Dried Blood Spots Research Articles

Blood Glucocerebrosidase Activity and α-Synuclein Levels in Patients with GBA1-Associated Parkinson's Disease and Asymptomatic <i>GBA1</i> Mutation Carriers

Introduction. Mutations in a GBA1 gene, which encodes a lysosomal enzyme called glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson's disease (PD). The pathogenesis of PD results from the death of dopaminergic neurons in the substantia nigra of the brain, which is associated with the aggregation of α-synuclein protein. However, not all GBA1 mutation carriers develop PD during their lifetime. The aim of this study was to evaluate GCase activity and α-synuclein levels in CD45+ blood cells of patients with PD associated with GBA1 mutations (GBА1-PD), asymptomatic carriers of GBA1 mutations (GBА1-carriers), and patients with sporadic PD (sPD), as well as correlation between the study parameters in the study groups. Materials and methods. The study included patients with GBА1-PD (n = 25) and sPD (n = 147), and GBА1-carriers (n = 16). A control group included healthy volunteers (n = 154). The level of α-synuclein in CD45+ cells was measured by enzyme-linked immunosorbent assay, and GCase activity in dried blood spots was detected by high-performance liquid chromatography with tandem mass spectrometry. Results. Increased level of α-synuclein protein was detected in CD45+ blood cells of patients with GBA1-PD, sPD, and GBA1-carriers compared to controls (p = 0.0043; p = 0.0002; p = 0.032, respectively). Decreased GCase activity was reported in GBA1-PD patients and GBA1-carriers compared to sPD patients (p = 0.0003; p = 0.003, respectively) and controls (p 0.0001; p 0.0001, respectively). However, negative correlation between α-synuclein levels and GCase activity was observed only in GBA1-PD patients, but not in GBA1-carriers. Conclusion. Our data suggest a possible functional relationship between the activity of GCase and the metabolism of α-synuclein in PD associated with GBA1 mutations.

A-059 Validation of self-collected capillary dried blood spot collection for clinical HbA1c testing

Abstract Background The increased prevalence of diabetes in socially disadvantaged populations requires solutions that are amenable to these communities. Unmanaged diabetes can cause cardiovascular disease, kidney damage, blindness, and limb amputation. Diabetes is traditionally diagnosed using fasting/oral glucose tolerance or hemoglobin A1c (HbA1c) tests; both of which require access to medical and laboratory facilities. Self-collected capillary dried blood spot (DBS) testing for HbA1c could alleviate health disparities by offering access to low cost and minimally invasive remote collection. The objective of this study was to validate self-collected capillary DBS specimens for clinical HbA1c testing. Methods DBS HbA1c was measured using the hemolysate application of the Roche cobas c513 Tina-quant Hemoglobin A1cDx Gen.3 assay. Calibration was performed according to the manufacturer’s package insert. Patient samples were prepared by collecting two 3mm DBS punches. Non-biological standards were prepared by placing two blank 3mm DBS punches into an empty tube and adding 10uL of standard. After 24 hours, samples were resuspended in 750uL of hemolyzing reagent and incubated for 75 minutes at room temperature (RT) while shaking at 800 RPM. Preliminary data showed that non-biological standards spotted onto DBS cards did not behave like real patient samples. A correction factor (CF) was derived (n=134) and challenged (n=20) to account for the systematic bias observed between matched patient DBS and venous specimens. Imprecision was established by running two patient DBS specimens as controls. Accuracy was evaluated using residual College of American Pathologists (CAP) proficiency testing (PT) material (n=32). Linearity was confirmed by running 5 levels of Maine Standards Linearity HbA1c Kit in triplicate. Stability of the DBS specimen and the hemolysate supernatant extracted from the DBS specimen were observed at RT. Results Specimens tested between venous and DBS HbA1c to derive the CF (n=134) had a bias of 0.26% (4.32%) and a linear equation of (y=0.938x +0.631; R=0.996). The resulting CF is: reported HbA1c% = (DBS HbA1c% - 0.631)/0.938. Matched venous and DBS specimens that challenged the CF (n=20) had a bias of -0.10% (-1.83%) and a linear equation of (y=1.06x -0.41; R=0.988). Imprecision was 2.3% and 1.3% for patient DBS control specimens with mean concentrations of 5.3% and 5.9%, respectively. Residual CAP PT samples (n=32) had an average bias of -0.05% (-0.40%). DBS HbA1c concentrations from 4.2-15.5% were linear (y=0.903x +1.15; R=0.999). The DBS specimen is stable for 60 days at RT and the hemolysate supernatant is stable for at least 10 hours at RT. Conclusions Self-collected capillary DBS specimens are acceptable for the clinical measurement of HbA1c, and effectively increase accessibility to diabetes screening and monitoring. Applying a CF to DBS patient samples removes the systematic bias observed between matched venous and DBS samples. Non-biological standards and capillary whole blood do not behave the same on DBS cards. Alternative PT for DBS specimens should be investigated as at-home collection advances.

