DBS Samples Research Articles

Mitochondrial HMG-CoA synthase deficiency.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) deficiency is a rare, potentially life-threatening autosomal recessive disorder resulting from mutations in the HMGCS2 gene, leading to impaired ketogenesis. We systematically reviewed the clinical presentations, biochemical and genetic abnormalities in 93 reported cases and 2 new patients diagnosed based on biochemical findings. Reported onset ages ranged from 3months to 6years, mostly before the age of 3. Children younger than one year old are more prone to a severe clinical course. In most patients, the initial metabolic decompensation occurs after an episode of gastroenteritis or gastroenteritis-like symptoms. Other commonly observed symptoms during the first clinical episode included poor intake, altered consciousness, dyspnea, seizures and hepatomegaly. Severity was correlated with the number of truncating mutations. Most patients presented with acute metabolic decompensation with hypoglycemia, dicarboxyluria and inadequate ketonuria. Dicarboxylic acid levels were elevated in 54/56 cases. The organic acid 4-hydroxy-6-methyl-2-pyrone (4HMP) was detected in 33/35 urine samples taken during the acute episodes, but typically only retrospectively. The plasma C2/C0 acylcarnitine ratio was abnormal in 16/18 (88.9%) of acute plasma samples, but only in 2/6 (33%) of DBS samples. Other metabolites that have been reported are hydroxyhexenoic acid, 3,5-dihydroxyhexanoic (1,5 lactone), glutaric acidand 3-OH-isovaleric acid. Laboratories should look for 4HMP in urinary organic acid analysis and an increased plasma C2/C0 acylcarnitine ratio to facilitate the diagnosis of HMGCS2 deficiency, especially in cases of metabolic decompensation with dicarboxyluria without adequate ketonuria.

Monitoring SARS-CoV-2 IgA, IgM and IgG antibodies in dried blood and saliva samples using antibody proximity extension assays (AbPEA)

Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from ø1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

Advancing towards practice: A novel LC-MS/MS method for detecting retinol in dried blood spots

BackgroundTo date, clinical laboratories face challenges in quantifying retinol from DBS samples. Disputes arise throughout the whole detection process, encompassing the storage condition, the release strategy as well as the selection of internal standards. MethodsWe incubated DBS with ascorbic acid solution. Then, retinol-d4 in acetonitrile was introduced to incorporate isotopic internal standard and promote protein precipitation. Afterward, sodium carbonate solution was added to ionize cytochromes (such as bilirubin), which amplified the difference of their hydrophobicity to retinol. Subsequently, cold-induced phase separation could be facilitated to separate retinol from the impurities. In the end, the upper layer was injected for LC-MS/MS analysis. ResultsBy comparing the detected retinol content in whole blood and DBS samples prepared from the same volume, we confirmed the established pretreatment was capable to extract most of retinol from DBS (recovery >90 %). Thereafter, we verified that within DBS, retinol possessed satisfying stability without antioxidation. Indoor-light exposure and storage duration would not cause obvious degradation (<10 %). Following systematic validation, the established method well met the criteria outlined in the relevant guidelines. After comparing with detected DBS results to the paired plasma samples, 54 out of 60 met the acceptance limit for cross-validation of ±20 %. ConclusionsWe realized precise quantification of retinol from one 3.2 mm DBS disc. By circumventing conventional antioxidation, liquid-liquid/solid-phase extraction and organic solvent evaporation, the pretreatment could be completed within 15 min consuming only minimal amounts of low-toxicity chemicals (ascorbic acid, acetonitrile, and sodium carbonate). We expect this contribution holds the potential to significantly facilitate the evaluation of patients' vitamin A status by using DBS samples in the future.

Maternal and neonatal IgG against Klebsiella pneumoniae are associated with broad protection from neonatal sepsis: a case-control study of hospitalized neonates in Botswana.

