Yes / The comet assay is a sensitive method used to detect DNA damage, measuring DNA breaks and alkali labile lesions in eukaryotic cells. Here, the use of whole blood in the alkaline gel electrophoresis method is described. Two hundred and seventy blood samples from individuals were examined: 120 healthy individuals, 65 suspected or pre-cancerous individuals and 85 cancer patients. Each sample was divided into two identical volumes in different falcon tubes. The blood was prepared and stored by adding the same amount of RPMI medium and 10% DMSO. Using the Student’s t-Test, the data showed a p value = 0.59 for Olive tail moment (OTM) and 0.16 for % tail DNA, and no statistically significant differences between the two methods, with or without treatment. In conclusion, using whole blood instead of isolated lymphocytes saves time, is still very sensitive and requires less than 20 µL of blood from each individual.

Biomaterials are commonly used in the medical clinic today; however, artificial materials can activate the cascade systems in the blood (complement-, coagulation-, contact- and fibrinolytic systems) as well as the platelets to various degrees. When an artificial surface comes in contact with blood, plasma proteins will be adsorbed to the surface within seconds. The composition of the layer of proteins differs between materials and is crucial for the hemocompatibility of the material. This thesis includes five projects. In Paper I the anticoagulants heparin and the thrombin inhibitor hirudin were evaluated in a whole blood model. Hirudin was found to be superior to low dose heparin since it did not affect the activation of the complement system nor the leukocytes. The most interesting observation was that expression of TF was seen on surface-attached monocytes in hirudin- treated blood but not heparin blood. In Paper II peptides from the streptococcal M-protein, which has affinity for the human complement inhibitor C4BP, were attached to a polymeric surface. When being exposed to blood the endogenous complement regulator was enriched at the surface of the material, via the M-peptides. With this new approach we created a self-regulatory surface, showing significant lowered material-induced complement activation. In Paper III apyrase, an enzyme which hydrolyzes nucleoside ATP and ADP, was immobilized on a polymer surface. Lower platelet activation and platelet-induced coagulation activation was seen for the apyrase-coated surface compared to control surfaces after exposure to whole human blood, due to the enzymes capability to degrade ADP released from activated platelets. In Paper IV and V we synthesized an array of polymeric materials which were characterized regarding physical-chemical properties, adsorption of plasma proteins, and hemocompatibility. The polymers showed widely heterogeneous protein adsorption. Furthermore, when the polymers were exposed to whole blood, two of the materials showed superior hemocompatibility (monitored as complement- and coagulation activation), compared to the reference poly(vinyl chloride).

Use of a smartphone as an optical detector for paper microfluidic devices has recently gained substantial attention due to its simplicity, ease of use, and handheld capability. Utilization of a UV light source enhances the optical signal intensities, especially for the particle immunoagglutination assay that has typically used visible or ambient light. Such enhancement is essential for true assimilation of assays to field deployable and point-of-care applications by greatly reducing the effects by independent environmental factors. This work is the first demonstration of using a UV LED (UVA) to enhance the Mie scatter signals from the particle immunoagglutination assay on the paper microfluidic devices and subsequent smartphone detection. Smartphone's CMOS camera can recognize the UVA scatter from the paper microfluidic channels efficiently in its green channel. For an Escherichia coli assay, the normalized signal intensities increased up to 50% from the negative signal with UV LED, compared with the 4% to 7% with ambient light. Detection limit was 10 colony-forming units/mL. Similar results were obtained in the presence of 10% human whole blood.

