The ethical and practical issues of obtaining a blood sample pose a significant challenge to performing pharmacokinetic studies in children, infants and neonates. Dried blood spot analysis, based on the collection of a micro blood sample has potential to overcome these difficulties. There are at present a limited number of reports on the utility of dried blood spot analysis in clinical pharmacokinetic studies. The studies described in this thesis were undertaken to investigate the accuracy and precision of dried blood spot sampling coupled with mass spectrometry detection for drug quantification, and clinically validate the robustness and feasibility of this technique for pharmacokinetic studies in preterm neonates. Dried blood spot methods were developed for application to pharmacokinetic studies of test drugs dexamethasone and caffeine. Investigations were focused on the blood collection system, analyte recovery and optimisation of the detection system. In-vitro validation results indicated developed methods were precise, accurate and selective in accordance with the Food and Drug Administration regulatory guidelines on the assessment of bioanalytical methods. Results were not significantly affected by small variations in the blood volume spotted or the presence of petroleum jelly, which is often used on the sampling site during capillary blood collection in neonates. Variability in haematocrit was determined to be the single most important factor affecting assay accuracy. Stability assessments by comparison with freshly prepared samples verified the suitability of sample drying, storage and post sample extraction conditions. An investigation of method transferability between different analytical instruments was undertaken with caffeine to provide an assessment of the robustness of dried blood spot analysis. Results generated from a single and triple quadrupole mass spectrometer were comparable with an expected lower limit of quantification with the latter technique most likely due to a greater ionisation and detection efficiency. Intravenous dexamethasone pharmacokinetics was determined in 5 preterm neonates receiving treatment for chronic lung disease. Individual pharmacokinetic analyses were performed using a one compartment model to estimate primary pharmacokinetic parameters, clearance (mean, 0.18 l/h/kg) and volume of distribution (mean, 1.33 l/kg). The whole blood derived mean estimates were similar to previous plasma clearance and volume estimates of 0.14 l/h/kg and 1.91 l/kg, respectively reported in neonates (n=7). This highlights the potential for dried blood spot analysis as an alternative to conventional plasma based methods for dexamethasone dose optimisation studies in neonates. The population pharmacokinetics of oral / intravenous caffeine was determined in 67 preterm neonates. A one compartment model was used to describe the blood concentration-time data. Model evaluation using a bootstrapping technique confirmed the robustness and stability of the developed model. Pharmacokinetic parameters derived from dried blood spot drug measurements were estimated with precision (relative standard error < 10%) and were comparable to estimates of plasma clearance (mean, 7.3 vs. 7.0 ml/h/kg) and volume of distribution (mean, 593 vs. 851 ml/kg) from a previous population study in neonates (n=110). Weight and postnatal age were the most influential covariates in the clearance model which is in agreement with previous population studies. These results demonstrate that dried blood spot analysis is a practical technique, with significant potential as a robust method for use in clinical pharmacokinetic studies in vulnerable populations such as preterms. Haematocrit related effects on paper will need to be accounted for if this potential is to be realised. Further investigations to determine the reproducibility of capillary blood sampling in neonates and the impact of using blood drug measurements on pharmacokinetic parameter estimation will be necessary before widespread use of the technique is possible.

Patients that have undergone organ transplantation are life-long treated with immunosuppressant drugs and these have to be monitored regularly to get the desired effect of suppressing the immune system. To monitor the drug concentration normally a venous blood sample is collected at a clinic but the use of dried blood spots (DBS) as a matrix for drug monitoring for immunosuppressant drugs will make home sampling possible for this patient group. The aim of this study was to develop and validate a bioanalytical method for quantifying cyclosporine A and tacrolimus in dried blood spots. The method consist of punching out a 5 mm disc from a blood spot , followed by extracting the spot in a 96-well hydrophobic filter plate with 150 µL extraction solution containing internal standard (ascomycin and cyclosporine A d12) in a methanol water solution (80:20v/v%). The extract is then centrifuged through the filter plate down in a 96-deep well plate and injected on the LC-MS/MS, with an analysis time of 2.5min. The method will be validated in accordance with the guidelines set by the European Medicines Agency with additions specific to DBS. The method is not fully validated but will be in due time. The validated parameters show a robust and fast analysing method that has the prospects of being used for analysing DBS samples for patients and in the future can possibly be used by patients in home environment.

