Dried Blood Spot Samples Research Articles

Real-Life Diagnostic Accuracy and Clinical Utility of Hepatitis B Virus (HBV) Nucleic Acid Testing Using the GeneXpert Point-of-Care Test System from Fresh Plasma and Dry Blood Spot Samples in The Gambia

The GeneXpert HBV Viral Load test is a simplified tool to scale up screening and HBV monitoring in resource-limited settings, where HBV is endemic and where molecular techniques to quantify HBV DNA are expensive and scarce. However, the accuracy of field diagnostics compared to gold standard assays in HBV-endemic African countries has not been well understood. We aim to validate the diagnostic performance of the GeneXpert HBV Viral Load test in freshly collected and stored plasma and dried blood spot (DBS) samples to assess turn-around-time (TAT) for sample processing and treatment initiation, to map GeneXpert machines and to determine limitations to its use in The Gambia. Freshly collected paired plasma and DBS samples (n = 56) were analyzed by the GeneXpert test. Similarly, stored plasma and DBS samples (n = 306, n = 91) were analyzed using the GeneXpert HBV test, in-house qPCR and COBAS TaqMan Roche. The correlation between freshly collected plasma and DBS is r = 0.88 with a mean bias of −1.4. The GeneXpert HBV test had the highest quantifiable HBV DNA viremia of 81.4% (n = 249/306), and the lowest was detected by in-house qPCR at 37.9% (n = 116/306) for stored plasma samples. Bland–Altman plots show strong correlation between GeneXpert and COBAS TaqMan and between GeneXpert and in-house qPCR with a mean bias of +0.316 and −1.173 log10 IU/mL, respectively. However, paired stored plasma and DBS samples had a lower mean bias of 1.831 log10 IU/mL, which is almost significant (95% limits of agreement: 0.66–3.001). Patients (n = 3) were enrolled in the study within a TAT of 6 days. The GeneXpert HBV test displayed excellent diagnostic accuracy by detecting HBV viremia in less than 10 IU/mL.

Unlocking the potential of dried blood spot sampling for SARS-CoV-2 antibody detection: A pathway to efficient pandemic surveillance

SummaryThe current SARS-CoV-2 pandemic has exerted unprecedented pressure on global healthcare systems, with an urgent demand for widespread testing to monitor virus spread and contribute to disease control. AimThis study aimed to validate the use of Dried Blood Spot (DBS) sampling for analyzing the immune response to SARS-CoV-2 infection, employing an accessible enzyme-linked immunosorbent assay (ELISA) for detecting IgG antibodies. MethodologyTraditional venous blood sampling presents several challenges, including discomfort to patients, risk of healthcare provider exposure, and the need for specialized equipment, which can limit mass testing capabilities. Our research involved the comparison of DBS and venous blood samples from 170 individuals, all of whom had PCR-confirmed SARS-CoV-2 infection, to assess the efficacy of DBS for seroprevalence studies and vaccine response evaluation. ResultsOur results confirmed that DBS sampling could detect SARS-CoV-2 IgG antibodies with 100 % accuracy, showcasing perfect agreement (Cohen's k value of 1.00) between DBS and venous serum samples in detecting the viral antibodies. However, the analysis indicated no significant correlation between the antibody levels in the two sampling methods. ConclusionThese results suggest that DBS is a viable, rapid alternative for serological studies, offering a solution to current testing limitations and enhancing our public health approach to combating future pandemics.

Neurodevelopment in the First 2 Years of Life Following Prenatal Exposure to Maternal SARS-CoV-2 Infection

The effects of prenatal exposure to SARS-CoV-2 infection on child development throughout the first 2 years of life are unknown. To evaluate whether prenatal exposure to SARS-CoV-2 infection was associated with child neurodevelopmental outcomes during the first 2 years of life. This cohort study used data from the longitudinal, population-based pan-Canadian Pregnancy During the COVID-19 Pandemic cohort, which recruited participants from April 2020 to July 2022. Children were categorized as exposed to prenatal SARS-CoV-2 infection if their birthing parent had a positive polymerase chain reaction test performed by a health authority or as a healthy negative comparison if their birthing parent did not have SARS-CoV-2 antibodies in their postpartum dried blood spot sample. Prenatal SARS-CoV-2 infection. The birthing parent reported on their child's temperament at ages 6 and 24 months, developmental milestones at ages 12 and 24 months, and social-emotional milestones at ages 12 and 24 months. A total of 896 children were included, with 96 children who had been exposed to a prenatal SARS-CoV-2 infection (mean [SD] gestational age at birth, 39.20 [1.50] weeks; 45 [47%] male) and 800 were healthy negative comparisons (mean [SD] gestational age at birth, 39.47 [1.54] weeks; 388 [49%] male). In analyses of covariance adjusted for prepregnancy medical conditions and household socioeconomic status, prenatal exposure to SARS CoV-2 infection was associated with slightly higher regulatory control scores, indicating more regulation, at age 6 months (difference in means, 0.19 [95% CI, 0.02-0.36]; P = .03; ηp2 = 0.01). No significant differences were observed for the other neurodevelopmental outcomes. In mixed models adjusted for the same covariates that aimed to examine change in outcomes over time, prenatal SARS-CoV-2 infection exposure was not associated with developmental change in any neurodevelopmental outcomes between ages 6 and 24 months. In this longitudinal cohort study of multiple aspects of child neurodevelopment between ages 6 and 24 months, negligible associations between prenatal exposure to SARS-CoV-2 infection and child outcomes were observed. Follow-up research is warranted to determine whether these predominantly null effects persist into later childhood.

