Application Of Dried Blood Spots Research Articles

Dried blood spots for doping controls-Development of a comprehensive initial testing procedure with fully automated sample preparation.

Currently, primarily urine, whole blood and serum samples are analyzed for doping-relevant substances in professional sports, but recently dried blood spots (DBS) have been introduced as complementary matrix, offering advantageous features, e.g. a minimally invasive sampling procedure. In order to cope with the increased application of DBS, a comprehensive initial testing procedure (ITP) was developed, optimized and validated, comprising a total of 233 substances representing all groups on the World Anti-Doping Agency's (WADA's) Prohibited List. The sample preparation was conducted by employing a fully automated system using an efficient flow-through extraction of a 4mm diameter spot followed by LC-HRMS/MS analysis. The procedure was successfully validated in terms of selectivity, limit of detection, reproducibility, carryover and robustness with respect to an alternative manual sample preparation, an alternative dried blood collection device and the sample extract stability, and was thus found to meet the required criteria of the relevant guidelines published by WADA for routine application. As a proof-of-concept, DBS samples were analyzed after the administration of the glucocorticoids prednisone and dexamethasone, as well as the stimulant pseudoephedrine and the beta-blocker propranolol. All substances were detected in post-administration samples for at least 4h and up to 24 h after intake, depending on the collection time period, using the developed testing procedure. In particular, for substances that are only banned in-competition, data obtained from DBS samples can be useful for the interpretation of adverse analytical findings. In conclusion, the developed ITP accounts for the anticipated increasing relevance of DBS in anti-doping analysis in the future and provides a foundation for optimized approaches for specific substance classes.

Determination of lithium in dried blood spots and dried plasma spots by graphite furnace atomic absorption spectrometry: Method development, validation and clinical application

Therapeutic drug monitoring (TDM) has become a standard of care for the mood stabilizer lithium (Li+). Dried Blood Spots (DBS) and Dried Plasma Spots (DPS) are promising alternative sampling strategies for TDM, which allows simple and cost-effective logistics in many settings, particularly in Developing Countries. DBS and DPS are of particular interest to Li + TDM for allowing the estimation of Li + erythrocyte levels. Thus, the aim of this study was to develop and validate an assay for the determination of Li+ in DBS and DPS by Graphite Furnace Atomic Absorption Spectrometry (GFAAS), and to evaluate its application in a clinical setting. Li+ was extracted from one 8 mm DBS disc punch with nitric acid 4.5% and from one 6 mm DPS disc punch with diluent solution (HNO3 1% + Triton 0.1%) and injected into GFAAS. The method was applied to Li + TDM in 43 patients with mood disorder. The assays were linear from 0.10 to 3.0 mEq L−1 (r > 0.99), precise, with CV 3.6–7.2% for DBS and 4.6–9.3% for DPS samples, and accurate, with accuracy values of 97–109% and 98–106% for DBS and DPS samples, respectively. Li+ was stable in dried samples during twenty days at up to 42 °C. The DBS assay accuracy and recovery were not influenced by blood hematocrit. The patients presented Li + serum concentrations of 0.18–1.1 mEq L−1 and 0.17 to 0.92 mEq L−1 in DBS and 0.15 to 0.99 mEq L−1 in DPS samples. DPS had comparable Li + concentrations to the ones found in fresh serum samples. With DBS samples it was possible to estimate the Li + erythrocyte to plasma concentration ratio (LiR). The findings of this study support the clinical application of DBS and DPS samples for the TDM of Li+.

Performance of a web-based application measuring spot quality in dried blood spot sampling

Background The dried blood spot (DBS) method allows patients and researchers to collect blood on a sampling card using a skin-prick. An important issue in the application of DBSs is that samples for therapeutic drug monitoring are frequently rejected because of poor spot quality, leading to delayed monitoring or missing data. We describe the development and performance of a web-based application (app), accessible on smartphones, tablets or desktops, capable of assessing DBS quality at the time of sampling by means of analyzing a picture of the DBS. Methods The performance of the app was compared to the judgment of experienced laboratory technicians for samples obtained in a trained and untrained setting. A robustness- and user test were performed. Results In a trained setting the app yielded an adequate decision in 90.0% of the cases with 4.1% false negatives (insufficient quality DBSs incorrectly not rejected) and 5.9% false positives (sufficient quality DBSs incorrectly rejected). In an untrained setting this was 87.4% with 5.5% false negatives and 7.1% false positives. A patient user test resulted in a system usability score of 74 out of 100 with a median time of 1 min and 45 s to use the app. Robustness testing showed a repeatability of 84%. Using the app in a trained and untrained setting improves the amount of sufficient quality samples from 80% to 95.9% and 42.2% to 87.9%, respectively. Conclusions The app can be used in trained and untrained setting to decrease the amount of insufficient quality DBS samples.

Evaluation of dried blood spots for hepatitis B and D serology and nucleic acid testing.

Areas with the highest burden of hepatitis B virus (HBV) infection are often low-middle-income countries with limited access to diagnosis due to isolation, affordability, and/or feasibility. Dried blood spots (DBSs) provide an alternative for remote areas where collection and transportation of serum is impractical. In this study, the application of DBS for serological and molecular detection of HBV and hepatitis D virus (HDV) was evaluated. Hepatitis B surface antigen was detected in 87 of 91 (95.6%) DBS. Seventeen of 21 (81%) had detectable HBeAg and 52 of 71 (73.2%) were anti-HBe positive. Anti-HD was detectable in 11 of 12 (91.6%) spiked control DBS after an initial failure to detect in patient DBS. HBV DNA was detected from 50 of 70 (71.4%) DBS with serum loads greater than 200 IU/mL in an in-house assay and 18 of 24 (75%) DBS with loads exceeding 389 IU/mL in a commercial assay. Using linear regression, HBV DNA loads from DBS were able to predict serum loads in 46 of 50 (92%) samples to within 1 log of actual serum load. HDV RNA was detected in 42 of 47 (89%) DBS with serum levels greater than 7200 IU/mL. DBSs are recommended for diagnosis of HBV, monitoring, and detection of high loads in pregnant women where peripheral blood testing remains unfeasible. Detection of HDV RNA from DBS may prove useful in endemic areas.

DETECTION OF MEASLES AND RUBELLA ANTIBODIES IN DRIED BLOOD SPOTS

This study aimed to determine the frequency of detection of measles and rubella antibodies in dried blood spots in Bulgaria.
 Material and Methods: Two types of clinical material, serum samples and dried blood spots (DBS), were tested from a total of 101 patients. Serological methods (indirect ELISA) were used for detection of specific viral markers (IgM and IgG antibodies) indicating acute or past measles and rubella infection.
 Results: In the present study, the patients were with median age of 39 years and divided into 11 age groups. The majority of patients were under 30 years of age and from the capital of Sofia. In 3 patients acute measles infection was confirmed by positive ELISA-IgM results for the serum samples and DBS. No acute rubella infection was detected. Measles and rubella IgG seroprevalence was determined as 83/101 (82%, 95% CI: 74.51÷89.49) and 79/101 (78%, 95% CI: 69.92÷86.08) in serum samples, and 79/101 (78%, 95% CI: 69.92÷86.08) and 73/101 (72%, 95% CI: 63.25÷80.75) in DBS, respectively. In combination with immunoenzymatic testing for measles and rubella IgM/IgG markers, coincidence of results for both types of clinical material was found in >90% of cases. No significant differences were found in the results in terms of gender and age.
 Conclusion: In recent years a variety of new and innovative applications of DBS are introduced in medicine, neonatology, virology, microbiology, etc. The optimisation of the DBS technique as an alternative approach to venepuncture in virology is very important for conducting seroepidemiological studies and to a certain extent for the surveillance of epidemic outbreaks.

Understanding of human metabolic pathways of different sub-classes of phenols from Arbutus unedo fruit after an acute intake.

Arbutus unedo is a small Mediterranean fruit, commonly named strawberry tree, which is a rich source of different sub-classes of phenolic compounds, the more representative being the gallic acid derivatives, including its mono and oligomeric forms esterified with quinic and shikimic acids. In addition, galloyl derivatives, particularly gallotannins, described in A. unedo, are part of a very selective phenolic group, present in a reduced number of plant-products. The aim of the present study is to provide a better understanding of human metabolic pathways of different sub-classes of phenols from the A. unedo fruit after an acute intake by healthy adults. Therefore, the A. unedo phenolic metabolites were studied in whole blood samples (0 to 24 h), urine (24 h) and feces (12 and 24 h). Special focus was placed on the application of dried blood spot (DBS) cards for the sample collection and for the analysis of phenolic metabolites in whole blood samples. The results of the blood analysis revealed two peaks for the maximum concentrations of the main phenolic metabolites. Furthermore, it is appropriate to highlight the application of DBS cards as an efficient and accurate way to collect blood samples in post-prandial bioavailability studies. The analysis of urine (24 h) gave a wide range of phenolic metabolites showing the extensive metabolism that A. unedo phenolic compounds underwent in the human body. The results of the study provide a relevant contribution to the understanding of the in vivo human bioavailability of phenolic compounds, especially galloyl derivatives, a singular phenolic sub-group present in the A. unedo fruit.

Application of dried blood spot in newborn screening for inherited metabolic diseases

Neonatal inherited metabolic diseases are a group of metabolic disorders caused by singe gene defect to cause a series of clinical symptoms.Neonatal dried blood spots have the advantages of simple preparation, safety, good stability, and show strong practicability in different screening methods for inherited metabolic diseases.With the development of screening methods, more and more diseases could be diagnosed by screening.The emergence of tandem mass spectrometry and molecular biological techniques promote the newborn screening and automation for inherited metabolic disease effectively.Inherited metabolic diseases induce great harm to the newborn, which could cause not only system organs damage, but also lead to death.Therefore, early screening is important for patients' prognosis.(Chin J Lab Med, 2015, 38: 220-222) Key words: Metabolism, inborn errors; Dried blood spot testing; Neonatal screening; Tandem mass spectrometry

Performance and storage integrity of dried blood spots for PCB, BFR and pesticide measurements

Dried blood spots (DBSs) can provide accurate and valuable estimates of exposure to environmental toxicants, and the use of information derived from archived newborn DBSs has enormous potential to open up new research on the impacts of early chemical exposure on disease. Broad application of DBS for the purpose of quantitative exposure estimation requires robust and validated methods. This study investigates the suitability of DBS analyses for population studies of exposure to three chemical groups: polychlorinated biphenyls (PCBs), brominated flame retardants (BFRs), and chlorinated pesticides. It examines background (matrix) contamination, recovery and extraction variability, sensitivity, and storage stability. DBS samples prepared using 50μL of adult blood were analyzed by GC/MS, and method performance was confirmed by using certified materials and paired DBS-blood samples from six volunteers. Several of the target compounds and their degradation products have not been previously measured in DBSs. All target compounds were detected in DBS samples collected from the volunteers. Sample DBS cards showed background contamination of several compounds. When stored at room temperature, target compounds, excluding PBDEs, were stable for up to one month. When refrigerated or frozen, stability was acceptable for all compounds up to one year, and multiyear storage appears acceptable at colder (e.g., −80°C) temperatures. Multicompartment models may be used to estimate or correct for storage losses. Considering concentrations of contaminants for adults and children reported in the literature, and experimental values of detection limits and background contamination, DBS samples are suitable for quantifying exposures to many PCBs, BFRs and persistent pesticides.