EVALUATION AND DETERMINATION OF THE SENSITIVITY AND SPECIFICITY OF A Treponema pallidum DRIED BLOOD SPOT (DBS) METHOD FOR SEROLOGIC DIAGNOSIS OF SYPHILIS Background: Syphilis is a sexually transmitted infection (STI) caused by Treponema pallidum subspecies pallidum. Syphilis is known as the "great imitator" due to the similarity of clinical signs and symptoms to other infectious diseases. The primary diagnosis of syphilis relies on clinical findings, including the examination of treponemal lesions, and/or serologic tests. Serologic tests are divided into nontreponemal and treponemal tests. Nontreponemal tests are useful for screening, while treponemal tests are used as confirmatory tests. Methods: A total of 200 serum and DBS specimens collected from patients at the Los Angeles Municipal Sexually Transmitted Disease Clinics were tested by the DBS and enzyme immunoassay (EIA) methods. These samples were sent to the Syphilis Diagnostics Laboratory, Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia for testing. Samples were blindly evaluated by the TREP-SPOTTM DBS and the TREP- SURETM EIA methods for the detection of anti-treponemal IgG- and IgM-class antibodies. Results: The sensitivity of the DBS method was 83% (95% CI, 73.89 - 89.50) and specificity was 100% (95% CI, 95.39 - 100)). The positive predictive value and negative predictive values were 100% (95% CI, 94.48 - 100) and 85% (95% CI, 77.43 - 91.0), respectively. The efficiency of the DBS method was 91.5%. The kappa value for the agreement between the DBS method and EIA assay was 0.83 (95% CI, 0.754 - 0.906). The correlation coefficient (r2) between the anti-treponemal antibody assay results obtained from DBS and serum samples was 0.94. Conclusion: DBS is an optimal choice to be used as a screening tool for the detection of anti-treponemal antibodies for the diagnosis of syphilis. The detection of anti-treponemal antibodies (TREP-SPOTTM DBS EIA) compared favorably to the results of serum-base assay (TREP-SURETM EIA), with an overall concordance of 91.5%. Dried blood spots are technically easier to obtain and are suitable blood samples for primary health care centers.

No description available.

A biomonitorização humana ou biomonitoramento (BH) é definido como a medida periódica de determinada substância química ou seu metabólito em fluidos biológicos, principalmente sangue e urina, de uma população com o objetivo de avaliar a exposição e os riscos à saúde. Tal método tem se tornado comum em países desenvolvidos, porém ainda é uma prática pouco utilizada no Brasil. Isso ocorre pela dificuldade de coleta, armazenamento e transporte das amostras, principalmente em regiões sem infraestrutura e de difícil acesso. Diante disso, alguns procedimentos alternativos de coleta de amostra vêm sendo propostos. Um destes procedimentos é o Dried Blood Spot (DBS) ou coleta e armazenamento de amostra em papel-cartão. Este método oferece uma série de vantagens sobre os procedimentos de coleta convencionais, principalmente por reduzir consideravelmente o volume de amostra coletada. Entretanto, pouco se sabe sobre a estabilidade dos analitos após a deposição da amostra no cartão e do risco de contaminação da amostra pelo substrato sólido. Além disso, procedimentos de extração dos analitos do papel, para posterior quantificação, ainda não estão totalmente estabelecidos. Neste sentido, o presente trabalho avaliou de forma sistemática o procedimento de coleta de sangue por DBS visando sua futura aplicação em programas de biomonitoramento no Brasil para determinação dos elementos químicos As, Cd, Cu, Hg, Mn, Pb, Se e Zn por espectrometria de massas com plasma indutivamente acoplado (ICP-MS). Para isso, no estudo foram utilizadas três diferentes marcas comerciais de cartão coletor: Whatman 903(TM), Munktell(TM), e DMPK-C(TM). Todas as marcas de cartão apresentaram baixas concentrações dos elementos químicos. Após a deposição da amostra no papel