Advancing the collection and storage of blood micro-volumes for downstream applications
Creators: name: STASKOVA, Lada
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 340399
subject: Macromolecular and materials chemistry not elsewhere classified

description: Blood is the sample that is used as the main biological source for diagnosis and screening. The current process for blood collection involves vein incision in the inner arm. Many find this process invasive and painful. Furthermore, the collection process relies on a trained health professional, which creates an extra burden for the patient who has to travel to a local collection centre.Such collection can be particularly challenging for children and the elderly. An alternative, less invasive method, which is used in newborn screening, called dried blood spot (DBS) method allows the collection of a small volume of blood onto the filter paper. The process requires a finger prick in adults or heel prick in kids and is performed using a lancet. The blood volume collected is minimal and the benefits of storing such a sample on filter paper reduce the cost of storage and transport. DBS is utilised in research, however, the use is limited in a clinical setting. From an analytical perspective the commonly discussed bias is the haematocrit (HCT), which limits thepossibility for accurate quantitative analysis. Different approaches are being developed to tackle the HCT related issue and improve the quantitative analysis yet maintaining the benefits of DBS. Several new blood collection and storage devices are being developed to allow easy and accurate blood collection, together with good quality samples, one of which is tested in this thesis. The blood collection device tested here is the hemaPEN®, which collects blood via four borosilicate capillaries and stores samples on pre-punched filter paper within the device.

Chapter 1 reviews the literature and outlines the importance of blood, clinical use of blood, current methods of blood collection including alternative methods and the benefits of DBS. The chapter further outlines the use of DBS method and identifies the issues associated with the current use of DBS. The final review identifies new emerging approaches in sample storage including different collection and storage devices.

Chapter 2 summaries the overall methods used in the project. The chapter outlines the processes used for hemaPEN®assisted collection, generating and analysing samples for method comparison, HCT bias and the comparison of DNA extraction kits across different substrates. Furthermore the chapter outlines the statistical methods.
 
Chapter 3 explains the utility of hemaPEN®from both the collection and analytical perspective. Firstly the chapter explains the results from assisted collection at the Royal Children’s Hospital Pathology Collection Centre, where the device was successfully used. The results concluded that most collectors could use the device given the instruction material but some collectors might require further practice. Furthermore, the device was tested from an analytical perspective in the newborn screening (NBS ) laboratory. The device was compared to strand DBS method across eleven different concentration levels at the average male HCT. Overall linear correlation with increasing concentration levels was observed in both methods indicating good reproducibly. However, when comparing the method agreement,a proportional bias was observed with larger difference at higher concentration levels. The difference in the methods was mostly due to the different volume collected and analysed which is associated with the known volumetric inaccuracy in standard DBS. 

Chapter 4 expands on the HCT bias associated with standard DBS method. The hemaPEN®device was used across five different haematocrit levels to test the HCT dependency when thevolume bias is corrected. The two methods were analysed in the NBS laboratory. By collecting accurate volumes of blood followed by the whole punch analysis, few analytes showed HCT independency in the hemaPEN®, indicating that volumetric correction can remove the HCT related bias.However, the results also suggested that by removing the volumetric bias associated with standard DBS, other areas of bias were uncovered outlining the complexity of HCT related bias. The other HCT dependant areas of bias such as HCT related extraction bias and the distribution of target analyte in whole blood must be understood when implementing the use of any DBS related workflow. 

Chapter 5 expands on the use of DBS in other fields such as genomic and epigenomics. DNA has been successfully extracted from DBS, however, the recovery is often effected by the spot size, sample age and extraction kit used. In this chapter, the different extraction kits and substrates both synthetic and non-synthetic were tested to maximise the yield. The results suggests that both extraction kit and substrate type can improve the yield. Chemically treated cellulose based substrate Whatman<sup>tm</sup>FTA<sup>tm</sup>Elute resulted in the highest DNA yield using the Quick-DNA<sup>tm</sup>extraction kit. Furthermore, the chapter concludes that the use of synthetic substrate resulted in isolating high molecular weight DNA. Further research to optimise such substrate and tailor its properties for DNA extraction could further improve DNA recovery. 

Chapter 6 outlines the conclusions for each chapter and summaries the overarching conclusions. The chapter highlights that to implement a novel device there are three areas that should require further testing;the usability from a collection perspective, either assisted or at home, use in workflow where DBS cannot be used and then move to optimising the methods to widen the use of such a device, in this case through substrate optimisation. The final discussion touches on the benefits of modifying the current medical system to a more patient-centric approach that would not only benefit the people but reduce hospital admission and medical burden.