Use of dried blood spots in doping control analysis of anabolic steroid esters

Dried blood spot (DBS) sampling, a technique for whole blood sampling on a piece of filter paper, has more than 50-years tradition, particularly in the diagnostic analysis of metabolic disorders in neonatal screening. Due to the minimal invasiveness, straightforwardness, robustness against manipulation and fastness DBS sampling recommends itself as an advantageous technique in doping control analysis. The present approach highlights the development of a screening assay for the analysis of eight anabolic steroid esters (nandrolone phenylpropionate, trenbolone enanthate, testosterone acetate, testosterone cypionate, testosterone isocaproate, testosterone phenylpropionate, testosterone decanoate and testosterone undecanoate) and nandrolone in DBS. The detection of the intact esters allows an unequivocal proof of the administration of conjugates of exogenous testosterone and its derivatives. Precise, specific and linear conditions were obtained by means of liquid chromatography high resolution/high accuracy mass spectrometry. Sensitivity in the low ppb range was accomplished by the preparation of the methyloxime derivatives of the target compounds. Labeled internal standards (d3-nandrolone, d3-nandrolone caproate and d3-nandrolone undecanoate) were applied to compensate for the broad range in chain length of the esters.The assay presented here outlines the application of DBS for the analysis of anabolic steroid esters in doping controls for the first time providing great potential to simplify the proof of exogenous administration of testosterone.

Evaluation of Dried Blood Spots for The Quantification of Therapeutic Monoclonal Antibodies and Detection of Anti-Drug Antibodies

Recently, the potential of dried blood spots (DBS) for small-molecule bioanalysis by LC-MS has been explored. The goal of this investigation was to evaluate the use of DBS for the quantification of biologics, where bioanalysis is with immunoassay. Therapeutic monoclonal antibodies were successfully eluted from DBS and detected by immunoassays, and the procedure could be validated in alignment with current guidelines. Accuracy, precision, selectivity and dilution linearity were all within the acceptance criteria currently used for the validation of binding assays with serum samples. Serum and DBS samples obtained in parallel during a PK research study in rats were analyzed for drug and anti-drug antibodies using AlphaLISA(®) technology. Drug concentrations in both sample types showed a strong correlation, and there was very good alignment in detection of immunogenicity positive animals. Using two examples, we have demonstrated that therapeutic monoclonal antibodies can be accurately quantified in DBS, and since anti-drug antibodies could also be successfully detected, there is scope for application of DBS to preclinical and clinical bioanalysis of monoclonal antibody drugs and anti-drug antibodies.

Validation of a Ligand Binding Assay Using Dried Blood Spot Sampling

Dried blood spots (DBS) technology has been introduced as a microsampling alternative to traditional plasma or serum sampling for pharmacokinetics or toxicokinetics evaluation. The application of DBS has been established for many small molecule drugs at discovery, nonclinical, and clinical stages. However, the application of DBS for large molecule therapeutics development is not yet well-established. This article describes the method validation of a ligand binding assay (LBA) for DBS sampling of a therapeutic monoclonal antibody-AMG 162 (Denosumab). The original serum LBA was modified for the DBS method. A fit-for-purpose method validation was performed to evaluate accuracy and precision, selectivity, dilutional linearity, and stability. In addition, the parameters relevant to DBS, such as spot volume, extraction recovery, whole blood stability, and hematocrit effects, were evaluated. The validation results demonstrated assay robustness with inter-assay precision of ≤ 19%, inter-assay accuracy of ≤ 9%, and total error of ≤ 24%. Selectivity, extraction recovery, dilutional linearity, and stability were demonstrated. The validation results revealed some limitations of the possible effect of blood hematocrit on therapeutic concentration measurements and the caution required using whole blood for standards and quality controls preparation. This is the first article to describe a thorough method validation of an LBA using DBS for a therapeutic monoclonal antibody. The lessons learned can serve as a model process for future method validation of other LBAs for large molecule therapeutics or biomarkers using the DBS sampling method.

