Dried Blood Spot Analysis Research Articles

B-248 Enhanced Detection of of Hemoglobin Variants in Clinical Research using Dried Blood Spot and HRAM MS

Abstract Background Hemoglobinopathies are one of the most prevalent genetic disorders, with more than 330,000 affected births occurring annually. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. However, there are challenges to differentiate isomers (i.e., between Hb C and Hb E), very similar masses, or new variants due to their lack of resolution. Mass spectrometry (MS) gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins. Here, we present the top-down analysis for enhanced detection of various hemoglobin variants using Orbitrap Exploris for clinical research. Methods Proteins were directly extracted from dried blood spots (DBS) and analyzed by Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option coupled to Vanquish HPLC system. A 3.2 mm disc was punched from the DBS cards to 96 well plate and 50 uL water was added to re-hydrate. For protein precipitation, 150 uL acetonitrile was added and incubated at -20 ⁰C for 15 minutes. After micro-centrifugation, 160 uL of supernatant was removed and 70 uL water was added to resuspend. For LC-MS analysis, 10 uL of supernatant was diluted 8 times with mobile phase A and transferred to another 96-well plate. LC separation was performed on MAbPac RP column 4 um, 1 x 100 mm, and data-dependent acquisition mode was operated. Results With direct protein extraction from DBS, the entire sample preparation time is less than 1 hour. The MAbPac column showed its capability of separating between different chains with minor overlap between normal beta and HbS beta chains. Data processing was performed by ProSightPD, resulting in ∼45% sequence coverage for both normal Hb and HbS beta chains. The mass difference between normal beta and Hb S beta chains is about 30 Da, which results in about 1.6 m/z difference at a charge state of 19. This mass difference can often be hindered by other proteins or interferences from the matrix if the mass analyzer doesn’t have sufficient resolving power. Here we successfully applied Orbitrap resolution at 120,000 to isolate these similar masses for accurate variants or subunits identification including normal alpha, normal beta, Hb S, and delta chains. The confirmation of the delta chain can be beneficial since its abundance is usually less than 0.5% in adults, which shows potential for detecting low abundant variants using this method. Also, the calibration curve was generated to demonstrate the method's feasibility which is not a typical quantitation method for hemoglobinopathies. For quantitation, three most abundant m/z values from 2 charge states were added to TraceFinder for peak area extraction and normal Hb beta chain was used as an alternative internal standard for data processing. The 3-day inter/intraday analysis generated great reproducibility with CV < 10% and LOQ was determined to be 2.5 mg/mL with R2 values >0.994. Conclusions Successful demonstration of a single-step protein extraction directly from DBS and top-down analysis of Hb variant for clinical research.

Dried blood spot sampling coupled with liquid chromatography-tandem mass for simultaneous quantitative analysis of multiple cardiovascular drugs

Dried Blood Spots (DBS) revolutionize therapeutic drug monitoring using LC-MS for the precise quantification of cardiovascular drugs (CDs), enabling personalized treatment adapted to patient-specific pharmacokinetics with minimal invasiveness. This study aims to achieve simultaneous quantification of eight CDs in DBS, overcoming physicochemical challenges. A two-step protein precipitation method was used for simple and precise sample preparation. The drugs were analyzed using LC-MS/MS in ESI positive-ion mode, showing high sensitivity and linearity, with a correlation coefficient (r2) exceeding 0.999, after being separated on a reversed-phase chromatography by gradient elution of DW-acetonitrile containing 0.1 % formic acid + 2 mM ammonium formate. The validation results indicate good selectivity, with no observed matrix effect and carry-over. The intra- and inter-day accuracy and precision were within 6 % for most drugs, except for digoxin and deslanoside at low therapeutic levels where the variation was within 20 %. Stability tests confirmed suitable DBS handling and storage conditions, indicating drug stability for at least 30 days at room temperature. The analysis of whole spot has demonstrated remarkable precision and reliability in all target drugs. The analysis of 3 mm internal diameter discs, punched in and out of DBS, presumed to contain 3 µL of blood, showed acceptable accuracy for most drugs, with less polar drugs like digoxin and deslanoside showing lower accuracy, indicating a need for further correction due to non-uniform drug distribution. Consequently, the developed LC-MS/MS method enables the quantification of multiple CDs in a single DBS analysis, while suggesting the potential for accuracy-based analysis.

