Direct Stimuli	Indirect Stimuli
Methacholine	Mannitol
Histamine	Adenosine (AMP)
Prostaglandins	Exercise
Leukotrienes	Eucapnic voluntary hyperventilation (EVH)
	Propranolol (β-blockers)
	Hypertonic saline

Figure 9-1 Direct versus indirect stimuli of the airways.
There are two categories of bronchoprovocation tests, those that act directly on smooth muscle and those that cause the airways to narrow indirectly by a release of endogenous mediators.

Histamine and methacholine act directly on the smooth muscle cells of the airways to cause bronchoconstriction and airway hyperresponsiveness (AHR). Indirect bronchoprovocation tests, such as mannitol and adenosine monophosphate (AMP), act through inducing the release of bronchoconstricting mediators. Hyperventilation, either at rest or during exercise, results in heat and water loss from the airway. This provokes a bronchospasm in susceptible patients. With each of these agents, pulmonary function variables are assessed before and after exposure to the challenge. FEV₁ is the variable most commonly used. Other flow measurements, as well as airway resistance (Raw) and specific conductance (sGaw), may also be evaluated before and after the challenge. Additional parameters that have been used to assess response to a bronchial challenge include breath sounds, transcutaneous PO₂ (tcPO₂, see Chapter 11), and forced oscillation measurements of resistance and reactance.

Methacholine Challenge

Bronchial challenge by inhalation of methacholine is performed by having the patient inhale increasing doses of the drug. All subjects will show a change in airway caliber with increasing concentrations of methacholine. Patients who have hyperresponsive airways demonstrate these changes at low doses of inhaled methacholine. This dose-response relationship permits the sensitivity of the airways to be quantified.

Spirometry, and sometimes sGaw, is measured after each dose. Most clinicians consider the test result positive when inhalation of methacholine precipitates a 20% decrease in FEV₁. The methacholine concentration at which this 20% decrease occurs is called the provocative concentration (PC₂₀). In some references it may be termed provocative dose (PD₂₀ ). In the doses usually used (Box 9-1), healthy subjects do not display decreases greater than 20% in FEV₁. Therefore, the methacholine challenge test is highly specific for airway hyperreactivity. Many patients who have asthma experience a 20% reduction in FEV₁ with doses of 8 mg/mL or less. However, bronchial hyperresponsiveness may also be seen in other pulmonary disorders such as COPD, cystic fibrosis, and bronchitis.

Box 9-1 Methacholine Dosing Schedules

Dose Quadrupling Schedule (4 × Increase)	Dose Doubling Schedule (2 × Increase)
0.0625 mg/mL	0.031 mg/mL
0.250 mg/mL	0.0625 mg/mL
1.0 mg/mL	0.125 mg/mL
4.0 mg/mL	0.25 mg/mL
16.0 mg/mL	1.0 mg/mL
	2.0 mg/mL
	4.0 mg/mL
	8.0 mg/mL
	16.0 mg/mL

9-1 How To…

Perform a Bronchoprovocation Inhalation Challenge Test

1. Tasks common to all procedures.

a. Calibrate and prepare equipment.

b. Review order.

c. Introduce yourself and identify patient according to institutional policies.

d. Describe and demonstrate procedure.

2. Verify compliance with pre-test instructions (Table 9-1) and note discrepancies. Test may need to be rescheduled.

3. Perform acceptable and repeatable baseline spirometry.

a. Determine eligibility to test (e.g., FEV₁ >65%-70% predicted or 1.5 L).

b. Determine FEV₁ target for PC₁₅ or PC₂₀, depending on the challenge agent.

c. Determine recovery FEV₁ (baseline FEV₁ × 0.90).

4. Formulate the dose concentrations based on the challenge agent and manufacturer’s instructions. Some agents may be formulated and not require additional mixing.

5. Deliver initial dose based on the protocol/agent selected (may include diluent or 0-mg drug).

a. Administer via dosimeter, tidal breathing, and/or dry powder inhaler.

b. Recalculate new target FEV₁ based on the “new baseline” FEV₁ obtained after the diluent or 0-mg administration (new baseline FEV₁× 0.8 of 0.85, depending on the challenge agent).

