ABSTRACT

The pulmonary diffusing capacity for carbon monoxide is a test that allows quantitative evaluation of the transfer of oxygen from the alveolar air to its union with hemoglobin through the alveolo-capillary membrane. Since its original description in 1957, the pulmonary diffusing capacity for carbon monoxide test has evolved thanks to the advent of rapid response gas analyzers, as well as standardization efforts and vast improvements in the software and reference values. Currently, the single-breath measurement method is strongly standardized and recommended for clinical purposes. The pulmonary carbon monoxide diffusing capacity with the single breath technique has implications for both the diagnosis and the follow-up and prognosis of patients with chronic diseases not limited to the respiratory system. This document is based on the 2005, 2017 and 2021 European Respiratory Society and American Thoracic Society standards to describe technical recommendations for rapid response gas analyzers-based with the single breath systems. The pulmonary carbon monoxide diffusing capacity is underutilized despite its clinically proven value, which ranks second only to spirometry testing. However, it holds particular relevance for patients with interstitial lung diseases, emphysema, and pulmonary vascular diseases.

KEYWORDS

respiratory function tests, pulmonary diffusing capacity, carbon monoxide, alveolar volume.

Abbreviations:

• ATPD = ambient temperature, atmospheric pressure, dry conditions.
• ATPS = ambient temperature, atmospheric pressure, saturated with water vapor conditions.
• ATS = American Thoracic Society.
• BHT = breath holding time.
• BTPS = body temperature, ambient pressure, saturated with water vapour conditions.
• CH3 = methane.
• COHb = carboxyhemoglobin.
• DLCO = pulmonary diffusion of carbon monoxide.
• DLCO_sb = single-breathing carbon monoxide lung diffusion.
• ILD = Interstitial lung disease.
• COPD = chronic obstructive pulmonary disease.
• ERS = European Respiratory Society.
• ERV = expiratory reserve volume.
• FRC = functional residual capacity.
• FVC = forced vital capacity.
• He = helium.
• IVC = inspiratory vital capacity.
• KCO = pulmonary transfer coefficient for carbon monoxide.
• LIN = lower limit of normal.
• Ne = neon.
• PiO2 = inspired oxygen pressure.
• RGA = rapid response gas analyzers.
• RV = residual volume.
• TI = inspiratory time.
• TLC = total lung capacity.
• TLCO = pulmonary transfer of carbon monoxide.
• VA = alveolar volume.
• VIN = inspiratory volume.
• V/Q = ventilation to perfusion ratio.

Introduction

The measurement of pulmonary diffusion of carbon monoxide (DLCO) has undergone significant evolution since its standardization in 1957 by Ogilvie et al.¹ The classical method used small samples of exhaled gas and required several minutes to measure the carbon monoxide concentration. In recent years, rapid response gas analyzers (RGA) have been developed that perform the measurement in less than 150 milliseconds. These advances coupled with rapid microprocessor calculations, and better and more numerous reference values, have led to a revolution in DLCO measurement.²

DLCO is a fundamental test for assessing gas exchange at the alveolocapillary membrane, playing a crucial role in the diagnosis, management and prognosis of various diseases not limited to the respiratory system.^3,4 Several techniques have been used to assess carbon monoxide transfer across the alveolocapillary membrane.⁵ These include: the multiple-breath (multibreath) method; the intrabreath method, which is performed when maximal inspiration is followed by a slow and uniform maximal exhalation, without a period of apnea in the maneuver;⁶ and the "one-breath" method developed by Krogh in 1910.⁷ The latter method is widely used and the most standardized.^8,9 In the "one-breath" or "single-breath" (DLCO_sb) method, a 10-second apnea period is performed during maximal inspiration.¹⁰ In addition to the measurement of DLCO_sb, simultaneous inhalation of inert gasses, such as helium (He), methane (CH3) or neon (Ne), allow calculation of alveolar volume (VA), total lung capacity (TLC) and residual volume (RV).

This review references the 2005 and 2017 update of the European Respiratory Society (ERS) and American Thoracic Society (ATS) standards,^2,8 as well as the ERS/ATS-2021¹¹ standard for pulmonary function test interpretation strategies. These standards seek to provide a technical update for DLCO systems based on RGA development and to describe new calculation standards that incorporate continuous gas analysis of the entire exhaled sample, as well as clinical and functional interpretation of test results.

DLCO measurement, performed under standardized conditions and under strict quality control, is a sensitive tool to detect changes in lung function, even less than 10%.¹² Its decrease may indicate chronic lung diseases such as chronic obstructive pulmonary disease (COPD) or interstitial lung disease (ILD), showing a direct correlation with the degree of emphysema, inflammation or fibrosis.^3,13,14 DLCO_sb also reflects abnormalities in pulmonary vascular diseases such as pulmonary hypertension,¹⁵ embolism and vasculitis, as well as extrapulmonary diseases such as hemoglobinopathies,¹⁶ obesity,¹⁷ musculoskeletal abnormalities and elevated carboxyhemoglobin (COHb) levels.¹⁸ This broad spectrum of clinical applications highlights the versatility of DLCO_sb as a sensitive indicator of multiple conditions affecting lung function and the overall health of the individual.

Physiological basis

DLCO is a test that measures the properties of the alveolocapillary membrane for oxygen exchange from alveolar air to erythrocytes in the alveolar capillaries, thus involving not only the physiological mechanism of pulmonary diffusion (Figure 1), but also ventilation, perfusion and the ventilation to perfusion ratio (V/Q). For this reason, especially in Europe, it is more appropriately called pulmonary transfer of CO (TLCO). If you would like to learn more about the physiological basis of pulmonary carbon monoxide diffusion, please refer to the supplementary material.