Sepsis is the leading postnatal cause of neonatal mortality worldwide. Globally Klebsiella pneumoniae is the leading cause of sepsis in hospitalized neonates. This study reports development and evaluation of ELISA for anti-Klebsiella IgG using dried blood spot samples and evaluates the association of anti-Klebsiella IgG (anti-Kleb IgG) antibodies in maternal and neonatal samples and the risk of neonatal sepsis. Neonates and their mothers were enrolled at 0-96 hours of life in the neonatal unit of a tertiary referral hospital in Gaborone, Botswana and followed until death or discharge to assess for episodes of blood culture-confirmed neonatal sepsis. Neonates with sepsis had significantly lower levels of Kleb-IgG compared to neonates who did not develop sepsis (Mann-Whitney U, p=0.012). Similarly, samples from mothers of neonates who developed sepsis tended to have less Kleb-IgG compared to mothers of controls (p=0.06). The inverse correlation between Kleb-IgG levels and all-cause bacteremia suggests that maternal Kleb-IgG is broadly protective through cross-reactivity with common bacterial epitopes. These data support the continued use of immunoglobulin assays using DBS samples to explore the role of passive immunity on neonatal sepsis risk and reaffirm the critical need for research supporting the development of maternal vaccines for neonatal sepsis.

Youth who acquired HIV perinatally have poorer viral suppression than those who acquired HIV later in life: findings from a population survey in Zimbabwe

Background: Perinatally acquired HIV may lead to worse health outcomes compared to later acquisition. We compared the demographic and clinical characteristics of youth diagnosed with HIV in childhood and adulthood, as a proxy for acquisition route (perinatal vs horizontal). Setting: Youth aged 18-24 years in 3 provinces in Zimbabwe Methods: In a representative population-based survey, participants were asked their HIV status, date of HIV diagnosis if positive, and whether they were diagnosed in childhood. A dried blood spot was taken to measure viral load. Multilevel mixed-effects generalized linear modelling was used to estimate the association between HIV acquisition time and viral non-suppression (≥1000 copies/ml). Results: 17,682 participants (60.8% female) were enrolled, 17553 (99.3%) gave a DBS sample, 1200 (6.8%) tested HIV antibody positive (7 indeterminate results) and 26 reported being HIV positive without confirmation. Of the 1226 participants living with HIV, 435 (35.5%) self-reported they were HIV-positive, of whom 196 (45.1%) were diagnosed in childhood (median age 7 years). A higher proportion of adult-diagnosed than child-diagnosed participants were female (91.2% vs 76.5%), had ever had sex (93.3% vs 61.5%), been married/cohabiting (59.4% vs 19.4%) and been pregnant (78.9% of women vs 40.0%). A lower proportion had viral suppression (39.3% vs 52.5%). Adjusting for sex, age, marital status and education, those diagnosed as children had higher odds of viral non-suppression (adjusted odds ratio=1.83, 95%CI 1.17-2.85, p=0.008). Conclusion: Youth who acquired HIV perinatally have differentiated care needs and greater risk of viral non-suppression compared to those who acquired HIV later.

Albendazole metabolites excretion in human saliva as a biomarker to assess treatment compliance in mass drug administration (MDA) anthelmintic programs.

Soil-transmitted-helminth (STH) infections continue to be a persistent global public health problem. Control strategies for STH have been based on the use of mass drug administration (MDA). Coverage and compliance assessment is critical to understanding the trueeffectiveness of albendazole(ABZ) in those MDA programs.The aims of this work wereto characterize the pattern ofalbendazole and metabolites excretion in human saliva, and to develop a saliva-based biomarker (HPLC drug/metabolite detection)useful to accurately estimate the coverage/compliance in MDA campaigns.The study subjects were 12 healthy volunteers treated with a single oral dose of ABZ (400 mg). Saliva and blood (dried blood spot, DBS) samples were taken previously and between 2 and 72 h post-treatment. The samples were analyzed by HPLC with UV detection, C18 reversed-phase column. ABZ sulphoxide was the main analyte recovered up to 72 h p.t. in blood and saliva. The concentration profiles measured in the blood (DBS samples) were higher (P < 0.05) than those in saliva, however,this ABZ-metabolite was recovered longer in saliva.Thein vivomeasurement of drugs/metabolites in saliva samples from ABZ-treated volunteers offers strong scientific evidence to support the use of saliva as a valid biological sample for assessing compliance in MDA programs.

