Alexander Daniel Edwards

Measuring platelet function is important for patient stratification to judge bleeding vs. thrombotic risk and for research into antiplatelet drugs to prevent cardiovascular disease. Variability in platelet function is not fully understood, and large studies of inter-individual variation are making gradual progress using laboratory measurements, but rapid and high-performance hematological tests are also needed. We present here a novel microfluidic technology for platelet function analysis that images light scatter by platelets, using a low-cost, open-source, high-throughput and customizable darkfield imaging system called Dark-Pi. The hardware consists of a camera and a simple LED light source controlled by a Raspberry Pi, with 3D-printed parts. Using the Dark-Pi, platelet aggregation was imaged within adenosine 5′-diphosphate-loaded microcapillaries, revealing clearly visible patterns. This darkfield cellular light scatter approach was previously developed for bacterial cells, and here we adapted and optimized it for directly monitoring platelet aggregation. Capturing high-quality time-resolved images of platelets undergoing activation within microcapillaries allowed us to measure changes in light scattering in platelet-rich plasma that correspond with aggregation measured using conventional laboratory methods. This novel prototype system shows that this approach may have potential for use in large-scale studies of platelet function, combining simplicity with low-cost components and using a disposable dip-and-test microfluidic format.

Background: Antimicrobial susceptibility tests (AST) are vital to guide antibiotic selection to treat bacterial infections, including urinary tract infections (UTI). Current laboratory AST takes at least 2 days. Direct testing of urine samples could speed up testing, with rapid reporting aiding timely treatment. Method: A scoping review was conducted to identify studies assessing the accuracy of direct-from-urine AST, and map the breadth of evidence. Studies that measured overall categorical agreement (CA) compared to a reference method were selected, and the reported accuracy, the type of AST method and study methodology assessed. Results: In total, 33 studies were identified that met the inclusion criteria (1976–2024). These reported overall CA for direct-from-urine AST ranging from 78.7% to 100% compared to a reference method. These studies varied in the AST method used; in urine sample collection, origin and processing; in the antibiotics evaluated; in study design and data analysis. The most common AST method evaluated was direct-from-urine disc diffusion. Most studies reported accuracy only for samples containing a single Gram-negative organism. Conclusion: Although increasing numbers of studies report the accuracy of direct-from-urine AST, heterogeneity prevented systematic comparison of methods or detailed meta-analysis. Many studies reported overall accuracy >90% CA for urine samples containing a single Gram-negative organism across several direct-from-urine AST methods, offering potential time saving compared with current methods. However, as single organism samples represent only a subset of urines taken from UTI patients, additional studies—that include analysis of samples containing mixed cultures and Gram-positive isolates—are still required.

Objectives: Antibiotic susceptibility testing (AST) results are needed more rapidly to support antimicrobial stewardship and improve patient outcomes. Diagnostic microbiology laboratories receive hundreds of urine samples daily from patients with suspected urinary tract infection (UTI) in both community and inpatient settings. Bacteriostatic boric acid preserves microbial contents during transport but may interfere with rapid AST methods. This study aimed to assess the accuracy of rapid microcapillary direct-from-urine (RMD) AST with suspected UTI patient urine, and to determine whether RMD AST is affected by boric acid. Methods: The overall accuracy of RMD AST was assessed with 352 diagnostic remnant urine samples collected with boric acid, for seven first-line antibiotics (ampicillin, amoxicillin/clavulanic acid, trimethoprim, nitrofurantoin, ciprofloxacin, cefalexin and cefoxitin). A further 90 urine samples were tested in duplicate with or without addition of bacteriostatic. Results: RMD AST showed a concordance with the reference method of 572/590 bacteria/antibiotic combinations (96.95%) for urine samples containing a single organism. The mean time to AST result was 5.85 h. When duplicate samples with or without boric acid were directly compared there was a categorical agreement of 158/160 (98.75%). Conclusions: The overall high accuracy of RMD AST for determining antimicrobial susceptibility to seven first-line antibiotics for UTI shows this method can deliver rapid results—without requiring additional processing—for urine samples routinely collected with boric acid from suspected UTI patients. The close agreement between duplicates with or without boric acid confirms this rapid direct method is unaffected by bacteriostatic collection.

The microfluidic measurement of capillary flow can be used to evaluate the response of biological samples to stimulation, where distance and velocity are altered. Melt-extruded multi-bored microfluidic capillaries allow for high-throughput testing with low device cost, but simple devices may limit control over sample flow when compared to the more complex “lab-on-a-chip” devices produced using advanced microfluidic fabrication methods. Previously, we measured the dynamics of global haemostasis stimulated by thrombin by dipping straight vertical microcapillaries into blood, but only the most rapid response could be monitored, as flow slowed significantly within 30 s. Here, we show an innovative method to extend both the stimulation process and flow measurement time without increasing the cost of the device by adding simple loops to the flexible extruded device. The loops enable longer time-scale measurements by increasing resistance to flow, thereby reducing the dependence on high stimulus concentrations for rapid reactions. The instantaneous velocity and equilibrium heights of straight and looped vertical microcapillary films were assessed with water, plasma and whole blood, showing that the loops create additional frictional resistances, reduce flow velocity and prolong residence times for increased time scales of the stimulation process. A modified pressure balance model was used to capture flow dynamics with the added loop. Looped devices loaded with thrombin and collagen showed an improved detection of blood stimulation responses even with lower stimulus concentrations, compared to straight vertical capillaries. Thrombin-activated blood samples in straight capillaries provided a maximum measurement zone of only 4 mm, while the looped design significantly increased this to 11 mm for much longer time scale measurements. Our results suggest that extending stimulation times can be achieved without complex microfluidic fabrication methods, potentially improving concentration–response blood stimulation assays, and may enhance the accuracy and reliability. We conclude adding a loop to low-cost extruded microfluidic devices may bring microfluidic devices closer to delivering on their promise of widespread, decentralized low-cost evaluation of blood response to stimulation in both research and clinical settings.

