Standardisation of the single-breath determination of carbon monoxide uptake in the lung

Definitions

The rate of CO uptake from the lungs is the product of alveolar partial pressure of CO in excess of any back pressure in the blood (the driving pressure) and a rate constant. This is for CO in the whole lung per unit of driving pressure. For practical reasons, using the single-breath method described below the CO uptake from the lung (K_CO) is measured as a concentration fall in alveolar CO per unit time per unit CO driving pressure (P_A,CO): When K_CO is multiplied by the volume of gas in the lung containing CO (alveolar volume (V_A)), the total uptake of CO by the lung per unit of time per unit driving pressure is obtained. This product, K_CO×V_A, has been termed transfer factor of the lung for CO by the European community and diffusing capacity of the lung for CO (D_L,CO) by the North American community. The former term recognises that the measurement of CO uptake reflects a number of processes (not just diffusion), and is a submaximal value and, thus, not truly a “capacity”. However, the latter term has considerable historical significance and, for the sake of uniformity, the ERS and ATS agreed to use the expression D_L,CO in this document.

The ERS recommends expressing D_L,CO in the SI units mmol·min⁻¹·kPa⁻¹, while the ATS prefers the traditional units mL (standard temperature, pressure and dry (STPD))·min⁻¹·mmHg⁻¹. In fact, this is not an important issue, providing the same set of units is used throughout all calculations. Values in SI units should be multiplied by 2.987 to obtain values in traditional units.

Determinants of CO uptake

The process of CO transfer from the environment to the pulmonary capillary blood includes: 1) bulk flow delivery of CO to the airways and alveolar spaces; 2) mixing and diffusion of CO in the alveolar ducts, air sacs and alveoli; 3) transfer of CO across the gaseous to liquid interface of the alveolar membrane; 4) mixing and diffusion of CO in the lung parenchyma and alveolar capillary plasma; 5) diffusion across the red cell membrane and within the interior of the red blood cell; and 6) chemical reaction with constituents of blood Hb 10–16.

The process of CO uptake can be simplified into two transfer or conductance properties: membrane conductivity (D_M), which reflects the diffusion properties of the alveolar capillary membrane; and the binding of CO and Hb. The latter can be represented as the product of the CO–Hb chemical reaction rate (θ) and the volume of Hb in alveolar capillary blood (V_c). Since these are conductances in series 14, these properties are related by: A number of physiological changes can affect D_M or θV_c to influence D_L,CO. As the lung inflates, D_M increases (due to unfolding membranes and increasing surface area), while V_c effects are variable (due to differential stretching and flattening of alveolar and extra-alveolar capillaries) 10, 17–24. The net effect of these changes is that D_L,CO tends to increase as the lung inflates. Exercise, the supine position and Mueller manoeuvres (inspiratory efforts against a closed glottis) can all recruit and dilate alveolar capillaries, thereby increasing V_c and D_L,CO 25–31. Alveolar-capillary recruitment also occurs in the remaining lung tissue following surgical resection, since the cardiac output now flows through a smaller capillary network. This causes a less than expected loss of V_c for the amount of lung tissue removed. In contrast, Valsalva manoeuvres (expiratory efforts against a closed glottis) can reduce V_c and thereby reduce D_L,CO 29.

The measurement of CO uptake is also affected by the distribution of ventilation with respect to D_M or θV_c (i.e. CO uptake can only be measured in lung units into which CO was inspired and subsequently expired) 15, 16, 32, 33. This is particularly important in diseases such as emphysema, where the inhaled CO may only go to the better-ventilated regions of the lung and the subsequently measured CO uptake will be determined primarily by uptake properties of those regions. Under these conditions, the tracer gas dilution used to calculate V_A will also reflect primarily regional dilution and underestimate the lung volume as a whole. The resulting calculated D_L,CO should thus be considered to be primarily reflecting the gas-exchange properties of the ventilated regions of the lung.

