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Abstract Background The screening of high-risk populations using dried blood spots (DBS) has allowed the rapid identification of patients with Pompe disease, mostly in Neurology departments. The aim of the study was to determine the prevalence of late-onset Pompe disease (LOPD) among patients not previously diagnosed or tested for this entity despite presenting possible signs or symptoms of the disease in Internal Medicine departments in Spain. Methods This epidemiological, observational, cross-sectional, multicenter study included a single cohort of individuals with clinical suspicion of LOPD seen at Internal Medicine departments in Spain. The diagnosis of LOPD was initially established on the basis of the result of DBS. If decreased enzyme acid-alpha-1,4-glucosidase (GAA) activity was detected in DBS, additional confirmatory diagnostic measurements were conducted, including GAA activity in lymphocytes, fibroblasts, or muscle and/or genetic testing. Results The diagnosis of LOPD was confirmed in 2 out of 322 patients (0.6%). Reasons for suspecting LOPD diagnosis were polymyositis or any type of myopathy of unknown etiology (in one patient), and asymptomatic or pauci-symptomatic hyperCKemia (in the other). The time between symptom onset and LOPD diagnosis was 2.0 and 0.0 years. Both patients were asymptomatic, with no muscle weakness. Additionally, 19.7% of the non-LOPD cases received an alternative diagnosis. Conclusions Our study highlights the existence of a hidden population of LOPD patients in Internal Medicine departments who might benefit from early diagnosis and early initiation of potential treatments.

Abstract INTRODUCTION The apolipoprotein E (APOE) ε4 allele is associated with high risk for Alzheimer's disease. It is unclear whether individual levels of the circulating apoE4 protein in ε4 carriers confer additional risk. Measuring apoE4 protein levels from dried blood spots (DBS) has the potential to provide information on genetic status as well as circulating levels and to include these measures in large survey settings. METHODS We developed a multiplex immunoassay to detect apoE4 protein levels in DBS from 15,974 participants, aged 50+ from Wave 6 of the Survey of Health, Ageing and Retirement in Europe (SHARE). RESULTS The apoE4 protein signal was presented in two separable distributions. One distribution corresponded to carriers of at least one copy of the ε4 allele. Fieldwork cofounders affected protein levels but did not explain individual differences. DISCUSSION Future research should investigate how genotype and apoE4 level interact with lifestyle and other variables to impact cognitive aging.

Abstract Introduction The exclusion of affected populations from Alzheimer's disease (AD) clinical research limits our understanding of disease heterogeneity and its impact on clinical care. While micro sampling with dried plasma spots (DPS) can promote inclusivity by enabling sample collection in remote areas, current techniques lack the sensitivity required for the quantification of phosphorylated tau at Thr181 (pTau‐181) in DPS extracts. Methods We developed an assay for pTau‐181 with reduced bead count and improved bead read efficiency (BRE) using a prototype Simoa instrument. This novel assay's performance was evaluated against standard pTau‐181 assays on two Simoa platforms, and DPS extracts were tested for pTau‐181 quantification feasibility. Results The novel assay quantifies pTau‐181 at concentrations up to 16x lower than traditional pTau‐181 assays on HD‐X and SR‐X platforms. DPS extracts tested with our low‐bead assay were quantified considerably above the lower limit of quantification (LLOQ), indicating the suitability of this assay for future DPS extract measurements. Discussion Implementing DPS sampling and pTau‐181 quantification could increase participation from underrepresented groups in AD research. However, additional assay optimization and an in‐depth study of preanalytical sample stability are essential for the transition to clinical applicability.

Chromatography–mass spectrometry was used to analyze dried blood spots collected from 6 female volunteers in a 3-day dry immersion (DI) test in order to detect proteins involved in acute adaptation of the female physiological systems. Sampling was performed before DI, every day in DI, and on the next day after DI completion. Proteomic analysis demonstrated an immediate reaction of proteins involved in carbohydrate metabolism and proteasome degradation of proteins. On DI day-2, we saw a reaction of immune response proteins and decreased level of PEDF, an angiogenesis inhibitor that controls many processes, including heart protection from injuries. On DI day-3, the response was joined by proteins participating in processes of the complement cascade and coagulation, platelet degranulation, hemostasis, extracellular matrix formation, glycolysis, water–salt balance, antioxidant system, and the neutrophils involving defense response. It should be noted that on DI day-3 we also found increased levels of histidine triad 1 nucleotide-binding protein 1 (HINT1) and myosin light chain 4 (MYL4) that play an important role in cardiac adaptation to a volume overload. Not all of the proteins returned to baseline levels 2 days after DI. Changes in the level of proteins involved in lipid metabolism were observed only after DI completion. [ABSTRACT FROM AUTHOR]

Purpose: Therapeutic drug monitoring of tacrolimus using trough concentration (Cmin) is mandatory to ensure drug efficacy and safety in solid organ transplantation. However, Cmin is just a proxy for the area under the curve of drug concentrations (AUC) which is the best pharmacokinetic parameter for exposure evaluation. Some studies suggest that patients may present discrepancies between these two parameters. AUC is now easily available through mini-invasive microsampling approach. The aim of this study is to evaluate the relationship between AUC and Cmin in patients benefiting from a complete pharmacokinetic profile using a microsampling approach. Methods: Fifty-one transplant recipients benefited from a complete pharmacokinetic profile using a microsampling approach, and their 24-h AUC were calculated using the trapezoidal method. The correlation with Cmin was then explored. In parallel, we estimated AUC using the sole Cmin and regression equations according to the post-transplantation days and the galenic form. Results: Weak correlations were found between 24-h AUC observed and the corresponding Cmin (R2 = 0.60) and between AUC observed and expected using the sole Cmin (R2 = 0.62). Therapeutic drug monitoring of tacrolimus using Cmin leads to over- or under-estimate drug exposure in 40.3% of patients. Conclusion: Tacrolimus Cmin appears to be an imperfect reflection of drug exposure. Evaluating AUC using a microsampling approach offers a mini-invasive strategy to monitor tacrolimus treatment in transplant recipients. [ABSTRACT FROM AUTHOR]

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Tenofovir diphosphate (TFV-DP) concentrations in dried blood spots (DBS) predict viral breakthrough, but their use remains understudied in real-world clinic settings. This pilot study examined acceptability, feasibility, and initial adherence outcomes of providing adherence feedback using TFV-DP concentrations on patient- and provider-levels in Cape Town, South Africa. We enrolled 60 persons with HIV (PWH) receiving tenofovir-containing ART attending a primary health clinic. They were randomized 1:1 to an intervention receiving TFV-DP concentration feedback by research staff vs. no feedback at monthly visits for 4 months. Acceptability among medical providers and level of clinical follow-up of TFV-DP results was examined. Patient acceptability was assessed descriptively. Mean electronic adherence (EA), as measured by WisePill device, and TFV-DP in DBS were compared between the two arms. All participants in the intervention group (100%) reported finding TFV-DP feedback helpful and 86% reported changing adherence behaviors. Medical providers indicated high acceptability of incorporating TFV-DP concentration feedback into the clinic, yet among 29 results < 1000 fmol/punch, only 2 were reviewed with no follow-up actions performed. In the intervention arm, mean TFV-DP concentrations were significantly higher (t = 2.5, p <.01) during follow-up and EA in upper quartile (96–100%) was greater compared to controls (x2 = 7.8, p ≤.05). This study found high acceptability among patients for receiving adherence feedback based on TFV-DP concentrations. TFV-DP and EA data demonstrated greater adherence in the intervention group. Providers indicated high acceptability of incorporating TFV-DP feedback into the clinic, but few providers reviewed results, which could impact clinic-level feasibility. [ABSTRACT FROM AUTHOR]

IntroductionUntargeted metabolomics holds significant promise for biomarker detection and development. In resource-limited settings, a dried blood spot (DBS)-based platform would offer significant advantages over plasma-based approaches that require a cold supply chain.ObjectivesThe primary goal of this study was to compare the ability of DBS- and plasma-based assays to characterize maternal metabolites. Utility of the two assays was also assessed in the context of a case-control predictive model in pregnant women living with HIV.MethodsUntargeted metabolomics was performed on archived paired maternal plasma and DBS from n = 79 women enrolled in a large clinical trial.ResultsA total of 984 named biochemicals were detected across both plasma and DBS samples, of which 627 (63.7%), 260 (26.4%), and 97 (9.9%) were detected in both plasma and DBS, plasma alone, and DBS alone, respectively. Variation attributable to study individual (R2 = 0.54, p

