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Recommendations and pitfalls in measurements of compounds within Exhaled Breath Condensate. John Hunt, MD Associate Professor of Pediatrics Pulmonology, Allergy & Immunology University of Virginia, USA For the ERS/ATS Joint Task Force on Exhaled Breath Condensate. Conflicts of Interest.
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Recommendations and pitfalls in measurements of compounds within Exhaled Breath Condensate John Hunt, MD Associate Professor of Pediatrics Pulmonology, Allergy & Immunology University of Virginia, USA For the ERS/ATS Joint Task Force on Exhaled Breath Condensate
Conflicts of Interest • Founder of Respiratory Research,Inc., which manufactures the RTube EBC collector. • Fan of American „Football“ and European FOOTBALL both of which my son is supposed to play each Saturday AM. Major Conflict.
Image stolen without permission from: The Sixth Sense starring Bruce Willis Exhaled Breath Condensate EBC NH3 NO3- NO2- H+ IL-X cysLT 8-isoPGF2α
EBC Assays: So easy, a caveman can do it. Cavemen are smart!!!!
EBC--Key Concept #1 • EBC is not a biomarker equivalent to exhaled nitric oxide. • It is not a biomarker at all • EBC is a matrix in which biomarkers with highly diverse chemical properties can be identified. • In this regard, EBC is equivalent to blood, urine, sweat, tears and other body fluids.
Key Concept #2 • EBC biomarkers are not clones of each other. • They each have individualized considerations pertaining to their assay.
Key Concept #3 • VALIDATE, VALIDATE, VALIDATE • EBC biomarker assays will lie to you if you don’t: • Call their bluff (false positives) • Check out their alibi (false negatives)
Storage of EBC prior to assay • Confirm storage stability of your biomarkers of interest in EBC in a pilot study. • To avoid cyclic freeze-thaws, aliquot samples into several tubes when many discrete assays are planned • Immediate storage at < - 70° or colder • Assay as rapidly as your mediator requires • Take your time: Nitrate, pH, DNA, cations(?) • Moderate rush: Nitrite, isoprostanes, LTs, conductivity, proteins. • Hurry as fast as you can: H2O2 • Just forget about it: RNA
And….Extremely rapid assay is required for EBC when seeking: • Quarks, Leptons, Neutrinos or • Weapons of Mass Destruction in Iraq
Consider: • Opportunities to stabilize your biomarker (or a reaction product) by adding a relevant analyte before freezing. • Assays for Thiobarbiturate reaction products and hydrogen peroxide specifically • Addition of purified albumin to protect protein biomarkers. • Addition of RNA’se inhibitor to condenser prior to collection. • VALIDATE, VALIDATE, VALIDATE
Why are you measuring your biomarker(s)?? • Good reasons • To learn about pathologic processes in lung disease • To learn about composition of the airway lining fluid • To validate them as biomarkers of lung disease potentially useful for diagnosis and prognosis, or as objective outcome measures in clinical studies. • Bad reasons: • To get an abstract accepted • To get a paper published • To fulfill research training requirements. • To do something stupid like starting a company
H2O2 • Volatile constituent • Reported by multiple laboratories. Dozens of papers. • Reported range generally < 1 uM • 45% intrasubject coefficient of variation • Assay rapidly (how rapidly??) • Colorimetric • Fluorometric • Rapid Biosensor assay (ECoCheck, Viasys)
Decay of unstable compounds [uranium 232] Time 15 millenia 30 millenia 60 millenia
Decompositoin of unstable compounds [H2O2] Time 15 min 30 min 60 min
Nitrogen Oxides • Nitrite, Nitrate, NO2-Tyrosine,S-nitrosothiols • Referred to collectively as “NOx”, “HiNOx”: • Higher Oxides of Nitrogen • Consider each of these separately. • Nitrite and nitrate are likely in part derived from oxidation of absorbed gaseous NO ex vivo. • Nitrite and nitrate are ubiquitous on all lab surfaces, pipette tips, latex gloves, fingers, microcentrifuge tubes, and EBC collection equipment and the inside of your nose.
Nitrite (NO2-) • Found at < 200 nM to 6 uM (higher levels highly associated with salivary contamination) • Commonly below detection limit of colorimetric assays. • Borderline usefulness of ion chromatography. • Fully within range of reductive chemilumiscence. • Slow decomposition is pH-dependent • Endotracheally collected samples have substantially lower nitrite levels than oral collections.
Nitrate (NO3-) • Found at 1 uM to 20 uM range. Higher suggests salivary contamination. • May be complementary to eNO, but not generally considered to associate with eNO. • No significant difference in levels between matched oral and intubated collections.
