Dried Blood Spot Extracts Research Articles

Detection of α-Galactosidase A Reaction in Samples Extracted from Dried Blood Spots Using Ion-Sensitive Field Effect Transistors.

Fabry disease is a lysosomal storage disorder caused by a significant decrease in the activity or absence of the enzyme α-galactosidase A. The diagnostics of Fabry disease during newborn screening are reasonable, due to the availability of enzyme replacement therapy. This paper presents an electrochemical method using complementary metal-oxide semiconductor (CMOS)-compatible ion-sensitive field effect transistors (ISFETs) with hafnium oxide-sensitive surfaces for the detection of α-galactosidase A activity in dried blood spot extracts. The capability of ISFETs to detect the reaction catalyzed by α-galactosidase A was demonstrated. The buffer composition was optimized to provide suitable conditions for both enzyme and ISFET performance. The use of ISFET structures as sensor elements allowed for the label-free detection of enzymatic reactions with melibiose, a natural substrate of α-galactosidase A, instead of a synthetic fluorogenic one. ISFET chips were packaged with printed circuit boards and microfluidic reaction chambers to enable long-term signal measurement using a custom device. The packaged sensors were demonstrated to discriminate between normal and inhibited GLA activity in dried blood spots extracts. The described method offers a promising solution for increasing the widespread distribution of newborn screening of Fabry disease.

B-027 Adaptation of Neonatal Dried Blood Spot Assays for Use on Automated Microplate Instruments

Abstract Background Newborn screening (NBS) is routinely conducted to identify various inherited metabolic disorders which can affect a child’s long-term health or survival. The assay methods used include enzyme-linked immunosorbent assays (ELISA) as well as enzymatic biochemical assays. Dried blood spots (DBS) prepared by applying whole blood to filter paper cards are typically used as the sample for these screening assays. In this study we have adapted an openly programmable automated ELISA processing instrument to perform these types of assays with a high level of accuracy and precision. Method The Awareness Technology (Palm City, FL) ChemWell-2 instrument was chosen for this study. This instrument is a two plate ELISA processing system which has the capability of performing the DBS extraction in an uncoated round bottom 96-well microplate and transferring a small volume of extracted sample to be assayed to a second flat bottom 96-well microplate. The following enzymatic assay kits manufactured by ZenTech (Liège, Belgium) were used in this study: total galactose, phenylalanine, glucose-6-phosphate dehydrogenase (G-6-PD) and biotinidase. Results In order to evaluate the assay performance controls and additional standard DBS were run as samples. Three to four different sample concentration levels were run four to five times in each assay. The runs were repeated 4 times each to determine inter-assay (between run) precision. The results are summarized in the table below: Conclusion Enzymatic NBS methods for total galactose, phenylalanine, G-6-PD and biotinidase including the DBS sample extraction step can be processed with clinically useful accuracy and precision using the Awareness Technology ChemWell-2 automated analyzer.

Insight into Increased Recovery and Simplification of Genomic DNA Extraction Methods from Dried Blood Spots.

There is no consensus on how to perform the manual extraction of nucleic acids from dried blood spots (DBSs). Current methods typically involve agitation of the DBSs in a solution for varying amounts of time with or without heat, and then purification of the eluted nucleic acids with a purification protocol. We explored several characteristics of genomic DNA (gDNA) DBS extraction such as extraction efficiency, the role of red blood cells (RBCs) in extraction and critical kinetic factors to understand if these protocols can be simplified while maintaining sufficient gDNA recovery. We found that agitation in a RBC lysis buffer before performing a DBS gDNA extraction protocol increases yield 1.5 to 5-fold, depending upon the anticoagulant used. The use of an alkaline lysing agent along with either heat or agitation was sufficient to elute quantitative polymerase chain reaction (qPCR) amplifiable gDNA in 5 minutes. This work adds insight into the extraction of gDNA from DBSs with the intention of informing a simple, standardized manual protocol for extraction.

