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Einleitung: Die essentiellen Spurenelemente Selen (Se) und Kupfer (Cu) haben Einfluss auf die Schilddrüsen- und Wachstumshormonachse und damit auf Reifungs- und Wachstumsprozesse des menschlichen Körpers. Doch über die Frage, ob tatsächlich ein direkter Zusammenhang von Selen- bzw. Kupferstatus und dem Spiegel von Schilddrüsenhormonen und Wachstumsfaktoren besteht, herrscht Uneinigkeit. Daher wurde in dieser Arbeit untersucht, ob die Serumkonzentrationen von Se und Cu mit der Schilddrüsenhormon- und Wachstumsachse korrelieren. Darüber hinaus scheint Selen eine Rolle bei verschiedenen Erkrankungen der Neugeborenenperiode wie der neonatalen Sepsis und der bronchopulmonalen Dysplasie zu spielen. Bislang wurde bei der Bestimmung des Selenstatus bei Neugeborenen häufig auf Nabelschnurblut zurückgegriffen, welches möglicherweise nicht exakt den kindlichen Status widerspiegelt. Venöse Blutentnahmen werden bei Kindern aufgrund der Schmerzhaftigkeit der Prozedur möglichst vermieden. Dagegen bietet das Aufbringen von Blut auf Filterpapierkarten die Möglichkeit der wenig invasiven Probengewinnung. Daher wurde eine neue Methode zur Bestimmung des Selenspiegels bei Neugeborenen aus Trockenblut (Dried Blood Spots, DBS) entwickelt, welche unter anderem für das Neugeborenen-Screening auf Anwendung getestet werden soll. Methodik: Untersuchung der Selen- und Kupferspiegel im Serum von zwei pädiatrischen Patientenkohorten (84 Kinder mit konnataler Hypothyreose und 66 kleinwüchsige Kinder im Alter von 3 bis 17 Jahren) mittels Total-Reflektions-Röntgenfluoreszenzanalyse (TXRF). Die Zusammenhänge von Spurenelementen und den Schilddrüsenhormonen TSH, T4, fT4, T3 sowie dem Wachstumsfaktor IGF-1 wurden mittels Pearson-Korrelation ermittelt. Zur Entwicklung der Methode Selenbestimmung aus DBS wurden korrespondierende Serumproben mit DBS-Proben von insgesamt 10 erwachsenen freiwilligen Probanden verglichen und verschiedene Parameter der DBS-Aufbereitung für die Spurenelementanalyse optimiert. Ergebnisse: Bei Kindern und Adoleszenten mit konnataler Hypothyreose bestand eine hochsignifikante positive Verbindung von Cu bzw. dem Cu/Se-Quotienten mit der Schilddrüsenhormonachse in Form von T3 und T4 (p ≤ 0,01). Bei Kindern mit Kleinwuchs zeigte sich dieser Zusammenhang nicht. Allerdings war hier eine hochsignifikante inverse Verbindung von Se mit dem Wachstumsfaktor IGF-1 zu verzeichnen (p ≤ 0,01). Es konnte eine geeignete Methode entwickelt werden, um eine Selenmessung mittels TXRF-Analyse aus DBS vorzunehmen, die äquivalent zur Serummessung ist. Die Selenmessungen aus DBS wichen um weniger als 10% von den Serumproben ab, wenn die Probenextraktion aus den DBS mittels einer neu entwickelten HCl- basierten Methode erfolgte. Schlussfolgerungen: Die Ergebnisse dieser Arbeit tragen zu einem besseren Verständnis der Interaktion von Spurenelementen mit der Schilddrüsen- und Wachstumshormonachse