A Novel Paper-Based Cytometer for the Detection and Enumeration of White Blood Cells According to their Immunophenotype

Total and differential white blood cell (WBC) counts are a vital metric used routinely by clinicians to aid in the identification of diseases. However, the equipment necessary to perform WBC counts restricts their operation to centralized laboratories, greatly limiting their accessibility. While several approaches have emerged to address this issue, the resulting handheld analyzers or microfluidic devices have costs and/or cumbersome external equipment that limit their widespread adoption as point-of-care cytometers. More established solutions for the development of point-of-care assays, namely lateral flow immunochromatographic assays and paper-based microfluidic devices, are inherently limited in their ability to support the detection of WBCs due to design constraints—the pore sizes of the materials used to fabricate these devices (e.g., nitrocellulose membranes or cellulosic chromatography papers) do not permit passive WBC transport via wicking. Herein, we identify a material capable of the unimpeded transport of WBCs in both the lateral and vertical directions: a coffee filter. This innovation facilitates the creation of the first paper-based cytometer. Through in situ labeling with an enzyme-labeled affinity reagent, our paper-based cytometer enumerates WBCs according to their immunophenotype. Using two cultured leukocyte lines (Jurkat D1.1 T cells and MAVER-1 B cells), we demonstrate the specific, colorimetric enumeration of each target cell population across the physiological range for total lymphocytes, 1000–4000 cells μL-1. This breakthrough demonstration paves the way for a new class of paper-based devices—those capable of controlled white blood cell transport, labeling, capture, and detection—thus expanding the opportunities for low-cost, point-of-care cytometers.