Blood Spot Assay Research Articles

Expert opinion on the diagnostic odyssey and management of late-onset Pompe disease: a neurologist's perspective.

This consensus statement by a panel of neurology experts aimed to provide a practical and implementable guidance document to assist clinicians with the best clinical practice in terms of diagnosis, treatment, and monitoring of late-onset Pompe disease (LOPD). The participating experts consider the clinical suspicion of LOPD by the physician to be of utmost importance in the prevention of diagnostic and therapeutic delay in LOPD patients. A diagnostic algorithm is proposed to facilitate the diagnosis of LOPD in patients presenting with unexplained proximal/axial weakness (with or without respiratory symptoms) or restrictive respiratory insufficiency with hyperCKemia and/or exercise intolerance as the red flag symptoms/signs that raise the index of suspicion for LOPD diagnosis. The diagnosis is based on the subsequent use of dried blood spot (DBS) assay, and the DBS assay can be confirmed by acid alpha-glucosidase (GAA) tissue analysis in leukocytes, fibroblasts, or muscle fibers and/or genetic mutation analysis. Accordingly, experts consider increased awareness among physicians about potential presenting characteristics with a high index of suspicion for LOPD to be crucial to suspect and consider LOPD in the differential diagnosis, while strongly suggesting the use of a diagnostic algorithm combined with DBS assay and confirmatory tests in the timely diagnosis of LOPD and implementation of best practice patterns.

Nigeria healthcare worker SARS-CoV-2 serology study: Results from a prospective, longitudinal cohort.

Healthcare workers, both globally and in Nigeria, have an increased risk for SARS-CoV-2 infection compared with the general population due to higher risk contacts, including occupational exposures. In addition, primary healthcare workers represent an important group for estimating prior infection to SARS-CoV-2 because they work at the first point-of-contact for most patients yet have not been included in prior COVID-19 seroepidemiology research in Nigeria. We sought to evaluate baseline seroprevalence, rates of seroconversion (IgG- to IgG+) and seroreversion (IgG+ to IgG-), change in IgG concentration at 3- and 6-month follow-up, and factors associated with seropositivity. From June 2020 to December 2020, we conducted a longitudinal seroepidemiology study among frontline health care workers in Nigeria using a validated dried blood spot assay. Among 525 participants, mean (SD) age was 39.1 (9.7) years, 61.0% were female, and 45.1% were community health workers. The six-month follow-up rate was 93.5%. Seropositivity rates increased from 31% (95% CI: 27%, 35%) at baseline to 45% (95% CI: 40%, 49%) at 3-month follow-up, and 70% (95% CI: 66%, 74%) at 6-month follow-up. There was a corresponding increase in IgG levels from baseline (median = 0.18 ug/mL) to 3-month (median = 0.35 ug/mL) and 6-month follow-up (median = 0.59 ug/mL, Ptrend < .0001). A minority of participants reported symptoms from February 2020 until baseline (12.2%) or during 3-month (6.6%) or 6-month (7.5%) follow-up. only 1 participant was hospitalized. This study demonstrated high baseline, 3-month and 6-month follow-up prevalence of IgG antibodies to SARS-CoV-2 during the first two waves of the COVID-19 pandemic in Nigeria among a cohort of unvaccinated frontline healthcare workers, including primary healthcare workers despite low symptomatology. These results may have implications in state- and national-level disease pandemic modeling. Trial registration: NCT04158154.

Evaluation of Dried Blood and Cerebrospinal Fluid Filter Paper Spots for Storing and Transporting Clinical Material for the Molecular Diagnosis of Invasive Meningococcal Disease.

To improve the storage and transport of clinical specimens for the diagnosis of Neisseria meningitidis (Nm) infections in resource-limited settings, we have evaluated the performance of dried blood spot (DBS) and dried cerebrospinal fluid spot (DCS) assays. DBS and DCS were prepared on filter paper from liquid specimens previously tested for Nm in the United Kingdom. Nm was detected and genogrouped by real-time PCR performed on crude genomic DNA extracted from the DBS (n = 226) and DCS (n = 226) specimens. Targeted whole-genome sequencing was performed on a subset of specimens, DBS (n = 4) and DCS (n = 6). The overall agreement between the analysis of liquid and dried specimens was (94.2%; 95% CI 90.8–96.7) for blood and (96.4%; 95% CI 93.5–98.0) for cerebrospinal fluid. Relative to liquid specimens as the reference, the DBS and DCS assays had sensitivities of (89.1%; 95% CI 82.7–93.8) and (94.2%; 95% CI 88.9–97.5), respectively, and both assays had specificities above 98%. A genogroup was identified by dried specimen analysis for 81.9% of the confirmed meningococcal infections. Near full-length Nm genome sequences (>86%) were obtained for all ten specimens tested which allowed determination of the sequence type, clonal complex, presence of antimicrobial resistance and other meningococcal genotyping. Dried blood and CSF filter spot assays offer a practical alternative to liquid specimens for the molecular and genomic characterisation of invasive meningococcal diseases in low-resource settings.