Testing, diagnosis, and treatment following the implementation of a program to provide dried blood spot testing for HIV and hepatitis C infections: the NSW DBS Pilot

BackgroundDried blood spot (DBS) testing provides an alternative to phlebotomy and addresses barriers to accessing healthcare experienced by some key populations. Large-scale evaluations of DBS testing programs are needed to understand their feasibility. This study evaluated the implementation of a state-wide DBS HIV and hepatitis C virus (HCV) testing pilot.MethodsThe New South Wales (NSW) DBS Pilot is an interventional cohort study of people testing for HIV antibody and/or HCV RNA from DBS samples in NSW, Australia. Participants at risk of HIV/HCV participated in testing via: 1) self-registration online with a DBS collection kit delivered and returned by conventional postal service; or 2) assisted DBS sample collection at 36 community health sites (including drug treatment and harm-minimisation services) and prisons. Participants received results by text (HIV antibody/ HCV RNA not detected) or a healthcare provider (HIV antibody/ HCV RNA detected). The RE-AIM framework was used to evaluate reach, effectiveness, adoption, and implementation.ResultsReach: Between November 2016 and December 2020, 7,392 individuals were tested for HIV and/or HCV (21% self-registration, 34% assisted in community, and 45% assisted in prison). Effectiveness: Of 6,922 people tested for HIV (19% men who have sex with men, 13% living outside major cities, 21% born outside Australia), 51% (3,521/6,922) had no HIV test in the past two years, 0.1% (10/6,922) were newly diagnosed with HIV, and 80% (8/10) initiated HIV treatment within six months. Of 5,960 people tested for HCV (24% women, 35% Aboriginal and/or Torres Strait Islander, 55% recently injected drugs), 15% had detectable HCV RNA (878/5,960), and 45% (393/878) initiated treatment within six months. Adoption: By the end of 2020, DBS via assisted registration was available at 36 community sites and 21 prisons. Implementation: 90% of DBS cards arriving at the laboratory had the three full spots required for testing; the proportion was higher in assisted (94%) compared to online (76%) registration.ConclusionsThis study demonstrated the feasibility of DBS testing for HIV and HCV in key populations including Aboriginal and Torres Strait Islander peoples, men who have sex with men, people who inject drugs, and demonstrated the utility of DBS in the prison setting.

Comprehensive characterization of protein modifications using mass spectrometry and dry blood spots.

The main objective of this study is to characterize and analyze modified peptides in DBS samples. This includes deciphering their specific PTMs and understanding their potential impact on the population or disease cohort under study. Using mass spectrometry-based proteomic approaches, we performed a comprehensive analysis of DBS samples. Our focus was on the identification and quantification of modified peptides. We also took advantage of recent advances in DBS mass spectrometry to ensure accurate detection and quantification. A comprehensive analysis identified 972 modified peptides in DBS samples. Of these, a subset of 211 peptides was consistently present in all samples, highlighting their potential biological importance and relevance. This indicates a diverse spectrum of PTMs in the proteome of DBS samples. Integration of mass spectrometry and proteomics has revealed a broad spectrum of modified peptides in DBS samples and highlighted their importance in biological processes and disease progression. Accurate detection of these PTMs may be critical for risk stratification and disease management. This study improves the understanding of molecular mechanisms underlying biological processes and disease development, providing important insights for clinical applications.

Genetic diversity of hepatitis B virus quasispecies in different biological compartments reveals distinct genotypes

The selection pressure imposed by the host immune system impacts hepatitis B virus (HBV) quasispecies variability. This study evaluates HBV genetic diversity in different biological fluids. Twenty paired serum, oral fluid, and DBS samples from chronic HBV carriers were analyzed using both Sanger and next generation sequencing (NGS). The mean HBV viral load in serum was 5.19 ± 4.3 log IU/mL (median 5.29, IQR 3.01–7.93). Genotype distribution was: HBV/A1 55% (11/20), A2 15% (3/20), D3 10% (2/20), F2 15% (3/20), and F4 5% (1/20). Genotype agreement between serum and oral fluid was 100% (genetic distances 0.0–0.006), while that between serum and DBS was 80% (genetic distances 0.0–0.115). Two individuals presented discordant genotypes in serum and DBS. Minor population analysis revealed a mixed population. All samples displayed mutations in polymerase and/or surface genes. Major population analysis of the polymerase pointed to positions H122 and M129 as the most polymorphic (≥ 75% variability), followed by V163 (55%) and I253 (50%). Neither Sanger nor NGS detected any antiviral primary resistance mutations in the major populations. Minor population analysis, however, demonstrated the rtM204I resistance mutation in all individuals, ranging from 2.8 to 7.5% in serum, 2.5 to 6.3% in oral fluid, and 3.6 to 7.2% in DBS. This study demonstrated that different fluids can be used to assess HBV diversity, nonetheless, genotypic differences according to biological compartments can be observed.

