The majority of laboratory testing errors originate in the pre-analytical phase. While the causes and frequencies of pre-analytical errors are well characterized, there are few studies investigating the cost of these errors. The objective of this research was to build a model to quantify the cost of pre-analytical errors occurring during inpatient complete blood count (CBC) testing at Sunnybrook Health Sciences Centre (Sunnybrook). The resultant cost model accounts for the costs of materials, resources, and personnel-time consumed in the CBC testing process. In 2011, pre-analytical errors in inpatient CBC testing cost Sunnybrook $43,462, and represented a loss of 775 employee hours due to laboratory test repetition and error-related activities. This cost model represents the minimum cost of a pre-analytical error, as costs extraneous to the laboratory were beyond the study scope. Future studies investigating downstream effects of pre-analytical errors and the costs associated with them should be conducted.

The majority of laboratory testing errors originate in the pre-analytical phase. While the causes and frequencies of pre-analytical errors are well characterized, there are few studies investigating the cost of these errors. The objective of this research was to build a model to quantify the cost of pre-analytical errors occurring during inpatient complete blood count (CBC) testing at Sunnybrook Health Sciences Centre (Sunnybrook). The resultant cost model accounts for the costs of materials, resources, and personnel-time consumed in the CBC testing process. In 2011, pre-analytical errors in inpatient CBC testing cost Sunnybrook $43,462, and represented a loss of 775 employee hours due to laboratory test repetition and error-related activities. This cost model represents the minimum cost of a pre-analytical error, as costs extraneous to the laboratory were beyond the study scope. Future studies investigating downstream effects of pre-analytical errors and the costs associated with them should be conducted.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Kateřina Totzauerová Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Free circulating nucleic acids in blood plasma Outline: The aim of this thesis was to compare various commercially available DNA extraction kits and choose the universally optimal method for routine isolation of free circulating DNA from blood plasma samples according to their optical characteristics, the qPCR results and yield of short fragments. Methods: Four commercially available methods of extraction have been chosen to compare: QIAmp DNA Blood Mini Kit, QIAmp DSP Virus Spin Kit (both Qiagen), NucleoSpin Plasma XS (Macherey-Nagel) and Agencourt Genefind v2 (Beckman Coulter). DNA was isolated from aliquots of a pooled blood plasma of healthy individuals. Plasmatic DNA was quantified after extraction by spectrophotometry, fluorimetry and quantitative polymerase chain reaction. Fragmentation analysis on selected samples by capillary electrophoresis was also realized. Results: The best yield and purity provided the method from Qiagen QIAmp DSP Virus Spin Kit. Average value of the concentration determined qPCR was 49.95 ± 23.57 ng/mL and it gave the highest values of the fluorescence of...

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Radka Vaňková Supervisor: doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Influence of Carrier Molecule Addition on Plasma Free Circulating Nucleic Acid Extraction Outline: The aim of this thesis was to compare the yields of cell-free DNA (cfDNA) extracted from blood plasma specimens with the addition of commercially available carrier molecules. According to the real-time polymerase chain reaction (qPCR) results, we chose the most optimal carrier molecules for routine isolation of cfDNA from blood plasma samples. Methods: cfDNA was isolated from aliquots of a pooled blood plasma by spin column extraction method NucleoSpin Plasma XS (Macherey-Nagel). We used 7 different types of carrier molecules as RNA carrier (Qiagen), Glycogen (Invitrogen), Poly (A) Polyadenylic acid (Roche), Linear Acrylamide (Invitrogen), Yeast transfer RNA (Ambion), Salmon sperm DNA (Invitrogen), Herring sperm DNA (Promega). After the extraction, plasmatic DNA with the addition of carrier molecules was quantified by spectrophotometry, fluorimetry and qPCR. Results: The best results were achieved with the addition of polyadenylic acid with final concentration of 1.55 μg/ml. The addition of carrier RNA and...

