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Background: In developing countries, diarrhoea is the most common cause of death for children under five years of age, with Giardia lamblia, Cryptosporidium and Entamoeba histolytica as the most frequent pathogenic parasites. Traditional microscopy for stool parasites has poor sensitivity and specificity, while new molecular methods may provide more accurate diagnostics. In poor regions with sample storage hampered by uncertain electricity supply, research would benefit from a method capable of analysing dried stools. Methods: A real-time multiplex PCR method with internal inhibition control was developed for detecting Giardia lamblia, Cryptosporidium hominis/parvum and Entamoeba histolytica directly from stool specimens. Applicability to dried samples was checked by comparing with fresh ones in a small test material. Finally, the assay was applied to dried specimens collected from Guinea-Bissauan children with diarrhoea. Results: The PCR's analytical sensitivity limit was 0.1 ng/ml for G. lamblia DNA, 0.01 ng/ml for E. histolytica DNA and 0.1 ng/ml for Cryptosporidium sp. In the test material, the assay performed similarly with fresh and dried stools. Of the 52 Guinea-Bissauan samples, local microscopy revealed a parasite in 15%, while PCR detected 62% positive for at least one parasite: 44% of the dried samples had Giardia, 23% Cryptosporidium and 0% E. histolytica. Conclusions: Our new multiplex real-time PCR for protozoa presents a sensitive method applicable to dried samples. As proof of concept, it worked well on stools collected from Guinea-Bissauan children with diarrhoea. It provides an epidemiological tool for analysing dried specimens from regions poor in resources.

Background Dried blood spots (DBSs) could allow patients to prepare their own samples at home and send them to the laboratory for therapeutic drug monitoring (TDM) of immunosuppressants. The purpose of this review is to provide an overview of the current knowledge about the impact of DBS-related preanalytical factors on TDM of tacrolimus, sirolimus and everolimus. Content Blood spot volume, blood spot inhomogeneity, stability of analytes in DBS and hematocrit (Hct) effects are considered important DBS-related preanalytical factors. In addition, the influence of drying time has recently been identified as a noteworthy preanalytical factor. Tacrolimus is not significantly influenced by these factors. Sirolimus and everolimus are more prone to heat degradation and exhibited variations in recovery which were dependent on Hct and drying time. Summary and outlook DBS-related preanalytical factors can have a significant impact on TDM for immunosuppressants. Tacrolimus is not significantly influenced by the studied preanalytical factors and is a viable candidate for DBS sampling. For sirolimus and everolimus more validation of preanalytical factors is needed. In particular, drying conditions need to be examined further, as current protocols may mask Hct-dependent effects on recovery. Further validation is also necessary for home-based self-sampling of immunosuppressants as the sampling quality is variable.

We present a technique for the fast screening of the lead concentration in whole blood samples using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The whole blood sample is deposited on a polymeric surface and wiped across a set of micro-grooves previously engraved into the surface. The engraving of the micro-grooves was accomplished with the same laser system used for LA-ICP-MS analysis. In each groove, a part of the liquid blood is trapped, and thus, the sample is divided into sub-aliquots. These aliquots dry quasi instantly and are then investigated by means of LA-ICP-MS. For quantification, external calibration against aqueous standard solutions was relied on, with iron as an internal standard to account for varying volumes of the sample aliquots. The 208Pb/57Fe nuclide ratio used for quantification was obtained via a data treatment protocol so far only used in the context of isotope ratio determination involving transient signals. The method presented here was shown to provide reliable results for Recipe ClinChek® Whole Blood Control levels I–III (nos. 8840–8842), with a repeatability of typically 3 % relative standard deviation (n = 6, for Pb at 442 μg L−1). Spiked and non-spiked real whole blood was analysed as well, and the results were compared with those obtained via dilution and sectorfield ICP-MS. A good agreement between both methods was observed. The detection limit (3 s) for lead in whole blood was established to be 10 μg L−1 for the laser ablation method presented here. Graphical Abstract Micro-grooves are filled with whole blood, dried, and analyzed by laser ablation ICP-mass spectrometry. Notice that the laser moves in perpendicular direction with regard to the micro-grooves Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-9717-3) contains supplementary material, which is available to authorized users.