Analysis of C-reactive protein from finger stick dried blood spot to predict high risk of cardiovascular disease

C-reactive protein (CRP) is an acute-phase protein involved in inflammation. Furthermore, CRP is an important biomarker used in diagnostics to predict risk of cardiovascular disease (CVD) in addition to monitoring bacterial and viral infections. To measure plasma CRP, venipuncture is still necessitated and has to be performed by trained phlebotomists. As a solution, dried blood spots (DBS) are used for minimally invasive at-home sampling of blood and can be send to diagnostic laboratories by regular mail. In this study, we included 53 patients that presented to the outpatient clinic of the University Medical Center Utrecht. Capillary finger stick was used to spot blood on a filter paper card and allowed to dry. After extraction of DBS, CRP was analyzed on an automated high-throughput chemistry analyzer. Additional validation steps regarding stability, effect of hematocrit, precision, and limits of blank and quantitation were conducted according to corresponding Clinical and Laboratory Standards Institute standards. An excellent regression analysis of R2 (95% confidence interval) = 0.986 (0.982–0.989) was found. This enabled correct classification for high CVD risk of all 25 cases with sensitivity (95% CI) of 1.00 (1.00–1.00) and specificity (95% CI) of 0.96 (0.89–1.03) and correct diagnosis of inflammation of 12/13 cases with sensitivity (95% CI) of 0.92 (0.77–1.07) and specificity (95% CI) of 1.00 (1.00–1.00). Furthermore, CRP was found to be stable for 31 days and observed hematocrit variation amongst patients was clinically acceptable. CRP from DBS can be accurately measured on an automated high-throughput chemistry analyzer and used to diagnose inflammation and classify high CVD risk. This method enables individuals to engage in at-home sampling of blood on DBS for (tele)diagnostics, screening programs, patient follow-up, and medication management.

Validation of a targeted metabolomics panel for improved second-tier newborn screening.

Improved second-tier assays are needed to reduce the number of false positives in newborn screening (NBS) for inherited metabolic disorders including those on the Recommended Uniform Screening Panel (RUSP). We developed an expanded metabolite panel for second-tier testing of dried blood spot (DBS) samples from screen-positive cases reported by the California NBS program, consisting of true- and false-positives from four disorders: glutaric acidemia type I (GA1), methylmalonic acidemia (MMA), ornithine transcarbamylase deficiency (OTCD), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). This panel was assembled from known disease markers and new features discovered by untargeted metabolomics and applied to second-tier analysis of single DBS punches using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a 3-min run. Additionally, we trained a Random Forest (RF) machine learning classifier to improve separation of true- and false positive cases. Targeted metabolomic analysis of 121 analytes from DBS extracts in combination with RF classification at a sensitivity of 100% reduced false positives for GA1 by 83%, MMA by 84%, OTCD by 100%, and VLCADD by 51%. This performance was driven by a combination of known disease markers (3-hydroxyglutaric acid, methylmalonic acid, citrulline, and C14:1), other amino acids and acylcarnitines, and novel metabolites identified to be isobaric to several long-chain acylcarnitine and hydroxy-acylcarnitine species. These findings establish the effectiveness of this second-tier test to improve screening for these four conditions and demonstrate the utility of supervised machine learning in reducing false-positives for conditions lacking clearly discriminating markers, with future studies aimed at optimizing and expanding the panel to additional disease targets.

Technical Study of Automated High-Throughput High-Sensitive Ceruloplasmin Assay on Dried Blood Spots-Reinstate the Potential Use for Newborn Screening of Wilson Disease.

