October 2002
Applied Optics
Chad Roller, Khosrow Namjou, James D. Jeffers, Mark Camp, Adam Mock, Patrick J. McCann, and Joe Grego

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Nitric oxide breath testing by tunable-diode laser absorption spectroscopy: application in monitoring respiratory inflammation

Chad Roller, Khosrow Namjou, James D. Jeffers, Mark Camp, Adam Mock, Patrick J. McCann, and Joe Grego

We used a high-resolution mid-IR tunable-laser absorption spectroscopy TLAS system with a single IV VI laser operating near 5.2 m to measure t he level of exhaled nitric oxide eNO in human breath. A method of internal calibration using simultaneous eNO and exhaled CO2 measurements eliminated the need for system calibration with gas standards. The results observed from internally calibrating the instrument for eNO measurements were compared with measurements of eNO calibrated to gas standards and were found to be similar. Various parameters of the TLAS system for eNO breath testing were examined and include gas cell pressure, exhalation time, and ambient NO concentrations. A reduction in eNO from elevated concentrations 44 parts in 109 to near-normal levels 20 parts in 109 from an asthmatic patient was observed after the patient had received treatment with an inhaled glucocorticoid anti-inflammatory medication. Such measurements can help in evaluating airway in- flammation and in monitoring the effectiveness of anti-inflammatory therapies. ?? 2002 Optical Society of America
OCIS codes: 000.1430, 140.3070, 300.1030, 300.6340.

1. Introduction
There has been much interest in the detection of exhaled nitric oxide eNO in medicine for directly assessing airway inflammation. Clinical eNO breath analysis provides the physician a simple and noninvasive window into the activities of disease, including asthma, chronic obstructive pulmonary disorder, and cystic fibrosis, in the lower airways. ¹ There have been numerous studies with which eNO was monitored in children and adults that focused mainly on the use of such measurements for assisting in initial investigations for the presence of airway inflammation associated with asthma and monitoring the effectiveness of anti-inflammatory glucocorticoid medications aimed at inhibiting nitric oxide NO producing synthase activity. ^2,3 Current diagnostic tests such as spirometry provide limited and only indirect information about the actual degree of in- flammation in the lower airways. ⁴ It is worth noting that there is a lack of objective and routine clinical diagnostic tests for assessing airway inflammation in children younger than 8 years of age.

Various technologies have been proposed for nextgeneration medical devices that will be capable of making routine eNO measurements of patients in a clinical setting. Chemiluminescence is the most recently proposed technique for this purpose and has given rise to numerous insightful reports, many of which are referenced in Ref. ⁵ Based on these reports, the American Thoracic Society has proposed standardized breath-collection procedures and the European Respiratory Society has stated that eNO serves as a valid biological marker of airway inflammation and can be used to evaluate the effectiveness of treatments. ^6,7 However, several factors have hampered the adoption of chemiluminescence in the United States, and these include the need for frequent calibrations and the necessity for the patient to maintain constant exhalation flows. ⁸ Although endogenous and exogenous water vapor, carbon dioxide, and ammonia can contribute to inaccurate eNO measurements with chemiluminescence instruments, ⁹ the reasons mentioned above are most likely responsible for the large variations of results reported in various clinical studies. ⁵ Because of the complexity of breath collection by chemiluminescence, the technique is not recommended for children younger than 8 years of age. Fourier-transform IR spectroscopy and gas chromatography coupled with mass spectroscopy are selective and sensitive but cannot perform rapid trace-gas measurements, which are desired for clinical use. To date, the U.S. Food and Drug Administration has not approved any medical device for NO breath monitoring, but it has assigned the product code MXA for such a device, and efforts to obtain approval are ongoing.

Mid-IR high-resolution spectroscopy in the 3 20- m region is an analytical technique that can permit rapid and selective measurements of eNO with the required sensitivity of 1 part in 10 ⁹ ppb . Tunable laser absorption spectroscopy TLAS is a common technique for laboratory and scientific field measurements of trace gases with absorption features residing in the mid-IR region of the electromagnetic spectrum. Two major factors have prevented the practical application of TLAS in clinical settings for routine breath analysis. First, the need for cryogenic cooling of some mid-IR lasers requires a large liquid-N2 supply, which is not readily available in most clinical settings. Second, the need for frequent calibrations with calibration gases can be burdensome and limits the operators of the instrument to trained personnel. A brief overview of candidate mid-IR spectroscopic gas sensing technologies for eNO breath measurements is now given.