In addition to these physiological and distributional effects on D_L,CO, a number of pathological states can affect D_M, θV_c, or both, and thereby affect D_L,CO (table 1⇓) 5, 6, 34–43. Measurement of D_L,CO is indicated when any of these pathological processes are suspected or need to be ruled out. Moreover, measuring changes in D_L,CO over time in these processes is a useful way of following the course of disease.

System design

Descriptions of the apparatus and general instructions for performing the single-breath diffusing capacity manoeuvre are available elsewhere 2, 44–48. Equipment in clinical use varies widely in complexity, but the basic principles are the same. All systems have a source of test gas (bag-in-box, spirometer, compressed gas cylinder), a method for measuring inspired and expired volume over time (spirometers with kymographs, pneumotachometers near the mouthpiece or near a bag-in-box), and gas analysers (single-sample analysers or continuous high-speed analysers). Single-sample gas-analyser systems usually display only volume over time (fig. 1a⇓). Continuous gas-analyser systems also provide a continuous tracing of CO and tracer gas concentrations during the test (fig. 1b⇓).

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Fig. 1—

Schematic of lung volume (a) and gas concentrations (b) during the single-breath diffusing capacity of the lung for carbon monoxide. The gas-sampling period occurs between the two dotted lines. -----: tracer gas; – – –: carbon monoxide. ^#: dead space washout; ^¶: sample collection. Modified from 1.

Equipment requirements

Infection control

The major goal of infection control is to prevent the transmission of infection to patients and staff during pulmonary function testing. The recommendations in the ATS/ERS documents for spirometry and general considerations for pulmonary function testing also apply to D_L,CO equipment and procedures 49, 56.

Patient conditions for measurement

Factors that affect V_c (e.g. exercise, body position, and Hb affinity for CO, such as alveolar oxygen partial pressure (P_A,O2), and carboxyhaemoglobin (COHb)) should be standardised. If clinically acceptable, the subject should not breathe supplemental oxygen for 10 min prior to a standard test. When using exercise or the supine position to assess the “recruitability” of D_L,CO 15, 25–28, the level of exercise and/or the duration of the supine position should be noted.

Before beginning the test, the manoeuvres should be demonstrated and the subject carefully instructed. The subject should be seated comfortably throughout the test procedure. The test should be performed at a stable comfortable temperature within manufacturer's equipment specifications.

COHb produces an acute and reversible decrease in D_L,CO 57–60, largely due to the effects on CO back pressure and the “anaemia effect” from decreased Hb binding sites for CO from the test gas. As cigarette smoking is the most common source of COHb, subjects should be asked to refrain from smoking or other CO exposures on the day of the test. The time of the last cigarette smoked should be recorded and noted for the interpretation. A correction for CO back pressure should be made for recent or heavy cigarette smoking (see Adjustment for carboxyhaemoglobin concentration and CO back pressure section). Manufacturers are encouraged to provide the capability to do this easily.

Inspiratory manoeuvre

Once the mouthpiece and nose clip are in place, tidal breathing should be carried out for a sufficient time to assure that the subject is comfortable with the mouthpiece. Deep inspirations should be avoided during this period as they can increase subsequent CO uptake 61. The D_L,CO manoeuvre begins with unforced exhalation to residual volume (RV). In obstructive lung disease, where exhalation to RV may require a prolonged period, a reasonable recommendation is that this portion of the manoeuvre should be limited to 6 s, a time consistent with using the forced expiratory volume in six seconds manoeuvre as a surrogate for VC 49. At RV, the subject's mouthpiece is connected to a source of test gas, and the subject inhales rapidly to TLC.

A submaximal inspired volume (i.e. less than the known VC) can affect CO uptake, depending upon whether it is a result of an initial suboptimal exhalation to RV (test performed at TLC) or whether it is due to a suboptimal inhalation from RV (test performed below TLC) 19–22. In the former case, the calculated V_A and D_L,CO will accurately reflect lung volume and the CO uptake properties of the lung at TLC. In the latter case, the V_A will be reduced and D_L,CO measurement will be affected (see Adjustment for lung volume section).