BackgroundThe retinol isotope dilution (RID) method has been used to evaluate vitamin A (VA) status in healthy adults and children in low- and middle-income countries (LMIC) and to assess the efficacy of various VA interventions.ObjectiveThe study was designed to examine whether dried serum spots (DSS) can be applied to RID when conducting VA total body store (TBS) assessments in community settings.MethodsFour days after an oral dose of 0.4 mg [13C10]retinyl acetate was administered to Filipino children (12-18 mo), a single blood draw was divided to isolate both serum and plasma. Serum (40 μL) was spotted and dried on Whatman 903 cards and shipped at ambient temperature whereas liquid plasma (LP) was frozen at -80°C and shipped on dry ice. The VA tracer to tracee ratio from DSS and LP was quantified by LC-MS/MS. Comparisons between DSS and LP paired samples (n = 72) were made for [13C10]retinol specific activity (SAp) by Pearson's correlation and for VA TBS by Bland-Altman analysis.ResultsThe sum of 3 coextracted DSS were required to consistently detect [13C10]retinol above the LC-MS/MS limit of quantitation (LOQ). [13C10]retinol SAp from DSS was highly correlated with SAp from LP (r = 0.945; P

Repeated blood sampling is required in certain clinical and research settings, which is currently performed by drawing blood from venous catheters requiring manual handling of each sample at the time of collection. A novel body-worn device for repeated serial samples, Fluispotter®, with automated extraction, collection, and storage of up to 20 venous dried blood spot samples over the course of 20 h may overcome problems with current methods for serial sampling. The purpose of this study was to assess the performance and safety of Fluispotter for the first time in healthy subjects. Fluispotter consists of a cartridge with tubing, a reservoir for flushing solution, pumps and filter paper, and a multi-lumen catheter placed in the brachial vein. We recruited healthy subjects for testing in an in-hospital setting. Fluispotter was attached by an anesthesiologist to 22 healthy subjects of which 9/22 (40.9%) participants had all 20 samples taken, which was lower than the goal of complete sampling in 80% of the subjects (P = 0.02). The main reason for sample failure was clogging of blood flow which was observed in 11/22 (50%) of the participants. No serious adverse events occurred, and the participants rated the pain from the insertion and the removal of catheter as very low. A cortisol profile showed nadir values at midnight and highest values at 05:00 h. Although full sampling was not successful in all participants, the Fluispotter technology proved safe and highly acceptable to the participants producing the expected cortisol profile without the requirement of staff during sample collection.

Abstract Background Malaria continues to cause burden in various parts of the world. Haiti, a Caribbean country, is among those aiming to eliminate malaria within a few years. Two surveys were conducted in Haiti during which we aimed to evaluate the performance of the simple and rapid procedure for ultra-rapid extraction–loop-mediated isothermal amplification (PURE-LAMP) method with dried blood spots as an alternative diagnostic method for malaria in the context of low to very low rates of transmission. Methods Febrile and afebrile people were recruited from three administrative divisions within Haiti: Nippes, Sud and Grand’Anse, during the summers of 2017 (early August to early September) and 2018 (late July to late August). Their blood samples were tested by microscopy, rapid diagnostic tests (RDT), PURE-LAMP and nested PCR to detect Plasmodium infection. Sensitivity, specificity, positive and negative predictive values and kappa statistics were estimated with the nested PCR results as the gold standard. Results Among 1074 samples analyzed, a positive rate of 8.3% was calculated based on the nested PCR results. Among febrile participants, the rates in 2017 and 2018 were 14.6% and 1.4%, respectively. Three positives were detected among 172 afebrile participants in 2018 by PURE-LAMP and nested PCR, and all three were from the same locality. There was no afebrile participants recruited in 2017. The PURE-LAMP, RDT and microscopy had respective sensitivities of 100%, 85.4% and 49.4%. All of the testing methods had specificities over 99%. Conclusions This study confirmed the high performance of the PURE-LAMP method to detect Plasmodium infection with dried blood spots and recommends its use in targeted mass screening and treatment activities in low endemic areas of malaria.

Background: Gaucher's and Fabry's disease are two of the most common treatable lysosomal storage diseases, and have a wide spectrum of clinical symptoms. Early detection is important, because timely initiation of treatments can improve the disease status and prevent complications. However disease manifestations develop in childhood, diagnosis is delayed until adulthood partly due to the limitations of the currently used diagnostic pathway. The aim of this research is to develop and validate a multiplex assay and defining reference ranges, which do not exist at this moment, to improve and facilitate the entire diagnostic work up and enable treatment in an earlier stage of disease. Methods and findings: Biomarkers glucosylsphingosine (GlcSph) and globotriaosylsphingosine (Lyso-Gb3) were detected and quantified using LC-MS/MS on dried blood spots. We developed an improved and new extraction method that allowed to measure GlcSph and Lyso-Gb3 in a multiplex analytical platform. After validation of the method, samples of 1480 individuals with normal enzymatic activity were collected to determine age and gender-related reference ranges.Our combination method showed a good linearity, precision, accuracy and limit of quantification with lack of carry-over following the specific international CLSI guidelines. The suggested protocol is robust, efficient, sensitive, specific, comprehensive and relatively cheap in order to accelerate the diagnostic process for both lysosomal storage diseases. The samples, with normal enzymatic activity, defined statistical relevant and clinical correct reference ranges for each specific age group by gender. Conclusion: We report a multiplex LC-MS/MS method and relevant reference ranges that are appropriate for the targeted screening, diagnosis and follow-up of Fabry and Gaucher disease.

Background: Suboptimal antiretroviral (ART) adherence can lead to virologic failure with consequent HIV-1 resistance. Tenofovir diphosphate (TFV-DP) in dried blood spots (DBS) is a powerful biomarker of cumulative adherence, predictive of future viremia. It has been associated with resistance in Persons With HIV (PWH) in South Africa and the US. We explored the relationship of TFV-DP concentrations with antiretroviral drug resistance at the time of treatment failure in SA. Methods: Adult PWH from health clinics in Cape Town, South Africa on efavirenz-based first-line ART containing tenofovir disoproxil fumarate (TDF) with an undetectable (< 50 copies/mL) HIV-1 viral load (VL) were prospectively enrolled in an observational cohort for 12 months. Monthly study visits included blood collection for HIV-1 VL and DBS for TFV-DP. The first confirmed viral breakthrough (VB) > 400 copies/mL triggered HIV-1 genotyping at the subsequent visit. An electronic adherence (EA) device monitored ART adherence in real-time, estimated as a percent for the 30-days prior to VB. Wilcoxon rank sum test was used to compare median [IQR] TFV-DP by genotype outcome. Results: Of 250 individuals, (n = 195, 78% women), 21 experienced VB, with a median of 5 [4;7] months on study, and a median EA of 33.3 [13.3;53.3]%. Demographic characteristics between those with and without VB were similar. Median VL at VB was 4.0 [3.2;4.5] log copies/mL. TFV-DP concentrations trended down towards the VB visit. Median TFV-DP concentrations were significantly higher in those HIV-1 genotype did not amplify due to being virally suppressed at the subsequent visit (n = 10; 380 [227–661] fmol/punch, p = 0.035; EA 45 [24.9; 59.2]%); than in those who were successfully genotyped with evidence of drug resistance (n = 5, 241 [150–247] fmol/punch, EA 20 [6.7;36.7]%) and in individuals who did not have resistance (n = 3, 39.9 [16.6; 93.9] fmol/punch; EA 33.3 [16–38]%). Three genotype collections were not done. Only non-nucleoside reverse transcriptase inhibitor-associated mutations were identified on resistance testing. (K103N, E138K, Y118H). Conclusion: TFV-DP in DBS showed a step-wise inverse relationship with VB and drug resistance, with evidence of low cumulative ART adherence in PWH who developed antiretroviral resistance. Monitoring TFV-DP concentrations could be a valuable tool for predicting future VB and future resistance. [ABSTRACT FROM AUTHOR]