Background: Detection of potential Fabry disease patients before appearance of life-threatening findings is of great importance, particularly in high-risk populations. This study was designed to determine prevalence of Fabry disease among Turkish patients undergoing peritoneal dialysis and hemodialysis for chronic renal failure (CRF). Methods: A total of 1,527 patients (mean (SD) age: 60.2 (14.2) years, 55.5% were males) on hemodialysis (n = 1,435) or peritoneal dialysis (n = 92) for CRF were included in this multicenter study conducted at 17 dialysis centers across Bursa province, Turkey. Prevalence of the disease was determined using combined enzymatic and genetic strategy with measuring the activity of alpha-galactosidase A (alpha-Gal A) and Sanger sequence analysis based genotyping in alpha-galactosidase A gene (GLA) in dried blood samples (DBS). Results: Overall alpha-Gal A activity was determined to be below the reference value in 130 (8.5%) of 1,527 patients. GLA genotyping confirmed the diagnosis of Fabry disease in 5 (0.3%) patients with low alpha-Gal A activity. All Fabry-positive patients were males corresponding to a 0.6% prevalence of disease in this gender. Conclusion: In conclusion, our findings, which were based on the use of DBS for both enzymatic activity and genotyping analyses, revealed the diagnosis of Fabry disease in 5 males corresponding to overall 0.3% prevalence of disease in the cohort and 0.6% prevalence among males. Our results support the likelihood of unrecognized Fabry disease in a non-negligible number of patients on dialysis and thus emphasize the value of screening studies in terms of detection of new cases and improved prognosis of the disease via early diagnosis and treatment. Sanofi-Aventis - Genzyme Corporation

About a century after its first described application by Ivar Bang, the potential of sampling via dried blood spots (DBS) as an alternative for classical venous blood sampling is increasingly recognized. Perhaps best known is the use of DBS in newborn screening programs, ignited by the hallmark paper by Guthrie and Susi half a century ago. However, it is only recently that both academia and industry have recognized the many advantages that DBS sampling may offer for bioanalytical purposes, as reflected by the strong increase in published reports during the last few years. Currently, major DBS applications include newborn screening for metabolic disorders, epidemiological surveys (e. g. HIV monitoring), therapeutic drug monitoring (TDM), as well as toxicology. In this review, we provide a comprehensive overview of the distinct subdisciplines of toxicology for which DBS sampling has been applied. DBS sampling for toxicological evaluation has been performed from birth until autopsy, aiming at the assessment of therapeutic drugs, drugs of abuse, environmental contaminants, toxins, as well as (trace) elements, with applications situated in fields as toxicokinetics, epidemiology and environmental and forensic toxicology. We discuss the strengths and limitations of DBS in the different subdisciplines and provide future prospects for the use of this promising sampling technique in toxicology.

Tuberculosis (TB) is a high-burden infectious disease, especially in low and middle-income countries. The efforts to eliminate this disease are challenged by the emergence of multidrug resistance and TB-HIV coinfection. The cumulative knowledge on pharmacokinetics/pharmacodynamics of antituberculosis agents has recently encouraged therapeutic drug monitoring (TDM) in patient care. However, logistical problems related to conventional sampling limit the application of TDM in research-oriented institutions. Dried blood spot (DBS) compared with conventional venous blood sampling has the advantages of easier sampling, storage and transportation, thus enabling the application of TDM even in remote areas. In addition, DBS with its lower biohazardous risk can be safely performed in a high HIV prevalence area, which also tends to have a high TB burden. Another benefit of DBS sampling is that it requires a smaller blood volume than conventional sampling and is highly recommended for application in pediatric TB. A limitation of DBS is that additional considerations are required for analysis, method development and validation. The accuracy of the DBS method is influenced by a number of factors that need to be thoroughly examined in method development and validation. Further, the agreement between DBS and plasma/serum concentrations is not always understood and further investigations are required