Dried blood spot analysis: Comparing diagnostic outcomes between uniform and layered samples in newborn screening

Dried blood spot analysis: Comparing diagnostic outcomes between uniform and layered samples in newborn screening

Proteome profiling of home-sampled dried blood spots reveals proteins of SARS-CoV-2 infections

BackgroundSelf-sampling of dried blood spots (DBS) offers new routes to gather valuable health-related information from the general population. Yet, the utility of using deep proteome profiling from home-sampled DBS to obtain clinically relevant insights about SARS-CoV-2 infections remains largely unexplored.MethodsOur study involved 228 individuals from the general Swedish population who used a volumetric DBS sampling device and completed questionnaires at home during spring 2020 and summer 2021. Using multi-analyte COVID-19 serology, we stratified the donors by their response phenotypes, divided them into three study sets, and analyzed 276 proteins by proximity extension assays (PEA). After normalizing the data to account for variances in layman-collected samples, we investigated the association of DBS proteomes with serology and self-reported information.ResultsOur three studies display highly consistent variance of protein levels and share associations of proteins with sex (e.g., MMP3) and age (e.g., GDF-15). Studying seropositive (IgG+) and seronegative (IgG-) donors from the first pandemic wave reveals a network of proteins reflecting immunity, inflammation, coagulation, and stress response. A comparison of the early-infection phase (IgM+IgG-) with the post-infection phase (IgM-IgG+) indicates several proteins from the respiratory system. In DBS from the later pandemic wave, we find that levels of a virus receptor on B-cells differ between seropositive (IgG+) and seronegative (IgG-) donors.ConclusionsProteome analysis of volumetric self-sampled DBS facilitates precise analysis of clinically relevant proteins, including those secreted into the circulation or found on blood cells, augmenting previous COVID-19 reports with clinical blood collections. Our population surveys support the usefulness of DBS, underscoring the role of timing the sample collection to complement clinical and precision health monitoring initiatives.

COVID-19 monitoring of school personnel through molecular salivary test and dried blood spot analysis.

When the coronavirus disease 2019 (COVID-19) pandemic broke out, most countries enforced school closures as a precautionary measure. Although COVID-19 is still present three years later, schools have been reopened. We aimed to test the association of molecular salivary testing (MST) and dried blood spot (DBS) analysis for community surveillance by investigating the immunological profile of a group of school staff during and following COVID-19 vaccination. We conducted the study in a school in Milan from April 2021, when school staff were administered the first dose of vaccine against SARS-CoV-2, until the school year ended in June 2022. Each participant provided samples for MST and DBS one month (T1, W1) after receiving their first dose of vaccine. Subsequently, they collected weekly MST samples for five weeks (W2-W6), plus a DBS sample in the last week (T2). Both samples were collected one (T3), four (T4), and seven months (T5) after the administration of the second vaccine dose in May 2021. A final DBS sample was collected one year (T6) after T3. Sixty participants provided 327 MSTs and 251 DBSs. None of the MST samples tested positive for SARS-CoV-2 RNA during the study period. A total of 201 DBS samples tested positive for the IgG semiquantitative analysis. Negative samples were found only at T1 (20.45%) and T2 (7.32%). We observed borderline results at T1 (4.55%), T2 (7.32%), and T4 (2.70%). The anti-SARS-CoV-2 average antibody ratio increased after the second dose between T2 and T3, and the trend peaked after the third dose between T4 and T6. We performed an immunoenzymatic assay of antibodies against nucleocapsid protein on samples collected at T1 from five participants who reported having been infected before the study and from four subjects with an abnormal increase in the antibody values at T4. Two samples tested positive in the first group and two in the second one. Our findings show that MST and DBS could be effective tools in the active surveillance of school personnel and that schools could be considered safe settings in view of SARS-CoV-2 infection. Vaccines might have contributed to case and/or symptom reduction.

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.

