Analytical Chemistry

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

Authors

Abstract

Total and differential white blood cell (WBC) counts are vital metrics 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. Established solutions for the development of point-of-care assays, namely lateral flow tests and paper-based microfluidic devices, are inherently limited in their ability to support the detection of WBCs—the pore sizes of materials used to fabricate these devices (e.g., membranes or chromatography papers) do not permit passive WBC transport via wicking. Herein, we identify a material capable of the unimpeded transport of WBCs in both lateral and vertical directions: a coffee filter. Through in situ labeling with an enzyme-labeled affinity reagent, our paper-based cytometer detects 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 expected physiological range for total lymphocytes, 1000–4000 cells µL-1. Additionally, we highlight a potential application of this type of device as a screening tool for detecting abnormal cell counts outside the normal physiological range and in sub-classes of cell types, which could aid in the identification of certain diseases (e.g., CD4+ T lymphocytes, an important biomarker for HIV disease/AIDS). These results pave 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.

Version notes

We expanded the range of cell counts for calibration curves for paper cytometers to detect T cells (via CD3) and B cells (via CD19). We also included a second demonstration with a paper cytometer to detect CD4+ T cells. Also included in the updated text are expanded discussions regarding the analytical performance of these assays.

Content

Thumbnail image of Murray_Paper Cytometer_MS.pdf

Supplementary material

Thumbnail image of Murray_Paper Cytometer_SI.pdf
Supporting Information for Murray et al. (Paper Cytometer)
Materials and Methods. Tables and figures depicting results from the material screen, figures and tables depicting results and analyses from the porosimetry characterization of select materials, flow cytometry results, the process for image analyses, results from statistical analyses, full calibration curves for enumerating T and B cells with and without shaded regions indicating the potential for semi- quantitative visual identification, full calibration curves for enumerating CD4+ T lymphocytes, calibration curve parameters for each assay, and results from an additional fluorescent cell mapping experiment. Supplementary references are also included.