A 3D-printed microfluidic lab-on-a-disc (LOAD) device was designed and manufactured using a low cost ( ̃£1600) consumer grade fused deposition modelling (FDM) Ultimaker 2+ 3D printer with imbedded microfluidic channels 1 mm wide, 400 μm depth and with a volumetric capacity of approximate 23 μl. FDM printers are not typically used, or are capable, of producing the fine detailed structures required for microfluidic fabrication; in addition 3D-printed objects can suffer from poor optical transparency. However, in this work, imbedded microfluidic channels were produced and the optical transparency of the device was improved though manufacture optimisation to such a point that optical colourimetric assays can be performed in a microfluidic cuvette device with sample path length of 500 μm and volumetric capacity of 190 μl. When acetone vapour treatment was used, it was possible to improve transparency of plastic samples by up to a further 30%. The LOAD device is capable of being spun using an unmodified optical disc drive (ODD), demonstrating the centrifugation based separation of plasma from whole blood in a low-cost FDM 3D-printed microfluidic LOAD device. A cholesterol assay and glucose assay was developed and optimised using cholesterol oxidase (ChOx) or glucose oxidase (GlOx) respectively and horseradish peroxidase (HRP) for the oxidative coupling of chromotropic acid (CTA) and 4-aminoantipyrine (AAP). This produced a blue quinoneimine dye with a broad absorbance peaking at 590 nm for the quantification of cholesterol/glucose in solution. The colourimetric enzymatic cascade assays were developed for use within low-cost FDM 3D-printed microfluidic devices to demonstrate the capabilities and functionality of the devices. For comparison, the assay was run in standard 96 well plates with a commercial plate reader. The results demonstrated that the quantification of 0-10 mM glucose solution using a 3D-printed microfluidic optical device had a performance comparable to a plate reader assay; glucose assay in whole blood samples R2 = 0.96.

<p>Pyrogens are substances that may induce fever in the human body. They can be parts of bacteria, virus or fungi and due to the reaction they may cause in the body, they are routinely looked for in the medical technology industries. A method called <em>in vitro</em> pyrogen test (IPT) has been developed to detect these pyrogens. It is based on the fever reaction in the human body and only requires blood in combination with a solution believed to contain pyrogens. If the result is positive, the production of cytokines is started. The cytokines of interest in the IPT method are those involved in the fever process and two of them are IL-1<em>β</em> and TNF-<em>α</em>, which are the cytokines used as markers of infection in this study. Since the production of cytokines is in proportion to the amount of pyrogens, the inflammation-inducing potential of the sample can be decided. Due to problems in standardizing the method, mainly because it handles with living blood cells, focus is still pointed at improving it. The aim of this study was to optimize parameters within the IPT method by analysing air samples taken in indoor surroundings believed to contain pyrogens. The different parameters included extraction of the filter from the air sampling, incubation of whole blood and sample extract and analysis of the incubation with ELISA (enzyme linked immunosorbent assay). More specific, some of the issues concerned extraction media, time and shaking intensity for the extraction, blood ratio for the whole blood incubation and cytokines suitable for the method.</p><p>A possible approach for the IPT method, when analysing air samples containing pyrogens, was reached.</p> / <p>Pyrogener kallas ämnen som framkallar feber och de kan exempelvis bestå av hela eller delar av bakterier, virus eller svamp (fungi). En metod som kallas för in vitro pyrogen test (IPT) har utvecklats för att detektera dessa pyrogener. Metoden bygger på att en lösning som misstänks innehålla pyrogener får komma i kontakt med blod från en människa. Efter en inkubering på mellan 4-24 timmar har blodet reagerat på eventuella pyrogener och bildat cytokiner, där mängden cytokiner är proportionell mot mängden pyrogener. De intressanta cytokinerna i den här studien var IL-1<em>β</em> och TNF-<em>α</em>, som båda är involverade i feberprocessen. Det har varit svårigheter med att standardisera metoden, mycket beroende på att det är levande celler som hela metoden bygger på, så syftet med den här studien var att förbättra in vitro pyrogen test. Luftprover tagna i inomhusmiljöer som misstänks innehålla pyrogener har använts i försöken att optimera varje steg i processen. De olika stegen inkluderade extraktion av filter som använts vid luftprovtagningen, inkubering med helblod och provextrakt och analys av inkuberingen med ELISA (enzyme linked immunosorbent assay). Några av de parametrar som undersöktes gällde extraktionsmedium, skaktid och skakintensitet under extraktionen, blodförhållande under helblodsinkuberingen och lämpliga cytokiner för metoden.</p><p>Studien resulterade i att en metodik, för att analysera luftprov innehållande pyrogener med<em> in vitro</em> pyrogen test, kunde tas fram.</p>