The impact of neonatal 17-hydroxyprogesterone cutoff determination in a public newborn screening program for congenital adrenal hyperplasia in Southern Brazil: 3 years' experience.

Congenital adrenal hyperplasia (CAH) occurs due to enzyme defects in adrenal steroidogenesis. The 21-hydroxylase deficiency accounts for 90-95% of cases, triggering accumulation of 17-hydroxyprogesterone (17-OHP). Early diagnosis through neonatal screening allows adequate treatment and reduced mortality. The purpose of the study was to determine 17-OHP cutoffs for the diagnosis of CAH in a public newborn screening program in Southern Brazil. A retrospective, descriptive, cross-sectional study was conducted to analyze 17-OHP levels in dried blood samples collected on filter paper of 317,745 newborns screened at a public newborn screening center from May 2014 to April 2017. Neonatal 17-OHP was measured in DBS samples using a time-resolved fluoroimmunoassay (GSP® kit 3305-0010; PerkinElmer). Different cutoffs were determined and stratified by birth weight. The incidence of CAH was 1:15,887 live births in the state of Rio Grande do Sul, with 20 cases of classical CAH diagnosed during the study period. Most newborns (80.73%) were white, and the prematurity rate was 9.8% in the study population. The combination of different percentiles, 98.5th for birth weight 2001-2500 g and 99.8th for the other birth weight groups, decreased false-positive results and increased specificity compared with current reference values to identify classical CAH cases. The local 17-OHP cutoffs determined were higher than those currently used by this screening program for all birth weight groups. The calculation of reference values from local population data and the combination of percentiles proved to be a valuable tool for proper diagnosis of CAH and reduction in the number of false positives.

Development and validation of a liquid chromatographic-tandem mass spectrometric assay for the quantification of cabotegravir and rilpivirine from dried blood spots

BackgroundDried blood spots (DBS) have been utilized as a blood plasma alternative for therapeutic drug monitoring and pharmacologic analysis. There are analytical and physiochemical considerations in bridging drug concentrations from plasma to DBS. Recently, the long-acting antiretroviral cabotegravir (CAB) has been approved for HIV prevention, and a co-packaged regimen of long-acting CAB and rilpivirine (RPV) has been approved for HIV treatment. Measurement of these drugs in blood collected as DBS may offer increased capacity and flexibility in translational applications. MethodsWhole blood was spiked with CAB and RPV and spotted on DBS cards. Following extraction and addition of isotopically labeled internal standards, samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. The method was validated according to regulatory recommendations, and the assay was evaluated in remnant samples from an HIV prevention trial in which paired DBS and plasma samples were collected. ResultsDBS CAB and RPV concentrations were linear from 25 to 20,000 ng/mL and 2–2500 ng/mL, respectively. Precision, accuracy, and matrix effect results were acceptable. DBS RPV demonstrated stability under all tested conditions; DBS CAB showed mean biases of − 23.5% when stored at room temperature for 36 days, and − 18.0% at 40 °C and 100% humidity for two days. DBS measurements for CAB and RPV were an average 54.0% and 14.1% lower, respectively, as compared to paired plasma samples. Derived conversion factors of 1.79 and 1.16 were applied to DBS CAB and RPV measurements, respectively, to estimate plasma concentrations. Estimated plasma CAB and RPV concentrations showed mean biases of 2.2% and 0.6%, respectively. In a CAB clinical trial, application of the conversion factor resulted in agreement between estimated plasma CAB concentrations from DBS and plasma CAB concentrations (y = 1.08x - 79.2, r = 0.932; mean bias of −3.2%; 95% CI: −48.2% to 41.9%). ConclusionsWe developed and validated a novel LC-MS/MS assay for the quantification of CAB and RPV from DBS, and identified conversion factors to estimate plasma concentrations from spotted blood.

