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Measuring cytokine levels in Exhaled Breath Condensate (EBC) continues to be a major objective of breath research. However, there are often substantial data reporting and interpretation differences between research groups making comparisons between studies difficult.

In his recent editorial entitled “Understanding new ‘‘exploratory’’ biomarker data: a first look at observed concentrations and associated detection limits” , Joachim D. Pleil of the National Exposure Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency outlines a simple technique to quantify and interpret biomarkers levels in EBC.

The distribution and range of cytokines in EBC as well as the detection limit of the instrument are often unknown in these exploratory biomarker data sets.  This can make direct comparisons within and between studies difficult. Dr. Pleil initially applies simple statistical tools in organizing these data. Given a list of measurements data for each particular analyte, he orders them from lowest to highest and determines which measurement values are positioned as minimum, maximum, and the 5th, 25th, 50th, 75th, 95th (etc.) percentiles. From this a simple table is constructed.

One of Dr. Pleil’s examples is a set of EBC cytokine measurements using a new immunochemistry platform (MesoScale Discovery, MSD, Gaithersburg, MD) detailed in a prior publication entitled “Analysis of inflammatory cytokines in human blood, breath condensate, and urine using a multiplex immunoassay platform” by Stiegel et. al. These data are tabulated in the editorial and show that:

  • Most EBC specimens had measurable levels of the selected cytokines above the estimated lower level of quantification (LLOQ)
  • Measurements ranged from 0.058 pg/ml to 222 pg/ml depending on the analyte
  • Quartile ranges for different cytokines varied substantially and offered some insight into their overall distributions
  • Cytokines ranked in order of prevalence were IL-10, IL-8, IL-4, IL-5, TNF-a, IFN-g, IL-12, IL-lb, IL-2, and IL-13 for this particular set of EBC specimens

Organizing the data set by percentiles makes it easy to rank these 10 cytokines for prevalence and concentration in EBC. This ranking can serve as a comparison point when interpreting cytokine data from different groups and allow a more accurate aggregate view of biomarker levels and distributions.

Dr. Pleil’s group is now working with a third-generation fully-automated robotic immunochemistry platform (SIMOA HD-1 analyzer, Quanterix Corporation, Lexington KY). They are preparing a new article evaluating ultra-low detection of cytokines in the very dilute EBC matrix that shows sub-pg/ml sensitivities. This methodology holds promise for high-throughput toxicity testing for linking in-vivo adverse outcome pathways (AOPs) with in-vitro molecular level analyses, a concept that the U.S. EPA has been developing for a few years now for rapid screening for chemicals in commerce (Pleil et al. 2012).

Dr. Pleil can be reached at pleil.joachim@epa.gov or at pleil@unc.edu with any questions about this research.

Pleil JD, Williams M, and Sobus JR, 2012. “Chemical Safety for Sustainability (CSS): Biomonitoring in vivo data for distinguishing among adverse, adaptive, and random responses from in vitro toxicology”, Toxicology Letters 215(3):201-207.

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