Background: The screening of high-risk populations using dried blood spots (DBS) has allowed the rapid identification of patients with Pompe disease, mostly in Neurology departments. The aim of the study was to determine the prevalence of late-onset Pompe disease (LOPD) among patients not previously diagnosed or tested for this entity despite presenting possible signs or symptoms of the disease in Internal Medicine departments in Spain. Methods: This epidemiological, observational, cross-sectional, multicenter study included a single cohort of individuals with clinical suspicion of LOPD seen at Internal Medicine departments in Spain. The diagnosis of LOPD was initially established on the basis of the result of DBS. If decreased enzyme acid-alpha-1,4-glucosidase (GAA) activity was detected in DBS, additional confirmatory diagnostic measurements were conducted, including GAA activity in lymphocytes, fibroblasts, or muscle and/or genetic testing. Results: The diagnosis of LOPD was confirmed in 2 out of 322 patients (0.6%). Reasons for suspecting LOPD diagnosis were polymyositis or any type of myopathy of unknown etiology (in one patient), and asymptomatic or pauci-symptomatic hyperCKemia (in the other). The time between symptom onset and LOPD diagnosis was 2.0 and 0.0 years. Both patients were asymptomatic, with no muscle weakness. Additionally, 19.7% of the non-LOPD cases received an alternative diagnosis. Conclusions: Our study highlights the existence of a hidden population of LOPD patients in Internal Medicine departments who might benefit from early diagnosis and early initiation of potential treatments. [ABSTRACT FROM AUTHOR]

LCP-tacrolimus displays enhanced oral bioavailability compared to immediate-release (IR-) tacrolimus. The ENVARSWITCH study aimed to compare tacrolimus AUC0-24 h in stable kidney (KTR) and liver transplant recipients (LTR) on IR-tacrolimus converted to LCPtacrolimus, in order to re-evaluate the 1:0.7 dose ratio recommended in the context of a switch and the efficiency of the subsequent dose adjustment. Tacrolimus AUC0-24 h was obtained by Bayesian estimation based on three concentrations measured in dried blood spots before (V2), after the switch (V3), and after LCP-tacrolimus dose adjustment intended to reach the pre-switch AUC0-24 h (V4). AUC0-24 h estimates and distributions were compared using the bioequivalence rule for narrow therapeutic range drugs (Westlake 90% CI within 0.90-1.11). Fifty-three KTR and 48 LTR completed the study with no major deviation. AUC0-24 h bioequivalence was met in the entire population and in KTR between V2 and V4 and between V2 and V3. In LTR, the Westlake 90% CI was close to the acceptance limits between V2 and V4 (90% CI = [0.96-1.14]) and between V2 and V3 (90% CI = [0.96-1.15]). The 1:0.7 dose ratio is convenient for KTR but may be adjusted individually for LTR. The combination of DBS and Bayesian estimation for tacrolimus dose adjustment may help with reaching appropriate exposure to tacrolimus rapidly after a switch. [ABSTRACT FROM AUTHOR]

Dried blood spot (DBS) microsampling is extensively employed in newborn screening (NBS) and neonatal studies. However, the impact of variable neonatal hematocrit (Ht) values on the results can be a source of analytical error, and the use of fixed Ht for calibration (Htcal) is not representative of all neonatal subpopulations. A computational approach based on neonatal demographics was developed and implemented in R® language to propose a strategy using correction factors to address the Ht effect in neonatal DBS partial-spot assays. A rational "tolerance level" was proposed for the Ht effect contribution to the total analytical error and a safe Ht range for neonatal samples, where the correction of concentrations can be omitted. Furthermore, an "alert zone" for a false positive or negative result in NBS was proposed, where the Ht effect has to be considered. Results point toward the use of Htcal values closely representative of populations under analysis and an acceptable level of percentage relative error can be attributed to the Ht effect, diminishing the probability of correction. Overall, the impact of the Ht effect on neonatal studies is important and future work may further investigate this parameter, correlated to other clinical variables potentially affecting results. [ABSTRACT FROM AUTHOR]

A common challenge when studying rare diseases or medical conditions is the limited number of patients, usually resulting in long inclusion periods as well as unequal sampling and storage conditions. The main purpose of this study was to demonstrate the challenges when comparing samples subject to different preanalytical conditions. We performed a global (commonly referred to as "untargeted") liquid chromatography‐high resolution mass spectrometry metabolomics analysis of blood samples from cases of sudden infant death syndrome and controls stored as dried blood spots on a chemical‐free filter card for 15 years at room temperature compared with the same blood samples stored as whole blood at −80°C before preparing new dried blood spots using a chemically treated filter card. Principal component analysis plots distinctly separated the samples based on the type of filter card and storage, but not sudden infant death syndrome versus controls. Note that, 1263 out of 5161 and 642 out of 1587 metabolite features detected in positive and negative ionization mode, respectively, were found to have significant 2‐fold changes in amounts corresponding to different preanalytical conditions. The study demonstrates that the dried blood spot metabolome is largely affected by preanalytical factors. This emphasizes the importance of thoroughly addressing preanalytical factors during study design and interpretation, enabling identification of real, biological differences between sample groups whilst preventing other factors or random variation to be falsely interpreted as positive results. [ABSTRACT FROM AUTHOR]

The use of dried blood spots (DBS) has gained interest in the field of therapeutic drug monitoring (TDM) due to its potential advantages, such as minimally invasive capillary blood collection, potential stabilization of drugs and metabolites at room or high temperatures, and lower biohazard, allowing for inexpensive storage and transportation. However, there are several drawbacks to the clinical use of DBS in TDM, mostly related to hematocrit (Hct) effects, differences between venous and capillary blood concentrations, among others, that must be evaluated during analytical and clinical method validation. This review focuses on the most recent publications on the applications of DBS sampling for TDM (2016–2022), with a special focus on the challenges presented by this alternative sampling strategy, as well as the opportunities for clinical applications. Real-life studies presenting clinical applications were reviewed. With the availability of method development and validation guidelines for DBS-based methods in TDM, higher levels of assay validation standardization have been achieved, expanding the clinical applications of DBS sampling in patient care. New sampling devices that overcome the limitations of classical DBS, such as the Hct effects, will further encourage the use of DBS in routine TDM. [ABSTRACT FROM AUTHOR]

Background Food insecurity has been linked to suboptimal antiretroviral therapy (ART) adherence in persons with HIV (PWH). This association has not been evaluated using tenofovir diphosphate (TFV-DP) in dried blood spots (DBSs), a biomarker of cumulative ART adherence and exposure. Methods Within a prospective South African cohort of treatment-naive PWH initiating ART, a subset of participants with measured TFV-DP in DBS values was assessed for food insecurity status. Bivariate and multivariate median-based regression analysis compared the association between food insecurity and TFV-DP concentrations in DBSs adjusting for age, sex, ethnicity, medication possession ratio (MPR), and estimated glomerular filtration rate. Results Drug concentrations were available for 285 study participants. Overall, 62 (22%) PWH reported worrying about food insecurity and 44 (15%) reported not having enough food to eat in the last month. The crude median concentrations of TFV-DP in DBSs differed significantly between those who expressed food insecurity worry versus those who did not (599 [interquartile range {IQR}, 417–783] fmol/punch vs 716 [IQR, 453–957] fmol/punch; P =.032). In adjusted median-based regression, those with food insecurity worry had concentrations of TFV-DP that were 155 (95% confidence interval, −275 to −35; P =.012) fmol/punch lower than those who did not report food insecurity worry. Age and MPR remained significantly associated with TFV-DP. Conclusions In this study, food insecurity worry is associated with lower TFV-DP concentrations in South African PWH. This highlights the role of food insecurity as a social determinant of HIV outcomes including ART failure and resistance. [ABSTRACT FROM AUTHOR]