In this study, we modified a fully automatic immunoassay on ceruloplasmin concentration on dried blood spots (DBS) to increase its analytical sensitivity in order to accurately differentiate newborns from true Wilson disease (WD) patients. Modifications to the assay parameters of the Roche/Hitachi Cobas c systems immunoturbidimetric assay are adjusted to lower the limit of quantitation to 0.60 mg/L from 30 mg/L. This enables sensitive measurement of ceruloplasmin in eluent after DBS extraction. In addition, reference intervals and receiver operating characteristic curve analysis for diagnostic cut-off were established using DBS of neonates and WD adult patients. After DBS whole blood calibration, the 95th percentile of the reference interval for newborns was 86-229 mg/L. The cut-off value of 54 mg/L was found to be the most optimal point for differentiating true adult WD from newborn controls. This test shows a high area under curve of 1.000 with 100% sensitivity and specificity in differentiating normal newborns from WD adult samples. However, the results should be further validated with true newborn WD patient samples together with the consideration of other factors that can also lead to low ceruloplasmin levels. This test shows application potential in newborn screening for WD, which can save lives through early identification and timely treatment.

Electro-DBS: A Simple Method to Rapidly Extract Genomic DNA from Dried Blood Spots

Dried blood spots (DBSs) have been used for more than 50 years and are used as a method of sample collection for pharmacological testing, genetic analyses, monitoring of viral infections, and more. Protocols for nucleic acid extraction from DBSs involve several steps that require specialized equipment and can be lengthy. Thus, we sought to explore ways to reduce the analytical burden of DBSs. We developed a DBS extraction method that uses the synergistic action of electrophoretic and diffusive transport mechanisms to extract genomic DNA (gDNA) through the DBS matrix. This method (which we termed "Electro-DBS") reduces the time of extraction from 40 min to 5 min and removes the need for heat, shaking, and DNA purification steps while maintaining gDNA quality and yield. We found that the electrophoretic transport speeds up gDNA elution, allowing for the diffusive transport of polymerase chain reaction (PCR) inhibitors to be minimized due to a reduced elution time. Overall, this work added mechanistic insight into the extraction of DNA from DBSs and developed a method that was ideal for point of care and automation.

Fully Automated Dried Blood Spot Extraction coupled to Liquid Chromatography-tandem Mass Spectrometry for Therapeutic Drug Monitoring of Immunosuppressants

Patients receiving immunosuppressant therapy, require intensive follow-up via therapeutic drug monitoring (TDM). This puts quite a burden on the patient involving frequent hospital visits and venipunctures and could (partially) be resolved by the use of dried blood microsamples (e.g. dried blood spots, DBS). One of the drawbacks of the use of DBS is the requirement for a dedicated, manual sample preparation. Fully automated DBS extraction systems, online coupled to standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) configurations, could provide a solution for that. The aim of this study was to evaluate the use of the DBS-MS 500, online coupled to an LC-MS/MS system, for the TDM of immunosuppressants using DBS. Two methods for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A, in both DBS and whole blood, were developed and validated based on international guidelines. For the DBS method also DBS-specific parameters were taken into account. Both methods proved to be accurate and reproducible with biases below 11% (20% for the LLOQ) and CVs (%) below 14% (with a single exception) (20% for the LLOQ) over a calibration range from 1 to 50 ng/mL for tacrolimus, sirolimus and everolimus and 20 to 1500 ng/mL for cyclosporin A. Reproducible (CV < 15%) IS-compensated relative recovery values were obtained. However, a hematocrit-dependent relative recovery was observed for DBS, with lower hematocrit values yielding higher relative recoveries (and vice versa). Relative to the reference hematocrit of 0.37, this difference exceeded 15% at hematocrit extremes (0.18 and 0.60). Application on venous left-over patient samples showed reasonable agreement between the results of both methodologies (8,6,9 and 9/10 mean DBS results within 20% of the mean whole blood result for tacrolimus, sirolimus, everolimus and cyclosporin A, respectively), although also here an impact of the hematocrit could be discerned. As a next step, larger patient sets are needed to allow a better insight on how (correction for) the hct effect affects the quantification of immunosuppressants via fully automated DBS analysis.