cartão verificou-se que a concentração dos elementos químicos manteve-se estável por um período de pelo menos 60 dias (temperatura ambiente e ao abrigo da luz). Foi otimizado o método de extração dos analitos do substrato, com melhor condição obtida após a imersão do papel (corte circular de diâmetro de 1/2´´) por 60 minutos em solução extratora (0,5% v/v HNO3 e 0,01% v/v Triton(TM) X-100) na proporção de 1:50 v/v, seguida de 10 segundos de agitação por vortex. Após a extração, a solução resultante contendo os analitos foi diretamente injetada no ICP-MS. Cabe também destacar que não foram observadas diferenças estatísticas nas concentrações dos elementos químicos com coleta de sangue da veia do antebraço (sangue venoso) ou do dedo (sangue capilar). Os resultados obtidos no presente estudo, devem contribuir para a implementação deste procedimento em análises de elementos químicos (biomonitoramento da população brasileira), principalmente considerando as dificuldades de coleta, armazenamento e transporte de amostras clínicas em nosso país por sua extensão territorial. Além disso, este procedimento pode facilitar estudos com populações vulneráveis e que vivem em áreas remotas e de difícil acesso. / Human biomonitoring or biomonitoring (BH) is defined as the measurement of a particular chemical or metabolites in biological fluids, especially blood and urine, in a population to assess the level of exposure and health risks. Human biomonitoring is a common activity in developed countries, but is still an uncommon practice in Brazil. It mainly occurs due to the difficulty of collection, storage and transport of samples, particularly in regions without infrastructure and difficult access. Therefore, some alternative procedures for sample collection have been proposed. One of the procedures is the Dried Blood Spot (DBS). This method offers distinct advantages over conventional sample collection procedures, including the reduced sample volume required for analysis. However, little is known about the stability of analytes after sample deposition on the card and the possible risk of sample contamination by the solid substrate. In addition, the extraction procedures of elements from the substrate surface before determination are not yet fully established. In this sense, the present study evaluated systematically the DBS blood collection procedure aiming at its future application in biomonitoring studies in Brazil to determine the chemical elements As, Cd, Cu, Hg, Mn, Pb, Se and Zn by inductively coupled plasma mass spectrometry (ICP-MS). For this, the study used three different brands of collecting cards: Whatman 903(TM), Munktell(TM), and DMPK-C(TM). All card brands presented low concentrations of chemical elements. After sample deposition on cardboard it was found that the concentration of chemical elements remained stable for at least 60 days (at room temperature and protected from light). It was optimized the method of analytes extraction from the substrate, with the best condition obtained after immersing the paper (circular cutting diameter of 1/2 \'\') for 60 minutes in an extraction solution containing 0,5% v/v HNO3 and 0,01% v/v Triton(TM) X-100 in the ratio 1:50 v/v, followed by 10 seconds of vortex. After extraction, the resulting solution containing the analytes was directly injected into the ICP-MS. It can also be pointed out that no statistical differences was found between the concentrations of elements determined in forearm vein blood (venous blood) and finger blood (capillary blood). Taken together, the results of the present study can contribute to the employment of the DBS procedure for the screening of chemical elements in the Brazilian population, especially considering the difficulties of collection, storage and transport of clinical specimens in our country. Moreover, studies in vulnerable populations living in remote areas and of difficult access should be simplified.

A biomonitorização humana ou biomonitoramento (BH) é definido como a medida periódica de determinada substância química ou seu metabólito em fluidos biológicos, principalmente sangue e urina, de uma população com o objetivo de avaliar a exposição e os riscos à saúde. Tal método tem se tornado comum em países desenvolvidos, porém ainda é uma prática pouco utilizada no Brasil. Isso ocorre pela dificuldade de coleta, armazenamento e transporte das amostras, principalmente em regiões sem infraestrutura e de difícil acesso. Diante disso, alguns procedimentos alternativos de coleta de amostra vêm sendo propostos. Um destes procedimentos é o Dried Blood Spot (DBS) ou coleta e armazenamento de amostra em papel-cartão. Este método oferece uma série de vantagens sobre os procedimentos de coleta convencionais, principalmente por reduzir consideravelmente o volume de amostra coletada. Entretanto, pouco se sabe sobre a estabilidade dos analitos após a deposição da amostra no cartão e do risco de contaminação da amostra pelo substrato sólido. Além disso, procedimentos de extração dos analitos do papel, para posterior quantificação, ainda não estão totalmente estabelecidos. Neste sentido, o presente trabalho avaliou de forma sistemática o procedimento de coleta de sangue por DBS visando sua futura aplicação em programas de biomonitoramento no Brasil para determinação dos elementos químicos As, Cd, Cu, Hg, Mn, Pb, Se e Zn por espectrometria de massas com plasma indutivamente acoplado (ICP-MS). Para isso, no estudo foram utilizadas três diferentes marcas comerciais de cartão coletor: Whatman 903(TM), Munktell(TM), e DMPK-C(TM). Todas as marcas de cartão apresentaram baixas concentrações dos elementos químicos. Após a deposição da amostra no papel cartão verificou-se que a concentração dos elementos químicos manteve-se estável por um período de pelo menos 60 dias (temperatura ambiente e ao abrigo da luz). Foi otimizado o método de extração dos analitos do substrato, com melhor condição obtida após a imersão do papel (corte circular de diâmetro de 1/2´´) por 60 minutos em solução extratora (0,5% v/v HNO3 e 0,01% v/v Triton(TM) X-100) na proporção de 1:50 v/v, seguida de 10 segundos de agitação por vortex. Após a extração, a solução resultante contendo os analitos foi diretamente injetada no ICP-MS. Cabe também destacar que não foram observadas diferenças estatísticas nas concentrações dos elementos químicos com coleta de sangue da veia do antebraço (sangue venoso) ou do dedo (sangue capilar). Os resultados obtidos no presente estudo, devem contribuir para a implementação deste procedimento em análises de elementos químicos (biomonitoramento da população brasileira), principalmente considerando as dificuldades de coleta, armazenamento e transporte de amostras clínicas em nosso país por sua extensão territorial. Além disso, este procedimento pode facilitar estudos com populações vulneráveis e que vivem em áreas remotas e de difícil acesso. / Human biomonitoring or biomonitoring (BH) is defined as the measurement of a particular chemical or metabolites in biological fluids, especially blood and urine, in a population to assess the level of exposure and health risks. Human biomonitoring is a common activity in developed countries, but is still an uncommon practice in Brazil. It mainly occurs due to the difficulty of collection, storage and transport of samples, particularly in regions without infrastructure and difficult access. Therefore, some alternative procedures for sample collection have been proposed. One of the procedures is the Dried Blood Spot (DBS). This method offers distinct advantages over conventional sample collection procedures, including the reduced sample volume required for analysis. However, little is known about the stability of analytes after sample deposition on the card and the possible risk of sample contamination by the solid substrate. In addition, the extraction procedures of elements from the substrate surface before determination are not yet fully established. In this sense, the present study evaluated systematically the DBS blood collection procedure aiming at its future application in biomonitoring studies in Brazil to determine the chemical elements As, Cd, Cu, Hg, Mn, Pb, Se and Zn by inductively coupled plasma mass spectrometry (ICP-MS). For this, the study used three different brands of collecting cards: Whatman 903(TM), Munktell(TM), and DMPK-C(TM). All card brands presented low concentrations of chemical elements. After sample deposition on cardboard it was found that the concentration of chemical elements remained stable for at least 60 days (at room temperature and protected from light). It was optimized the method of analytes extraction from the substrate, with the best condition obtained after immersing the paper (circular cutting diameter of 1/2 \'\') for 60 minutes in an extraction solution containing 0,5% v/v HNO3 and 0,01% v/v Triton(TM) X-100 in the ratio 1:50 v/v, followed by 10 seconds of vortex. After extraction, the resulting solution containing the analytes was directly injected into the ICP-MS. It can also be pointed out that no statistical differences was found between the concentrations of elements determined in forearm vein blood (venous blood) and finger blood (capillary blood). Taken together, the results of the present study can contribute to the employment of the DBS procedure for the screening of chemical elements in the Brazilian population, especially considering the difficulties of collection, storage and transport of clinical specimens in our country. Moreover, studies in vulnerable populations living in remote areas and of difficult access should be simplified.

No description available.

A method for extraction of therapeutic proteins from dried blood spots (DBS) followed by quantification on Gyrolab(TM) has been developed. The method makes it possible to measure the concentration of the analyte in the range 100-6000 ng/mL. The procedure can generate full analytical information from 15 μL blood originally sampled from a subject. The modest sample requirements allows for sampling a full pre-clinical pharmacokinetic profile from a single mouse. This may allow for reduced usage of animals during preclinical development of new therapeutic proteins in accordance with the 3R’s, replace, refine and reduce.

A method for extraction of proteins from dried blood spots (DBS) for analysis using Proseek is developed and evaluated. DBS, as sample format, possesses a number of desirable advantages over for example plasma samples. These advantages include for example minimal patient invasiveness, sampling simplicity and non regulated sample transportation. Highly reproducible quantitative detection of 92 proteins is demonstrated from a 1.2 mm in diameter DBS disk. The DBS inter spot analysis precision (7% coefficient of variance) is comparable to plasma inter assay precision (6% coefficient of variance). Coefficient of variance is the ratio between standard deviation to mean value for the analysed replicates. Proseek analysis of DBS could possibly reveal a unique opportunity to examine health related issues in extremely premature infants hopefully resulting in increased survival rates in the future.

INTRODUÇÃO: As mucopolissacaridoses (MPS) são doenças de depósito lisossômico, caracterizadas pela deficiência de enzimas lisossômicas envolvidas na degradação dos glicosaminoglicanos (GAGs). O acúmulo anormal dessas macromoléculas no interior dos lisossomos provoca alterações estruturais e funcionais, de caráter multissistêmico e progressivo. Os GAGs acumulados também são excretados na urina, onde podem ser identificados através de diversos métodos bioquímicos. Estas doenças estão presentes em todos os grupos étnicos e a incidência conjunta das MPS, é estimada entre 1:10.000 a 1:25.000 nascidos vivos. (Baehner, 2005). A causa das MPS é a deficiência de uma enzima específica na rota de degradação dos GAGs. As MPS são classificadas segundo o tipo de substrato (GAGs) acumulado e a enzima específica deficiente. Na síndrome de Morquio A, ou mucopolissacaridose tipo IVA (MPS IVA), o substrato acumulado é o queratan sulfato e a enzima deficiente é a N-acetilgalactosamina-6-sulfatase. (GALNS). Os pacientes afetados por MPS IVA apresentam baixa estatura, disostose múltipla, opacidade de córnea, entre outros sinais e sintomas. O desenvolvimento psicomotor e mental é normal. O método de detecção inicial das MPS baseia-se na identificação dos GAGs, que são excretados em excesso na urina destes pacientes. A presença de queratan sulfato na eletroforese ou a detecção de níveis aumentados na dosagem quantitativa, direciona a investigação laboratorial para a MPS IV. O diagnóstico definitivo se estabelece através da medida da atividade enzimática em leucócitos ou fibroblastos, onde se constata a deficiência enzimática. OBJETIVOS: Este estudo teve como objetivo principal, tornar disponível um novo método, mais simples, rápido e acessível, para o diagnóstico bioquímico de mucopolissacaridose tipo IVA, utilizando amostras de sangue impregnadas em papel filtro (SIPF). MATERIAIS E MÉTODOS: Amostras de SIPF e leucócitos de 35 pacientes de ambos os sexos, com idade entre 3 e 47 anos, com diagnóstico previamente estabelecido de MPS IVA, pelo método convencional, em leucócitos e /ou fibroblastos, foram analisadas. Para o estabelecimento dos valores de referência, foram estudadas amostras de leucócitos e de SIPF de 54 indivíduos saudáveis (18-50 anos), de ambos os sexos. Após assinatura do termo de consentimento, amostras de sangue periférico de pacientes e controles, foram coletadas, para a obtenção de leucócitos e sangue impregnado em papel filtro.