Niemann-Pick type C disease as proof-of-concept for intelligent biomarker panel selection in neurometabolic disorders.

Using Niemann-Pick type C disease (NPC) as a paradigm, we aimed to improve biomarker discovery in patients with neurometabolic disorders. Using a multiplexed liquid chromatography tandem mass spectrometry dried bloodspot assay, we developed a selective intelligent biomarker panel to monitor known biomarkers N-palmitoyl-O-phosphocholineserine and 3β,5α,6β-trihydroxy-cholanoyl-glycine as well as compounds predicted to be affected in NPC pathology. We applied this panel to a clinically relevant paediatric patient cohort (n=75; 35 males, 40 females; mean age 7 years 6 months, range 4 days-19 years 8 months) presenting with neurodevelopmental and/or neurodegenerative pathology, similar to that observed in NPC. The panel had a far superior performance compared with individual biomarkers. Namely, NPC-related established biomarkers used individually had 91% to 97% specificity but the combined panel had 100% specificity. Moreover, multivariate analysis revealed long-chain isoforms of glucosylceramide were elevated and very specific for patients with NPC. Despite advancements in next-generation sequencing and precision medicine, neurological non-enzymatic disorders remain difficult to diagnose and lack robust biomarkers or routine functional testing for genetic variants of unknown significance. Biomarker panels may have better diagnostic accuracy than individual biomarkers in neurometabolic disorders, hence they can facilitate more prompt disease identification and implementation of emerging targeted, disease-specific therapies. Intelligent biomarker panel design can help expedite diagnosis in neurometabolic disorders. In Niemann-Pick type C disease, such a panel performed better than individual biomarkers. Biomarker panels are easy to implement and widely applicable to neurometabolic conditions.

Development and Validation of a Dried Blood Spot Assay Using UHPLC-MS/MS to Identify and Quantify 12 Antihypertensive Drugs and 4 Active Metabolites: Clinical Needs and Analytical Limitations.

As nonadherence to antihypertensive drugs (AHDs) can increase the risk of cardiovascular events, hospitalization, and higher costs, there is a need for a reliable, objective, and easy method to assess nonadherence in patients. The dried blood spot (DBS) sampling method used to measure drug concentrations meets these requirements. For detecting nonadherence, identification is more important than quantification. Owing to their use in clinical practice, it is important to measure multiple AHDs with a single method. Therefore, we developed and validated a single DBS method for 17 commonly used AHDs and 4 active metabolites using ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Analytical validation of the DBS assay was performed in accordance with the guidelines on bioanalytical method validation of the European Medicines Agency and US Food and Drug Administration as well as the International Association of Therapeutic Drug Monitoring and Clinical Toxicology guidelines. We validated 12 of the 17 AHDs according to the European Medicines Agency and Food and Drug Administration requirements for bioanalytical method validation. Eleven AHDs were validated for both identification and quantification of drug concentrations, whereas nifedipine was only validated for identification. However, 5 of the 17 AHDs were excluded due to suboptimal validation results. Lercanidipine was excluded due to nonlinearity, and all 4 AHDs measured in the negative mode of UHPLC-MS/MS were not in accordance with one or more of the acceptance criteria and were therefore excluded. The described method accurately measured AHDs in DBS and can be used to determine nonadherence in patients. However, method validation revealed a challenging balance between analytical limitations and clinical needs when analyzing multiple drugs using the same method.