Indications and contraindications of the test

In general, the main indication for DLCO_sb testing is the diagnostic evaluation and follow-up of lung parenchymal diseases (Table 1). The updated contraindications for the DLCO_sb test are listed in Table 2.^2,3,19

DLCO equipment and Supplies

DLCO equipment should meet the international technical recommendations issued by ATS/ERS 2017,² with the following recommended minimum requirements for volume measurements and rapid gas analyzer, which can be found in the equipment user manual and supplementary material.

Pre-test preparation

Preparation of technical personnel prior to testing

During the COVID-19 pandemic, disease transmission became more prominent in order to mitigate risks. Rigorous implementation of safety and disinfection measures are required (Table 3).²⁰

Quality control and equipment calibration^2,21

• 1. Daily calibration check: start with zero flow before each maneuver. Verify volume calibration with a 3 L syringe, performing at least three different flows (low, medium and high) between 0.5 and 12 L/s, meeting an accuracy requirement ≤ 2.5%.
• 2. Weekly procedures or in case of problems: should be performed with a calibrated 3 L syringe, ensuring that the VA calculation is within ± 300 mL of the expected value and DLCO is < 0.166 mmol/min/kPa or < 0.5 mL/min/mmHg. The biological control test should not have deviations > 12% or > 3 mL/min/mmHg, because it could indicate quality control problems.
• 3. Monthly testing: leak testing of the 3 L calibration syringe is recommended. If it does not return within 10 mL of full fill, it should be sent for repair. Also, a linearity evaluation of the gas analyzer should be performed using known dilutions of the test gas or using a high precision test gas. However, automation of linearity by manufacturers is preferred.
• 4. General recommendations: In the absence of a high accuracy DLCO and gas simulator, system checks should be performed using a 3 L calibration syringe in ambient temperature, atmospheric pressure, saturated with water vapour (ATPS) conditions, with VA reporting in ambient temperature, atmospheric pressure, dry (ATPD) conditions instead of body temperature, ambient pressure, saturated with water vapour (BTPS) conditions. A digital calibration option should be available to verify the computational algorithms of the system. This option should use simulated flow data, CO concentration and tracer gas concentration from standardized maneuvers with a known DLCO.

Instructions for the patient

To minimize variability, the following pre-test specifications, pre-test patient instructions (Table 4) and patient preparation for the test (Table 5) should be considered.

Procedure^2,8,22,23

• 1. In the diffusing equipment system the patient data will be placed, for the interpretation of DLCO_sb values an adjustment for equipment dead space and barometric pressure (altitude) is required, which should be done by the equipment software before calculating the predicted values.
• 2. Perform spirometry to obtain a forced vital capacity (FVC) maneuver according to the latest international standards.
• 3. The individual is positioned correctly, holding the mouthpiece and positioning the nose clip appropriately. A new mouthpiece with a filter should always be used on each patient and check that there are no leaks through the mouthpiece or nose.
• 4. As illustrated in Figure 2, start with two to three breaths at tidal volume, maintaining a stable functional residual capacity (FRC).²⁴
• 5. From FRC, the patient is asked to exhale in a relaxed manner until RV (expiratory reserve volume maneuver, ERV), where a plateau (< 25 mL) of at least one second should be achieved, and then the valve is activated.
  
  
  • a. In obstructive patients, where exhalation to RV may require more time, it is recommended that this part of the maneuver be limited to < 12 s, allowing this group of patients to exhale enough to achieve maximum vital capacity on the subsequent inhalation.

• 6. In VR, the subject is asked to inhale rapidly to TLC (where the mouthpiece is connected to the gas source).
• 7. The maximum inspiratory vital capacity (IVC) maneuver should be performed in less than four seconds, reaching a volume ≥ 90% of the FVC measured by spirometry previously (with a minimum tolerance of 85% for a B quality and 80% for a C quality).
• 8. The patient is asked to maintain a period of apnea for 10 ± 2 s, avoiding leaks and Valsalva or Müller maneuvers (expiratory or inspiratory effort against a closed glottis, respectively).
• 9. The subject is instructed to perform an unforced exhalation, without interruptions or hesitations.
• 10. In rapid systems (RGA), exhalation should be continued to RV, which improves VA measurement.
• 11. In case of a failed maneuver, instructions and demonstration should be repeated if necessary.
• 12. The time between maneuvers should be at least four minutes, to allow adequate tracer gas removal, in cases of severe airflow obstruction up to 10 minutes of waiting time is recommended.
• 13. A minimum of two maneuvers that meet acceptability and repeatability criteria must be completed, with a maximum of five attempts, in order to avoid increasing COHb in the blood (five DLCO_sb maneuvers increases 3-3.5% of COHb in the blood).²⁴

A submaximal inspiratory volume of the sample gas less than the known vital capacity may affect carbon monoxide inhalation, depending on whether it was from a suboptimal exhalation to RV (performed at TLC) or was from a suboptimal inhalation from RV (maneuver achieved below TLC). In the first case, VA and calculated DLCO_sb reliably reflect lung volume and lung properties at TLC. In the second case, VA is reduced and DLCO measurement is affected.

Inspiration must be rapid, as the DLCO_sb calculation assumes instantaneous lung filling, this explained because when the lungs fill more slowly, they decrease the amount of time the lung is in full inspiration, with a consequent reduction in carbon monoxide entry.

