Thread-based microfluidics research has so far focused on utilizing and manipulating the wicking properties of threads to form controllable microfluidic channels. In this study we aim to understand the separation properties of threads, which are important to their microfluidic detection applications for blood analysis. Confocal microscopy was utilized to investigate the effect of the microscale surface morphologies of fibers on the threads separation efficiency of red blood cells. We demonstrated the remarkably different separation properties of threads made using silk and cotton fibers. Thread separation properties dominate the clarity of blood typing assays of the ABO groups and some of their weak subgroups (Ax and A3). The microfluidic thread-based analytical devices (?TADs) designed in this work were used to accurately type different blood samples, including 89 normal ABO and 6 weak A subgroups. By selecting thread with the right surface morphology, we were able to build ?TADs capable of providing rapid and accurate typing of the weak blood groups with high clarity.