The Royal Canadian Mounted Police (RCMP) Forensic Laboratory Services (FLS) needed a method to confirm positive lysergic acid diethylamide (LSD) immunoassay screening results. As a result, an ultra performance liquid chromatography tandem mass spectrometry (UPLC¢â/MS/MS) method was validated for the confirmation and quantitation of LSD, iso-LSD, N-demethyl-LSD (nor-LSD), and 2-oxo-3-hydroxy-LSD (O-H-LSD). The method was validated in urine and whole blood, where linearity, accuracy, precision, sensitivity, stability, selectivity, recovery, matrix effects, and reproducibility were evaluated. <p>The method involved a liquid-liquid extraction (LLE) of the analytes and the deuterated internal standard from 1 mL of urine or whole blood with dichloromethane:isopropyl alcohol after being basified. The average recovery for all analytes was ¡Ã 62%, and the matrix effect was found to be insignificant. MS/MS analysis was conducted with a triple quadrupole mass spectrometer by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The lowest limit of quantitation (LLOQ) was 20 pg/mL for LSD and iso-LSD, and 50 pg/mL for nor-LSD and O-H-LSD. The method was linear, accurate, precise, selective, and reproducible from 20 to 2000 pg/mL for LSD and iso-LSD, and from 50 to 2000 pg/mL for nor-LSD and O-H-LSD with an r2 ¡Ã 0.99. <p>The refrigerated and frozen long term stability was investigated for 90 days. LSD was stable at all temperatures for 90 days. Iso-LSD in blood was also stable at all temperatures for 90 days, but iso-LSD in urine showed an initial decrease followed by a gradual increase back to day 0 concentrations. Nor-LSD was stable at all temperatures up to day 14, with >43% decrease by day 30, with no additional decrease for the next 60 days. O-H-LSD in urine was stable at all temperatures for 90 days, but by day 90 O-H-LSD in whole blood stored refrigerated decreased in concentration by >37%. Additionally, a case sample that was stored at -50¡ÆC for ten years was found to still contain measurable amounts of each compound. <p>The method was applied to blind samples and a case that screened positive with immunoassay. Retention time, relative retention time, and ion ratios were used as identification parameters and found to correctly identify the analytes 100% of the time with no false positives. The case sample showed that the concentration of O-H-LSD was 4 times greater than LSD in urine. Furthermore, both the detection of O-H-LSD in a blood case sample, and LSD in a vitreous humor case sample were the first to be documented.

The Royal Canadian Mounted Police (RCMP) Forensic Laboratory Services (FLS) needed a method to confirm positive lysergic acid diethylamide (LSD) immunoassay screening results. As a result, an ultra performance liquid chromatography tandem mass spectrometry (UPLC¢â/MS/MS) method was validated for the confirmation and quantitation of LSD, iso-LSD, N-demethyl-LSD (nor-LSD), and 2-oxo-3-hydroxy-LSD (O-H-LSD). The method was validated in urine and whole blood, where linearity, accuracy, precision, sensitivity, stability, selectivity, recovery, matrix effects, and reproducibility were evaluated. <p>The method involved a liquid-liquid extraction (LLE) of the analytes and the deuterated internal standard from 1 mL of urine or whole blood with dichloromethane:isopropyl alcohol after being basified. The average recovery for all analytes was ¡Ã 62%, and the matrix effect was found to be insignificant. MS/MS analysis was conducted with a triple quadrupole mass spectrometer by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The lowest limit of quantitation (LLOQ) was 20 pg/mL for LSD and iso-LSD, and 50 pg/mL for nor-LSD and O-H-LSD. The method was linear, accurate, precise, selective, and reproducible from 20 to 2000 pg/mL for LSD and iso-LSD, and from 50 to 2000 pg/mL for nor-LSD and O-H-LSD with an r2 ¡Ã 0.99. <p>The refrigerated and frozen long term stability was investigated for 90 days. LSD was stable at all temperatures for 90 days. Iso-LSD in blood was also stable at all temperatures for 90 days, but iso-LSD in urine showed an initial decrease followed by a gradual increase back to day 0 concentrations. Nor-LSD was stable at all temperatures up to day 14, with >43% decrease by day 30, with no additional decrease for the next 60 days. O-H-LSD in urine was stable at all temperatures for 90 days, but by day 90 O-H-LSD in whole blood stored refrigerated decreased in concentration by >37%. Additionally, a case sample that was stored at -50¡ÆC for ten years was found to still contain measurable amounts of each compound. <p>The method was applied to blind samples and a case that screened positive with immunoassay. Retention time, relative retention time, and ion ratios were used as identification parameters and found to correctly identify the analytes 100% of the time with no false positives. The case sample showed that the concentration of O-H-LSD was 4 times greater than LSD in urine. Furthermore, both the detection of O-H-LSD in a blood case sample, and LSD in a vitreous humor case sample were the first to be documented.