Unawareness; The Reason of Delayed Diagnosis of Niemann Pick Disease Type C and the Birth of Another Involved Sibling

Background: Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral lysosomal storage disorder resulting from mutations in either the NPC1 or the NPC2 gene. It shows a broad spectrum of clinical phenotypes and a variable age at diagnosis. As most patients have normal routine examinations (MRI, cerebrospinal fluid, electrophysiology, and so on), its diagnosis is often a challenge, and the start of treatment delays for several years. Case Report: We reported a 9-year-old boy who presented with Stuttered speech, hepatosplenomegaly, and up and downward gaze palsy whose Niemann Pick disease was not diagnosed during infancy due to unawareness. Dried blood spot assay and genetic study confirmed the diagnosis of Niemann Pick disease type C. Conclusion: Due to the high rate of consanguineous marriages and NPC presentation with atypical phenotypes, more educational programs for pediatricians, hematologists, neurologists, endocrinologists, and clinicians help the appropriate and timely diagnosis and prevent another involved sibling.

An improved functional assay in blood spot to diagnose Barth syndrome using the monolysocardiolipin/cardiolipin ratio

Barth syndrome is an X‐linked disorder characterized by cardiomyopathy, skeletal myopathy, and neutropenia, caused by deleterious variants in TAFAZZIN. This gene encodes a phospholipid‐lysophospholipid transacylase that is required for the remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, individuals with Barth syndrome have a deficiency of mature CL and accumulation of the remodeling intermediate monolysocardiolipin (MLCL). Diagnosis typically relies on mass spectrometric measurement of CL and MLCL in cells or tissues, and we previously described a method in blood spot that uses a specific MLCL/CL ratio as diagnostic biomarker. Here, we describe the evolution of our blood spot assay that is based on the implementation of reversed phase‐UHPLC separation followed by full scan high resolution mass spectrometry. In addition to the MLCL/CL ratio, our improved method also generates a complete CL spectrum allowing the interrogation of the CL fatty acid composition, which considerably enhances the diagnostic reliability. This addition negates the need for a confirmatory test in lymphocytes thereby providing a shorter turn‐around‐time while achieving a more certain test result. As one of the few laboratories that offer this assay, we also evaluated the diagnostic yield and performance from 2006 to 2021 encompassing the use of both the original and improved assay. In this period, we performed 796 diagnostic analyses of which 117 (15%) were characteristic of Barth syndrome. In total, we diagnosed 93 unique individuals with Barth syndrome, including three females, which together amounts to about 40% of all reported individuals with Barth syndrome in the world.

Reproductive hormone measurement from minimally invasive sample types: Methodological considerations and anthropological importance.

Energetic investment in human reproduction has long been recognized as costly, influencing developmental, physiological, and behavioral patterns in males and females. These effects are largely coordinated through the actions of reproductive hormones (eg, testosterone, estradiol, and progesterone). Here, the utility and limitations of minimally invasive sampling techniques are explored, providing a novel perspective on how reproductive hormone measurements can enhance reproductive endocrinology research. Salivary steroid measures are most commonly used, although several dried blood spot and urine assays are also available, and researchers continue to explore the efficacy of other sample types. These relatively simple measures have facilitated the collection of multiple samples from a single participant, allowing researchers to more accurately track the diurnal and cyclical variation exhibited by many reproductive hormones. Ultimately, the ability to collect fine-grained participant data allows biological anthropologists to better test questions central to human reproductive ecology, life history theory, and public health. For example, fieldwork using these techniques suggests that testosterone profile variation across populations is influenced by energetic constraints and reproductive status. Moreover, hormone concentrations shape the development of sex characteristics, with implications for evolutionary questions related to sexual selection. Hormone levels also can be used to identify a range of medical concerns (eg, suppressed hormone production levels linked with psychosocial stress). These findings highlight how minimally invasive collection techniques can be applied to test diverse evolutionary hypotheses and identify important health concerns. Still, more work is needed to standardize collection and laboratory analysis procedures, thereby enabling more direct data comparisons between researchers.

Clinical utilization of dried blood spot nitisinone (NTBC) and succinylacetone (SA) concentrations in hereditary tyrosinaemia type 1 - A UK centre experience.