Dried blood spot analysis for the quantification of vancomycin and creatinine using liquid chromatography – tandem mass spectrometry: Method development and validation

BackgroundVancomycin is a widely used antibiotic for the treatment of gram-positive bacterial infections, especially for methicillin-resistant Staphylococcus aureus (MRSA) infections. Due to a small therapeutic range and large inter-patient variability, therapeutic drug monitoring (TDM) of vancomycin is required to minimize toxicity and maximize treatment efficacy. Venous blood sampling is mostly applied for TDM of vancomycin, although this widely used sampling method is more invasive compared to less painful alternatives, such as the dried blood spot (DBS) method, which can be performed at home. MethodWe developed an UPLC-MS/MS method for the quantification of vancomycin and creatinine in DBS. A fast sample preparation and short analysis run time of 5.2 min were applied, which makes this method highly suitable for clinical settings. Validation was performed according to international (FDA and EMA) guidelines. ResultsThe validated concentration range was found linear for creatinine from 41.8 µmol/L to 722 µmol/L and for vancomycin from 3.8 mg/L to 76.6 mg/L (r2 > 0.990) and the inaccuracies, imprecisions, hematocrit effects, and recoveries were < 15 % for both compounds. No significant carryover effect was observed. ConclusionHence, we successfully validated a quantification method for the simultaneous determination of creatinine and vancomycin in DBS.

A New Test Method for Biochemical Analysis of Plasmalogens in Dried Blood Spots and Erythrocytes From Patients With Peroxisomal Disorders.

Measurement of plasmalogens is useful for the biochemical diagnosis of rhizomelic chondrodysplasia punctata (RCDP) and is also informative for Zellweger spectrum disorders (ZSD). We have developed a test method for the simultaneous quantitation of C16:0, C18:0, and C18:1 plasmalogen species and their corresponding fatty acids in dried blood spots (DBS) and erythrocytes (RBC) by using capillary gas chromatography-mass spectrometry (GC-MS). Normal reference ranges for measured markers and 10 calculated ratios were established by the analysis of 720 and 473 unaffected DBS and RBC samples, respectively. Determination of preliminary disease ranges was made by using 45 samples from 43 unique patients: RCDP type 1 (DBS: 1 mild, 17 severe; RBC: 1 mild, 6 severe), RCDP type 2 (DBS: 2 mild, 1 severe; RBC: 2 severe), RCDP type 3 (DBS: 1 severe), RCDP type 4 (RBC: 2 severe), and ZSD (DBS: 3 severe; RBC: 2 mild, 7 severe). Postanalytical interpretive tools in Collaborative Laboratory Integrated Reports (CLIR) were used to generate an integrated score and a likelihood of disease. In conjunction with a review of clinical phenotype, phytanic acid, and very long-chain fatty acid test results, the CLIR analysis allowed for differentiation between RCDP and ZSD. Data will continue to be gathered to improve CLIR analysis as more samples from affected patients with variable disease severity are analyzed. The addition of DBS analysis of plasmalogens may allow for at-home specimen collection and second-tier testing for newborn screening programs. This article is protected by copyright. All rights reserved.

Dried blood spot analysis for elements of nutritional concern as demonstrated in studies of Galápagos land iguanas (Conolophus species)

BackgroundDried blood spot (DBS) technology is valuable in providing simple means of storing blood samples from wildlife with small blood volumes. Methods designed for heavy metal analysis on DBS become more useful if extended to elements of nutritional significance. Purpose(1) Development of procedures for measuring Mn, Fe, Co, Cu, Zn, Se and Mo in DBS; (2) use the designed methods in health assessments of Galápagos land iguanas (Conolophus species). ProceduresElements were measured by inductively coupled plasma/mass spectrometry (ICP-MS) following acid digestion of whole blood or DBS from the same animal for direct comparison. Study animals comprised free-ranging iguanas from separate islands in the Galápagos archipelago. Main FindingsDBS spikes (Mn, Fe, Co, Cu, Zn, Se and Mo) demonstrated accuracy to ∼100 ppb; reporting limits were set there except for Fe and Zn which were set at 1000 ppb. Plasma samples – generally preferable for nutritional element diagnostics – were submitted from Galápagos land iguanas along with DBS as part of a large-scale health assessment. In plasma versus DBS concentration comparisons, Fe, Cu, Se and Mn correlated well with R^2 values of 0.799, 0.818, 0.896 and 0.899, respectively, and slopes ranging 0.88 – 1.3. Co and Zn showed greater scatter. Mo had insufficient points above its reporting limit and offered advantages for toxicity assessments. Bland-Altman diagrams showed flat scatter between 2x standard deviation boundaries with no undue trends except for Mn which had few points above its reporting limit. Bias, defined as the average difference [DBS – plasma] divided by the average value, was relatively low throughout, with values of − 19.3 % (Fe), − 48.7 % (Co), − 19.6 % (Cu), − 6.9 % (Zn), − 21.4 % (Se) and + 40.7 % (Mn). Normal distribution assessment of iguana Cu, Zn, Se and Fe plasma values showed unanticipated divergences between two species. ConclusionsThe DBS approach for nutritional element analysis offers a suitable methodology for determining crucial elements Mn, Fe, Co, Cu, Zn, Se, and Mo in veterinary samples. Analyses of samples from Conolophus species revealed interesting divergences particularly for Cu, Zn, Se and Fe, elements generally associated with defense against oxidative stress.