Background Patient safety is an undisputable part of healthcare. The use of clinical practice guidelines, usually based on evidence-based practice/best practice, promotes patient safety and high quality care, reduces unnecessary patient suffering, and healthcare costs. Analysing results from venous blood specimen collection is one of the most commonly used services within healthcare, and a substantial number of decisions on diagnosis, treatment, and treatment evaluation are based on the results. Hence, the accuracy of these tests are vitally important. Earlier research has demonstrated that healthcare staff report suboptimal adherence to venous blood specimen collection guidelines together with the need for improved practices. Blood sample collection is carried out by several professionals, among them registered nurses and, as a consequence, nursing students too. University nursing students learn and practice venous blood specimen collection in one of their first semesters. After initial skill training at clinical skill laboratories, they continue to perform the task during clinical placements in various clinical settings. Few or no studies have been performed on nursing students, hence it seemed important to assess guideline adherence to venous blood specimen collection among university students as well as to further explore adherence to guidelines among healthcare staff. Therefore, the overall aim for this thesis was to explore adherence to, and factors influencing venous blood specimen collection guidelines practice among university nursing students and healthcare staff. Methods The thesis includes four studies. Study I-III had a quantitative, cross-sectional design, study IV had a qualitative approach. Study I included 164 healthcare staff from 25 primary healthcare centres. Study II included 101 nursing students in their 5th and 6th semesters, and study III included 305 nursing students in their 2nd, 4th, and 6th semesters. To assess adherence to venous blood specimen collection guidelines, data were collected using the Venous Blood Specimen Questionnaire, completed with background variables (I, II, III) and additional scales (III). Descriptive statistics, multilevel and multiple logistic regression analyses were used to analyse the data. In study IV, data were collected through five focus group interviews among 6th semester nursing students (n=26). Data were analysed using qualitative content analysis. Results Workplace affiliation was found to explain variances in reported adherence between different primary healthcare centres. Associations between reported venous blood specimen collection practices and individual as well as workplace factors were revealed. Nursing students were found to increasingly deviate from guideline adherence during their education. Also among students, several associations between guideline adherence and other iv factors were revealed. Reported research use at clinical practice was associated with higher levels of adherence, as were higher capability beliefs regarding both evidence-based practice and academic ability. Analyses from focus group interviews summarised students’ reflections on deviations from VBSC guidelines in the overall theme ‘Striving to blend in and simultaneously follow guidelines’. Conclusion Both healthcare staff at primary healthcare centres and nursing students demonstrate decreasing levels of guideline adherence with time. Factors influencing adherence are both individual as well as contextual. This indicate that both students and staff are subjected to socialisation processes that influences levels of adherence. In order to enhance venous blood specimen collection practices and thereby patient safety, actions must be taken - both in healthcare clinical contexts and by educators. The use of models in practical skill training, and in the ambition to bridge the theory-practice gap may be the path to success. It is reasonable to assume that collaboration between, on the one hand, education representatives and on the other, supervising RNs in clinical settings, will be fruitful. Finally, by empowering students their self-efficacy may be strengthened, and hence their ability to maintain guideline adherence.

Medical Laboratories Accreditation is covered by ISO 15189:2012 - Medical Laboratories — Requirements for Quality and Competence. In Portugal, accreditation processes are held under the auspices of the Portuguese Accreditation Institute (IPAC), which applies the Portuguese edition (NP EN ISO 15189:2014). Accordingly, Medical Laboratories accreditation processes now require the estimate of measurement uncertainty (MU) associated to the results. The Guide to the Expression of Uncertainty in Measurement (GUM) describes the calculation of MU, not contemplating the specific aspects of medical laboratory testing. Several models have been advocated, yet without a final consensus. Given the lack of studies on MU in Portugal, especially on its application in the medical laboratory, it is the objective of this thesis to reach to a model that fulfils the IPAC’s accreditation regulations, in regards to this specific requirement. The study was based on the implementation of two formulae (MU-A and MU-B), using the Quality Management System (QMS) data of an ISO 15189 Accredited Laboratory. Including the laboratory’s two Cobas® 6000–c501 (Roche®) analysers (C1 and C2) the work focused three analytes: creatinine, glucose and total cholesterol. The MU-B model formula, combining the standard uncertainties of the method’s imprecision, of the calibrator’s assigned value and from the pre-analytical variation, was considered the one best fitting to the laboratory's objectives and to the study's purposes, representing well the dispersion of values reasonably attributable to the measurand final result. Expanded Uncertainties were: Creatinine - C1 = 9,60%; C2 = 5,80%; Glucose - C1 = 8,32%; C2 = 8,34%; Cholesterol - C1 = 4,00%; C2 = 3,54 %. ...[cont.].

The pre-analytical phase is known for being the most important step in the laboratory process to reach reliable test results. If handling, transport or preparation of the sample is performed incorrectly the results can deviate from the true value. Today, sampling tubes contains various additives to stabilize concentration levels. The aim of this study was to test a new sampling tube containing fluoride/citrate for glucose, lactate and homocysteine. It was also of interest to evaluate the stability of those three analytes in lithium-heparin, sodium-fluoride/potassium oxalate and fluoride/citrate tubes. To perform the sustainability study, a method verification was done for homocysteine in plasma. The study was performed in a hospital laboratory on the routine instrument Roche Cobas 6000 analyzer. Blood was drawn from 20 patients and was analyzed at the hospital laboratory in Gävle. The blood samples were transported frozen to the laboratory in Hudiksvall and were used in the method verification. For the sustainability study, blood was drawn from 10 healthy volunteers in lithium-heparin, sodium-fluoride/potassium oxalate and fluoride/citrate tubes. The method verification was approved. The results showed that glucose was stable for up to 72 hours in Vacuette Glycaemia tube with fluoride/citrate and this tube also gave more accurate results. Lactate and homocysteine were also stable in fluoride/citrate, but needs further studies. All three analytes were more stable if the sample tubes were centrifuged as soon as possible after blood collection. Fluoride/citrate tubes were stable without centrifugation directly.

In clinical diagnostics and research, blood samples are one of the most frequently used materials. Nevertheless, exploring the chemical composition of human plasma and serum is challenging due to the highly dynamic influence of pre-analytical variation. A prominent example is the variability in pre-centrifugation delay (time-to-centrifugation; TTC). Quality indicators (QI) reflecting sample TTC are of utmost importance in assessing sample history and resulting sample quality, which is essential for accurate diagnostics and conclusive, reproducible research. In the present study, we subjected human blood to varying TTCs at room temperature prior to processing for plasma or serum preparation. Potential sample QIs were identified by Ultra high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) based metabolite profiling in samples from healthy volunteers (n = 10). Selected QIs were validated by a targeted MS/MS approach in two independent sets of samples from patients (n = 40 and n = 70). In serum, the hypoxanthine/guanosine (HG) and hypoxanthine/inosine (HI) ratios demonstrated high diagnostic performance (Sensitivity/Specificity > 80%) for the discrimination of samples with a TTC > 1 h. We identified several eicosanoids, such as 12-HETE, 15-(S)-HETE, 8-(S)-HETE, 12-oxo-HETE, (±)13-HODE and 12-(S)-HEPE as QIs for a pre-centrifugation delay > 2 h. 12-HETE, 12-oxo-HETE, 8-(S)-HETE, and 12-(S)-HEPE, and the HI- and HG-ratios could be validated in patient samples.

Le sepsis est une dérégulation de la réponse de l’hôte à une infection, associé à un dysfonctionnement des organes engageant le pronostic vital du patient. Plus de 30 millions de cas et 5 millions de décès sont estimés par an dans le monde. Le diagnostic du sepsis est basé sur des signes cliniques non spécifiques et la longue procédure d’identification des pathogènes responsables de l’infection. L’objectif de cette étude est de développer et d’évaluer le potentiel de la spectroscopie vibrationnelle appliquée au sérum pour améliorer le diagnostic du sepsis. Les défis inhérents à la nature de l’échantillon et à la technique de même que certains paramètres pré-analytiques ont été évalués pour assurer la qualité des données. Les variations de contenu en eau des échantillons après séchage pouvant affecter la discrimination des données, ont été corrigées en testant différentes méthodes. Enfin, des sérums de patients septicémiques (n=380) collectés avant chirurgie et jusqu’à 3 jours avant et le jour du diagnostic ont été analysés. Les échantillons du groupe contrôle (n=353) collectés suivant la même cinétique, provenant de patients ayant un profil similaire en termes d’âge, de sexe, de procédure chirurgicale subie mais n’ayant pas développé de sepsis et des échantillons (n=180) de patients atteints d’un syndrome de réponse inflammatoire systémique collectés avant chirurgie et le jour du diagnostic ont également été analysés. Les données acquises ont été exploitées par méthodes chimiométriques pour discriminer des zones spectrales reflétant des différences de composition moléculaire avec des sensibilités et spécificités supérieures à 70% malgré l’influence de l’eau résiduelle. / Sepsis is a dysregulated host response to an infection that causes life-threatening organ dysfunction. Each year, over 30 million cases and 5 million deaths are estimated worldwide. Diagnosis of sepsis is based on non-specific clinical signs and time consuming positive identification of the causative pathogen. The objective of this study is to develop and evaluate the potential of vibrational spectroscopy applied to human serum to improve diagnosis of sepsis. Challenges of serum spectroscopy inherent to the sample nature and preparation as well as to the technique have been assessed to determine the most suitable methodological approach. Then, some aspects of the pre-analytical phase have been addressed in order to standardise protocols in sample handling and preparation for spectral acquisitions to ensure quality and reproducibility of spectral data collected. Different methods have been tested to correct water content variations in dried serum, which can impact on data discrimination. Finally, based upon the developed methodology, patient serum samples (n=380) collected before surgery, up to 3 days before sepsis diagnosis, and on the day of sepsis diagnosis have been analysed. Control serum samples (n=353) from age/ sex/ procedure-matched patients who did not go on to develop sepsis have been also analysed over similar timeframes post-surgery as well as samples (n=180) from patients with systemic inflammatory response syndrome. Spectral data acquired have been interrogated by chemometric methods to identify spectral zones reflecting differences in molecular composition allowing discrimination with over 70% of sensitivities and specificities despite water interferences.

