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Volumetric absorptive microsampling (VAMS) is a recently developed sample collection method that enables single-drop blood collection in a minimally invasive manner. Blood biomolecules can then be extracted and processed for analysis using several analytical platforms. The integration of VAMS with conventional mass spectrometry (MS)-based metabolomics approaches is an attractive solution for human studies representing a less-invasive procedure compared to phlebotomy with the additional potential for remote sample collection. However, as we recently demonstrated, VAMS samples require long-term storage at −80 °C. This study investigated the stability of VAMS samples during short-term storage and compared the metabolome obtained from capillary blood collected from the fingertip to those of plasma and venous blood from 22 healthy volunteers. Our results suggest that the blood metabolome collected by VAMS samples is stable at room temperature only for up to 6 h requiring subsequent storage at −80 °C to avoid significant changes in the metabolome. We also demonstrated that capillary blood provides better coverage of the metabolome compared to plasma enabling the analysis of several intracellular metabolites presented in red blood cells. Finally, this work demonstrates that with the appropriate pre-analytical protocol capillary blood can be successfully used for untargeted metabolomics studies.

Introduction: Indirect estimation of reference intervals (RIs) is straightforward and inexpensive procedure for determination of intra-laboratory RIs. We applied the indirect approach to assess RIs for haematological parameters in capillary blood of pre-school children, using results stored in our laboratory database. Materials and methods: We extracted data from laboratory information system, for the results obtained by automatic haematology analyser in capillary blood of 154 boys and 146 girls during pre-school medical examination. Data distribution was tested, and logarithmic transformation was applied if needed. Reference intervals were calculated by the nonparametric percentile method. Results: Reference intervals were calculated for: RBC count (4.2-5.4 x1012/L), haemoglobin (114-146 g/L), MCH (25.0-29.4 pg), MCHC (321-368 g/L), RDW-SD (36.1-43.5 fL), WBC count (4.5-12.3 x109/L), neutrophils count (1.7-6.9 x109/L) and percentage (29.0-69.0%), lymphocytes count (1.6-4.4 x109/L) and percentage (21.9-60.7%), PLT (165-459 x109/L), MPV (8.1-11.4 fL) and PDW (9.2-14.4%). Gender specific RIs were calculated for mo-nocytes count (male (M): 0.2-1.6 x109/L; female (F): 0.1-1.4 x109/L) and percentage (M: 2.5-18.3%; F: 1.8-16.7%), haematocrit (M: 0.34-0.42 L/L; F: 0.34-0.43 L/L), MCV (M: 73.4-84.6 fL; F: 75.5-84.2 fL) and RDW (M: 12.1-14.3%; F: 11.7-13.9%), due to observed gender differences in these parameters (P = 0.031, 0.028, 0.020, 0.012 and 0.001; respectively). Estimated RIs markedly varied from the literature based RIs that are used in the labora-tory. Conclusions: Indirect method employed in this study enables straightforward assessment of RIs in pre-school children. Herein derived RIs differed from the literature-based ones, indicating the need for intra-laboratory determination of RIs for specific populations and sample types.

