Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults

2.4.1 Histamine and methacholine

Challenge tests with histamine and methacholine, performed in a carefully standardized manner, are safe. Therefore, the precautions during these tests are those that are recommended in general in any clinical lung function laboratory. This includes oxygen and bronchodilators [39]. Personnel should be trained in the management of acute severe asthma [17, 39].

If the test is performed in the context of epidemiological surveys, it can be carried out even if a physician is not present provided that:

1 a standardized protocol is used and the starting dose should be low;

2 baseline FEV₁ is ≥ predicted value minus 3·SD of the predicted value [33] (§ 2.2) and ≥2 l;

3 no significant bronchoconstriction occurs after the nebulisation of diluent;

4 a trained and experienced technician performs the test;

5 oxygen and a β₂-adrenergical agent can be readily used;

6 the responsible physician or a community physician informed of the protocol and qualified to manage bronchoconstriction can rapidly (within 10 min) see the subject in case of emergency.

If the test is performed in the hospital, a doctor experienced in challenge tests should be present in the hospital and readily available if needed, as criteria no. 2 and 3 listed above might not be fulfilled.

Under all circumstances, the patient must not be left unattended at any time. The patient should be instructed to discontinue inhalation if symptoms become troublesome. At the end of the test the patient should only leave the testing area after his/her bronchoconstriction has adequately improved, either spontaneously or by inhaled bronchodilator (towards FEV₁ values >90% of baseline) [40]. The patient should also receive proper instructions in case of relapse of bronchoconstriction during the first 24 h after the test.

2.4.2 Allergens or occupational sensitizers

If challenges with allergens or occupational sensitizers are carried out, in addition to the above (§ 2.4.1), cardiopulmonary resuscitation equipment must be available in the room, together with ready to use oxygen, inhaled and intravenous bronchodilators, intravenous anti-histamines, intravenous steroids and adrenaline (see § 3.5 and § 3.6) [17, 39]. The inhaled doses must be carefully standardized according to the present recommendations (§ 3.5.2–3.5.4, § 3.6.2–3.6.4). The tests can only be performed by a specifically trained technician, and a doctor, experienced in this type of challenge and in acute severe asthma, must be present in the room during the challenge. After the challenge, the doctor should be at close call in the laboratory. The patient is monitored in the laboratory for at least 7 h, and lung function (e.g. PEF) should be measured repetitively during the first 24 h. Severe airways obstruction should be treated adequately [17, 39].

2.4.3 Other inhalation challenges

The very stringent precautions identified in § 2.4.2 are also required for many other inhalation challenges, particularly those tests that have not been fully standardized. This includes tests with any other pharmacological or sensitizing agents. With regard to the physical challenges, there is general consensus that standardized exercise tests are safe (see § 3.4). However, the experience with other physical tests is still relatively limited. Therefore, it is recommended that the safety equipment should be similar as that for allergen challenge, and that a doctor, experienced in this type of challenge, is readily available. If a late response can be expected, lung function should be monitored as after allergen challenge (§ 2.4.2). Alternatively, if bronchoconstriction is adequately improved (see § 2.4.1) the observation period in the laboratory following these challenges may be shorter than for allergens, according to the recommendations for histamine and methacholine.

3.1.1 Background

Pharmacological challenges with aerosolized solutions of carbachol [55] or histamine [56, 57] were introduced on both sides of the Atlantic about half a century ago. The procedures were further developed by De Vries et al. [58] and Orehek [59], and subsequent worldwide application of histamine and methacholine inhalation challenge tests was based on the work of Hargreave et al. [60]. Currently, these pharmacological challenges are the first choice for airway responsiveness measurements in clinical practice as well as in research (§ 2).

Histamine is one of the major inflammatory mediators involved in asthma, producing airways obstruction by smooth muscle contraction, and to some extent by increased microvascular permeability and/or stimulation of (non)cholinergic activity [61]. Carbachol and methacholine are synthetic muscarinic agonists that are more stable than acetylcholine itself and not degradable by cholinesterase [59]. Even though carbachol challenges have been used in asthma [62], most current experience exists for methacholine [60]. The solubility of methacholine allows administration of higher doses than with histamine, without side effects [50]. This may be particularly useful in epidemiological studies. Remarkably, histamine and methacholine provide concordant results (comparable PC or PD values) although they are not fully interchangeable (§ 3.1.8).

3.1.2 Solutions

Histamine acid phosphate (histamine di-phosphate)

Standardized solutions of histamine are usually made from histamine di-phosphate powder (HDP, molecular weight: 307) and phosphate-buffered saline (PBS). Phosphatebuffered saline is used as the diluent because, at higher histamine concentrations, unbuffered solutions become sufficiently acid to alter the response of the airways [63]. PBS and histamine di-phosphate solutions need to be prepared in a carefully standardized manner, particularly paying attention to the molecular water content of the various salts (e.g. HDP.1H₂O). Recently, detailed recommendations on this have been supplied (table 1) [64].

To make a solution of 32 mg·mL⁻¹ histamine: weigh 32.00 g HDP (or 33.88 g HDP.1H₂O) and add 1000 mL of sterile PBS. Filter through a 0.22 μm filter, put into a sterile vial and autoclave. Histamine di-phosphate does not dissolve easily in phosphate-buffered saline and the higher concentrations may precipitate out when stored at 4°C. These solutions should be shaken well before use.

3.1.3 Aerosol generation

Airway responsiveness is defined as the response of the airways to a provoking agent. It is essential for a reliable assessment of responsiveness that both the dose of the provoking agent and the response are measured accurately. Unless both are carefully standardized, results are unreliable and cannot be related to previously established reference values [60]. It is of overriding importance that the aerosol generation for one type of challenge is consistent between and within subjects, so that the same dose is delivered in an identical way on different occasions.

The current standardization of the dose refers to the amount of provocative agent administered to the mouth. The factors that determine the deposition of aerosols in the airways are [73]: the number and size of the droplets delivered to the mouth, air temperature and relative humidity, airways geometry and breathing pattern. Due to oropharyngeal deposition the actual dose that enters the lungs can only be estimated.

The doses of aerosol for the three most commonly used histamine/methacholine test procedures, described below, are generated by jet nebulizers. In this section we examine variables that need to be standardized to ensure that the dose is accurate [74]. These methods are theoretically suitable for adults as well as for children. However, one is reminded that with the tidal breathing and dosimeter method children are inhaling the same dose as adults [75]. There is evidence that for similar doses, the response to histamine and methacholine may be greater in children that in adults [75]. Not only should this be taken into consideration when selecting starting doses for children, but also when interpreting the results. To date it is unclear as to whether the dose in children should be size-corrected [75].

Apparatus between nebulizer and mouth

Aerosols evaporate, impact or deposit in apparatus placed between the nebulizer and mouth. In general, the distance between the nebulizer and the mouth should be kept to a minimum. Modifications of the face mask or the mouthpiece, tubing, and valve box may seriously affect the dose delivered at the mouth (fig. 2) [80].

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Figure 2–

Experimental set-up of a jet nebulizer connected to the central chamber of an in- and expiratory three-way valve box with an expiratory aerosol filter. The subject is connected to the mouthpiece. See tidal breathing method (§ 3.1.4). Modified from Juniper et al. [84], with permission.

3.1.4 Tidal breathing method [82–84]

Aerosols of histamine/methacholine are generated by a validated jet nebulizer with MMAD between 1 and 4 μm, and calibrated to give an output as estimated by weight loss of 0.13 mL·min⁻¹. Aerosols are delivered either through a face mask, held loosely against the face, or through a Hans Rudolph valve box with a mouthpiece [85]. The latter system allows absorption of the expirate in a low resistance aerosol filter attached to the expiratory port of the valve box (fig. 2). Each aerosol is inhaled by quiet tidal breathing at spontaneous frequency through the mouth for 2 min, using a nose clip. The first aerosol inhaled is the diluent and this is followed at 5 min intervals by doubling concentrations of histamine or methacholine from 0.03–32 mg·mL⁻¹ (or higher concentrations if necessary in research). The FEV₁ is measured before the test and at 30 and 90 s after each inhalation, the lowest technically satisfactory [33] recording will be used in the analysis. The lowest value reflects maximal bronchoconstriction at a certain dose. It has been chosen in order to take into account slight variations in the time course of bronchoconstriction induced by the agents [86], and because deep inspirations may remove bronchoconstriction [45]. The test is stopped when the FEV₁ has fallen by 20% or more from baseline. The results are expressed as the concentration of methacholine/histamine causing a 20% fall in FEV₁ (PC₂₀) (§ 3.1.7 and 3.1.8). The reproducibility of the test is described in § 4.3.

To shorten the test procedure, low concentrations may be omitted in some patients. However, it should be emphasized that this can only be recommended when the administrator of the test has extensive experience. The starting concentration is calculated from the baseline FEV₁, the response to diluent, and current medication usage [84, 87] (§ 2.3) in the following way:

1 FEV₁/VC>80% and FEV₁>70% predicted and FEV₁ falls <10% after the diluent inhalation and the patient's symptoms are well controlled, use starting concentrations between 0.125 and 2.0 mg·mL⁻¹, depending on the medication being taken:

Medication Starting concentration

Inhaled or ingested corticosteroids 0.125 mg·mL⁻¹

Daily bronchodilators 0.25 mg·mL⁻¹

Occasional bronchodilators (< once/day) 1.0 mg·ms⁻¹

No medication 2.0 mg·mL⁻¹

2 FEV₁/VC<80% or FEV₁<70% predicted and FEV₁ falls <10% after the diluent inhalation and the patient's symptoms are well-controlled, use starting concentrations between 0.03 and 0.125 mg·mL⁻¹, depending on the used medication:

Medication Starting concentration

Inhaled or ingested corticosteroids 0.03 mg·mL⁻¹

Other or no medication 0.125 mg·mL⁻¹

3 If a patient's FEV₁ falls by 10% or more after the diluent inhalation, or if asthma symptoms do not appear to be well controlled, do not omit any concentrations - start all patients at 0.03 mg·mL⁻¹.

If, after the first concentration of histamine or methacholine, there has been no significant fall in the FEV₁ (less than 5% from best baseline) and there is no clinical evidence of any bronchoconstriction (chest tightness, cough or wheezing), the next dose may be omitted. Again, this can only be done if the technician is highly experienced. For example: if, after 0.03 mg·mL⁻¹, there are no symptoms and the fall in FEV₁ is less than 5%, the next concentration may be 0.125 mg·mL⁻¹; if this produces no significant change in FEV₁ and there are still no symptoms, then 0.5 mg·mL⁻¹ may be given. As soon as there is any evidence of symptoms or the FEV₁ is falling: do not omit any further concentrations. Even after a fall of 5% in the FEV₁, the next concentration sometimes gives quite a precipitous fall. If concentrations are omitted, it is important to stress before every 2-min inhalation that the subjects should remove the face mask/mouthpiece as soon as they experience any breathing or chest discomfort.

The test may also be shortened by reducing the nebulization time from 2 min to 0.5 min [58]. This worsens the reproducibility of the test results [88], even though a recent study did report similar reproducibility for the PC₂₀ obtained by 0.5 min and 2 min inhalation [89]. Furthermore, the ratio of the PC₂₀ between the two methods has been reported to be 3.1, in stead of the expected value of 4 [89]. Therefore, at present the 2 min tidal breathing method is recommended.

3.1.5 Dosimeter method [90–93]

Aerosols of methacholine or histamine are generated by a jet nebulizer. A dosimeter is an electrical valve system, which enables one to administer aerosol during inspiration only. A flow sensor in the expiratory port triggers a solenoid which exposes the nebulizer to compressed air at 138 kPa (20 p.s.i.) for about 0.6 s, to give a calibrated output per puff of 9.0 μl [92]. The bolus of aerosol is inhaled through the mouth from the outlet of the nebulizer during an inspiratory capacity breath over 5 s, without breath holding at total lung capacity (TLC) [92]. This is done 5 times per concentration (total output 45 μl) without delay. The first aerosol is diluent followed by doubling concentrations of histamine or methacholine from 0.03 to 32 mg·mL⁻¹ (or larger if necessary).

The doses are given at 5 min intervals. FEV₁ is usually measured 30 and 90 s following each inhalation as for the tidal breathing method. The test is stopped when FEV₁ has fallen by 20% or more. The tests may be shortened according to the recommendations in § 3.1.4. Results should not be calculated from cumulative breath units (1 unit = 1 inhalation of aerosol from a 1.0 mg·mL⁻¹ solution) [90], because the non-standardized nebulizer outputs in the literature (ranging between 1.0 μl [94], 2.0 μl [95], 7.1 μl [96], 8.9 μl [91] and 9.0 μl [92] per puff) make between-centre comparisons in terms of breath units very unreliable. It is recommended to express the results in cumulative nebulized mol histamine or methacholine to cause a 20% fall in FEV₁ (PD20) (§ 3.1.7 and 3.1.8). Alternatively, the results can be presented as PC20, which has been reported to be similar between tidal breathing and dosimeter method when the present standardization is accomplished [92]. The reproducibility of the method is described in § 4.3.

3.1.6 Yan method [94]

This is a hand-operated method, delivering aerosols during inspiration only. Aerosols are generated by five calibrated DeVilbiss 40 glass, handheld, nebulizers (stoppers removed) (2.2–3.8 μl per squeeze). Saline and histamine or methacholine solutions of 3.15, 6.25, 25 and 50 mg·mL⁻¹ are placed in each nebulizer. After assessment of baseline FEV₁, the saline nebulizer is placed between the teeth and the patient exhales to FRC. At the beginning of an inspiratory capacity manoeuvre to TLC lasting 1–2 s, the operator gives the nebulizer bulb one firm squeeze. The breath is held at TLC for 3 s, whereafter the subject exhales outside the nebulizer. This is repeated twice for saline but the number of breaths for each concentration of histamine varies according to the dose being administered, which ranges from 0.03 to 7.8 μmol histamine di-phosphate or from 0.05 to 12.3 μmol methacholine chloride (cumulative), or greater if needed [94]. FEV₁ is measured 60 s after each dose and the test is stopped when the FEV₁ has fallen by 20% or more from post-saline. Results are expressed as the cumulative dose (in μmol) causing a 20% fall in FEV₁ (PD20). The reproducibility of PD20 is described in § 4.3.

3.1.7 Calculation of the response

The airway narrowing response can be calculated as % fall in FEV₁ in two ways: taking either the post-diluent or baseline value as a reference.

3.1.8 Expression of the response

Calculation of PC20 or PD20

PC₂₀ is used for the tidal breathing method, whereas PD₂₀ is the preferred index for the dosimeter and Yan method. Other outcome variables are discussed in § 4.1.

figure 3 shows the calculation of the PC₂₀ in mg·mL⁻¹ for the tidal breathing method. The method is exactly the same (including the log transformation) for the dosimeter and the 'Yan' method (PD₂₀ μmol). Plot the % fall in FEV₁ against the concentration or dose of methacholine/histamine on a log scale. The PC₂₀ or PD₂₀ is obtained by linear interpolation between the last two points, according to the formula [84]: where:

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Figure 3–

Example of the calculation of the PC₂₀ (provocative concentration at 20% fall in FEV₁ from baseline) from a concentration-response curve to inhaled histamine or methacholine in one subject. The PC₂₀ is obtained by linear interpolation between the adjacent data-points. Modified with permission from reference [84].