bei. Außerdem wurde die Möglichkeit geschaffen eine wenig invasive Bestimmung des Selenstatus aus kleinen, gut lagerbaren Trockenblutproben vorzunehmen. Weitere Untersuchungen in größeren Gruppen und mit anderen klinischen Fragestellungen sind notwendig, um das volle Potential dieser Methode auszuschöpfen., Introduction: The essential trace elements selenium and copper affect the thyroid and growth hormone axis with effects on maturation and growth processes of the human body. However it is controversially discussed whether there is a direct link between the selenium or copper status and the levels of thyroid hormones and growth factors. Therefore, this work investigated whether the serum concentrations correlate. In addition, selenium may have an effect on various diseases of the newborn period such as sepsis and bronchopulmonary dysplasia. So far umbilical cord blood was often used to determine the selenium status in newborns. This may not accurately reflect the child's status. Because of the painful procedure venous blood collection should be avoided in children where possible. In contrast application of blood on filter paper cards is less invasive. Therefore, a new method for determination of selenium levels in newborns from dried blood spots (DBS) was developed. This method was tested to be utilized within the newborn screening. Methods: Study of selenium- and copper levels in serum of two pediatric patient cohorts (84 children with congenital hypothyroidism and 66 children with dwarfism from 3 to 17 years) using total reflection X-ray fluorescence spectroscopy (TXRF). Interactions between trace elements and the thyroid hormones TSH, T4, fT4, T3 and the growth factor IGF-1 were determined by Pearson correlation. To develop the method of selenium measurement from DBS, corresponding serum samples were compared with DBS samples from 10 adult volunteers. Various parameters of DBS treatment for trace element analysis were optimized. Results: Children and adolescents with congenital hypothyroidism showed a highly significant positive correlation between copper or copper/selenium ratio with the thyroid hormone axis (T3 and T4, p ≤ 0.01). In children with dwarfism a highly significant inverse correlation between selenium and IGF-1 was demonstrated (p ≤ 0.01). An accurate method was established to measure selenium using TXRF analysis of DBS, which is equivalent to serum measurements. Measurement of selenium from DBS deviated by less than 10% from serum using the newly developed HCl-based extraction method. Conclusion: The results of this study contribute to a better understanding of the interaction of trace elements with the thyroid and growth hormone axis. In addition, a less invasive method of measuring selenium levels was developed based on small and well storable DBS samples. Further investigations in larger groups and with other clinical issues are necessary in order to elucidate the full potential of the novel method.