Moxifloxacin (MFX) is a potential oral agent use in the treatment of multidrug-resistance tuberculosis (MDR-TB). Due to variability in pharmacokinetics and in vitro susceptibility of causative bacteria, therapeutic drug monitoring (TDM) of MFX is recommended. Conventional plasma sampling for TDM is facing logistical challenges, especially in limited resource areas, and dried blood spots (DBS) sampling may offer a chance to overcome this problem. The objective of this study was to develop a LC-MS/MS method for determination of MFX in dried blood spots (DBS) that is applicable for TDM. The influence of paper type, the hematocrit (Hct) and the blood volume per spot (V(b)) on the estimated blood volume in a disc (V(est)) was investigated. The extracts of 8mm diameter discs punched out from DBS were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS) with cyanoimipramin as internal standard. The method was validated with respect to selectivity, linearity, accuracy, precision, sensitivity, recovery and stability. The effect of Hct and V(b) on LC-MS/MS analytical result was also investigated. The relationship between MFX concentrations in venous and finger prick DBS and those in plasma was clinically explored. V(est) was highly influenced by Hct while the effect of V(b) appeared to be different among paper types. Calibration curves were linear in the range of 0.05-6.00 mg/L with inter-day and intra-day precisions and biases of less than 11.1%. The recovery was 84.5, 85.1 and 92.6% in response to blood concentration of 0.15, 2.50 and 5.00 mg/L, respectively. A matrix effect of less than 11.9% was observed. MFX in DBS was stable for at least 4 weeks at room condition (temperature of 25°C and humidity of 50%). A large range of Hct value produced a significant analytical bias and it can be corrected with resulting DBS size. A good correlation between DBS and plasma concentrations was observed and comparable results between venous DBS and finger prick DBS was attained. This fully validated method is suitable for determination of MFX in dried blood spot and applicable for TDM.

SUMMARY Congenital infection with cytomegalovirus (CMV) is the main cause of sensorineural hearing loss and psychomotor impairment which can develop at birth or later in infant’s life. Because of a lack of nation-wide serological screening for pregnant women and accepted antiviral therapy during pregnancy in a high seroprevalence rate population of Poland, we introduced the regional screening programme for CMV infection in neonates from the Poznan Province to diminish a risk of the symptomatic disease. The aims of the study were: (i) to determine the prevalence of specific anti-CMV antibodies in populations of pregnant women and newborns from the Poznan area, (ii) to increase the early postnatal detection of congenital CMV infections, and (iii) to evaluate a risk of perinatal or early postnatal infections with CMV in newborns or infants born to seropositive mothers. Serological testing was performed in 4.192 live born neonates, using dried blood filter-paper specimens. The seropositivity rate in the studied population of neonates and pregnant women was 78.6%. The incidence of perinatal and early postnatal CMV infections was evaluated to be 3.1% or 1 per 25 neonates born to seropositive mothers. Congenital CMV infections confirmed by a presence of specific IgM antibodies were diagnosed in 5 newborns, which represent 1 case per 838 successive deliveries. In a clinical pattern of cytomegalovirus disease respiratory disorders and low birth weight were most frequently observed, and neurological signs, hepatitis, haemorrhagies or jaundice were sporadically diagnosed. Implementation of mass immunodiagnostic screening for congenital CMV infection, combined with other obligatory neonatal tests for metabolic errors, congenital malformations and endocrine disorders seems to be a valuable third line prophylactic strategy to prevent a late development of clinically overt cytomegalovirus disease.

