Dried Blood Spot Samples Research Articles

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.

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.

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.

Utility of an Archival Dried Blood Spot (DBS) Collection from HIV-Infected Individuals with and without Cancer in a Resource-Limited Setting.

The frequency of virus-associated cancers is growing worldwide, especially in resource-limited settings. One of the biggest challenges in cancer research among people living with HIV (PLWH) has been understanding how infection with both HIV and Kaposi sarcoma-associated herpesvirus (KSHV) promotes the pathogenesis of Kaposi sarcoma (KS), the most common cancer among PLWH worldwide and a significant public health problem in regions with high prevalence of HIV such as Sub-Saharan Africa (SSA). The AIDS and Cancer Specimen Resource (ACSR) provides samples for research, including dried blood spots (DBS) that were collected from large clinical epidemiology studies of KSHV and KS in PLWH conducted more than a decade ago in SSA. Here, we validated the quality of DNA derived from DBS samples from SSA studies and provided evidence of quantitative recovery of inflammatory cytokines using these DBS samples through comparison with paired frozen plasma. Significant differences in DNA, protein yields, and inflammatory biomarker levels were also observed between PLWH with/without KS. Establishing the fitness of DBS samples for studies of KS pathogenesis extends the number of projects that can be supported by these ACSR special collections and provides evidence that DBS collection for future KS research is a practical option in resource-limited settings.

Comparison between standard hematological parameters and blood doping biomarkers in dried blood spots within the athlete population of Swiss Sport Integrity.

The study demonstrated the feasibility of incorporating RNA biomarkers, specifically 5-aminolevulinic acid synthase (ALAS2) and carbonic anhydrase 1 (CA1), to improve the hematological module of the Athlete Biological Passport (ABP) in routine antidoping context. The aim was to investigate the implementation of reticulocyte (RET) related biomarkers, specifically ALAS2 and CA1, using quantitative reverse transcription polymerase chain reaction (RT-qPCR) on dried blood spots (DBS) from elite athletes. Hemoglobin changes over time in DBS samples was measured as well. Combining hemoglobin and messenger RNA (mRNA) analyses allowed to monitor alterations of the established marker, "DBS OFF-score". Ten athletes were selected for sampling by the Swiss national antidoping organization, Swiss Sports Integrity (SSI). Samples were collected, transported and analyzed for ABP following the World Anti-Doping Agency (WADA) procedures and spotted onto Protein Saver DBS cards. Most athletes exhibited stable biomarker levels, except for one individual involved in ski mountaineering, who demonstrated a sustained increase in ALAS2 compared to the individual baseline. This elevation could be due to blood withdrawal or other factors, such as doping with substances outside the targeted test menu. In this study, RNA-biomarkers were successfully analyzed in routine blood samples, and the project demonstrated promising results for the implementation of ALAS2 and CA1 in routine analysis to complement the ABP.

Integrated Serosurveillance for Onchocerciasis, Lymphatic Filariasis, and Schistosomiasis in North Darfur, Sudan.

Sudan is endemic for multiple neglected tropical diseases, including trachoma, onchocerciasis (OV), lymphatic filariasis (LF), and schistosomiasis (SCH). In 2019, dried blood spot samples were collected for a baseline trachoma serosurvey in three localities (El Seraif, Kotom, and Saraf Omrah) in North Darfur State. None were classified previously as OV- or LF-endemic, although low levels of SCH had been identified in all three. Approximately 30 households from 25 communities in each locality were selected by multistage cluster random sampling. Collections of DBSs were analyzed by multiplex bead assay for antibodies to multiple pathogens. This paper presents data on OV (Ov16), LF (Wb123, Bm14, Bm33), and SCH (soluble egg antigen [SEA], Sm25) antibodies among 8,322 individuals from 2,119 households. The survey-adjusted seroprevalence estimates for Ov16 were <0.3% in all localities. Lymphatic filariasis-antigen seroprevalences were discordant. Seroprevalence estimates ranged from 4.6-6.0% (Wb123), 0.99-1.4% (Bm14), and 29.2-33.3% (Bm33). Schistosomiasis seroprevalence estimates among school-aged children ranged from 2.7-8.0% (SEA) and 10.9-15.6% (Sm25). Ov16 seropositivity was low and supported the localities' classification as nonendemic. The results suggested LF exposure, but discordance between antigens, challenges defining seropositivity thresholds, and the absence of programmatic guidance based on antibody serology alone for Wuchereria bancrofti indicate a need for remapping surveys to confirm transmission. Schistosomiasis antibody levels were high enough to warrant further mapping to guide treatment decisions. The lack of gold standards limited interpretation of results, particularly for LF, but in resource-challenged areas, integrated serological surveillance offers the possibility of efficient monitoring of exposure to multiple diseases.