With the advancements in analytical and molecular techniques, Dried Blood Spots (DBS) are re-emerging as attractive and cost-effective alternatives for global health surveillance. The use of DBS has been well-characterized in the neonatal screening of metabolic diseases, therapeutic screening as well as in epidemiological studies for biomonitoring. Malaria is one such infectious disease where DBS use can expedite molecular surveillance for assessing drug resistance and for refining drug usage policies. In India, malaria cases have reduced significantly over the past decade but to achieve malaria elimination by 2030, country-wide DBS-based screening should be conducted to identify the presence of molecular markers of artemisinin resistance and to study parasite reservoirs in asymptomatic populations. DBS has wide applications in genomics, proteomics, and metabolomic studies concerning both host and pathogen factors. Hence, it is a comprehensive tool for malaria surveillance that can capture both host and parasite information. In this review, we elucidate the current and prospective role of DBS in malaria surveillance and its applications in studies ranging from genetic epidemiology, parasite and vector surveillance, drug development and polymorphisms to ultimately how they can pave the roadmap for countries aiming malaria elimination

Introduction: Adherence to medication is an important determinant of outcomes in chronic diseases like heart failure. Drug assays provide objective adherence biomarkers. Dried blood spots (DBS) are appealing samples for drug assays due to less demanding transportation and storage requirements. Objectives: To analytically validate a LC-MS/MS method for the simultaneous quantification of carvedilol, enalaprilat, and perindoprilat in DBS and evaluate the feasibility of using the method as an adherence determining assay. To validate the assay further clinically by establishing correlation and agreement between plasma and DBS samples from a pharmacokinetic pilot study. Methods: The method was validated over a concentration range of 1.00–200 ng/mL according to FDA guidelines. Adherence tracking ability of the assay was evaluated using a pharmacokinetic pilot study. Correlation and agreement were evaluated through Deming regression and Bland-Altman analysis, respectively. Results: Accuracy, precision, selectivity, and sensitivity were proven with complete and reproducible extraction recovery at all concentrations tested. Stability of the analytes in the matrix and throughout sample processing was proven. The full range of concentrations of the pharmacokinetic pilot study could be quantified for enalaprilat, but not for carvedilol and perindoprilat. The difference between the observed and calculated plasma concentrations was less than 20 % of their mean for >67 % of samples for all analytes. Conclusions: The assay is suitable as a screening tool for carvedilol and perindoprilat, while suitable as an adherence determining assay for enalaprilat. Equivalence between observed and predicted plasma concentrations proves DBS and plasma concentrations can be used interchangeably.

Objectives: This study aimed to determine the agreement between a rapid diagnostic test (RDT) and a dried blood spot (DBS)-based electrochemiluminescence immunoassay (ECLIA) of hepatitis B surface antigen and to compare the costs of conducting serosurveys using RDTs and DBS in a field setting. Methods: A serosurvey was conducted in the South Central Coast region of Vietnam in May 2019. Participants aged 1-39 years were recruited using a four-stage random sampling method and tested for hepatitis B surface antigen using an RDT kit (Alere Determine) and a DBS-based ECLIA. The agreement between the RDT and the DBS-based ECLIA was assessed using cross-tabulation and Cohen kappa. Cost data were categorized by input (personnel, transportation, field consumables, laboratory consumables, and capital item/overhead) and survey phase (survey preparation, data/biospecimen collection, laboratory testing, and coordination). Results: A total of 2072 participants were analyzed. There was a 99% agreement between the RDT and the DBS-based ECLIA results, with a Cohen kappa of 0.9. The estimated cost of conducting a serosurvey by DBS was UD $75,291, whereas RDT was $53,182. Conclusion: RDTs and DBS-based ECLIA provide test results with high agreements. RDTs are a better option in terms of cost, whereas the DBS-based ECLIA may be useful when evaluating multiple infectious diseases.

Dried blood spots (DBSs) provide an alternative sample input for serologic testing. We evaluated DBSs for the ARCHITECT® hepatitis B surface antigen (HBsAg) NEXT, hepatitis B e-antigen (HBeAg), anti-hepatitis B core antigen (anti-HBc II), HIV antigen/antibody (Ag/Ab) Combo and AdviseDx SARS-CoV-2 IgG II assays. Assay performance with DBSs was assessed with or without assay modification and compared with on-market assay with plasma samples. DBS stability was also determined. HBsAg NEXT and HIV Ag/Ab Combo assays using DBSs showed sensitivity and specificity comparable to that of on-market assays. Modified HBeAg, anti-HBc II and SARS-CoV-2 IgG II DBS assays achieved performance comparable to on-market assays. Use of DBSs as input for high-throughput serologic assays is expected to have significant implications for improving population surveillance and increasing access to diagnostic testing.

Background: Malaria continues to cause burden in various parts of the world. Haiti, a Caribbean country, is among those aiming to eliminate malaria within a few years. Two surveys were conducted in Haiti during which we aimed to evaluate the performance of the simple and rapid procedure for ultra-rapid extraction–loop-mediated isothermal amplification (PURE-LAMP) method with dried blood spots as an alternative diagnostic method for malaria in the context of low to very low rates of transmission. Methods: Febrile and afebrile people were recruited from three administrative divisions within Haiti: Nippes, Sud and Grand'Anse, during the summers of 2017 (early August to early September) and 2018 (late July to late August). Their blood samples were tested by microscopy, rapid diagnostic tests (RDT), PURE-LAMP and nested PCR to detect Plasmodium infection. Sensitivity, specificity, positive and negative predictive values and kappa statistics were estimated with the nested PCR results as the gold standard. Results: Among 1074 samples analyzed, a positive rate of 8.3% was calculated based on the nested PCR results. Among febrile participants, the rates in 2017 and 2018 were 14.6% and 1.4%, respectively. Three positives were detected among 172 afebrile participants in 2018 by PURE-LAMP and nested PCR, and all three were from the same locality. There was no afebrile participants recruited in 2017. The PURE-LAMP, RDT and microscopy had respective sensitivities of 100%, 85.4% and 49.4%. All of the testing methods had specificities over 99%. Conclusions: This study confirmed the high performance of the PURE-LAMP method to detect Plasmodium infection with dried blood spots and recommends its use in targeted mass screening and treatment activities in low endemic areas of malaria. [ABSTRACT FROM AUTHOR]

Here, we report on the first direct comparative evaluation of two different non-contact hematocrit prediction strategies, NIR and UV-vis spectroscopy. 4 Capiau, S, Wilk, L, De Kesel, P, Aalders, M, Stove, C. Correction for the hematocrit bias in dried blood spot analysis using a nondestructive, single-wavelength reflectance-based hematocrit prediction method. Keywords: dried blood spots; hematocrit prediction; near-infrared; therapeutic drug monitoring; UV-vis spectroscopy EN dried blood spots hematocrit prediction near-infrared therapeutic drug monitoring UV-vis spectroscopy e87 e90 4 04/25/23 20230515 NES 230515 To the Editor, The last decade has witnessed an increased use of dried blood spots (DBS) in different fields, such as for the therapeutic drug monitoring (TDM) of immunosuppressants. [Extracted from the article]

This study set out to examine pre-analytical factors affecting the frequency of positive results in newborn screening for biotinidase deficiency. This investigation was prompted by an increase in the annual screen positive rate for biotinidase deficiency in Ontario from 2.65x10−4 in 2016 to 6.57x10−4 in 2017. Season and trend decomposition was used to separate seasonality from an underlying trend in the time series of biotindase activity measurements for the period 2014–01-12 to 2019–07-27 (n = 798,770). This analysis revealed a marked seasonal effect (winter = median + ⩽ 17 MRU, summer = mean - ⩽ 20 MRU) and a non-linear negative trend. Seasonal temperature was correlated with biotinidase results (Pearson's r = 0.79) but not with the observed negative trend (Pearson's r = 0.0025). Time series analysis of biotinidase results grouped by print lot of filter paper revealed that recently printed filter paper cards inhibit biotinidase and that this inhibition resolved over time. This study demonstrates that biotindase activity is inhibited by both increased seasonal temperature and collection on newly printed filter cards. [ABSTRACT FROM AUTHOR]