C₁ = second last concentration (<20% FEV₁ fall),

C₂ = last concentration (>20% FEV₁ fall),

R₁ = % fall FEV₁ after C₁,

R₂ = % fall FEV₁ after C₂.

Extrapolation of dose-response curves should be avoided, although it may be appropriate over a dose-range not exceeding one doubling dose [97].

At the recommended nebulizer output a normal PC₂₀ for histamine and methacholine in adults is ≥8 mg·mL⁻¹ (with a «grey zone» between 4 – 16 mg·mL⁻¹) [60] and a normal PD₂₀ is ≥7.8 μmol [94, 98]. These are arbitrary cut-off points (§ 2.1). It means that at these levels of airway responsiveness variable airways obstruction is unlikely at this point. The agreement between the tidal breathing, dosimeter, and Yan method is good [92, 94, 99], although numerical results do not always correspond due to differences in the reported units. In asthma the PC₂₀ is similar for histamine and methacholine [83, 87], whereas the PD₂₀ has been reported to be slightly lower for histamine as compared to methacholine in asthmatic children [75, 98] (§ 3.1.3) as well as in adults with COPD [100].

If two challenge tests are carried out within hours of each other, the bronchoconstrictive response to the second test may be less than to the first test, particularly when high doses are being applied [101, 102]. To minimize this tachyphylactic effect, histamine tests should be done at least 6 h apart. Methacholine tests should preferably be separated by >24 h, even though the methacholine tachyphylaxis, as observed in normals [102], could not be confirmed in asthmatics [103].

3.2.1 Background

Already in 1968 De Vries et al. [58] and later, in 1980, Allegra and Bianco [104] reported that the inhalation of an aerosol of distilled water could induce an increase in specific airways resistance (sRaw) in patients with asthma. Thereafter, Schoeffel et al. [105] reported that hypertonic as well as hypotonic aerosols of saline could cause a reduction in FEV₁. Over the next 10 years a number of investigators used these aerosols for the assessment of bronchial hyperresponsiveness, and the challenge procedure is now widely used [106–109].

It is now generally accepted that non-isotonic aerosols induce airway narrowing indirectly by causing the release of endogenous mediators which cause bronchial smooth muscle contraction and airway oedema. The release of mediators is thought to be directly caused by the change in osmolarity [110]. Histamine is likely to be an important mediator released because specific antihistamines are very effective in inhibiting the airway response to both hyper- and hypotonic aerosols [109, 111, 112]. However, it is likely that other mediators such as the leukotrienes and prostaglandins are also involved. Further, there may be a neural component involving release of sensory neuropeptides or a parasympathetic reflex [113].

Sodium cromoglycate [114], nedocromil sodium [115] and furosemide [116, 117], drugs which are thought to affect mast cell release of mediators and/or the noncholinergic neural activity, are very effective at inhibiting airway responses to non-isotonic aerosols. The anticholinergic agents have been shown to be inconsistent in their effect and changes in baseline FEV₁ due to their action as bronchodilators make the findings difficult to interpret [114, 118, 119]. The sensitivity to hypertonic saline is reduced with daily administration of corticosteroids given as aerosols [120]. This usually occurs after 400–2000 μg has been taken daily for 2–8 weeks.

The responsiveness to 4.5% saline has been shown in asthmatics to correlate with numbers of mast cells obtained by bronchial biopsy [121]. The airways response to distilled water in patients with transplanted lungs has also been shown to correlate with airway inflammation [122].

There now seems to be sufficient evidence to suggest that the severity of the airway response to these aerosols reflects the involvement of inflammatory cells and their mediators in the airways [106]. Thus, in addition to demonstrating the capacity of the airways to narrow in response to the endogenous release of inflammatory mediators, challenges with these aerosols, when followed over time, may be useful in evaluating both the acute and the chronic effect of medications used in the treatment of airway inflammation.

Tests with non-isotonic aerosols are generally considered to be safe. However, there is one report of acute, fatal bronchoconstriction in response to a standardized distilled water challenge (Fabbri, personal communication). Therefore, extensive precautions must be taken (§ 2.4).

3.2.2 Solutions

The most commonly used solutions are distilled water and 4.5% saline. Some laboratories use 3.6% saline, and a concentration of 2.7% saline is usually sufficient to induce a reduction in FEV₁ in very sensitive patients. Dose-response curves are obtained by increasing the time of exposure to the single saline concentration. Alternatively, a dose-response curve can be obtained by doubling the concentration (0.9, 1.8, 3.6, 7.2, 14.4%) [112]. This is less practical for routine purposes and should be regarded as a research procedure.

A concentration of 4.5% saline is recommended in preference to the other solutions for several reasons. The test is shorter when this concentration of saline is used compared with lower concentrations, and 80% of clinically recognised asthmatics have a 20% fall in FEV₁ after 15 mL has been nebulized or less. A person who responds to 4.5% saline usually also has exercise-induced asthma. The osmolarity is slightly above sea water, and the test is also used for screening SCUBA divers.

3.2.3 Aerosol generation

Ultrasonic nebulizers are recommended for generation of non-isotonic aerosols because they produce aerosols which are more dense than conventional jet nebulizers. The mass-median aerodynamic droplet size is usually between 2 and 10 μm and this is reduced to less than 5 μm when the breathing circuit is attached. It is recommended that a large two-way valve (Hans Rudolph 2700) be used and that the tubing attaching this to the nebulizer should have a smooth internal bore and be of constant length and diameter.

It is important to choose a nebulizer that has a reproducible output of at least 1.2 mL·min⁻¹ with the breathing circuit attached. Further, it is necessary to have a nebulizer with a bowl or canister which can be easily detached for weighing. A volume capacity of 100–200 mL for the bowl is recommended. The temperature of the solution increases with time, but this is minimised with these relatively large volumes, and the small increases in temperature appear to have little or no effect on the response. Preferably use the same volume fill for each subject and the same setting for the nebulizer output. The output of the nebulizer and the total dose of aerosol delivered to each subject can be measured by weighing the bowl and tubing, but not the valve, before and after challenge. The valve is not weighed because of the production of saliva during the test.

For any one individual, the output of the nebulizer during the challenge is linearly related to time. Thus, to obtain the dose delivered for each exposure, it is only necessary to know the output. The output is expressed in mL per min. This can be obtained simply by dividing the total dose delivered during the challenge, measured by a change in weight, over the total time of exposure to the aerosol. The dose of aerosol, in mL, delivered for each interval can be calculated from the time of exposure. For clinical purposes 1 g of 4.5% NaCl is considered as 1 mL and a correction (multiply by 1.03) is not made for specific gravity.

3.2.4 Protocol

At present it is recommended that the dose of aerosol be increased by increasing the length of each challenge interval. Although the nebulizer output or saline concentration could be increased to achieve a higher dose, this may cause cough and be distressing to the patient (see § 3.2.2). In order to prevent any problems with electrical charge in some ultrasonic nebulizers, 0.03% saline can be used instead of distilled water in case of hypotonic challenge. For hypertonic challenge 4.5% saline is recommended.

Measurements of FEV₁ or specific airways resistance (sRaw) [2, 48] are made in duplicate or triplicate before and between 60 and 90 s after each exposure to the aerosol. The exposure times are doubled as follows: 30 s, 1 min, 2, 4 and 8 min. If the fall in FEV₁ is greater than 10% of baseline the previous exposure time is repeated rather than increased. The challenge is stopped after 15 mL is nebulized or when there is a 20% reduction in FEV₁ or a doubling (100% increase) in sRaw.

Although a 20% fall in FEV₁ is generally accepted as abnormal, on the basis of the findings in healthy non-asthmatic subjects, a value of 15% or more should probably be regarded as abnormal [123–126]. A broncho-dilator is always administered by aerosol at the end of each challenge.