Interlaboratory Harmonization Study and Prospective Evaluation of the PURE–Trypanosoma cruzi–Loop-Mediated Isothermal Amplification Assay for Detecting Parasite DNA in Newborn's Dried Blood Spots

Timely diagnosis of vertical Trypanosoma cruzi infections involves microscopy-based detection of circulating parasites from peripheral blood, which lacks sensitivity and is operator dependent. Consequently, most children born to T. cruzi-infected mothers are required to undergo serological testing after nine months, which risks loss to follow-up. Alternatively, the Loop-mediated isothermal amplification (LAMP) test for T. cruzi DNA offers high analytical and clinical performance and easy to use in low-complexity laboratories. Recently, we optimized this technique using an ultra-rapid DNA extraction method combined with the LAMP in dried blood spots (DBS) on FTA cards. The procedure has been implemented in ten public maternities across Paraguay, Bolivia, and Argentina, involving the training of 14 technicians. Operators' performance was evaluated using a standardized DBS testing panel for harmonization, including negative controls and DBS samples artificially contaminated with T. cruzi at 50 and 20 cells/mL. There was strong agreement (ĸ = 0.924) for controls and 50 cells/mL samples, and good agreement (ĸ = 0.718) across all testing panels, even at the detection limit of the test. A prospective study collected 306 DBS from 222 newborns at birth and/or two months, detecting T. cruzi microscopically in four cases. LAMP identified eight positive cases and perfectly aligned with Real Time PCR (ĸ = 1), demonstrating higher sensitivity than microscopic observation for early detection of infection in infants.

Application of a Novel UPLC-MS/MS Method for Analysis of Rivaroxaban Concentrations in Dried Blood Spot and Plasma Samples Collected from Patients with Venous Thrombosis.

Despite a higher safety profile compared to vitamin K antagonists, rivaroxaban therapy is still connected with multiple adverse effects, such as a high risk of bleeding. Thus, therapeutic drug monitoring (TDM) of rivaroxaban concentrations is suggested. An alternative to plasma samples can be dried blood spots (DBS), which minimize the cost of sample storage and transport. In this study, we developed a UPLC-MS/MS method for the analysis of rivaroxaban in DBS and plasma samples. Chromatographic separation was achieved on a Zorbax Eclipse Plus C18 column (2.1 × 100 mm; 3.5 µm, Agilent Technologies Inc., Santa Clara, CA, USA) with a mobile phase consisting of water and acetonitrile, both containing 0.1% formic acid. The analytes were detected using a positive ionization mode by multiple reaction monitoring. We validated the method according to ICH guidelines. The precision and accuracy were satisfactory. Extraction recovery was approximately 57% and 66% for DBS and plasma samples, respectively. A high correlation between rivaroxaban concentrations in plasma and DBS samples collected from patients was confirmed with Deming regression. The suitability of both sampling techniques for the rivaroxaban TDM was also verified by Bland-Altman plots based on DBS-predicted and observed plasma concentrations. In addition, we found a significant relationship between rivaroxaban concentrations and coagulation parameters, including prothrombin time (PT) and international normalized ratio (INR).

Clinical, Biochemical, and Molecular Characteristics of Filipino Patients with Tyrosinemia Type 1.

Hereditary tyrosinemia type I (HT1), or hepatorenal tyrosinemia, is an amino acid disorder which may cause hepatic failure as well as renal and neurologic comorbidities. Early detection of this disorder is possible with newborn screening (NBS). The objective of this study is to describe the clinical, biochemical, and molecular characteristics of Filipino patients diagnosed with HT1 through the expansion of the Philippine NBS program in 2014. There were a total of 16 patients with confirmed HT1 from then until September 2022. Clinical and biochemical data during confirmation and initial evaluation, as well as molecular data, were obtained from the patients' medical records. The cohort included children between the ages of 18 and 54 months at the time of data collection. The mean age at treatment initiation was 26.8 days. The mean succinylacetone level from dried blood spot sampling using tandem mass spectrometry (MS) was 11.1 µmol/L. Biochemical confirmatory tests via plasma amino acid analysis showed mean levels of tyrosine, phenylalanine, and methionine of 506.1 µmol/L, 111.5 µmol/L, and 125.4 µmol/L, respectively. Upon urine organic acid (UOA) analysis, succinylacetone was detected in all except for one patient, who was managed prior to UOA analysis. The most common clinical characteristics were abnormal clotting times (62.5%), elevated alpha fetoprotein (37.5%), anemia (31.3%), and metabolic acidosis (31.3%). The most common genotype was homozygous c.122T>C p.Leu41Pro in 64.3% of patients. The allelic frequency of this pathogenic variant is 71.4%. The inclusion of HT1 in the Philippine NBS program allowed early diagnosis and management of HT1 patients.