Blood Spot Assay Research Articles

Development of sensitive, specific, point of care enzyme-linked immunosorbent assay combined with DBS and hand-held ELISA reader for the rapid detection of hepatitis C virus in resource-limited settings

Background: Hepatitis C virus cause liver infection which can lead to liver cirrhosis and hepatocellular carcinoma. Identification of hepatitis C virus infection initially depends on the antibody screening test. Aim and Objectives: We developed highly specific and sensitive recombinant protein-based HCV ELISA and combined the assay with Dried Blood Spot (DBS) assay and hand-held ELISA reader to establish its use in limited resource areas as well as in field level. Materials and Methods: A total of 610 specimens were collected for assay validation purpose. To check the proficiency of our diagnostic strategy, we compared our test results with standard HCV chemiluminescence enzyme immune assay. The Institutional Ethical Committee approved this study (IEC). NCSS 11 statistical software analysed all the data. The correlation-regression was done to compare the assay. The level of significance of this study was p<0.05. Results: Among 60 known reactive specimens, 59 showed reactive by HCV ELISA and chemiluminescence enzyme assay. Therefore sensitivity for both the assay recorded 98.33%. Out of 150 known non-reactive samples, both the method showed the nonreactive result. Thus specificity calculated 100%. While HCV ELISA tested with DBS, we found no significant difference in specificity and sensitivity of the assay (r2=0.9940). ELISA optical density value obtained by Hand-held ELISA reader showed better performance as compared to Tulip ELISA plate reader (r2=0.9921). While performed field study, out of 400 specimens, 6 showed reactive by both the methods. Therefore specificity and sensitivity was 100% for in-house HCV ELISA as compared to chemiluminescence assay. In this case, also we found a good performance of DBS assay (r2=0.9476). There was a good correlation between O.D. obtained by hand-held ELISA reader and Tulip ELISA plate reader (r2=0.9923). Conclusion: According to our study report, in-house HCV ELISA is highly specific and sensitive method. Addition of DBS assay and hand-held ELISA reader makes it possible to run the assay in limited resource settings as well as in field level or outside the standard laboratory setup. This unique HCV diagnostic strategy can reduce the global burden of HCV infection.

A dried blood spot assay for paclitaxel and its metabolites

After being used for decades in clinical screening, dried blood spots (DBS) have recently received considerable attention for their application in pharmacokinetic and toxicokinetic studies in rodents. The goal of this study was to develop and apply a DBS-based assay for a pharmacokinetic study of paclitaxel (PTX) and its metabolites in SCID/Beige mice. A fast and sensitive UHPLC–MS/MS method has been developed for the simultaneous determination of PTX, its three metabolites (6α-hydroxy-paclitaxel, 3′-p-hydroxy-paclitaxel, and 6α,3′-p-dihydroxy-paclitaxel) and its stereoisomer 7-epi-paclitaxel. The 10μL DBS sample was extracted with methanol for 20min at 37°C. After dilution of the extracts with water in a ratio of 1:1, the analytes were separated on a reversed-phase 2.1mm I.D. column using gradient elution. The total run time was 2.5min. The analytes were detected by use of multiple reaction monitoring mass spectrometry. The extraction recoveries of the compounds were all greater than 60%, resulting in a quantification limit of 1ng/ml. The calibration curves ranged from 1 to 1000ng/ml. The intra-day and inter-day imprecision (%CV) across three validation runs over four quality control levels were less than or equal to 14.6%. The accuracy was within ±11.9% in terms of relative error. The described method is advantageous in terms of its ease-of-use and speed compared to other published PTX assays. The method’s usefulness was demonstrated by applying it to a preclinical pharmacokinetic investigation of PTX and its metabolites in SCID/Beige mice with an intraperitoneal administration of 50mg/kg Abraxane®.

Penicillin Dried Blood Spot Assay for Use in Patients Receiving Intramuscular Benzathine Penicillin G and Other Penicillin Preparations To Prevent Rheumatic Fever.