β-Glucans are components of fungal cell walls and potent stimulants of innate immunity. The majority of research on biological activities of glucans has focused on β-(1→3)-glucans, which have been implicated in relation to fungal exposure-associated respiratory symptoms and as important stimulatory agents in anti-fungal immune responses. Fungi - and bacteria and plants - produce a wide variety of glucans with vast differences in the proportion and arrangement of their β-(1→3)-, -(1→4)- and -(1→6)-glycosidic linkages. Thus far, the pro-inflammatory potential of different β-glucans has not been studied within the same experimental model. Therefore, we compared the potency of 13 different glucan preparations to induce in vitro production of IL-1β, IL-6, IL-8 and TNF-α in human, whole blood cultures. The strongest inducers of all cytokines were pustulan [β-(1→6)-glucan], lichenan [β-(1→3)-(1→4)-glucan], xyloglucan [β-(1→4)- glucan] and pullulan [α-(1→4)-(1→6)-glucan]. Moderate-to-strong cytokine production was observed for curdlan [β-(1→3)-glucan], baker's yeast glucan [β-(1→3)-(1→6)-glucan] and barley glucan [β-(1→3)-(1→4)-glucan], while all other glucan preparations induced very low, or no, detectable levels of cytokines. We therefore conclude that innate immunity reactions are not exclusively induced by β-(1→3)-glucans, but also by β-(1→6)- and β-(1→4)-structures. Thus, not only β-(1→3)-glucan, but also other β-glucans and particularly β-(1→6)-glucans should be considered in future research.

β-Glucans are components of fungal cell walls and potent stimulants of innate immunity. The majority of research on biological activities of glucans has focused on β-(1→3)-glucans, which have been implicated in relation to fungal exposure-associated respiratory symptoms and as important stimulatory agents in anti-fungal immune responses. Fungi - and bacteria and plants - produce a wide variety of glucans with vast differences in the proportion and arrangement of their β-(1→3)-, -(1→4)- and -(1→6)-glycosidic linkages. Thus far, the pro-inflammatory potential of different β-glucans has not been studied within the same experimental model. Therefore, we compared the potency of 13 different glucan preparations to induce in vitro production of IL-1β, IL-6, IL-8 and TNF-α in human, whole blood cultures. The strongest inducers of all cytokines were pustulan [β-(1→6)-glucan], lichenan [β-(1→3)-(1→4)-glucan], xyloglucan [β-(1→4)- glucan] and pullulan [α-(1→4)-(1→6)-glucan]. Moderate-to-strong cytokine production was observed for curdlan [β-(1→3)-glucan], baker's yeast glucan [β-(1→3)-(1→6)-glucan] and barley glucan [β-(1→3)-(1→4)-glucan], while all other glucan preparations induced very low, or no, detectable levels of cytokines. We therefore conclude that innate immunity reactions are not exclusively induced by β-(1→3)-glucans, but also by β-(1→6)- and β-(1→4)-structures. Thus, not only β-(1→3)-glucan, but also other β-glucans and particularly β-(1→6)-glucans should be considered in future research.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18847号 / 医博第3958号 / 新制||医||1007(附属図書館) / 31798 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 小泉 昭夫, 教授 藤渕 航 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM