Increased consumption of n-3 long chain polyunsaturated fatty acids (LCPUFA) is associated with higher n-3 LCPUFA status in the circulation, which has in turn been associated with a number of health benefits in humans (Calder et al. 2006; Makrides et al. 2009; Einvik et al. 2010). The conventional approach to assay n-3 fatty acid status in humans involves invasive venous blood collection and expensive, time consuming multi-step processes as that limit its application in large-scare clinical trials and routine population screening (Riséet al. 2007). Recently, efforts have been made to adopt the dried blood spot (DBS) as a quick, inexpensive and minimally invasive alternative for the measurement of fatty acid status in humans (Marangoni et al. 2004). However, the existing DBS approaches have had only limited success in providing an accurate tool for the measurement of n-3 LCPUFA status in humans. This has been due to the presence of fatty acid contaminants in blood collection papers which are released during sample processing (Nishio et al. 1986; Ichihara et al. 2002),and the failure to prevent significant oxidative loss of the n-3 LCUFA in DBS sample during transportation and storage (Min et al. 2011; Bell et al. 2011). This thesis aimed to develop a novel DBS technique which would overcome these limitations and enable the technology to be used for the accurate evaluation of n-3 LCPUFA status in human subjects. Firstly, a wide range of potential collection matrices and lab consumables were tested to determine which contained the lowest contaminant levels. A range of antioxidants and chelating agents were then tested with DBS in order to identity the optimal combination of these factors for protecting the LCPUFA in DBS from oxidation. The protection system which provided optimal protection consisted of a combination of an antioxidant and a chelating agent applied to silica gel coated blood collection paper, and this resulted in more than 90% of the original n-3 LCPUFA content (expressed as a weight percentage in blood total lipids) in the DBS being retained following 2 months of storage at room temperature (20-25°C). This system (termed "PUFAcoat") represents a significant improvement in LCPUFA stability in DBS compared with previously reported standard DBS protection systems. For example, the standard Fluka system (Fluka blood collection kit) uses a single antioxidant (butylated hydroxytoluene, BHT) as protectant, and normal chromatography paper as a collection paper which retains only ~60% of the n-3 LCPUFA content in the applied DBS over the same time period (Min et al. 2011). To explore the mechanisms underlying the protective effect of the "PUFAcoat" and to improve my understanding of the processes causing the rapid breakdown of LCPUFA in DBS, a novel in vitro system (comprising an oil blend on collection paper) was developed. Using this model I established that iron-induced oxidation was the principle driver of the rapid loss of then-3 LCPUFA absorbed on the blood collection paper, and that iron chelating agent in the "PUFAcoat" system eliminated this process by binding the irons in the DBS samples. The clinical validity of the "PUFAcoat" system was established in a human study that compared the fatty acid spectrum obtained from my DBS method (using capillary blood) with those obtained by traditional analytical techniques (using venous blood fractions). This study demonstrated strong correlations in fatty acid status between my DBS method and conventional measurements, which indicate the potential of use of my DBS method as an appropriate alternative to conventional assessments. Morever, this clinical study showed that the n-3 LCPUFA status obtained using my DBS method reflected the habitual dietary n-3 fatty acid intakes of the study population. This thesis is the first report of a protection system that is capable of stabilising the n-3 LCPUFAs in human DBS samples over 2 months storage at room temperature. Thus, my newly developed DBS method offers a significant improvement in the useability and reliability of the DBS technique for assessing n-3 LCPUFA status in humans. My DBS method has significant potential for use in large-scale clinical testing and population based screening diagnostics which focused on the role of n-3 fatty acid status in human health.

Omega-3 long chain polyunsaturated fatty acid (n-3 LCPUFA) blood levels are a potential risk factor for coronary heart disease, particularly sudden cardiac death. Venipuncture sampling for fatty acid profiling is invasive, requires highly qualified personnel and requires a multi-step protocol to isolate blood fractions. Alternately, the use of whole blood for fatty acid profiling improves analytical throughput and allows sample collection in field research locations by enabling dried blood spotting (DBS). Dried blood spots are advantageous in comparison to venous blood sampling as they require small blood volumes and is relatively inexpensive to collect. However, FA profiles in DBS are commonly expressed qualitatively (% of total fatty acids) and not quantitatively ([mu]g/mL) as finger-tip prick (FTP) sampling usually results in the collection of an unknown volume of blood. Quantitation can be effected by preexisting fatty acid contaminants on DBS collection materials and oxidative losses of sensitive fatty acids such as n-3 LCPUFA due to the increased surface area of DBS samples. Fatty acid quantitation could detect hypo- and hyper-lipidemia in samples that a qualitative only assessments would miss. To address these issues, the relationship between blood volume and blood spot area on 903 Protein Saver Cards (903 PSC) was examined to determine the blood volume associated with a 6mm hole punch and the Mitra Microsampling Device, a product designed to collect 10 [mu]L of blood regardless of hematocrit, was assessed for FA and lipidomic analyses. To determine if fatty acid contaminants were present, the 903 PSC, the Mitra tips and Whatman chromatography paper (also commonly used to collect blood spots) were examined using gas chromatography and ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Finally, the stability of the fatty acids in a DBS sample on Mitra and 903 PSC stored at ambient, 4C, -20C and -80C temperatures with and without antioxidant for 3 months was examined. It was determined that the 6mm punch of 903 PSC contained 9.6 [mu]L of blood and that FA profiles determined from the Mitra samples were comparable to FA profiles from wet blood controls. The Mitra tips could also be used to provide similar lipidomic profiles. The 903 PSC, the Mitra tips and the Whatman paper all contained palmitic and stearic acid as free fatty acids (FFA) while the Mitra tips also had palmitoyl and stearoyl lysophosphatidylcholines (LysoPC) present. With DBS storage, the n-3 LCPUFA biomarkers were the most stable with -80C storage followed by 4C or ambient room temperatures while samples stored at -20C storage had the lowest stability in both antioxidant and no antioxidant conditions, which mirrored previous research examining whole blood storage. In conclusion, quantitative fatty acid determinations of DBS samples are possible. Blood volumes can be estimated using a defined hole punch on the commonly used 903 PSC, or defined by using a Mitra sampling device. Analysis of blank sampling devices is recommended to assess the potential impact of fatty acid and fatty acyl contaminants in any DBS collection materials to be used. Finally, storage conditions need to be a consideration with DBS sample collection as preventative steps such as storage temperature and the use of antioxidants can improve sample stability and ensure data integrity.