Nitrotyrosine • Marker of nitrative/oxidative activity in the airways • Assay most commonly used is a sandwich immunoassay • 2 antibody binding sites • Not going to happen on one little amino acid • Sandwich ELISA identifies NITRATED PROTEINS, not the isolated nitrated amino acid. • Assay detects down to 4 ng/ml (of what?) • Identified range < 14 ng/ml (of what?) • Mass spectroscopy assay may be sensitive to pM range. • Few data are available.
Nitrosothiols • Relevant endogenous bronchodilators found in airway lining fluid. • Commercial colorimetric assay probably not as sensitive as claimed (25 nM). • Chemiluminescence assay available (20 nM sensitivity) • Uncertain stability in EBC. • Few data are available
Adenosine • One of the most technically validated biomarkers in EBC (primarily one group) • Assay—HPLC based, reproducible • Intraassay variability of <10% • Range of levels 0-20 nM • Biomarker with active effects in respiratory system.
Prostaglandins and Thromboxanes • Several independent groups have demonstrated these compounds in EBC using discrete assays. • Assays by competitive ELISA and RIA • HPLC based techniques • Specificity of antibodies always a potential issue. • Low pg/ml range • Reportedly small effect size
Leukotrienes • A lung biomarker in which there is strong interest because of potential clinical relevance • Identified in various assays by various groups in low pg/ml range • EIA is Competitive ELISA. Intrasample repeat assay correlation is acceptable. Interassay (different assay techniques) may reveal marked discrepancies however. Specificity a concern. • Beware if you are new to this assay!
8-isoprostane • Same issues as for LT’s • A lung biomarker in which there is strong interest because it serves as strong evidence of excessive oxidative processes • Identified in various assays by various groups in low pg/ml range • Assay is Competitive EIA. Intrasample repeat assay correlation is acceptable. Interassay (different assay techniques) may reveal marked discrepancies however. Specificity a concern. • At least one group has clearly validated their EIA against LCMS. • Beware if new to this assay!
Lipid peroxidation products • Aldehydes, nexanal, others • Higher levels probably represent injurious oxidative activity • Assay by LC/MS • Levels reported: 40 nM to 1 uM range • Few labs reporting.
EBC pH • EBC pH determined by captured volatiles • Acids and bases in the respiratory fluid have differential volatility depending on pH of source fluid • Gas standardization/deaeration is commonly used • Although CO2 is relevant to airway fluid pH, CO2 has no significant differential volatility based on pH of source fluid, and most (but not all) investigators consider it a source of noise in EBC. • gas standardizing to a known CO2 concentration
EBC pH • Colorimetric assays, various probes • Validation and reference ranges fairly well established • Intrasubject CV in health: between 3 and 12% • Within range of available assays always • (But this may be a trick!) • Oral ammonia contaminates EBC, does not lead to acidification, but at higher concentrations may decrease the sensitivity of EBC to a lower airway acidity. • Beware of freezing and sublimation (removal of acid from the EBC) • Open storage tubes only after thaw and shaking • Not all pH probes are effective for EBC pH • Consider Ross-type pH electrodes. • Or probes with high fluid leak across reference electrode.
Ammonia (NH3/ NH4+) • Volatile constituent • Dominant source is oropharyngeal (80%) • Avoided in intubated subjects • Tells little about lungs. May decrease sensitivity of EBC pH assays to detect true lower airway acidification.
Cytokines, chemokines (and proteins in general) • High interest to immune researchers, pharmaceutical companies • Protein compounds. Nonvolatile. • Assay trouble has confounded literature • ELISA usually used.. • Levels at or below detection limit • “Matrix effect” may contribute to false positives
Cytokines 2 • Opportunities • Concentration: Lyophilization/resuspension. • Multiplex bead arrays for assay • Potential for thousands of assays on one non-invasive sample • Non-invasive proteomics
Assessing salivary contamination • Amylase activity assay • Remember, it will be highly diluted. • Assays needs to be able to identify presence of amylase at 20,000 fold dilution of time-matched saliva samples. • Validate Validate Validate. • What is the stability of Amylase in your freezer? • Can amylase be formed in diseased peripheral lung tissue? • Etc..
Measurement of dilution • Quite important, unless • You are measuring volatiles (dilution is irrelevant in that setting) • You are measuring ratios of related compounds • Nitrate/nitrite ratio • IFN gamma/IL-4 ratio • GSH/GSSG ratio • Multiple methods, none well validated yet • Dilution factor assessment requires validation just like any other measurement in EBC before conclusions should be made.
Conclusion re: EBC assays • Validate, Validate, Validate • Validate again. • EBC is EASY to Collect. But Hard to Assay! • The biomarkers are there if you look hard enough. • Don’t expect more from an exhaled biomarker than you would from any clinical test. • Clinical tests have tons of problems too! • So we need to continue to use our brains. Like the Cavemen do.