Dried blood spot monitoring of nitisinone and succinylacetone in hereditary tyrosinaemia type 1 patients is not widely available in the United Kingdom. Currently, biochemical monitoring utilizes urinary succinylacetone, blood spot tyrosine and phenylalanine monitoring, which can lack in convenience and accuracy, respectively. We report the development of a dried blood spot assay for nitisinone and succinylacetone and analysed retrospective clinical and biochemical data for hereditary tyrosinaemia type 1 patients from a single UK centre. A total of 13 hereditary tyrosinaemia type 1 patients were evaluated. Eleven presented with liver dysfunction (two with associated renal tubulopathy) and two were detected by early sibling screening. All patients (age 0.03-22 months) were commenced on a tyrosine-/phenylalanine-restricted diet and nitisinone at diagnosis. Ten patients were on twice daily dosing and three were on single daily dosing at the start of monitoring. One patient from each dosing group swapped between dosing regimens at 20 years of age and 8 months of age, respectively. A total of 684 dried blood spot samples were analysed; 80% of nitisinone concentrations were between 9.2 and 27 µmol/L when succinylacetone was <0.3 µmol/L. Patients on twice daily dosing regimens had significantly higher nitisinone concentration compared with those on once daily dosing (P < 0.0001). The median dose required in the twice daily doing group was significantly lower when compared with once daily dosing. Dried blood spot monitoring for nitisinone and succinylacetone concentrations in hereditary tyrosinaemia type 1 patients is a rapid and convenient method which allows physicians to individualize treatment plans and observe adherence to treatment.

A dried blood spot assay with HPLC-MS/MS for the determination of larotrectinib in mouse blood and its application to a pharmacokinetic study.

Larotrectinib is a first-generation tropomyosin kinase inhibitor, approved for the treatment of solid tumors. In this paper, we present a validated dried blood spot (DBS) method for the quantitation of larotrectinib from mouse blood using HPLC-MS/MS, which was operated under multiple reaction monitoring mode. To the DBS disc cards, acidified methanol enriched with internal standard (IS; enasidenib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of larotrectinib and the IS was achieved on an Atlantis dC18 column using 10 mm ammonium formate-acetonitrile (30:70, v/v) delivered at a flow-rate of 0.80 ml/min. Under these optimized conditions, the retention times of larotrectinib and the IS were ~0.93 and 1.37 min, respectively. The total run time was 2.50 min. Larotrectinib and the IS were analyzed using positive ion scan mode and parent-daughter mass to charge ion (m/z) transitions of 429.1 → 342.1 and 474.1 → 267.1, respectively, were used for the quantitation. The calibration range was 1.06-5,080 ng/ml. No matrix effect or carryover was observed. Hematocrit did not influence DBS larotrectinib concentrations. All of the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mouse pharmacokinetic study.

High seroprevalence for SARS-CoV-2 among household members of essential workers detected using a dried blood spot assay.

ObjectiveSerological testing is needed to investigate the extent of transmission of SARS-CoV-2 from front-line essential workers to their household members. However, the requirement for serum/plasma limits serological testing to clinical settings where it is feasible to collect and process venous blood. To address this problem we developed a serological test for SARS-CoV-2 IgG antibodies that requires only a single drop of finger stick capillary whole blood, collected in the home and dried on filter paper (dried blood spot, DBS). We describe assay performance and demonstrate its utility for remote sampling with results from a community-based study.MethodsAn ELISA to the receptor binding domain of the SARS-CoV-2 spike protein was optimized to quantify IgG antibodies in DBS. Samples were self-collected from a community sample of 232 participants enriched with health care workers, including 30 known COVID-19 cases and their household members.ResultsAmong 30 individuals sharing a household with a virus-confirmed case of COVID-19, 80% were seropositive. Of 202 community individuals without prior confirmed acute COVID-19 diagnoses, 36% were seropositive. Of documented convalescent COVID-19 cases from the community, 29 of 30 (97%) were seropositive for IgG antibodies to the receptor binding domain.ConclusionDBS ELISA provides a minimally-invasive alternative to venous blood collection. Early analysis suggests a high rate of transmission among household members. High rates of seroconversion were also noted following recovery from infection. Serological testing for SARS-CoV-2 IgG antibodies in DBS samples can facilitate seroprevalence assessment in community settings to address epidemiological questions, monitor duration of antibody responses, and assess if antibodies against the spike protein correlate with protection from reinfection.

Pre-Analytical and Clinical Validation of a Dried Blood Spot Assay for Asymmetric Dimethylarginine and L-Arginine.

Asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) synthesis. It is a risk marker for cardiovascular events and mortality in patients with cardiometabolic diseases and in population-based studies. Plasma or serum analysis of ADMA may be hampered by pre-analytical sample handling. We validated a dried blood spot (DBS) assay for ADMA and L-arginine and show here that this assay has excellent variabilities and reproducibilities. Filter paper is impregnated with the arginase inhibitor nor-NOHA (Nω-hydroxy-nor-Arginine) to avoid L-arginine degradation. Clinical validation of this DBS assay confirms elevated ADMA concentration in hemodialysis patients as compared to healthy controls, higher ADMA concentrations in men versus women, and elevated L-arginine concentration in subjects supplemented with L-arginine. The DBS assay was used in a cohort study involving 100 primarily healthy subjects in the Andean region to assess the impact of chronic intermittent hypoxia on ADMA and L-arginine; ADMA DBS concentration at sea level was prospectively associated with pulmonary hypertension after six months of exposure to 3500 m. In a cohort of 753 individuals, L-arginine/ADMA ratio significantly decreased with increasing number of traditional cardiovascular risk factors. Analysis of ADMA and L-arginine in DBS is a reliable and reproducible method for quantitation of these markers in field studies.

Validation and clinical application of dried blood spot assay for quantitative assessment of edoxaban in healthy adults.

Aim: Dried blood spot (DBS) is a sampling approach that offers several advantages over plasma and whole blood (WB) sampling, but several factors, such as hematocrit and temperature, can adversely affect quantitation. Methodology & results: In an open-label, three-way crossover study in healthy subjects, we explored the correlation between DBS, WB and plasma samples, and between DBS samples from finger-prick and venipuncture blood for measuring edoxaban and its metabolite M-4 using LC-MS/MS. The methods were validated comprehensively. The incurred sample reanalysis experiments demonstrated quantitation reproducibility in all three matrices. Overall, there was a good correlation (near perfect concordance for edoxaban) among plasma, WB and DBS measurements. M-4 concentrations in DBS and WB were lower than in plasma. Conclusion: These results indicate using DBS may be used as an alternative methodology to measure edoxaban pharmacokinetics.

Clinical application of a dried blood spot assay for sirolimus and everolimus in transplant patients.

Background Monitoring of immunosuppressive drugs such as everolimus and sirolimus is important in allograft rejection prevention in transplant patients. Dried blood spots (DBS) sampling gives patients the opportunity to sample a drop of blood from a fingerprick at home, which can be sent to the laboratory by mail. Methods A total of 39 sirolimus and 44 everolimus paired fingerprick DBS and whole blood (WB) samples were obtained from 60 adult transplant patients for method comparison using Passing-Bablok regression. Bias was assessed using Bland-Altman. Two validation limits were pre-defined: limits of analytical acceptance were set at >67% of all paired samples within 20% of the mean of both samples and limits of clinical relevance were set in a multidisciplinary team at >80% of all paired samples within 15% of the mean of both samples. Results For both sirolimus and everolimus, Passing-Bablok regression showed no differences between WB and DBS with slopes of 0.86 (95% CI slope, 0.72-1.02) and 0.96 (95% CI 0.84-1.06), respectively. Only everolimus showed a significant constant bias of 4%. For both sirolimus and everolimus, limits of analytical acceptance were met (76.9% and 81.8%, respectively), but limits or clinical relevance were not met (77.3% and 61.5%, respectively). Conclusions Because pre-defined limits of clinical relevance were not met, this DBS sampling method for sirolimus and everolimus cannot replace WB sampling in our center at this time. However, if the clinical setting is compatible with less strict limits for clinical relevance, this DBS method is suitable for clinical application.

A dried blood spot-based method to measure levels of tartrate-resistant acid phosphatase 5b (TRACP-5b), a marker of bone resorption.

A number of basic questions about bone biology have not been answered, including population differences in bone turnover. In part, this stems from the lack of validated minimally invasive biomarker techniques to measure bone formation and resorption in field-based population-level research. The present study addresses this gap by validating a fingerprick dried blood spot (fDBS) assay for tartrate-resistant acid phosphatase 5b (TRACP-5b), a well-defined biomarker of bone resorption and osteoclast number. We adapted a commercially available enzyme-linked immunosorbent assay (ELISA) kit from MyBiosource for the quantitative determination of TRACP-5b levels in serum and plasma for use with DBS. We used a rigorous process of assay modification and validation, including the use of a matched set of 189 adult plasma, fDBS, and venous DBS (vDBS) samples; parameters evaluated included precision, reliability, and analyte stability. Plasma and DBS TRACP-5b concentrations showed a linear relationship. There were no systematic differences in TRACP-5b levels in fDBS and vDBS, indicating no significant differences in TRACP-5b distribution between capillary and venous blood. Parallelism and spike-and-recovery results indicated that matrix factors in DBS do not interfere with measurement of TRACP-5b levels from DBS using the validated kit. Intra- and interassay CVs were 5.0% and 12.1%, respectively. DBS samples should preferably be stored frozen but controlled room temperature storage for up to a month may be acceptable. This DBS-based ELISA assay adds to the methodological toolkit available to human biologists and will facilitate research on bone turnover in population studies.