Utilization and validation of dried blood spot-based metabolomics in plasma-derived diagnostic models

Metabolomics has rapidly advanced in life sciences, enhancing our understanding of physiological conditions. Plasma stands out as a predominant sample type in metabolomics studies. Dried blood spot (DBS) has been recognized as a valuable strategy due to its unique characteristics, such as minimal invasiveness, small sample volume, and easy transport. Most large cohort studies construct the diagnostic model using plasma-based metabolomics rather than DBS. However, the comparability of metabolite measurements between DBS and plasma samples, as well as the integration of DBS-based metabolomics into established plasma-derived diagnostic models, remains unclear. We collected plasma and DBS samples from the same 12 healthy controls under fasting and non-fasting conditions and performed ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS)-based metabolomics. We identified 277 metabolites present in both plasma and DBS samples, with 229 (82.7 %) showing a strong correlation between the two sample types. Furthermore, pre-processing datasets from plasma and DBS samples before combining them effectively diminished inconsistencies between plasma- and DBS-based metabolomics without compromising the inherent classification within the data. For the clinical application, the plasma samples were obtained from the gastric cancer patients (n = 204) and the healthy controls (n = 128) to serve as the training cohorts. Additionally, DBS samples were collected from different participants of gastric cancer (n = 19) and healthy controls (n = 12) to validate the diagnostic model. Finally, DBS-based metabolomics were integrated into established plasma-derived gastric cancer diagnostic models, yielding an area under the ROC curve (AUC) of 0.934. Overall, incorporating DBS-based metabolomics into an established plasma-derived diagnostic model holds promise, offering valuable opportunities and insights for diagnostic research and clinical practice.

GC-MS uncovers unique metabolic markers of drug-resistant epilepsy in capillary but not venous dried blood spots

This study compared the effectiveness of capillary dried blood spots (DBS) versus venous DBS in detecting metabolic changes related to drug-resistant epilepsy (DRE). DBS samples were collected from 142 epilepsy patients (58 drug-resistant, 84 drug-responsive) via venipuncture or fingerstick capillary sampling. Metabolomic analysis using gas chromatography-mass spectrometry compared DBS metabolite profiles between the two groups. While venous DBS profiles showed no distinct patterns, capillary DBS profiles revealed clustering patterns in principal components analysis, with the first two principal components explaining 14.5%, and 13.5% of the total variance, respectively. Orthogonal PLS-DA confirmed group discrimination (R2Y=0.989, Q2=0.742). Drug-resistant patients exhibited elevated capillary DBS levels of glutamine, pyruvic acid, and serine, and decreased palmitic acid compared to drug-responsive patients. Pathway analysis revealed disruptions in amino acid metabolism, neurotransmission, and cellular energy regulation. Elevated glutamine levels may contribute to an imbalance between excitatory glutamate and inhibitory GABA neurotransmission, key factors in epileptogenesis and drug resistance. Capillary DBS, likely enriched with arterial blood supply to the brain, appears to better capture central nervous system metabolic disturbances compared to venous DBS containing systemic contributions. This minimally invasive capillary DBS approach offers effective metabolic profiling of brain conditions like DRE, for monitoring disease progression and treatment response, enhancing personalized patient management in epilepsy.

Feasibility of dried blood spot collection for caffeine pharmacokinetic studies in microgravity: Insights from parabolic flight campaigns.