68 f. / Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2013-04-05T17:50:29Z No. of bitstreams: 1 Dissertacao - Wdson Costa Santos.PDF: 1066509 bytes, checksum: 0409b0432c52d5bd01e90ebcf214766a (MD5) / Approved for entry into archive by Ana Hilda Fonseca(anahilda@ufba.br) on 2013-06-06T14:53:28Z (GMT) No. of bitstreams: 1 Dissertacao - Wdson Costa Santos.PDF: 1066509 bytes, checksum: 0409b0432c52d5bd01e90ebcf214766a (MD5) / Made available in DSpace on 2013-06-06T14:53:28Z (GMT). No. of bitstreams: 1 Dissertacao - Wdson Costa Santos.PDF: 1066509 bytes, checksum: 0409b0432c52d5bd01e90ebcf214766a (MD5) Previous issue date: 2012 / CAPES / No presente trabalho foram investigados os efeitos sobre a recuperação de elementos causados por modificações em processos prévios a amostragem laboratorial e lixiviação dos analitos das polpas de abóbora e batata. Inicialmente, foi avaliado o efeito da inversão na ordem de operações do procedimento de tratamento preliminar das amostras brutas das polpas sobre as recuperações dos elementos. As amostras das polpas foram secas para posterior trituração (PA) ou as amostras foram trituradas antes de serem submetidas à secagem (PB). Os resultados obtidos empregando ambos os procedimentos foram comparados para 95% de confiança e eles mostraram diferenças significativas para alguns elementos determinados na amostra de batata. As concentrações de K, P e Mn, na polpa de batata, determinadas aplicando o primeiro procedimento de pré-tratamento (PA) foram 13,6±0,4 mg K g-1, 1,95±0,04 mg P g-1 e 5,7±0,2 μg Mn g-1, enquanto 12,2±0,3 mg K g-1, 1,62±0,03 mg P g-1 e 2,6±0,2 μg Mn g-1 foram determinadas quando a mesma amostra bruta foi submetida ao pré-tratamento PB para a obtenção da amostra teste. Em contraposição, no caso da amostra bruta de polpa de abóbora, os resultados para os elementos (K, P, Mn, Mg) foram maiores quando a polpa foi previamente esmagada antes da secagem. Em um estudo independente, a eficiência da extração de metais de amostras de polpas trituradas de abóbora ou batata para soluções diluídas de HNO3 foi avaliada variando o modo (agitação mecânica ou irradiação ultrassônica) e o período (de 10 a 30 min) de agitação, bem como a concentração da solução de HNO3 na solução de lixiviação (de 0,7 a 2,1 mol L-1). Os resultados foram também comparados com aqueles obtidos nas digestões assistidas por micro-ondas das amostras teste v em meio nítrico-peróxido. Foi observado que para ambas as amostras teste (abóbora e batata) e independentemente do modo ou período de agitação que a eficiência de extração de Ca, K, Mg e Mn foi diminuída para solução para 2,1 mol L-1 HNO3. Uma melhora no nível de recuperação de P foi obtida quando a lixiviação foi realizada com soluções diluídas de HNO3 (0,7 e 1,4 mol L-1) e sob irradiação ultrassônica. Esse efeito foi relacionado à presença de átomos de fósforo em moléculas estruturais de células de abóbora e batata, bem como à cominuição (ou fragmentação) das partículas submetidas a um campo ultrassônico intenso. Contudo, efeitos indesejados foram também observados para alguns elementos quando submetidos às lixiviações assistidas com ultrassom, como exemplificado pelo teor de cálcio determinado após a lixiviação da amostra teste de batata ter atingido valor 2,3 vezes maior que o valor obtido após a mineralização da amostra em micro-ondas. Muitos desses efeitos foram relacionados à erosão da superfície interna frascos irradiados. / Salvador