SUMMARY The strategic approach for preventing congenital toxoplasmosis is strictly related to the incidence of primary T. gondii infection during pregnancy in a studied population. Early postnatal diagnosis by mass testing of newborns is an option in areas where obligatory serological screening in pregnant women has not been implemented but it requires sensitive immunodiagnostic methods followed by a good confirmatory analysis. The aims of the regional neonatal screening programme were (i) analysis of the prevalence of congenital T. gondii infection at birth in the West Poland Province, (ii) determination of the value of the serological examination of filter-paper blood specimens collected at birth for the diagnosis of congenital toxoplasmosis, and (iii) evaluation of the duration of T. gondii-specific immunoglobulin A and immunoglobulin M antibodies in infants’ sera. The neonates born in the obstetric clinics of the University Gynaecology-Obstetrics Hospital in Poznan (Poland) and in the maternity wards of the 10 main district hospitals from the West Poland region were systematically screened for congenital T. gondii infection. Peripheral blood from newborns was collected by a non-invasive heel-stick puncture during the first 3 days of life, absorbed onto Guthrie cards and analysed for anti-T. gondii specific IgM (1996-1998) or both IgA and IgM antibodies (1998-2000) by non-commercial immunocapture ELISAs. When the screening result was positive, the diagnosis of congenital infection was confirmed by testing serum samples from the suspected neonate and the mother using a Western blot IgM-IgG comparative immunological profile analysis and traditional serological techniques (ELISA, ISAGA) for anti-Toxoplasma IgA, IgM and IgG specific antibodies. From June 1996 to April 2000, 45,169 filter-paper specimens from liveborn neonates were screened: 27,516 samples were tested for specific IgM and the next 17,653 Guthrie cards were analysed by the combined IgA/IgM assay. The prevalence of anti-Toxoplasma IgM in filter-paper eluates at birth was 1 per 2,117 liveborn neonates (0.47/1000) or 1 per 1,185 infants (0.84/1000) born to seronegative women with a potential risk of primary T. gondii infection during pregnancy. For the joint detection of IgA and IgM, these values significantly increased to 1 per 929 neonates (1.08/1000) or 1 per 520 pregnancies at risk (1.92/1000) respectively, comparing to the seropositivity rate of 43.7% in a pregnant women population in the studied area. In newborns untreated prenatally, the diagnostic sensitivity of the IgM ELISA using neonatal Guthrie cards was not more than 86.7% and that of the combined IgA/IgM ELISA was 95%; the diagnostic specificity of the both methods was calculated to be 99.9%. Congenital T. gondii infection was finally diagnosed in 35 neonates, mostly asymptomatic at birth. Conclusions: (i) The neonatal screening for anti-Toxoplasma IgA and/or IgM antibodies is a good sensitivity method for an early postnatal diagnosis of congenital toxoplasmosis in newborns untreated prenatally. (ii) In the absence of obligatory nation-wide screening during pregnancy followed by an early prenatal treatment, this valuable technique may be considered a preventive option in areas of a high annual number of births associated with a high seroprevalence of T. gondii infection.

Background: The relation between hematocrit, substance concentration, extraction recovery and spot formation of tacrolimus, sirolimus, everolimus, ascomycin, temsirolimus and cyclosporin A was investigated for Whatman 31 ET CHR, Whatman FTA DMPK-C, Whatman 903, Perkin Elmer 226 and Agilent Bond Elut DMS DBS cards. Results & Discussion: We found that all DBS cards showed the same hematocrit and concentration-dependent recovery patterns for sirolimus, everolimus and temsirolimus. At high concentrations, the total hematocrit effects were much more pronounced than at low concentrations for tacrolimus, sirolimus, everolimus, ascomycin and temsirolimus. Conclusion: The tested card types showed differences in performance, especially at extreme concentrations and hematocrit values. It may be useful to investigate the performance of different types of DBS cards prior to analytical method validation.

In vivo studies of hepatic carbohydrate metabolism in (genetically modified) conscious mice are hampered by limitations of blood and urine sample sizes. We developed and validated methods to quantify stable isotope dilution and incorporation in small blood and urine samples spotted onto filter paper. Blood glucose and urinary paracetamol-glucuronic acid were extracted from filter paper spots reproducibly and with high yield. Fractional isotopomer distributions of glucose and paracetamol-glucuronic acid when extracted from filter paper spots were almost identical to those isolated from the original body fluids. Rates of infusion of labeled compounds could be adjusted without perturbing hepatic glucose metabolism. This approach was used in mice to find the optimal metabolic condition for the study of hepatic carbohydrate metabolism. In fed mice, no isotopic steady state was observed during a 6-h label-infusion experiment. In 9-h-fasted mice, isotopic steady state was reached after 3 h of label infusion and important parameters in hepatic glucose metabolism could be calculated. The rate of de novo glucose-6-phosphate synthesis was 143 +/- 17 mumol kg(-1) min(-1) and partitioning to plasma glucose was 79.0 +/- 5.2%. In 24-h-fasted mice, abrupt changes were noticed in whole body and in hepatic glucose metabolism at the end of the experiment. (C) 2003 Elsevier Inc. All rights reserved.