Therapeutic drug monitoring for astronauts faces limitations in conventional blood sampling and sample management onboard the international space station. Here, we explore the feasibility of dried blood spot (DBS) collection during parabolic flights (PF) to overcome these constraints. We assessed the feasibility of blood deposition on blotting paper for preanalytical aspects in a PF using synthetic blood. Subsequently, DBS sampling validation was carried out in another PF campaign. Twenty volunteers participated in a pharmacokinetic study on caffeine and its metabolite, paraxanthine, conducted during parabolic flights. After >18 h caffeine washout, coffee (115 mg), tea (30 mg) or dark chocolate (11 mg) were ingested by the participants. DBS samples were collected at baseline, during weightlessness and post-flight. Caffeine and paraxanthine were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Genotyping for cytochrome P450-1A2 (CYP1A2) was performed and a metabolic ratio by area under the curve for caffeine and paraxanthine (AUCPAX/AUCCAF) for CYP1A2 was determined. A user experience survey was also conducted. Full in-flight pharmacokinetic study was feasible in seventeen volunteers with three unable to perform the sampling due to motion sickness. Nineteen participants (twelve males and seven females) completed pharmacokinetic profiles, with repeated pharmacokinetic studies for six participants. CYP1A2 genotyping resulted in eight ultrarapid, eleven intermediate, and one poor metabolizer. Among the women, four were on oestrogen contraceptives, a known inhibitor of CYP1A2, and were considered as poor metabolizers. Expected differences in kinetic profiles, consistent with consumption habits, the ingested dose and the genotypic/phenotypic information, were observed. The mean caffeine AUC for coffee, tea and chocolate were 9419ng.h.mL-1 (95% confidence interval [CI]: 6222-12 616, n = 10), 6917ng.h.mL-1 (95% CI: 2729-11 105), n = 7) and 3039ng.h.mL-1 (95% CI: 1614-4142, n = 12), respectively. The mean paraxanthine AUC were 10 566ng.h.mL-1 (95% CI: 6242-14 890, n = 10), 4011ng.h.mL-1 (95% CI: 2305-5716, n = 7) and 3638ng.h.mL-1 (95% CI: 1589-40 859, n = 12), respectively. The mean metabolic ratio in oestrogen-treated women was 0.53 (95% CI: 0.35-0.71) compared to 1.19 (95% CI: 0.99-1.33) in others. The mean metabolic ratio was 1.02 (95% CI: 0.81-1.23, n = 15) on the ground and 1.10 (95% CI: 0.70-1.41, n = 13) during the parabolic flights, with no significant difference observed between the two conditions. Overall participants were satisfied with the usability of the method. DBS collection was safe, stable, feasible and well accepted in weightlessness. This method would offer valuable insights into human metabolism adaptation during long-term spaceflight, addressing space pharmacology challenges.

On the Use of Image Analysis for Hematocrit Evaluation in Dried Blood Spots

Dried blood spots (DBSs) are formed by collecting a small sample of blood on specialized filter paper and allowing it to dry naturally. Various domains of life sciences and drug research extensively use DBSs as a sampling technique. The “Hematocrit (Ht) effect” affects assay bias, and several strategies have been put forth to deal with it, including the correction of quantified concentrations using an appropriate correction factor. The approach was previously applied, following the utilization of an image processing algorithm developed in Matlab® to derive a reliable equation correlating DBS areas to Ht% values. The present work looks more closely at the application of image analysis to the evaluation of Ht in DBS samples. Utilizing image analysis software, DBS samples with known Ht values were processed. Preparation of cards has followed a previously developed protocol for the appropriate formation of uniform area DBSs, irrespective of Ht. The resulting areas showed close resemblance to the respective theoretical areas calculated by applying the correlation equation. Following that, the equation was utilized to determine the Ht values for each sample, and a comprehensive comparison of measured versus calculated Ht was carried out using various statistical approaches for method comparison. The results demonstrated a strong correlation, suggesting the method’s viability in estimating Ht for unknown DBS samples.

A comparative study of extraction free detection of HBV DNA using sodium dodecyl sulfate, N-lauroylsarcosine sodium salt, and sodium dodecyl benzene sulfonate