Valsalva or Müller maneuvers can affect the calculation of DLCO_sb by decreasing or increasing intrathoracic blood volume, resulting in an increase or decrease in DLCO_sb, respectively for each maneuver. Figure 3 shows some artifacts that may be observed during the maneuver.

Results review

Acceptability criteria²

• 1. Obtain an inspiratory volume (VIN) ≥ 90% of the largest FVC in the same test session; if this is not achieved, we can obtain an A quality with a VIN ≥ 85% of the largest FVC in the same test session along with a VA within 200 mL or 5% (whichever is greater) of the largest VA from other acceptable maneuvers.
• 2. Inspiratory time (IT) less than 4 seconds (obtain 85% of the test gas inhaled in < 4 seconds).
• 3. Stable breath holding time (BHT) for 10 ± 2 seconds with no evidence of leaks or Valsalva/Müller maneuvers during this time.
• 4. On classical analyzers the exhalation time must be greater than 4 seconds (i.e., sample collection is completed within 4 seconds of the onset of exhalation). In rapid response analyzers the exhalation should continue up to the residual volume, with a maximum exhalation time of 12 seconds, which provides a better measurement of VA.

Repeatability evaluation

The variability of DLCO_sb depends more on technical than biological factors. The DLCO_sb test should have at least two repeatable maneuvers in two units of DLCO_sb in mL/min/mmHg (equivalent to 0.67 unit in mmol/min/kPa). It is considered that more than 95.5% of patients can achieve this repeatability criterion.^2,9

Quality control of DLCO_sb maneuvers

A grade A maneuver is one that meets all acceptability criteria, therefore, the average DLCO_sb of two repeatable grade A maneuvers should be reported. If after repeating the test, the operator cannot obtain two repeatable grade A maneuvers, then the values are reported with the warning to the interpreter that the test session was not optimal (Table 6).²

DLCO_sb report

Special considerations and limitations for DLCO_sb

• 1. For interpretation of DLCO_sb results, equipment dead space adjustment should be performed.¹¹ The dead space adjustment should include the respiratory circuit proximal to the sampling point, the filter, and the gas analyzer mouthpiece, which should be < 200 mL. Smaller dead space volumes are recommended for pediatric-aged patients and adults with a vital capacity of less than 2 L.²⁵
• 2. DLCO_sb increases at higher altitudes because of lower oxygen competition due to lower PiO2.²⁶
• 3. Hemoglobin, COHb and backpressure concentrations or increased carbon monoxide outflow resistances may affect the DLCO_sb test, and should be considered when interpreting.^11,16
• 4. Diurnal variation in the DLCO result has been reported (1.2%/hour drop from 9:30 a.m. to 5:50 p.m.).²⁷
• 5. There is a change of up to 13% during menstrual cycles. The highest value of DLCO_sb is just before menstruation and the lowest value on the third day of menstruation.²⁸
• 6. There may be a reduction of up to 15% of the DLCO_sb value within 90 minutes of ingesting alcohol.^29,30
• 7. Smoking affects test results; a prevalence of a DLCO_sb below the lower limit of normal (LLN) in patients without airway obstruction has been observed in 26.7% when they are active smokers, and 14.4% in those who have quit smoking.³¹
• 8. An increase in DLCO_sb during pregnancy (first trimester) has been described but not consistently found in other studies.^32,33
• 9. The Valsalva maneuver can decrease DLCO_sb because it decreases the amount of blood in the pulmonary capillaries.¹²
• 10. In subjects with obstructive disease, bronchodilator use increases DLCO_sb by up to 6%, so the use of these drugs should be recorded by the technician.³⁴ However, recent studies have found no significant effects on DLCO with doses lower than 1,000 µg of salbutamol, so the use of bronchodilators before DLCO testing is not inadvisable.²

Adjustments in the DLCO_sb value

There are physiological factors that can affect the DLCO_sb measurement, inducing changes in opposite directions, therefore, current standards recommend four adjustments: Hb, COHb, the inspired pressure of oxygen (PiO₂) or altitude adjustment and VA adjustment. It is suggested to adjust for these factors in the predicted value of DLCO_sb rather than the measured value. This predicted value is calculated from measurements in healthy individuals without disease, with normal Hb and COHb levels, performed at rest and breathing at room air. If any of these conditions are not met, corresponding adjustments to the predicted value are advised and can be found in the supplementary material.^2,11

Basic interpretation process

• 1. The primary measurements are the pulmonary transfer coefficient for carbon monoxide (KCO) (carbon monoxide concentration change measured over time per unit volume and pressure) and VA, its product (DLCO = KCO × VA) is the key index interpreted for gas transfer.
• 2. Define the alveolocapillary membrane diffusion pattern according to the DLCO_sb concentrations proposed by the 2022 ATS/ERS interpretation strategies technical standard,¹¹ algorithm in Figure 4.
• 3. For severity grading it is recommended to use the DLCO_sb Z-score, i.e., the DLCO measurement expressed in standard deviations outside that predicted by reference values for individuals of the same height, age and sex generating the following categories:
  
  
  • a. Normal DLCO: ± 1.645 SD.
  • b. Mild decrease: from –1.645 to –2.5 SD.
  • c. Moderate decrease: from –2.51 to –4.0 SD.
  • d. Severe decrease: < –4.1 SD.