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: <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: <5.0 ng/mL), and total Galactose (Gal) and Galactose-1-Phosphate (Gal-1-P) levels were 1.68 mg/dL (reference value: <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 (<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.

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.

A-348 Portable aptamer-based home monitoring system for blood phenylalanine measurement

Abstract Background Aptatek BioSciences, Inc. has developed a home monitoring tool for blood phenylalanine (phe) levels. The Aptatek system integrates a proprietary aptamer assay approach with a user-friendly handheld, smartphone-enabled test platform. PKU (phenylketonuria), is an inborn error of phenylalanine (Phe) metabolism If undiagnosed or untreated, phe accumulates in all body tissues, including the blood and brain, where it is especially neurotoxic and when untreated it can cause significant cognitive impairment and neurological defects. PKU management targets maintaining blood Phe concentration within the therapeutic range (120-360uM) to avoid severe neurological sequelae, mainly through nutritional therapy. Methods PheCheck™ includes a portable hand-held reader, software accessible as a mobile phone application, and a disposable single-use lateral flow cassette, blood collection, cap, and buffer chamber. PheCheck™ detects phe by binding of the phe in a small blood sample using small molecule-specific DNA aptamers. A patient (or caregiver) collects 5μl of capillary blood using a lancet to lance the fingertip and use the collection cap to collect the specimen. Once collected, the specimen is placed into the buffer chamber where the blood is diluted and processed in preparation for a lateral flow assay. The buffer chamber is placed on top of a lateral flow cassette to release the solution into the cassette. The lateral flow cassette is inserted into a small electronic reader after specimen incubation. The reader will detect a fluorescent signal from the strip and report the quantitative Phe concentration present in the specimen to the integrated mobile phone application. The reader has a simple design with a single operating push button, USB data connection port, and replaceable battery compartment. The reader reads the output of the lateral flow cassette and reports the result to a Smartphone app. The app is designed to either display the quantitative results or store the results without displaying the quantitative value. Results Twelve patients were trained on the PheCheck system and observed using the system for self-testing. Patients also prepared dried blood spot cards for comparative testing using LC-mass spectrometry. Patients were asked to return to the clinic within two weeks to retest. The blood phenylalanine levels measured by the PheCheck system were within targeted standard deviation of the dried blood spots demonstrating the feasibility of portable home-based testing for routine monitoring of blood phenylalanine levels in phenylketonuria patients. Such a portable system would enable real time monitoring and adjustment of diet or therapy for chronic diseases such as PKU. Conclusions Aptatek has developed a DNA aptamer based portable monitor for measuring blood phenylalanine levels. The system quantitatively measures phenylalanine from a 5-microliter finger stick blood sampler. Aptamer-based detection of phenylalanine provides reliable quantitation across a wide clinical concentration range and encouraging performance relative to gold standard testing of venous blood draw or dried blood spots. Analysis of samples from patients on Palynziq indicate that the PheCheck system could provide more reliable testing than venous blood draws. The system shows strong promise for routine home monitoring of blood phe levels by patients.