From "wet" matrices to "dry" blood spot sampling strategy: a versatile LC-MS/MS assay for simultaneous monitoring caffeine and its three primary metabolites in preterm infants.

To update traditional "wet" matrices to dried blood spot (DBS) sampling, based on the liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) technique, and develop a method for simultaneous analyzing caffeine and its three primary metabolites (theobromine, paraxanthine, and theophylline), supporting routine therapeutic drug monitoring (TDM) for preterm infants. DBS samples were prepared by a two-step quantitative sampling method, i.e.,volumetric sampling of a quantitative 10 μL volume of peripheral blood and an 8 mm diameter whole punch extraction by a methanol/water (80/20, v/v) mixture containing 125 mM formic acid. Four paired stable isotope labeled internal standards and a collision energy defect strategy were applied for the method optimization. The method was fully validated following international guidelines and industrial recommendations on DBS analysis. Cross validation with previously developed plasma method was also proceeded. The validated method was then implemented on the TDM for preterm infants. The two-step quantitative sampling strategy and ahigh recovery extraction method were developed and optimized. The method validation results were all within the acceptable criteria. Satisfactory parallelism, concordance, and correlation were observed between DBS and plasma concentrations of the four analytes. The method was applied to provide routine TDM services to 20 preterm infants. A versatile LC-MS/MS platform for simultaneous monitoring caffeine and its three primary metabolites was developed, fully validated, and successfully applied into the routine clinical TDM practices. Sampling method switching from "wet" matrices to "dry" DBS will facilitate and support the precision dosing of caffeine for preterm infants.

Diagnosis and Management of Late-Onset Pompe Disease

Pompe disease is a lysosomal storage disorder, with onset between the first weeks after birth and adulthood, depending on its phenotype. It can affect multiple organ systems and presents itself with a wide variety of symptoms. Thus, recognizing Pompe disease is difficult. Especially since enzyme replacement therapy for Pompe disease was introduced (in Germany in 2006), early diagnosis by means of enzyme activity determination from dried blood spot analysis and genetic verification has become important for outcome and quality of life. When facing an obscure muscular disorder, it is crucial to consider Pompe disease. This article provides an overview about Pompe disease and focuses on the diagnosis of the late onset type. The most important aspects of interdiciplinary care for patients with Pompe disease are presented. Additionally, it contains a section focusing on psychosocial challenges for children with Pompe disease and their families, which may include mental disorders and social retreat, and gives advice on how to support parents of affected children.

Endogenous, non-reducing end glycosaminoglycan biomarkers are superior to internal disaccharide glycosaminoglycan biomarkers for newborn screening of mucopolysaccharidoses and GM1 gangliosidosis

Measurement of enzymatic activity in newborn dried blood spots (DBS) is the preferred first-tier method in newborn screening (NBS) for mucopolysaccharidoses (MPSs). Our previous publications on glycosaminoglycan (GAG) biomarker levels in DBS for mucopolysaccharidosis type 1 (MPS-I) and MPS-II demonstrated that second-tier GAG biomarker analysis can dramatically reduce the false positive rate in NBS. In the present study, we evaluate two methods for measuring GAG biomarkers in seven MPS types and GM1 gangliosidosis. We obtained newborn DBS from patients with MPS-IIIA-D, -IVA, -VI, -VII, and GM1 gangliosidosis. These samples were analyzed via two GAG mass spectrometry methods: (1) The internal disaccharide biomarker method; (2) The endogenous non-reducing end (NRE) biomarker method. This study supports the use of second-tier GAG analysis of newborn DBS by the endogenous NRE biomarker method, as part of NBS to reduce the false positive rate.

Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry.

Dried blood spot samples are simple to prepare and transport, enabling safe and accessible diagnostics, both locally and globally. We review dried blood spot samples for clinical analysis, focusing on liquid chromatography-mass spectrometry as a versatile measurement tool for these samples. Dried blood spot samples can provide information for, for example, metabolomics, xenobiotic analysis, and proteomics. Targeted analyses of small molecules are the main application of dried blood spot samples and liquid chromatography-mass spectrometry, but emerging applications include untargeted metabolomics and proteomics. Applications are highly varied, including analyses related to newborn screening, diagnostics and monitoring of disease progression and treatment effects of virtually any disease, as well as studies into the physiology and effects of diet, exercise, xenobiotics, and doping. A range of dried blood spot products and methods are available, and applied liquid chromatography-mass spectrometry instrumentation is varied with regard to liquid chromatography column formats and selectivity. In addition, novel approaches such as on-paper sample preparation (e.g., selective trapping of analytes with paper-immobilized antibodies) are described. We focus on research papers published in the last 5 years.

Dried Blood Spot Analyses for the Diagnosis of Anti-cytokine Autoantibodies

BackgroundHigh-titer neutralizing anti-cytokine autoantibodies have been shown to be involved in several acquired diseases, including pulmonary alveolar proteinosis, cryptococcal meningitis, and disseminated/extrapulmonary Nocardia infections (anti-GM-CSF autoantibodies), disseminated mycobacterial disease (anti-IFN-γ autoantibodies), and some cases of severe COVID-19 infection (anti-type 1 interferons). Currently, patient blood samples are shipped via courier and require temperaturecontrolled conditions for transfer. This method is expensive and requires patients to have access to medical personnel to draw the blood. However, the well-established technique of collecting blood on a paper card as a dried blood spot (DBS) for diagnosis offers a point of care alternative which can be performed with a simple finger prick. This method is less invasive, cheaper, and allows for easy transport of patient samples. Methods30 uL of whole blood from patients was blotted on filter paper and stored at 4C until use. The filter paper was hole punched and each punched spot was eluted with 150 uL of a 0.05% Tween PBS solution at room temperature overnight. The eluate was screened for anti-cytokine autoantibodies using a particle-based approach. Patient plasma was also screened in conjunction for comparison. ResultsWe confirmed the presence of autoantibodies in the DBS eluate from 4 previously diagnosed patients with anti-GM-CSF autoantibodies and 2 patients with anti-IFN-γ autoantibodies. Functional studies showed the DBS eluate from a patient with anti-GM-CSF autoantibodies was able to block GM-CSF-induced STAT-5 phosphorylation in normal PBMC. As a proof of concept and to increase the number of patients evaluated, we also confirmed the presence of anti-cytokine autoantibodies using dried plasma eluate from 9 patients with known anti-GM-CSF autoantibodies and 9 patients with anti-IFN-γ autoantibodies. Levels detected in DBS analyses were comparable to the levels found in plasma from the same patients not subjected to blotting and elution. Temperature studies showed that the autoantibodies were detected at similar levels when stored at 4C, 25C, and 40C for a week. ConclusionsThe diagnosis of pathogenic anti-cytokine autoantibodies should be considered in the context of unusual or adult-onset infections, and screening for this diagnosis can be performed with dried blood spot testing.

Dried blood spot eluates are suitable for testing of SARS-CoV-2 IgG antibodies targeting Spike protein 1 and Nucleocapsid protein

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.

