Characterizing asthma from a drop of blood using neutrophil chemotaxis

Abstract

Asthma is a chronic inflammatory disorder that affects more than 300 million people worldwide. Asthma management would benefit from additional tools that establish biomarkers to identify phenotypes of asthma. We present a microfluidic solution that discriminates asthma from allergic rhinitis based on a patient's neutrophil chemotactic function. The handheld diagnostic device sorts neutrophils from whole blood within 5 min, and generates a gradient of chemoattractant in the microchannels by placing a lid with chemoattractant onto the base of the device. This technology was used in a clinical setting to assay 34 asthmatic (n = 23) and nonasthmatic, allergic rhinitis (n = 11) patients to establish domains for asthma diagnosis based on neutrophil chemotaxis. We determined that neutrophils from asthmatic patients migrate significantly more slowly toward the chemoattractant compared with nonasthmatic patients (P = 0.002). Analysis of the receiver operator characteristics of the patient data revealed that using a chemotaxis velocity of 1.55 μm/min for asthma yields a diagnostic sensitivity and specificity of 96% and 73%, respectively. This study identifies neutrophil chemotaxis velocity as a potential biomarker for asthma, and we demonstrate a microfluidic technology that was used in a clinical setting to perform these measurements.

Keywords: KOALA; diagnostics; microfluidics; passive pumping.

Conflict of interest statement

Conflict of interest statement: E.K.-H.S., E.B., and D.J.B. have patent applications pending on technology cited in this work. D.J.B. has ownership in Ratio, Inc., and Bellbrook Labs, LLC. E.K.-H.S., E.B., and D.J.B. have ownership in Salus Discovery, LLC.

Figures

Fig. 1.

Overview of different diagnostic techniques and the role of neutrophils in the pathology of asthma. (A) Summary of the role of neutrophils in the pathology of asthma, showing neutrophil adhesion and transendothelial migration; chemotaxis mediated by macrophages and T-helper cells; and neutrophilia in the lung tissue that leads to airway remodeling and airflow obstruction. (B) Proposed microfluidic method (more details in Fig. S1) for phenotyping asthma patients by measuring upstream of the asthma pathology with rapid neutrophil sorting on a P-selectin–coated surface (1); neutrophil chemotaxis monitored with high-throughput microscopy and automatically tracked with software (2); and asthma characterization on the basis of chemotaxis outputs (3). (C) Traditional clinical asthma diagnostic methods occur downstream of the asthma pathophysiology by measuring the effect of leukocyte inflammation on airway obstruction, nitric oxide output, or clinical symptoms.

Fig. 2.

Characterization of diagnostic chip for performing neutrophil chemotaxis from a drop of blood. (A) Capture of neutrophils, or polymorphonuclear leukocytes (PMNs), from clinical blood samples before and after laminar flow wash steps; capture efficiency was 89% (n = 4). (B) Increase in neutrophil capture on P-selectin–coated microchannels with additional passes of blood across the microchannel substrate (n = 3). (C) Chemoattractant (CA) dose–response for neutrophils obtained from healthy human patients, measuring the absolute migration speed (a), chemotactic index (b), and chemotaxis velocity (c); time-lapse imaging for 120 min (n = 3). Note: no observable neutrophil migration in controls (gel without CA). (D) Neutrophil chemotaxis in a linear gradient of fMLP with a source concentration of 100 nM; time-lapse imaging for 90 min (n = 3). All chemotaxis data tracked and analyzed with JEX. Error bars show SEM.

Fig. 3.

Neutrophil chemotaxis for blood samples obtained from the clinic and comparison with FeNO measurements. (A) No statistically significant difference in neutrophil migration speed (i) and chemotactic index (ii) for asthmatic and nonasthmatic patients; neutrophil chemotaxis velocity (iii) is significantly lower for asthmatic patients (n = 23) compared with nonasthmatic patients (n = 11). *P = 0.002; dark line shows median; boxes show 25th and 75th percentile; endlines show minimum and maximum. (B) Comparison of neutrophil chemotaxis velocity and an emerging clinical diagnostic test (FeNO) showing higher neutrophil chemotaxis velocity for nonasthmatic patients correlating with lower FeNO values. (C) Diagnosis based on multiple chemotaxis outputs, as shown in Fig. 1B; other chemotaxis outputs (chemotactic index and absolute migration speed) do not improve diagnostic performance. All chemotaxis data tracked and analyzed with JEX. Error bars show SEM.

Fig. 4.

Performance of microfluidic assay compared with traditional methods. (A) ROC curve for the neutrophil chemotaxis velocity patient data, showing the optimal sensitivity and specificity at a diagnostic cutoff of 1.545 μm/min. (B) Comparison of our method to techniques reported in the literature (see Table S1 for additional details).