A-320 Determination of Guanidinoacetate Methyltransferase Enzyme Biomarkers in Newborn Dried Blood Spot by Tandem Mass Spectrometry-A Two Tier Approach

Abstract Background Guanidinoacetate methyltransferase enzyme (GAMT) deficiency is a rare, inherited metabolic disorder that affects the synthesis of creatine. Creatine is an essential metabolite for regenerating energy, particularly in the brain and muscles. Creatine is synthesized from guanidinoacetate (GUAC) by the GAMT enzyme. Mutations in the GAMT gene result in a shortage of the GAMT enzyme that causes a deficiency of creatine and accumulation of GUAC in the body, which is a neurotoxin. Following a recommendation by the Recommended Uniform Screening Panel and in line with California State statute, all California newborns will be screened for GAMT deficiency beginning July 2024. Here, we developed and validated a quantitative two-tier tandem mass spectrometry (MS/MS) assay for the detection of GAMT biomarkers (GUAC, creatine and creatinine) in newborn dried blood spot (DBS). Methods This method punches a 3.2 mm disc of controls and newborn patient DBS into a 96-well plate. 100 μL of extraction solution, containing internal standards of GAMT biomarkers, is added to each well and incubated at 40°C for 45 minutes. The extract is transferred, evaporated, and derivatized into a 3N-butanol-HCl solution at 60°C for 30 minutes. The excess HCl is evaporated to dryness. The conversion of GUAC and creatine to their butyl esters has improved their detection sensitivity by MS/MS. The butyl esters are reconstituted into the mobile phase and shaken for 10 minutes at 27°C. The final 10 μL extract was analyzed by flow injection analysis (FIA)-MS/MS in multiple reaction monitoring (MRM) positive ion mode with a total run time of 1.5 minutes per sample. The presumptive positive samples from initial analysis are prepared following the same method, and a 5 μL extract is resolved through an ultra-performance liquid chromatography (UPLC) column and analyzed by UPLC-MS/MS in MRM positive ion mode with a total run time of 3.5 minutes per sample. The validation was performed by assessing the linearity, accuracy, imprecision, carryover, limit of detection (LOD) and lower limit of quantitation (LLOQ), matrix effect, analytical measurement range (AMR), percent total allowable error (TEa), and reference range. Results This method is linear for GUAC, creatine and creatinine over a measured range of 1.17-40.00, 121.20-857.36 and 41.34-387.46 µmol/L respectively. The R2 values=1.000, 0.997, 0.9998; slope=0.999, 0.9945, 0.9982; intercept=0.0398, 4.881, 1.7027; mean recoveries=99-104%, 100-104%, 100-104%; intra-assay precision (n=20) =4.05-5.95%, 4.62-5.31%, 3.27-5.19%, inter-assay precision (n=40)=3.7-12.6%, 4.3-7.6%, 3.8-7.4%; TEa=6-21%, 7-17%, 6-17%; LOD=0.12, 0.54, 1.11 µmol/L; AMR=0.33-1300, 1.11-2700, 3.50-4500 µmol/L; median patient results observed from 2293 normal patient=1.48, 494.02, 89.97 μmol/L for GUAC, creatine, and creatinine respectively. The tier-1 and tier-2 assay screening cutoff for GUAC at the population 95th percentile is 2.53 and 4.55 μmol/L respectively. Conclusions The tier-1 and tier-2 methods has been developed and validated to determine GAMT biomarkers in newborn DBS, making it suitable for routine screening of ∼2000 newborn specimens per day by tier-1 and followed by more specific tier-2 to separate GAMT biomarkers from potential isobaric interfering compounds. This two-tier approach greatly improved the test performance and significantly reduced false positive rate.

Newborn screening for SCID: the very first prospective pilot study from Türkiye.

The measurement of T-cell receptor excision circle (TREC) is used for newborn screening (NBS) in dried blood spot (DBS) samples from Guthrie card for severe combined immunodeficiency (SCID). Here, we report the results of first newborn screening pilot program for SCID conducted in Türkiye. The study was carried out together with Ankara University School of Medicine and The Ministry of Health, Public Health General Directorate, Pediatric and Adolescent Health Department. TREC measurements were performed in randomly selected Guthrie card samples obtained from 20253 babies born between October 2018 and October 2020. The TREC analyses were performed together with beta Actin (β-Actin) via RT-PCR (Real Time Polymerase Chain Reaction). TRECs found to be normal (≥15 copies/µl) in 98,6% of the newborns (n: 19975) but low (<15 copies/µl) in 1.4% (n:278) at the initial analyses. TRECs were retested in 278 suspected infants and found to be normal in 160 (0.8%) while low in 118 (0.58%). New DBS were obtained from the babies with low TRECs (new sample test). TRECs were normal in 108 (0.53%) of the new sample tests and low in 10 (0.049%). Two among 10 babies who had abnormal (undetectable) TRECs were diagnosed as SCID; ADA (P1) and RAG1 (P2) defects were confirmed respectively. They both received curative treatments [gene therapy (P1) and HSCT (P2)]. The remaining 6 of 8 newborns with abnormal TRECs were found normal after clinical and laboratory immune work-up, while medical records of other two revealed early postnatal death due to extreme prematurity. In the light of this study the incidence of SCID was detected at least 1/10000 live births in Türkiye. This study shows the feasibility and usefulness of initiating SCID screening in Türkiye.