Introduction: Lysosomal storage disorders (LSD) are inherited diseases caused, in the majority of them, by the deficiency of lysosomal enzymatic activities. Ob-jectives: We aimed to analyze the usefulness of DBS samples for diagnosis of 4 LSDs, with the availability of a large quantity of patient samples. Design and methods: Blood samples from previously diagnosed patients with Fabry, Gaucher, Hunter, and Maro-teaux-Lamy syndromes and normal control indi-viduals, were collected and dispen-sed in filter paper, and used for enzymatic activity determination. Re-sults: Diagnosis of hemi/homo-zygous patients with Fabry, Hunter and Maroteaux-Lamy diseases using DBS samples showed ideal parameters of 100% sen-sitivity and specificity. DBS assay for Gaucher dis-ease would need a posterior confirmatory step. Con-clusions: Leukocyte measu-rement is the only reli-able way to diagnose Gaucher disease. For Hunter, Fabry and Maroteaux-Lamy disorders discrimina-tion between patients and controls seems adequate by DBS., Laboratorio de Investigaciones del Sistema Inmune

Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)24H] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The ‘contact’ LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS. Graphical Abstract ᅟ

Background In large epidemiological studies it is often challenging to obtain biological samples. Self-sampling by study participants using dried blood spots (DBS) technique has been suggested to overcome this challenge. DBS is a type of biosampling where blood samples are obtained by a finger-prick lancet, blotted and dried on filter paper. However, the feasibility and efficacy of collecting DBS samples from study participants in large-scale epidemiological studies is not known. The aim of the present study was to test the feasibility and response rate of collecting self-sampled DBS and saliva samples in a population–based study of women above 50 years of age. Methods We determined response proportions, number of phone calls to the study center with questions about sampling, and quality of the DBS. We recruited women through a study conducted within the Norwegian Breast Cancer Screening Program. Invitations, instructions and materials were sent to 4,597 women. The data collection took place over a 3 month period in the spring of 2009. Results Response proportions for the collection of DBS and saliva samples were 71.0% (3,263) and 70.9% (3,258), respectively. We received 312 phone calls (7% of the 4,597 women) with questions regarding sampling. Of the 3,263 individuals that returned DBS cards, 3,038 (93.1%) had been packaged and shipped according to instructions. A total of 3,032 DBS samples were sufficient for at least one biomarker analysis (i.e. 92.9% of DBS samples received by the laboratory). 2,418 (74.1%) of the DBS cards received by the laboratory were filled with blood according to the instructions (i.e. 10 completely filled spots with up to 7 punches per spot for up to 70 separate analyses). To assess the quality of the samples, we selected and measured two biomarkers (carotenoids and vitamin D). The biomarker levels were consistent with previous reports. Conclusion Collecting self-sampled DBS and saliva samples through the postal services provides a low cost, effective and feasible alternative in epidemiological studies.

Early life exposures and metabolic programming are associated with later disease risk. In particular lipid metabolism is thought to play a key role in the development of the metabolic syndrome and insulin resistance in later life. Investigative studies of metabolic programming are limited by the ethics and practicalities of sample collection in small infants. Dried blood spots on filter paper, derived from heel pricks are considered as the most suitable option for this age group. We validated a novel lipid profiling method, based on high resolution mass spectrometry to successfully determine the lipid composition of infants using dried blood spots. The spotting and air drying of blood on paper has noticeable effects on many of the lipids, leading to lipid oxidation and hydrolysis, which demand careful interpretation of the obtained data. We compared the lipid profiles from plasma or whole blood samples and the results from dried blood spots to determine if these revealed the same inter-subject differences. The results from dried blood spots were no less reproducible than other lipid profiling methods which required comparatively larger sample volumes. Therefore, lipid profiles obtained from dried blood spots can be successfully used to monitor infancy lipid metabolism and we show significant differences in the lipid metabolism of infants at age 3 versus 12 months.

This study evaluated the ability of the Murex HIV Ag/Ab Combination assay to detect human immunodeficiency virus (HIV) antibodies in 12 617 dried blood spots (DBSs) on filter paper. The assay had an overall sensitivity of 99.6% and a specificity of 99.9%. In view of its ability to detect p24 antigen and both HIV-1 and HIV-2 antibodies in samples collected in the form of DBSs, the Murex Ag/Ab Combination assay is suitable for use as a standard screening assay for seroprevalence studies, as well as for routine diagnostic use in clinical laboratories.

