Hands-free cytometry of whole blood: controlled antibody release from hydrogels for on-chip cell staining

Point-of-care (POC) diagnostics are designed for use at or near the patient. Shifting complex diagnostics performed in centralized laboratories to robust and easy-to-use POC diagnostics can reduce cost and time needed for tests, allow early detection of diseases for prompt treatment and extend care to under-served populations. The success of POC diagnostics relies on the development of automated systems for rapid analysis of blood samples. Rapid advancement in Lab-on-a-chip technologies enables the integration of multiple analytical functions into self-contained microdevices for diverse biological assays. In such devices, reshaping the conventional benchtop sample preparation into an on-chip format is essential. Over the last decade, various microfluidic concepts using sophisticated microstructure, employing regulated fluid and implementing external field have been developed to minimize the user’s intervention in the sample preparation process. However, most development strategies still employ external equipment or partially requires manual operation for the sample preparation, very few concepts can be applied into routine clinical practice.\ud \ud In this thesis, a POC immunostaining based cell identification and enumeration (InstantCount) assay of whole blood was developed. This new concept (InstantCount) was realized by incorporating controlled antibody release systems in simply designed cell counting chips for complete on-chip sample preparation (mixing and staining) and performing fluorescence imaging of stained samples for automated image analysis (cell counting). Passive and active antibody release schemes enabled by specifically prepared hydrogel matrices were implemented in the cell counting chips for homogenous staining. The acquired cell counts (e.g. CD4 counting) from the clinical study in the University Medical Centre Utrecht were validated to be accurate. Moreover, the fabrication hydrogel/antibody integrated counting chips in a fully automated manner was achieved. Therefore, our new cell counting concept, fabricated by low-cost/high-throughput drop-on-demand printing technique, allowing for intervention-free sample preparation and ensuring accurate testing, fulfils all the basic criteria for a practical and affordable CD4 counting assay in POC settings. \ud \ud The current study narrows down the scope by limiting the release matrix to hydrogel material and focusing only on immunostaining based cell counting, which only requires one-step mixing in the sample preparation. However, by exploring more in material science and taking advantage of novel material dispensing techniques, this concept has the potential to achieve multistep bioassays requiring sequential reagent release and multiplex biosensing requiring spatial reagent release