Analytical Validation Strategies for DBS Biomarker Assays

In recent years biomarker assays have gained precedence both in the clinical and pharmaceutical laboratory. In the pharmaceutical arena biomarkers play multifaceted roles in the safety and efficacy studies of drug development. Alternatively, in the clinic, biomarker assays are useful in screening, diagnosis, detection, prognosis and prediction of diseases and disorders. The bio analytical validation of biomarker assays has been a topic of wide interest. The major bottle neck in this area is the paucity of a fixed regulatory guidance. The various phases in the development of new biomarker assays are pre-analytical considerations, analytical considerations and biomarker assay implementation. The focus of this review has been to address in detail the pre-analytical and analytical considerations in the development of new biomarker assays in a well-known matrix like dried blood spots both in the pharmaceutical and clinical laboratory. The validation parameters discussed include specificity, sensitivity, matrix effect, precision, accuracy, linearity, dilution linearity, parallelism, carry over, proficiency testing and reference intervals. Additional parameters for dried blood spot assays include hematocrit, blood spot volume, chromatographic effect, spot to spot carry over and DBS vs whole blood/plasma concentration.

Longitudinal Validity and Reliability of Brief Smartphone Self-Monitoring of Diet, Stress, and Physical Activity in a Diverse Sample of Mothers.

BackgroundMultiple strategies can be used when self-monitoring diet, physical activity, and perceived stress, but no gold standards are available. Although self-monitoring is a core element of self-management and behavior change, the success of mHealth behavioral tools depends on their validity and reliability, which lack evidence. African American and Latina mothers in the United States are high-priority populations for apps that can be used for self-monitoring of diet, physical activity, and stress because the body mass index (BMI) of mothers typically increases for several years after childbirth and the risks of obesity and its’ sequelae diseases are elevated among minority populations.ObjectiveTo examine the intermethod reliability and concurrent validity of smartphone-based self-monitoring via ecological momentary assessments (EMAs) and use of daily diaries for diet, stress, and physical activity compared with brief recall measures, anthropometric biomeasures, and bloodspot biomarkers.MethodsA purposive sample (n=42) of primarily African American (16/42, 39%) and Latina (18/42, 44%) mothers was assigned Android smartphones for using Ohmage apps to self-monitor diet, perceived stress, and physical activity over 6 months. Participants were assessed at 3- and 6-month follow-ups. Recall measures included brief food frequency screeners, physical activity assessments adapted from the National Health and Nutrition Examination Survey, and the nine-item psychological stress measure. Anthropometric biomeasures included BMI, body fat, waist circumference, and blood pressure. Bloodspot assays for Epstein–Barr virus and C-reactive protein were used as systemic load and stress biomarkers. EMAs and daily diary questions assessed perceived quality and quantity of meals, perceived stress levels, and moderate, vigorous, and light physical activity. Units of analysis were follow-up assessments (n=29 to n=45 depending on the domain) of the participants (n=29 with sufficient data for analyses). Correlations, R2 statistics, and multivariate linear regressions were used to assess the strength of associations between variables.ResultsAlmost all participants (39/42, 93%) completed the study. Intermethod reliability between smartphone-based EMAs and diary reports and their corresponding recall reports was highest for stress and diet; correlations ranged from .27 to .52 (P<.05). However, it was unexpectedly low for physical activity; no significant associations were observed. Concurrent validity was demonstrated for diet EMAs and diary reports on systolic blood pressure (r=−.32), C-reactive protein level (r=−.34), and moderate and vigorous physical activity recalls (r=.35 to.48), suggesting a covariation between healthy diet and physical activity behaviors. EMAs and diary reports on stress were not associated with Epstein–Barr virus and C-reactive protein level. Diary reports on moderate and vigorous physical activity were negatively associated with BMI and body fat (r=−.35 to −.44, P<.05).ConclusionsBrief smartphone-based EMA use may be valid and reliable for long-term self-monitoring of diet, stress, and physical activity. Lack of intermethod reliability for physical activity measures is consistent with prior research, warranting more research on the efficacy of smartphone-based self-monitoring of self-management and behavior change support.

