Lack of standardization and sampling procedures has led to differing results and conclusions. Although a number of groups have suggested a standardized practise in breath analysis, it is still not being commonly used and so it has not been established in the breath community [57]. A problem is in part associated with the fact that no one method may be suitable, depending on the disease and volatiles.
Following an established method for collecting breath samples is crucial to have reproducible results. The difficulty in breath collection is that exhaled breath is not an homogenous sample. For this reason, breath should be sampled under specific protocol and controlled sampling procedures [58]. The challenge in breath sampling is the separation and collection of the alveolar breath from the dead space (volume of breath contained in the upper airways). The aim in breath analysis for biomarker discovery is to be able to determine when the alveolar phase is reached. For this CO2 levels could be
monitored during breath sampling.
For PTR-MS, and especially where breath to breath analysis is being done in real- time it is possible to monitor for instance acetone levels to ascertain when the alveolar
Chapter 3. Breath analysis for clinical diagnosis
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phase has been reached. However, this is limited because the PTR-MS can not be brought to a patient.
The sampling of VOCs in breath can be undertaken following two different methods. In the off-line analysis, the sample is collected in bags, canisters, syringes or using pre- concentration methods such as needle traps. In contrast, for on-line breath gas sampling, patients are able to breath directly into the instrument and the sample is analysed in real time [59]. In this way, certain problems due to sample decomposition and contamination are avoided.
3.4.1 Use of capnography for alveolar breath sampling
Capnography is a technique that gives us an excellent picture of the respiratory process and can be used to improve gas-sampling methods. Capnography provides a wave display of the carbon dioxide CO2 during the respiratory cycle. In medicine is used during
anaesthesia and intensive care as a non-invasive analysis providing and recording CO2
partial pressure measurements in exhaled breath over time.
It is important to note that a capnograph will display an erratic waveform when there are air leaks in the airways system, tubing obstructions, disconnections and CO2
recording apparatus fails to function normally [60]. For instance, the shape of CO2
waveform is monitored in order to detect any malfunction in the system. The capnogram displays a uniform and stable shape when CO2 measurements are acceptable. Whereas a
capnograph shows an irregular and anomalous shape of CO2 waveform when there is a
Chapter 3. Breath analysis for clinical diagnosis
46
The end of exhalation, called end-tidal CO2, (Et-CO2), is correlated with the alveolar
phase in exhaled breath. Et- CO2 represents the maximum concentration of CO2 or partial
pressure expressed as a percentage or in units of mmHg. The Et- CO2 is measured by a
sensor located between the patient airway and the capnograph.
The technology is based on infrared spectroscopy (IR). CO2 absorbs in the IR part
of the electromagnetic spectrum at very specific wavenumbers (usually the line at 2350 cm-1 is used) [61]. Detecting these energy absorptions, using an appropriate photo detector sensitive in this spectral region, allows CO2 concentrations in a gas sample to be
easily determined.
The capnograph monitor displays the CO2 waveform which is called capnogram,
showing the relationship of CO2 concentration versus time. The normal values are 5% to
6% CO2 and 35-45 mmHg [62].
Figure 3.3 shows a normal capnogram and the phases in the expiratory and inspiratory cycle. The first phase point A to B, represents the end of inspiration. The phase from B to C represents the beginning of expiration, exhalation of CO2 free gas from
the anatomical and apparatus dead space. This is the first step in exhalation. From point C to D the volunteer begins to exhale additional CO2 from the connecting airways and
alveoli of the lungs. This phase represents air from the alveoli and CO2 remains relatively
constant because mainly alveolar gas is exhaled and is defined as the alveolar plateau. The Et-CO2 is measured at the end of the plateau (D) correspond with the highest
concentration of CO2. Once the majority of air is exhaled from the airways, the inhalation
begins and the increasing slope of the waveform will begin to slow down (D-E). Oxygen fills the airways and CO2 levels decreases back to zero.
Chapter 3. Breath analysis for clinical diagnosis
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Uncontrolled breath sampling has been shown to be unreliable. For the correct analysis of exhaled breath it is crucial to monitor and collect only the alveolar phase of the volunteer breath. Previous investigations have determined the impact of sampling methods on the analysis of VOCs in exhaled breath. A higher concentration of volatile organic compounds in alveolar breath samples is found compared to mixed exhaled breath samples [63].
Figure 3.3 Phases in the expiratory and inspiratory cycle.
Et -CO 2 ( m m H g) Alveolar Plateau Inspiration
A
B
C
D
E
Normal Capnogram Normal Et-CO2: 35-45 mmHgEnd-Tidal CO 2 A-B Baseline B-C Expiratory Upstroke C-D Expiratory Plateau D-E Inspiration Real time
Expira
Expiration InspirationChapter 3. Breath analysis for clinical diagnosis
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Figure 3.4 shows a picture of the capnometer used in this study to control the expiratory and inspiratory phases. It is a mainstream capnogard model 1265 from Novametrix Medical Systems, INC (Wallingford, USA).
Figure 3.4 Capnogard 1265 from Novametrix.
In figure 3.5 a screen close-up is shown in which the phases in the expiratory cycle can be clearly identified.
Chapter 3. Breath analysis for clinical diagnosis
49
This capnograph is a small and light portable instrument that completes a self-testing and auto-calibration within minutes. The capnogard provides a screen display with information of the end tidal CO2 with an alarm set event.