Red Blood Cell Agglutination for Blood Typing Within Passive Microfluidic Biochips.

Pre-transfusion bedside compatibility test is mandatory to check that the donor and the recipient present compatible groups before any transfusion is performed. Although blood typing devices are present on the market, they still suffer from various drawbacks, like results that are based on naked-eye observation or difficulties in blood handling and process automation. In this study, we addressed the development of a red blood cells (RBC) agglutination assay for point-of-care blood typing. An injection molded microfluidic chip that is designed to enhance capillary flow contained anti-A or anti-B dried reagents inside its microchannel. The only blood handling step in the assay protocol consisted in the deposit of a blood drop at the tip of the biochip, and imaging was then achieved. The embedded reagents were able to trigger RBC agglutination in situ, allowing for us to monitor in real time the whole process. An image processing algorithm was developed on diluted bloods to compute real-time agglutination indicator and was further validated on undiluted blood. Through this proof of concept, we achieved efficient, automated, real time, and quantitative measurement of agglutination inside a passive biochip for blood typing which could be further generalized to blood biomarker detection and quantification.

Real time observation and automated measurement of red blood cells agglutination inside a passive microfluidic biochip containing embedded reagents.

The process of agglutination is commonly used for the detection of biomarkers like proteins or viruses. The multiple bindings between micrometer sized particles, either latex beads or red blood cells (RBCs), create aggregates that are easily detectable and give qualitative information about the presence of the biomarkers. In most cases, the detection is made by simple naked-eye observation of agglutinates without any access to the kinetics of agglutination. In this study, we address the development of a real-time time observation of RBCs agglutination. Using ABO blood typing as a proof-of-concept, we developed i) an integrated biological protocol suitable for further use as point-of-care (POC) analysis and ii) two dedicated image processing algorithms for the real-time and quantitative measurement of agglutination. Anti-A or anti-B typing reagents were dried inside the microchannel of a passive microfluidic chip designed to enhance capillary flow. A blood drop deposit at the tip of the biochip established a simple biological protocol. In situ agglutination of autologous RBCs was achieved by means of embedded reagents and real time agglutination process was monitored by video recording. Using a training set of 24 experiments, two real-time indicators based on correlation and variance of gray levels were optimized and then further confirmed on a validation set. 100% correct discrimination between positive and negative agglutinations was performed within less than 2min by measuring real-time evolution of both correlation and variance indicators.