Due to these effects, it is important that the V_I be as close to the known VC as possible. Data from a large patient population have shown that the V_I during D_L,CO measurements averages ∼90% of the VC 19, but that as many as 32% of subjects may fall below this target 62. A more recent study of >6,000 D_L,CO measurements in a university laboratory demonstrated that 72, 86 and 92% of these patients could achieve V_I targets of 90, 85 and 80%, respectively, of the known VC 63. Since it appears that V_I reductions of as much as 15% of the known VC will reduce the D_L,CO <5% 19, a V_I target of 85% of the largest-known VC seems both reasonable and attainable.

The inspiration should be rapid, since the D_L,CO calculations assume “instantaneous” lung filling 24, 64–70. Slower lung filling decreases the amount of time the lung is at full inspiration with a consequent reduction in CO uptake. Although various sample timing techniques address the issue of lung filling and emptying time, it is still reasonable to expect that 85% of V_I should be inspired in <4.0 s. If longer inspiratory times are needed to achieve the 85% V_I goal, this should be noted on the test report.

Condition of the breath-hold and expiratory manoeuvre

Valsalva (expiratory efforts against a closed airway) and Muller manoeuvres (inspiratory efforts against a closed airway) during the breath-hold, by decreasing and increasing thoracic blood volume, respectively, will decrease and increase D_L,CO, respectively 29, 71, 72 The intrapulmonary pressure during the breath hold should thus be near atmospheric, and this is best accomplished by having the subject voluntarily maintain full inspiration using only the minimal effort necessary. The breath-hold time should be 10±2 s, a target easily achieved in the vast majority of subjects 62.

As with inspiration, the D_L,CO calculation assumes instantaneous lung emptying 24, 64–69. Although various sample timing techniques address the fact that emptying is not instantaneous, it is still reasonable to expect that the expiratory manoeuvre should be smooth, unforced, without hesitation or interruption, and total exhalation time should not exceed 4 s (with sample collection time <3 s). In subjects who require a longer expiratory time to provide an appropriate alveolar gas sample, the expiratory time should be noted in the test report. Common errors that can occur during the inspiration, breath-hold and expiration manoeuvres are given in figure 2⇓.

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Fig. 2—

Potential problems with the single-breath diffusing capacity of the lung for carbon monoxide breathing manoeuvre that can lead to measurement errors. ·······: stepwise inhalation or exhalation; – - –: exhaled gas leak; – -- –: inhalation too slow; – – –: exhaled volume larger than inhaled volume; ------: transient overshoot from high flows and changing gas temperatures. Adapted from 2.

Washout and sample collection volume

The D_L,CO calculations (see Calculations section) require alveolar gas samples. During expiration, a volume of gas must be expired and discarded to clear anatomic and mechanical V_D before the alveolar sample is collected (fig. 1⇑). Contamination of the alveolar gas sample with V_D gas will cause an underestimation of true CO uptake. In general, the washout volume should be 0.75–1.0 L (BTPS). If the patient's VC is <2.00 L, the washout volume may be reduced to 0.50 L. Newer devices can provide a graphical display of exhaled gas concentrations to assure that V_D gas is not present in the alveolar sample (fig. 1⇑). Using such an analyser, Huang et al. 71 showed that the standard approach noted above adequately cleared V_D in >90% of adults.

The sample gas volume (V_S) is the volume of gas used to analyse alveolar CO and tracer gas concentrations at the end of the breath-hold. In subjects with good gas mixing and uniform ventilation and CO uptake properties, virtually any gas sample after V_D washout will be a good reflection of the lung as a whole. However, in subjects with poor gas mixing or marked sequential emptying of various lung regions, the gas sample collected will only reflect the properties of the regions contributing to that sample. V_S collection time will also affect the measurement of breath-hold time (see below). In order to standardise the collection process, a V_S of 0.50–1.00 L should be collected for analysis. In patients with VC <1 L, a V_S <0.50L may be used if it can be assured that the V_D has been cleared.