This study aimed to develop an extraction-free method for quantitative and qualitative detection of HBV DNA compared to traditional nucleic acid extraction. Paired serum and dried blood spot (DBS) samples from 67 HBsAg-positive and 67 HBsAg-negative individuals were included. Two samples with known HBV DNA titers (~ 109 copies/mL) were examined by extraction-free detection using three surfactants (0.2 to 1% of Sodium dodecyl sulfate:SDS, N-Lauroylsarcosine sodium salt:NL, Sodium dodecyl benzene sulfonate:SDBS), two stabilizing agents (0.1 or 0.01% 2-Mercaptoethanol:2ME and 3.5 or 7% Bovine Serum Albumin:BSA) and two Taq polymerases (Fast Advanced and Prime Direct Probe). HBV DNA in all 67 HBsAg-positive and 67 HBsAg-negative serum and DBS samples was directly quantified by Rt-PCR using 0.4% SDS or 0.4% NL with Fast Advance or Prime Direct Probe Taq. Extraction-free amplification was also performed. Detection limits were varied by different surfactants and Taq. SDS combined with Fast Advanced Taq showed lower detection limits, while SDS with Prime Direct Probe Taq outperformed NL or SDBS-based detection. Adding 2ME to SDS improved detection limit with Prime Direct Probe Taq but not significantly compared to SDS alone. BSA did not significantly enhance detection limits but provided insights into sample stability. The senitivity and specificity of 0.4% SDS and NL in combination with either Fast advanced or Prime Direct Probe Taq polymerase in serum samples were > 90% and 100% resepctively, while it was > 80% and 100% respectively in DBS samples. Extraction-free HBV DNA amplification provided 100% identity with original genomes. Our study suggests that SDS, NL or SDBS-based extraction-free HBV DNA detection strategies using Prime Direct Probe Taq have potential to simplify and accelerate HBV DNA detection with high sensitivity and specificity in both serum and DBS samples, with implications for resource-limited settings and clinical applications. Utilizing surfactants with 2ME is optional, and further research and validation are necessary to broaden its application in real-world diagnostics.

A Non-targeted Proteomics Newborn Screening Platform for Inborn Errors of Immunity

PurposeNewborn screening using dried blood spot (DBS) samples for the targeted measurement of metabolites and nucleic acids has made a substantial contribution to public healthcare by facilitating the detection of neonates with genetic disorders. Here, we investigated the applicability of non-targeted quantitative proteomics analysis to newborn screening for inborn errors of immunity (IEIs).MethodsDBS samples from 40 healthy newborns and eight healthy adults were subjected to non-targeted proteomics analysis using liquid chromatography-mass spectrometry after removal of the hydrophilic fraction. Subsequently, DBS samples from 43 IEI patients were analyzed to determine whether patients can be identified by reduced expression of disease-associated proteins.ResultsDBS protein profiling allowed monitoring of levels of proteins encoded by 2912 genes, including 1110 listed in the Online Mendelian Inheritance in Man database, in healthy newborn samples, and was useful in identifying patients with IEIs by detecting reduced levels of disease causative proteins and their interacting proteins, as well as cell-phenotypical alterations.ConclusionOur results indicate that non-targeted quantitative protein profiling of DBS samples can be used to identify patients with IEIs and develop a novel newborn screening platform for genetic disorders.

Hoping to Adhere? Examining the Relationship Between Hope and Pre-exposure Prophylaxis Willingness, Adherence, and Persistence Among Young Women in South Africa and Zimbabwe (HPTN 082).

Hope is a powerful psychological construct which is linked to positive health. Greater hope is associated with improved antiretroviral therapy adherence; however, less is known about the impact of hope on oral pre-exposure prophylaxis (PrEP) outcomes. HIV Prevention Trials Network 082, was an open-label PrEP study among young women (ages 16-25) in South Africa and Zimbabwe. Hope was measured at baseline and follow-up using a subset of the Hope for the Future Scale (score range 6-24) and PrEP willingness was measured using a subscale of the HIV Prevention Readiness Measure (score range 6-30). Intracellular tenofovir-diphosphate (TFV-DP) concentrations were obtained from dried blood spot samples at weeks 13, 26, and 52; high PrEP adherence was defined as TFV-DP concentrations ≥ 700 fmol/punch. Persistence was defined as TFV-DP > 16 fmol/punch at weeks 26 and 52. Linear regression and generalized estimating equations were used to assess the relationship between hope and PrEP willingness, adherence, and persistence. The median age of participants (n = 432) was 21years (interquartile range [IQR]: 19-22). The mean hope score at baseline was 21.0 (SD = 3.4). Although hope was positively associated with PrEP willingness (β = 0.22, 95% CI 0.15, 0.37), it was not associated with high PrEP adherence (aRR = 1.00, 95% CI 0.96, 1.05), or persistence at follow-up (aRR = 1.02, 95% CI 0.99, 1.05). While cultivating hope may be an important strategy in building willingness to take oral PrEP, it may not be enough to sustain PrEP adherence or persistence.

Serological and molecular analysis of Leishmania infection in a recent outbreak of visceral leishmaniasis in South Omo Zone, Ethiopia.