• 4. It is also useful to compare VA with TLC measured by body plethysmography to analyze whether maldistribution of the test gas that may contribute to lower DLCO_sb (i.e., carbon monoxide uptake can only be analyzed for regions where the test gasses are distributed). The normal value for the VA/TLC ratio in adults is approximately 0.85-0.90.³⁵ Values significantly below this suggest that deficiencies in the gas mixture are likely to contribute to a low measured DLCO_sb. In the absence of lung volume data by plethysmography, the presence of a steep downward slope in the inert gas tracing during exhalation suggests the possibility of gas maldistribution; however, there are no ideal ways to adjust for these conditions.¹¹
• 5. Quality grading according to Table 6.
• 6. The choice of reference equation may affect the final interpretation. Each laboratory must select the most appropriate equation for the methods and population selected. This is essential since large differences between reference equations have been described.^11,26,36
• In Mexico we have two reference equations, which were mostly performed in Mexico City (2,240 m above sea level), in a pediatric population (4 to 20 years of age) by Gochicoa et al.³⁷ and in an adult population (22 to 83 years of age) by Vázquez et al. the latter includes adjustment for altitude.³⁸
• 7. For interpretation, the relevant adjusted values for altitude (PiO2), Hb value and COHb should be considered.

Conclusions

DLCO is a pulmonary function test that assesses gas exchange and plays a crucial role in the diagnosis, monitoring and prognosis of multiple diseases. It is crucial to note that DLCO cannot be assessed in isolation; its constituent components, such as VA and KCO, need to be considered. Ignoring these variables may result in the loss of relevant clinical information. Furthermore, the importance of performing an integrated analysis of DLCO in conjunction with other pulmonary functional tests and available clinical data is emphasized.

Conflict of interests: the authors declare that they have no conflict of interests.

Funding: this manuscript did not receive any financial support.

Supplementary materials

Pulmonary diffusion of carbon monoxide: updates in recommendations and procedure

Physiological bases

The extraordinary affinity of carbon monoxide (CO) for hemoglobin allows this gas to be useful for evaluating gas exchange in the alveolocapillary membrane. This measurement reflects both the diffusion of CO and the rate of absorption by hemoglobin (Hb). There are several processes by which the transfer or uptake of CO from the outside to the Hb is interfered with, these processes are determined by Fick's diffusion law, which describes the flow of a gas through a semipermeable barrier, formula 1:

Gas flow (ṿ) = (A/T) × (P1 – P2) × K

(Formula 1)

The amount of gas transferred per unit of time (ṿ) is directly proportional to the diffusion area or surface (A), the gas diffusion constant (K) and the partial pressure gradient of the gasses across the membrane (P1 – P2); and inversely proportional to the thickness of the membrane (T). Applying this equation to pulmonary gas transfer, P1 and P2 are the gas concentrations in the alveolus and pulmonary capillary, respectively. Since it is not possible to specify the alveolar area (A), the membrane thickness (T) and the diffusion constant (K) of the alveolocapillary membrane for the entire lung, these variables are replaced by a single constant (DL), which represents the diffusion capacity for the lung as a whole³⁹ (Figure 5).

Measurement of CO transfer capacity is preferred over oxygen (O₂) for several reasons. Although both gases diffuse easily through the alveolocapillary membrane and combine with Hb, CO has a higher affinity than O₂; that is, about 210 times more related by Hb. The measurement of CO, being easily detectable, provides a more precise evaluation of pulmonary diffusion properties. When CO is measured, the P2 in the equation is assumed to be zero due to the high affinity of CO for Hb,^40,41 in contrast, the partial pressure of oxygen (PO₂) in the capillary increases as the erythrocyte It travels along the pulmonary capillary, so that, under normal conditions of rest and cardiac output, the oxygen pressure in the alveolus and capillary comes to a near equilibrium when the erythrocyte is only one-third the length of the capillary. At this point, no more O₂ can be transferred. However, if more blood flows through the capillary, more O₂ can be taken up, making O₂ uptake both "diffusion-limited" and "perfusion-limited".⁴²

Although there are situations in which O₂ transfer may be diffusion limited, ventilation-perfusion imbalance and shunt are much more important causes of resting hypoxemia than changes in membrane thickness. alveolocapillary.^43,44 On the other hand, diffusion can more easily reach its maximum and limit oxygen transfer during exercise and at altitude.

The initial partial pressure of CO in the alveolus (PACO) can be analyzed assuming that CO is diluted to the same extent as the inhaled inert gas (such as helium), which is used to calculate the instantaneous dilution of inhaled CO by volume residual. DLCO is expressed as the volume of CO (in milliliters) transferred per minute per millimeter of mercury of alveolar partial pressure of CO (mL/min/mmHg).

As illustrated in Figure 5, the CO diffusion pathway requires passing through the alveolocapillary membrane. Roughton and Forster⁴⁰ simplified this process into two steps: 1) diffusion of CO, described as the membrane component (Dm), and 2) binding of CO to Hb, described as the chemical reaction rate of COHb (θ) multiplied by pulmonary capillary blood volume (Vc), formula 2:

DLCO = Dm + θVc

(Formula 2)

This basic equation for DLCO is a conductance, flow divided by pressure change (ṿ /ΔP). The uptake of CO can be simplified to two properties of gas conductance. First, the CO conductance across the alveolocapillary membrane (Dm), which reflects the diffusion capacity of the membrane; and second, the binding capacity of CO to Hb (θVC). These two conductances are in series and are summarized in formula 3:

1/DLCO = (1/Dm) + (1/θVc)

(Formula 3)

Starting from formula 3, the conductances through which the molecules of a gas in the alveolocapillary membrane have to pass are represented as the reciprocals of the resistances, so that they can be added in series.