Analysis of tamoxifen and its metabolites in dried blood spot and volumetric absorptive microsampling: comparison and clinical application

This research was conducted to develop the Dried Blood Spot (DBS) and Volumetric Absorptive Microsampling (VAMS) method in the analysis of Tamoxifen (TAM) and its metabolites endoxifen (END), 4-hydroxytamoxifen (4-HT), and N-desmethyltamoxifen (NDT) using Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). This method was then applied to monitor TAM and its metabolites in breast cancer patients. The UPLC-MS/MS method was developed and validated with propranolol as the internal standard. The recovery and matrix effects on DBS and VAMS were investigated. The validation requirements were fulfilled by the methodology of analysis and sample preparation described in this study. Both VAMS and DBS extraction recoveries were satisfactory, with low variability. Extraction recovery in the VAMS sample was found to be slightly higher than in the DBS sample. Sample stability in DBS and VAMS was demonstrated for up to 2 months. Both of these methods were successfully applied for the analysis of TAM and metabolites in clinical patients. The mean concentrations obtained from the two methods were not significantly different.

P311 Intensive dried blood spot sampling shows a higher drug exposure throughout the first 24 weeks of therapy in ustekinumab-treated Crohn’s disease patients achieving endoscopic remission

Abstract Background Various studies have shown an association between ustekinumab serum concentrations and treatment response in patients with Crohn’s disease (CD). However, the time point at which this concentration is most informative for long-term outcome is unknown. Dried blood spot (DBS) sampling allows remote collection of blood for measurements at various time points. By use of this sampling method, the current study aimed to evaluate the exposure-response relationship of ustekinumab throughout the first 24 weeks of treatment and to investigate the time points at which ustekinumab levels could identify patients achieving endoscopic remission. Methods This interim-analysis included DBS (n = 332) and serum (n = 60) samples collected from 13 ustekinumab-treated CD patients at 25 time points during the first 6 months of therapy. All patients received an intravenous infusion of ~6 mg/kg ustekinumab followed by subcutaneous dosing of 90 mg every 8 weeks. Ustekinumab concentrations were measured in serum and DBS extracts using an ustekinumab ELISA (apDia, Turnhout, Belgium). Endoscopic remission was defined as simple endoscopic score for CD (SES-CD) ≤3 after 6 months of therapy. The Mann-Whitney U-test was used to compare unpaired data. Results Three out of 13 patients (23.1%) achieved endoscopic remission. Ustekinumab concentrations in serum correlated with those in DBS extracts (Spearman r = 0.97, p &amp;lt;0.0001). Measurement of the ustekinumab concentration in 38 simultaneously collected DBS and serum samples showed an average DBS-to-serum conversion factor of 2.5±0.7 (mean±SD) with an acceptable interpatient variability (CV ≤29%). Concentration-time profiles showed that patients in remission have a higher area under the curve (AUC), hence a higher drug exposure, than patients not achieving remission (906 vs 466 μg*day/mL, p=0.007, Figure). A negative correlation was observed between the AUC and SES-CD at week 24 (Spearman r = -0.74, p=0.0048). Ustekinumab concentrations in DBS extracts at the intermediate time points week 6, week 11 and week 12 after treatment start exhibited the most significant difference between remitters and non-remitters (Table). In contrast, no significant difference was observed in ustekinumab levels in DBS extracts at week 8, week 16 and week 24 between remitters and non-remitters. Conclusion Intensive DBS sampling showed that ustekinumab-treated CD patients in endoscopic remission have a higher drug exposure throughout the first 24 weeks of treatment than patients not in remission. DBS is an easy sampling method that allows accurate monitoring of ustekinumab exposure over time and could help to identify underexposed patients which might benefit from treatment optimization.