Rheumatic heart disease (RHD) remains an important global health challenge. Administration of benzathine penicillin (BPG) every 3 to 4 weeks is recommended as a secondary prophylaxis to prevent recurrent episodes of acute rheumatic fever and subsequent RHD. Following intramuscular injection, BPG is hydrolyzed to penicillin G (benzylpenicillin). However, little is known of the pharmacokinetics (PK) of BPG in pediatric populations at high risk of RHD or of the pharmacokinetic-pharmacodynamic relationship between penicillin exposure and clinically relevant outcomes. Dried blood spot (DBS) assays can facilitate PK studies in situations where frequent venous blood sampling is logistically difficult. A liquid chromatography-mass spectroscopy assay for penicillin G in plasma and DBS was developed and validated. Application of the DBS assay for PK studies was confirmed using samples from adult patients receiving penicillin as part of an infection management plan. The limit of quantification for penicillin G in DBS was 0.005 mg/liter. Penicillin G is stable in DBS for approximately 12 h at room temperature (22°C), 6 days at 4°C, and >1 month at -20°C. Plasma and DBS penicillin G concentrations for patients receiving BPG and penicillin G given via bolus doses correlated well and had comparable time-concentration profiles. There was poor correlation for patients receiving penicillin via continuous infusions, perhaps as a result of the presence of residual penicillin in the peripherally inserted central catheter, from which the plasma samples were collected. The present DBS penicillin G assay can be used as a surrogate for plasma concentrations to provide valid PK data for studies of BPG and other penicillin preparations developed to prevent rheumatic fever and RHD.

Dried Blood Spot Thyroglobulin as a Biomarker of Iodine Status in Pregnant Women.

Thyroglobulin (Tg) could be a sensitive biomarker of iodine nutrition in pregnant women (PW). A dried blood spot (DBS) assay would simplify collection and transport in field studies. Our aims were to (1) establish and test a reference range for DBS-Tg in PW; (2) determine whether co-measurement of Tg antibodies (Abs) is necessary to define population iodine status. Standardized cross-sectional studies of 3870 PW from 11 countries. For the DBS-Tg reference range, we included TgAb-negative PW (n = 599) from 3 countries with sufficient iodine intake. We measured the urinary iodine concentration and DBS thyroid-stimulating hormone, total thyroxin, Tg, and TgAb. In the reference population, the median DBS-Tg was 9.2 μg/L (95% confidence interval, 8.7 to 9.8 μg/L) and was not significantly different among trimesters. The reference range was 0.3 to 43.5 μg/L. Over a range of iodine intake, the Tg concentrations were U-shaped. Within countries, the median DBS-Tg and the presence of elevated DBS-Tg did not differ significantly between all PW and PW who were TgAb-negative. A median DBS-Tg of ∼10 μg/L with <3% of values ≥44 μg/L indicated population iodine sufficiency. Concurrent measurement of TgAb did not appear necessary to assess the population iodine status.

Physical activity, sedentary behaviors, and Epstein-Barr virus antibodies in young adults

This study aimed to elucidate the associations between physical activity, sedentary behaviors, and Epstein-Barr virus (EBV) antibody levels (as an indirect marker of cell-mediated immunity, CMI). This study made use of a 14-year longitudinal study with a representative sample of adolescents in the US. A total of 3361 participants (42.1% male) aged 11 to 21years at baseline who completed Wave I (1994–1995), Wave III (2001−2002), and Wave IV (2008) surveys of the National Longitudinal Study of Adolescent Health (Add Health) were analyzed. Physical activity and sedentary behaviors at Waves I and III were assessed using interviewer-administered questionnaire. EBV viral capsid antigen (VCA) IgG antibody levels at Wave IV were analyzed from dried blood spot assays. Adjusted for confounders, among males, one additional day spent per week on strenuous sports at Wave III were associated with a decrease of 4.09AU/ml in EBV antibody levels (p=0.012), while one additional hour spent per week viewing videos at Wave I was associated with an increase of 0.83AU/ml in EBV antibody levels (p=0.026). Among females, one additional day spent per week on individual sports at Wave III were associated with a decrease of 4.63AU/ml in EBV antibody levels (p=0.014), while sedentary behaviors were not associated with EBV antibody levels. To conclude, physical activity and sedentary behaviors were associated with CMI among males and physical activity was associated with CMI among females.