The Dm depends on: 1) surface area and thickness of the alveolocapillary membrane, 2) the thickness and surface area of the erythrocyte membrane contained in the alveolar capillaries and 3) the thickness of the plasma barrier, including all its components. The product of θVC is also called reactive conductance. Theta (θ) is the product of the ratio of the chemical reaction between CO and Hb, expressed as a ratio of 1 mL of blood (with a standard hemoglobin concentration); and Vc is the volume of Hb in the alveolar capillary blood.

Understanding this formula is important for interpretation purposes. Alveolar recruitment due to lung hyperinflation affects Dm, while capillary recruitment, as occurs in changes in body position, for example, supine position or during the Müller maneuver (deep inspiration with closed glottis), increases θVc.

DLCO equipment and consumables

DLCO equipment should meet the international technical recommendations issued by the American Thoracic Society and the European Respiratory Society (ATS/ERS 2017),⁷ with the following recommended minimum requirements for volume measurements and rapid gas analyzer, which can be found in the equipment user manual:

• 1. The equipment must meet the flow and volume measurement requirements established by ATS/ERS 2019 for spirometry.⁸ Flow measurement accuracy should be in the range of –10 to +10 L/s, which should be within ± 2%.
• 2. Calibration with 3 L syringe, with specified maximum error of ± 0.5% (i.e., 2.985 to 3.015 L), the calibration volume should be within ± 2.5%, which is equivalent to an error tolerance ≤ 75 mL. This volume measurement accuracy must be maintained over the entire range of gas composition and concentration.
• 3. The response time from 0 to 90% should be ≤ 150 ms.
• 4. The CO and tracer gas analyzer should have a linear response from zero concentration to full concentration of the test gas. The error in the linear response of the analyzer should not exceed more than 0.5% on the full scale.
• 5. The gas analyzer output should be accurate to within ± 1% of full scale.
• 6. The gas analyzer should be stable throughout the test, maintaining a minimum zero offset (measured in ppm and percent) and minimum gain offset. The gas analyzer offset should be ≤ 10 ppm in 30 seconds for carbon monoxide and ≤ 0.5% of full scale in 30 seconds for tracer gas.
• 7. The presence of carbon dioxide (CO₂) and water vapor should not interfere with the gas analyzer. If so, the equipment should remove these gasses before the sample passes through the analyzer or the equipment makes adjustments to the gas measurement according to the concentration of CO₂ and H₂O vapor present.
• 8. Circuit resistance should be < 1.5 cmH₂O/L/s at a flow rate of 6 L/s, if the test gas tank uses a flow demand regulator, the maximum inspiratory pressure across the circuit and valve should be < 10 cmH₂O.
• 9. The device timer should be accurate to 1% (100 ms over 10 seconds).
• 10. Monitor and report tracer gas and CO concentrations at end-expiration (alert operator if flushing is incomplete).
• 11. Ensure correct alignment of gas concentration signals and flow signal.
• 12. The equipment should measure the anatomical dead space using the Fowler method; failure to do so may result in an estimate of the anatomical dead space, but with the risk of inaccurate results.⁹
• 13. Display a graph of gas concentration versus exhaled volume to confirm the dead space washout point and report the amount of manual adjustment if performed.
• 14. Report DLCO adjusted for the change in P_AO₂ due to barometric pressure.
• 15. Ability to enter simulated digital test data and calculate DLCO, VA, TLC, VD.
• 16. Compensate for end-expiratory gas concentrations prior to test gas inhalation in the calculation of VA and DLCO.
• 17. The equipment dead space volume (DV) for both the inspired test gas and the alveolar sample should be known, its role in all data computation algorithms should be identified and documented. For adults, the VD should be < 200 mL, including the breathing circuit proximal to the gas analyzer sampling point, filter, and mouthpiece. Smaller dead space volumes are recommended for pediatric population and persons with a vital capacity (VC) < 2 L.
• 18. The system should be free of leaks.
• 19. For the digitized signal to accurately follow the gas concentration signal and provide adequate opportunity for signal processing for data alignment, the minimum signal sampling rate should be ≥ 100 Hz per channel with > 14 bits of resolution; however, a rate of 1,000 Hz is recommended.
• 20. The accuracy of the barometric pressure sensor should be within ± 2.5%.
• 21. Must have the capability to perform a quality check (with a 3 L syringe, under ATPS conditions and inhalation of ~2 L of test gas), the equipment must calculate total volume (VA) of 3 ± 0.3 L and DLCO of < 0.5 mL/min/mmHg or < 0.166 mmol/min/kPa.

Other equipment and consumables

• 1. Gas mixture tank for medical use; example: 0.27-0.33% CO, 9-11% helium, 18-25% oxygen and the rest nitrogen.
• 2. Computer and printer, according to device requirements.
• 3. Scales for weight and height measurement and tape measure for arm extension measurement, when required.
• 4. Environmental thermometers with an accuracy of 1 °C.
• 5. Disposable in-line filter nozzle with > 99% efficiency for filtration of viruses, bacteria and mycobacteria; dead space < 100 mL and resistance less than 1.5 cmH₂O at a flow rate of 6 L/s.
• 6. Infection control attachments:
• 7. Access to hand washing and disinfectant gel.
• 8. Surgical mask for general protection, and when N95 mask is required it must have a leakage of less than 10% and a filtration efficiency of > 95% at a flow of 50 L/min.