Ethiopia has a high burden of visceral leishmaniasis. Recently, there was a significant increase in cases in the South Omo Zone. This study aims to assess the prevalence of Leishmania donovani infection and its associated factors. A household-based cross-sectional study was carried out in January 2023 in the South Omo Zone in Ethiopia. Dried blood spot samples were collected from 382 randomly selected study participants. Direct agglutination test (DAT) and kinetoplast DNA real-time PCR tests were performed to detect L. donovani infection. Participants' sociodemographic, clinical and risk factors for L. donovani infection data were collected using questionnaires. Bivariate and multivariate logistic regressions were used to analyze the data. Febrile cases were checked for malaria with a multiplex PCR assay. Overall prevalence of L. donovani infection among the sampled population was 32.5% (n=124), of which 41.1% (n=51) was detected by PCR, 33.9% (n=42) by DAT and 25.0% (n=31) by both tests. The majority of the positives were from the Logira (28.2%; n=35) and Dilbayne (29.0%; n=36) villages. Participants residing in Logira (adjusted OR [AOR]: 5.80; 95% CI 1.85 to 18.15) and Dilbayne (AOR: 3.38; 95% CI 1.15 to 9.96) villages and owning cows (AOR: 2.31; 95% CI 1.03 to 5.15) showed an association with Leishmania infection. Plasmodium falciparum was detected in 3.4% (n=2) of 59 febrile participants. The prevalence of L. donovani infection in the South Omo Zone is high. Further research on the role of cows in the transmission cycle is needed to design the best strategy to control Leishmania infection in the South Omo Zone. Such interventions should focus on the Logira and Dilbayne villages, where most of the infections were identified.

Short- and long-term stability of SARS-CoV-2 antibodies on dried blood spots under different storage conditions.

Dried blood spots (DBS) are broadly used for different serological analyses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assessment. In order to biobank DBS samples, an understanding of the preservation of SARS-CoV-2 antibodies is needed. Therefore, we assessed the stability of SARS-CoV-2 antibodies on DBS during short- and long-term storage under different storage temperatures. Seven sample donors were enrolled, each donating twenty 6 mm DBS to assess anti-spike (S1) SARS-CoV-2 IgG antibodies (EUROIMMUN). Baseline samples were analyzed on the day of collection. The remainder of the samples was stored in grip seal bags kept in a cryobox at room temperature/4°C until 2 months after collection and at -20°C until 2 years after collection. Samples were analyzed at regular intervals within the total storage duration and after one and five freeze-thaw cycles. A pooled coefficient of variation was calculated for each storage temperature. We found that anti-S1 SARS-CoV-2 antibodies collected on DBS saver cards remain stable during short-term storage at RT, 4°C, and -20°C (at least to 2 months) and long-term storage at -20°C (at least 2 years). Moreover, up to five freeze-thaw cycles can occur without impacting the anti-S1 SARS-CoV-2 antibody level. The inter-assay coefficient of variation lies between 10 and 15%. As DBS can be preserved for both shorter periods of time at RT and longer periods of time at -20°C, they are a perfect application for studies that require sample shipment by mail, self-sampling studies, studies in limited resource settings and biobanking.IMPORTANCEDried blood spots (DBS) are currently widely used as a microsampling technique for different qualitative and quantitative serological assessments. Yet, there is a lack of long-term stability and storage condition studies. In our study, first, we assessed the stability of SARS-CoV-2 antibodies on DBS up to 2 years post-collection. We believe that our data are not only important for future COVID-19 research but also for studies on other infections/diseases using DBS-based serology.

Evaluating vitamin D status in Chinese pre-school children using dried blood spots coupled with liquid chromatograph coupled with mass spectrometer.

Vitamin D is an essential micronutrient for multiple physiological processes, and its deficiency remains a world-wide public health problem that cannot be ignored. Dried blood spot (DBS) is a convenient tool in large-scale epidemiological studies, but its application in evaluating vitamin D status in Chinese population is still scarce. Herein, we aimed to determine the vitamin D status in Chinese pre-school children using DBS coupled with LC-MS/MS method. We first developed a sensitive and reliable method for the determination of 25-hydroxyvitamin D (25(OH)D) in DBS samples using an ultra-high-performance liquid chromatograph coupled with triple quadrupole mass spectrometer (UHPLC-QQQ-MS/MS). Next, we conducted a pilot study to compare the 25(OH)D concentration in DBS and serum samples. Finally, the assay method was used to evaluate vitamin D status in Chinese pre-school children. The present method was validated to be reliable and robust for the determination of 25(OH)D in DBS samples. Comparable consistency was observed between the 25(OH)D concentration in DBS and serum samples. A total of 3826 DBS samples collected from children aged 1-7 years were determined. The median concentration of 25(OH)D was 19.57 ng/mL (interquartile range 14.73-24.36 ng/mL), and decreased from 1 to 7 years of age. In addition, 13.51% of male children and 15.12% female children are found to be deficient in 25(OH)D. DBS coupled with LC-MS/MS is a feasible strategy to evaluate vitamin D status in epidemiological studies. And vitamin D deficiency remains a common health problem in Chinese pre-school children.