V okviru doktorskega dela sem razvila in ovrednotila tehniko suhe lise plazme za kvantitativno določevanje topiramata v študijah farmakokinetike in bioekvivalence. Tehnika suhe lise plazme je izpeljana iz tehnike suhe lise krvi. Slednjo so v zadnjih dveh letih bioanalitiki, farmakokinetiki in klinični raziskovalci na novo odkrili in jo uvedli v študije farmakokinetike in bioekvivalence kot alternativo za tradicionalno vzorčenje in določevanje učinkovin iz tekoče humane plazme. Tehnika odvzema vzorcev krvi na papir, znana kot suha lisa krvi (v nadaljevanju DBS – Dried Blood Spots), se je začela uporabljati v zgodnjih šestdesetih letih prejšnjega stoletja v presejalnih testih za novorojenčke. Danes pri izvajanju farmakokinetičnih in bioekvivalenčnih študijah DBS nudi v primerjavi z običajno plazmo veliko prednosti, ki se nanašajo na praktične, etične in stroškovne aspekte. DBS omogoča manj invazivno vzorčenje (vbod v prst ali peto namesto uporabe običajne venske kanile) in etične prednosti odvzema manjšega volumna krvi (manj kot 100 µL v primerjavi z več kot 1,0 mL krvi pri običajnem vzorčenju krvi). Poleg tega omogoča lažji transport in enostavnejše ter cenejše shranjevanje (ni potrebe po suhem ledu ali zamrzovalnikih). V obdobju obujanja DBS tehnike so se obravnavala predvsem vprašanja iz vidika njenih praktičnih prednosti, tehničnih izboljšav ter morebitnih analiznih težav in so ji šele pozneje namenili pozornost s stališča farmakokinetike in farmakodinamike. Tedaj so ugotovili, da matrica krvi in s tem DBS tehnika ni primerna za določevanje zdravilnih učinkovin v vseh vrstah študij. DBS tehnika tako še ni regulatorno priznana kot samostojna metoda za kvantitativno določevanje zdravilnih učinkovin pri registraciji humanih in veterinarskih zdravil. Pri farmakokinetičnih študijah običajno kot biološko matrico za določevanje zdravilnih učinkovin uporabljamo tekočo plazmo. Zgoraj omenjena tehnika suhe lise plazme (v nadaljevanju DPS – Dried Plasma Spots) nudi enake prednosti kot DBS tehnika, poleg tega pa je plazma že uveljavljena biološka matrica za analizo večine zdravilnih učinkovin. Pri tem se mi je porajalo vprašanje, ali lahko tehnika suhe lise plazme popolnoma zamenja klasično metodo določevanja zdravilnih učinkovin iz temeljne matrice, tekoče plazme. Odgovor sem iskala na primeru spojine topiramat. Topiramat (TPM) je antiepileptično zdravilo, ki ga uporabljamo za zdravljenje različnih vrst epilepsije pri odraslih in otrocih, za preprečevanje pogosto ponavljajočih se napadov migrene pri odraslih ter kot dodatno zdravilo za zdravljenje Lennox-Gastautovega sindroma. Topiramat spodbuja delovanje inhibitornih nevrotransmiterjev (GABA), blokira Na-kanale in šibko inhibira karbonsko anhidrazo. Vezava na karboanhidrazo, ki je v veliki meri prisotna v eritrocitih, pa lahko vpliva na farmakokinetiko zdravilne učinkovine. Topiramat ima tendenco vezave na karboanhidrazo v eritrocitih, kar pomeni, da se koncentracije v krvi v odvisnosti od časa lahko razlikujejo od plazemskega profila, ter da je razmerje koncentracij kri - plazma večje od 1. Namen raziskave je bil na podlagi literature, DBS ter DPS vzorcev študije spremljanja terapevtskega učinka oceniti, katera suha biološka matrica je najprimernejša za njegovo kvantitativno določevanje v bioloških vzorcih študij farmakokinetike in bioekvivalence. Doktorska naloga je sestavljena iz treh raziskav. Razvila in preizkusila sem tri bioanalizne metode za določevanje topiramata v bioloških matricah. V prvi fazi sem razvila in preizkusila metodo za kvantitativno določevanje topiramata iz suhe lise plazme. V drugem in tretjem delu raziskave sem razvila in preizkusila metodo iz tekoče humane plazme ter metodo določevanja topiramata iz suhe lise krvi. Za kvantitativno določitev koncentracij topiramata sem uporabila metodo devteriranega internega standarda (topiramat-d12) in tekočinsko kromatografijo s tandemskim masnim spektrometrom. Spojine sem ionizirala z elektrorazprševanjem (ESI) in dolo In my doctoral studies I have developed and evaluated dried plasma spot technique for determining topiramate in pharmacokinetic and bioequivalence studies. Dried plasma spot technique derives from dried blood spot technique. In the last two years the latter was rediscovered by bioanalysts and pharmacokineticians and introduced in pharmacokinetic and bioequivalence studies as an alternative for traditional sampling and determination of substances from liquid human plasma. The collection of whole blood samples on paper, known as the dried blood spot technique (DBS), dates back to the early 1960s, when it was used for detecting metabolic problems in newborns. Today DBS offers a number of advantages over the conventional whole blood in pharmacokinetic and bioequivalence studies. These advantages are related to ethical, practical, and cost aspects. DBS provides the ethical benefits of reduced blood volume (less than 100 μL compared to more than 1.0 mL blood, which is usually obtained during the conventional blood sample collection) and less invasive sampling (finger or heel prick rather than conventional venous cannula). Furthermore, it provides easier transfer and simpler and cheaper storage (no need for dry ice or freezers). When DBS technique was being reintroduced, the questions from the perspective of its practical advantages, technical improvements and possible analysis problems were considered. It was only until later that the technique got attention from the viewpoint of pharmacokinetics and bioanalysis. At that point it was discovered that blood matrix and consequently DBS technique is not useful for determining active substances in all types of studies. Therefore, DBS technique hasn’t been regulatory acknowledged as an independent method for the quantitative determination of active substances when it comes to the registration of human and veterinary drugs. In pharmacokinetic studies, liquid plasma is usually used as biological matrix for the determination of active substances. The aforementioned dried plasma spot technique (from now on DPS – Dried Plasma Spot) offers the same advantages as DBS technique alongside with the advantage of plasma being already an acknowledged matrix for the majority of active substances. As a result, I considered, a question whether DPS technique can completely replace the method of determining active substances from the basic matrix, liquid plasma. The answer to that question was sought on the case of the compound topiramate. Topiramate is an anti-epileptic drug that is used for the treatment of various forms of epilepsy in adults and children and for the prevention of frequently recurring migraines in adults as well as for the treatment of Lennox-Gastaut syndrome. Topiramate enhances the action of inhibitory neurotransmitters (GABA), blocks voltage-sensitive sodium channels and mildly inhibits carbonic anhydrase (CA) isoenzymes. Binding to CA, which is highly concentrated in erythrocytes, may affect drug pharmacokinetics. Topiramate’s tendency to bind to CA in erythrocytes implies that blood concentration-time profile may be different from plasma profile and the blood-to-plasma ratio might be greater than 1. The purpose of the research was to determine which is the most appropriate dried matrix for the quantitative determination of topiramate in biological samples of pharmacokinetic and bioequivalence studies, on the basis of literature and DBS and DPS samples from the monitoring research on the therapeutic effect. The doctoral dissertation consists of three parts of experimental work. In the first part, the method for quantitative determination of topiramate from dried plasma spot was developed and validated. In the second and third part of the experimental work, the method for quantitative determination of topiramate from liquid human plasma and the method for quantitative determination of topiramate from dried blood spot were developed and validated. For quantitative determination of TPM concentration the method with deuterated internal standard (topiramate-d12) and