An informative and comprehensive book on the applications and techniques of dried blood spot sampling Dried blood spot (DBS) sampling involves the collection of a small volume of blood, via a simple prick or other means, from a study subject onto a cellulose or polymer paper card, which is followed by drying and transfer to the laboratory for analysis. For many years, this method of blood sample collection has been extensively utilized in some important areas of human healthcare (for example, newborn screening for inherited metabolic disorders and HIV-related epidemiological studies). Because of its advantages over conventional blood, plasma, or serum sample collection, DBS sampling has been valued by the pharmaceutical industry in drug research and development. Dried Blood Spots: Applications and Techniques features contributions from an international team of leading scientists in the field. Their contributions present a unique resource on the history, principles, procedures, methodologies, applications, and emerging technologies related to DBS. Presented in three parts, the book thoroughly examines: Applications of DBS sampling and associated procedures and methodologies in various human healthcare studies Applications and perspectives of DBS sampling in drug research and development, and therapeutic drug monitoring New technologies and emerging applications related to DBS sampling and analysis Dried Blood Spots: Applications and Techniques is a valuable working guide for researchers, professionals, and students in healthcare, medical science, diagnostics, clinical chemistry, and pharmaceuticals, etc.

An informative and comprehensive book on the applications and techniques of dried blood spot sampling Dried blood spot (DBS) sampling involves the collection of a small volume of blood, via a simple prick or other means, from a study subject onto a cellulose or polymer paper card, which is followed by drying and transfer to the laboratory for analysis. For many years, this method of blood sample collection has been extensively utilized in some important areas of human healthcare (for example, newborn screening for inherited metabolic disorders and HIV-related epidemiological studies). Because of its advantages over conventional blood, plasma, or serum sample collection, DBS sampling has been valued by the pharmaceutical industry in drug research and development. Dried Blood Spots: Applications and Techniques features contributions from an international team of leading scientists in the field. Their contributions present a unique resource on the history, principles, procedures, methodologies, applications, and emerging technologies related to DBS. Presented in three parts, the book thoroughly examines: Applications of DBS sampling and associated procedures and methodologies in various human healthcare studies Applications and perspectives of DBS sampling in drug research and development, and therapeutic drug monitoring New technologies and emerging applications related to DBS sampling and analysis Dried Blood Spots: Applications and Techniques is a valuable working guide for researchers, professionals, and students in healthcare, medical science, diagnostics, clinical chemistry, and pharmaceuticals, etc.

Estimation of the Time Since Death remains the foremost authoritative book on scientifically calculating the estimated time of death postmortem. Building on the success of previous editions which covered the early postmortem period, this new edition also covers the later postmortem period including putrefactive changes, entomology, and postmortem r

Fatty acid profiling provides information on dietary intakes and an understanding of lipid metabolism. High throughput techniques such as fingertip prick (FTP) sampling has gained popularity in recent years as a simplified method for basic research, and could be further used to assess disease risk in the population, and other similar high-throughput techniques have the potential to assist in the monitoring and labeling of fatty acids in the food supply. With the advancement of high-throughput sample analysis techniques, a more complete understanding of storage stability is required as a larger volume of samples are produced with equal amounts of time to analyze them. Energy-assisted analysis techniques have the potential to help ameliorate some of these issues. Presently, FTP blood, whole blood and salmon storage stability is assessed under various storage conditions, and both microwave-assisted direct transesterification and indirect ultrasound-assisted extraction techniques are assessed. It is determined that storage of FTP blood and whole blood samples at -20°C results in significant and nearly complete highly unsaturated fatty acid (HUFA) degradation compared to all other temperatures examined. This degradation is determined to be the result of hemolysis and subsequent iron release from erythrocytes initiating fatty acid peroxidation reactions. Direct transesterification of FTP blood is reduced from as long as three hours to one minute with microwave-assisted energy and fatty acid extraction from ground flaxseed is reduced to 40 minutes from as long as 24 hours without compromising fatty acid profiles. Results of the current study provides insight into the storage stability of food sample and blood samples collected via high-throughput techniques, and provides support for the utilization of further high-throughput energy-assisted analytical methods that can help to minimize the potentially detrimental effects that long-term storage can have on fatty acid profiles.