1884. Evaluating SARS-CoV-2 Surveillance Strategies at the United States Naval Academy: A Comparison of Saliva and Dried Blood Spot Serosurveillance Against Molecular-Confirmed Case Detection

Abstract Background Congregate military populations remain at risk of SARS-CoV-2 outbreaks and the optimal surveillance approach in such settings remains unclear. We enrolled midshipmen at the United States Naval Academy (USNA) in a setting of frequent PCR screening use of prevention strategies. Methods Dried blood spots (DBS) and saliva were collected in August 2020, December 2020, February 2021 (saliva only) and April/May 2021 to measure anti-SARS-CoV-2 spike (S) and nucleoprotein (NP) IgG. COVID-19 vaccine history and records of SARS-CoV-2 PCR tests and routine asymptomatic screening assays were obtained from the USNA Brigade Medical Clinic. Attack rates were compared with cumulative frequencies of infections. Concordance of saliva and DBS anti-NP and anti-S IgG positivity was determined using Cohen’s kappa coefficient. Results The study enrolled 181 midshipmen. COVID-19 vaccinations were administered in March/April 2021. Samples were collected for 101 participants in August, 73 in December, 57 in February (saliva only), and 63 in April/May. In August, 17 (17%) participants showed evidence of SARS-CoV-2 infection based on anti-S IgG values from DBS and/or saliva. By December 2020, anti-S seroconversion was observed for 5 more based on DBS and/or saliva. By May 2021, 100% of participants were anti-S IgG seropositive after vaccination based on DBS and/or saliva; 48% of participants had seroconverted to anti-NP IgG. Among participants with both DBS and saliva samples, a coefficient of 0.64 showed substantial agreement between anti-S IgG results in August and perfect agreement in December (Table 1). DBS and saliva results for anti-NP IgG were in perfect agreement through December and in substantial agreement in May (0.68, Table 2). Prior to vaccination in March/April 2021, 4/48 of participants had at least one documented SARS-CoV-2 PCR positive result (Table 3). Cumulative PCR test positivity concordance with DBS seroconversion was 37.5% and 60% for anti-S IgG and anti-NP IgG, respectively. Conclusion There was a substantive SARS-CoV-2 attack rate before vaccination; all vaccinees mounted an anti-S IgG response in blood. We note high agreement between DBS and saliva for IgG measurement. Serology-based surveillance identified substantially more SARS-CoV-2 infections than PCR screening. Disclosures Jitendrakumar Modi, MD, GlaxoSmithKline: I am a paid speaker for GSK. I do not speak for their flu brand. Timothy H. Burgess, MD, MPH, AstraZeneca: The HJF, in support of the USU IDCRP, was funded to conduct or augment unrelated Phase III Mab and vaccine trials as part of US Govt. COVID19 response Simon Pollett, MBBS, Astra Zeneca: The HJF, in support of the USU IDCRP, was funded to conduct or augment unrelated Phase III Mab and vaccine trials as part of US Govt. COVID19 response Mark P. Simons, PhD, AstraZeneca: The HJF, in support of the USU IDCRP, was funded to conduct or augment unrelated Phase III Mab and vaccine trials as part of US Govt. COVID19 response.

Opportunistic Community Screening of Chronic Chagas Disease Using a Rapid Diagnosis Test in Pharmacies in Barcelona (Catalonia, Spain): Study Protocol and Pilot Phase Results.

Objectives: This study aimed to report the protocol and results from the pilot phase of an opportunistic CP-based CD screening program in Barcelona, Spain. Methods: Three strategies according to recruitment approach were designed: passive, active and active-community. The study process consisted of signing the informed consent form, recording the patient's data in a web-based database system, and performing the rapid test and blood collection on dry paper. Results: Nineteen pharmacies participated and 64 patients were included during the pilot phase of the study. The rapid diagnostic test (RDT) was positive in 2/64 (3.13%) cases. Of the 49 DBS samples that arrived at the laboratory, 22 (45%) were collected incorrectly. After quantitative and qualitative assessment of the program, the dry paper sample and passive strategy were ruled out. Conclusion: DBS sampling and the passive strategy are not suitable for CD screening in community pharmacies. There is a need to expand the number of participating pharmacies and individuals to determine whether conducting a RDT in community pharmacies is an effective screening method to increase access to CD diagnosis in a non-endemic area.