Актуальність теми дослідження. На сьогоднішній день з розвитком цифрових та комп’ютерних технологій біометричні інформаційні системи здатні розпізнавати людей за їх статичними анатомічними особливостями (відбитку пальців, райдужної оболонки, форми обличчя, долонь та інших частин тіла) та за динамічними показниками поведінки та реакцій організму (за голосом, швидкістю клавіатурного почерку, мікровібрацією пальців та інше). Дані біометричні методи є досить простими у реалізації але не забезпечують належного захисту. Актуальність роботи обумовлена активною інформатизацією суспільства та необхідністю захисту баз даних від сторонніх осіб полягає у розробці модулю біометричної ідентифікації для автоматизованої системи аналізу крові, що дозволить забезпечити високий захист від навмисної підміни результатів дослідження та виключення появи помилок від людського фактору. Метою роботи підвищення якості біометричної ідентифікації людини для використання в автоматизованій системі забору крові шляхом створення алгоритму обробки сигналу роботи серця знятого датчиками електрокардіограми та фотоплетизмограми. Для досягнення поставленої мети необхідно вирішити такі задачі: 1. Здійснити порівняльний аналіз основних методів та засобів проведення проколу шкіри пальця для забору капілярною крові. 2. Провести аналіз основних методів та засобів для реєстрації сигналу електрокардіограми та фотоплетизмограми. 3. Виконати аналіз основних алгоритмів для обробки сигналу та виділення вагомих характеристик для подальшої біометричної ідентифікації. 4. Обґрунтувати необхідність та переваги розробки модулю біометричної ідентифікації для автоматизованої системи аналізу крові. 5. Розробити структурно-функціональну схему роботи автоматизованої системи для забору крові з біометричною ідентифікацією пацієнта. 6. Обґрунтувати вибір елементної бази для реєстрації сигналу роботи серця. 7. Провести обробку сигналу та розробити алгоритм відділення ознак для створення шаблону. 8. Розробити блок-схему алгоритму, The relevance of master’s thesis. For today in modern society conducting of laboratory diagnostics of biochemical blood analysis is a necessary component for determining the state of the organism and establishing a reliable diagnosis. Existing methods and tools for studying blood parameters can be divided into invasive and non-invasive, but the most accurate are the methods that require sampling. As a result of the study of arterial and venous blood, vascular trauma occurs, accompanied by a painful sensation and there is a high risk of infection. In the works (Mingacheva AA, Mishanich T.V.) it is shown that capillary blood can be used for monitoring the health of a person and has great potential for diagnostics of an organism's condition. The disadvantages of the invasive method of capillary blood capture are the occurrence of errors in the procedures for obtaining biological samples obtained during the stages described in the works (Skvortsova RG, Ludupova E.Yu, etc.): identification of the patient, skin perforation, blood collection, transportation to the laboratory, analysis, processing and data transmission. The main percentage of errors arises mainly as a result of the impact on the process of sampling a human factor (random or targeted). The urgency of the work is to develop a perforation module for the automated system of capillary blood collection, which will provide protection from errors at the preanalytical stage, as well as exclude the impact on the human factor. Proceeding from this, the accuracy and reliability of the obtained results increases, and therefore the development of this device is an urgent task. The main goal of the work is to improve the capillary blood collection capability and reduce errors at the preanalytical stage of laboratory research by creating an automated capillary blood collection system. To achieve this goal, the following tasks need to be addressed: 1. To carry out a comparative analysis of the basic methods and means of car

На сьогоднішній день у сучасному суспільстві проведення лабораторної діагностики біохімічного аналізу крові є необхідною складовою для визначення стану організму та встановлення достовірного діагнозу. Існуючі методи та засоби дослідження параметрів крові можна поділити на інвазивні та неінвазивні, але найбільш точними є методи які вимагають забір зразка. В наслідок дослідження артеріальної і венозної крові відбувається травматизація судин, що супроводжується больовим відчуттям та існує високий ризик зараження. В роботах (Мингачева А.А., Мишанич Т.В.) показано, що капілярна кров може використовують для моніторингу стану здоров’я людини та має великий потенціал для діагностики стану організму. Недоліки інвазивного методу забору капілярної крові полягають у виникненні похибок отриманих на етапах процедури забору біологічного зразка описані в роботах (Скворцова Р.Г., Лудупова Е.Ю. та ін.): ідентифікація пацієнта, перфорація шкіри, взяття крові, транспортування до лабораторії, аналіз, обробка та передача даних. Основний відсоток похибок виникає переважно в результаті впливу на процес забору зразка людського фактору (випадкового або ціліспрямованого). Актуальність роботи полягає у розробці модулю перфорації для автоматизованої системи забору капілярної крові, що дозволить забезпечити захист від похибок на преданалітичному етапі, а також виключити вплив на зразок людського фактору. Виходячи з цього підвищується точність та достовірність отриманих результатів, а отже розробка даного приладу є актуальною задачею., For today in modern society conducting of laboratory diagnostics of biochemical blood analysis is a necessary component for determining the state of the organism and establishing a reliable diagnosis. Existing methods and tools for studying blood parameters can be divided into invasive and non-invasive, but the most accurate are the methods that require sampling. As a result of the study of arterial and venous blood, vascular trauma occurs, accompanied by a painful sensation and there is a high risk of infection. In the works (Mingacheva AA, Mishanich T.V.) it is shown that capillary blood can be used for monitoring the health of a person and has great potential for diagnostics of an organism's condition. The disadvantages of the invasive method of capillary blood capture are the occurrence of errors in the procedures for obtaining biological samples obtained during the stages described in the works (Skvortsova RG, Ludupova E.Yu, etc.): identification of the patient, skin perforation, blood collection, transportation to the laboratory, analysis, processing and data transmission. The main percentage of errors arises mainly as a result of the impact on the process of sampling a human factor (random or targeted). The urgency of the work is to develop a perforation module for the automated system of capillary blood collection, which will provide protection from errors at the preanalytical stage, as well as exclude the impact on the human factor. Proceeding from this, the accuracy and reliability of the obtained results increases, and therefore the development of this device is an urgent task.