Dried Blood Spot Technique for the Monitoring of Ambrisentan, Bosentan, Sildenafil, and Tadalafil in Patients with Pulmonary Arterial Hypertension.

Endothelin receptor antagonists (ERA) and phosphodiesterase 5 inhibitors (PDE5I) are long-term therapeutics for the treatment of pulmonary arterial hypertension (PAH). Their interindividual pharmacokinetic variability is remarkably large, and despite the seriousness of the disease, nonadherence is occurring. Therefore, methods to monitor sufficient circulating drug levels are essential. The objectives of this study were to develop and validate dried blood spot (DBS) assays for the quantification of ambrisentan, bosentan, sildenafil, tadalafil, and their main metabolites. We also quantified the influence of different hematocrit levels and assessed the correlation of simultaneously taken capillary whole blood (DBS) and venous plasma samples. The aliquot punches were extracted by liquid/liquid extraction followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) quantification methods. All assays fulfilled the requirements of the FDA and EMA guidelines for assay validation with a lower limit of quantification of 2.5 ng/mL for the ERAs, 5 ng/mL for sildenafil, and 10 ng/mL for tadalafil. All analytes were stable for at least 147 days when stored on DBS filter paper cards at room temperature in the dark. Due to poor distribution into erythrocytes, drug concentrations in DBS were always lower than in plasma, resulting in conversion factors of 1.58 for ambrisentan and sildenafil and 1.52 for bosentan and tadalafil.

A simple dried blood spot method for clinical pharmacological analyses of etoposide in cancer patients using liquid chromatography and fluorescence detection

BackgroundTherapeutic drug monitoring of etoposide is not part of the routine clinical practice, however, measuring etoposide plasma concentration may be useful to prevent chemotherapy related adverse drug reactions. This paper describes the development and validation of a dried blood spot (DBS) assay for the determination of etoposide in blood samples of lung cancer patients. MethodsThe whole blood spot was cut out of the DBS card followed by sonication assisted liquid drug extraction. Extraction solution was evaporated and re-dissolved. A high-performance-liquid-chromatography method with fluorimetric detection ( λex=230nm; λem=330nm) was used. ResultsMethod met the validation criteria in terms of selectivity, linearity (0.5–20.0μg/mL), accuracy (≥96.1%), precision (≤10.1%) and stability (long term 4weeks at room temperature and 40°C). Haematocrit did not influence DBS etoposide concentration. Good correlation between measured plasma and DBS concentrations was observed. The equation considering only haematocrit value was used for conversion of DBS to plasma concentration. ConclusionsDBS sampling method showed comparable results to plasma samples. Therefore, it can be concluded that the developed and validated DBS method, which is more patient-friendly and requires less sample handling, is a reliable alternative to conventional plasma methods for measuring etoposide concentration in clinical pharmacological analyses.

Targeted screening for the detection of Pompe disease in patients with unclassified limb-girdle muscular dystrophy or asymptomatic hyperCKemia using dried blood: A Spanish cohort

We aimed to screen for Pompe disease in patients with unclassified limb-girdle muscular dystrophy (LGMD) or asymptomatic hyperCKemia using dried blood spot (DBS) assays. Subsequently, we aimed to calculate the diagnostic delay between initial symptom presentation and the diagnosis. A prospective, multicenter, observational study was conducted in 348 patients: 146 with unclassified LGMD and 202 with asymptomatic or paucisymptomatic hyperCKemia. We quantified levels of acid alpha-glucosidase (GAA) from dried blood spots analyzed fluorometrically. The test was positive in 20 patients, and Pompe disease was confirmed by genetic testing in 16. Undiagnosed Pompe disease was detected in 7.5% of patients with LGMD and in 2.5% of patients with persistent, idiopathic elevation of serum creatine kinase. The c.-32-13 T > G mutation was found most commonly. The diagnostic delay was 15 years on average. In conclusion, DBS tests are useful and reliable screening tools for Pompe disease. We recommend the dried blood spot test to be included in the diagnostic work-up of patients with unclassified myopathies with proximal weakness and/or hyperCKemia of unknown cause and, when positive, to define the diagnosis, it will have to be confirmed by biochemical and/or molecular genetic analysis.

Combining rapid diagnostic tests and dried blood spot assays for point-of-care testing of human immunodeficiency virus, hepatitis B and hepatitis C infections in Burkina Faso, West Africa

AbstractPeople screened for human immunodeficiency virus (HIV) using rapid diagnostic tests (RDTs) in Africa remain generally unaware of their status for hepatitis B (HBV) and hepatitis C (HCV) infections. We evaluated atwo-step screening strategy in Burkina Faso, using both HIV RDTs and Dried Blood Spot (DBS) assays to confirm an HIV-positive test, and to test for HBV and HCV infections. HIV counselling and point-of-care testing were performed at a voluntary counselling and testing centre with HBV, HCV status and HIV confirmation using DBS specimens, being assessed at a central laboratory. Serological testing on plasma was used as the reference standard assay to control for the performance of DBS assays. Nineteen out of 218 participants included in the study were positive for HIV using RDTs. A fourth-generation HIV ELISA and immunoblot assays on DBS confirmed HIV status. Twenty-four out of 25 participants infected with HBV were found positive for hepatitis B surface antigen (HBsAg) using DBS. One sample with a low HBsAg concentration on plasma was not detected on DBS. Five participants tested positive for HCV antibodies were confirmed positive with an immunoblot assay using DBS specimens. Laboratory results were communicated within 7 days to participants with no loss to follow up of participants between the first and second post-test counselling sessions. In conclusion, DBS collection during HIV point-of-care testing enables screening and confirmation of HBV, HCV and HIV infections. Diagnosis using DBS may assist with implementation of national programmes for HBV, HCV and HIV screening and clinical care in middle- to low-income countries.

Development and validation of a dried blood spot assay for the quantification of ribavirin using liquid chromatography coupled to mass spectrometry

Efficient, inexpensive and sensitive assays for the measurement of drugs are of interest for pharmacokinetic and pharmacodynamics (PK-PD) analysis. Dried blood spots (DBS) are a unique bioanaltyical matrix with the potential to fulfill this interest for the measurement of numerous analytes. Here we describe the development and validation of a reversed-phase high performance liquid chromatographic (LC), tandem mass spectrometry (MS/MS) assay for the determination of ribavirin (RBV) in DBS. A 3mm punch from spotted and dried whole blood was extracted in methanol utilizing isotopically labeled internal standard for LC-MS/MS analysis. Validation was performed over a range of 0.05μg/mL to 10.0μg/mL and the method was shown to be precise (coefficient of variation ≤15%) and accurate (within ±15% of control). These acceptance criteria were met for hematocrit ranges of 20-54%, for center versus edge punches and for spot volumes from 10 to 60μL. RBV was stable for up to 140 days at room temperature and -20°C as well as for three freeze/thaw cycles. Correlation of RBV in DBS versus in plasma yielded r(2)≥0.98 demonstrating that DBS can be used as an alternative to plasma for PK-PD studies in human subjects.

Blood spot versus plasma chitotriosidase: A systematic clinical comparison

ObjectivesThis study aimed to evaluate the agreement between blood spot and plasma chitotriosidase using the economic substrate 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside, and to investigate the utility of the blood spot assay for the wide scale screening for lysosomal storage disorders among the clinically suspected. Design and methodsBlinded blood spot samples were compared with the corresponding plasma levels in 199 children (56 with confirmed diagnoses of ten different lysosomal storage disorders, 73 normal controls and 70 pathological controls). Several performance criteria (limit of detection, linearity, within-run and day-to-day precision and sample stability) were also evaluated. ResultsPlasma assay performed better by most criteria; however, blood spot performance was quite satisfactory. Quantitative values of the two methods can't be used interchangeably based on their 95% limits of agreement. Diagnostic sensitivity and specificity derived from ROC curves were 75.0 and 85.3% for the plasma assay and 71.4 and 79.0% for the blood spot assay, respectively. Cohen's kappa was 0.72 (95% CI: 0.616–0.821) denoting a good categorical agreement between the two methods. ConclusionThe clinical use of blood spot chitotriosidase for the screening of lysosomal storage disorders can be quite practical, provided proper cut-off values are determined for each lab.

Evaluation of a Dried Blood Spot Assay to Measure Prenatal Screening Markers Pregnancy-Associated Plasma Protein A and Free β-Subunit of Human Chorionic Gonadotropin

First-trimester prenatal screening for aneuploidy by use of dried blood spots (DBSs) may offer practical benefits in settings where the instability of intact human chorionic gonadotropin (hCG) is problematic. We evaluated a DBS pregnancy-associated plasma protein A (PAPP-A) and free β-subunit of hCG (free hCGβ) dual assay and compared it to serum screening. Hematocrit-corrected DBS PAPP-A and free-hCGβ concentrations were measured and compared with serum concentrations in 252 first-trimester samples. Serum intact hCG was also measured and, with serum free hCGβ, was used to fit a model to predict serum-equivalent DBS free-hCGβ concentrations. In a separate experiment, we investigated the effects of temperature and relative humidity during the blood spot drying process. The DBS assay for PAPP-A performed similarly to the serum assay, whereas free-hCGβ DBS measurements were consistently higher than in serum. Purifying blood spots of intact hCG suggested that the free-hCGβ DBS assay is measuring a composite of free hCGβ and additional β-subunits from intact hCG. The drying experiment showed that increased temperature and relative humidity during the drying process resulted in increased free hCGβ and reduced PAPP-A. Despite measuring additional free hCGβ compared to the serum assay, DBS analysis has a role in first-trimester combined screening for trisomy 21.

Effect of neutral and acid pH on the fluorescence of 4-methylumbelliferone and the implications for dry blood spot assays

Effect of neutral and acid pH on the fluorescence of 4-methylumbelliferone and the implications for dry blood spot assays

Filter Paper Blood Spot Enzyme Linked Immunoassay for Insulin and Application in the Evaluation of Determinants of Child Insulin Resistance

BackgroundIn large-scale epidemiology, bloodspot sampling by fingerstick onto filter paper has many advantages, including ease and low costs of collection, processing and transport. We describe the development of an enzyme-linked immunoassay (ELISA) for quantifying insulin from dried blood spots and demonstrate its application in a large trial.MethodsWe adapted an existing commercial kit (Mercodia Human Insulin ELISA, 10-1113-01) to quantify insulin from two 3-mm diameter discs (≈6 µL of blood) punched from whole blood standards and from trial samples. Paediatricians collected dried blood spots in a follow-up of 13,879 fasted children aged 11.5 years (interquartile range 11.3–11.8 years) from 31 trial sites across Belarus. We quantified bloodspot insulin levels and examined their distribution by demography and anthropometry.ResultsMean intra-assay (n = 157) coefficients of variation were 15% and 6% for ‘low’ (6.7 mU/L) and ‘high’ (23.1 mU/L) values, respectively; the respective inter-assay values (n = 33) were 23% and 11%. The intraclass correlation coefficient between 50 paired whole bloodspot versus serum samples, collected simultaneously, was 0.90 (95% confidence interval 0.85 to 0.95). Bloodspot insulin was stable for at least 31 months at −80°C, for one week at +30°C and following four freeze-thaw cycles. Paediatricians collected a median of 8 blood spots from 13,487 (97%) children. The geometric mean insulin (log standard deviation) concentrations amongst 12,812 children were 3.0 mU/L (1.1) in boys and 4.0 mU/L (1.0) in girls and were positively associated with pubertal stage, measures of central and peripheral adiposity, height and fasting glucose.ConclusionsOur simple and convenient bloodspot assay is suitable for the measurement of insulin in very small volumes of blood collected on filter paper cards and can be applied to large-scale epidemiology studies of the early-life determinants of circulating insulin.

S19-04 Psychological First Aid following Major Disasters – the Boxing Day Tsunami, Sri Lanka, 2004

This study aimed to elucidate the associations between physical activity, sedentary behaviors, and Epstein-Barr virus (EBV) antibody levels (as an indirect marker of cell-mediated immunity, CMI). This study made use of a 14-year longitudinal study with a representative sample of adolescents in the US. A total of 3361 participants (42.1% male) aged 11 to 21 years at baseline who completed Wave I (1994–1995), Wave III (2001 − 2002), and Wave IV (2008) surveys of the National Longitudinal Study of Adolescent Health (Add Health) were analyzed. Physical activity and sedentary behaviors at Waves I and III were assessed using interviewer-administered questionnaire. EBV viral capsid antigen (VCA) IgG antibody levels at Wave IV were analyzed from dried blood spot assays. Adjusted for confounders, among males, one additional day spent per week on strenuous sports at Wave III were associated with a decrease of 4.09 AU/ml in EBV antibody levels (p = 0.012), while one additional hour spent per week viewing videos at Wave I was associated with an increase of 0.83 AU/ml in EBV antibody levels (p = 0.026). Among females, one additional day spent per week on individual sports at Wave III were associated with a decrease of 4.63 AU/ml in EBV antibody levels (p = 0.014), while sedentary behaviors were not associated with EBV antibody levels. To conclude, physical activity and sedentary behaviors were associated with CMI among males and physical activity was associated with CMI among females.

First trimester Down syndrome screening with dried blood spots using a dual analyte free beta hCG and PAPP‐A immunofluorometric assay

To determine the effectiveness of first trimester Down syndrome screening with dried blood spots using a dual analyte free beta human chorionic gonadotrophin (hCG)/pregnancy-associated plasma protein A (PAPP-A) immunofluorometric assay. An initial retrospective study of 54 Down syndrome cases and 1064 control specimens was performed followed by a series of 146,513 specimens from routine screening. Detection rates at a fixed 5% false-positive rate were determined separately based on reference data from the retrospective study set and then adjusted based on the routine screening study set. On the basis of the retrospective analysis, the estimated detection rate using free beta hCG, PAPP-A and maternal age varied from 78% at 9 weeks of pregnancy to 70% at 13 weeks of pregnancy. Using a combined protocol, including NT, the detection rate varied from 92 to 90% between 9 and 13 weeks of gestation. Adjusting distribution parameters based on the routine screening dataset reduced the detection rate by at most 1%. Analysis of free beta hCG and PAPP-A using a dual analyte dried blood spot assay is an effective tool in Down syndrome screening, adding an important option for those considering implementation or modification of existing prenatal screening programs.

Space–time clustering of elevated thyroid stimulating hormone levels

Previous studies of congenital hypothyroidism (CHT) have reported an increasing incidence which may suggest that environmental factors play an aetiological role. If so, then cases may exhibit space-time clustering, where cases occur at similar times and close proximities to other cases. In this study we investigated whether space-time clustering of elevated thyroid stimulating hormone (TSH) in newborns exists. All infants born in the Northern Region of England are screened by measuring levels of circulating TSH using a blood spot assay. Data on 207 cases of elevated TSH values, as a proxy for CHT, in newborns born from 1994 to 2006 inclusive were available and analysed using rigorous space-time clustering statistical methods. Analysis showed statistically significant evidence of space-time clustering. The strength of clustering was most marked for cases born within 0.1-0.7 year (1-8 months) of one another. This is the first study to find significant space-time clustering of cases of elevated TSH levels in newborns, a surrogate for space-time clustering of CHT. Whilst the reasons for the clustering are unclear, it would appear from this analysis that transient environmental exposures are likely to be involved, although environmental determinants of genetic mutations and epigenetic factors cannot be ruled out. Further research is required to a) validate these results in other populations and b) to assess in more detail the potential environmental determinants of increased CHT risk.

Dried blood spot assay for estimation of metronidazole concentrations in rats and its application in single animal drug pharmacokinetic study

An HPLC-UV-dried blood spot (DBS) method for the estimation of metronidazole (MTZ) in rat whole blood is reported. Method employs Ahlstrom 226 sample collection paper and DBS samples were prepared by spotting with 30 μl of whole blood (spiked calibration standards/quality control samples/in vivo study samples). A 6 mm disc was punched from each DBS and extraction was carried out using water containing the internal standard (tinidazole). The calibration for MTZ was linear over 2.5–50 μg/ml concentration range. Accuracy (% bias) and precision (expressed as % Coefficient of variation) values for within and between day were <20% at the lower level quality control sample (LQC) and <15% at all other concentrations tested. The limit of quantification (LOQ) of the method was 2.5 μg/ml. The validated method was applied for the analysis of in vivo pharmacokinetic (PK) study samples after intravenous administration of MTZ to a rat. Whole blood PK parameters observed in this study were in compliance with literature based PK parameters. The DBS sampling approach was found to be useful in a single animal pharmacokinetic study.

Clinical Evaluation of a Dried Blood Spot Assay for Atazanavir

Current procedures for obtaining and measuring plasma concentrations of HIV protease inhibitors (PIs) are technically challenging. Dried blood spot (DBS) assays offer a way to overcome many of the obstacles. We sought to develop a DBS assay for quantitation of the PI atazanavir (ATV) and to compare this method with a previously validated plasma assay. We prospectively enrolled 48 patients with well-controlled HIV disease who had been on ATV for at least 7 days. ATV was quantified from plasma by use of high-performance liquid chromatography (HPLC). A reversed-phase ultrahigh-performance liquid chromatography (UPLC) assay was utilized for DBS samples. The concentrations of ATV quantified in a DBS matrix showed very strong agreement with those measured in plasma (r(2) = 0.988). The mean difference in ATV concentration between the two methods was -10.8% (95% confidence interval [95% CI], -7.65% to -13.95%), indicating that the DBS method has a slight negative bias. A majority (97.8%) of the differences in concentration between the two assays fell within ±2 standard deviations. ATV concentrations were lower in subjects who had detectable HIV RNA in plasma (mean, 543 ng/ml) than in those with HIV RNA of <50 copies/ml (mean, 1,582 ng/ml) (P = 0.03, Wilcoxon rank-sum test). In conclusion, our study demonstrated that ATV quantitation in a DBS matrix is feasible and accurate. DBS use offers a convenient alternative for measuring plasma concentrations of ATV and may have utility in monitoring of drug concentrations in clinical practice and in future studies.

Increasing Incidence, but Lack of Seasonality, of Elevated TSH Levels, on Newborn Screening, in the North of England

Previous studies of congenital hypothyroidism have suggested an increasing incidence and seasonal variation in incidence, which may suggest nongenetic factors involved in aetiology. This study describes the incidence of elevated thyroid stimulating hormone (TSH) values in newborns, a surrogate for congenital hypothyroidism, measured as part of the screening programme for congenital hypothyroidism, over an eleven-year period (1994–2005), and assesses whether seasonal variation exists. All infants born in the Northern Region of England are screened by measuring levels of circulating TSH using a blood spot assay. Data on all 213 cases born from 1994 to 2005 inclusive were available. Annual incidence increased significantly from 37 per 100,000 in 1994 to a peak of 92.8 per 100,000 in 2003. There was no evidence of seasonal variation in incidence. The reasons for the increasing incidence are unclear, but do not appear to involve increasing exposure to seasonally varying factors or changes in measurements methods.