Microfluidic devices for the preparation of blood plasma samples in circulating nucleic acid based medical applications

Abstract

Separation of plasma from blood is the first sample processing step in numerous medical diagnostics, including a novel class of tests that target cell-free nucleic acids (cfNAs), such as the non-invasive prenatal tests (NIPT). Conventionally, blood plasma separation (BPS) is achieved by centrifugation and followed by a number of labour-intensive bench-top procedures. Thus, developing an alternative BPS technology based on microfluidics would open the way for integration of the entire sample processing route in a single automated device, increasing sample processing throughput and robustness. Rapid extraction of cfNAs would also improve their stability facilitating further analysis. Plasma samples in cfNA-based diagnostics need to meet strict quality criteria. Currently, all the industrial and academic operators require over 1 ml of plasma for the cfNA-based tests. However, microfluidic systems typically enable miniaturisation and smaller footprints via the extraction of much lower volumes (0.1-100 µl). Thus, combining high sample quality and large output volume was the main challenge for the development of a suitable microfluidic solution here. This thesis presents a novel coupling of hydrodynamic and sedimentation-based microfluidic cell removal in a single “hybrid” BPS system. The device output was characterised in relation to several parameters: (1) blood dilution, (2) chip geometry, (3) extraction yield, (4) cell removal, (5) haemolysis and (6) cell-free DNA (cfDNA) concentration. The quality of separated plasma was shown to be compatible with advanced cfDNA analysis methods used in NIPT (digital PCR and sequencing). Its fetal and maternal cfDNA content was demonstrated to be the same as in the control samples obtained via NIPT gold standard protocol. The design of the BPS system, most importantly its hydrodynamic module, enables the use of cost-effective mass production methods as well as the coupling of additional sample processing modules. The industrialisation of the prototype, together with the validation of its use in cfDNA workflows, will enable the future implementation of the developed system as a component of an integrated sample processing tool in cfNA-based diagnostics.