Introduction
Sepsis is an acute inflammatory response to pathogens by an immune-compromised host body. Sepsis may be defined as the body’s systemic inflammatory response (SIRS) to an infection caused by pathogens. If left untreated, it may lead to shock, multi-organ failure, and death – especially if not recognized early and treated promptly. Sepsis is the final common pathway to death from most infectious diseases worldwide, including viral infections such as SARS-CoV-2 / COVID-191. The definition of sepsis (Sepsis 3) was updated in 2016 and is currently defined as a life threatening condition caused by a dysregulated host response due to microbial infection leading to host organ dysfunction2. The term ‘septic shock’ was also defined in the same study as a subset of sepsis, where a substantial increase in mortality occurs due to profound cellular, metabolic, and circulatory abnormalities. The development of sepsis starts from a site of local infection where bacteria enter into the blood stream. The most common bacteria causing sepsis includeE.coli and Staphylococcus among others such asPseudomonas , Klebsiella , Candida, Acinetobacteretc3, 4. The detailed etiology of organisms causing sepsis highlighting the frequency of infection and their corresponding mortality rate has been studied and reported extensively3, 5. According to 2018 statistics from the World Health Organization (WHO) 6, 7, about 30 million people are affected by sepsis worldwide, among which approximately 3 million are new born and 1.2 million are children. Of those affected, 6 million people die worldwide. More than 500 thousand new born babies die each year, and one in ten maternal death occur. A recent study reported that sepsis-related deaths globally are double to what was previously estimated 8, 9. Sepsis also remains the most common cause of in-hospital deaths in the United States, costing the country $24 billion a year in 201310. In a majority of the cases, the causative pathogen is bacteria3. Rapid diagnostics and therapy administration play a vital role in sepsis-related mortality11. Sepsis can lead to septic shock, multiple organ failure, and ultimately death, with an associated rise in mortality of 7% for every hour delay in the administration of appropriate antibiotics12. Currently, the “gold standard” for diagnosis of blood stream infections (BSI) is by blood cultures. It can take 24-72h before a complete answer can be reached, including the antibiotic resistance profile 13-15. In addition, detection of fastidious bacteria and fungi, which often require longer time to grow, is challenging 16. For faster turnaround time, nucleic acid-based techniques (NAT) are promising, e.g. IRIDICA, SeptiFast, SeptiTest or U-dHRM17. These kits combine lysis buffers for fast blood sample pre-treatment and DNA extraction, with PCR analysis enabling the detection of bacterial DNA, but with low sensitivity and specificity. This is mainly due to background from the human DNA17. Although tremendous improvements have been made over the past few years, including the introduction of next generation sequencing technologies, sample preparation remains the bottleneck for further expansion of molecular diagnostics into the clinical settings.
Among emerging technologies, microfluidics is very promising tool and has the potential to enrich the pathogens from blood sample in a seemingly integrated fashion. The ability to precisely manipulate blood cells has attracted considerable research in the field and several attempts have been made to address sample preparation for sepsis diagnostics. Common methods either use the surface property to isolate bacteria using affinity separation 18-21, or other properties, such as their shape, size, deformability, density, electric or magnetic susceptibility, and hydrodynamic properties22-38. In general, there is often a trade-off between efficiency and throughput. To this end, inertial microfluidics is attractive since the passive, size-based, technology exploits inherent hydrodynamic forces that scale with increased flow rate and typically operate at extremely high volumetric flow rates (∼mL/min). However, while the technology has been utilized extensively to precisely focus and separate mammalian cells including circulating tumor cells39, separation of bacteria has been challenging. Initial work in inertial microfluidics were mostly focused on separation of bacteria from diluted red blood cells 28. Using a spiral device, Hou et al. recently used a sheath flow at the inlet to push the blood cells toward one side of the inlet wall and performed size-based differential migration of blood cells to separate the bacteria 37. Here, the bacteria and blood cells both migrate to opposite walls due of the half and full Dean cycles respectively and bacteria could be extracted at an efficiency of 70%. Although the authors achieved high throughput, influence of Dean on both big and small sized particles, could have led to a compromise in separation efficiency.
The main challenge in inertial microfluidics-based bacteria separation is due to the narrow size difference between bacteria and blood cells (\(\sim\) 3–15 µm). Size of bacteria is range from \(\sim\)1-2 µm while the size of blood cells are: 6 to 8 µm for red blood cells (RBCs)40, 7 to 30 µm for white blood cells (WBCs)41, 42 and 2 to 4 µm for platelets42. One way of improving the size resolution is to use the rheological properties of viscoelastic fluid itself to manipulate the cells. In elasto-inertial microfluidics, the combination of fluid elasticity and fluid inertia enable size-based focusing of particles43-45. Using a straight rectangular channel, we recently reported the separation of bacteria from whole blood by selectively migrating blood cells away from the walls towards the center line of the channel while bacteria remain in the streamline and could be separated 30. While the method offers capabilities for precise cell manipulation, the relatively low separation efficiency and limited throughput is hindering practical implementation. The relatively low volumetric flow rate is an inherent limitation of elasto-inertial microfluidics since the synergetic effect of the elastic forces and inertial forces are ideal at moderate flow rates. In this work, we utilize PEO (Polyethylene Oxide) as an elasticity enhancer and report on blood cell focusing and bacteria separation in spiral channels at high separation efficiency (>80%) and total volumetric flow rates (~ mL/min range). The total volumetric flow rate is defined as the summation of sample and sheath flow rates. Using a two inlet and two outlet spiral device, diluted blood sample is pinched towards the outer wall by the PEO buffer. As the hydrodynamic forces develop, the blood cells remained fully focused throughout the channel length, while smaller bacteria follow the Dean vortices and are effectively separated and collected at the inner outlet. This phenomenon of blood cells staying fully focused at the outer wall throughout the channel length, unlike in inertial microfluidics, provides rapid and continuous filtration of blood cells and allowing only the smaller bacteria to migrate efficiently to the opposite wall for separation. First, the focusing and separation phenomena of the spiral device were studied using microparticles. Using 1 μm particles as a model for bacteria, it was possible to separate the particles from 7 μm particles at a an extremely high total flow rate (sheath + sample = 1 mL/min). In addition, we also observed that it was possible to separate 100% of particles with sizes >= 3µm from 1µm particles. Thereafter, the optimized spiral geometry was utilized to separate bacteria from diluted blood. First, blood cells spiked with 1 μm particles were investigated to identify the optimal dilution conditions where the blood cells remain close to the outer wall while the 1 μm particles readily migrate towards the inner wall for separation. Finally, usingE.coli bacteria spiked into diluted blood sample as a model for sepsis, bacteria separation is demonstrated at an efficiency of 82 to 90% depending on the blood dilution (1:2 to 1:10 dilution).