2. Mid-IR Laser Gas Sensing Technologies
Mid-IR sources for TLAS systems that are currently available include quantum cascade QC distributedfeedback DFB lasers and IV VI diode lasers. QC lasers can be operated either in continuous-wave cw mode at cryogenic temperatures or by pulsing of the pump current with duty cycles that are typically less than 1% at temperatures ranging from 40 ??C to above room temperature. There is a significant reduction in average optical output power when the QC laser is pulsed, but average optical output powers in the milliwatt range can still be achieved without the need for liquid-N2 sources. Pulsed QC lasers have demonstrated room-temperature operation, and Namjou et al.10 reported measurements of N2O in the low parts in 106 ppm range by pulsing the laser and using second-harmonic 2f detection. NO trace-gas measurements at concentrations of less than 10 ppb with rapid integration times (less than or equal to 5 s) for pulsed QC DFB lasers operated at near room temperature in the 5.1 to 5 m region can be difficult to achieve. Pulsing the QC laser current can result in laser linewidth broadening caused by frequency chirp reducing the effective sensitivity. However, the frequency chirp effect when QC lasers are pulsed could be minimized by incorporation of advanced data-acquisition techniques. Research into improving mid-IR QC laser technology is ongoing, and room-temperature cw QC lasers operating at 9.1 m have recently been demonstrated with maximum optical output powers of 17 mW. ¹¹ Extension of this achievement toward fabrication of room-temperature cw QC lasers operating at shorter wavelengths at which NO absorption lines can be measured is a worthwhile objective.

IV-VI diode lasers also known as lead-salt lasers have been used to make sensitive measurements of many molecular species. Specifically, cryogenically cooled IV VI lasers operated in cw mode have been used to measure CO, NO, CO2, NH3, and CH4 in the exhaled breath of human subjects. ^12,13 IV-VI lasers with emission wavelengths of 4-8m composed of conventional double-heterostructure p n junctions are commercially available. As with QC lasers, research into improving mid-IR IV-VI laser technology is ongoing. For example, it has been shown ¹⁴ that one can achieve a significant increase in operating temperatures by removing the growth substrate from an epitaxially grown laser structure ^15,16 and applying a second heat sink to improve heat dissipation in the active region. Other recent achievements that illustrate the possibility of room-temperature cw laser emission include demonstration of above-roomtemperature cw photoluminescence from IV-VI multiple-quantum-well structures ^17-19 and aboveroom- temperature operation near 4.1 m of an optically pumped vertical-cavity surface-emitting laser. ²⁰ The results of this theoretical and experimental work show that significant improvements in IV-VI laser technology are possible, and commercialization of novel mid-IR IV VI lasers with improved operating characteristics is a realistic expectation. Further development of IV- VI laser technology, as with QC laser technology, will permit significant reductions in the size and cost of chemical sensing instruments designed for trace-gas measurements and clinical breath analysis.

An alternative to waiting for improvements in the operating temperatures of mid-IR laser sources is to eliminate the need for liquid-N2 refills by using a closed-cycle cryogenic refrigerator to cool both the mid-IR laser and the mid-IR detector in the TLAS system. That is the method employed and described in the research reported in this paper. With this in mind, the TLAS techniques described here are potentially suitable for either IV-VI or QC lasers.

Of equal importance to the tunable laser in a mid-IR high-resolution spectroscopy system is the choice of gas cell and detection technique. A long optical path length is needed for sensitive measurements, and a small gas-cell volume is needed for fast gas exchange rates and rapid measurements. Cavity ring-down spectroscopy CRDS gas cells can achieve optical path lengths of several kilometers and gas cell volumes near ~15 cm3. The CRDS technique measures the decay times of cavity modes and has been used to measure 48.4 0.7 ppb NO in pure N2 with integration times of ~8 s. ²¹ In the study described in Ref. 21, CRDS with cw QC lasers was not able to observe eNO in breath near 1921.6 cm-1 because of strong interference from CO2 absorption lines nearby at 1921.575 and 1921.641 cm-1, which has high concentrations in breath of approximately 4% CO2. NO absorption lines located near 1900 cm-1, where there is a larger separation between NO and CO2 absorption features, ²¹ may be better candidates for breath analysis applications that use CRDS. Herein, all techniques other than CRDS that are discussed are based on intensity measurements, whereas CRDS is based on cavity decay times and is independent of laser optical output power fluctuations.

