br Conclusion br Acknowledgments br

Conclusion
Acknowledgments
Introduction Blood typing is important in clinical practice; in particular, ABO and Rh(D) blood typing are commonly performed prior to blood transfusions to prevent hemolysis caused by type mismatched. Manual test methods, including those using a slide or a tube, and fully automated test instruments are widely used at present [1]. In recent years novel methods have been proposed to improve the sensitivity, usability, or cost of conventional blood typing. These methods include optical sensor-based [2–5], microfluidic device-based [6–8], and paper-based [9–11] techniques. Methods that are suit for on-site use [5,6,9–11] are candidates for point-of-care blood typing. A method needs to fully satisfy criteria, including sensitivity, usability, and low cost, to be used for point-of-care blood typing. However, such a method has not been developed. Recently, we developed a method to rapidly and sensitively detect hemagglutination [12]. The method, herein called the restrictive channel method, uses a microfluidic channel paired with an evanescent field optical sensor. The channel functions as a spatial restraint against agglutinated red blood Tenovin-6 (RBCs), resulting in signal differences between agglutinated and non-agglutinated samples and thus allowing hemagglutination detection. In the previous report, we used a single-channel microfluidic chip and manual sample-reagent mixing before injection into the channel. Although hemagglutination detection has been demonstrated, multiplexing and automation of the measurements, which are essential for point-of-care blood typing, have not yet been established. The development of such a system holds promise for improving conventional blood typing methods and merits further investigation. In this paper, we present a microfluidic chip for simultaneous ABO and Rh(D) forward blood typing tests based on the restrictive channel method. To enable simultaneous and semi-automated measurements on microfluidic chips, a “mixer” for sample-reagent mixing and a “measurement chamber” on the microfluidic chips were designed. A waveguide-mode sensor, i.e., an evanescent field optical sensor equipped with a slab optical waveguide, was used with the microfluidic chips. Five measurement channels were included on the microfluidic chips to allow testing with anti-A, anti-B, anti-D, Rh control reagents, and a reference blank. ABO and Rh(D) blood typing using our measurement system were demonstrated and are described here. A droplet of diluted blood (40μL) was sufficient for distinguishing blood types.
Material and methods
Results and discussion
Conclusion A multi-channel microfluidic chip for simultaneous hemagglutination measurements was developed. To conduct parallel sample filling and sample-reagent mixing using a simple fluidic system, an appropriate mixer and measurement chamber were designed. A mixing hole encapsulated with freeze-dried reagents was employed as a mixer, which allowed sample-reagent mixing by passing the samples through the holes. The measurement chambers were equipped with capillary stop valves at their ends to facilitate parallel sample filling. The capillary valves, including four sequential valves with an opening width of 20μm, showed almost 100% stopping efficiency for dozens of measurements. Based on these results, a five-channel microfluidic chip was designed and tested. The chip was tested using freeze-dried anti-A, anti-B, anti-D, and Rh control reagents, which were encapsulated in the mixing holes to conduct ABO and Rh(D) forward blood typing. Diluted blood samples (40μL) were injected into the chip and simultaneous blood typing measurements were conducted in each of the chambers. Hemagglutination was detected using the restrictive channel method, and ABO and Rh(D) blood types were determined within 5min. Further improvements of the microfluidic chip can be implemented to increase the stability and accuracy of hemagglutination detection, including the introduction of a reservoir to increase mixing performance.