Detection of SARS-CoV-2 IgG antibodies in dried blood spots

The COVID-19 pandemic led to development of numerous serologic tests. To obviate the need for phlebotomy services, we validated dried blood spots (DBS) for anti-SARS-CoV-2 serologic testing. Immunoglobulins were extracted from 3 mm DBS punches and the extracts were analyzed using the Euroimmun anti-SARS-CoV-2 IgG ELISA. Various pre-analytical factors were studied. Results were favorable for DBS stored for at least 67 days at or below 37°C. Comparison between paired serum and DBS specimens tested by the Euroimmun ELISA showed 96.8% and 81.3% positive and negative agreement, respectively, indicating that confirmatory testing of positive Euroimmun results on DBS extracts is necessary to achieve clinical accuracy. Our findings suggest that any SARS-CoV-2 antibody assay that requires pre-dilution of serum is amenable to DBS as an alternate specimen type that is relatively low cost, self-collectable, stable, can be shipped by standard mail and could be used to establish the seroprevalence of large populations.

Dried blood spots for the quantitative evaluation of IgG isotypes and correlation with serum samples in HIV-exposed uninfected (HEU) infants

BackgroundThe determination of IgG levels and their subclasses can provide clinically relevant information on the status of the immune system. Here we determined the sensitivity and reproducibility of the quantification of IgG subclasses from Dried Blood Spots (DBS) in Malawian uninfected infants exposed to HIV (HEU). MethodsSixty paired samples of serum and DBS from HEU infants were used. Samples were collected from 1, 6, and 24-month old infants. IgGs concentrations from both serum and DBS were analyzed by BN ProSpec Siemens assay, using a different setting for sample dilutions. The reproducibility of the DBS method was tested on 10 samples run twice, starting from the DBS extraction process. To assess the systematic, proportional, and random differences, we computed the Passing-Bablok regression, and the Bland-Altman analysis to estimate the total mean bias between the two tests. ResultsThe IgG isotypes concentrations from serum and DBS showed significant differences in all the comparisons. Generally, the DBS method underestimated IgG subclasses' values showing a recovery range between 51.2% and 77.6%. Passing Bablok regression on age-based groups showed agreement for IgG, IgG1, and IgG2, but not for IgG3 and IgG4. The mean bias obtained with the Bland Altman test varied largely depending on IgG isotypes (−0.02–2.21 g/l) Coefficient of variation <7.0% was found in the repeated tests for IgG, IgG1, IgG3, and IgG4, while it was 12.4% for IgG2. ConclusionsVarying degrees of differences were seen in the IgGs measurement in the two different matrices. In IgGs analysis, the DBS method offers promise for population-based research, but the results should be carefully evaluated and considered as a relative value since they are not equivalent to the serum concentrations.

Low levels of Lysosomal Acid Lipase (LAL) activity increases necroinflammation in adult patients with biopsy-proven metabolic associated fatty liver disease

Introduction and objectiveMetabolic associated fatty liver disease (MAFLD), characterized by intra-hepatic fat accumulation, will soon be the leading cause of end-stage liver disease. Lysosomal Acid Lipase (LAL) is a key enzyme in lipid metabolism. We investigated its activity in patients with biopsy-proven MAFLD. MethodsProspective cross-sectional study in patients with biopsy-proven MAFLD. Blood LAL-activity (pmol/punch/h) was measured with dried blood spot extracts using Lalistat 2. Demographic, clinical, and laboratory data were collected. Results101 adult patients were recruited. Among them, 11.9% had a diagnosis of MAFLD without steatohepatitis and 88.1% had MAFLD with steatohepatitis. The median of LAL-activity in patients with MAFLD was 76.8 pmol/punch/h. MAFLD patients with steatohepatitis showed an increase in gamma-glutamyl transferase (p = 0.042), insulin (p = 0.001), homeostatic model assessment for insulin resistance (HOMA-IR, p = 0.001) and advanced liver fibrosis (p < 0.001), compared to cases of MAFLD without steatohepatitis. There was no statistical difference in LAL-activity between the cases (p = 0.296). When considering LAL-activity above and below 77 pmol/punch/h as a cut-off value, patients with reduced LAL-activity had a significant increase in necroinflammatory activity according to the METAVIR score (p = 0.040), and NAFLD activity score (NAS, p = 0.031) compared to cases with higher LAL-activity. ConclusionOur findings suggest that reduced LAL-activity is associated with increased necroinflammatory activity and severity of the NAS. A better knowledge of the role of LAL may provide new insights into the pathogenesis and progression of MAFLD.