3.2.5 Expression of the response

The dose-response curve is constructed by plotting the change in FEV₁, expressed as a percentage of the baseline value or the predicted value, against the cumulative dose of aerosol delivered, expressed in mL (fig. 4). The dose is calibrated according to § 3.2.3. About 15% of the measured weight loss will be deposited in the intrapul-monary airways. A value for PD₁₅, and PD₂₀ can be obtained by linear interpolation. Within the asthmatic population the values for PD₂₀ are log normally distributed so the PD₂₀ values are log transformed before a statistical analysis is carried out.

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Figure 4–

Cumulative dose of water or 4.5% saline used to provoke a fall in FEV₁, expressed as a % of the pre-challenge level or as a % of predicted FEV₁. The cumulative dose is measured by weighing the nebulizer and tubing, but not the valve, before and after challenge. The cumulative time is recorded. The output of the nebulizer is calculated in mL per min. The dose for each exposure can be calculated from the time.

To assess changes in sensitivity after an intervention the values for PD₂₀ are compared using log transformation. For an individual the fold difference in PD₂₀ after an intervention can also be calculated after log transformation [120]. Because baseline lung function can be altered by treatment, or change spontaneously over time, a reactivity index (slope of the dose-response curve) can be useful. This is defined as the change in FEV₁, expressed as a percentage of the predicted value, per unit dose of aerosol [120]. To compare the responses after an intervention the index is compared over the same values of percent predicted FEV₁. This index can also be expressed as a fold difference. For subjects who do not reach a 15 or 20% fall in FEV₁, the maximum % fall in FEV₁ and the maximum dose of aerosol delivered should be reported. A person is considered to have a severe response if the PD₂₀ is <2 mL, a moderate response if it is 2.1–6.0 mL, and a mild response if the PD₂₀ is 6–20 mL [123].

3.3.1 Background

Airway responsiveness can be measured by the inhalation of stimuli which cause bronchoconstriction through the release of mediators from cells within the airways, as well as by the inhalation of these bronchoconstrictor mediators. One such method is by isocapnic hyperventilation of cold and/or dry air. Although the original description of hyperventilation induced bronchoconstriction was made in 1946 [127], renewed interest in this method for inducing bronchoconstriction occurred because of the recognition that cooling and/or drying of the airways is the mechanism of exercise-induced bronchoconstriction. Airway narrowing can result from cooling and subsequently rewarming of the airway mucosa, as well as from local hyperosmolarity due to drying (reviewed in § 3.4). The degree of airway hyperresponsiveness to isocapnic hyperventilation of cold dry air is moderately correlated with the degree of airway hyperresponsiveness to inhaled methacholine [128] and histamine [129] in asthmatic subjects.

Isocapnic hyperventilation of cold/dry air uses a naturally occurring stimulus to provoke bronchoconstriction rather than a chemical stimulus. Whether this implies a better safety profile still needs to be confirmed. Isocapnic hyperventilation can be administered in a dose-response fashion, and, for this reason may in some instances have advantages over exercise for measuring airway responsiveness. When giving doubling doses, the shape of the dose-response curve obtained in asthmatics is similar to other bronchoconstrictor stimuli: a lower threshold, increased slope and absence of a plateau response, when compared to non-asthmatics [130].

3.3.2 Production and conditioning of air

The most convenient source of air is to use dry compressed air, which is cooled by being passed over a cooling coil, through which methanol cooled to −35 °C is passed. This results in inspired air cooled to −12 to −15 °C. In many laboratories, however, just dry air at room temperature is used, because this is adequate to provoke bronchoconstriction in most asthmatics, and avoids the complexities of having to cool the air. This technique leads to similar results as those with cold dry air, even though in animals studies differences between these methods have been observed [131]. Subjects inhale the cold and/or dry air through a valve. A Hans Rudolph valve can be used for this purpose, which can be divided into an inspiratory and expiratory port, if inspired and expired air temperature is being measured. If this method is used, end-tidal CO₂ needs to be continuously measured in the expired air, using a capnograph, and CO₂ added to the inspired air to keep the subject eucapnic.

3.3.3 Protocol

After measurement of baseline spirometry, the subject breathes at increasing minute volumes, beginning at 7.5 l·min⁻¹, increasing to 15, 30, 60 l·min⁻¹ and maximum minute ventilation, each period of ventilation lasting 3 min. The inspired volumes can be achieved by targeting, using a device which gives a visual display of the inspired volume. The rate of breathing is dictated either by the technician or by a metronome. The tidal volumes and rates which can be used to achieve the desired minute ventilation are: 0.75 l at 10 breaths/min; 1.5 l at 10 breaths/min; 2.0 l at 15 breaths/min. After each step the patient breathes room air, and the FEV₁ is measured at 30 s, 90 s, then at 3 min and 5 min and every 2 min until the lowest technically satisfactory value [33] is reached. The challenge is stopped once a 20% fall in FEV₁ occurs. A major limitation with the method, however, is that the maximal challenge that can be given is limited to the maximal voluntary ventilation that can be achieved by the subject being studied. On the other hand, achieving maximal ventilation may be an advantage of this technique over exercise. Although this method produces a cumulative bronchoconstrictor effect, the maximal bronchoconstriction obtained is not significantly greater than if maximal minute ventilation alone is used as the stimulus [132]. A simplified method, in which CO2 is added in a fixed amount to the inspired air (FI.CO2 = 0.049), prevents hypocapnia over a wide range of minute ventilations from 40 to 105 l·min⁻¹ [133]. This eliminates the need to measure end-tidal CO₂. However, this FI.CO₂ may cause hypercapnia at lower minute ventilations used in the challenge procedures (7.5, 15, 30, l·min⁻¹). Therefore, with this technique the ventilation loads of 30%, 60% and 100% predicted maximal voluntary ventilation are used to overcome the possibility of hypercapnia. If patients with severely increased airway hyperresponsiveness are being tested, who may develop significant bronchoconstriction at these lower minute ventilations, it is necessary to monitor end-tidal CO₂.

An even more simplified technique has been used in paediatrics: a single-step 4 min isocapnic voluntary hyperventilation at 75% of the maximal voluntary ventilation (MVV, as determined from pre-challenge FEV₁) of cold dry air (−10 °C) [134]. Since in adults there are no comparative studies of this test with the multiple-step protocol, at this stage, the dose-response approach is recommended.

3.3.4 Expression of the response

The response is measured as the change in FEV₁ from the baseline values. A dose-response curve is obtained by plotting the incremental increase in minute ventilation (V′E) against the change in FEV₁, (usually 10% or 20% fall), and the response expressed as the PV′E,10 or PV′E,20 (the provocative minute ventilation causing a 10% or 20% fall in FEV₁, respectively). If cold dry air is being used as the stimulus, some investigators have calculated the respiratory heat exchange (RHE) [135]. This requires the measurement of the inspired and expired air temperature, using rapidly responding thermocouples, and water content. If dry air is used, then the water content is 0 mg·l⁻¹. The expired air is assumed to be fully saturated, and therefore the water content can be obtained from standard graphs of air temperature and humidity. A dose-response curve is obtained by plotting the incremental increase in RHE (kcal·min⁻¹) against the change in FEV₁, and the results expressed as the RHE giving the predetermined fall in FEV₁. However, in most instances, when just dry air is used as the stimulus, reporting minute ventilation is sufficient and the respiratory heat exchange does not need to be calculated.

A fall in FEV₁ of at least 10% is generally required to indicate a positive result to the challenge. The magnitude of this response is greater than 2 standard deviations above the response seen in normal subjects [135, 136].

3.4.1 Background

It is now 30 years since Jones [137] described the effects of exercise on the airways resistance in children with asthma. This exercise-induced bronchoconstriction is usually referred to as exercise-induced asthma (EIA), even though it is generally accepted that exercise is not an inducer, but an inciter of asthma. By the early 1970's, Godfrey and his colleagues had identified many of the factors which determined the severity of the airway response [138]. These included the type of exercise, its intensity and duration, and the interval since exercise had last provoked an attack of asthma. By the late 70's it was recognised that the most important determinant was the level of ventilation which was reached and sustained during exercise. It was noted that exercise in itself was not necessary to provoke an attack of asthma, and hyperventilation alone could achieve the same response [139]. At the same time the effects of temperature and water content of the inspired air were being investigated. It was discovered that exercise-induced asthma could be completely inhibited when air at body temperature and water vapour was inhaled during exercise [140]. Thus it was concluded that the stimulus for EIA had to be the loss of either heat or water or both from the airways during exercise.

Initially it was considered that cooling of the airways was the stimulus [141] but later studies demonstrated that water loss was more important than heat loss. From these studies it was proposed that transient hyperosmolarity of the airway surface liquid as a result of evaporative water loss was the stimulus to EIA [142]. It was also demonstrated in vitro that an increase in osmolarity provided an ideal environment for the release of mast cell mediators [143]. These mediators have the potential to cause the airways to narrow by contraction of bronchial smooth muscle. A more recent proposal has suggested that vascular engorgement and oedema of the bronchial circulation is more important than bronchial smooth muscle contraction in causing the airways to narrow [144, 145]. At present there is no direct evidence to support either of these two hypotheses, and further studies are required to clarify this important issue [146].

Exercise provokes an attack of asthma in 70 to 80% of the population with clinically recognized asthma. In clinical situations exercise tests are not very sensitive, but are highly specific for the diagnosis of asthma, and are particularly useful in children [147–149]. It may not be very practical in older patients. In field studies, exercise tests are less specific for the diagnosis of asthma [150], showing a prevalence of positive responses between 7 and 16% [150, 151]. EIA may occur at any age and is equally common in adults and children. The signs and symptoms of EIA do not differ from other forms of acute asthma except that they are of short duration. In addition to increasing airways resistance exercise can induce a transient hyperinflation and arterial hypoxaemia. The majority of persons recover spontaneously from an attack of EIA within 30 min while a minority require oxygen and bronchodilators given by nebulization. In 50% of persons EIA is followed by a refractory period of about 1 hour [152]. The occurrence of a late asthmatic response, several hours after exercise is rare and their association with exercise itself is still a controversial issue [153, 154].

EIA is prevented in most asthmatic patients by premedication with sodium cromoglycate, nedocromil sodium or a β-adrenoceptor agonist [152, 155–158]. In some cases a combination of both types of medication is required. In difficult cases the dose of these agents is doubled and ipratropium bromide added.

3.4.2 Experimental set-up

In the laboratory exercise should be carried out on either a bicycle ergometer or a motor-driven treadmill. There are definite advantages in using a bicycle in that the head is stable and it is easier to administer dry gas and collect expired air. Further, the work intensity during cycling, but not running, is independent of body weight, making it easier to predict the workload required to achieve the target ventilation.

The ventilation in litres per minute should be measured at least during the last 4 min of exercise. The time of exercise should be 6 to 8 min.

Heart rate should be monitored continuously and in the case of persons over 40 yr of age an electrocardiogram should be taken throughout exercise and for 5 min after its completion.

Changes in airways resistance are best measured indirectly by documenting changes in FEV₁ or PEF. Although PEF is easier to measure, FEV₁ is recommended as it reflects a larger proportion of the flow-volume curve.

It is strongly recommended that air inspired during exercise has a water content as low as possible and less than 10 mg per litre (relative humidity less than 50% between 20–25°C). The best way to achieve this is to have the subject inspire compressed medical air at room temperature via a demand valve and regulator. Although some laboratories generate air of subfreezing temperatures there is no need to have expensive equipment to condition the inspired air. Providing the inspired air is dry it is only necessary to increase the time of the exercise to increase the severity of the airway response.

Medication and baseline lung function alter the response to exercise and need to be taken into consideration when planning the protocol. Measurements of FEV₁ should be made on arrival in the laboratory and the stability and reproducibility of the value established. The best values should preferably not vary by more than 10%; the absolute value should be within 80% of the subject's usual value and preferably better than 75% of their predicted value. The subject should have withheld the medications according to recommendations in § 2.3.

3.4.3 Protocol

The highest of the FEV₁ measurements taken immediately before exercise is recorded and used in the calculations. The work intensity is selected for the subject to achieve between 40% to 60% of their predicted maximum voluntary ventilation (which can be taken as FEV₁ predicted·35) during the last 4 min of exercise. The oxygen consumption to achieve this ventilation can be estimated from the equation relating these measurements in children and in adults [156]. The work load to achieve this oxygen consumption is then determined from the equation. For the first minute the work load is set to 60% of the target load. This is increased to 75% in the second min, 90% in the third min and 100% in the 4th minute. When the subject has achieved the target level of ventilation or reached the ventilatory or working capacity the work load is sustained for 4 min. Minor adjustments may need to be made to this work load but this protocol is good both for general laboratory use and for research [157, 158]. A pilot exercise study may be needed, since it seems unnecessary to have responses above 30% fall in FEV₁ if repeated tests are required.

To select a work load for running on a treadmill it is necessary to know the weight of the subject. It is usual to aim for a speed and slope on the treadmill which will induce 30–45 mL of oxygen consumption per kilogram. Running at 5–9 km·h⁻¹ (3–5 mph) up a 10% incline is usually sufficient work for most subjects. Once the target ventilation is achieved it should be sustained for 4 min if possible.

The subject must have the nose clipped to ensure mouth breathing. The measurements of FEV₁ are made in duplicate after exercise at 1, 3, 5, 7, 10 and 15 min and the highest value at each time is recorded [156]. A bronchodilator is always given by aerosol upon completion of the protocol.

3.4.4 Expression of the response

The % fall index is the most widely used term to express the severity of EIA (fig. 5). It is calculated by subtracting the lowest value of the measurement of FEV₁ recorded after exercise and expressing it as a percentage of the value recorded immediately before exercise. A fall in FEV₁ of 10% or more of the pre-exercise value should be considered abnormal when tests are being performed in the laboratory. However, in the field a fall of 15% or more is used [155]. If measurements are made during exercise (this is now unusual) the % rise index can be calculated. This is calculated by subtracting the value recorded immediately before exercise from the highest value measured during exercise and expressing it as a percentage of the value measured before exercise. The exercise lability index is the sum of the % fall and the % rise and is an excellent index for characterising airway responsiveness to exercise (fig. 5) [138].

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Figure 5–

Changes in peak expiratory flow (PEF) and forced expiratory volume in one second (FEV₁) recorded during and after exercise in an asthmatic person who has some airflow limitation at rest. The highest, lowest, and pre-exercise values are used to calculate the % rise and % fall. Modified from Anderson [152], with permission.

It is recommended that in addition to the % fall index, the pre-exercise value for FEV₁ and the lowest value after exercise be reported in % predicted. In this way the clinical relevance of response to therapy is more easily ascertained [159].

The ventilation required to induce the observed % fall in FEV₁ is also reported and is useful for assessing the effect of therapy. Reporting values for heat and water loss has little meaning in clinical practice. The recording of a potential late response is not recommended, because the interpretation of a fall in lung function at about 3–8 h after exercise is still unclear [153, 154].