Global Metabolomics Using LC-MS for Clinical Applications.

Metabolomics can be used for a multitude of purposes, including monitoring of treatment effects and for increasing the knowledge of the pathophysiology of a wide range of diseases. Global (commonly referred to as "untargeted") metabolomics is hypothesis-generating and provides the opportunity to discover new biomarkers. Being versatile and having a high degree of selectivity and sensitivity, liquid chromatography-mass spectrometry (LC-MS) is the most common technique applied for metabolomics. We here present our global metabolomics LC-electrospray ionization-MS/MS method. The sample preparation procedures for plasma, serum, dried blood spots, urine, and cerebrospinal fluid are simple and nonspecific to reduce the risk of analyte loss. The method is based on reversed-phase chromatography using a diphenyl column. The high-resolution Q Exactive Orbitrap MS with data-dependent acquisition provides MS/MS spectra of a wide range of analytes. Our method covers a large part of the metabolome regarding hydrophobicity and compound class.

B-176 Rapid Diagnosis of 21-Hydroxylase Deficiency Using LC-MS/MS for Urinary Unconjugated Steroid Analysis

Abstract Background Urinary steroid profiling through gas chromatography-mass spectrometry (GC-MS) serves as a potent diagnostic tool for detecting abnormal steroid production linked to congenital enzyme deficiencies and steroid-related diseases. This technique involves deconjugating and extracting both conjugated and unconjugated steroids, followed by derivatization and analysis via GC-MS. It provides comprehensive insights into steroid metabolism, aiding in the identification of genetic disorders and conditions with atypical steroid levels. However, the procedure is intricate and time-consuming, particularly the deconjugation of steroids for analysis. The present study aimed to assess the usability of LC-MS/MS assays measuring unconjugated Δ4- and Δ5-steroids in urine for diagnosing steroid abnormalities. Methods The Biotage ISOLUTE SLE+ (Biotage, Sweden) was used for sample preparation, with dichloromethane for elution. LC-MS/MS analysis was performed on an LCMS8060NX (Shimadzu, Kyoto, Japan), using a Shim-Pack Velox Biphenyl column 2.1mm*150mm* 1.8μm (Shimadzu GLC, Tokyo, Japan). The mobile phase consisted of a 0.2mmol/L ammonium fluoride aqueous solution and a 0.2mmol/L ammonium fluoride methanol solution. We targeted 38 steroids, including four steroid medications, and used 19 stable isotope-labeled steroids for quantification. For the measurements, 30 neonatal urine samples were used (IRB Approval Number 20221210), which were suspected of 21-hydroxylase deficiency (21OHD) based on clinical symptoms or screening for 17α-hydroxyprogesterone (17OHP) using Dried Blood Spot. Of these, 18 had been confirmed as 21OHD cases by exceeding the GC-MS diagnostic criterion for Pregnanetriolone (&amp;gt;0.06ng/g Creatinine). Results Blood 21-deoxycortisol (21DOF) is a reliable marker for diagnosing 21OHD, which is generated from 17OHP through an alternative biochemical route. In consideration of that, we measured the urinary concentration of unconjugated 21-deoxycortisone (21DOE, precurcer of Pregnanetriolone), into which 21DOF is putatively converted in the kidney. In cases where 21OHD was excluded by GC-MS diagnosis, 21DOE was undetectable, falling below the sensitivity levels. On the other hand, 21DOE was detected in all samples confirmed 21OHD cases, consistent with the conversion of 21DOF to 21DOE in the kidney. Conclusions The present study suggested the usability of LC-MS/MS assays measuring urinary unconjugated 21DOE for diagnosing 21OHD. This approach provides a faster, less complex method for diagnosis, eliminating the need for the deconjugation process. To establish the value of urinary unconjugated steroids as diagnostic markers for steroid abnormalities, efforts to accumulate further findings are underway using clinical samples.