Fully Automated Optical Hematocrit Measurement From Dried Blood Spots.

The impact of the hematocrit (HCT) on the dried blood spot's (DBS) spreading area is one of the most important hurdles which prevents the full acceptance of quantitative microsampling strategies. Several destructive- and non-destructive strategies to assess the HCT from a DBS post-sampling have been presented. Unfortunately, the current methods are either labor-intensive, require a complicated algorithm, or are not automatable. Here, we present a novel setup that permits the fully automated reflectance analysis to measure the HCT from a DBS. The underlying principle is based on the concept presented by Capiau et al. for the non-destructive single-wavelength measurement of the HCT. The novel module was embedded within the DBS-MS 500 platform to enable high-throughput analysis of hematocrit values in combination with automated DBS extraction. The novel setup was assessed and optimized for the probe to card distance, stability, anti-coagulant, spotting volume, scan number, calibration variability, accuracy, and precision. It showed excellent inter-day (≤3.7%) and intra-day (≤1.16%) precision, as well as high accuracy when analyzing authentic samples 101%±7% (range:87%-127%). Besides, the simple and straightforward application of an HCT correction for DBS was demonstrated during a pharmacokinetic study with diclofenac involving three subjects. Thereby, the sample's HCT and the HCT impact on the analyte was assessed and compensated. In conclusion, the novel setup enables quantitative analysis of non-volumetric samples in an automated fashion without compromising the concept of cost-effective, minimally invasive sampling.

A feasibility study of metabolic phenotyping of dried blood spot specimens in rural Chinese women exposed to household air pollution.

Exposure-response studies and policy evaluations of household air pollution (HAP) are limited by current methods of exposure assessment which are expensive and burdensome to participants. We collected 152 dried blood spot (DBS) specimens during the heating and non-heating seasons from 53 women who regularly used biomass-burning stoves for cooking and heating. Participants were enrolled in a longitudinal study in China. Untargeted metabolic phenotyping of DBS were generated using ultra-high performance liquid chromatography coupled with mass spectrometry to exemplify measurement precision and assessment for feasibility to detect exposure to HAP, evaluated by season (high pollution vs. low pollution) and measured personal exposure to fine particulate matter <2.5 μm diameters (PM2.5) and black carbon (BC) in the 48-h prior to collecting the DBS specimen. Metabolites e.g., amino acids, acyl-carnitines, lyso-phosphorylcholines, sphinganine, and choline were detected in the DBS specimens. Our approach is capable of detecting the differences in personal exposure to HAP whilst showing high analytical reproducibility, coefficient of variance (CV) <15%, meeting the U.S. Food and Drug Administration criteria. Our results provide a proof of principle that high-resolution metabolic phenotypic data can be generated using a simple DBS extraction method thus suitable for exposure studies in remote, low-resource settings where the collection of serum and plasma is logistically challenging or infeasible. The analytical run time (19 min/specimen) is similar to most global phenotyping methods and therefore suitable for large-scale application.

Analysis of Acrylamide in Dried Blood Spots of Lung Cancer Patients by Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry.

Acrylamide (AA) is a carcinogenic substance found in food, cigarette smoke and in an environment exposed to acrylamide. This study aims to analyze AA levels in dried blood spot (DBS) samples of lung cancer patients with smoking record, without smoking record, and also in the negative blank. Analysis of AA levels was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) and DBS extraction using protein precipitation techniques. Mass detection was done using positive electron spray ionization (ESI) and multiple reaction monitoring (MRM) type with m/z values of 71.99 > 55.23 for acrylamide and m/z 260.16 > 116.04 for propranolol as the internal standard. AA levels in lung cancer patients with smoking record is in the range of 4.670 μg/mL to 11.986 μg/mL. AA levels in lung cancer patients without smoking record is in the range of 2.041 μg/mL to 12.702 μg/mL. Data on AA levels on negative blanks is in the range of 2.72 μg/mL to 3.51 μg/mL. The results of the independent sample t-test (p > 0.05) showed that AA levels in patients with smoking record and those without smoking record did not differ significantly. Then, the Mann-Whitney test was performed between the lung cancer group and the negative blank group and a significant difference was found between the two groups (p < 0.05).