Loss to follow-up of HIV-exposed infants for confirmatory HIV test under Early Infant Diagnosis program in India: analysis of national-level data from reference laboratories

BackgroundEarly Infant Diagnosis was launched in India in 2010 and its effect on the diagnosis of HIV-exposed infants needs to be assessed. The present study was done to find out the median age at DBS sample collection for early infant diagnosis and its trend over years, the median age at diagnosis of HIV among the HIV-exposed infants with DNA PCR tests, and the proportion of infants who completed testing cascades after detection of HIV-1 in a sample.MethodsDNA PCR data (from 2013 to 2017) maintained at all regional reference laboratories in India was collated with each infant identified by a unique code. Cohort analysis of the infant data was used to find the median age at sample collection and diagnosis. The outcomes of testing in each cascade and the overall outcomes of testing for infants were prepared.ResultsThe median age at sample collection for the four years combined at all India level was 60 days (48–110 days). The median age at diagnosis of HIV was 285 days (174–418 days). HIV-1 was detected in samples of 1897 (6.3%) infants out of 30,216 infants who had a DNA PCR test, out of whom 1070 (56.4%) completed the testing cascade and the rest were lost to follow-up.ConclusionThe data highlights delay in diagnosis; both due to delay in sample collection and turn-around-times. Loss to follow-up of HIV-exposed infants with virus detection is a significant concern to the Early Infant Diagnosis and tracking systems need to be strengthened.

Fully automated dried blood spot sample handling and extraction for BoHV-1 antibody testing by ELISA

This study is the first proof of concept of the DBS technology for Bovine alphaherpesvirus 1 (BoHV-1) antibody detection by ELISA after fully automated DBS extraction. DBS were prepared from nine BoHV-1 seropositive plasma samples spiked with erythrocytes. Spots were extracted automatically on a DBS-MS 500 HCT autosampler, as well as manually using a 3.2 mm puncher. DBS were equally prepared from 20 bovine seronegative EDTA-blood samples and extracted automatically. Extracts were tested in a commercial BoHV-1 antibody ELISA and results were compared with those from liquid plasma. Eight seropositive DBS samples were additionally tested in the ELISA after storage for four weeks at different conditions. After automated extraction all DBS samples yielded qualitatively correct results and were in full accordance with those obtained from liquid plasma. Automated extraction using a 6 mm extraction head was more sensitive than a 4 mm head. Stability of DBS was highest at − 20 °C and decreased with increasing temperature. Even after four weeks at 37 °C, most seropositive samples yielded a positive result in the ELISA. The minimal invasiveness, biosafety, and simplicity of DBS collection together with automated extraction represents an interesting, high-throughput compatible alternative to liquid blood samples for BoHV-1 monitoring or eradication programs.

Optimization of DBS-self sampling for PEth in a drinking experiment with standard DBS cards and with a new design of volumetric DBS card

Due to insufficient sample quality when using capillary-blood self-sampling in a drinking experiment for phosphatidyl ethanol (PEth) determination, we implemented an additional quality criterion for DBS and tested a new paper-based DBS card. We finally present results of PEth concentrations in capillary blood in a drinking study with blood alcohol concentrations (BAC) of up to 0.75 g/kg. We recently reported on a drinking experiment with BACs up to 0.5 g/kg, after long-term abstinence, where PEth was not detectable in any DBS sample with a cut-off of 20 ng/mL. Therefore, we adjusted the target BAC in further drinking experiments up to 0.75 g/kg. DBS sampling has been described as “easy”. When using commercial DBS cards for self-sampling with an imprinted circle, we found that some subjects had problems placing a full-sized droplet on the DBS card in an appropriate way. Instead, there some touched the card surface with the finger once a small drop had formed on the fingertip, and filled the printed circle on the card by repeatedly touching the card's surface. Thus, the blood is not sucked vertically through the entire card, which results in the sample not being quantitatively evaluable. We have introduced an important quality criterion for evaluable DBS cards: results can only be valid if the blood-soaked area on the front and on the back of the DBS-card is sufficient for extraction. This can be seen by the size of the extraction circle imprinted by the autosampler extraction tool (CAMAG, using a 4 mm diameter) after extraction on both sides. In practice, this means that the blood-penetrated circular area on the front and on the back of the card must have a minimum diameter of 6 mm if an extraction tool with 4 mm diameter is used. In order to optimise DBS self-collection, a newly designed card with a paper comb with four teeth per card (DBSv, Protzek, Lörrach, Germany) was evaluated – in parallel to the use of standard DBS cards. A preanalytical criterion for the validity of DBS self-sampling by a standard filter-paper DBS card (without the use of a volumetric device) was defined – with respect to full penetration of the DBS-card from the front to the back by the collected blood. In the drinking experiment, PEth was found positive (above the cut-off of 20 ng/mL) after a single drinking event with target BACs of up to 0.75 g/kg. PEth concentrations reached up to 119 ng/mL PEth 16:0/18:1 (starting from PEth negative before start of drinking) and declined to below 40 ng/mL within five days. Although four weeks of abstinence from ethanol were required prior to the first drinking event, half of the volunteers still had positive PEth values (above 20 ng/mL) at the time of the drinking event and had to be eliminated from the study. Furthermore, the new DBSv card (Protzek, Lörrach, Germany) allowed a volumetric collection of approx. 20 μL blood per tooth, circumventing the problem, that not enough blood is collected per spot. We could assure the quality of DBS self-sampling by an additional quality requirement as well as by new volumetric DBSv cards. In the drinking experiment, PEth-concentrations above 20 ng/mL were obtained by single drinking up to a BAC of 0.75 g/kg.