Adjustment for hemoglobin

Because Hb is the binding site for CO, DLCO_sb can change significantly depending on the Hb concentration in the blood. Better results are obtained with Hb measured on the same day, particularly in suspected polyglobulia, anemia or long-term measurements. Using these relationships and expressing Hb in g/dL, the predicted DLCO in adolescents and adult men can be adjusted using the following equation:

DLCO [predicted for Hb] = DLCO [predicted] × (1.7 × Hb/(10.22 + Hb))

While that of children under 15 years of age and women is adjusted using the following equation:⁴⁵

DLCO [predicted for Hb] = DLCO [predicted] × (1.7Hb/(9.38 + Hb))

Adjustment for carboxyhemoglobin

CO binds to Hb, and DLCO depends on the amount of Hb, therefore, DLCO is reduced if COHb increases. Adjustment for COHb is not routinely required, but is recommended, if COHb levels are suspected to be high, usually in smokers. Smokers have COHb of 5-10%, while nonsmokers < 3%. If COHb is < 2% no adjustment is required. Adjustment of DLCO for COHb is performed following the following equation.^45,48,49

DLCO [predicted for COHb] = DLCO [predicted] × (102-COHb%)

It should be remembered that CO inhalation in the single-breath maneuver causes COHb to increase by 0.6 to 0.7% for each maneuver.^45,50

Alveolar Oxygen Pressure Adjustment (P_AO₂)

Oxygen and CO compete for the same binding sites with Hb, so P_AO₂ affects DLCO. If P_AO₂ is high, DLCO decreases and vice versa. The first adjustment to this level is a concentration of 21% oxygen in the test gas. The DLCO value will change by approximately 0.35% for every 1 mmHg change in P_AO₂ or approximately 2.6% for every 1 kPa change in P_AO₂.⁴⁶

Altitude adjustment

Altitude also affects P_AO₂. The higher the altitude, the higher the DLCO because P_AO₂ decreases. Adjustment for altitude could be made in two ways:

• 1. DLCO [adjusted PB] = DLCO (0.505 + 0.00065 PB)
• 2. Altitude-adjusted DLCO = measured DLCO × [1 + 0.0031 (PiO₂-150)].

Where estimated IOP₂ = 0.21 (barometric pressure –47), or predicted values can be adjusted.

The P_AO₂ = 0.21 (PB-47)

Example: BP in Mexico City averages 585 mmHg, therefore:

IOP₂ = 113 mmHg

The adjustment in Mexico City would correspond to:

DLCO CDMX = DLCO (0.885)

REFERENCES

1. Ogilvie CM, Forster RE, Blakemore WS, Morton JW. A standardized breath holding technique for the clinical measurement of the diffusing capacity of the lung for carbon monoxide 1. J Clin Invest. 1957;36(1 Pt 1):1-17. Available in: http://www.jci.org/articles/view/103402

2. Graham BL, Brusasco V, Burgos F, Cooper BG, Jensen R, Kendrick A, et al. 2017 ERS/ATS standards for single-breath carbon monoxide uptake in the lung. Eur Respir J. 2017;49(1):1600016. Available in: https://doi.org/10.1183/13993003.00016-2016

3. Neder JA, Berton DC, Muller PT, O'Donnell DE. Incorporating lung diffusing capacity for carbon monoxide in clinical decision making in chest medicine. Clin Chest Med. 2019;40(2):285-305. Available in: https://doi.org/10.1016/j.ccm.2019.02.005

4. Baldini M, Chiapella MN, Fernandez A, Guardia S, De Vito EL, Sala H, et al. Capacidad de difusión de monóxido de carbono, pautas para su interpretación. 4 ed. Buenos Aires: Medicina; 2020. p. 359-730.

5. Cotes JE, Chinn DJ, Quanjer PH, Roca J, Yernault JC. Standardization of the measurement of transfer factor (diffusing capacity). report working party standardization of lung function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. Eur Respir J Suppl. 1993;16:41-52. Available in: http://www.ncbi.nlm.nih.gov/pubmed/8499053

6. Huang YCT, O'Brien SR, MacIntyre NR. Intrabreath diffusing capacity of the lung in healthy individuals at rest and during exercise. Chest. 2002;122(1):177-185. Available in: https://linkinghub.elsevier.com/retrieve/pii/S0012369216462963

7. Hughes JMB, Bates DV. Historical review: the carbon monoxide diffusing capacity (DlCO) and its membrane (Dm) and red cell (Θ Vc) components. Respir Physiol Neurobiol. 2003;138(2-3):115-142. Available in: https://linkinghub.elsevier.com/retrieve/pii/S1569904803002131

8. Macintyre N, Crapo RO, Viegi G, Johnson DC, van der Grinten CPM, Brusasco V, et al. Standardisation of the single-breath determination of carbon monoxide uptake in the lung. Eur Respir J. 2005;26(4):720-735. Available in: https://doi.org/10.1183/09031936.05.00034905

9. Wise RA, Teeter JG, Jensen RL, England RD, Schwartz PF, Giles DR, et al. Standardization of the single-breath diffusing capacity in a multicenter clinical trial. Chest. 2007;132(4):1191-1197. Available in: https://linkinghub.elsevier.com/retrieve/pii/S001236921536712X

10. Crapo RO, Jensen RL, Wanger JS. Single-breath carbon monoxide diffusing capacity. Clin Chest Med. 2001;22(4):637-649. Available in: https://doi.org/10.1016/s0272-5231(05)70057-5

11. Stanojevic S, Kaminsky DA, Miller MR, Thompson B, Aliverti A, Barjaktarevic I, et al. ERS/ATS technical standard on interpretive strategies for routine lung function tests. Eur Respir J. 2022;60(1):2101499. Available in: https://doi.org/10.1183/13993003.01499-2021