A dried blood spot assay with UPLC–MS/MS for the simultaneous determination of E6005, a phosphodiesterase 4 inhibitor, and its metabolite in human blood

E6005, a novel phosphodiesterase 4 inhibitor, is currently under clinical development for the treatment of atopic dermatitis. To support pediatric clinical trials, the dried blood spot assay for simultaneous determination of E6005 and its main metabolite, ER-392710 (M11), has been developed using ultra-performance liquid chromatography with tandem mass spectrometry. E6005 and M11, in 25 μL blood spotted onto FTA™ DMPK-C cards, were extracted by water/acetonitrile (1:1, v/v), and then chromatographed on a reversed phase column under gradient elution. The mass transitions, m/z 473.1 → 163.0 for E6005 and m/z 459.1 → 149.0 for M11, with corresponding stable isotope internal standard, m/z 477.2 → 167.0, and m/z 463.2 → 153.0, were monitored. E6005 and M11 were quantifiable from 1 to 200 ng/mL as free base. Accuracy and precision of the two analytes in the intra- and inter-batch reproducibility were within ±8.0% and 15.7%, respectively. Extraction recoveries of the analytes were 73% or more and hematocrit ranging from 26.9% to 51.8% did not impact the analytes’ accuracy. Various stability assessments, including possible conversion of E6005 to M11, were thoroughly performed, and bench-top stability was ensured up to 160 days. The DBS method was applied to determine E6005 and M11 concentrations in blood samples supporting a pediatric clinical trial.

Understanding protective plasma levels of penicillin and monitoring antibody responses for the prevention of GAS infection in a paediatric population in New Zealand

Abstract Acute Rheumatic Fever (ARF) is an autoimmune condition resulting from untreated Group A Streptococcal (GAS) infection of the upper respiratory tract and possibly skin. Repeated untreated and severe episodes of ARF can cause permanent cardiac damage known as Rheumatic Heart Disease (RHD). Rates of ARF in New Zealand and Australia are among the highest in the world, particularly amongst Māori and Pacific and Indigenous Aboriginal children. Monthly injections of Benzathine Penicillin G (BPG) are given intramuscularly to prevent GAS infections that can lead to ARF and cause RHD. Early studies exploring the Pharmacokinetics (PK) properties of BPG were conducted in healthy, fit, young military recruits without ARF/RHD. BPG bioavailability, metabolism may differ in paediatric populations groups with ARF who have comorbidities and vary in Body Mass Index. This project seeks to determine the PK characteristics in a paediatric population currently receiving monthly injections of BPG while at the same time monitoring antibody immune responses to possible GAS infections. This is a prospective cohort study in a paediatric population of Wellington based children and teens (aged 5–21 years of predominantly Pasifika and Māori heritage) with a previous diagnosis of ARF and are receiving monthly BPG injections via community services. Participants will contribute finger prick blood samples over time for Dried Blood Spot assays designed to quantify penicillin G levels in the blood and measure streptococcal serology. Routine throat swab samples will also be collected with ASOT antibody levels monitored for GAS breakthrough infections. This is the first population PK study exploring use of BPG in Māori and Pacific children previously diagnosed with ARF.

Incorporating dried blood spot LC-MS/MS analysis for clinical development of a novel oncolytic agent.

Design and execution of a dried blood spot (DBS-LC-MS/MS) assay for pharmacokinetic analyses in oncology patients. The methodology was validated to collect and store DBS samples from multiple clinical sites, and analyze blood with diverse hematocrit ranges (25-55) to match the potential patient population. Bridging data comparing DBS and plasma showed high degree of concordance with DBS:plasma ratios of 0.81, demonstrating no preferential uptake or association with cellular components of the blood. Pharmacokinetic analysis supporting clinical development was performed using 20μl of blood collected as DBS. Incurred sample reanalysis showed high correlation. Successful validation of a DBS method and implementation in the clinic enabled pharmacokinetic analysis during the clinical development of a novel oncolytic agent in oncology patients.