Development of a fast and robust liquid chromatography-mass spectrometry-based metabolomics analysis method for neonatal dried blood spots

Dried blood spot (DBS) samples have been widely used in many fields including newborn screening, with the advantages in transportation, storage and non-invasiveness. The DBS metabolomics research of neonatal congenital diseases will greatly expand the understanding of the disease. In this study, we developed a liquid chromatography-mass spectrometry-based method for neonatal metabolomics analysis of DBS. The influences of blood volume and chromatographic effects on the filter paper on metabolite levels were studied. The levels of 11.11 % metabolites were different between 75 μL and 35 μL of blood volumes used for DBS preparation. Chromatographic effects on the filter paper occurred in DBS prepared with 75 μL whole blood and 6.67 % metabolites had different MS responses when central disks were compared with outer disks. The DBS storage stability study showed that compared with − 80 °C storage, storing at 4 °C for 1 year had obvious influences on more than half metabolites. Storing at 4 °C and − 20 °C for short term (< 14 days) and − 20 °C for longer term (1 year) had less influences on amino acids, acyl-carnitines and sphingomyelins, but greater influences on partial phospholipids. Method validation showed that this method has a good repeatability, intra-day and inter-day precision and linearity. Finally, this method was applied to investigate metabolic disruptions of congenital hypothyroidism (CH), metabolic changes of CH newborns were mainly involved in amino acid metabolism and lipid metabolism.

Coordinating smoking cessation treatment with menstrual cycle phase to improve quit outcomes (MC-NRT): study protocol for a randomized controlled trial

BackgroundWomen experience greater difficulty achieving smoking abstinence compared to men. Recent evidence suggests that hormonal fluctuations during different phases of the menstrual cycle can contribute to lower smoking abstinence rates following a quit attempt among women. However, these findings are limited by small sample sizes and variability among targeted smoking quit dates. This clinical trial aims to clarify whether targeting the quit date to the follicular or luteal phase of the menstrual cycle can improve smoking abstinence.MethodsParticipants will enroll in an online smoking cessation program providing nicotine replacement therapy (NRT) and behavioral support. We will randomize 1200 eligible individuals to set a target quit date: (1) during the mid-luteal phase, (2) during the mid-follicular phase, or (3) 15–30 days after enrollment with no regard to the menstrual cycle phase (usual practice). Participants will receive a 6-week supply of combination NRT consisting of a nicotine patch plus their choice of nicotine gum or lozenge. Participants will be instructed to start using NRT on their target quit date. Optional behavioral support will consist of a free downloadable app and brief videos focusing on building a quit plan, coping with cravings, and relapse prevention, delivered via e-mail. Smoking status will be assessed via dried blood spot analysis of cotinine concentration at 7 days, 6 weeks, and 6 months post-target quit date.DiscussionWe aim to overcome the limitations of previous studies by recruiting a large sample of participants and assigning target quit dates to the middle of both the follicular and luteal phases. The results of the trial can further elucidate the effects of the menstrual cycle on smoking cessation outcomes and whether it is beneficial to combine menstrual cycle phase timing strategies with accessible and low-cost NRT.Trial registrationClinicalTrials.gov NCT05515354. Registered on August 23, 2022.

A Pilot Study of Biliary Atresia Newborn Screening Using Dried Blood Spot Matrix Metalloproteinase-7.

Timely diagnosis is a critical challenge and is associated with improved survival of biliary atresia (BA) patients. We aimed to measure matrix metalloproteinase-7 (MMP-7) levels in BA patients within 3 days of birth using the dried blood spot (DBS) method and evaluate its potential as a screening tool. The study enrolled 132 patients, including 25 patients diagnosed with BA and 107 non-BA patients with other congenital or perinatal conditions from the National Taiwan University Children Hospital. The stored DBS samples collected from 48 to 72 hours of life were retrieved from newborn screening centers. MMP-7 on the DBS was quantified using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA). The MMP-7 levels of BA patients on the DBS were significantly higher than those of non-BA patients (19.2 ± 10.4 vs 5.6 ± 2.7 ng/mL, P value < 0.0001). MMP-7 levels in non-BA patients, including 5 patients with hepatobiliary structural anomaly, 9 patients with intrahepatic cholestasis, and 93 patients with other perinatal diseases, were 11.6 ± 4.2 ng/mL, 6.9 ± 3.0 ng/mL, and 5.2 ± 2.1 ng/mL, respectively. The DBS MMP-7 level showed good accuracy for identifying BA, with an area under the curve of 93.7% [95% confidence interval (CI): 87.7%-99.7%]. The MMP-7 cutoff at 8.0 ng/mL showed a sensitivity of 92.0% (95% CI: 75.0%-98.6%) and specificity of 92.5% (95% CI: 85.9%-96.1%) for detecting BA from other congenital or perinatal diseases. MMP-7 DBS analysis can be used to distinguish BA from other conditions as early as 3 days of age.