Introduction: Rifampicin (RIF) and clarithromycin (CLR) are common drugs for the treatment of infections like Mycobacterium tuberculosis and Mycobacterium ulcerans. Treatment for these diseases are long-term and the individual pharmacokinetic variation, drug-drug interactions or non-adherence may introduce sub-therapeutic exposure or toxicity. The application of therapeutic drug monitoring (TDM) can be used to ensure efficacy and avoid toxicity. With the use of dried blood spot (DBS), TDM may be feasible in rural areas. During DBS method development, unexpected interactions or matrix effects may be encountered due to endogenous components in the blood. Another complication compared to plasma analysis is that RIF can form chelate complexes with ferric ions or can bind with hemes, which are potentially present in the extracts of dried blood spots.Methods: The investigation focused on the interaction between RIF and the endogenous components of the DBS. The use of ethylenediaminetetraacetic acid (EDTA) and deferoxamine (DFX) as chelator agents to improve recoveries and matrix effects were investigated. A rapid analytical method was developed and validated to quantify RIF and CLR and their active metabolites desacetyl rifampicin (DAc-RIF) and 14-hydroxyclatythromcin (14OH-CLR) in DOS samples. A clinical application study was performed in tuberculosis patients by comparing DBS concentrations with plasma concentrations.Results: The interaction between RIF and the DBS matrix was avoided using the complexing agents EDTA and DFX, which improved recoveries and matrix effects. The developed sample procedure resulted in a simple and fast method for the simultaneous quantification of RIF, CLR and their metabolites in DOS samples. High stability was observed as all four substances were stable at ambient temperature for 2 months. Deming regression analysis of the clinical application study showed no significant differences for RIF, DAc-RIF, CLR and 14OH-CLR between patient plasma and DBS analysis. The slopes of the correlation lines between DBS and plasma concentrations of RIF, DAc-RIF, CLR and 14OH-CLR were 0.90, 0.99, 0.80 and 1.09 respectively. High correlations between plasma and DBS concentrations were observed for RIF (R-2=0.9076), CLR (R-2=0.9752) and 14OH-CLR (R-2=0.9421). Lower correlation was found for DAc-RIF (R-2 of 0.6856).Conclusion: The validated method is applicable for TDM of RIF, CLR and their active metabolites. The stability of the DBS at high temperatures can facilitate the TDM and pharmacokinetic studies of RIF and CLR even in resource limited areas. The role of EDTA and DFX as complexing agents in the extraction was well investigated and may provide a solution for potential applications to other DBS analytical methods. (C) 2013 Elsevier B.V. All rights reserved.

Background: This study evaluates HbA1c measurements from dried blood spots collected on filter paper and compares HbA1c from filter paper (capillary blood) with HbA1c measured in venous blood.Methods: Patient satisfaction was evaluated using a questionnaire. The performance with the filter paper method was assessed by comparing HbA1c results from EDTA-blood samples obtained via dried blood spots with HbA1c results obtained with freshly hemolyzed blood (routine HbA1c). Adult patients visiting the outpatient clinic for HbA1c analyses were recruited for the evaluation of dried blood spot sampling at home. Laboratory personnel collected a capillary blood sample on filter paper as well as a venous EDTA-blood sample. The participants collected another capillary blood sample at home and sent the dried filter paper back to the laboratory. Samples were analyzed with an immunoturbidimetric assay.Results: Between-filter coefficient of variation was 1.8%. Filter paper HbA1c increased slightly during storage, particularly during the first 5 days. Filter paper HbA1c highly correlated with routine HbA1c (r=0.987). The evaluation of samples collected at home showed comparable HbA1c values by filter paper and routine sampling methods (n=93). Eighty-three percent of participants said they would like the filter method to be brought into practice.Conclusions: Home HbA1c sampling on filter paper is an acceptable sampling alternative for analysis of HbA1c. Clin Chem Lab Med 2009;47:1259-64.