(SIPF). Os ensaios enzimáticos foram realizados nas amostras de leucócitos e SIPF, simultaneamente, para comparar os resultados. RESULTADOS: Os resultados obtidos nos ensaios enzimáticos de todos os pacientes apresentando MPS IVA, confirmaram a deficiência da atividade enzimática em ambos materiais (leucócitos e SIPF) com uma diferença estatisticamente significativa em relação ao grupo controle. (Mann-Witney U test, p< 0,001). Neste estudo, a medida de GALNS em amostras de SIPF permitiu a identificação dos pacientes com MPS IVA, com sensibilidade de 100 %. Os testes de estabilidade realizados nas amostras de SIPF indicaram que amostras coletadas para a medida da atividade de GALNS devem ser mantidas a 4ºC sempre que possível, sendo estáveis nesta temperatura por mais de 30 dias. CONCLUSÕES: Nas condições utilizadas, amostras de SIPF se mostraram adequadas para a identificação segura de pacientes com MPS tipo IVA. O método que utiliza amostras de SIPF é mais acessível e rápido, simplificando a etapa de coleta e transporte, podendo ser utilizado para detectar pacientes afetados, especialmente em áreas de difícil acesso para a coleta e transporte de amostras líquidas. / INTRODUCTION: Mucopolysaccharidosis (MPS) are lysosomal deposit diseases characterized by lysosomal enzymes deficiency involved in the degradation of glycosaminoglycans (GAGs). The abnormal accumulation of these macromolecules inside the lysosomes provokes structural and functional alterations multi-systemically and progressively. The accumulated GAGs are also excreted in the urine, where they may be identified through many different biochemical methods. These diseases occur among all ethnical groups and the combined incidence of MPS is estimated at 1:10.000 to 1:25.000 live births. (Baehner, 2005). The MPS’ cause is the deficiency of a specific enzyme in the GAGs degradation route. The MPS are classified according to a type of substrate accumulated (GAGs) and the deficiency of a specific enzyme. In Morquio syndrome A or Mucopolysaccharidosis type IVA (MPS IVA), the accumulated substrate is the keratan sulfate and the deficient enzyme is the N-acetylgalactosamine-6-sulfatase (GALNS). The patients affected by MPS IVA present short stature, dysostosis multiplex, corneal opacity, among others signs and symptoms. The cognitive and mental developments are normal. The MPS initial detection method is based on the identification of the GAGs which are excreted in the patients’ urine. The presence of the keratan sulphate in the electrophoresis or the detection of the increased levels in the quantitative dosage directs the laboratory investigation to MPS IV. The definitive diagnosis is established through measuring the enzymatic activity in leukocytes or fibroblasts, in which the enzymatic deficiency is proved. OBJECTIVE: This study’s main purpose is to offer an original, simpler, faster and more accessible method for biochemical diagnosis of Mucopolysaccharidosis type IVA using dried blood samples (DBS). MATERIALS AND METHODS: DBS and leukocytes from 35 patients from both sexes between 3 and 47 years of age with previously established diagnosis of MPS IVA through the conventional method in leukocytes and/or fibroblasts were analyzed. In order to establish reference values DBS and leukocytes samples from 54 healthy people (18-50 years of age) from both sexes were studied. After signing a paper consent form, peripheral blood samples from patients and controls were collected for obtaining leukocytes and dried blood samples (DBS). To validate the method, we made a simultaneous GALNS assay in leukocytes and DBS. RESULTS: The results obtained in the enzymatic assays from all patients presenting MPS IVA confirmed the deficiency of enzymatic activity in both materials (leukocytes and DBS) with a significant statistical difference in relation to the control group. (Mann-Witney U tes, p< 0,001). In this study, the quantity of GALNS in DBS allowed the identification of patients with MPS IVA with sensibility of 100%. The stability tests indicate that DBS samples collected for measuring the activity of GALNS must be kept at 4ºC whenever possible, being stable in this temperature for more than 30 days. CONCLUSION: In the used conditions, DBS were adequate for a safe identification of patients with MPS type IVA. The method which utilizes DBS is cheaper and faster, what simplifies the collection and transportation stage and can be used to detect affected patients especially in difficult access areas for the collection and transportation of liquid samples.