of cases of MAT II/III deficiency display the dominant mutation of the MAT1A gene, c.791G>A (p.Arg264His). These genetic profiles and others were determined as diagnostic confirmation for 24 of the IEM screened. CONCLUSION: This data shows the molecular epidemiology of patients with confirmed IEM diagnosis identified by neonatal screening. Some diseases out of the scope of the PNSP were also detected as a differential diagnosis after biochemical suspicion in the dried blood spot sample. The retrospective analysis of the PNSP allows for an overview of 18 years of achievements accomplished by the national screening for IEM since MS/MS was implemented. For some pathologies with low incidence, it's difficult to trace a discernible pattern. However, presenting de novo mutations for these diseases might provide insights on how to approach different phenotypes. The aim of this work is to establish the molecular epidemiology of metabolic diseases screened.

A simple method for direct mercury analysis in dried blood spots (DBS) samples for human biomonitoring studies.

Human exposure to mercury can have serious health effects, especially in vulnerable groups such as children and fetuses. The use of dried blood spot (DBS) samples to collect capillary blood greatly facilitates sample collection and fieldwork, being a less invasive alternative to blood collection by venipuncture, needing a small volume of sample, and does not require specialized medical staff. Moreover, DBS sampling reduces logistical and financial barriers related to transport and storage of blood samples. We propose here a novel method to analyze total mercury in DBS samples in a Direct Mercury Analyzer (DMA) that allow the control of the volume of the DBS samples. This method has shown good results in terms of precision (<6% error), accuracy (<10% coefficient of variation) and recovery (75-106%). The applicability of the method in human biomonitoring (HBM) was demonstrated in a pilot study involving 41 adults aged 18-65. Mercury concentrations of DBS samples from capillary blood collected by finger prick (real DBS samples) were determined in the DMA and compared with those determined in whole blood (venous blood) by ICP-MS, the method usually used in HBM. The sampling procedure was also validated by comparison of real DBS samples and DBS generated artificially in the laboratory by depositing venous samples in cellulose cards (laboratory DBS). There were no statistically significant differences in the results obtained using both methodologies (DMA: Geometric Mean (confidence interval 95%) = 3.87 (3.12-4.79) µg/L; ICP-MS: Geometric Mean (confidence interval 95%) = 3.46 (2.80-4.27) µg/L). The proposed method is an excellent alternative to be applied in clinical settings as screening methodology for assessing mercury exposure in vulnerable groups, such us pregnant woman, babies and children.

At-home dried blood spot (DBS) collection to increase population heterogeneity representation in pediatric research: An ECHO study.

Self-collection of dried blood samples (DBS) in the participant's home provides an alternative to university/hospital visits for research and has the potential to improve the representation of population heterogeneity in research. This study aimed to assess the feasibility of guardian and/or self-DBS collection in healthy youth in the lab and home. Guardians/youth [N = 140; females = 63; M age = 8.73, SD age = 3.56] who enrolled in a longitudinal study of typical development were asked during a lab visit to provide a DBS. Upon providing a sample, the participants were asked if they would be willing to self-collect in the home and return the sample via the post office. Of those asked to provide a sample in the lab, 82% consented and 18% declined, with a significant difference in age but no significant difference in sex, ethnicity, race, or family income. Of those who provided a sample in the lab, 75% were willing to self-collect DBS in the home, with no significant difference in demographic variables between them. We report a quality assessment and DNA extraction results from a subset of samples. The results demonstrate a high feasibility of DBS collection from healthy youth for research purposes both in the laboratory and in the home across different demographic variables. Developmental researchers should consider including this approach in their studies to increase population heterogeneity representation.

Validation and application of volumetric absorptive microsampling (VAMS) dried blood method for phenylalanine measurement in patients with phenylketonuria.

BACKGROUND: Frequent blood phenylalanine (Phe) measurement is required for phenylketonuria (PKU) patients for diagnosis and disease status monitoring. Though various methods are available for blood Phe measurement, there is a lack of validated quantitative methods for measuring Phe with less than 15% variability. A method to allow at home blood sample collection for the PKU community is in high demand. METHODS: A volumetric absorptive microsampling (VAMS) dried blood collection high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and fully validated for blood Phe measurement in compliance with regulatory guidances. The method accuracy, precision, stability, selectivity, matrix and hematocrit effects were assessed. A venous plasma collection HPLC-MS/MS method was developed and validated as a reference method. 311 matching VAMS and plasma samples were collected from 24 PKU subjects in a Phase 2 clinical study. Phe measurements using the two methods were compared. RESULTS: Both VAMS and the plasma sample collection methods met the acceptance criteria for Good Laboratory Practice (GLP) bioanalytical analysis. Comparisons showed a high Pearson's correlation of 0.9813. The Passing-Bablok analysis showed that the difference was estimated to be less than 5% and Bland Altman analysis indicated that the difference was proportional with Phe concentration and for the majority of samples (88.85%) the measurement was within ±20% difference. Following 7 days treatment with 60 or 20 mg/kg/day PTC923 (Sepiapterin) or 20 mg/kg/day sapropterin, PKU patients exhibited respectively -206.4, -146.9, and -91.5 µmol/L reductions of blood Phe as measured by the VAMS method. CONCLUSIONS: Concordant results were obtained using VAMS and plasma methods, which demonstrated that VAMS is a reliable method for clinical applications to monitor blood Phe for PKU patients.

A fast and validated LC-MS/MS method to quantify lenvatinib in dried blood spot.

A new LC-MS/MS method for the quantification of lenvatinib (LENVA) in venous Dried Blood Spot (DBS) samples has been presented. This method is characterized by a short run time (4 min), requires a volumetric sampling of 10 µL and extraction of the entire spot to avoid hematocrit (Hct) and spot volume effects. The quantification method was successfully validated in the range of 5.00-2000 ng/mL on two different DBS filter papers (Whatman 31 ET CHR and Whatman 903) according to European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines, European Bioanalysis Forum (EBF), and International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) recommendations. During the validation process, the following parameters were evaluated: recovery (≥ 77% for both filter papers), absence of matrix effect, process efficiency (close to 72% for Whatman 31 ET CHR and close to 77% for Whatman 903), Hct effect (CV ≤ 6.3% and accuracy within 96-112%), linearity (r ≥ 0.998 for Whatman 31 ET CHR and r ≥ 0.999 for Whatman 903), intra- and inter-day precision (CV ≤ 8.8%) and accuracy (92.8-108%), selectivity and sensitivity, reproducibility with incurred samples reanalysis (ISR), and stability. This method was applied to quantify venous DBS samples from patients with hepatocellular carcinoma treated with LENVA enrolled in a cross-validation study (CRO-2018-83). A good correlation between LENVA plasma concentration determined by standard procedure and the new developed DBS LENVA method (R2 ≥ 0.996) has been observed.