3.5.1 Background

The airway response to allergen and chemical sensitizers (§ 3.6) is more complex than that to the bronchoconstrictive triggers covered above in § 3.1 to 3.4. The early asthmatic response (EAR) is an episode of airflow obstruction, predominantly airway smooth muscle contraction, maximal between 10 and 20 min after inhalation and resolving within 90 to 120 min [160]. The late asthmatic response (LAR) is an episode of airflow obstruction, probably caused by both airway smooth muscle contraction and inflammation, occurring between 3 and ≥8 h after inhalation [38, 160]. Allergen-induced increase in airway responsiveness to histamine or methacholine ΔPC₂₀), likely an indirect measure of allergen-induced inflammation, occurs between 3 h [161] and several days [162] after allergen exposure, chiefly in subjects manifesting late asthmatic responses. Controlled standardized allergen inhalation tests have a limited, if any, role clinically. They may be helpful in confirming allergic asthmatic responses in exceptional cases where immunotherapy is contemplated. However, they are of great value in research, both for investigating the pathophysiology of asthma [161–163], and studying new, particularly prophylactic, pharmacological agents [164, 165]. The allergen inhalation test protocol outlined below takes into account the three aspects noted above: EAR, LAR, and change in PC₂₀ [160, 162–165]. Standardization is directed primarily towards within-subject reproducibility, which is particularly relevant to research studies. Under all circumstances extensive precautions are needed for safety requirements (§ 2.4).

3.5.2 Solutions and dose range

Solutions for allergen inhalation are made from the best available commercial aqueous allergen standardized extracts. These must be carefully standardized according to recent recommendations [166]. The stock solutions should preferably have a concentration of 10,000 biological units (BU) or 10 histamine equivalent prick test (HEP) per mL (1000 BU·mL⁻¹ = 1 HEP). In weight/volume (w/v) this ranges approximately from 1:10–1:20 w/v (pollen and animal danders) to 1:50–1:100 w/v (house-dust mite). Stock solutions are diluted 1:8 with sterile buffered carbol isotonic saline containing 0.5% phenol. Thereafter, serial doubling dilutions (1:16, 1:32,… ≥1:1024) are made. The weakest allergen solution used for allergen inhalation is determined by the skin test endpoint (§ 3.5.4) in the individual undergoing challenge [167], and may be as dilute as 1:65,536. The most concentrated solution used for inhalations is usually the 1:8 dilution; this represents 1250 BU·mL⁻¹ or 1.25 HEP (1:80 w/v dilution for pollen extracts to 1:800 w/v dilution for house-dust mite extract). Stability of particularly the weaker allergen concentrations is important, and is assured by freshly mixing allergen dilutions before each inhalation challenge.

3.5.3 Aerosol generation

Aerosols are generated with a jet nebulizer (driving pressure up to 344 kPa or 50 p.s.i.) calibrated with airflow between 4 and 9 l·min⁻¹, to give a mass loss of 0.13 g·min⁻¹ (approximately equivalent to 0.13 mL·min⁻¹). A closed system should be used to prevent inadvertent allergen exposure and sensitization of laboratory personnel. The nebulizer is connected to a Hans-Rudolph box and valve system, and to the patient by a mouthpiece. Two ventilator breathing circuit filters are placed in series on the expiratory line to trap the uninspired nebulizate, and the exhalate.

3.5.4 Protocol

A control day is important to ensure stability of FEV₁ (within 10% of baseline) over the 8 to 10 h of the study. On the control day, diluent is inhaled for 2 min at 10 min intervals on three occasions. FEV₁ is measured initially in triplicate and in duplicate 10 min after each inhalation, and following the last inhalation, every 10 min for the first hour, at 90 min and 2 hours, and then hourly up to 7 h after challenge. On the control day, duplicate prick skin tests with the doubling dilutions of allergen for inhalation are done in order to determine the skin test endpoint, i.e. the weakest concentration producing a wheal of 2 mm or 2 mm larger than control. A histamine or methacholine PC20 is measured at 7 h. The severity of the early asthmatic response (PC20,allergen) can be predicted from the skin test endpoint and the histamine or methacholine PC20 [167]. This is highly relevant for safety purposes. It is being done with the following formula: where SS = the skin test endpoint.

On the allergen challenge day, allergen inhalations are commenced starting two to four concentrations below the predicted PC_20,allergen. Doubling concentrations of allergen are inhaled for 2 min at 10 min intervals until the FEV₁ measured at 10 min has fallen by ≥15%, or until the top concentration has been reached. If the FEV₁ falls by <10%, the next concentration is inhaled. If the FEV₁ falls between 10–15%, the next concentration should be inhaled for 1.5 min rather than 2 min. Only if the fall in FEV₁ is still <15% should the concentration be inhaled for 2 min. This is an effort to minimize the maximal fall in FEV₁ after inhaled allergen. Since the EAR progresses for ≥20 min, even the slightest of symptoms occurring during allergen inhalation is an indication for immediate discontinuation of inhalation. The FEV₁ is monitored for at least 7 h as on the control day. It should be emphasized that this monitoring is always required, because even in the absence of an EAR a LAR can not be excluded. The patient then receives written instructions about the medical care during the first 24 h after the challenge (§ 2.4). In exceptional cases, only the EAR is of interest. Then the patient should be given a β-agonist either or not combined with inhaled steroids following the EAR. After full recovery from the EAR the patient is allowed home with adequate instructions on measurements of PEF and medical care during the first 24 h.

Allergen-induced increase in airway responsiveness can be addressed by measuring histamine or methacholine PC₂₀ at 3 h (between the early and late response), or the next day at 24 to 32 h after inhalation. Repeat measurements of PC₂₀ may be made over several days if indicated.

Repeat allergen inhalation tests, generally under experimental conditions, are done with the same dose of allergen throughout, since the cumulative allergen dosage determines the response of the subjects [168]. Both for safety and consistency in timing of pre-allergen medications, one should strive to have three inhalations up to and including the final concentration of allergen delivered for each test. Repeat tests are done a ≥7 day intervals when the baseline FEV₁ is stable (within ±10%), the histamine or methacholine PC20 measured before the test is stable (within ± one doubling dilution), and there has been no natural allergen exposure or respiratory tract infection for ≥4 wk [164, 167]. In contrast to repeat histamine/methacholine challenges under experimental conditions, e.g. in situations where the early response is being inhibited by an agent such as a β2-agonist, one should not aim to achieve a fall in FEV₁ of 20% during the EAR because of the possibility of severe prolonged late responses. It also needs emphasis that the so-called anti-inflammatory drugs have profound effects on early and late asthmatic responses [164]. Continuous personal attendance by a physician is required during the challenge, and for the duration of the early asthmatic response. Complete cardiopulmonary resuscitation equipment must be readily available including particularly parenteral adrenaline (drawn up and ready to use) and inhaled β-agonists (see § 2.4). These tests should only be carried out in a hospital setting.

3.5.5 Expression of response

The best FEV₁ at each time is retained for analysis. A typical example of time-response curves is illustrated in figure 6. The EAR is defined as the maximum per cent reduction in FEV₁ (% fall from baseline) occurring in the first hour after challenge. The magnitude of the LAR is defined as the maximum per cent reduction in FEV₁ occurring between 3 and 7 h after challenge. An alternative way to express the EAR is the PC_20,allergen [167]. In addition, some investigators express the EAR and LAR as the area under the time-response curve [169]: the advantage of this type of analysis is the inclusion of all data-points on the time-response curve. Allergen-induced increase in airway responsiveness is generally defined either as the difference in log PC₂₀ or as the pre/post PC₂₀ ratio using logarithmic transformation for statistical analyses.

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Figure 6–

Mean (± SEM between subjects) time-response curves of FEV₁ (squares, upper panel) and of expiratory flow obtained from partial flow-volume curves (V′40p) (triangles, lower panel) after an allergen challenge with inhaled house-dust mite extract in 10 atopic asthmatic subjects on 2 days (open and closed symbols) separated by a 2 weeks interval. When using the present standardization, the reproducibility of the early and late asthmatic response appears to be good. Modified from Bel et al. [249], with permission.