Background Mucopolysaccharidosis type I (MPS I) is a genetic disease caused by the deficiency of α-L-iduronidase (IDUA) activity. MPS I is classified into three clinical phenotypes called Hurler, Scheie, and Hurler-Scheie syndromes according to their clinical severity. Treatments for MPS I are available. Better outcomes are associated with early treatment, which suggests a need for newborn screening for MPS I. The goal of this study was to determine whether measuring IDUA activity in dried blood on filter paper was effective in newborn screening for MPS I. Methods We conducted a newborn screening pilot program for MPS I from October 01, 2008 to April 30, 2013. Screening involved measuring IDUA activity in dried blood spots from 35,285 newborns using a fluorometric assay. Results Of the 35,285 newborns screened, 19 did not pass the tests and had been noticed for a recall examination. After completing further recheck process, 3 were recalled again for leukocyte IDUA enzyme activity testing. Two of the three had deficient leukocyte IDUA activity. Molecular DNA analyses confirmed the diagnosis of MPS I in these two newborns. Conclusions It is feasible to use the IDUA enzyme assay for newborn screening. The incidence of MPS I in Taiwan estimated from this study is about 1/17,643.

Background: Asymptomatic malaria parasitaemia has been reported in areas with high malaria transmission. It may serve as a reservoir for continued transmission, and furthermore complicates diagnostics, as not all individuals with a positive malaria test are necessarily ill due to malaria, although they may present with malaria-like symptoms. Asymptomatic malaria increases with age as immunity to malaria gradually develops. As mortality and morbidity of malaria is higher among younger children it is important to know the prevalence of asymptomatic malaria parasitaemia in this population in order to interpret laboratory results for malaria correctly. Methods: A total of 108 children that had neither been treated for malaria nor had a fever the previous four weeks were recruited consecutively at a maternal and child health clinic (MCHC) in Dar es Salaam, Tanzania. A rapid diagnostic test (RDT) for malaria and dried blood spot (DBS) on filter paper were taken from each child. Social and clinical data were recorded. DNA was extracted from the DBS of study participants by a method using InstaGene™ matrix. PCR targeting the Plasmodium mitochondrial genome was performed on all samples. Results: Median age was 4.6 months (range 0.5-38). All the RDTs were negative. PCR was negative for all study subjects. Conclusion: The study suggests that asymptomatic malaria may not be present in apparently healthy children up to the age of three years in Dar es Salaam, Tanzania. However, because of the small sample size and low median age of the study population, the findings cannot be generalized. Larger studies, including higher age groups, need to be done to clarify whether asymptomatic malaria parasitaemia is present in the general population in the Dar es Salaam area. publishedVersion

Background. Gaucher Disease (GD) is a hereditary lysosomal storage disorder characterized by the accumulation of glucosylceramide, mainly in the cells of the reticuloendothelial system, due to a deficiency of the enzyme acid β-glucosidase (GBA). Diagnosis is usually based on measurement of GBA activity in peripheral leukocytes. The purpose of this study was to evaluate the ability of screening for GBA and chitotriosidase activity using Dried Blood Spots on Filter Paper (DBS-FP) to identify individuals at high risk for GD in high-risk populations such as that of Tabuleiro do Norte, a small town in Northeastern Brazil. Methods. Between June 1, 2007 and May 31, 2008, 740 consented residents and descendants of traditional families from Tabuleiro do Norte were submitted to screening with DBS-FP. Subjects with GBA activity

Introduction: Diagnosis of HIV infection in infants is difficult due to the presence of maternal antibodies; only nucleic acid assays are very helpful in early detection. Filter papers are especially useful for blood collection in resource-poor settings with limited access to diagnostic facilities. Materials & Methods: DBS samples were collected from the infants born to HIV seropositive mothers who had received single dose nevirapine at onset of labor. The samples were directly spotted onto the Whatman 903 cards from heel, big toe or finger prick depending on the age of the infants. A total of 766 infant samples were collected on dried blood spots (DBS) and transported to the Department of Experimental Medicine (DEM), Chennai, for testing from different government hospitals of rural and urban parts of Tamil Nadu, South India. According to National AIDS Control Organization's (NACO) protocol DNA was extracted from all these DBS and PCR was performed using the Roche kit version 1.5. Results: Fifteen infants were found to be HIV positive and 751 were HIV negative; all these 15 positive infants and 49 negative infants who were in the age group between 10 and 18 months were repeated with another DBS and compared with whole blood. The DBS results were concordant with the whole blood method and the sensitivity and specificity were 100%.

The discovery of a point-mutation, adenine-to-guanine, at position 985 in the gene coding for MCAD (G985), gave the basis for an easy and specific polymerase chain reaction test. We tested the specificity of such a PCR based assay and detected correctly G985 and A985 in sequence verified cDNA clones. We showed that the G985 mutation is present in genomic DNA from 48 of 50 patients with confirmed MCAD deficiency, originating from various European countries, Australia and the USA. On the basis of this high frequency of the G985 mutation among patients, we improved and optimized the assay with respect to reliability and convenience for routine diagnostic and screening purposes. As little as 2 microliters blood from filter-paper blood-spots (Guthrie spots) is sufficient for the test.