Over 2.5 million neonatal dried blood spots (DBS) are stored at the Danish National Biobank. These samples offer extraordinary possibilities for metabolomics research, including prediction of disease and understanding of underlying molecular mechanisms of disease development. Nevertheless, Danish neonatal DBS have been little explored in metabolomics studies. One question that remains underinvestigated is the long-term stability of the large number of metabolites typically assessed in untargeted metabolomics over long time periods of storage. Here, we investigate temporal trends of metabolites measured in 200 neonatal DBS collected over a time course of 10 years, using an untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS) based metabolomics protocol. We found that a majority (71%) of the metabolome was stable during 10 years of storage at −20 °C. However, we found decreasing trends for lipid-related metabolites, such as glycerophosphocholines and acylcarnitines. A few metabolites, including glutathione and methionine, may be strongly influenced by storage, with changes in metabolite levels up to 0.1–0.2 standard deviation units per year. Our findings indicate that untargeted metabolomics of DBS samples, with long-term storage in biobanks, is suitable for retrospective epidemiological studies. We identify metabolites whose stability in DBS should be closely monitored in future studies of DBS samples with long-term storage. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the difference in drug exposure of rifampicin in native versus non-native Paraguayan populations using dried blood spots (DBS) samples collected utilizing a limited sampling strategy. This was a prospective pharmacokinetic study that enrolled hospitalized tuberculosis (TB) patients from both native and non-native populations receiving oral rifampicin 10 mg/kg once-daily dosing. Steady-state DBS samples were collected at 2, 4, and 6 h after intake of rifampicin. The area under the time concentration curve 0–24 h (AUC0–24) was calculated using a Bayesian population PK model. Rifampicin AUC0–24 < 38.7 mg*h/L was considered as low. The probability of target attainment (PTA) was calculated using AUC0–24/MIC > 271 as a target and estimated MIC values of 0.125 and 0.25 mg/L. In total, 50 patients were included. Native patients (n = 30) showed comparable drug exposure to the non-natives (n = 20), median AUC0–24 24.7 (17.1–29.5 IQR) and 21.6 (15.0–35.4 IQR) mg*h/L (p = 0.66), respectively. Among total patients, only 16% (n = 8) had a rifampicin AUC0–24 > 38.7 mg*h/L. Furthermore, PTA analysis showed that only 12 (24%) of the patients met a target AUC0–24 /MIC ≥ 271, assuming an MIC of 0.125 mg/L, which plummeted to 0% at a wild-type MIC of 0.25 mg/L. We successfully used DBS and limited sampling for the AUC0–24 estimation of rifampicin. Currently, our group, the EUSAT-RCS consortium, is preparing a prospective multinational, multicenter phase IIb clinical trial evaluating the safety and efficacy of high-dose rifampicin (35 mg/kg) in adult subjects using the DBS technique for AUC0–24 estimation. [ABSTRACT FROM AUTHOR]

Background: Phosphatidylethanol 16:0/18:1 (PEth), found in whole blood, is a biomarker for alcohol consumption with high sensitivity, specificity, and a long detection window. The TASSO‐M20 device is used to self‐collect capillary blood from the upper arm and has advantages over finger stick methods. The purpose of this study was to (1) validate PEth measurement using the TASSO‐M20 device, (2) describe the TASSO‐M20 for blood self‐collection during a virtual intervention, and (3) characterize PEth, urinary ethyl glucuronide (uEtG) and self‐reported alcohol in a single participant over time. Methods: PEth levels in blood samples dried on TASSO‐M20 plugs were compared to those in (1) liquid whole blood (N = 14) and (2) dried blood spot cards (DBS; N = 23). Additionally, the self‐reported drinking, positive or negative uEtG results (dip card cutoff ≥300 ng/mL), and observed self‐collection of blood with TASSO‐M20 devices for PEth levels were obtained over time during virtual interviews of a single contingency management participant. High‐performance liquid chromatography with tandem mass spectrometry detection was used to measure PEth levels for both preparations. Results: PEth concentrations from dried blood on TASSO‐M20 plugs and liquid whole blood were correlated (0 to 1700 ng/mL; N = 14; r2 = 0.988; slope = 0.951) and in a subgroup of samples with lower concentrations (N = 7; 0 to 200 ng/mL; r2 = 0.944, slope = 0.816). PEth concentrations from dried blood on TASSO‐M20 plugs and DBS were correlated (0 to 2200 ng/mL; N = 23; r2 = 0.927; slope = 0.667) and in a subgroup of samples with lower concentrations (N = 16; 0 to 180 ng/mL; r2 = 0.978, slope = 0.749). Results of the contingency management participant indicate that changes in PEth levels (TASSO‐M20) and uEtG concentrations were consistent with each other and with changes in self‐reported alcohol use. Conclusions: Our data support the utility, accuracy, and feasibility of using the TASSO‐M20 device for blood self‐collection during a virtual study. The TASSO‐M20 device had multiple advantages over the typical finger stick method, including consistent blood collection, participant acceptability, and less discomfort as indicated by acceptability interviews. [ABSTRACT FROM AUTHOR]

Iron supplementation is not considered as a doping method; however, it can affect the levels of several biomarkers of the hematologic module of the athlete biological passport (ABP), such as the reticulocyte percentage (%RET) and hemoglobin (HGB) level. Thus, iron injection could be a confounding factor in antidoping analyses. Previous studies have suggested that the HGB level and the expression levels of reticulocyte‐related‐mRNAs, such as 5′‐aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1), could be promising biomarkers for the ABP and detectable in dried blood spots (DBSs). Therefore, in this study, we examined the impact of iron injection on the levels of these potential biomarkers in DBSs. Reticulocyte‐related‐mRNAs analyses were performed by RT‐qPCR. Ferritin level in DBS was measured with enzyme‐linked immunosorbent assay (ELISA) method. Notably, there were no significant effects of iron supplementation on the levels of ALAS2 and CA1 mRNAs but by contrast, the %RET and immature reticulocyte fraction (IRF) measured in whole blood increased significantly following iron injection. As expected, iron supplementation increased the ferritin level significantly in both serum and DBS samples. In conclusion, these findings reinforce the specificity of reticulocyte‐related mRNAs in DBSs as biomarkers of blood doping to target in antidoping analyses. [ABSTRACT FROM AUTHOR]

Dried blood spots (DBS) provide easy handling and are thus a beneficial tool for data collection, e.g. for epidemiological studies. The suitability of DBS for the assessment of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was analyzed with regards to the use in future studies addressing seroprevalence in the population.121 volunteers gave a venous blood sample and capillary blood samples on two DBS cards (PerkinElmer and Ahlstrom-Munksjö) via self-sampling under supervision. All samples were analyzed using the Anti-SARS-CoV-2 ELISA (IgG) and the Anti-SARS-CoV-2 NCP ELISA (IgG) from EUROIMMUN performed on the EUROIMMUN EUROLabWorkstation ELISA. Correlation coefficients between ELISA results based on the different sampling methods were calculated.Results of DBS analysis for SARS-CoV-2 IgG S1 and NCP highly correlated with the serum values (r = 0.96). In addition, the calculation of the phi coefficient showed no significant difference between the qualitative results of both sampling methods (rφ = 0.98–1.0). Further analysis of DBS eluates after prolonged storage of 6–8 h also showed a high correlation with serum results (r = 0.97 and r = 0.93, respectively).The study results indicate suitability of DBS for the analysis of antibodies against SARS-CoV-2 S1 and NCP. For DBS eluate, a stability of 6–8 h for measurement of SARS-CoV-2 antibodies can be assumed.

In this study, we compare next-generation sequencing (NGS) approaches (targeted panel (tNGS), whole exome sequencing (WES), and whole genome sequencing (WGS)) for application in newborn screening (NBS). DNA was extracted from dried blood spots (DBS) from 50 patients with genetically confirmed inherited metabolic disorders (IMDs) and 50 control samples. One hundred IMD-related genes were analyzed. Two data-filtering strategies were applied: one to detect only (likely) pathogenic ((L)P) variants, and one to detect (L)P variants in combination with variants of unknown significance (VUS). The variants were filtered and interpreted, defining true/false positives (TP/FP) and true/false negatives (TN/FN). The variant filtering strategies were assessed in a background cohort (BC) of 4833 individuals. Reliable results were obtained within 5 days. TP results (47 patient samples) for tNGS, WES, and WGS results were 33, 31, and 30, respectively, using the (L)P filtering, and 40, 40, and 38, respectively, when including VUS. FN results were 11, 13, and 14, respectively, excluding VUS, and 4, 4, and 6, when including VUS. The remaining FN were mainly samples with a homozygous VUS. All controls were TN. Three BC individuals showed a homozygous (L)P variant, all related to a variable, mild phenotype. The use of NGS-based workflows in NBS seems promising, although more knowledge of data handling, automated variant interpretation, and costs is needed before implementation.