If continuous analysers with graphical displays are used, computerised or visual inspection of the expired CO and tracer gas curves may be used to adjust washout and the V_S if needed (fig. 1⇑) 71. These adjustments may be useful in subjects with VC <1 L who are unable to meet the minimum V_D washout and V_S recommended previously (e.g. paediatric patients, or adult patients with severe restrictive processes). These adjustments may also be useful in subjects with a large V_D in whom the recommended value range of 0.75–1.0 L is inadequate. For these adjustments to be achieved properly, the displays must represent actual gas concentrations that occurred at the mouth, synchronised for delays in gas transport and adjusted for gas-analyser response. In making such adjustments, the start of the V_S (end of the washout) must clearly be at a point where the tracer gas has started to plateau after the immediate fall from its inspiratory concentration, and the CO curve has ceased its immediate fall and started a smooth gradual decline (fig. 1⇑). Furthermore, reports must indicate that manual adjustments were used to select washout volumes and V_S, so the interpreter can review and verify the adjustments.

Inspired gas composition

The test gases used to calculate D_L,CO include a tracer gas to measure V_A, as well as CO. The remainder of the test gas mixture includes O₂ and N₂.

The tracer gas should be relatively insoluble and chemically and biologically inert. Since the tracer gas is used to determine the initial alveolar CO concentration, as well as the V_A from which CO uptake is occurring, its gaseous diffusivity should be similar to CO. It should not interfere with the measurement of CO concentration. The tracer gas should not ordinarily be present in alveolar gas or else be present at a known, fixed concentration (e.g. argon).

Commonly used tracer gases are helium (He) and methane (CH₄). While He meets most of the previous criteria, its gaseous diffusivity is considerably higher than CO. CH₄ is commonly used as a tracer gas for systems that continuously sample expired gas. Its gaseous diffusivity is closer to CO, but it has a slightly higher liquid solubility than He. As new tracer gases are introduced, manufacturers should demonstrate that they produce V_A and D_L,CO values equivalent to those measured using He, as this is the tracer gas that is used to derive most of the available reference equations.

The inspired CO should nominally be 0.3%. However, as ratios are more important than absolute values, exact concentrations are not critical. The assumption in calculating CO uptake is that capillary blood does not contain CO. Thus, corrections are needed in patients who have significant COHb (see Adjustment for COHb concentration and CO back pressure section).

Since P_A,O2 fluctuates over the ventilatory cycle 72 and can affect CO uptake by affecting θ, a more stable P_A,O2 during the D_L,CO manoeuvre would seem desirable and, theoretically, can be achieved with a test gas fraction of inspired oxygen (F_I,O2) of 0.17. Most current systems use either a F_I,O2 of 0.21 (with fractional concentrations of tracer gases such as CH₄ of <0.01), or gas mixtures containing CO and 10% He with “balance air” (an effective F_I,O2 of 0.19). Since D_L,CO will increase 0.31 to 0.35% for each 0.133 kPa (1 mmHg) drop in P_A,O2 73, 74, the increase in D_L,CO that would be expected as the F_I,O2 is decreased from 0.21 to 0.17 (P_A,O2 decreased ∼3.7 kPa (∼28 mmHg)) is 8–9%. It is recommended that laboratories use gas mixtures with inspired oxygen partial pressure (P_I,O2) values similar to the reference set used in the interpretation (table 4⇓) 75–82, or make appropriate adjustments of measured or predicted D_L,CO for the P_I,O2.

By measuring D_L,CO at several different levels of P_A,O2, the two components of D_L,CO (D_M and V_c) can be distinguished. This is accomplished by using the Roughton–Forster relationship noted previously (equation 2) and varying θ (the reaction rate of O₂ and Hb) by altering the P_I,O2. Subsequently, 1/D_L,CO is plotted against 1/θ at the different P_I,O2 levels. The slope of this relationship is 1/V_c and the intercept is 1/D_M.

Interval between tests

At least 4 min should be allowed between tests to allow an adequate elimination of test gas from the lungs. The subject should remain seated during this interval. In patients with obstructive airway disease, a longer period (e.g. 10 min) should be considered. Several deep inspirations during this period may help to clear test gases more effectively. If continuous monitoring of expired gas concentrations is available, the washout of tracer gas from the previous test may be confirmed by observing end-tidal gas concentrations before beginning the next test.

Miscellaneous factors

There may be diurnal variation in D_L,CO, since one study has found that D_L,CO fell 1.2–2.2% per hour throughout the day 83. The reason for the change was not clear and was not explained by CO back pressure or changes in V_A, V_I or breath-hold time. One explanation is a combination of changes in CO back pressure and diurnal variation in Hb concentration 84. A 13% change in D_L,CO during the menstrual cycle has been reported 85. The highest value was observed just before the menses, and the lowest was on the third day of menses. It is not clear, however, if this is simply a Hb effect or whether it reflects other physiological processes (e.g. hormonal changes on pulmonary vascular tone). Ingestion of ethanol has been reported to decrease D_L,CO 86. The mechanisms involved are not clear, although it is known that some fuel-cell CO analysers are sensitive to exhaled ethanol and ketones. In obstructive lung disease subjects, after administration of a bronchodilator, D_L,CO may increase up to 6% 87. Bronchodilators can affect V_A, vasomotor tone, etc., and their use prior to testing could conceivably optimise these factors. Use of a bronchodilator should be noted in the interpretation 88.

Calculating breath-hold time

The “breath-hold time” or time of transfer during which CO changes from its initial to final concentration is in the denominator of the D_L,CO equation (equation 7). As noted previously, the single-breath measurement of CO uptake assumes an “instantaneous” lung filling and emptying process. However, both inspiration and expiration require up to several seconds, and these periods of changing gas volume in the lung must be accounted for in the calculations. For purposes of standardisation, the method by Jones and Meade 68 (fig. 3⇓) is recommended, since it has the theoretical appeal of empirically accounting for the effects of inspiratory and expiratory time. This method has also been shown to adequately address inspiratory flows as low as 1 L·s⁻¹, breath-hold times as short as 5 s, and expiratory flows as low as 0.5 L·s⁻¹ in normal subjects 64.

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Fig. 3—

Schematic illustration of different methods of measuring breath-hold time for the single-breath diffusing capacity of the lung for carbon monoxide. The method by Ogilvie (––––––) 48 measures breath-hold time from the beginning of inspiration to the beginning of alveolar sample collection. The method by Jones and Meade (···········) 68 includes 0.70 of inspiratory time and half of sample time. The Epidemiologic Standardization Project (– – – –) measures breath-hold time from the time of 50% of inspired volume (V_I) to the beginning of alveolar sample collection. t_I: time of inspiration (-----; defined from the back-extrapolated time 0 to the time that 90% of the V_I has been inhaled); TLC: total lung capacity; RV: residual volume. ^#: dead space washout; ^¶: sample collection. Adapted from 1.

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With the approach taken by Jones and Meade 68, breath-hold time equals the time starting from 0.3 of the inspiratory time to the middle of the sample collection time. As in spirometry, the back-extrapolation technique should be used to establish time zero 48, 49. The time when 90% of the V_I has been inspired is a reasonable end point for defining inspiratory time (fig. 3⇑).

A theoretically more accurate way to account for volume changes over time during inspiration and expiration is to use three separate equations for D_L,CO during inspiration, breath hold and expiration (the “three-equation” technique) 24, 64. This algorithm is commercially available and may be particularly useful in subjects unable to rapidly fill or empty their lungs. However, clinical experience with this approach is limited.

Other breath-hold timing algorithms may be appropriate in maintaining consistency (e.g. longitudinal studies), but these measurements should be recognised as less suitable recommendations.