Validation and clinical application of a method to quantify efavirenz in cervicovaginal secretions from flocked swabs using liquid chromatography tandem mass spectrometry

Background : A liquid chromatography tandem mass spectrometry method to quantify drugs in dried cervicovaginal secretions from flocked swabs was developed and validated using the antiretroviral efavirenz as an example. Methods: Cervicovaginal swabs (CVS) were prepared by submerging flocked swabs in efavirenz-spiked plasma matrix. Time to full saturation, weight uniformity, recovery and room temperature stability were evaluated. Chromatographic separation was on a reverse-phase C18 column by gradient elution using 1mM ammonium acetate in water/acetonitrile at 400 µL/min. Detection and quantification were on a TSQ Quantum Access triple quadrupole mass spectrometer operated in negative ionisation mode. The method was used to quantify efavirenz in CVS samples from human immunodeficiency virus (HIV)-positive women in the VADICT study (NCT03284645). A total of 98 samples (35 paired intensive CVS and DBS pharmacokinetic samples, 14 paired sparse CVS and DBS samples) from 19 participants were available for this analysis. Results: Swabs were fully saturated within 15 seconds, absorbing 128 µL of plasma matrix with coefficient of variation (%CV) below 1.3%. The method was linear with a weighting factor (1/X) in the range of 25-10000 ng/mL with inter- and intra-day precision (% CV) of 7.69-14.9%, and accuracy (% bias) of 99.1-105.3%. Mean recovery of efavirenz from CVS was 83.8% (%CV, 11.2) with no significant matrix effect. Efavirenz remained stable in swabs for at least 35 days after drying and storage at room temperature. Median (range) CVS efavirenz AUC 0-24h was 16370 ng*h/mL (5803-22088), C max was 1618 ng/mL (610-2438) at a T max of 8.0 h (8.0-12), and C min was 399 ng/mL (110-981). Efavirenz CVS:plasma AUC 0-24h ratio was 0.41 (0.20-0.59). Conclusions: Further application of this method will improve our understanding of the pharmacology of other therapeutics in the female genital tract, including in low- and middle-income countries.

Validation and clinical application of a method to quantify efavirenz in cervicovaginal secretions from flocked swabs using liquid chromatography tandem mass spectrometry.

Background : A liquid chromatography tandem mass spectrometry method to quantify drugs in dried cervicovaginal secretions from flocked swabs was developed and validated using the antiretroviral efavirenz as an example. Methods: Cervicovaginal swabs (CVS) were prepared by submerging flocked swabs in efavirenz-spiked plasma matrix. Time to full saturation, weight uniformity, recovery and room temperature stability were evaluated. Chromatographic separation was on a reverse-phase C18 column by gradient elution using 1mM ammonium acetate in water/acetonitrile at 400 µL/min. Detection and quantification were on a TSQ Quantum Access triple quadrupole mass spectrometer operated in negative ionisation mode. The method was used to quantify efavirenz in CVS samples from human immunodeficiency virus (HIV)-positive women in the VADICT study (NCT03284645). A total of 98 samples (35 paired intensive CVS and DBS pharmacokinetic samples, 14 paired sparse CVS and DBS samples) from 19 participants were available for this analysis. Results: Swabs were fully saturated within 15 seconds, absorbing 128 µL of plasma matrix with coefficient of variation (%CV) below 1.3%. The method was linear with a weighting factor (1/X) in the range of 25-10000 ng/mL with inter- and intra-day precision (% CV) of 7.69-14.9%, and accuracy (% bias) of 99.1-105.3%. Mean recovery of efavirenz from CVS was 83.8% (%CV, 11.2) with no significant matrix effect. Efavirenz remained stable in swabs for at least 35 days after drying and storage at room temperature. Median (range) CVS efavirenz AUC 0-24h was 16370 ng*h/mL (5803-22088), C max was 1618 ng/mL (610-2438) at a T max of 8.0 h (8.0-12), and C min was 399 ng/mL (110-981). Efavirenz CVS:plasma AUC 0-24h ratio was 0.41 (0.20-0.59). Conclusions: Further application of this method will improve our understanding of the pharmacology of other therapeutics in the female genital tract, including in low- and middle-income countries.