12. Jensen RL, Crapo RO. Diffusing capacity: how to get it right. Respir Care. 2003;48(8):777-782. Available in: http://www.ncbi.nlm.nih.gov/pubmed/12890298

13. Balasubramanian A, Putcha N, MacIntyre NR, Jensen RL, Kinney G, Stringer WW, et al. Diffusing capacity and mortality in chronic obstructive pulmonary disease. Ann Am Thorac Soc. 2023;20(1):38-46. Available in: https://doi.org/10.1513/annalsats.202203-226oc

14. Raghu G, Remy-Jardin M, Richeldi L, Thomson CC, Inoue Y, Johkoh T, et al. Idiopathic pulmonary fibrosis (an Update) and progressive pulmonary fibrosis in adults: an official ATS/ERS/JRS/ALAT Clinical Practice Guideline. Am J Respir Crit Care Med. 2022;205(9):e18-e47. Available in: https://doi.org/10.1164/rccm.202202-0399st

15. Sivova N, Launay D, Wémeau-Stervinou L, De Groote P, Remy-Jardin M, Denis G, et al. Relevance of partitioning DLCO to detect pulmonary hypertension in systemic sclerosis. PLoS One. 2013;8(10):e78001. Available in: https://doi.org/10.1371/journal.pone.0078001

16. Mohsenifar Z, Brown HV, Schnitzer B, Prause JA, Koerner SK. The effect of abnormal levels of hematocrit on the single breath diffusing capacity. Lung. 1982;160(6):325-330. Available in: https://doi.org/10.1007/bf02719308

17. Holley AB, Carbone T, Holtzclaw AW, Huprikar NA, Wagner R, Morris MJ. Obesity-related changes in diffusing capacity and transfer coefficient of the lung for carbon monoxide and resulting patterns of abnormality across reference equations. Ann Am Thorac Soc. 2023;20(7):969-975. Available in: https://doi.org/10.1513/annalsats.202207-640oc

18. Coburn RF, Forster RE, Kane PB. Considerations of the physiological variables that determine the blood carboxyhemoglobin concentration in man. J Clin Invest. 1965;44(11):1899-1910. Available in: https://doi.org/10.1172/jci105296

19. Cooper BG. An update on contraindications for lung function testing. Thorax. 2011;66(8):714-723. Available in: https://doi.org/10.1136/thx.2010.139881

20. Schonffeldt-Guerrero P, Gochicoa-Rangel L, Aguirre Franco C, Arce SC, Rodríguez Flores C. ALAT 2023 Recommendations for Performing Respiratory Function Studies. Arch Bronconeumol. 2023;59(10):619-620. Available in: https://doi.org/10.1016/j.arbres.2023.04.004

21. Haynes JM, Ruppel GL, Kaminsky DA. Should diffusing capacity quality control be treated like other laboratory devices? Eur Respir J. 2021;58(6):2102642. Available in: http://www.ncbi.nlm.nih.gov/pubmed/34737225

22. Graham BL, Steenbruggen I, Miller MR, Barjaktarevic IZ, Cooper BG, Hall GL, et al. Standardization of Spirometry 2019 Update. An Official American Thoracic Society and European Respiratory Society Technical Statement. Am J Respir Crit Care Med. 2019;200(8):e70-e88. Available in: https://doi.org/10.1164/rccm.201908-1590st

23. DeCato TW, Hegewald MJ. Breathing red: Physiology of an elevated single-breath diffusing capacity of carbon monoxide. Ann Am Thorac Soc. 2016;13(11):2087-2092. Available in: https://doi.org/10.1513/annalsats.201605-355cc

24. Zavorsky GS. The rise in carboxyhemoglobin from repeated pulmonary diffusing capacity tests. Respir Physiol Neurobiol. 2013;186(1):103-108. Available in: https://doi.org/10.1016/j.resp.2013.01.001

25. Stanojevic S, Graham BL, Cooper BG, Thompson BR, Carter KW, Francis RW, et al. Official ERS technical standards: Global Lung Function Initiative reference values for the carbon monoxide transfer factor for Caucasians. Eur Respir J. 2017;50(3):1700010. Available in: https://doi.org/10.1183/13993003.00010-2017

26. Gray G, Zamel N, Crapo RO. Effect of a simulated 3,048 meter altitude on the single-breath transfer factor. Bull Eur Physiopathol Respir. 1986;22(5):429-431.

27. Kirla KT, Nemes S, Betts J, Kristensson C, Mo J, Asimus S, et al. Diurnal variation in DLCO and non-standardized study procedures may cause a false positive safety signal in clinical trials. Respir Med. 2022;191:106705. Available in: https://doi.org/10.1016/j.rmed.2021.106705

28. Sansores RH, Abboud RT, Kennell C, Haynes N. The effect of menstruation on the pulmonary carbon monoxide diffusing capacity. Am J Respir Crit Care Med. 1995;152(1):381-384. Available in: https://doi.org/10.1164/ajrccm.152.1.7599851

29. Simeone F, Wiese J, Glindmeyer H, Lasky J. The effects of ethanol ingestion on the accuracy of pulmonary diffusing capacity measurement. Chest. 2005;128(6):3875-3880. Available in: https://doi.org/10.1378/chest.128.6.3875

30. Peavy HH, Summer WR, Gurtner G. The effects of acute ethanol ingestion on pulmonary diffusing capacity. Chest. 1980;77(4):488-492. Available in: https://doi.org/10.1378/chest.77.4.488