6. Measure FEV₁ (airways resistance optional).

a. If greater than target FEV₁, move to next dose level.

b. If less than target FEV₁, stop test and administer a bronchodilator.

7. Measure postbronchodilator FEV₁.

a. If greater than target recovery FEV₁ and the subject is symptom free, they can be dismissed.

b. If less than target recovery FEV₁, follow lab protocol, which may include administering another dose of bronchodilator.

8. Calculate PC₁₅ or PC₂₀ dose.

9. Report data and note comments related to test quality.

Patients to be tested should be asymptomatic, with no coughing or obvious wheezing. Recent upper or lower respiratory tract infections may alter airway responsiveness, so bronchial challenge testing may need to be deferred. Their baseline FEV₁ should be normal or at least greater than 60%–70% of their expected value. For patients with known obstruction or restriction, FEV₁ should be close to their highest previously observed value. Obvious airway obstruction (e.g., FEV_1% less than the lower limit of normal) is a relative contraindication. If the patient has an FEV₁ less than 1.0–1.5 L, there is a risk that a large drop in FEV₁ after a methacholine challenge might leave the individual with a compromised lung function. Bronchial challenge may be indicated in obstructed patients if the clinical question is related to the degree of responsiveness. Box 9-2 lists the absolute and relative contraindications to a methacholine challenge.

If the patient has been taking bronchodilators, they should be withheld according to the schedule listed in Table 9-2. Other medications or substances can affect the validity of the challenge as well. All medications being taken at the time of testing should be recorded to assist in the evaluation of the test results.

Table 9-2

Withholding Medications Before Bronchial Challenge

Short-acting β agonist agents (inhaled)	8 hours
Long-acting β agonist agents (inhaled)	48 hours (some may require longer)
Standard β agonist agents (oral)	12 hours
Long-acting β agonist agents (oral)	24 hours
Anticholinergic agents (ipratropium)	24 hours
Standard theophylline preparations	12–24 hours
Sustained-action theophylline preparations	48 hours
Cromolyn sodium	8 hours
Nedocromil	48 hours
Antihistamines	72–96 hours
Corticosteroids (inhaled or oral)	Patients challenged while taking a stable dosage*
Leukotriene modifiers	24 hours
Caffeine-containing drinks (cola, coffee)	6 hours
β-blocking agents	May increase the response

^*Corticosteroids may decrease bronchial hyperreactivity.

Baseline spirometry is performed to establish that the patient’s FEV₁ is greater than 60%–70% of predicted or the previously observed best value. Patients who demonstrate obstruction based on reduced FEV_1% or other flows typically do not require challenge testing. However, obstructed patients may be tested to establish the degree of hyperreactivity. Patients who have a restrictive process (i.e., reduced FEV₁, FVC [forced vital capacity], and TLC) may also be tested for coexisting airway hyperresponsiveness. If a patient is unable to perform acceptable and repeatable baseline spirometry (i.e., FEV₁), changes after an inhalation challenge may be impossible to interpret. In these situations, other parameters (such as sGaw or oscillatory resistance) that are less dependent on patient effort may be preferable as an endpoint.

Two methods of delivering methacholine to the airway have been recommended by the American Thoracic Society (ATS): the five-breath dosimeter method and the 2-minute tidal breathing method. A dosimeter can provide a true “quantitative” challenge test by delivering a consistent volume of the drug. The dosimeter (or nebulizer) is activated during inspiration, either automatically (by a flow sensor) or manually (by the technologist). A standardized driving pressure (typically 20 psi) and activation time (0.5–0.8 seconds) allows a fixed volume of aerosol to be generated for each breath. By limiting the period of aerosol production, the last part of the inhalation carries the aerosol into the lung. The tidal breathing method is somewhat simpler because only a nebulizer is used.

A small-volume nebulizer is used to generate the methacholine aerosol (Figure 9-2).