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

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

Dried blood spot improves global access to aquaporin-4-IgG testing for neuromyelitis optica.

This study aimed to evaluate the diagnostic accuracy of dried blood spot (DBS) compared with conventional serum Aquaporin-4-IgG (AQP4-IgG) testing. Prospective multicenter diagnostic study was conducted between April 2018 and October 2023 across medical centers in the United States, Uganda, and the Republic of Guinea. Neuromyelitis optica spectrum disorder (NMOSD) patients and controls collected blood on filter paper cards along with concurrent serum samples. These samples underwent analysis using flow cytometric live-cell-based assays (CBA) and enzyme-linked immunosorbent assay (ELISA) to determine AQP4 serostatus. The accuracy of AQP4-IgG detection between DBS and serum (gold standard) was compared. Among 150 participants (47 cases, 103 controls), there was a strong correlation between DBS and serum samples (Spearman's correlation coefficient of 0.82). The AUC was 0.97 (95% CI: 0.92-0.99). AQP4-IgG detection through DBS showed 87.0% sensitivity (95% CI: 0.74-0.95) and 100% specificity (95% CI: 0.96-1.00) using CBA, and 65.2% sensitivity (95% CI: 0.43-0.84) and 95.2% specificity (95% CI: 0.76-0.99) using ELISA. Serum ELISA demonstrated 69.6% sensitivity (95% CI: 0.47-0.87) and 98.4% specificity (95% CI: 0.91-0.99). The stability of DBS in detecting AQP4-IgG persisted over 24 months for most cases. The DBS represents a viable alternative for detecting AQP4-IgG in resource-limited settings to diagnose NMOSD, offering high sensitivity and specificity comparable to serum testing. Moreover, DBS has low shipping costs, is easy to administer, and is suitable for point-of-care testing.

ONT sequencing identifies a high prevalence of crt sensitive, triple mutant dhfr and single mutant dhps parasites within an ANC population in Nigeria.

Malaria in pregnancy is a major public health issue, particularly among vulnerable populations in malaria-endemic sub-Saharan African countries. To mitigate its risks, WHO recommends sulphadoxine-pyrimethamine (SP) for chemoprevention and artemisinin-based combination therapy (ACT) to treat uncomplicated Plasmodium falciparum malaria. These interventions have helped to alleviate the risk associated with malaria in pregnancy; however, in the context of the emergence of SP- and ACT-resistant P. falciparum, maintained efficacy is under threat. Molecular surveillance is a reliable tool to monitor the emergence of resistance where molecular markers are known. Thus, the objective of the study was to use a multiplexed amplicon Oxford Nanopore sequencing approach to assess the molecular markers for antimalarial resistance among pregnant women in Nigeria. Dried blood spots (DBS) were collected from pregnant women who received IPTp-SP at the enrollment and follow-up visits. P. falciparum genomic DNA was extracted by the Chelex® method and Pf18S qPCR was used to detect parasite DNA in each sample. With nested PCR assays, fragments of Pfdhps, Pfdhfr, Pfmdr1, Pfcrt, Pfk13 and Pfama1 genes were amplified and multiplexed amplicon-based sequencing was conducted on the minION Oxford Nanopore Technology. In total, 251 pregnant women were enrolled in the study and 457 DBS samples were collected. P. falciparum genomic DNA was detected in 12% (56/457) of the samples, 31 at baseline and the remaining during the follow-up visits. Pfama1, pfk13, Pfdhps, Pfdhfr, Pfmdr1 and Pfcrt were successfully sequenced in a single run. Notably, k13 artemisinin resistance mutations were absent, the frequencies of Pfdhfr and Pfdhps SP resistance haplotypes, IRN for pyrimethamine resistance and ISGKA/IAGKA associated with sulphadoxine resistance were 82% (36/44) and 64% (27/42), respectively, and the Pfcrt CVIET resistant haplotype was at approximately 22% (7/32). Here a multiplexed amplicon-based ONT assay established that triple mutant Pfdfhr-IRN, double mutant Pfdhps-SG haplotypes and the chloroquine sensitive strain were prevalent among pregnant women in Nigeria.