The integration of Raspberry Pi miniature computer systems with microfluidics has revolutionised the development of low-cost and customizable analytical systems in life science laboratories. This review explores the applications of Raspberry Pi in microfluidics, with a focus on imaging, including microscopy and automated image capture. By leveraging the low cost, flexibility and accessibility of Raspberry Pi components, high-resolution imaging and analysis have been achieved in direct mammalian and bacterial cellular imaging and a plethora of image-based biochemical and molecular assays, from immunoassays, through microbial growth, to nucleic acid methods such as real-time-qPCR. The control of image capture permitted by Raspberry Pi hardware can also be combined with onboard image analysis. Open-source hardware offers an opportunity to develop complex laboratory instrumentation systems at a fraction of the cost of commercial equipment and, importantly, offers an opportunity for complete customisation to meet the users’ needs. However, these benefits come with a trade-off: challenges remain for those wishing to incorporate open-source hardware equipment in their own work, including requirements for construction and operator skill, the need for good documentation and the availability of rapid prototyping such as 3D printing plus other components. These advances in open-source hardware have the potential to improve the efficiency, accessibility, and cost-effectiveness of microfluidic-based experiments and applications.

High throughput, automated, time-lapse imaging for multiparameter haemostasis.

The time-consuming nature of current methods for detecting antimicrobial resistance (AMR) to guide mastitis treatment and for surveillance, drives innovation towards faster, easier, and more portable technology. Rapid on-farm testing could guide antibiotic selection, reducing misuse that contributes to resistance. We identify challenges that arise when developing miniaturized antibiotic susceptibility tests (AST) for rapid on-farm use directly in milk. We experimentally studied three factors: sample matrix (specifically milk or spoiled milk); the commensal bacteria found in fresh bovine milk; and result time on the performance of miniaturised AST. Microfluidic “dip-and-test” devices made from microcapillary film (MCF) were able to monitor Gram-negative bacterial growth colourimetrically even in the presence of milk and yoghurt (used to simulate spoiled milk samples), as long as this sample matrix was diluted 1:5 or more in growth medium. Growth detection kinetics using resazurin was not changed by milk at final concentrations of 20% or lower, but a significant delay was seen with yoghurt above 10%. The minimum inhibitory concentration (MIC) for ciprofloxacin and gentamicin was increased in the presence of higher concentrations of milk and yoghurt. When diluted to 1% all observed MIC were within range, indicating dilution may be sufficient to avoid milk matrix interfering with microfluidic AST. We found a median commensal cell count of 6 × 10⁵ CFU/mL across 40 healthy milk samples and tested if these bacteria could alter microfluidic AST. We found that false susceptibility may be observed at early endpoint times if testing some pathogen and commensal mixtures. However, such errors are only expected to occur when a susceptible commensal organism is present at higher cell density relative to the resistant pathogen, and this can be avoided by reading at later endpoints, leading to a trade-off between accuracy and time-to-result. We conclude that with further optimisation, and additional studies of Gram-positive organisms, it should be possible to obtain rapid results for microfluidic AST, but a trade-off is needed between time-to-result, sample dilution, and accuracy.

Innovation in infection based point-of-care (PoC) diagnostics is vital to avoid unnecessary use of antibiotics and the development of antimicrobial resistance. Several groups including our research team have in recent years successfully miniaturised phenotypic antibiotic susceptibility tests (AST) of isolated bacterial strains, providing validation that miniaturised AST can match conventional microbiological methods. Some studies have also shown the feasibility of direct testing (without isolation or purification), specifically for urinary tract infections, paving the way for direct microfluidic AST systems at PoC. As rate of bacteria growth is intrinsically linked to the temperature of incubation, transferring miniaturised AST nearer the patient requires building new capabilities in terms of temperature control at PoC, furthermore widespread clinical use will require mass-manufacturing of microfluidic test strips and direct testing of urine samples. This study shows for the first-time application of microcapillary antibiotic susceptibility testing (mcAST) directly from clinical samples, using minimal equipment and simple liquid handling, and with kinetics of growth recorded using a smartphone camera. A complete PoC–mcAST system was presented and tested using 12 clinical samples sent to a clinical laboratory for microbiological analysis. The test showed 100% accuracy for determining bacteria in urine above the clinical threshold (5 out of 12 positive) and achieved 95% categorical agreement for 5 positive urines tested with 4 antibiotics (nitrofurantoin, ciprofloxacin, trimethoprim and cephalexin) within 6 h compared to the reference standard overnight AST method. A kinetic model is presented for metabolization of resazurin, demonstrating kinetics of degradation of resazurin in microcapillaries follow those observed for a microtiter plate, with time for AST dependent on the initial CFU ml⁻¹ of uropathogenic bacteria in the urine sample. In addition, we show for the first time that use of air-drying for mass-manufacturing and deposition of AST reagents within the inner surface of mcAST strips matches results obtained with standard AST methods. These results take mcAST a step closer to clinical application, for example as PoC support for antibiotic prescription decisions within a day.