Telomere length analysis from minimally‐invasively collected samples: Methods development and meta‐analysis of the validity of different sampling techniques

Telomeres are the protective caps of chromosomes. They shorten with cell replication, age, and possibly environmental stimuli (eg, infection and stress). Short telomere length (TL) predicts subsequent worse health. Although venous whole blood (VWB) is most commonly used for TL measurement, other, more minimally invasive, sampling techniques are becoming increasingly common due to their field-friendliness, allowing for feasible measurement in low-resource contexts. We conducted statistical validation work for measuring TL in dried blood spots (DBS) and incorporated our results into a meta-analysis evaluating minimally invasive sampling techniques to measure TL. We isolated DNA extracts from DBS using a modified extraction protocol and tested how they endured different shipping conditions and long-term cryostorage. We then included our in-house DBS TL validation statistics (correlation values with VWB TL and age) in a series of meta-analyses of results from 24 other studies that published similar associations for values between TL measured in DBS, saliva, and buccal cells. Our modified DBS extraction technique produced DNA yields that were roughly twice as large as previously recorded. Partially extracted DBS DNA was stable for 7 days at room temperature, and still provided reliable TL measurements, as determined by external validation statistics. In our meta-analysis, DBS TL had the highest external validity, followed by saliva, and then buccal cells-possibly reflecting similarities/differences in cellular composition vs VWB. DBS DNA is the best proxy for VWB from the three minimally-invasively specimen types evaluated and can be used to expand TL research to diverse settings and populations.

Evaluating an easy sampling method using dried blood spots to determine vedolizumab concentrations

An association between vedolizumab (VDZ) trough concentrations and therapeutic outcome has been observed in patients with inflammatory bowel diseases. VDZ samples are typically collected via venous sampling for therapeutic drug monitoring (TDM), but can alternatively be collected via dried blood spot (DBS) samples, which can be used for intensive sampling to investigate pharmacokinetic profiles. Therefore, we have developed a DBS method for determining VDZ concentrations and validated this method by comparing VDZ measurements in paired DBS and venous patient samples. First, VDZ was spiked in citrated whole blood and spotted on filter paper. After drying, DBS were extracted and VDZ concentrations were determined in the extracts using ELISA. For clinical validation, 41 paired DBS and serum samples were collected from 19 VDZ-treated patients. VDZ concentrations measured in DBS extracts strongly correlated with serum concentrations (Pearson r = 0.978, p < 0.0001). No significant impact of the hematocrit value was observed on the VDZ DBS/serum concentration ratios. Additionally, the VDZ DBS/serum ratio was calculated in nine individual patients, which was, in general, shown to be stable over time. VDZ DBS sampling is a robust method that can be used as an alternative to venous blood collection for TDM of VDZ. VDZ concentrations in DBS highly correlated with VDZ serum concentrations over a broad concentration range, allowing DBS to be used for intensive sampling to gain more insight in VDZ pharmacokinetics.

Paper-based molecularly imprinted-interpenetrating polymer network for on-spot collection and microextraction of dried blood spots for capillary electrophoresis determination of carbamazepine.