Fe oxide-impregnated paper (Pi-paper) is used as an artificial Phosphorus (P) sink to study P availability in soils and runoff. Pi-papers were introduced as they would mimic P uptake by plant roots by decreasing the P concentration in solution to negligibly low levels and thus enhancing P desorption from the soils solid phase. The rate of transfer of P from soil to Pi-paper would thus be limited by the soil P desorption rate. The maximum desorption rate is indeed achieved when the original method is used in which (at least) four Pi-papers per gram soil are placed in a soil suspension (soil–solution ratio of 0.025 kg L− 1). In several studies this method has however been adapted depending on the research question of interest without investigating the effect of this adaptation to the processes involved in the transfer of P from soil to Pi-paper. The aim of this study is to improve our understanding of the processes that occur in the Pi-paper–solution–soil system and so to extend the theoretical basis of this method. Insight is gained in these processes by comparing the experimentally determined P transfer from soil to Pi-paper with P transfer that is modeled based on the measured P concentration in solution and the (kinetic) Langmuir equation of the Pi-paper. P adsorption by a Pi-paper from standard solutions is not instantaneous but can be described with a kinetic Langmuir equation that is a characteristic of the Pi-paper. Over time, the Pi-paper reaches equilibrium with the solution, and the kinetic Langmuir equation can be rewritten to a Langmuir equation. Regardless if there is equilibrium or not, P adsorption to the Pi-paper is a function of the P concentration in solution. By adding Pi-paper to a soil suspension, a re-distribution of P takes place between the reactive surface area of the soil and the new reactive surface area of the Pi-paper that initially contains no adsorbed phosphate. As opposed to the original method where the desorption rate was limiting the overall P transfer, the adsorption rate to the Pi-paper is limiting when one Pi-paper per gram soil is placed in a soil suspension (soil–solution ratio of 0.1 kg L− 1). In this situation, the P concentration in solution is found to be in equilibrium with the soil's solid phase. With increasing contact time (> ~ 24 h) the whole system approaches equilibrium. With each successive Pi-paper newly added, more P is removed from the soil system and the decrease of P in solution will be governed by the soil P desorption isotherm. Varying the number of Pi-papers and the soil to solution ratio thus has a large effect on the transfer rate between soil and Pi-paper and if either the soil desorption rate, the Pi-paper adsorption rate or a combination limits this transfer rate. With increased insight in the P transfer between soil and Pi-paper sink, it becomes possible to tailor the experimental design to help answer the research question one is interested in.

Contains fulltext : 80034.pdf (Publisher’s version ) (Closed access) BACKGROUND: The mucus layer is an important dynamic component of the epithelial barrier. It contains mucin glycoproteins and other compounds secreted by the intestinal epithelium, such as secretory IgA. However, a standardized in vivo sampling technique of mucus in humans is not yet available. AIM: To assess the validity and feasibility of mucin and protein determinations in human colonic mucus collected under physiological conditions. SUBJECTS AND METHODS: Triplicate colonic mucus samples were collected in 11 healthy volunteers using cytology brushes during sigmoidoscopy. As an indication of the quantity of collected mucus, total protein and mucin concentrations were determined by measuring oligosaccharide equivalents and monosaccharides. Also secretory IgA and sialic acid concentrations were determined and proteomic analysis was performed using surface enhanced laser desorption/ionization-time of flight-mass spectrometry. RESULTS: Mean values of secretory IgA and sialic acid corrected for the amount of mucus ranged from 0.16 to 1.81 g secretory IgA/mmol oligosaccharide equivalents and from 12.6 to 48.6g sialic acid/mmol oligosaccharide equivalents. Proteomic analysis of mucus is feasible and cluster analysis showed subject specific profiles. CONCLUSION: Using cytology brushes, human colonic mucus can be sampled and under physiological conditions. These samples could give information on the composition and quality of the mucus layer.