31. Garcia-Rio F, Miravitlles M, Soriano JB, Cosío BG, Soler-Cataluña JJ, Casanova C, et al. Prevalence of reduced lung diffusing capacity and CT scan findings in smokers without airflow limitation: a population-based study. BMJ Open Respir Res. 2023;10(1):e001468. Available in: https://doi.org/10.1136/bmjresp-2022-001468

32. Gazioglu K, Kaltreider NL, Rosen M, Yu PN. Pulmonary function during pregnancy in normal women and in patients with cardiopulmonary disease. Thorax. 1970;25(4):445-450. Available in: https://doi.org/10.1136/thx.25.4.445

33. Milne JA, Mills RJ, Coutts JR, Macnaughton MC, Moran F, Pack AI. The effect of human pregnancy on the pulmonary transfer factor for carbon monoxide as measured by the single-breath method. Clin Sci Mol Med. 1977;53(3):271-276. Available in: https://doi.org/10.1042/cs0530271

34. Balasubramanian A, MacIntyre NR, Henderson RJ, Jensen RL, Kinney G, Stringer WW, et al. Diffusing capacity of carbon monoxide in assessment of COPD. Chest. 2019;156(6):1111-1119. Available in: https://doi.org/10.1016/j.chest.2019.06.035

35. Hughes JMB, Pride NB. Examination of the carbon monoxide diffusing capacity (DLCO) in relation to its KCO and VA components. Am J Respir Crit Care Med. 2012;186(2):132-139. Available in: https://doi.org/10.1164/rccm.201112-2160ci

36. McGrath MW, Thomson ML. The effect of age, body size and lung volume change on alveolar-capillary permeability and diffusing capacity in man. J Physiol. 1959;146(3):572-582. Available in: https://doi.org/10.1113/jphysiol.1959.sp006212

37. Gochicoa-Rangel L, Del-Río-Hidalgo R, Álvarez-Arroyo MR, Martínez-Briseño D, Mora-Romero U, Martínez-Valdeavellano L, et al. Diffusing capacity of the lung for carbon monoxide in Mexican/Latino children. Quality control and reference values. Ann Am Thorac Soc. 2019;16(2):240-247. Available in: https://doi.org/10.1513/annalsats.201712-922oc

38. Vázquez-García JC, Pérez-Padilla R, Casas A, Schonffeldt-Guerrero P, Pereira J, Vargas-Domínguez C, et al. Reference values for the diffusing capacity determined by the single-breath technique at different altitudes: the Latin American Single-Breath Diffusing Capacity Reference Project. Respir Care [Internet]. 2016;61(9):1217-1223. Available in: http://www.ncbi.nlm.nih.gov/pubmed/27587868

39. DeCato TW, Hegewald MJ. Breathing red: physiology of an elevated single-breath diffusing capacity of carbon monoxide. Ann Am Thorac Soc. 2016;13(11):2087-2092. Available in: https://doi.org/10.1513/annalsats.201605-355cc

40. Hughes M. The Roughton-Forster equation for pulmonary diffusion: how it happened. Eur Respir J. 2022;60(1):2200789. Available in: https://doi.org/10.1183/13993003.00789-2022

41. Hughes JMB. Assessing gas exchange. Chron Respir Dis. 2007;4(4):205-214. Available in: https://doi.org/10.1177/1479972307084446

42. Hsia CCW. Recruitment of lung diffusing capacity: update of concept and application. Chest. 2002;122(5):1774-1783. Available in: https://doi.org/10.1378/chest.122.5.1774

43. Chaouat A, Adir Y. Diffusing capacity for carbon monoxide is a reflection of the pulmonary microcirculation, but not only. Chest. 2020;158(2):455-457. Available in: https://doi.org/10.1016/j.chest.2020.03.033

44. Hughes JMB. The single breath transfer factor (Tl,co) and the transfer coefficient (Kco): a window onto the pulmonary microcirculation. Clin Physiol Funct Imaging. 2003;23(2):63-71. Available in: https://doi.org/10.1046/j.1475-097x.2003.00482.x

45. Graham BL, Brusasco V, Burgos F, Cooper BG, Jensen R, Kendrick A, et al. 2017 ERS/ATS standards for single-breath carbon monoxide uptake in the lung. Eur Respir J. 2017;49(1):1600016. Available in: https://doi.org/10.1183/13993003.00016-2016

46. Graham BL, Steenbruggen I, Miller MR, Barjaktarevic IZ, Cooper BG, Hall GL, et al. Standardization of spirometry 2019 Update. An Official American Thoracic Society and European Respiratory Society Technical Statement. Am J Respir Crit Care Med. 2019;200(8):e70-e88. Available in: https://doi.org/10.1164/rccm.201908-1590st

47. Jones RS, Meade F. A theoretical and experimental analysis of anomalies in the estimation of pulmonary diffusing capacity by the single breath method. Q J Exp Physiol Cogn Med Sci. 1961;46:131-143. Available in: https://doi.org/10.1113/expphysiol.1961.sp001525

48. Coburn RF, Forster RE, Kane PB. Considerations of the physiological variables that determine the blood carboxyhemoglobin concentration in man. J Clin Invest. 1965;44(11):1899-1910. Available in: https://doi.org/10.1172/jci105296

49. Jensen RL, Crapo RO. Diffusing capacity: how to get it right. Respir Care. 2003;48(8):777-782. Available in: http://www.ncbi.nlm.nih.gov/pubmed/12890298

50. Zavorsky GS. The rise in carboxyhemoglobin from repeated pulmonary diffusing capacity tests. Respir Physiol Neurobiol. 2013;186(1):103-108. Available in: https://doi.org/10.1016/j.resp.2013.01.001