Calculating the alveolar volume

V_A represents an estimate of lung gas volume into which CO is distributed and then transferred across the alveolar capillary membrane 3, 4. Thus, it is critical in the measurement of D_L,CO. As noted previously, V_A is measured simultaneously with CO uptake by calculating the dilution of an inert Tr. For normal subjects, this calculated single-breath determination of V_A (V_A,sb) plus estimated V_D closely matches TLC determined by plethysmography 19, 70. However, poor gas mixing in patients with maldistribution of inspired volume (e.g. obstructed airways patients) can markedly reduce Tr dilution and, thus, lead to values for V_A,sb that are markedly less than a V_A determined from the actual total thoracic gas volume (V_TG). The observed CO uptake is also affected by poor gas mixing under these conditions, and will primarily reflect the CO transfer properties of the regions into which the test gas is distributed. It has been suggested that a separately determined V_A from a more accurate technique (e.g. multiple-breath technique (V_A,mb) or plethysmography (V_A,plethys)) could be substituted for V_A,sb under these conditions to “correct” for the effects of maldistribution. However, the D_L,CO calculation (equations 4 and 7) is based on the volume of gas into which the Tr (and CO) distributes, and not the total V_TG. Moreover, substituting a larger, separately determined V_A,mb or V_A,plethys assumes that D_M and V_c properties in the unmeasured lung regions are similar to those in the measured lung regions, an assumption that is difficult to justify. Due to these considerations, a separately measured V_A,mb or V_A,plethys should not be substituted for V_A,sb. Instead, when the V_A,sb is markedly less than a separately determined V_A,mb or V_A,plethys, this should be reported and the ratio of V_A,sb to V_A,mb or V_A,plethys reported. For the subsequent interpretation of D_L,CO, it should then be noted that the maldistribution of inspired gas probably contributed to any observed reduction in measured D_L,CO.

The volume of distribution for the tracer gas can be determined from values for V_I, F_I,Tr and F_A,Tr, and knowing the conditions of the inspired and expired gases. Since the amount of tracer gas in the lung (alveolar plus dead space) equals the amount of inspired tracer gas, and the dead space tracer gas fraction is the same as the inspired fraction (all expressed at BTPS): Although V_A is usually expressed under BTPS conditions, it must be converted to STPD conditions to calculate D_L,CO in equation 7.

It is essential that V_D is considered in the calculation of V_A. V_D occurs in two areas: instrument V_D (i.e. volume of the mouthpiece, filters and connections within the valving system); and anatomic V_D (i.e. the volume in the conducting airways that does not participate in gas exchange). Instrument V_D should be specified by the manufacturer, but may vary as the user alters the system (e.g. addition of a filter).

There are various methods to estimate anatomic V_D. Examples include a fixed value of 150 mL 1 (although this does not work well for small adults or children), and another of 2.2 mL×kg body weight 47 (although this does not work well for very obese subjects). In studies deriving the commonly used reference equations (table 4⇑), the most commonly used technique was to assume 2.2 mL×kg body weight. However, some investigators ignored anatomic V_D 79, 80, 82, and one used age+2.2mL×kg body weight 78. If the body mass index is <30, the current authors recommend using an estimate for anatomic V_D of 2.2 mL×kg body weight. In more obese subjects or if the weight is unknown, V_D (mL) can be estimated using the following equation: where height is measured in cm, or: where height is measured in inches.

In single-sample systems, the sample-bag residual volume (sometimes called a sample-bag dead space) dilutes the sample gas and alters the measured concentrations of expired gases. The size and direction of the error depends on V_S, the residual volume of the sample bag and its connectors (V_SRV), and V_SRV gas content. V_SRV could contain test gas, room air or expired gas from a subject (after a D_L,CO test). When V_SRV contains room air, its effect is to reduce the measured concentrations of expired gases. The following equation adjusts for this: Estimates of the potential change in D_L,CO in existing systems when no adjustment is made for sample-bag dead space range from 0.3–8%, depending on sample-bag size and V_SRV 89.

Manufacturers should report instrument and sample-bag dead space. Both of these must be flushed with room air (or, if D_M and V_c are to be calculated, appropriate levels of oxygen) before the single-breath manoeuvre so that it will not contain expiratory gas from a previous subject. V_SRV should be <2% of the V_S or 10 mL, whichever is larger.