Over the past 10 years, the use of saliva as a diagnostic fluid has gained attention and has become a translational research success story. Some of the current nanotechnologies have been demonstrated to have the analytical sensitivity required for the use of saliva as a diagnostic medium to detect and predict disease progression. However, these technologies have not yet been integrated into current clinical practice and work flow. As a diagnostic fluid, saliva offers advantages over serum because it can be collected noninvasively by individuals with modest training, and it offers a cost-effective approach for the screening of large populations. Gland-specific saliva can also be used for diagnosis of pathology specific to one of the major salivary glands. There is minimal risk of contracting infections during saliva collection, and saliva can be used in clinically challenging situations, such as obtaining samples from children or handicapped or anxious patients, in whom blood sampling could be a difficult act to perform. In this review we highlight the production of and secretion of saliva, the salivary proteome, transportation of biomolecules from blood capillaries to salivary glands, and the diagnostic potential of saliva for use in detection of cardiovascular disease and oral and breast cancers. We also highlight the barriers to application of saliva testing and its advancement in clinical settings. Saliva has the potential to become a first-line diagnostic sample of choice owing to the advancements in detection technologies coupled with combinations of biomolecules with clinical relevance.

This thesis describes bioanalytical methods for measuring melatonin and some anti-infective drugs in biological fluids. Solid-phase extraction (SPE) or protein precipitation was used for enrichment and purification of the analytes and Liquid Chromatography (LC) was used to analyze the samples. Developed methods were validated according to international guidelines. Melatonin is a hormone secreted by the pineal gland with a robust circadian rhythm. Bioanalytical methods for determination of melatonin in plasma and saliva have been developed which were used for monitoring melatonin levels in volunteers and patients suffering from sleep related diseases. Eflornithine (DFMO) is a chiral drug used for the treatment of human African trypanosomiasis. A bioanalytical method for determination of the DFMO enantiomers in plasma, after precolumn derivatization with o-phtalaldehyde and N-acetyl-L-cystein has been developed. The method has been used to study the L- and D-DFMO pharmacokinetics, in order to investigate the possible development of an oral treatment of DFMO. A method for simultaneous determination of three antiretroviral drugs i.e. Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) in dried blood spots (DBS) was developed. The method was used for drug determination in two subjects after receiving standard antiretroviral treatment. The method seemed well suitable for the determination of 3TC and NVP and in some extent for AZT. Lumefantrine (LF) is one of the active components in a new fixed drug combination recommended by the WHO as a replacement to older drugs that has lost their effect. A method for the determination of LF in DBS was developed. The method is suitable for monitoring of drug treatment in rural settings. Tafenoquine is a new promising antimalarial drug under development. A method for the determination of Tafenoquine in plasma and in DBS is described. The method may be useful in future clinical studies in laboratory environment as well as in rural s, Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 703

This thesis describes bioanalytical methods for measuring melatonin and some anti-infective drugs in biological fluids. Solid-phase extraction (SPE) or protein precipitation was used for enrichment and purification of the analytes and Liquid Chromatography (LC) was used to analyze the samples. Developed methods were validated according to international guidelines. Melatonin is a hormone secreted by the pineal gland with a robust circadian rhythm. Bioanalytical methods for determination of melatonin in plasma and saliva have been developed which were used for monitoring melatonin levels in volunteers and patients suffering from sleep related diseases. Eflornithine (DFMO) is a chiral drug used for the treatment of human African trypanosomiasis. A bioanalytical method for determination of the DFMO enantiomers in plasma, after precolumn derivatization with o-phtalaldehyde and N-acetyl-L-cystein has been developed. The method has been used to study the L- and D-DFMO pharmacokinetics, in order to investigate the possible development of an oral treatment of DFMO. A method for simultaneous determination of three antiretroviral drugs i.e. Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) in dried blood spots (DBS) was developed. The method was used for drug determination in two subjects after receiving standard antiretroviral treatment. The method seemed well suitable for the determination of 3TC and NVP and in some extent for AZT. Lumefantrine (LF) is one of the active components in a new fixed drug combination recommended by the WHO as a replacement to older drugs that has lost their effect. A method for the determination of LF in DBS was developed. The method is suitable for monitoring of drug treatment in rural settings. Tafenoquine is a new promising antimalarial drug under development. A method for the determination of Tafenoquine in plasma and in DBS is described. The method may be useful in future clinical studies in laboratory environment as well as in rural s, Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 703

This thesis describes bioanalytical methods for measuring melatonin and some anti-infective drugs in biological fluids. Solid-phase extraction (SPE) or protein precipitation was used for enrichment and purification of the analytes and Liquid Chromatography (LC) was used to analyze the samples. Developed methods were validated according to international guidelines. Melatonin is a hormone secreted by the pineal gland with a robust circadian rhythm. Bioanalytical methods for determination of melatonin in plasma and saliva have been developed which were used for monitoring melatonin levels in volunteers and patients suffering from sleep related diseases. Eflornithine (DFMO) is a chiral drug used for the treatment of human African trypanosomiasis. A bioanalytical method for determination of the DFMO enantiomers in plasma, after precolumn derivatization with o-phtalaldehyde and N-acetyl-L-cystein has been developed. The method has been used to study the L- and D-DFMO pharmacokinetics, in order to investigate the possible development of an oral treatment of DFMO. A method for simultaneous determination of three antiretroviral drugs i.e. Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) in dried blood spots (DBS) was developed. The method was used for drug determination in two subjects after receiving standard antiretroviral treatment. The method seemed well suitable for the determination of 3TC and NVP and in some extent for AZT. Lumefantrine (LF) is one of the active components in a new fixed drug combination recommended by the WHO as a replacement to older drugs that has lost their effect. A method for the determination of LF in DBS was developed. The method is suitable for monitoring of drug treatment in rural settings. Tafenoquine is a new promising antimalarial drug under development. A method for the determination of Tafenoquine in plasma and in DBS is described. The method may be useful in future clinical studies in laboratory environment as well as in rural s, Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 703

This thesis describes bioanalytical methods for measuring melatonin and some anti-infective drugs in biological fluids. Solid-phase extraction (SPE) or protein precipitation was used for enrichment and purification of the analytes and Liquid Chromatography (LC) was used to analyze the samples. Developed methods were validated according to international guidelines. Melatonin is a hormone secreted by the pineal gland with a robust circadian rhythm. Bioanalytical methods for determination of melatonin in plasma and saliva have been developed which were used for monitoring melatonin levels in volunteers and patients suffering from sleep related diseases. Eflornithine (DFMO) is a chiral drug used for the treatment of human African trypanosomiasis. A bioanalytical method for determination of the DFMO enantiomers in plasma, after precolumn derivatization with o-phtalaldehyde and N-acetyl-L-cystein has been developed. The method has been used to study the L- and D-DFMO pharmacokinetics, in order to investigate the possible development of an oral treatment of DFMO. A method for simultaneous determination of three antiretroviral drugs i.e. Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) in dried blood spots (DBS) was developed. The method was used for drug determination in two subjects after receiving standard antiretroviral treatment. The method seemed well suitable for the determination of 3TC and NVP and in some extent for AZT. Lumefantrine (LF) is one of the active components in a new fixed drug combination recommended by the WHO as a replacement to older drugs that has lost their effect. A method for the determination of LF in DBS was developed. The method is suitable for monitoring of drug treatment in rural settings. Tafenoquine is a new promising antimalarial drug under development. A method for the determination of Tafenoquine in plasma and in DBS is described. The method may be useful in future clinical studies in laboratory environment as well as in rural s, Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 703

A bioanalytical method for the determination of lumefantrine in 100 μl blood applied onto sampling paper, by solid-phase extraction and liquid chromatography, has been developed and validated. Whatman 31 ET Chr sampling paper was pre-treated with 0.75 M tartaric acid before sampling capillary blood to enable a high recovery of lumefantrine. Lumefantrine was extracted from the sampling paper, then further purified using solid-phase extraction and finally quantified with HPLC. The between-day variation was below 10% over the range 0.4-25 μM. The lower limit of quantification was 0.25 μM in 100 μl capillary blood. No decrease in lumefantrine concentration in dried blood spot is seen after 4 months storage at 22 °C. The method was also evaluated in field samples from patients in Tanzania after treatment with lumefantrine/artemether. Lumefantrine could be estimated accurately enough to assess bioavailability and treatment compliance on day 7 (i.e. 4 days after the last dose) after a standard regimen with the lumefantrine/artemether combination.

The aim of the KOALA Birth Cohort Study in the Netherlands is to identify factors that influence the clinical expression of atopic disease with a main focus on lifestyle (e.g., anthroposophy, vaccinations, antibiotics, dietary habits, breastfeeding and breast milk composition, intestinal microflora composition, infections during the first year of life, and gene-environment interaction). The recruitment of pregnant women started in October 2000. First, participants with 'conventional lifestyles' (n = 2343) were retrieved from an ongoing prospective cohort study (n = 7020) on pregnancy-related pelvic girdle pain. In addition, pregnant women (n = 491) with 'alternative lifestyles' with regard to child rearing practices, dietary habits (organic, vegetarian), vaccination schemes and/or use of antibiotics, were recruited through organic food shops, anthroposophic doctors and midwives, Steiner schools, and dedicated magazines. All participants were enrolled between 14 and 18 wk of gestation and completed an intake questionnaire on family history of atopy and infant care intentions. Documentation of other relevant variables started in the pregnant mother and covered the first and third trimester as well as early childhood by repeated questionnaires at 14-18, 30, and 34 wk of gestation and 3, 7, 12, and 24 months post-partum. A subgroup of participants, including both conventional and alternative lifestyles, was asked to consent to maternal blood sampling, breast milk and a faecal sample of the infant at 1 month post-partum, capillary blood at age 1 yr, venous blood and observation of manifestation of atopic dermatitis during home visits at the age of 2 yr (using the UK working party criteria and the severity scoring of atopic dermatitis index), and buccal swabs for DNA isolation from child-parent trios. From the start, ethical approval and informed consent procedures included gene-environment interaction studies. Follow-up at 3 and 7 months post-partum was completed with hi

The aim of the developed assay is to determine drug use and self medication in areas where achange in treatment policy has taken place. The assay will be a complement to interviewing patients and will increase reliability of the survey. Several of the drugs included in this screening assay have long half-lifes e.g. chloroquine, sulfadoxine, mefloquine, andlumefantrine and are detectable at least 7 days after administration. The sample collection is very simple, using 100 μl capillary whole blood applied onto Whatman 31 ET Chr filter paper.The drugs are extracted from the dried blood spot with two different extraction methods (due to the big differences in physico-chemical properties). Solid phase extraction is used as sample clean-up and separation is performed with gradient-LC with MS ion-trap detection. Detection limits (S/N > 5:1) at 50 ng/ml or better were achieved for all drugs except lumefantrine (200 ng/ml), Manuscript