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...

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.

Bakgrund: Provmaterial för joniserat kalcium är känsligt för pH-förändringar och med tanke på svårstuckna patienter är det betydelsefullt att undersöka lägre fyllnadsnivåers påverkan på analysresultatet. På grund av olika pre-analytiska faktorer kan tidsgränsen (4 timmar) för analys av standardbikarbonat överskridas. Förvaring av post-analytiska serumprover medför att kompletteringsanalyser kan beställas. Begränsad dokumentation finns om avkorkade serumprovers stabilitet i rumstemperatur. Syfte: Syftet var att undersöka hur lägre fyllnadsnivåer av serum påverkar analysresultatet för joniserat kalcium, om standardbikarbonatsprover på helblod kan analyseras senare än 4 timmar och hur länge serumprover kan stå i rumstemperatur utan kork för eventuella kompletteringsanalyser. Metod: Koncentrationen av analyten joniserat kalcium i serumprover med fyllnadsnivåerna 1 mL och 2 mL jämfördes med maximalt fyllda provrör. Kylskåpsförvarade helblodsprover analyserades för standardbikarbonat efter 4-7 timmar. Avkorkade serumprover analyserades för 10 biokemiska analyter efter att ha stått i rumstemperatur 2-8 timmar. Genomsnittlig procentuell avvikelse jämfördes med en analytisk och biologisk imprecisionsgräns för att bedöma analyters stabilitet. Resultat och slutsatser: Analysresultat av joniserat kalcium i lägre fyllnadsnivåer var tillförlitliga. Stabiliteten av standardbikarbonatsproverna kunde inte bedömas och därmed kunde inte en eventuell tidsgränsändring rekommenderas. De biokemiska analyterna var stabila upp till 8 timmar i rumstemperatur. / Background: Ionized calcium concentrations decrease when samples are exposed to air. Due to pre-analytical factors, the 4 hour time limit for analysis of standard bicarbonate, can sometimes be exceeded. There is limited documentation about additional analyses on post-analytic decapped serum samples stored at room temperature. Aim: The aim was to examine how lower sample volumes affect the concentration of ionized calcium, if the time limit for analysis of standard bicarbonate on whole blood can be prolonged and how long decapped serum samples can be stored at room temperature for eventual additional analyses. Methods: The concentration of ionized calcium was analyzed on serum samples filled with 1 mL and 2 mL and were compared to maximally filled samples. Refrigerated whole blood samples were analyzed for standard bicarbonate after 4-7 hours. Ten biochemical analytes were measured in decapped serum samples after 2-8 hours of storage at room temperature. The mean percentage deviation was compared to an analytical and biological imprecision limit to determine analyte stability. Results and conclusions: Ionized calcium concentrations in lower sample volumes were reliable. The stability of standard bicarbonate could not be determined, therefore a longer possible time limit could not be recommended. The biochemical analytes were stable for 8 hours.