3.6.1 Background

3.6.2 Agents

3.6.3 Dose delivery

Agents in powder or dust form

The method of tipping powder from one tray onto another, used in the past, should not be used anymore, because it does not allow for monitoring of the concentration of particles in the air. Indeed, as for all occupational agents, the levels of dust to which the subjects are exposed should never exceed the short-term exposure limit (TLV-STEL) set by the American Conference for Governmental Industrial Hygienists (ACGH) [187]. A recently developed apparatus makes exposure to steady and low concentrations of particles possible and permits monitoring of the diameter of the particles (fig. 7) [188–190]. This consists of a particle generator, an exposure chamber with elimination system, and photometer- and cascade-impactor monitoring devices, all being automated using closed-loop feedback, regulated by computer programme [189, 190].

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Figure 7–

Aerosolization device made in three parts: 1) the particles generator on the left end where the powder is vibrated, taken to the rotating plate by an endless screw and sucked out; 2) the exposure of aerosol delivery chamber, a long plexiglass cylinder with three holes in the centre, one for the facemask, one for the photometer probe and one for the cascade impactor probe; 3) the recording instruments: the photometer and the cascade impactor. Modified from Cloutier and Malo [189], with permission.

Vapours

Challenges with vapours or gases have so far been performed by various investigators in challenge rooms that are similar to each other (fig. 8) [191–193]. The rooms are usually well ventilated with a circuit near the ceiling, so that the air in the antechamber and challenge rooms can be completely changed within a few minutes.

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Figure 8–

The challenge rooms used at the Hôpital du Sacré-Coeur (Montreal, Canada) for the last few years. There are two antechambers and two challenge rooms. The ventilation system in the ceiling of the rooms is shown by transparency. Two large plexiglass windows allow the technician located in the antechamber at the top of the figure to have a direct access to the ventilation and solenoid valve regulators to control aerosolization within the challenge rooms. For challenges with polyisocyanates, a MDA-7100 monitor is placed in room 1, adjacent to room 2 where the subject sits. The small sampling tube crosses the plexiglass window between rooms 1 and 2.

After the control days required for assessment of reproducibility of lung function measurements, and to exclude airflow obstruction induced by nonspecific mechanisms, subjects are usually exposed to increasing concentrations of the vapour or gas. For polyisocyanates, for example, exposure may be performed in one of the following ways depending on the type of isocyanate:

1 for TDI: 20–100 mL of pure commercial TDI (80% 2.4-TDI and 20% 2.6-TDI) is deposited in a small cup within a bottle, and air is passed through and dispersed in the exposure room;

2 for HDI (hexamethyl diisocyanate), the commercial preparation the subject is exposed to at work (from 20% to 75% of HDI/HDI biuret, depending on the product) is nebulized with the diluent (1:3 concentration) using a plastic or glass nebulizer;

3 for MDI (methylene diphenyl diisocyanate): the commercial preparation containing 40% to 50% of MDI and 50% to 60% of polyethylene polyphenyl isocyanate is heated in a metal cup to approximately 80°C.

The isocyanate concentration may be monitored by automatic monitor (MDA-7050 or MDA-7100). The monitor is usually put in the adjacent challenge room as contamination of the recording paper can occur if the monitor is located in the same room as the source of isocyanate. The tip of the sampling tube of the MDA-7100 monitor crosses the window and is put at a distance equivalent to that which separates the source of polyisocyanates from the subject's mouth. By controlling the ventilation in the room and the output of the nebulizer, the technician can stabilize the concentration of polyisocyanates in the challenge room and keep it below the threshold limit value for short term exposure level (e.g. TLV-STEL which is 20 ppb for isocyanates) [187]. The humidity in the chamber should be kept close to 50% (the reading of polyisocyanates by the MDA-7100 monitor is affected by humidity). A ventilator can homogenize the isocyanate in the air.

A self-contained apparatus including a closed-circuit mixing chamber has recently been developed which allows delivery of controlled concentrations of gaseous isocyanates at the mouth of the subject, without the need of a cumbersome exposure chamber [194]. This alternative approach is promising, and deserves further validation.

3.6.4 Level and duration of exposure

After the control day of assessment of reproducibility of lung function, the subjects are usually exposed to a control chemical (e.g. for TDI challenge it would be the chemical that is usually mixed with polyisocyanates) for a total of 15 min in a challenge room. This allows to verify that the reaction to the «sensitizer» is specific and not due to nonspecific irritation.

The principle is to generate a dose-response curve by increasing the duration and/or the concentration of exposure on different days. The duration of exposure to a very low concentration should be increased progressively for the first day of exposure (e.g. 1, 5, 10, 15 min exposure to 1 ppb TDI). If there is no asthmatic reaction (see below), the duration and/or the concentration of exposure may be increased in different days (e.g. 1, 5, 10, and 15 min to 5, 10, 20 ppb TDI, respectively) during the following 3 days.

In principle, the overall concentration should not exceed the threshold limits value for short-term exposure (TLV-STEL) recommended by the ACGIH [187].

The duration and intensity of exposure to the suspected offending agent should be dictated by clinical history. If there is any indication of a severe immediate reaction, short exposure under controlled conditions should be considered. The level of bronchial obstruction and nonspecific airway responsiveness are also helpful in determining the duration and level of the initial exposure [195].

3.6.5 Expression of the response

FEV₁ is more sensitive than the simpler PEF in detecting the response [196]. To be considered positive, investigators generally require a fall of 20% or more in FEV₁ at the time of an immediate reaction with progressive recovery in the first hour, or a sustained fall in FEV₁ of at least 20% in the case of non-immediate reactions, both in the absence of significant changes on the day of exposure to the control product.

FEV₁ is usually assessed for at least 8 h, i.e. every 10 min for the first hour, every 30 min for the second hour and then hourly. Subjects are also asked to continue measuring at least PEF during the evening or during the night in order to investigate whether airway obstruction occurs after the usual period of 8 h monitoring.

An increase in histamine or methacholine airway responsiveness typically occurs after late reactions, but it may also occur after immediate asthmatic reactions [185, 197–201]. Airway responsiveness may also increase without significant changes in FEV₁. In these cases a longer exposure to the specific agent can induce bronchoconstrictive reaction [200].

3.7.1 Adenosine 5′-monophosphate

Adenosine 5′-monophosphate (AMP) is considered to be an “indirect” stimulus of airway narrowing, since it exerts its effects through the release of mediators from immunologically activated airway mast cells and probably also through activation of neuronal reflexes [204]. AMP is rapidly metabolised to adenosine, which stimulates several subtypes of purinoceptors. Due to its solubility, AMP enables relatively high doses of adenosine to be given by inhalation in vivo. Asthmatics and atopic non-asthmatic subjects usually respond to AMP with acute airway narrowing, whereas nonatopic normal subjects do not [205, 206]. Repeated challenges with AMP lead to substantial tachyphylaxis and cross-tachyphylaxis with e.g. exercise. Furthermore, complex interaction with hypertonic saline and allergen challenges has been described. AMP is increasingly used in testing the involvement of cellular mechanisms in airway hyperresponsiveness [204].

