Fluorescent labeling, sensing, and differentiation of leukocytes from undiluted whole blood samples

In this paper, we demonstrated leukocyte labeling, sensing and differentiation from undiluted human whole blood samples with microfabricated devices. A challenging issue in leukocyte sensing from blood samples is the required high-dilution level, which is used mainly to prevent interference from the overwhelmingly outnumbered erythrocytes. Dilution is undesirable for micro hemacytometers. It not only increases sample volume and processing time, but also requires mixing and buffer storage for on-chip implementation. Unlike commercial bulk instruments and previous efforts by other groups, we completely eliminated the requirement for dilution by staining leukocytes specifically with fluorescent dye acridine orange (AO) in undiluted human whole blood and then sensing them in microfluidic devices. Green fluorescent signal centered at 525 nm is used for leukocyte count and red fluorescent signal centered at 650 nm is used for leukocyte differentiation. Throughput of 1000 leukocytes per second was achieved.