Inspired gas conditions

Though inspired gas is often assumed to be measured at ambient temperature and pressure, saturated with water vapour conditions, this is only true in systems in which the test gas is transferred to a water-sealed spirometer before it is inspired. In most cases, the test gas inspired from a bag-in-box system, through a pneumotachometer from a bag, or a compressed gas cylinder with a demand valve is a dry gas (<10 ppm H₂O) and, thus, at ambient temperature and pressure, dry conditions. The inspired volume needs to be converted to BTPS conditions to use in equations 7, 8 and 9. It is recommended the V_I (BTPS) be reported, and manufacturers should specify and document inspired gas conditions for each instrument.

CO₂, H₂O and temperature adjustment for V_A calculations

Exhaled gas contains CO₂ and H₂O, which were not present in the test gas mixture. As noted previously, some systems remove one or both of these if they interfere with analyser function, and this will raise both CO and tracer gas concentrations. Under these circumstances, adjustments are required for the increase in F_A,Tr to calculate V_A (table 5⇓). However, no adjustment for the increase in alveolar inspired CO fraction at time t (F_A,CO,t) and F_A,Tr is necessary in calculating the rate of CO uptake, since the concentration factor appears in both the numerator and the denominator of the expression (F_A,CO,0/F_A,CO,t) and therefore cancels.

Exhaled gas is initially at body temperature. Some systems allow this to cool (gas volume contracts), whereas others will provide heat to maintain the temperature. Adjustments to BTPS conditions may be required depending upon the system design (table 5⇑).

All of these adjustments should be documented by the manufacturer for their particular system.

Acceptability, repeatability and number of tests

Acceptable tests are defined in table 6⇓. Repeatability describes the variability on repeated testing with no change in test conditions 90, 91. In a large university-based laboratory study, a coefficient of variation of repeated measurements in normal subjects was 3.1%, and this increased only slightly (from 4.0 to 4.4%) in patients with abnormal spirometry patterns 63. In contrast, an inter-session D_L,CO variability of up to 9% (reproducibility) has been documented in normal individuals in repeated measurements over a period of 1 yr 92.

Since most intra-session variability is technical rather than physiological, the mean of acceptable tests is reasonable to report. In this report, there should be at least two acceptable tests that meet the repeatability requirement of either being within 3 mL CO (STPD)·min⁻¹·mmHg⁻¹ (or 1 mmol·min⁻¹·kPa⁻¹) of each other or within 10% of the highest value. In a large university-based laboratory study, >95% of the patients could meet this criteria 63.

The average of at least two acceptable tests that meet this repeatability requirement should be reported (i.e. outliers excluded). While it is recommended that at least two D_L,CO tests should be performed, research is needed to determine the actual number of tests required to provide a reasonable estimate of average D_L,CO value for a given person. As noted below, five tests will increase COHb by ∼3.5% 84, which will decrease the measured D_L,CO by ∼3–3.5%. Thus, more than five tests are not recommended at the present time.

Adjustments to the measurement of D_L,CO prior to interpretation

D_L,CO depends upon a number of physiological factors. Besides varying with age, sex, height and possibly race, D_L,CO also changes with Hb, lung volume, COHb, P_I,O2 (e.g. altitude), exercise and body position. Although these effects may cause changes in D_L,CO in opposite directions 93, all should be considered in interpreting the observed CO uptake. Moreover, specific adjustments for three of these factors (Hb, COHb and P_I,O2) should always be made to ensure appropriate interpretation (see below). Consideration could also be given to adjust for a submaximal inspiration resulting in a less than expected V_A.

Adjustments for P_A,O2

As noted previously, P_A,O2 affects the measurement of D_L,CO. P_A,O2 changes will occur as a consequence of supplemental O₂ breathing (higher P_A,O2) or performing D_L,CO assessments at altitude (lower P_A,O2). As mentioned before, D_L,CO will change by ∼0.35% per mmHg change in P_A,O2 73, 74 or by ∼0.31% per mmHg decrease in P_I,O2. Adjustments to the predicted D_L,CO in a subject on supplemental O₂ may be made using a measured P_A,O2 and assuming a normal P_A,O2 on room air at a sea level of 100 mm Hg, as follows: If the adjustment is being made for altitude, assuming a P_I,O2 of 150 mmHg at sea level:

Adjustment for COHb concentration and CO back pressure

COHb can affect the measured uptake in the following two ways 98–100. First, by occupying Hb binding sites, CO produces an “anaemia effect”. Secondly, CO partial pressure in the blood will reduce the driving pressure for CO transport from alveolar gas to capillary blood.