Newborn screening (NBS) is an important public health program that aims to identify pre-symptomatic healthy babies that will develop significant disease if left undiagnosed and untreated. The number of conditions being screened globally is expanding rapidly in parallel with advances in technology, diagnosis, and treatment availability for these conditions. In Hong Kong, NBS for inborn errors of metabolism (NBSIEM) began as a pilot program in October 2015 and was implemented to all birthing hospitals within the public healthcare system in phases, with completion in October 2020. The number of conditions screened for increased from 21 to 24 in April 2016 and then to 26 in October 2019. The overall recruitment rate of the NBS program was 99.5%. In the period between October 2015 and December 2022, 125,688 newborns were screened and 295 were referred back for abnormal results. The recall rate was reduced from 0.26% to 0.12% after the implementation of second-tier testing. An inherited metabolic disorder (IMD) was eventually confirmed in 47 infants, making the prevalence of IMD in Hong Kong 1 in 2674. At the time of the NBS result, 78.7% of the newborns with IMD were asymptomatic. There were two deaths reported: one newborn with methylmalonic acidemia cobalamin B type (MMACblB) died after the initial crisis and another case of carnitine palmitoyltransferase II deficiency (CPTII) died at 18 months of age after metabolic decompensation. The most common IMD noted were disorders of fatty acid oxidation metabolism (40%, 19 cases), closely followed by disorders of amino acid metabolism (38%, 18 cases), with carnitine uptake defect (19.1%, 9 cases) and citrullinemia type II (17%, 8 cases) being the two most common IMD picked up by the NBSIEM in Hong Kong. Out of the all the IMDs identified, 19.1% belonged to diverse ethnic groups. False negative cases were reported for citrullinemia type II and congenital adrenal hyperplasia during this period.

Spinal muscular atrophy 5q (SMA) is one of the most common neuromuscular inherited diseases and is the most common genetic cause of infant mortality. SMA is associated with homozygous deletion of exon 7 in the SMN1 gene. Recently developed drugs can improve the motor functions of infants with SMA when they are treated in the pre-symptomatic stage. With aim of providing an early diagnosis, newborn screening (NBS) for SMA using a real-time PCR assay with dried blood spots (DBS) was performed from January 2022 through November 2022 in Saint Petersburg, which is a representative Russian megapolis. Here, 36,140 newborns were screened by the GenomeX real-time PCR-based screening test, and three genotypes were identified: homozygous deletion carriers (4 newborns), heterozygous carriers (772 newborns), and wild-type individuals (35,364 newborns). The disease status of all four newborns that screened positive for the homozygous SMN1 deletion was confirmed by alternate methods. Two of the newborns had two copies of SMN2, and two of the newborns had three copies. We determined the incidence of spinal muscular atrophy in Saint Petersburg to be 1 in 9035 and the SMA carrier frequency to be 1 in 47. In conclusion, providing timely information regarding SMN1, confirmation of disease status, and SMN2 copy number as part of the SMA newborn-screening algorithm can significantly improve clinical follow-up, testing of family members, and treatment of patients with SMA.

Metabolism of chemicals from the diet, exposures to xenobiotics, the microbiome, and lifestyle factors (e.g., smoking, alcohol intake) produce electrophiles that react with nucleophilic sites in circulating proteins, notably Cys34 of human serum albumin (HSA). To discover potential risk factors resulting from in utero exposures, we are investigating HSA-Cys34 adducts in archived newborn dried blood spots (DBS) that reflect systemic exposures during the last month of gestation. The workflow includes extraction of proteins from DBS, measurement of hemoglobin (Hb) to normalize for blood volume, addition of methanol to enrich HSA by precipitation of Hb and other interfering proteins, digestion with trypsin, and detection of HSA-Cys34 adducts via nanoflow liquid chromatography-high-resolution mass spectrometry. As proof-of-principle, we applied the method to 49 archived DBS collected from newborns whose mothers either actively smoked during pregnancy or were nonsmokers. Twenty-six HSA-Cys34 adducts were detected, including Cys34 oxidation products, mixed disulfides with low molecular weight thiols (e.g., cysteine, homocysteine, glutathione, cysteinylglycine), and other modifications. Data were normalized with a novel method ("scone") to remove unwanted technical variation arising from HSA digestion, blood volume, DBS age, mass spectrometry analysis, and batch effects. Using an ensemble of linear and nonlinear models, the Cys34 adduct of cyanide was found to consistently discriminate between newborns of smoking and nonsmoking mothers with a mean fold change (smoking/nonsmoking) of 1.31. These results indicate that DBS adductomics is suitable for investigating in utero exposures to reactive chemicals and metabolites that may influence disease risks later in life.

Abstract Mucopolysaccharidoses (MPSs) are a group of genetic alterations whose effect is the progressive intralysosomal accumulation of glycosaminoglycans. Affected individuals are deficient in one or more lysosomal enzymes which, depending on the MPS, may cause coarse facial features, short stature, multiple skeletal dysplasia, joint stiffness, or developmental delay. Their diagnosis is mostly performed late or incorrectly, and it represents a challenge since it requires specialized tests only performed in major cities. This makes it difficult for patients to have access to physicians since their geographical location is distant and therefore, the use of samples collected in solid‐phase represents an advantage for the study of high‐risk populations. In addition, epidemiological information about rare diseases, especially in Latin America, is scarce or inconsistent. Our aim was to report the experience of 20 years of selective screening by assessing enzyme activity and reporting incidence values of MPS in Colombia. This study validated a group of fluorometric endpoint techniques in 8239 patients. The samples were dried blood spots (DBS) collected on filter paper and leukocyte extracts. Reference values in the Colombian population for α‐l‐iduronidase, iduronate 2‐sulfatase, α‐N‐acetylglucosaminidase, N‐acetylglucosamine‐6‐sulfate sulfatase, β‐galactosidase, arylsulfatase B, and β‐glucuronidase were established in leukocyte extracts, and patients reference ranges were updated in the case of DBS samples. Incidence values were calculated for each MPS and the distribution of cases across the country is also shown. This study offers very useful information for the health system, the scientific community, and it facilitates the diagnosis of these disorders. This is indispensable when seeking to develop new diagnostic or treatment approaches for patients.

Abstract Background Strategies to support adherence are constrained by the lack of tools to objectively monitor medication intake in low-resource settings. Pharmacologic measures are objective, but pharmacy refill data is more accessible and cost-efficient. This study compared short-term and long-term efavirenz (EFV) drug levels with pharmacy refill adherence data (PRA) and evaluated their ability to predict viral suppression among people living with HIV in Nigeria. Methods Paired hair and dried blood spot (DBS) samples were obtained from 91 adults living with HIV receiving 600 mg EFV-based antiretroviral therapy (ART) and EFV concentrations were measured via validated methods using liquid-chromatography-mass-spectrometry. PRA was estimated from pharmacy records, based on the number of days a patient collected medication before or after the scheduled pick-up date. PRA was categorized into ≤ 74%, 75–94% and ≥ 95%, defined as poor, medium and high adherence, respectively. HIV viral loads closest to the hair sampling time (within 6 months) were also abstracted. Receiver Operating Characteristics (ROC) curve analyses compared the ability of adherence metrics to predict viral suppression. Results Based on PRA, 81% of participants had high adherence while 11% and 8% had medium and poor adherence, respectively. The median (IQR) EFV concentrations were 6.85 ng/mg (4.56–10.93) for hair and 1495.6 ng/ml (1050.7–2365.8) for DBS. Of the three measures of adherence, hair EFV concentration had the highest Area Under Curve (AUC) to predict viral suppression. Correlations between EFV concentrations in DBS and hair with PRA were positive (r = 0.12, P = 0.27 and r = 0.21, P = 0.05, respectively) but not strong. Conclusions EFV concentrations in hair were the strongest predictor of viral suppression and only weakly correlated with pharmacy refill adherence data in Nigeria. This study suggests that resource-limited settings may benefit from objective adherence metrics to monitor and support adherence.