Influence of Hematocrit and Storage Temperature on the Stability of Dried Blood Samples in Serological Analyses of Tetanus, Diphtheria, and Pertussis.

Dried blood spots (DBSs) enable the study of serological markers of various pathogens without the need to obtain serum/plasma through venipuncture. Sixty-four blood samples were prepared on Whatman™ 903 cards using specimens obtained by venipuncture to study the detection of serological markers of diphtheria, tetanus, and pertussis in DBSs, and their stability 4 months post-collection. An automated chemiluminescent immunoassay was used to investigate diphtheria, tetanus, and pertussis IgG levels from both DBSs and plasma samples. An optimal cut-off value for DBSs was calculated to improve the performance of diphtheria and tetanus serological markers in DBSs, achieving high sensitivity (95% and 98%, respectively) and specificity (91.7% and 92.3%, respectively). No protection against pertussis was found in the population studied. The correlation observed between the plasma and the DBSs processed after sample collection was high (0.967-0.970) for all antibodies studied except pertussis (0.753), both considering hematocrit before sample elution or not. The correlation between DBSs and plasma for diphtheria and tetanus remained strong following a 4-month delay in DBS processing at 4 °C (0.925-0.964) and -20 °C (0.924-0.966), with only a minor decrease observed for diphtheria at room temperature (0.889), while maintaining a strong correlation for tetanus (0.960). For pertussis, the correlation between DBSs and plasma was drastically reduced after delaying its processing for 4 months at any temperature. To summarize, hematocrit levels within the normal range do not affect the processing of DBSs in the study of serological markers of diphtheria, tetanus, and pertussis. The DBS stability for serological diagnosis of diphtheria and tetanus is adequate when samples are stored at -20 °C for a period of 4 months. The pertussis serological marker does not appear to remain stable after 4 months, even when the DBS is stored frozen at -20 °C.

Blood biomarkers of trans-fatty acid intake among Nigerian adults in the Federal Capital Territory: a cross-sectional study

BackgroundIntake of trans-fatty acids (TFAs) is an established risk factor for cardiovascular disease. In April 2023, Nigeria passed regulations limiting TFA content in foods, fats, and oils, but the current level of TFA exposure in the Nigerian population is unknown. We assessed TFA biomarkers in dried blood spots from Nigerian adults in the Federal Capital Territory before policy enforcement to establish a baseline for future evaluation. MethodsWe used gas chromatography to measure TFA content in dried blood spots from adults participating in a cross-sectional household survey using a representative sampling frame. Individual TFA (t-16:1, t-18:1, and t- 18:2) and their total were expressed as percentage of total fatty acids. We assessed differences in TFA levels between subgroups based on sex, age, body mass index (BMI), education, income, and local government area using multivariable-adjusted linear regression models. Mean TFA levels were compared with samples from individuals in 30 countries. ResultsIn 213 adults (62% females; mean age: 36 y, mean BMI: 25.9 kg/m2), the mean TFA level in dried blood spots was 0.61% of total fatty acids (range: 0.23%–1.31%). In multivariable-adjusted models, TFA levels were higher in younger adults {<30 y compared with ≥42 y, 0.07% [95% confidence interval (CI): 0.00, 0.15], P = 0.047}, those without a high school degree [compared with higher education, 0.08% (95% CI: 0.01, 0.16), P = 0.023], and residents of Abuja Municipal Area Council [compared with residents in Gwagwalada, 0.12% (95% CI: 0.05, 0.20), P = 0.001]. Total TFA levels were comparable with international samples, but t-16:1 and t-18:1 appeared lower, whereas t-18:2 appeared greater (52% of all TFA), in the Nigerian samples. ConclusionsThese results provide a baseline assessment of TFA exposure in Nigerian adults to evaluate implementation and effect of national regulation passed in 2023. The observed subgroup differences may help identify subpopulations for targeted interventions to reduce TFA intake.