Simultaneous detection of myostatin-targeting monoclonal antibodies in dried blood spots and plasma using liquid chromatography-tandem mass spectrometry with field asymmetric ion mobility spectrometry

Transforming growth factor-β superfamily members, such as myostatin, growth/differentiation factor 11, and activin A, negatively regulate skeletal muscle mass. Inhibitors targeting these cytokines or activin receptor type IIB have the potential to treat muscular diseases and enhance physical performance. However, because of their effects on muscle mass and potential misuse, they are strictly prohibited in sports. Given the high potential for misuse as a doping agent in sports, effective analytical methods for these prohibited antibodies targeting these specific cytokines or their receptor are critically needed. In this study, we aimed to develop and validate a multitarget method to detect the prohibited transforming growth factor-β superfamily-targeting monoclonal antibodies, such as landogrozumab, domagrozumab, and the activin receptor type IIB-targeting antibody, bimagrumab, in human plasma and dried blood spot (DBS) samples using liquid chromatography-tandem mass spectrometry. Antibodies were purified from both the DBS and plasma samples using protein G magnetic beads and field-asymmetric ion mobility spectrometry (FAIMS) to minimize interference, followed by liquid chromatography-tandem mass spectrometry analysis. The validation process included tests for specificity, selectivity, linearity, limit of detection (LOD), limit of identification, precision, recovery, carryover effect, and matrix effect. The LODs for the target antibodies were identical in both DBS and plasma samples at 0.1 µg/mL for landogrozumab heavy and light chains, as well as 0.25 µg/mL for the domagrozumab light chain and 0.25 µg/mL for the bimagrumab heavy chain. However, the heavy chain of domagrozumab exhibited an LOD of 0.5 µg/mL in DBS and 1 µg/mL in plasma. The analytical method demonstrated strong linearity, with R² values greater than 0.99 for both plasma and DBS, and no carryover effect. Precision (CV%) was below 15 % at both middle (1 or 5 µg/mL; specific to the heavy chain of domagrozumab in plasma) and high (10 µg/mL) concentrations and was less than 20 % at the LOD. The selectivity and specificity indicated no interference in the analysis of target mAbs in different blood samples. Recovery was 31.6–49.8 % for DBS and 51.4–85.3 % for plasma, with no significant matrix effect. This study provides an effective method for doping analysis and novel protein detection.

Dried Blood Spots Sampling and Population Pharmacokinetic Modeling for Dosing Optimization of Piperacillin in Chinese Neonates.

Piperacillin is commonly used off-label in neonates for the treatment of bacterial infections. This study aimed to assess a dried blood spots (DBS)-based microsampling strategy for supporting population pharmacokinetics and treatment optimization of piperacillin in Chinese neonates. DBS samples from neonatal patients were collected at predefined intervals. Drug blood concentrations were quantified using a validated ultra-high-performance liquid chromatography-tandem mass spectrometry method. A population pharmacokinetic model was developed using a nonlinear mixed-effects modeling approach. The pharmacokinetic/pharmacodynamics (PK/PD) target was 75% of the time with the unbound drug plasma concentration above the minimum inhibitory concentration (fT>MIC), with a toxicity threshold of unbound drug plasma trough concentration above 64 mg/L. A total of 45 piperacillin samples from 24 neonates were collected. The pharmacokinetics of piperacillin was described using a one-compartment model with postmenstrual age (PMA) as the most significant covariate on clearance. Simulations showed that dosing regimens achieving >90% PK/PD target attainment with <10% risk of possible toxicity were: PMA 33-35 weeks (50 mg/kg q12h), 35-37 weeks (50 mg/kg q8h), and 37-41 weeks (50 mg/kg q6h). In conclusion, Using DBS sampling, we developed a population pharmacokinetic model of piperacillin in Chinese neonates, incorporating PMA to determine optimal dosing regimens.

