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

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 subclasses 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.

Generating signals at converging liquid fronts to create line-format readouts of soluble assay products in three-dimensional paper-based devices

“…Nonetheless, the lateral flow device architecture largely limits testing to analytes that can be captured by immobilized molecular recognition agents (e.g., antibodies 1 or nucleic acids 2 ) and testing is predominantly qualitative (i.e., presence or absence of a test line), both of which inherently restrict how broadly LFTs can be applied as a platform for POC measurements. 3,4 As an alternative to LFTs, paper-based microfluidic devices or microfluidic paper analytical devices (µPAD) have drawn appeal due to their potential to support a broader test menu than LFTs (e.g., cell counts, nucleic acid amplification, immunoassays), [5][6][7][8][9][10] compatibility with a wide range of assay readout methods (e.g., colorimetric, fluorescent, electrochemical, chemiluminescent), [11][12][13][14][15] ability to support device complexity (e.g., evaporative concentration, origami or "pop-up" assemblies, and fluid control valves) 5,16,17,18 and utilize creative options for generating a user interface (e.g., wicking distance, timed readout, zones filled, and text appearance). [19][20][21][22][23] The first paper-based microfluidic device, and similarly many subsequent prototypes, demonstrated the detection of analytes (e.g., glucose and albumin) using chemistries that formed soluble colorimetric reaction products within the void volume of patterned paper zones.…”

Generating signals at converging liquid fronts to create line-format readouts of soluble assay products in three-dimensional paper-based devices

“…Nonetheless, the lateral flow device architecture largely limits testing to analytes that can be captured by immobilized molecular recognition agents (e.g., antibodies 1 or nucleic acids 2 and testing is predominantly qualitative (i.e., presence or absence of a test line), both of which inherently restrict how broadly LFTs can be applied as a platform for POC measurements. 3,4 As an alternative to LFTs, paper-based microfluidic devices or microfluidic paper analytical devices (μPAD) have drawn recent appeal due to their potential to support a broader test menu than LFTs (e.g., cell counts, nucleic acid amplification, immunoassays), [5][6][7][8][9][10] compatibility with a wide range of assay readout methods (e.g., colorimetric, fluorescent, electrochemical, chemiluminescent), [11][12][13][14][15] ability to support device complexity (e.g., evaporative concentration, origami or "pop-up", fluid control valves) 5,[16][17][18] and utilize creative options for generating a user interface (e.g., wicking distance, timed readout, zones filled, and text appearance). [19][20][21][22][23] The first paper-based microfluidic device, and similarly many subsequent prototypes, demonstrated the detection of analytes (e.g., glucose and albumin) using chemistries that formed soluble colorimetric reaction products within the void volume of patterned paper zones.…”

“…3,4 As an alternative to LFTs, paper-based microfluidic devices or microfluidic paper analytical devices (μPAD) have drawn recent appeal due to their potential to support a broader test menu than LFTs ( e.g. , cell counts, nucleic acid amplification, immunoassays), 5–10 compatibility with a wide range of assay readout methods ( e.g. , colorimetric, fluorescent, electrochemical, chemiluminescent), 11–15 ability to support device complexity ( e.g.…”