Reported History of Measles and Long-term Impact on Tetanus Antibody Detected in Children 9-59 Months of Age and Receiving 3 Doses of Tetanus Vaccine in the Democratic Republic of the Congo.

Recent studies suggest measles-induced immune amnesia could have long-term immunosuppressive effects via preferential depletion of memory CD150+ lymphocytes, and associations with a 2-3 year period of increased mortality and morbidity from infectious diseases other than measles has been shown in children from wealthy and low-income countries. To further examine the associations previous measles virus infection may have on immunologic memory among children in the Democratic Republic of the Congo (DRC), we assessed tetanus antibody levels among fully vaccinated children, with and without a history of measles. We assessed 711 children 9-59 months of age whose mothers were selected for interview in the 2013-2014 DRC Demographic and Health Survey. History of measles was obtained by maternal report and classification of children who had measles in the past was completed using maternal recall and measles IgG serostatus obtained from a multiplex chemiluminescent automated immunoassay dried blood spot analysis. Tetanus IgG antibody serostatus was similarly obtained. A logistic regression model was used to identify association of measles and other predictors with subprotective tetanus IgG antibody. Subprotective geometric mean concentration tetanus IgG antibody values were seen among fully vaccinated children 9-59 months of age, who had a history of measles. Controlling for potential confounding variables, children classified as measles cases were less likely to have seroprotective tetanus toxoid antibody (odds ratio: 0.21; 95% confidence interval: 0.08-0.55) compared with children who had not had measles. History of measles was associated with subprotective tetanus antibody among this sample of children in the DRC who were 9-59 months of age and fully vaccinated against tetanus.

Utilizing quantitative dried blood spot analysis to objectively assess adherence to cardiovascular pharmacotherapy among patients at Kenyatta National Hospital, Nairobi, Kenya.

The burden of cardiovascular disease (CVD) is rising in Kenya and non-adherence to cardiovascular pharmacotherapy is a growing global public health issue that leads to treatment failure, an increased risk of cardiac events and poor clinical outcomes. This study assessed adherence to selected cardiovascular therapy medications among CVD patients attending outpatient clinics at Kenyatta National Hospital, Kenya by determining drug concentration(s) in patient dried blood spot (DBS) samples. Patients who had been taking one or more of the five commonly prescribed CVD medications (amlodipine, atenolol, atorvastatin, losartan, and valsartan) for at least six months were enrolled. Each patient completed a short questionnaire about their medication history and then provided a finger-prick blood spot sample from which drug concentrations were determined by liquid chromatography-high resolution mass spectrometry analysis. Two hundred and thirty-nine patients (62.3% female) participated in the study. The median number of medications used by patients was 2 (IQR 75%-25% is 3-1). Less than half (117; 49.0%) of patients were adherent to their prescribed CVD pharmacotherapy. Binary regression analysis revealed a significant correlation between non-adherence and the number of medications in the treatment regimen (Odds Ratio (OR) 1.583; 95%CI: 0.949-2.639; P-value = 0.039) and that gender was not an independent predictor of medication adherence (OR 1.233; 95%CI: 0.730-2.083; P-value = 0.216). Valuable information about adherence to each medication in the patient's treatment regimen was obtained using quantitative DBS analysis showing that adherence to CVD medications was not uniform. DBS sampling, due its minimally invasive nature, convenience and ease of transport is a useful alternative matrix to monitor adherence to pharmacotherapies objectively, when combined with hyphenated mass spectrometry analytical techniques. This information can provide physicians with an evidence-based novel approach towards personalization and optimization of CVD pharmacotherapy and implementing interventions in the Kenyan population, thereby improving clinical outcomes.