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9.5 Off-line measurement
NO determinations can be measured from samples collected into a reservoir system and later
fed through a cheminiluminescence analyzer. Similar to early online experiments, while the
absolute levels of NO measured by reservoir collection varied between investigators, the
findings across disease entities remained consistent (Alving, Weitzberg et al. 1993; Persson,
Wiklund et al. 1993; Kharitonov, Yates et al. L994; Kharitonov, Yates et al. 1995; Massaro,
Gaston et al. 1995; Deykin, Halpern et al. 1998).
A number of standardisation issues arose with this technique. Firstly, the concern regarding
NO reactivity and that inert collection bags would be required. Other factors that could affect
stability included temperature, light exposure, damage to bags, leaks and pressure changes.
Mylar or tedlar bags were early reported as the most stable (Kharitonov, Alving et al. 1997)
with NO levels consistent up to 12 hours post collection (Massaro, Gaston et al. 1995;
Massaro, Mehta et al. 1996; Canady, Platts-Mills et al. 1999; Jobsis, Schellekens et al. 2001).
Some investigators reported continued stability for 24-48 hours @aredi, Loukides et al. 1998;
Djupesland, Qian et al. 2001), while others found an increase in the NO concentration
(Silkoffl Stevens et al. 1999). NO collected in a reservoir containing silica gel was stable for
24 hours @aredi, Ioukides et al. 1998). One $oup
used a tube collection system showing
better online and off-line agreement than bag collections for both exhaled and nasal levels
@jupesland, Qian et al. 2001). Reservoir stability also depends on temperature. NO results
appeared to be stable for nine hours at 4o to 37oC but increased between nine and 48 hours in
samples with initially low concentrations. It was not stable at higher temperatures and the use
of a drying agent did not improve the stability (Bodini, Pijnenburg et al. 2003). One study
evaluated 185 breaths in bags that were re-used 10-20 times flushing three times with NO free
air between uses and stored at 22oC. Over a four day period, the samples with low NO
increased in a linear fashion, and those with high NO from asthmatic subjects showed a
gradual decrease (Silkoff, Stevens et al. L999; Kharitonov, Gonio et al. 2OO3). Higher
temperatures made the changes more rapid and lower temperatures made it less likely to
occur. The size of the balloon was not thought to be critical but was recommended to be
similar to, or larger than, the subjects' vital capacity (Linn, Avila et al. 2004).
Secondly, uncertainty existed as to whether the collection should be a single exhaled breath,
several 'single exhaled breaths' or tidal breathing. The reproducibility with single breaths
appeared to be fair or good with intra-subject coefficients of variation down to 5Vo (Gaston,
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