7.1 Single-Breath Carbon Monoxide Diffusing Capacity (DLCO) Testing Protocol

7.1 Single-Breath Carbon Monoxide Diffusing Capacity (DLCO) Testing Protocol

Determining the diffusing capacity of the lung for carbon monoxide ($DL_{CO}$)—also referred to internationally as the transfer factor ($TL_{CO}$)—is a cornerstone diagnostic modality in pulmonary function testing. While spirometry and lung volume determinations quantify the mechanical and volumetric properties of the lungs, $DL_{CO}$ evaluates the functional capability of the alveolar-capillary membrane to transfer gas from the alveoli into the pulmonary capillary blood stream.

Carbon monoxide is the ideal test gas for assessing diffusing capacity because its uptake across the alveolar-capillary membrane is diffusion-limited rather than perfusion-limited. Because carbon monoxide binds to hemoglobin with an affinity approximately 210 times greater than that of oxygen, the partial pressure of free dissolved CO in pulmonary capillary plasma remains virtually zero during a brief test maneuver. Consequently, the rate of CO uptake across the alveolar-capillary membrane is directly proportional to the surface area available for gas exchange, the physical diffusion properties of the membrane barrier, and the total mass of functional hemoglobin in pulmonary capillary blood.

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Test Gas Composition

Standard single-breath $DL_{CO}$ testing ($DL_{CO}\text{-SB}$, historically termed the modified Krogh technique) utilizes a precisely certified gas mixture containing four primary components:

1. Carbon Monoxide ($0.3% \text{ CO}$ or $3,000 \text{ ppm}$): Serves as the primary indicator gas to measure alveolar-capillary transfer.
2. Inert Tracer Gas ($10% \text{ Helium [He]}$ or $0.3% \text{ Methane [CH}_4\text{]}$): An insoluble, non-absorbable gas used to calculate the alveolar volume ($V_A$) at full inspiration via single-breath gas dilution and to determine the initial alveolar CO concentration ($F_{A,CO,0}$) prior to diffusion.
3. Oxygen ($21% \text{ O}_2$): Maintained at standard ambient room-air concentration to ensure normoxia and prevent competitive displacement of CO at hemoglobin binding sites. (Special hyperoxic mixtures using $98% \text{ O}_2$ are utilized only when separating membrane diffusing capacity $D_M$ from pulmonary capillary blood volume $V_c$).
4. Nitrogen ($N_2$) Balance: Forms the remaining balance of the gas mixture.

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Patient Preparation and Pre-Test Guidelines

To prevent artifactual alterations in $DL_{CO}$ measurements, pulmonary technologists must enforce strict pre-test patient preparation protocols prior to testing:

• Abstinence from Smoking and Vaping: The patient must abstain from smoking cigarettes, cigars, pipes, or electronic vaping devices for at least 4 hours (ideally 12 to 24 hours) prior to testing. Smoking elevates blood carboxyhemoglobin ($COHb$) levels, creating a "back-pressure" effect that reduces the partial pressure gradient for CO uptake and artificially lowers measured $DL_{CO}$.
• Discontinuation of Supplemental Oxygen: If clinically safe, supplemental oxygen therapy must be discontinued for at least 15 to 20 minutes prior to $DL_{CO}$ testing. Breathing elevated $F_{I,O2}$ increases alveolar oxygen partial pressure ($P_{A,O2}$), which competitively inhibits CO binding to hemoglobin and falsely depresses measured $DL_{CO}$. If a patient cannot tolerate $O_2$ withdrawal, the supplemental $F_{I,O2}$ must be recorded, and the testing report must note the hyperoxic conditions.
• Avoidance of Heavy Exercise: Strenuous physical exertion must be avoided for at least 2 hours prior to the test. Intense exercise increases cardiac output and pulmonary capillary blood volume ($V_c$), which transiently elevates $DL_{CO}$.
• Medication Withholding: Routine bronchodilators do not significantly alter $DL_{CO}$ in non-asthmatic patients, but standard pre-test bronchodilator withholding schedules are often maintained if spirometry is performed during the same session.

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Step-by-Step Clinical Testing Protocol

Executing an acceptable single-breath $DL_{CO}$ maneuver requires strict adherence to standardized sequence and timing:

1. Baseline Phase and Positioning: The patient sits comfortably upright in an ergonomic testing chair with a nose clip applied. The patient breathes tidal room air through a low-resistance two-way valve assembly until a stable end-expiratory tidal level (FRC) is established.
2. Exhalation to Residual Volume: Upon prompt, the patient performs an unforced exhalation down to Residual Volume (RV). This exhalation should be complete, smooth, and accomplished within $\le 6.0\text{ seconds}$ (or $\le 12.0\text{ seconds}$ in patients with severe airflow obstruction).
3. Rapid Maximal Inspiration ($V_I$): At RV, the valve rapidly switches the patient to the $DL_{CO}$ test gas reservoir. The patient inspires maximally to Total Lung Capacity (TLC) as rapidly as possible.
   • Volume Requirement (the 90%/85% rule): The inspired volume ($V_I$) should be $\ge 90%$ of the largest previously recorded Vital Capacity obtained in the same session. A $V_I$ between 85% and 90% is still acceptable only if the resulting alveolar volume ($V_A$) is within 200 mL or 5% of the largest $V_A$ from another acceptable maneuver. Below 85%, the maneuver is rejected. (Older references quote a flat "$\ge 85%$" rule; the 2017 ERS/ATS DLCO standard tightened it to 90% with the 85% conditional exception.)
   • Time Requirement: At least 85% of $V_I$ must be inhaled within 4.0 seconds (target $< 2.5$ seconds in healthy adults). Slow inspiration invalidates the maneuver because meaningful CO uptake occurs before the formal breath-hold begins.
4. Breath-Hold Period at TLC: Once TLC is reached, the patient holds their breath at full inspiration for 8 to 12 seconds (target $10 \pm 0.5\text{ seconds}$).
   • Physiological Caution: The patient must remain relaxed during the breath-hold. They must avoid performing a Valsalva maneuver (forced expiration against a closed glottis), which increases intrathoracic pressure, squeezes blood out of the pulmonary capillaries, and falsely decreases $DL_{CO}$. Conversely, a Müller maneuver (forced inspiration against a closed glottis) generates negative intrathoracic pressure, engorges pulmonary capillaries, and falsely increases $DL_{CO}$.
5. Exhalation and Sample Collection: After the breath-hold, the patient exhales smoothly and without hesitation.
   • Washout Volume Phase: The initial $0.75\text{ L to } 1.0\text{ L}$ of exhaled gas is diverted and discarded into a washout container (or cleared past the gas sampling port). This volume represents anatomical and equipment dead space containing gas that did not participate in alveolar exchange.
     • Special Exception: If the patient's Vital Capacity is $< 2.0\text{ Liters}$, the required washout volume is reduced to $0.50\text{ Liters}$ to ensure adequate gas remains for the alveolar sample.
   • Alveolar Sample Collection: Immediately following dead-space washout, an alveolar sample of $0.50\text{ L to } 1.0\text{ L}$ is collected into a sampling bag or analyzed continuously via rapid-response multi-gas analyzers. Per the 2017 ERS/ATS standard, sample collection must be completed within 4 seconds of the start of exhalation, which in practice means the whole washout-plus-sample sequence has to be executed briskly.

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Breath-Hold Timing Methods: The Jones-Meade Standard

Accurate calculation of $DL_{CO}$ depends critically on precisely defining the breath-hold time ($t$). Three historical methods have been used to measure breath-hold duration, but current ATS/ERS guidelines explicitly specify the Jones-Meade Method as the universal gold standard:

1. Jones-Meade Method (ATS/ERS Standard): Breath-hold time begins at $30% \text{ (0.30)}$ of the inspiratory time ($t_I$) and ends at the midpoint of the alveolar sample collection time ($t_S$). By incorporating $30%$ of inspiration and half of sample collection, the Jones-Meade formula accounts for the small amounts of CO diffusion occurring during gas entry and exhalation. $t_{\text{Jones-Meade}} = (t_{\text{inspiration}} - 0.30 \times t_I) + t_{\text{hold}} + (0.50 \times t_S)$
2. Ogilvie Method (Historical): Measured breath-hold time from the beginning of inspiration to the beginning of alveolar sample collection. This method overestimates total contact time and yields slightly lower $DL_{CO}$ values.
3. Epidemiology Standardization Project (ESP / Ferris) Method: Measured time from the end of inspiration (completion of $V_I$) to the beginning of alveolar sample collection. This method underestimates total contact time and yields higher $DL_{CO}$ values.

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Repeatability and Quality Assurance Criteria

To achieve clinical acceptability and repeatability during a testing session:

• Number of Trials: At least two acceptable maneuvers must be obtained.
• Repeatability Standard: The two acceptable $DL_{CO}$ values must agree within $2.0\text{ mL CO/min/mmHg}$ ($0.67\text{ mmol/min/kPa}$) of each other. This is an absolute criterion only — the 2017 ERS/ATS standard dropped the older "3 mL/min/mmHg or 10%" alternative. The stale percentage rule wrongly passes pairs such as 40.0 and 37.0 mL/min/mmHg, which differ by 3.0 and therefore fail the current standard.
• Maximum Trial Limit: No more than 5 maneuvers should be performed in a single testing session. Performing excess trials causes cumulative accumulation of carbon monoxide in pulmonary capillary blood (increasing $COHb$ levels), which falsely depresses subsequent $DL_{CO}$ values.
• Inter-Test Rest Interval: A mandatory delay of at least 4 minutes breathing room air must separate successive $DL_{CO}$ trials. This delay allows complete washout of inspired tracer gas from the lungs and clears transiently elevated alveolar CO concentrations. In patients with severe obstructive airway disease or slow-ventilating compartments, this interval should be extended to 6 to 10 minutes.