Figure 9-2 Nebulizer and dosimeter.
A dosimeter that controls the flow of gas to the nebulizer is also shown. The dosimeter provides a timer so that nebulization occurs for a specific time (e.g., 0.6 seconds), depending on the nebulizer used and its specific output. This dosimeter senses the patient’s inspiratory effort and triggers flow automatically; some require manual actuation. (Courtesy nSpire Health, Inc. Longmont, CO)

The nebulizer should generate an aerosol with a particle size in the range of 1.0–3.6 μm (mass median aerodynamic diameter). This particle range promotes deposition in the medium and small airways. For the tidal breathing method, the nebulizer output should be 0.13 mL/min; for use with a dosimeter (five-breath method), the output should be 0.009 mL for each 0.6-second actuation of the dosimeter. Because the output of each nebulizer varies by manufacturer and can change over time, it should be measured. This can be done by weighing the nebulizer on an accurate scale before and after a sham administration of the drug. The output should be measured with the protocol to be used for testing. For the five-breath dosimeter method, more breaths may be needed to measure the small output. The nebulizer should also be “primed” with an actuation before patient testing to alleviate the electrostatic properties of a plastic nebulizer or spacer. The delivered dose of methacholine is standardized by using a fixed number of breaths (5) or breathing for a fixed length of time (2 minutes).

PF Tip 9-1

Despite different techniques for administering methacholine, patients who truly have asthma usually display a 20% decrease in FEV₁. The lower the dose of methacholine, the more sensitive, or hyperresponsive, the patient’s airways are. Detecting a 20% decrease in FEV₁ requires that acceptable and repeatable baseline spirometry is obtained.

Two dosing routines are commonly used for a methacholine challenge (see Box 9-2). One routine uses a quadrupling (4×) increase in methacholine concentration, and the other method uses a doubling dose (2×). For each of these regimens, the highest dose is 16 mg/mL, and the dilutions can be easily prepared from a stock solution, starting with 100 mg of dry methacholine (Table 9-3). The stock solution is prepared by dissolving the powdered drug in a saline diluent. A preservative (0.4% phenol) may be added to the solution but is not required. Methacholine concentrations from 0.025–25.0 mg/mL are stable after mixing and may be kept for 5 months if refrigerated at 2°C–8°C (36-46°F). An alternate dosing scheme is provided with the FDA-approved form of methacholine (Provocholine, Methapharm, Ontario, Canada). This dosing schedule uses methacholine concentrations of 0.025, 0.25, 2.5, 10, and 25 mg/mL and is designed for use with the five-breath dosimeter method. Methacholine should be prepared by a pharmacist or individual trained in preparing drugs using a sterile technique. Appropriate precautions should be taken when handling dry powdered methacholine. Vials of methacholine should be carefully marked with labels that clearly identify the concentration.

Table 9-3

Preparation of Methacholine for Two Common Dosing Schedules *

Methacholine	Diluent (0.9% NaCl)	Dilution
Doubling Dosage
100 mg (dry powder)	6.25 mL	16.0 mg/mL
3 mL of 16.0 mg/mL	3 mL	8.0 mg/mL
3 mL of 8.0 mg/mL	3 mL	4.0 mg/mL
3 mL of 4.0 mg/mL	3 mL	2.0 mg/mL
3 mL of 2.0 mg/mL	3 mL	1.0 mg/mL
3 mL of 1.0 mg/mL	3 mL	0.5 mg/mL
3 mL of 0.5 mg/mL	3 mL	0.25 mg/mL
3 mL of 0.25 mg/mL	3 mL	0.125 mg/mL
3 mL of 0.125 mg/mL	3 mL	0.0625 mg/mL
3 mL of 0.625 mg/mL	3 mL	0.031 mg/mL
Quadrupling Dosage
100 mg (dry powder)	6.25 mL	16.0 mg/mL
3 mL of 16.0 mg/mL	9 mL	4.0 mg/mL
3 mL of 4.0 mg/mL	9 mL	1.0 mg/mL
3 mL of 1.0 mg/mL	9 mL	0.25 mg/mL
3 mL of 0.25 mg/mL	9 mL	0.0625 mg/mL

^*For each schedule, 6.25 mL of saline are added to dry powdered methacholine. Subsequent dilutions then use 3 mL or 9 mL of saline added to 3 mL of the previous dilution.

