EPO transgene detection in dried blood spots for antidoping application

The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-μl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-μl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-μl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.     

Optimizing detection of erythropoietin receptor agonists from dried blood spots for anti-doping application

The World Anti-Doping Agency (WADA) has recently implemented dried blood spots (DBSs) as a matrix for doping control. However, specifications regarding the analysis of the class of prohibited substances called erythropoietin (EPO) receptor agonists (ERAs) from DBSs are not yet described. The aim of this study was to find optimal conditions (sample volume and storage) to sensitively detect endogenous erythropoietin (hEPO) and prohibited ERAs from DBSs and compare detection limits to WADA-stipulated minimum required performance levels (MRPLs) for ERAs in serum/plasma samples. Venous whole blood was spotted onto Whatman 903 DBS cards with primarily 60 μl of blood, but various volumes from 20 to75 μl were tested. All samples were immunopurified with MAIIA EPO Purification Gel kit (EPGK) and analysed with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western blot. Sixty-microliter DBSs allowed the detection of the four main ERAs (BRP, NESP, CERA and EPO-Fc) at concentrations close to WADA's MRPLs described for 500 μl of serum/plasma. Different storage temperatures, from −20°C to 37°C, were evaluated and did not affect ERA detection. A comparison of the detection of endogenous EPO from the different anti-doping matrices (urine, serum and DBSs produced from upper arm capillary blood) from five participants for 6 weeks was performed. Endogenous EPO extracted from DBSs showed intra-individual variations in male and female subjects, but less than in urine. Doping controls would benefit from the stability of ERAs on DBSs: It can be a complementary matrix for ERA analysis, particularly in the absence of EPO signals in urine.     

No pain,just gain: Painless,easy, and fast dried blood spot collection from fingertip and upper arm in doping control

This study aimed to determine and compare the perception, painfulness, and usability of the minimally invasive dried blood spot (DBS) collections from fingertip versus upper arm from different athlete populations: males and females representing sports dependent on hand/arm, sports less dependent on hand/arm and para-athletes. To accomplish this, 108 national level athletes from Denmark were recruited (♀ = 49, ♂ = 59, 25 ± 6 years; mean ± SD) and 11 Doping Control Officers (DCOs) collected manual fingerprick DBS (HemaSpot HF) and automated upper-arm DBS (Tasso-M20) from each athlete. Athletes and DCOs responded to questionnaires regarding the perception of sample collection procedures. On a 0–10 scale, the athletes reported a low pain score and a very good general experience for both sampling sites, but following upper-arm DBS collection, the associated pain was rated lower (−0.4 ± 1.6, p < 0.05), and the general experience rated better (+0.6 ± 2.3, p ≤ 0.001) than after the fingerprick DBS collection. The DCOs rated the general experience with the upper-arm DBS collection better (+1.6 ± 1.1, p ≤ 0.01) than the fingerprick DBS collection, partly because problems occurred more frequently during the DBS collection from the fingertip (28%) than from the upper arm (6%). In conclusion, it appears that DBS sampling is affiliated with minimal sensation of pain and is preferred by both DCOs and athletes, independent of gender and discipline, over conventional sample collection methods. Collection of DBS from the upper arm was preferred over fingerprick by both athletes and DCOs.     

干血点采样技术和干血浆采样技术在麦考酚酸药动学分析中的应用

目的探讨干血点采样技术（dried blood spots,DBS）和干血浆采样技术（dried plasma spots,DPS）在麦考酚酸药动学分析中的应用。方法色谱柱为Ecosil C18（150mm×4.6mm,5μm）,流动相为0.01moL/L甲酸水（0.1%甲酸）-乙腈-甲醇（25：45：30）,体积流量为0.4mL/min,柱温为30℃,进样量10μL;质谱检测采用ESI离子源,正离子MRM方式检测;考察可能的影响因素并进行临床研究。结果 DBS法和DPS法方法学和临床实验数据均良好。结论 DBS方法和DPS法较一般处理方法简便,用血量少,储存运输方便,在一些药物的药动学分析中能代替一般的处理方法。     

干血点采样技术和干血浆采样技术在麦考酚酸药动学分析中的应用

目的 探讨干血点采样技术（dried blood spots,DBS）和干血浆采样技术（dried plasma spots,DPS）在麦考酚酸药代动力学分析中的应用。方法 色谱柱为Ecosil C₁₈（150 mm×4.6 mm,5 μm）,流动相为0.01 moL/L甲酸水（0.1%甲酸）- 乙腈-甲醇（25∶45∶30）,体积流量为0.4 mL/min,柱温为30 ℃,进样量10 μL;质谱检测采用ESI离子源,正离子MRM方式检测;考察可能的影响因素并进行临床研究。结果 DBS法和DPS法方法学和临床实验数据均良好。结论 DBS方法和DPS法较一般处理方法简便,用血量少,储存运输方便,在一些药物的药动学分析中能代替一般的处理方法。     

Current and future applications of dried blood spots in viral disease management

Almost five decades after their first application in diagnostics, dried blood spot (DBS) cards remain to be of key interest in many research areas and clinical applications. The advantages of sample stability during transport and storage, can now be combined with the high sensitivity of novel diagnostic techniques for the measurement and analysis of nucleic acids, proteins and small molecules which may overcome the limitations of the small samples sizes in DBS cards. Here we present a survey of the literature on the use of DBS cards for diagnosis, monitoring and epidemiological studies of virus infections other than HIV, including CMV, HBV, HCV, HAV, HEV, HTLV, EBV, HSV, measles-, rubella- and dengue-virus. The minimal invasiveness of sampling and the relative ease of handling and storing DBS cards is expected to offer additional opportunities to measure and analyze biomarkers of viral disease in resource poor settings or when limited amount of blood can be obtained. Large retrospective studies of virus infections in newborns using stored DBS cards have already been undertaken for screening of congenital infections. In addition, DBS cards have been used prospectively for prevalence studies, outbreak surveillance, mass screening for viral infections, follow-up of chronic infection and its treatment in resource-limited areas. We do not expect that current wet sampling techniques of plasma or serum will be replaced by DBS sampling but it allows extension of sampling in persons and settings that are currently difficult to access or that lack suitable storage facilities. In conclusion, DBS card sampling and storage will aid adequate outbreak management of existing and emerging viral diseases.     

Single‐dose administration of clenbuterol is detectable in dried blood spots

Clenbuterol is a β₂‐agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time‐consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 μg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post‐ingestion, with additional urine collections on days 7 and 10. Using LC–MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post‐ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7–10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 μg.     

Rapid assay of topiramate in dried blood spots by a new liquid chromatography-tandem mass spectrometric method

Topiramate (TPM) is a new antiepiletic drug with efficacy in several types of seizures. Therapeutic drug monitoring of TPM is essential for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate the TPM levels during the treatment. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. Performance comparison between this method and the commercially available fluorescence-polarization immunoassay (FPIA) was made.The analysis was performed in selected reaction monitoring (SRM) mode. The calibration curve in matrix using D₁₂-topiramate was linear in the concentration range of 0.0166–1.66 mg/L (0.5–50 mg/L in DBS) of topiramate with correlation coefficient value of 0.9985. In the concentration range of 0.5–50 mg/L, the coefficients of variation in DBS were in the range 2.13–11.85% and the accuracy ranged from 93.93% to 110.67%.There was no significant differences between the concentrations (ranging 0.5–50 mg/L) measured both FPIA on venous samples and LC-MS/MS assay on simultaneous DBS samples.The sensitivity and specificity of tandem mass spectrometry allow now high throughput topiramate analysis (the improvement was three times in comparison with FPIA). This new assay has favourable characteristics being highly precise and accurate. FPIA also proved to be precise and accurate, but is not always suitable for the sample collection in neonates in whom obtaining larger blood samples is not convenient or possible.     

滤纸干血斑点法测定苯妥英血药浓度

本文用滤纸干血斑点法测定苯妥英血药浓度是将病人全血直接滴加在定量滤纸上，晾干，制成干血斑点（ＤＢＳ，以便邮寄或递送）。测定时将干血斑点剪下，洗脱，用ＦＰＩＡ法测定。方法学测得回收率为１０３±３％，日内、日间差ＲＳＤ均＜６％。测定１５例病人苯妥英ＤＢＳ浓度，并与全血、血清浓度对照，结果ＤＢＳ与全血浓度无显著性差异（Ｐ＞０．０５），与血清浓度比值为０．６０±０．０６，相关性为ｒ＝０．９７７５。本法简便、快速、准确，样品稳定性好，易于保存，特别适用于偏远地区和当地无条件测苯妥英血药浓度的患者。     

Automated system for on-line desorption of dried blood spots applied to LC/MS/MS pharmacokinetic study of flurbiprofen and its metabolite

This paper illustrates the development of an automated system for the on-line bioanalysis of dried blood spots (on-line DBS). In this way, a prototype was designed for integration into a conventional LC/MS/MS, allowing the successive extraction of 30 DBS toward the analytical system without any sample pretreatment.     

EBF recommendation on the validation of bioanalytical methods for dried blood spots

Over the last few years bioanalysts, pharmacokineticists and clinical investigators have rediscovered the technique of dried blood spots. The revival has provided pharmaceutical R&D a wealth of opportunities to optimize the drug-discovery and development process with respect to animal and patient ethics, new scientific insights and costs savings. On the bioanalytical front, multiple experiments have been performed and a lot of experience has been gained. Nevertheless, the technique still has a number of bioanalytical challenges. The European Bioanalysis Forum discussed the advantages and hurdles of the technique and summarized their current thinking in a recommendation on the validation of bioanalytical methods for dried blood spots, which can be used as a cornerstone for further discussions and experiments.     