Cavity enhanced spectroscopy takes advantage of low-loss dielectric mirrors in the IR and can achieve optical path lengths up to 1 km and cell volumes below 20 cm3. A 670-m optical path-length cavity enhanced spectroscopy gas cell has been used with quasi-cw QC lasers duty cycle, 50% at cryogenic temperatures to measure NO, but it was not able to achieve the required sensitivities for measurements of eNO in human breath. ²² In the same cryogenically cooled quasi-cw QC laser system, the cavity enhanced spectroscopy cell was replaced with a multipass Herriott cell of 100-m optical path length and 3.5-L cell volume. This system was capable of measuring eNO in human breath. The minimum detectable limits for the cryogenically cooled QC laser system coupled with a Herriott cell were reported to be 2.6 ppb, with a measurement integration time of 200 s. ²

TLAS systems equipped with IV-VI lasers in conjunction with either a Herriott or a multipass White cell have been used in studies that measured eNO in human breath. ^23,24 IV-VI lasers have suitable characteristics for breath analysis, such as sufficient single-mode powers 500 W , narrow spectral linewidths 100 MHz ^225,26 and wide tunability 200 cm-1 with temperature tuning and 3 cm-1 with injection current tuning . These characteristics give TLAS instruments with IV-VI lasers high sensitivities, high molecular selectivity, rapid response times, and the ability to measure multiple trace gases simultaneously. Until QC lasers demonstrate sensitive and near real-time measurements of eNO at thermoelectrically cooled or higher laser operating temperatures, IV-VI lasers will be the most attractive and cost-effective mid-IR tunable diode lasers to use for breath analysis, especially when cryogenic temperatures are unavoidable.
The narrow linewidths of IV-VI lasers allow measurements of CO2 and NO to be made simultaneously in the region near 1912.8 cm-1. Because optical output powers from IV-VI lasers can vary over time, CO2 can be used to normalize absorption magnitudes and calculate eNO concentrations without the need for calibration procedures that use calibration gases or added optics to measure reference spectra.

It is useful to compare TLAS systems equipped with QC lasers and IV-VI lasers designed for performing eNO measurements in adults. The cryogenically cooled TLAS system with a cw QC laser described in Ref. ²² incorporated a 100-m optical path-length Herriott cell and achieved an estimated minimum detection limit for NO of 2.6 ppb for a 200-s integration time. The TLAS system equipped with a cwIV-VI laser with an 8-m optical path-length Herriott cell as described in Ref. ²⁴ achieved an estimated similar minimum detection limit of 1 ppb with an 0.5-s integration time. Table 1 compares the operational characteristics of the two systems. The most noticeable difference between the QC laser system and the IV-VI laser system is the much shorter integration time for the IV-VI laser system, which is sufficiently short to permit real-time measurements of eNO during an exhalation. Breath analysis with long integration times is possible when off-line techniques are used, and chemiluminescence experiments comparing on-line real-time and offline eNO measurements have suggested that either technique is suitable. ¹ However, real-time measurements have the distinct advantage of giving instant feedback for verification of correct breath donations.

To reach cryogenic temperatures, TLAS systems that use IV-VI lasers generally require liquid N2, which results in the need for frequent refills and an available liquid-N2 source. Replacing liquid-N2 Dewars with a closed-cycle cryogenic refrigerator capable of continuous operation eliminates the need for liquid-N2 refills and allows for long periods of convenient operation. Modern closed-cycle refrigerators can achieve continuous maintenance-free operation over a 5-year period, a more than adequate operational time for successful deployment in a clinical setting.

In this paper we describe a liquid-N2-free TLAS system equipped with a IV-VI laser operating near 5.2 m for the purpose of analyzing eNO and exhaled CO2 (eCO2) simultaneously in expired breath. The system required no consumables other than disposable mouthpieces for breath analysis. Absorption measurements were performed with a 107-m multipass White cell with a 16-L volume. A closed-cycle cryogenic refrigerator was used to maintain cryogenic laser operating temperatures below 120 K. These refrigerators can dissipate 5 W of power at typical laser heat-sink temperatures of 90 K. IV VI lasers are well suited for cooling with such a system because they typically generate less than 1 W of waste heat. By contrast, QC lasers with their large compliance voltages often generate more than 10 W of waste heat. ^27,28 The system further takes advantage of the ability of a single IV VI laser to measure H2O, CO2, and NO simultaneously, which eliminates the need for calibration gases, reference cells, and reference detectors. ²³ A breath-collection apparatus was fabricated to collect and sample breath in close accordance to the recommendations suggested by the American Thoracic Society. Daily breath measurements from five human volunteers over a period of 10 working days were performed. Daily eNO concentrations measured from these five individuals calculated with eCO2 end-tidal absorption magnitudes as a reference were compared with concentrations obtained by comparison with a calibrated NO gas standard. The effect of elevated NO levels in the ambient air on calculated eNO concentrations when eCO2 was used as an internal reference was also studied. To test the flexibility of the internal calibration scheme and to simulate measurements of a child s breath, we performed experiments in which an adult s breath was measured at different exhalation times from 5 to 20 s. Finally, suggestions for future research to establish better-standardized methodologies for implementing internal calibration for eNO by use of eCO2 are given.

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