Application of DBS for The Quantitative Assessment of A Protein Biologic Using On-Card Digestion LC–MS/MS or Immunoassay

Dried blood spot (DBS) sampling has recently gained popularity in the bioanalysis community for quantitation of small molecules. Since the pharmaceutical industry continues to increase investment in biopharmaceuticals, DBS technologies were investigated to determine if immunoassay and/or LC-MS/MS techniques would be amenable for quantitation of a large protein therapeutic (>70 kDa). Methods were successfully qualified for the protein therapeutic utilizing DBS technology. DBS methods in rat blood were qualified for this therapeutic protein using either immunoassay or enzymatic digestion directly off the DBS card followed by UHPLC-MS/MS separation and detection. Both qualifications were carried out in accordance with current acceptable practices defined by international acceptance criteria. Card selection was critical to both DBS methods. The advantages gained by DBS technology can successfully be applied to the quantitative assessment of biologics. This UHPLC-MS/MS method illustrates that digestion of large molecules directly off blood spot cards allows quantitation of these molecules. In addition, DBS technologies are amenable to immunoassay analysis. The immunoassay was 20-fold more sensitive than the UHPLC-MS/MS method, however the UHPLC-MS/MS assay had a much broader dynamic range.

Application of DBS for Quantitative Assessment of the Peptide Exendin-4; Comparison of Plasma and DBS Method by UHPLC–MS/MS

An investigation was performed in order to establish if dried blood spots (DBS) could be applied to the quantitation of biopharmaceuticals in biological matrices and perform equivalently in terms of accuracy, precision and stability to traditional plasma methods. A method was successfully validated for the peptide Exendin-4 (39 amino acids in length) utilizing DBS technology. The validated DBS method resulted in a more sensitive and simplistic method than an existing monkey plasma method and required tenfold less sample volume. The final DBS method resulted in a 10-2000-ng/ml linear calibration range using approximately 5 µl of dried blood, compared with the plasma method in which 150 µl of plasma coupled with SPE sample preparation resulted in a 20-2000-ng/ml linear calibration range. Although not needed for DBS, SPE was required for the plasma method to reduce endogenous matrix interferences and achieve desired LLOQ. Matrix stability was also enhanced by the implementation of the DBS platform when compared with either plasma or whole blood. DBS technology can be utilized for the quantitation of biopharmaceuticals and offer advantages over traditional plasma-based methods.

Ultrafast and high-throughput mass spectrometric assay for therapeutic drug monitoring of antiretroviral drugs in pediatric HIV-1 infection applying dried blood spots

Kaletra® (Abott Laboratories) is a co-formulated medication used in the treatment of HIV-1-infected children, and it contains the two antiretroviral protease inhibitor drugs lopinavir and ritonavir. We validated two new ultrafast and high-throughput mass spectrometric assays to be used for therapeutic drug monitoring of lopinavir and ritonavir concentrations in whole blood and in plasma from HIV-1-infected children. Whole blood was blotted onto dried blood spot (DBS) collecting cards, and plasma was collected simultaneously. DBS collecting cards were extracted by an acetonitrile/water mixture while plasma samples were deproteinized with acetone. Drug concentrations were determined by matrix-assisted laser desorption/ionization-triple quadrupole tandem mass spectrometry (MALDI-QqQ-MS/MS). The application of DBS made it possible to measure lopinavir and ritonavir in whole blood in therapeutically relevant concentrations. The MALDI-QqQ-MS/MS plasma assay was successfully cross-validated with a commonly used high-performance liquid chromatography (HPLC)–ultraviolet (UV) assay for the therapeutic drug monitoring (TDM) of HIV-1-infected patients, and it showed comparable performance characteristics. Observed DBS concentrations showed as well, a good correlation between plasma concentrations obtained by MALDI-QqQ-MS/MS and those obtained by the HPLC-UV assay. Application of DBS for TDM proved to be a good alternative to the normally used plasma screening. Moreover, collection of DBS requires small amounts of whole blood which can be easily performed especially in (very) young children where collection of large whole blood amounts is often not possible. DBS is perfectly suited for TDM of HIV-1-infected children; but nevertheless, DBS can also easily be applied for TDM of patients in areas with limited or no laboratory facilities.