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

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

Microcolumn and coelution hydration of oil seal blood spot for efficient screening of newborn α-thalassemia via chip isoelectric focusing

BackgroundThe global prevalence of α-thalassemia necessitates effective newborn screening strategies due to its severe clinical consequences. Traditional methods such as liquid chromatography (LC), capillary electrophoresis (CE), and isoelectric focusing (IEF) face limitations, including low separation efficiency, poor sensitivity for detecting Hb Bart’s, and time-intensive operations, particularly with dried blood spots (DBS). These limitations hinder timely and accurate screening. This study addresses the need for a more efficient, sensitive, and rapid method for detecting Hb Bart’s in newborns. ResultsWe enhanced IEF separation and sensitivity by designing a microfluidic IEF (mIEF) system with shortened columns and employing a coelution sample loading technique using oil-sealed blood spots for rapid sample pretreatment. Our experiments demonstrated significant improvements: the total analysis time was reduced from 1110 minutes (IPG IEF) and 46 minutes (LC) per batch to 36 minutes per batch. For individual samples, the focusing time decreased from 6 minutes (previous mIEF) to 3 minutes, with the microcolumn length shortened by 50%, from 30 mm to 15 mm. The developed method showed excellent consistency with clinical Bart’s detection and PCR diagnosis, achieving 100% sensitivity and 98% specificity for α-thalassemia screening. Significance and NoveltyOur novel mIEF method provides an efficient, sensitive, and rapid tool for screening newborns for α-thalassemia. This advancement addresses the limitations of traditional techniques, improving early diagnosis and intervention strategies and ultimately enhancing health outcomes for at-risk newborns.

Micronutrient intake and status in young vegans, lacto-ovo-vegetarians, pescatarians, flexitarians, and omnivores.

Whether youth who follow plant-based diets in Nordic countries meet their dietary needs for micronutrients remains unclear. This study aims to evaluate micronutrient intake and status in Norwegian youth following vegan, lacto-ovo-vegetarian, pescatarian, flexitarian and omnivore diets. Cross-sectional design, with healthy 16-to-24-year-olds (n = 165). Participants were asked to complete a questionnaire and four 24-hour dietary recalls. Dried blood spots (DBS) and spot-urine samples were collected for analysis of methyl malonic acid (MMA) (n = 65), haemoglobin (Hb) (n = 164) and urinary iodine concentration (UIC) (n = 163). Vegans reported highest habitual supplement usage of multivitamin (58%), B12 (90%) and macroalgae consumption (32%), while flexitarians reported highest habitual usage of omega-3 supplements (56%). For daily supplement usage, vegans reported highest use of multivitamins (42%), B12 (79%), iodine (37%) and iron (63%). Increased risk of inadequate intake (energy-adjusted) were found for vitamin D (60% within lacto-ovo-vegetarians), selenium (70% within lacto-ovo-vegetarians, 65% within omnivores), and iodine (63% within vegans). Median MMA levels suggest low risk of insufficient B12 status across all groups (MMA 0.04‒0.37µmol/l) and 2% had MMA levels indicating possible B12 deficiency and 8% had elevated levels. Median Hb levels indicated low risk of anemia across all groups (≥ 12.0 females, ≥ 13.0g/dl males), though 7% had Hb values indicating risk of mild anemia and 4% risk of moderate anemia. The median UIC indicates mild iodine deficiency in all groups (UIC < 100µg/l), except vegans, who were moderately iodine deficient (UIC < 50µg/l). Our study indicated that the participating youth had low risk of inadequate intake of most micronutrients, partly due to high supplement usage. However, for iodine, vitamin D, and selenium higher risk of inadequate intake was found. UIC corroborated the low iodine intake among vegans. Thus, we suggest iodine status of youth in Norway should be monitored, especially among young fertile women who omits dietary iodine sources, until a mandatory iodine fortification program is implemented. Furthermore, we suggest that food education on how to secure sufficient nutrients from food in general should be provided to the Norwegian youth population, especially how to secure adequate intake of vitamin D, selenium and iodine.

Peer-Delivered HIV Self-Testing, Sexually Transmitted Infection Self-Sampling, and Pre-exposure Prophylaxis for Transgender Women in Uganda: A Randomized Trial.