Underserved, low-income, rural and certain migrant populations have greater risks and higher incidences of Coronavirus disease 2019 (COVID-19) than more privileged populations. Current in-person testing methods have limitations, namely exposure risk, a requirement of accessible transportation to healthcare facilities, and economic barriers. Dried blood spots (DBS) samples are widely used for diagnostics in many infectious diseases including Rabies, HIV, Ebola viruses and newborn screening. Our goal was to determine the accuracy and reliability of measuring COVID-19 IgG in DBS compared to paired plasma samples in a population with known infection status and then apply this method to screen an underserved minority population with high risk for COVID-9 infection (unvaccinated, pregnant, low income, Hispanic women). To optimize the assay, we tested 22 nonpregnant women, 12 with positive prior PCR testing for SARS-CoV2 infection and 10 with negative PCR results. After the assay was optimized, we tested the assay in a vulnerable population with a high risk for infection, who were 52 Hispanic pregnant women without prior PCR testing or vaccination. DBS assay results in both groups showed an agreement of 100% with paired plasma samples. The availability of a DBS assay could enable people who may not have access or transportation to healthcare facilities to use DBS as a COVID-19 testing vehicle.

Monitoring cortisol replacement therapy in congenital adrenal hyperplasia (CAH) patients is vital to avoid serious adverse events such as adrenal crises due to cortisol underexposure or metabolic consequences due to cortisol overexposure. The less invasive dried blood spot (DBS) sampling is an advantageous alternative to traditional plasma sampling, especially in pediatric patients. However, target concentrations for important disease biomarkers such as 17α-hydroxyprogesterone (17-OHP) are unknown using DBS. Therefore, a modeling and simulation framework, including a pharmacokinetic/pharmacodynamic model linking plasma cortisol concentrations to DBS 17-OHP concentrations, was used to derive a target morning DBS 17-OHP concentration range of 2–8 nmol/L in pediatric CAH patients. Since either capillary or venous DBS sampling is becoming more common in the clinics, the clinical applicability of this work was shown by demonstrating the comparability of capillary and venous cortisol and 17-OHP concentrations collected by DBS sampling, using a Bland-Altman and Passing-Bablok analysis. The derived target morning DBS 17-OHP concentration range is a first step towards providing improved therapy monitoring using DBS sampling and adjusting hydrocortisone (synthetic cortisol) dosing in children with CAH. In the future, this framework can be used to assess further research questions, e.g., target replacement ranges for the entire day. [ABSTRACT FROM AUTHOR]

Fabry disease (FD) is an X-linked lysosomal storage disorder where impaired α-galactosidase A enzyme activity leads to the intracellular accumulation of undegraded glycosphingolipids, including globotriaosylsphingosine (lyso-Gb3) and related analogues. Lyso-Gb3 and related analogues are useful biomarkers for screening and should be routinely monitored for longitudinal patient evaluation. In recent years, a growing interest has emerged in the analysis of FD biomarkers in dried blood spots (DBSs), considering the several advantages compared to venipuncture as a technique for collecting whole-blood specimens. The focus of this study was to devise and validate a UHPLC-MS/MS method for the analysis of lyso-Gb3 and related analogues in DBSs to facilitate sample collection and shipment to reference laboratories. The assay was devised in conventional DBS collection cards and in Capitainer®B blood collection devices using both capillary and venous blood specimens from 12 healthy controls and 20 patients affected with FD. The measured biomarker concentrations were similar in capillary and venous blood specimens. The hematocrit (Hct) did not affect the correlation between plasma and DBS measurements in our cohort (Hct range: 34.3–52.2%). This UHPLC-MS/MS method using DBS would facilitate high-risk screening and the follow-up and monitoring of patients affected with FD. [ABSTRACT FROM AUTHOR]

To broaden access to HIV viral load monitoring (VLM), the use of blood samples from dried blood spots (DBS) or point-of-care (POC) devices, could be of great help in settings where plasma is not easily accessible. The variety of assays available makes the choice complex. This systematic review and meta-analysis aims to estimate the sensitivity and specificity of DBS and POC devices to identify patients in virological failure using World Health Organization (WHO) recommendations (viral load ≥1000 copies/mL), compared with plasma, for the assays currently available. Four databases were searched for articles, and two reviewers independently identified articles reporting sensitivity and specificity of DBS and/or POC to identify patients in virological failure. We excluded articles that used other thresholds as well as articles with a total number of participants below 50 to avoid reporting bias. Heterogeneity and factors associated with assays' performances were assessed by I2 statistics and metaregression. The protocol of this review follows the PRISMA guidelines. Out of 941 articles, 47 were included: 32 DBS evaluations and 16 POC evaluations. Overall, when using DBS, the Abbott RT HIV-1, Roche CAP-CTM, NucliSENS BioMerieux and Aptima assays presented sensitivity and specificity exceeding 85%, but reported results were highly heterogeneous. Factors associated with better performances were high volume of blood and the use of the same assay for DBS and plasma VLM. Regarding the POC devices, SAMBA I, SAMBA II, and GeneXpert devices presented high sensitivity and specificity exceeding 90%, with less heterogeneity. DBS is suitable VLM, but performances can vary greatly depending on the protocols, and should be performed in trained centers. POC is suitable for VLM with less risk of heterogeneity but is more intensive in costs and logistics. [ABSTRACT FROM AUTHOR]

Objectives: Conduction of pharmacokinetic (PK) study in pediatric patients is challenging due to blood sampling limits. The dried blood spots (DBS) method represents a potential matrix for microsampling in support of PK studies in children. Herein, we used the Capitainer® qDBS device to develop a DBS method that can collect an exact 10 µL volume of blood on a paper card. This DBS method was developed to simultaneously quantify the concentrations of eight antibiotics, including sulbactam, tazobactam, ampicillin, meropenem, cefotaxime, cefoperazone, piperacillin, and metronidazole using ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Methods: The prepared DBS samples were extracted in methanol containing acetaminophen as the internal standard at 20 °C on a block bath shaker at 500 rpm for 30 min. The extracted antibiotics were eluted on an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) using gradient elution with a total chromatographic run time of 6.5 min. The precursor and product ions of the analytes were detected by use of the multiple reaction monitoring (MRM) mode. Results: No interfering peaks at the respective retention times of the analytes were observed in DBS samples. The lower limits of quantification (LLOQ) for the antibiotics were between 0.25 and 2.0 μg/mL, and satisfactory accuracies (intra/inter-assay bias −16.7 to +13.6%) and precisions (intra/inter-assay coefficient of variations 1.5–15.6%) were obtained for the analytes. As a proof of concept, the method was applied to DBS samples obtained from neonatal patients treated with ampicillin and piperacillin/sulbactam. Conclusions: The DBS method is simple and robust, and it can be used in children with limited blood sampling. [ABSTRACT FROM AUTHOR]

Biological bases of autism spectrum disorder (ASD) include both genetic and epigenetic causes. Patients with ASD show anomalies in the profile of certain plasma amino acids, including neuroactive amino acids. Monitoring plasma amino acids may be relevant for patient care and interventions. We evaluated the plasma amino acid profile in samples extracted from dry blood spots by electrospray ionization-tandem mass spectrometry. Fourteen amino acids and eleven amino acid ratios were examined in patients with ASD and intellectual disability (ID), and neurotypical control subjects (TD). The amino acid profile in the ASD group showed reduced levels of ornithine (p = 0.008), phenylalanine (p = 0.042) and tyrosine (p = 0.013). The statistically significant amino acid ratios were Leu+Val/Phe+Tyr (p = 0.002), Tyr/Leu (p = 0.007) and Val/Phe (p = 0.028), such differences remaining significant only in the comparison between ASD and TD. Finally, a positive correlation emerged between the score of the restricted and repetitive behavior on ADOS-2 and the citrulline levels in the ASD group (p = 0.0047). To conclude, patients with ASD may show a distinguishable metabolic profile useful for studying their metabolic pathways in order to develop screening tests and targeted therapies. [ABSTRACT FROM AUTHOR]

