Non-invasive lung disease diagnostics from exhaled microdroplets of lung fluid: perspectives and technical challenges.

The combination of ultra-sensitive assay techniques and recent improvements in the instrumentation used to collect microdroplets of lung fluid (MLF) from exhaled breath has enabled the development of non-invasive lung disease diagnostics that are based on MLF analysis. In one example of this approach, electrospun nylon filters were used to collect MLFs from patients with pulmonary tuberculosis. The filters were washed to obtain liquid probes, which were then tested for human immunoglobulin A (h-IgA) and fractions of h-IgA specific to ESAT-6 and Psts-1, two antigens secreted by Mycobacterium tuberculosis. Probes collected for 10 min contained 100-1500 fg of h-IgA and, in patients with pulmonary tuberculosis, a portion of these h-IgA molecules showed specificity to the secreted antigens. Separate MLFs and their dry residues were successfully collected using an electrostatic collector and impactor developed especially for this purpose. Visualization of MLF dry residues by atomic force microscopy made it possible to estimate the lipid content in each MLF and revealed mucin molecules in some MLFs. This exciting new approach will likely make it possible to detect biomarkers in individual MLFs. MLFs emerging from an infection site ('hot' microdroplets) are expected to be enriched with infection biomarkers. This paper discusses possible experimental approaches to detecting biomarkers in single MLFs, as well as certain technological problems that need to be resolved in order to develop new non-invasive diagnostics based on analysing biomarkers in separate MLFs.

A collection system for dry solid residues from exhaled breath for analysis via atomic force microscopy.

Exhaled air contains sub-micron droplets of lung liquid, which potentially bear biomarkers of lung diseases. After dehydration they form dry residue particles (DRPs). As a first step in developing techniques to characterize individual DRPs, a new electrostatic collector was designed in which DRPs are charged within a unipolar corona charger, concentrated in a cone funnel, and deposited onto a limited area of a highly oriented pyrolytic graphite surface. The collector captures 80%-90% of DRPs at an optimal flow rate of 0.15 l min-1. Atomic force microscopy (AFM) revealed flattened round particles 20-50 nm high, with notable protrusions at their surface suggestive of an inhomogeneous internal structure. Exposure to humid air resulted in the DRPs spreading over the surface, with a 50%-200% decrease in their heights and an increase in their lateral dimensions so that their volume decreased by only 10% ± 3%. Exposure to saturated chloroform vapor resulted in drainage of 10%-15% of the DRP volume (presumably lipids), forming collar-shaped rings around each particle but leaving the core size and structure unchanged. AFM measurements combined with laser counter measurements of the DRP concentrations were used to estimate that one liter of air exhaled by volunteers contained less than 100 pg of dry residue material.