Exposure to ordinary environmental CO and endogenous production of CO as a byproduct of Hb catabolism commonly results in measured COHb levels of 1–2% 98. The 1–2% baseline COHb levels that are attributable to endogenous production of CO and ordinary environmental exposures are already incorporated into reference values based on healthy nonsmoking subjects. Cigarette smoke and other environmental sources, however, can produce measurable levels of CO back pressure and COHb that may need to be considered in the measurement of CO uptake 99. Small increases in COHb also occur when CO is inspired in the D_L,CO test. Frey et al. 84, for example, found that COHb increased by ∼0.7% with each single-breath D_L,CO test.

CO back pressure can be measured in expired gas before a D_L,CO manoeuvre or estimated using one of several available techniques 100–103. For example, CO back pressure can be calculated from COHb from the following equation:

D_L,CO can then be recalculated after subtracting the estimated CO back pressure from both the initial and final alveolar CO. Units must be consistent before making the subtraction. However, this method will not adjust D_L,CO for the “anaemia” effect of COHb.

Several studies have evaluated both the empirical and theoretical effects of COHb on D_L,CO and incorporated both the back pressure and the “anaemia” effects of COHb. In general, a 1% increase in COHb reduces the measured D_L,CO by ∼0.8–1% from both effects 13, 14. Using this approach, the following equation empirically reduces predicted D_L,CO by 1% for each per cent COHb >2%: An adjustment for COHb is not required, but is recommended for interpretative purposes when COHb is elevated/suspected. No adjustment is required if COHb <2%, since reference equations already incorporate this.

Adjustment for lung volume

As noted previously, D_L,CO decreases as the lung deflates as a function of both membrane and capillary configuration changes 17–24, 104–111. The relationship is complex, however, and is probably nonlinear 108, 110. In normal subjects with experimental reductions in V_I (and, thus, V_A), adjustment equations for this effect have been derived 18, 19, 109, 111 and a recent representative example consists of the following: where V_Am represents measured V_A and V_Ap represents predicted V_A at normal TLC.

It should be noted that this D_L,CO adjustment for a reduced V_I (and V_A) from a submaximal effort is substantially less than a 1:1 D_L,CO/V_A adjustment (i.e. the fall in D_L,CO as lung volumes are reduced is much less than the fall in V_A). As a consequence, the D_L,CO/V_A ratio will rise with a reduced V_I from a submaximal effort. Thus, if this ratio is used to adjust (“correct”) D_L,CO for the effects of a reduced V_A from a submaximal V_I, it will markedly “overcorrect”.

It is important to emphasise that the V_A effects on D_L,CO discussed above were derived from studies in normal subjects with submaximal V_I. These V_A effects (and consequent D_L,CO adjustments for V_A) have not been validated in lung diseases where lung pathology has reduced CO uptake properties, as well as V_I and V_A. In some of these diseases (e.g. status post-pneumonectomy), the reduction in D_L,CO may be less than the reduction in V_A (high D_L,CO/V_A); in others (e.g. pulmonary vascular disease), the reduction in D_L,CO may be greater than the reduction in V_A (low D_L,CO/V_A) 17. In many disease states, however, the ratio of pathological reductions in D_L,CO and V_A may be quite variable and of unclear physiological or clinical significance. Thus, although the D_L,CO/V_A relationship can be used to describe the relative reductions in CO uptake properties and alveolar gas volumes in lung disease 17, 19, 107, 112, drawing more specific clinical or pathological conclusions based upon V_A (or any other volume) adjustments should be made with caution. This is especially true if the adjustment leads to the implication that CO uptake properties of the lung are normal. Further study is clearly needed on the interactions of CO uptake and alveolar gas volume in lung disease before more specific volume-adjustment recommendations can be made.