Optimizing the Phenylalanine Cut-Off Value in a Newborn Screening Program.

Phenylketonuria (PKU) was the first disorder for which newborn screening (NBS) was introduced in the early 1960s. Slovenia started the NBS program for PKU in 1979, and the fluorimetric method was implemented in 1992, with a phenylalanine (Phe) cut-off set at 120 mol/L. This value has been in use for almost thirty years and has never been revised. We aimed to analyze the DBS samples and review the data from a large nationwide cohort of newborns to optimize the cut-off values for HFA screening to minimize the number of false positives while maintaining the highest level of sensitivity by detecting all those who needed to be treated. In the first prospective part of the study, we analyzed samples of all newborns in Slovenia in 2019 and 2020, and in the second retrospective part, we reviewed data from all known patients with hyperphenylalaninemia (HFA) in Slovenia born from 2000 to 2018. We defined true screening-positive cases as those that required a low-Phe diet. The sensitivity, specificity and positive predictive values of the modeling elevation of the Phe cut-off value from 120 µmol/L to 200 µmol/L were assessed. The number of recalls at the cut-off of 120 µmol/L was 108 out of 37,784 samples at NBS (2019–2020). Six newborns were defined as true positives and 102 samples as false positives. If the cut-off value was adjusted to 160 µmol/L, only 12 samples exceeded it and all six true positive newborns would be detected. Among the 360,000 samples collected at the NBS between 2000 and 2018, 72 HFA patients in need of a low-Phe diet were found. All the diagnosed cases would have been detected if the cut-off was set to 160 µmol/L. We demonstrated in a large group of newborns (400,000 in 20 years) that using the fluorimetric approach, a cut-off value of 160 µmol/L, rather than 120 mol/L, is safe and that there were no missing true positive patients who required treatment. By increasing the cut-off, this method becomes more precise, resulting in a significantly reduced rate of false positives and thus being less burdensome on both families and the healthcare system.

Qualitative and quantitative detection of SARS-CoV-2 antibodies from dried blood spots

IntroductionDried blood spot (DBS) sampling is a minimally invasive method for specimen collection with potential multifaceted uses, particularly for serosurveillance of previous SARS-CoV-2 infection. In this study, we assessed DBS as a potential specimen type for assessing IgG and total (including IgG and IgM) antibodies to SARS-CoV-2 in vaccinated and naturally infected patients. MethodsSix candidate buffers were assessed for eluting blood from DBS cards. The study utilized one hundred and five paired plasma specimens and DBS specimens from prospectively collected SARS-CoV-2 vaccinated individuals, remnants from those with PCR confirmed SARS-CoV-2 infections, or remnants from those without history of infection or vaccination. All specimens were tested with the Siemens SARS-CoV-2 total assay (COV2T) or IgG assay (sCOVG). ResultsThe lowest backgrounds were observed with water and PBS, and water was used for elution. Relative to plasma samples, DBS samples had a positive percent agreement (PPA) of 94.4% (95% CI: 94.9–100%) for COV2T and 79.2 (68.4–87.0) for sCOVG using the manufacturer’s cutoff. The NPA was 100 % (87.1–100.0 and 85.13–100) for both assays. Dilution studies revealed 100% (95% CI: 90.8–100%) qualitative agreement between specimen types on the COV2T assay and 98.0% (88.0–99.9%) with the sCOVG using study defined cutoffs. ConclusionDBS specimens demonstrated high PPA and NPA relative to plasma for SARS-CoV-2 serological testing. Our data support feasibility of DBS sampling for SARS-CoV-2 serological testing.