Track: Bioanalytics - Biomolecular - Biomarker Quantification - New Matrices
Category: Poster Abstract
Non-invasive Measurement of Lower Respiratory Cytokine Biomarkers with PExA and Ultrasensitive Immunoassays
Purpose: Asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), lung transplant rejection, and even SARS-CoV-2 infection, is characterized by inflammation mediated by cytokines and dysfunction of small airways. However, no standardized non-invasive biomarkers are available to assess small airway inflammation. Previous non-invasive respiratory tract collections such as exhaled breath condensate are diluted with water and contaminated with oral cavity proteins. Particles in exhaled air (PExA) from respiratory tract lining fluid of small airways consist of lipids and proteins making them an ideal non-invasive biomarker candidate. However, methods have not been created to measure cytokines in PExA due to their expected ultra-low abundance. We aimed to measure cytokines in PExA by combining microsampling techniques typically used for blood with ultrasensitive immunoassays. Methods: We collected PExA using a specialized instrument in 3 healthy volunteers. A constant 100 L of breath was collected and corrected for acquired particle mass. The PExA were collected onto a 25 mm Millipore membrane. To optimize detection of cytokines, we utilized punch out technology typically used for dried blood spots (DBS). The membrane was punched into 10 identical 1.2 mm plugs to attain 25 ng of PExA for each sample and placed into a polypropylene tube. Each plug was extracted into 25 ul of buffer. Several extraction buffers and temperatures were compared, and final samples were extracted using phosphate-buffered saline (PBS) containing 1% bovine serum albumin, 0.05% Tween-20, and protease inhibitor. We measured IgG, IL-6, TNF-a, and IL-10 in extracted samples using ultrasensitive MesoScale Discovery and Quanterix Simoa biomarker kits following manufacturer protocols. Results: IgG was detected in all PExA samples and IL-10 and TNF-a were detectable in 25% of PExA samples. IL-6 was below the limit of detection of the assay. PBS containing buffer outperformed acetonitrile or ammonia containing DBS extraction buffers. Further studies are underway to optimize intra-sample variability and determine the correlation of PExA cytokines with respiratory diseases. Conclusion: PExA collection and analysis with ultrasensitive immunoassays is a valid non-invasive method to monitor cytokines sampled from the lower respiratory tract.