7701 Comparative Analysis of Dried Blood Spot and Serum ELISAs for AMH, FSH, and LH in Patients Seeking IVF Treatment

Abstract Disclosure: T. Kumar: None. R. Chauhan: None. B. Kalra: None. A.S. Patel: None. S. Chauhan: None. A. Kumar: None. Relevance: Dried Blood Spot (DBS) technology has not been widely implemented in the diagnostic industry due to its limitations in obtaining reliable, quantitative data from small blood samples. However, recent advancements in methods, i.e., sensitive antibodies, homogeneous and reproducible paper quality, and painless needles have renewed the interest in the DBS collection technique. Objective: The aim of the study is to validate the hormone measurements using DBS technology and compare the correlation matrix between AMH, LH, FSH, inhibin B, Antral Follicle Counts (AFC), age, and BMI in patients seeking infertility workup. Materials and Methods: Matched serum and DBS samples from thirty-nine patients between the ages of 18 and 45 years were collected on day 3 of the cycle. The subjects were visiting the clinic for initial fertility workup and subjects on medications for ovarian suppression were excluded. The samples were tested on Ansh Labs DBS AMH (AL-129), FSH (AL-187), and LH (AL-190), PCOCheck AMH (N-N terminus, AL-196), picoAMH (C-N terminus, AL-124-r), US Inhibin B (AL-195) ELISAs. Baseline AFC obtained via transvaginal ultrasound, BMI, and age were collected. Spearman’s correlations, and regression analysis using Passing and Bablok were calculated to examine the relationship between AFC, AMH, inhibin B, FSH LH, age, and BMI. Results: DBS samples were highly correlated to their matched serum specimens. The slope and Spearman’s rank correlation (rs) between matched serum and DBS specimens for AMH, FSH, and LH were 1.17x, 1.07x, 1.07x, and 0.98, 0.93, and 0.97, respectively. DBS AMH strongly correlated with AFC. The Spearman’s rank correlation (rs) between AFC and DBS AMH, DBS LH, and DBS FSH were 0.80, -0.11, and -0.31, respectively. A strong correlation was observed between age and AMH, LH and FSH. A weak correlation was observed between BMI and AMH, LH and FSH. The Spearman’s rank correlation (rs) between age and DBS AMH, LH, and FSH were -0.34, 0.28, and 0.25, respectively. DBS AMH, LH, and FSH correlation with BMI were -0.06, 0.05, and 0.09, respectively. Inhibin B had a strong correlation to age (rs -0.33), BMI (rs -0.67), AFC (rs 0.44), DBS AMH (rs 0.40), DBS FSH (rs -0.19), DBS LH (rs-0.11), respectively. AMH method comparison between N-N and N-C terminal ELISA using 39 serum samples yielded slopes of 0.85x and Spearman’s rank correlation of 0.97. Conclusions: DBS and serum measurements for AMH, LH, and FSH are very accurate and precise. They can be used interchangeably. The strong relationship between BMI and Inhibin B will require further investigation and may open new avenues in obesity and reproductive endocrinology. Presentation: 6/1/2024

A-314 Influence of EDTA on TSH and 17-OHP in Newborn Screening: A Case Study

Abstract Background A 9-month-old infant presented with developmental motor delay. Comprehensive evaluation including microarray and brain MRI revealed no specific abnormalities except for hypotonicity. The patient's perinatal history, including newborn screening test (NST), showed no remarkable findings. Additionally, the infant exhibited facial dysmorphism and microcephaly, leading to another NST to explore potential metabolic diseases. Upon NST, Thyroid-Stimulating Hormone (TSH) levels were 1.86 and 1.27 mlU/L (duplicate testing results; reference value: &amp;lt;12 mlU/L), 17-Hydroxyprogesterone (17-OHP) levels were 0.55 and 0.48 ng/mL (duplicate testing results; reference value adjusted for body weight: &amp;lt;5.0 ng/mL), and total Galactose (Gal) and Galactose-1-Phosphate (Gal-1-P) levels were 1.68 mg/dL (reference value: &amp;lt;8 mg/dL). Methods Upon inspection of the dried blood spot (DBS) sample, the DBS used for the tests partially appeared to be inadequately collected on the absorbent filter paper, which could cause errors related to the disc and specimen itself. Consequently, re-sampling and re-testing were necessary to ensure accuracy. A second NST was performed with the new DBS samples. In the second NST results, most parameters were within the allowable ranges. However, the TSH levels were notably below the detectable limit (&amp;lt;0.7 mlU/L), and the 17-OHP levels were significantly elevated at 332ng/mL, diverging markedly from the initial NST results. To ensure reliability of the measured test results, TSH levels were analyzed using both the existing PerkinElmer equipment and compared with results from a Cobas 8000 system (Roche) and mass spectrometry. Similarly, 17-OHP levels were also compared using mass spectrometry. Results Upon comparing the TSH results, both analytical methods (PerkinElmer and Roche) yielded identical outcomes. However, significant discrepancy was observed when compared to mass spectrometry results. Review of the relevant testing batches showed no significant quality control issues with the NST, leading to clinical suspicion of ethylene-diamine-tetraacetic acid (EDTA)-induced interference. According to existing literature, when NST are conducted using the lanthanide fluorescence method, distortions can occur in DBS containing EDTA, depending on the concentration of EDTA present. In such cases, the EDTA-induced interference may result in false-negative TSH and false-positive 17-OHP results. These discrepancies can be differentiated using standard MS/MS testing. With regards to our case, upon enquiry, the second DBS sample was revealed to be collected from an existing EDTA sample rather than directly from the patient. Thus, adequate DBS discs from the initial sample were obtained and re-analyzed for NST using mass spectrometry, with final results reporting no specific abnormalities. Conclusions The discrepancies observed in TSH and 17-OHP levels between the first and second set of DBS samples were found to be unrelated to other laboratory factors, such as quality control issues. Instead, they were conclusively identified as false-negative and false-positive results associated with interference due to the use of EDTA-containing collection bottles. Mass spectrometry analysis was able to confirm the interference. Clinical awareness should be promoted in order to prevent EDTA-induced interference in NST.