3.7.2 Leukotrienes

Leukotrienes are pro-inflammatory metabolites of arachidonic acid through the 5-lipoxygenase pathway. Since the cysteinyl leukotrienes (LTC₄, LTD₄, and LTE₄) are considered to be of major importance in bronchial asthma [207], these agents have been given by inhalation in many research studies. They exert their effects through specific receptors, acting on smooth muscle, endothelial cells, nerve fibres, and secretory cells. Therefore, airway narrowing to these substances has a “direct” as well as an “indirect” component. Asthmatic subjects are hyperresponsive to leukotrienes, particularly LTE₄ [208], and in some studies leukotrienes have been shown to induce transient airway hyperresponsiveness to other stimuli [209, 210]. A highly efficient challenge methodology has been described [210], which has been shown to produce reproducible results [211]. Leukotriene provocation tests are used in pathophysiological research, particularly in testing the increasingly available leukotriene-antagonists or synthesis inhibitors.

3.7.3 Platelet-activating factor

Platelet-activating factor (PAF) is also a pro-inflammatory mediator of potential importance in asthma [212]. It acts through various subtype PAF-receptors on inflammatory cells, endothelium, and secretory cells. The acute bronchoconstriction after PAF inhalation might be due to the secondary release of leukotrienes [212]. PAF has been shown to induce airway hyperresponsiveness in normal subjects [213], but this could not be confirmed by other investigators [214]. There is rapid tachyphylaxis to PAF inhalation.

3.7.4 Bradykinin

Bradykinin is a potent pro-inflammatory peptide [215] leading to airway narrowing through an “indirect” mechanism. When given by inhalation, it causes acute bronchoconstriction in asthmatics [216], which is most likely due to smooth muscle contraction, vasodilation, oedema, and/or airway secretion secondary to stimulation of C-fibre nerve endings and the release of sensory neuropeptides. Therefore, this challenge is currently used to examine the role of axon reflexes under various experimental conditions, e.g. following allergen challenge in asthma [217].

3.7.5 SO₂ and sodium metabisulphite

SO2 is considered to be a stimulus to investigate the role of cholinergic and/or noncholinergic neural pathways in airway narrowing. Instead of administering gaseous SO2 [58, 218] it is much simpler to aerosolize a SO₂-generating solution: sodium metabisulphite [219]. The challenge with this preservative causes acute airway narrowing in vivo in a reproducible way [220], and is thereby suitable for research studies. SO₂ and metabisulphite challenges may be used to distinguish asthma from COPD [220], but this needs further investigation.

3.7.6 Tachykinins

Tachykinins are sensory neuropeptides localized in airway C-fibre nerve endings, which are believed to play a role in bronchial asthma [222]. These pro-inflammatory peptides are released by local axon-reflexes, causing smooth muscle contraction, vasodilation, microvascular leakage, and hypersecretion, through specific NK-receptors [222]. Therefore, tachykinins can be considered to stimulate both «direct» and «indirect» mechanisms of airway narrowing. Among the tachykinins, neurokinin A and substance P are being used for inhalation challenge testing in research, either by traditional jet nebulizer [223] or by highly efficient nebulization [224]. Relatively high doses are needed to produce bronchoconstriction in normal subjects, because of endogenous neuropeptide-degrading enzyme activity [224]. In male tachykinins have not been shown to induce subsequent airway hyperresponsiveness to other agents.

3.7.7 Propranolol

Another alternative challenging agent is the β-blocker propranolol. When given by inhalation propranolol induces bronchoconstriction in asthmatic patients but not in normal subjects [225, 226]. Many patients with COPD do not respond to propranolol [25, 221]. The mechanism of bronchoconstriction to propranolol is probably related to a blockade of inhibitory β-receptors on cholinergic nerves. This would enhance acetylcholine release, particularly in asthma, because asthmatics may have impaired function of the inhibitory muscarinic autoreceptors [6]. This mechanism explains the effective reversal of propranolol-induced bronchoconstriction by anticholinergic agents [225, 226]. Since propranolol blocks the effect of β-adrenergic bronchodilators, anti-cholinergic drugs are an essential component of the safety precautions during this type of challenge.

5.1.1 General

- measurements of airwayresponsiveness have a good safety record, provided the procedures are carefully standardized;

- the presently standardized laboratory-protocols are the methods of first choice for pharmacological and physical challenges, as well as for challenges with sensitizing agents; they allow comparison of the results with previous data in the literature;

- the term «nonspecific» airway responsiveness is ambiguous, and should be identified with the challenging agent, since the mechanisms of bronchoconstriction varies between the distinct stimuli;

- hyperresponsiveness should be regarded as an abnormality of airway function, which is associated with various features of inflammation;

- newly developed and potentially better methods should be evaluated on their repeatability and their agreement with currently standardized challenges.

5.1.2 Clinical usage

- airway responsiveness measurements with pharmacological agents are particularly useful for the exclusion of asthma in the clinical setting, they are less useful in confirming and distinguishing the diagnoses of asthma and COPD;

- hyperresponsiveness is not synonymous with the diagnosis of asthma, but can provide additional information to clinical symptoms and peak flow measurements on the potential of variable airways obstruction in epidemiological and clinical setting;

- a 10–20% fall in FEV₁ in response to isocapnic hyperventilation or exercise is consistent with the diagnosis of asthma; however, a negative response to these stimuli does not exclude asthma;

- the combination of airway hyperresponsiveness and recent wheezing (in the past year) can be regarded as a definition of asthma for epidemiology;

- in clinical practice histamine and methacholine tests are most extensively validated, and thereby the challenges of first choice in adults; in paediatrics exercise tests can be a better alternative;

- physical challenges (exercise, cold air, non-isotonic aerosols) reflect the involvement of a cascade of «indirect» components of airway narrowing, such as cellular and neurogenic mechanisms;

- inhalation tests with common allergens should be regarded as a research tool;

- challenge tests with occupational sensitizers should only be performed in specialized centres;

- the safety requirements for inhalation challenge tests vary between the different types of stimuli, being most stringent for tests with allergens and occupational sensitizers;

5.1.3 Materials and methods

- output should be calibrated for each individual nebulizer; in clinical practice this can be estimated adequately by weighing;

- for research purposes the same nebulizer should be used at exactly the same setting throughout the study;

- there are three standardized methods for pharmacological challenges (tidal breathing, dosimeter, and Yan method); the results have similar reproducibility, and the choice between them depends on the type of study (clinical or epidemiological) and local facilities and experience;

- for the tidal breathing method, 2 min inhalation of doubling doses (nebulizer output 0.13 mL·min⁻¹) is recommended;

- for the dosimeter method, doubling doses should be administered by inhaling 5 puffs of 9 μl per dose;

- FEV₁ is the lung function index of first choice during challenge tests in clinical practice.

- hypotonic- and hypertonic aerosol challenge should be performed with ultrasonically nebulized distilled water and 4.5% saline, respectively, with 1.2 mL·min⁻¹ aerosol output and doubling exposure times;

- isocapnic hyperventilation can be performed by doubling steps of minute ventilation with cooled dry air (water content 0 mg·l⁻¹, −15°C) or dry air at room temperature; alternatively dry air at room temperature can be used at 30, 60, and 100% of maximal voluntary ventilation;

- during exercise challenge the subject should attain a level of ventilation between 40%–60% of the subjects predicted maximal voluntary ventilation during the last 4 min of a 6–8 min test;

- in allergen challenge tests the starting allergen dose is calculated from the histamine or methacholine PC₂₀ and the result of allergen prick skin tests («skin test endpoint»);

- until European consensus about the exposure limits during challenge tests with occupational sensitizers is reached, the American limits (TLV-STEL) are recommended.

5.1.4 Analysis and interpretation

- when analyzing dose-response curves obtained by inhalation challenge tests, the position of the curve (PC or PD value) is the index of choice in clinical practice;

- the so-called «two-point slope» enables analysis of dose-response curves in the general population, and is a promising index of airway responsiveness in epidemiological studies; its repeatability needs further studies, particularly in adults;

- there is a complex interaction between baseline lung function and airway responsiveness, that appears to be different between asthma and COPD; therefore, challenge tests are hard to interpret in the presence of airways obstruction.