On the road of dried blood spot sampling for antidoping tests: Detection of GHRP-2 abuse

Dried blood spots (DBSs) sampling is gaining support by the antidoping community because of simplicity and cost-effective characteristics, especially in collection, transport, and storage. Nevertheless, DBS applicability demands specific studies for each of the analytes proposed for testing. Here, GHRP-2 has been selected as a representing member of the growth hormone-releasing peptides (GHRPs) family to provide further evidence of DBS suitability for GHRPs abuse detection in sport testing. An analytical procedure to extract GHRP-2 and its main metabolite (AA-3) from DBS and to detect them by liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed. The method has been validated for the detection of GHRP-2. Specificity and identification capabilities have been assessed in agreement with antidoping guidelines. The low AA-3 levels found in DBS samples prevented its effective application for the determination of this metabolite. The limit of detection (LoD) for GHRP-2 has been established at 50 pg/ml. Long-term stability (>2 years) has been confirmed. The procedure has been successfully applied to actual DBS samples from an administration study with a single intravenous dose of GHRP-2 (100 μg) being detected up to 4 h after drug injection. GHRP-2 concentrations have been higher in venous blood DBS than in capillary blood DBS. Despite the observed differences, a similar detection window has been achieved independently of the type of blood used. In summary, this study provides specific evidence supporting DBS usefulness to detect GHRP-2, and potentially other GHRPs family members, for antidoping tests.     

干血点采样技术及其在药动学研究中的应用

2011年8月3日,美国食品药品管理局(FDA)通告:孕妇在妊娠早期长期大剂量(400～800mg/d)使用抗真菌药氟康唑(商品名Diflucan,大扶康)可能发生罕见的、明显的婴儿出生缺陷。同时声明,氟康唑单次、低剂量(150mg)治疗念珠菌引起的阴道宫颈感染,不会发生该风险。氟康唑为氟代三唑类广谱抗真菌药物,1990年在美国上市,主要用于各种念珠菌、隐球菌病及各种真菌引起的脑膜炎及艾滋病患者口腔、     

Development and clinical applications of the dried blood spot method for therapeutic drug monitoring of anti‐epileptic drugs

Anti‐epileptic drugs (AEDs) have various pharmacokinetic profiles, inter‐individual variabilities, high possibilities of drug‐drug interactions and narrow therapeutic indices. To provide optimal treatment for patients, therapeutic drug monitoring (TDM) is necessary. However, TDM requires sufficient quantities of blood samples to measure drug concentrations. Therefore, TDM could be a burden, particularly in paediatric cases. A good alternative that overcomes these disadvantages is the dried blood spot (DBS) method, which is simple, convenient to use and less invasive, requiring a lower quantity of blood than traditional blood sampling methods. However, the DBS method is affected by haematocrit (Hct) levels to varying extents depending on the drug properties. In addition, different papers with varying characteristics are available for use when applying the DBS method. Therefore, it has not yet been applied to TDM in clinical practice. To achieve this, several steps are required, including method development, method validation and clinical validation. Currently, the development status of the DBS method is different for each AED and unclear. Therefore, we assessed the development status of the following 19 AEDs in 26 studies: lamotrigine, valproic acid, levetiracetam, phenytoin, topiramate, carbamazepine, carbamazepine epoxide, gabapentin, phenobarbital, pregabalin, clobazam, clonazepam, ethosuximide, felbamate, monohydroxycarbamazepine, nitrazepam, rufinamide, vigabatrin and zonisamide. Among them, carbamazepine, lamotrigine, topiramate and valproic acid have been developed such that they are nearly available for TDM. In addition, Whatman 903 Protein Saver Cards and concentration analysis by liquid chromatography with triple quadrupole mass spectrometer were used most often.     

The effects of iron injection on blood doping biomarkers in dried blood spots

Iron supplementation is not considered as a doping method; however, it can affect the levels of several biomarkers of the hematologic module of the athlete biological passport (ABP), such as the reticulocyte percentage (%RET) and hemoglobin (HGB) level. Thus, iron injection could be a confounding factor in antidoping analyses. Previous studies have suggested that the HGB level and the expression levels of reticulocyte-related-mRNAs, such as 5′-aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1), could be promising biomarkers for the ABP and detectable in dried blood spots (DBSs). Therefore, in this study, we examined the impact of iron injection on the levels of these potential biomarkers in DBSs. Reticulocyte-related-mRNAs analyses were performed by RT-qPCR. Ferritin level in DBS was measured with enzyme-linked immunosorbent assay (ELISA) method. Notably, there were no significant effects of iron supplementation on the levels of ALAS2 and CA1 mRNAs but by contrast, the %RET and immature reticulocyte fraction (IRF) measured in whole blood increased significantly following iron injection. As expected, iron supplementation increased the ferritin level significantly in both serum and DBS samples. In conclusion, these findings reinforce the specificity of reticulocyte-related mRNAs in DBSs as biomarkers of blood doping to target in antidoping analyses.