Five-Breath Dosimeter Method

Methacholine is prepared according to the desired dosing scheme. Methacholine may be stored under refrigeration but should be brought to room temperature before administration. Baseline spirometry is performed.

The patient begins by inhaling five breaths of nebulized diluent, usually normal saline. The diluent step is optional but provides a means of checking that the patient understands the procedure and that the system is working properly. If the diluent step is performed, the FEV₁ following diluent becomes the “control,” and the target FEV₁ for a positive test is 80% of this value. If the diluent step is omitted, the target FEV₁ is 80% of the baseline spirometry value. The breaths should be slow and deep, and the patient should wear a noseclip. The patient should inspire from FRC to TLC. The dosimeter should be triggered as inspiration begins; this may be done manually or automatically. The nebulizer should be activated for 0.5–0.8 seconds, depending on the output of the nebulizer to deliver 90 μl of drug per actuation. Inspiration should last about 5 seconds, with a 5-second breath hold at TLC to maximize aerosol deposition. Inhalations are repeated for five breaths, lasting 2 minutes or less.

Spirometry is then repeated at approximately 30 and 90 seconds after the last inhalation. A timer or stopwatch is useful for staging the maneuvers. The FVC maneuver should be acceptable and may be repeated, if necessary. The number of attempts should be limited to three or four efforts so that two acceptable maneuvers are obtained within 5 minutes. Full flow-volume loop maneuvers are useful for detecting changes in inspiratory flow that may occur (e.g., vocal cord dysfunction). If Raw and sGaw are also measured, the patient should be seated in the plethysmograph and the door closed as soon as spirometry has been completed. With practice and careful timing, spirometry and resistance measurements can be completed within about 5 minutes after each dose of methacholine.

The largest FEV₁ after each dose should be reported. If airway resistance/conductance measurements are made, the average of two acceptable panting maneuvers should be reported. If FEV₁ decreases less than 20%, or specific conductance (sGaw) decreases less than 35%–40%, the next highest dose is administered. If FEV₁ decreases more than 20%, the challenge is complete. Signs and symptoms (e.g., coughing, wheezing, chest tightness) related to asthma should be recorded. A β agonist bronchodilator should be administered and spirometry repeated after a 10-minute delay.

Two-Minute Tidal Breathing Method

In this method, normal relaxed breathing is used as the patient inhales the aerosol. Methacholine is usually prepared in 10 doses of doubling concentrations (see Box 9-1 and Table 9-3). If the methacholine has been refrigerated, it should be allowed to come to room temperature for 30 minutes. A nebulizer capable of delivering 0.13 mL/min (±10%) driven by compressed air should be used. An accurate flowmeter allows adjustment to the flow necessary to deliver the desired volume.

The patient should hold the nebulizer upright and breathe quietly through the mouthpiece with a noseclip in place. A facemask may be used in place of a mouthpiece, but the noseclip should not be omitted (noseclip may be placed over the mask). A filter may be placed on the expiratory limb of the nebulizer circuit to limit the amount of methacholine released in aerosol form in the testing area. A timer or stopwatch should be used to ensure that the breathing interval is exactly 2 minutes long.

As in the dosimeter method, spirometry is repeated at 30 and 90 seconds after the end of the 2-minute tidal breathing interval. If a diluent step is included, the target FEV₁ (for a positive response) is 80% of the largest value obtained after the diluent. If the diluent step is omitted, the target FEV₁ is 80% of the baseline value. Patients with highly reactive airways may have a positive response (e.g., a 20% decrease in FEV₁) to the diluent. The FVC maneuvers should be completed within about 3 minutes. If Raw or sGaw is to be measured, those measurements should be performed as quickly as possible after spirometry. If FEV₁ decreases less than 20%, the next highest dose should be administered. If FEV₁ decreases by 20% or more, the challenge is complete. A β agonist bronchodilator should be administered to reverse the bronchospasm and spirometry repeated after 10 minutes.