Carbamazepine is an antiepileptic drug with a narrow therapeutic index, which requires an efficient method for blood level monitoring. Finger-prick dried blood spot (DBS) collection is an alternative microsampling technique, which is less invasive than conventional venipuncture. Paper-based molecularly imprinted-interpenetrating polymer networks (MI-IPN) were developed as blood collection devices, which allowed for selective on-spot microextraction of carbamazepine from DBS. A hybrid of homogeneous polystyrene and silica gel polymer was synthesized and coated on a Whatman® Grade 1 filter paper. Proteins and other interferences in the blood samples were eliminated by using the MI-IPN collection devices, and the resulting DBS extracts were suitable for direct injection into the capillary electrophoretic instrument. The lower limit of quantitation of 4μg/mL in capillary blood was achieved by the sweeping-micellar electrokinetic chromatography method using a KCl-containing matrix, which was sufficient for the therapeutic drug monitoring purposes. Method accuracies were in the range of 88.4 ± 4.5% to 94.5 ± 2.7% with RSD values ≤ 5.1%. The developed paper-based MI-IPN provided superior extraction efficiencies (92.2 ± 2.5%) in comparison with commercially available DBS collection cards, i.e., Whatman® 903 protein saver card (59.8 ± 2.8%) and GenCollect™ 2.0 card (47.2 ± 1.4%). The paper-based MI-IPN devices for DBS collection and on-spot extraction were characterized by simple fabrication, low costs, disposability, and reduction in sample preparation steps, and their further developments might open new perspectives in clinical applications, such as in therapeutic drug monitoring. Graphical abstract.

P601 Development and validation of dried blood spot sampling as a tool to identify the best time point to measure predictive ustekinumab serum concentrations in patients with Crohn’s disease

Abstract Background Therapeutic drug monitoring of ustekinumab (UST), a monoclonal antibody directed against the p40 subunit of IL12/23, can serve as a tool to identify underexposed Crohn’s disease (CD) patients, as UST concentrations have been linked to treatment response. Drug concentrations are most often measured when a patient is losing response. To reduce the risk of loss of response, drug concentrations could be measured upfront to predict the chance of achieving response later on and optimising treatment if necessary. Identification of the optimal time point to measure UST levels that have a predictive value for long-term outcome requires studies with multiple sampling. Dried blood spot (DBS) sampling allows convenient and remote collection of a blood drop through a small finger prick. The current study aimed to develop and validate the DBS method in order to identify the best time point to measure UST concentrations to predict long-term outcome in CD patients and explore the pharmacokinetic profile. Methods UST concentrations (0.2–80 μg/ml) were spiked in citrated whole blood and 40 μl was spotted onto protein saver cards. After punching a 6mm disc, DBS was extracted and the UST concentration was measured with an in-house developed MA-UST56A2D11/MA-UST56C1H12 ELISA. The extraction efficiency, accuracy, precision and the effect of anti-UST antibodies on UST detection were evaluated. Additionally, the effect of the spotted blood volume, the stability of the DBS card at room temperature (RT) and DBS extract at -20°C was examined. To evaluate the correlation between UST in DBS extracts and serum, DBS and serum were simultaneously collected from 8 UST-treated CD patients at two different time points. Results Spiking UST to citrated whole blood and subsequent spotting and extraction revealed an average extraction efficiency of 65 ± 9% (n = 11). The accuracy and imprecision of all concentrations tested were 92–109% and 11–18%, respectively. Addition of anti-UST antibodies to spiked UST samples caused a similar decrease in UST concentration in DBS extracts as in serum. Spotted blood volumes between 15 and 50 μl demonstrated similar recoveries. Storing the DBS card at RT for up to 2 weeks and DBS extract at -20°C for 2 months did not impair recovery. UST concentrations in DBS extracts (range: 0.55–12.1 μg/ml) from CD patients correlated strongly with those in serum (Pearson r = 0.982, p &amp;lt; 0.0001). Conclusion DBS is a robust method that facilitates multiple sampling for the determination of UST concentrations in CD patients. To explore the pharmacokinetic profile of UST and identify the best time point during induction to predict the long-term outcome, a prospective study in which multiple DBS are collected in active CD patients initiating UST is warranted.