Dried blood spots (DBS) are a reliable tool to diagnose viremic hepatitis C virus (HCV) infection. We evaluated the clinical performance of a DBS‐based molecular assay for the assessment of cure and reinfection after on‐site treatment at a harm reduction center (HRC). Genotyping from DBS samples was also assessed to discriminate reinfection from treatment failure. People who inject drugs (PWID) from an ongoing test‐and‐treat pilot at the largest HRC in Barcelona were included in the study. HCV‐RNA detection from DBS collected after treatment (with follow‐up at 12, 36, and 60 weeks) was compared with a molecular point‐of‐care test using finger‐stick blood (GeneXpert). Baseline and follow‐up DBS samples were genotyped by NS5B sequencing or commercial real‐time PCR. Among treated patients, 193 follow‐up DBS samples were tested. The DBS‐based assay showed 100% specificity (129/129), and sensitivity ranged from 84.4% to 96.1% according to different viral load cut‐offs (from detectable to 3000 IU/mL). Sensitivity as test of cure (follow‐up 12) ranged from 85.1% to 97.4%. Among the 64 patients with recurrent viremia, 10.9% had low viral loads (≤1000 IU/mL); HCV genotyping allowed us to classify 73.5% of viremic cases either as reinfection or as treatment failure. DBS samples are useful to assess cure and differentiate reinfection from relapse after HCV antiviral treatment in the real world, facilitating decentralization of treatment and posttreatment follow‐up in PWID. However, a fraction of patients presented with low viral loads, limiting viremia detection and genotyping in DBS and, therefore, repeat testing is recommended. [ABSTRACT FROM AUTHOR]

For decades, blood testing has been an integral part of routine doping controls. The breadth of information contained in blood samples has become considerably more accessible for anti-doping purposes over the last 10 years through technological advancements regarding analytical instrumentation as well as enhanced sample collection systems. Particularly, microsampling of whole blood and serum, for instance as dried blood spots (DBS), has opened new avenues in sports drug testing and substantially increased the availability and cost-effectiveness of doping control specimens. Thus, microvolume blood specimens possess the potential to improve monitoring of blood hormone and drug levels, support evaluation of circulating drug concentrations in competition, and enhance the stability of labile markers and target analytes in blood passport analyses as well as peptide hormone and steroid ester detection. Further, the availability of the fraction of lysed erythrocytes for anti-doping purposes warrants additional investigation, considering the sequestering capability of red blood cells (RBCs) for certain substances, as a complementary approach in support of the clean sport. [ABSTRACT FROM AUTHOR]

Also in 2021/2022, considerable efforts were invested into advancing human sports drug testing programs, recognizing and taking into account existing as well as emerging challenges in anti‐doping, especially with regard to substances and methods of doping specified in the World Anti‐Doping Agency's 2022 Prohibited List. In this edition of the annual banned‐substance review, literature on recent developments published between October 2021 and September 2022 is summarized and discussed. Focus is put particularly on enhanced analytical approaches and complementary testing options in human doping controls, appreciating the exigence and mission in anti‐doping and, equally, the contemporary "new normal" considering, for example, the athlete's exposome versus analytical sensitivity and applicable anti‐doping regulations for result interpretation and management. [ABSTRACT FROM AUTHOR]

Abstract Introduction Currently, strategies for improving alpha1 antitrypsin deficiency (AATD) diagnosis are needed. Here we report the performance of a multinational multiplex-based genotyping test on dried blood spots and buccal swabs sent by post or courier and with web registration for subjects with suspected AATD in Argentina, Brazil, Chile, Colombia, Spain, and Turkey. Methods This was an observational, cross-sectional analysis of samples from patients with suspected AATD from March 2018 to January 2022. Samples were coded on a web platform and sent by post or courier to the central laboratory in Northern Spain. Allele-specific genotyping for the 14 most common mutations was carried out with the A1AT Genotyping Test (Progenika-Grifols, Spain). SERPINA1 gene sequencing was performed if none of the mutations were found or one variant was detected in heterozygous status and the AAT serum level was

• HIV diagnostic algorithms using multiple rapid serologic tests may lead to misclassification. • Dried blood spots (DBS) are simple and inexpensive to collect and transport. • DBS-based HIV-1 nucleic acid detection methods were compared to determine their suitability to confirm HIV-1 infection in adults with suppressed or low-level virus loads. • Cepheid Xpert HIV-1 Qual Assay [96.9 % (158/163)] and Roche Cobas Ampliprep/Cobas TaqMan HIV-1 test, v.2.0 (CAP/CTM) with sample pre-extraction buffer [95.7 % (156/163) were similarly sensitive. • CAP/CTM with PBS sample preparation [68.2 % (107/157)] and Hologic Aptima HIV-1 Quant Dx [69.2 % (99/143)] assay were significantly less sensitive. The WHO HIV testing algorithm for high prevalence populations recommends the use of three different serologic assays, though this approach may lead to diagnostic misclassification. The study objective was to compare dried blood spot (DBS)-based HIV-1 nucleic acid detection methods to determine their suitability to confirm the diagnosis of HIV-1 in adults generally with suppressed or low-level plasma HIV-1 RNA. Four methods were evaluated: Cepheid Xpert HIV-1 Qual Assay (Xpert), Hologic Aptima HIV-1 Quant Dx assay (Aptima), Roche Cobas Ampliprep/Cobas TaqMan HIV-1 test, v.2.0 (CAP/CTM) with guanidinium-based sample pre-extraction buffer (SPEX), or CAP/CTM with phosphate-buffered saline (PBS). Testing was performed on 163 DBS samples collected from participants with HIV-1 in the AIDS Clinical Trial Group (ACTG) A5230 study (73 samples) and the Peninsula AIDS Research Cohort (PARC) study (90 samples). Xpert and SPEX CAP/CTM [96.9 % (158/163):95.7 % (156/163); P = 0.75) showed similar sensitivity. However, PBS CAP/CTM and Aptima demonstrated significantly lower sensitivity, 68.2 % (107/157) and 69.2 % (99/143), respectively, compared to Xpert and SPEX CAP/CTM (P < 0.0001 for all comparisons). Overall agreement between Xpert and SPEX CAP/CTM was 93.9 % (153/163), including 152 DBS samples in which both methods detected HIV-1 nucleic acids. Xpert and SPEX CAP/CTM provide sensitive performance for the detection of HIV-1 nucleic acids using DBS collected from adults living with HIV-1, including those with suppressed virus loads. Given the cost and side-effects associated with inappropriate life-long antiretroviral therapy, these assays may play a role in diagnosing HIV-1 infection in individuals with suspected false-positive serologic testing. [ABSTRACT FROM AUTHOR]

• Epstein–Barr virus (EBV) DNA detection in dried blood spots (DBS) performed a specificity of 100 %. • The sensitivity of EBV DNA detection in DBS increased with elevating viral load. • There was a good correlation in EBV DNA quantification between DBS and whole blood samples. • Our research firstly demonstrated the feasibility of using DBS for qualitative and semi-quantitative detection of EBV DNA. Epstein–Barr virus (EBV) is a ubiquitous and oncogenic virus that is associated with various malignancies and non-malignant diseases and EBV DNA detection is widely used for the diagnosis and prognosis prediction for these diseases. The dried blood spots (DBS) sampling method holds great potential as an alternative to venous blood samples in geographically remote areas, for individuals with disabilities, or for newborn blood collection. Therefore, the objective of this study was to assess the viability of detecting EBV DNA load from DBS. Matched whole blood and DBS samples were collected for EBV DNA extraction and quantification detection. EBV DNA detection in DBS presented a specificity of 100 %. At different EBV DNA viral load in whole blood, the sensitivity of EBV DNA detection in DBS was 38.78 % (≥1 copies/mL), 43.18 % (≥500 copies/mL), 58.63 % (≥1000 copies/mL), 71.43 % (≥2000 copies/mL), 82.35 % (≥4000 copies/mL), and 92.86 % (≥5000 copies/mL), respectively. These results indicated that the sensitivity of EBV DNA detection in DBS increased with elevating viral load. Moreover, there was good correlation between EBV DNA levels measured in whole blood and DBS, and on average, the viral load measured in whole blood was about 6-fold higher than in DBS. Our research firstly demonstrated the feasibility of using DBS for qualitative and semi-quantitative detection of EBV DNA for diagnosis and surveillance of EBV-related diseases. [ABSTRACT FROM AUTHOR]