Peer-delivered HIV self-testing (HIVST) and sexually transmitted infection self-sampling (STISS) may promote adherence to oral pre-exposure prophylaxis (PrEP), but no studies have analyzed this approach among transgender women (TGW) in sub-Saharan Africa. The Peer study was a cluster randomized trial in Uganda (October 2020-July 2022; NCT04328025). Ten TGW peer groups, each with 1 TGW peer and 8 TGW, were randomized 1:1 to receive quarterly in-clinic HIV testing with PrEP refills as standard-of-care (SOC) or SOC plus monthly peer delivery of oral-fluid HIVST, STISS, and PrEP refills (intervention). Participants were followed for 12 months. The primary outcome was PrEP adherence. We screened 85 TGW and enrolled 82 (41 per arm). The median age was 22 years (interquartile range [IQR] 20-24). Twelve-month retention was 88% (72/82). At the 3, 6, 9, and 12-month clinic visits, 10%, 5%, 5%, and 0% of TGW in the intervention arm had TFV-DP levels ≥700 fmol/punch, versus 7%, 15%, 7%, and 2% in the SOC arm, respectively (P = 0.18). At all visits, any detectable TFV-DP levels were significantly higher in SOC than the peer delivery group (P < 0.04). PrEP adherence was associated with sex work (incidence rate ratio 6.93; 95% CI: 2.33 to 20.60) and >10 years of schooling (incidence rate ratio 2.35; 95% CI: 1.14 to 4.84). There was a strong correlation between tenofovir detection in dried blood spots and urine (P < 0.001). No HIV seroconversions occurred. Peer-delivered HIVST and STISS did not increase low levels of oral PrEP adherence among TGW in Uganda. Long-acting PrEP formulations should be considered for this population.

Implementation of newborn screening for mucopolysaccharidosis type IVA and long-term monitoring in Taiwan

PurposeMucopolysaccharidosis IVA (MPS IVA) is a rare lysosomal storage disorder arising from a deficiency in N-acetylgalactosamine-6-sulfatase. MethodsFrom September 2019 to October 2023, a total of 264,843 Taiwanese newborns underwent screening for MPS IVA using dried blood spots and tandem mass spectrometry. ResultsAmong the 95 referred infants, 9 (9%) were confirmed to have MPS IVA (group 1), 18 (19%) were highly suspected to have MPS IVA (group 2), 61 (64%) were identified as heterozygotes of MPS IVA (group 3), and 7 (7%) were determined not to have MPS IVA (group 4). A total of 34 different GALNS (HGNC:4122) gene variants were identified through our MPS IVA newborn screening program. The most prevalent variant was c.857C>T p.(Thr286Met), found in 33 cases (29%), followed by c.953T>G p.(Met318Arg) in 22 cases (19%). Intravenous enzyme replacement therapy was initiated in 5 patients at ages ranging from 0.3 to 1.7 years. The estimated incidence of MPS IVA in this screening program was 3.4 per 100,000 live births. ConclusionBecause of the progressive nature of MPS IVA, an early diagnosis facilitated by newborn screening and prompt initiation of enzyme replacement therapy before irreversible organ damage occurs may result in improved clinical outcomes.

Quantitative determination by UHPLC-MS/MS of 18 common drugs of abuse and metabolites, including THC and OH-THC, in volumetric dried blood spots: a sustainable method with minimally invasive sampling

The increased use of drugs of abuse urges forensic toxicologists to create quick, simple, minimally invasive sampling techniques for biological fluids combined with analytical methods assuring accurate results. To this purpose, a method was developed aimed at quantifying 18 drugs of abuse and metabolites in DBS. Validation of the method was conducted by spiking blank whole blood with the analytes on Capitainer® B cards. The extracts were analyzed by a targeted UHPLC-MS/MS method. Linear calibration was achieved in the range of 1–100 ng/mL for: amphetamine, MDA, MDMA, methamphetamine, cocaine, codeine, benzoylecgonine, cocaethylene, morphine, 6-MAM, buprenorphine, methadone, EDDP, ketamine, norbuprenorphine norketamine, THC, and OH-THC. Experimental LOD was found at 0.5 ng/mL for all analytes except for norbuprenorphine, THC and THC-OH which yielded a LOD of 1 ng/mL. Intra- and inter-day accuracy was satisfactory with bias% resulting within 5%. Evaluation of intra- and inter-day precision yielded CV% values within 20%, for all compounds except EDDP. Average extraction recovery calculated at low (2 ng/mL) and high (75 ng/mL) concentration levels was 63% while average matrix effect determined at the same levels was found to be within 85% − 115% for all analytes except from codeine (70%) and MDMA (131%). The method was applied to authentic blood samples spotted onto the DBS card and the minimum value detected was 1.3 ng/mL. HPLC-MS/MS proved capable to identify all the targeted analytes at low concentrations in the small blood volumes obtainable from DBS cards, which in turn confirmed to be effective and sustainable micro-sampling devices.