Spirometry or plethysmographic measurements are the most commonly used endpoints for bronchial challenge tests. For each parameter, the percent of decrease is calculated as follows:

< ?xml:namespace prefix = "mml" />V·maxFRCFRC

% Decrease = x−yx × 100

where:

x = control FEV₁ (baseline or after diluent)

y = current FEV₁ after methacholine inhalation

This change is sometimes reported as a negative value (e.g., −20%) to indicate a fall in the FEV₁.

A 20% or greater decrease in the FEV₁ is considered a positive response. The decrease should be sustained. Additional spirometry efforts may be necessary to distinguish an actual decrease from variability in the maneuvers. The same equation may be used to calculate changes in airway resistance or specific conductance. A decrease of 35%–45% in sGaw is consistent with increased bronchial responsiveness. In patients suspected of having vocal cord dysfunction (VCD), complete flow-volume (F-V) loops may be helpful. VCD is sometimes mistaken for asthma in patients referred for bronchial challenge testing. Limitation of inspiratory flow (with little or no change in FEV₁) is usually observed in VCD.

Several methods of quantifying the results of the challenge are commonly used. The concentration of methacholine that results in a 20% decrease (PC₂₀) can be calculated from the last and second-to-last doses administered:

V·maxFRCFRC

PC20=antilog [logC1+(logC2−logC1)(20−R1)R2−R1]

where:

C₁ = second-to-last methacholine concentration

C₂ = final methacholine concentration (causing 20% or greater decrease)

R₁ = percent decrease in FEV₁ after C₁

R₂ = percent decrease in FEV₁ after C₂

PC₂₀ calculated this way provides a single index of bronchial responsiveness. PC₂₀ may also be identified directly from a graph in which change in FEV₁ is plotted against the log concentration of methacholine (Figure 9-3).

Figure 9-3 Methacholine challenge test.
Results of data gathered during a bronchial challenge test are shown. The dosage of the challenge agent (methacholine, in this case) is plotted on a logarithmic scale on the x-axis. FEV₁ (or other variable) is plotted on the y-axis. The first point represents the baseline FEV₁ (4.0 L, in this example). The control value (FEV₁ after inhalation of the diluent) is plotted next. FEV₁ after each dose of methacholine is plotted until a 20% or greater decrease occurs. In this example, FEV₁ decreased by more than 20% with a dose of 1 mg/mL. A vertical line drawn from the point at which the dose response curve crosses the 20% line defines the PC₂₀. The patient is then given an inhaled bronchodilator to reverse the effect of the provocative agent, and the response is plotted.

Provocative concentrations for other variables, such as sGaw, can be calculated similarly by substituting the appropriate percentage for 20 in the preceding equation and substituting the percent decrease for that variable for R₁ and R₂. Note that this calculation requires that at least two concentrations of methacholine have been given. If FEV₁ decreases 20% after the diluent or the first dose of methacholine, PC₂₀ should be reported as less than the lowest concentration administered. If FEV₁ does not decrease by at least 20% after the highest dose, PC₂₀ should be reported as “greater than 16 mg/mL.”

See Interpretive Strategies 9-1. Airway responsiveness to methacholine can be described using the PC₂₀. Most patients referred for bronchial challenge testing have a history or symptoms suggestive of asthma but not a definite diagnosis. For these patients, if FEV₁ decreases less than 20% at the highest dose (PC₂₀>16 mg/mL), bronchial responsiveness is probably normal and asthma is unlikely. For patients whose FEV₁ decreases 20% or more at low doses of methacholine (PC₂₀<1.0 mg/mL), the diagnosis of asthma is highly likely. For patients with PC₂₀ values from 1–16 mg/mL, the diagnosis of asthma must be considered, based on the pre-test probability of asthma, the history of symptoms, and other possible causes for bronchial hyperreactivity. In practice, patients who have a PC₂₀ greater than 8–16 mg/mL often do not have asthma. Patients who have a negative methacholine challenge (PC₂₀>16 mg/mL) may have asthma that has been suppressed by anti-inflammatory medications or occupational asthma that is triggered by a specific agent. Conversely, some individuals who have PC₂₀ values less than 8 mg/mL may not have asthma. Patients with allergic rhinitis and smokers with COPD often have bronchial hyperreactivity but not asthma.