Microdroplet Phase Change Leads to Substantial Variation in Exhaled Breath Condensate Sampling

Exhaled breath condensate (EBC) has been proposed as a promising non-invasive diagnostic biofluid for 50 years, yet no EBC-based test has reached clinical deployment. A major barrier to clinical translation is its measurement variability, which can span orders of magnitude, far exceeding the 10% clinical tolerance. Whether this variability originates from human biology or physical measurement error remains unknown. To separate these contributions, we examined how EBC biomarkers are transported during the condensation and evaporation of EBC droplets, a physical process common to all EBC samplers. Contrary to the previous assumption that biomarkers accumulate steadily, we found that microscale droplets can lose significant volatile content within each breath cycle and generate substantial cumulative measurement errors. We further developed physical models to quantify EBC biomarker transport. Together, these findings establish a basis for characterizing EBC physical measurement variability and provide an actionable path toward EBC's clinical deployment.