Efficacy of Internet Recruitment and HIV Self-Testing for Diagnosing HIV Infections Among Black and Hispanic/Latino MSM and Transgender Women in 11 US States, 2020-2021.

We evaluated internet platforms for distributing HIV self-tests (HIVSTs) to Black or African American (Black) and Hispanic or Latino men who have sex with men (MSM) and transgender women (TGW). We recruited MSM and TGW from general interest, dating, and lesbian, gay, bisexual, and transgender platforms. Two HIVSTs were mailed to all MSM and TGW. Surveys (screening, baseline, 4-month, and results reporting) were completed online. After 4 months, participants were mailed another HIVST and a dried blood spot card. All HIVST interpretations and images of HIVST devices were reported online. Of 2093 MSM and 102 TGW, most were recruited through general interest and dating platforms. Over 50% were 18-29 years old, most identified as gay or bisexual. Overall, 45% had not tested for HIV in the past 12 months, and 9.1% of MSM reported a positive (reactive for HIV antibodies) HIVST result, with the highest percentage among Black MSM (11.5%). Dating platforms recruited higher percentages of MSM who recorded positive results compared with MSM from general interest platforms during the intervention period (11.9% vs 5.5% (P < 0.0001)), and MSM who had never tested for HIV reported a greater percentage of positive HIVST results compared with MSM who had been tested for HIV before enrollment (16.1% vs. 7.1%; P < 0.0001). MSM were able to correctly interpret and report HIVST results. Of TGW, 7% reported a positive HIVST result. Internet dating and general interest platforms can be key to increasing awareness of infection among BMSM, HMSM, and TGW persons, including those who do not use existing HIV services. www.clinicaltrials.gov Identifier: NCT04219878.

Effective Newborn Screening for Type 1 and 3 Primary Hyperoxaluria

IntroductionNewborn Screening programs for a defined set of eligible diseases have been enormously successful, but genomic newborn screening allowing for detection of additional treatable disorders has not been broadly implemented. All three types of primary hyperoxaluria (PH1-3) are rare autosomal recessive diseases caused by distinct defects of glyoxylate metabolism that are diagnosed genetically with certainty. Early diagnosis and treatment are mandatory to avoid renal failure or sequalae associated with persistent hyperoxaluria. MethodsThe prospective pilot study was undertaken within the framework of the German Newborn Screening. DNA samples extracted from dried blood spot cards were screened by multiplex PCR for the two most prevalent mutations: AGXT c.508G>A (PH1) and HOGA1 c.700+5G>T (PH3). Heterozygous AGXT/HOGA1 carriers received repeated spot urine analyses and, in case of persistent hyperoxaluria, complete Sanger sequencing of AGXT and HOGA1 gene, respectively. ResultsBetween March 15th 2022 and June 30th 2023 additional screening for PH1 and PH3 was performed in 77199 out of 222638 newborns included in the regular newborn screening program. No homozygous individuals, but 274 potential carriers for the AGXT mistargeting and 287 potential carriers for the HOGA1 splice variant were identified. Further work up revealed two already symptomatic compound heterozygous infants, one with PH1 (genotype c.508G>A; c.33delC) and one with PH3 (genotype: c.700+5G>T;c.134C>G). A second symptomatic PH1 patient (father of an identified carrier; genotype: c.508G>A;c.508G>A) was uncovered via family history. ConclusionThis pilot study demonstrated the efficacy of a genomic neonatal screening program for primary hyperoxaluria even in relatively small cohorts.

Validation of Dried Blood Spot Polyunsaturated Fatty Acids as Biomarkers of Polyunsaturated Fatty Acid Intake in Breast Cancer Survivors

Validation of Dried Blood Spot Polyunsaturated Fatty Acids as Biomarkers of Polyunsaturated Fatty Acid Intake in Breast Cancer Survivors