Lung Function and Bronchial Challenge Testing for the Allergist

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1 Lung Function and Bronchial Challenge Testing for the Allergist Klaus F. Rabe, Adrian Gillissen, and Zuzana Diamant General Introduction The diagnosis and management of respiratory allergies in general, bronchial asthma, and rhinitis inevitably requires objective measurement of respiratory function. A wide variety of lung function equipment is commercially available, but varying expertise in its use and general reservations towards the complexity of some measurements likely have hampered the widespread and optimal use of state-of-the-art technology in general allergy practice. Curiously, there are almost no specific guidelines for the standards, scope or interpretation, or the quality assurance of lung function testing issued from the large allergy societies. Bronchoprovocation testing with direct bronchoconstrictor stimuli, with allergen, or with clinical models is relevant for the diagnosis of allergies and asthma, in particular when baseline lung function is normal. These methods need a standardized approach and involve reliable functional assessment of lung function to be safe, reproducible, and of adequate quality. Lately the functional assessment of airway function has been complemented by noninvasive techniques to measure components in exhaled breath, breath condensate, and in sputum. These techniques have in part been introduced into clinical algorithms, they are increasingly used in respiratory research, and they complement the classical techniques of physiological lung function measurements. This chapter, therefore, strives to give a concise overview of relevant techniques for lung function testing, spanning from simple peak flow measurements to classical physiological techniques to novel developments for airway inflammometry. K.F. Rabe ( ) Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands K.F.Rabe@lumc.nl A. Gillissen Robert-Koch-Hospital, St. George Medical Center, Leipzig, Germany Z. Diamant Centre for Human Drug Research, Leiden, The Netherlands R. Pawankar et al. (eds.), Allergy Frontiers: Diagnosis and Health Economics, 101 DOI / _6, Springer 2009

2 102 K.F. Rabe et al. Measurement of Lung Function Pulmonary function tests provide measures of lung volumes, flow rates, gas exchange, and respiratory muscle function. Since Hutchinson first developed the spirometer in 1846, measurements of the dynamic lung volumes and of maximal flow rates have been used in the detection and quantification of diseases affecting the airways and the lung parenchyma. Basic pulmonary function tests that are available in the ambulatory setting include peak flow and spirometry [1, 2]. These tests provide physiologic measures of pulmonary function and can be used to quickly narrow a differential diagnosis and suggest a subsequent strategy of additional testing or therapy. Additional lung function tests include body plethysmography to measure the amount of thoracic gas volume, which is important to quantify hyperinflation and the airway resistance, esophageal pressure measurements to determine pressure volume relationships, blood gas analysis, and exercise testing [3, 4]. These provide a more detailed description of physiologic abnormalities and the underlying pathology. The choice and sequence of testing are guided by information from the history, the physical information, and after all by the technical availability. Peak Flow The maximal peak expiratory flow is measured with a peak-flow meter, which is a very user-friendly device, portable, made from plastic, and therefore inexpensive, although electronic versions are available [5]. Peak-flow measurements can be performed by the patients themselves, and can therefore be used for self-management purposes, diagnosis, or to detect environmental exposure situations causing airway obstruction. Peak-flow measurements provide a simple, quantitative measure of limitation of exhaled airflow typically seen in obstructive airway diseases. Daily monitoring can be used to detect worsening of lung function in the absence for symptoms, to assess variations in lung function throughout the day, to identify triggers, to make appropriate medication decisions, and to monitor the patient s response to therapy. Peak-flow measurements can be done in adults and also in children over 5 years of age. Optimal peak-flow assessment requires some degree of patient education: The patients should know that it is a key measure to monitor asthma at home [6]. The device and the breathing technique must be explained. It is important that the patient perform the peak-flow measurement always in the same body position, preferably when standing straight. After taking a deep breath, the patient must blow as hard as possible without bending over. Most commonly, peak flow is measured first thing in the morning prior to the treatment. The number on the indicator scale is the maximal exhaled airflow. These steps should be repeated two or three times, and the largest number measured should be recorded in a peak-flow meter protocol.

3 Lung Function and Bronchial Challenge Testing for the Allergist Peak Flow (l / min) Budesonide 200µg bid + Formoterol 9µg bid date Fig. 1 Before therapy high daily variability of peak flows indicate poorly controlled asthma During the day peak-flow measurements can be repeated as often as necessary. The best peak-flow number is important for the patient because it is the comparator for the best achievable value, which serves as a reverence value for monitoring the effects of changes over time [7]. A decrease of 20% of the personal-best number (or 60 ml/min) or a diurnal variation of >20% (2 /day reading: >10%) may indicate (a) an asthma attack or (b) it shows an inadequately controlled disease [8] (Fig. 1). A fall in peak flow, especially when accompanied by symptoms such as increased cough, shortness of breath, or wheezing may signal the onset of an exacerbation, requiring immediate treatment to prevent complications. High diurnal variability is an important feature of poorly controlled asthma. There are two ways to calculate the peak-flow variability: The difference between the maximum and the minimum value for the day, expressed as a percentage of the mean daily peak-flow value and averaged over 1 2 weeks, or The minimum morning pre-bronchodilator peak flow over 1 week, expressed as a percentage of the recent best min/max (%). This has been suggested to be the best index of airway lability for clinical practice because it requires only a 1 / day reading, it is easy to calculate, and it correlates best with airway hyperresponsiveness (AHR). However, peak-flow measurements have also the following disadvantages [9]: The degree of airflow limitation can be underestimated, particularly as airflow limitation and gas trapping worsens. Values vary considerably according to a person s age, sex, body size, and between different peak-flow meters. Peak-flow meters cannot be calibrated [10].

4 Name... Date... Peak Flow Rate Time am pm am pm am pm am pm am pm am pm am pm am pm am pm am pm am pm am pm am pm am pm Day Fig. 2 An example of a peak-flow meter protocol. The best of the three is the reading to record. Mark this with a cross on the chart Figure 2 shows an example of a chart the patient can use for recording his peakflow values on a day-to-day basis. Spirometry The simplest form of pulmonary function testing is spirometry, which in principle measures how quickly air can be expelled from the lungs [11]. Spirometry is performed by blowing into a spirometer, which measures timed expired and inspired volumes. A spirogram is thus either a volume time curve or a flow volume curve, e.g., expressed as a peak expiratory flow or as a function of volume (Fig. 3). Although highly dependent on individual factors, these curves are reproducible for any individual, but vary considerably between different lung diseases and disease stages. Furthermore, the spirographic values are dependent on the factors age, height, and gender (see below). The following parameters are usually measured (Fig. 4): Vital capacity (VC): It is the maximum volume of air which can be exhaled or inspired during either a forced (FVC) or a slow (VC) maneuver. Force expired volume in one second (FEV ): It is the volume expired in the first 1 second of maximal expiration after a maximal inspiration and is a useful measure of how quickly full lungs can be emptied. FEV 1 is the most widely used parameter in asthma and chronic obstructive pulmonary disease (COPD). Besides the symptoms, FEV 1 and FEV 1 /FVC determine the severity of both diseases. The extent of FEV 1 decline is a strong predictor of poor disease outcome and indicates poor asthma management and vice versa [2, 3]. FEV /FVC or FEV /VC: The ratio of FEV is expressed as a percentage of FVC or VC and gives a clinically useful index of airflow limitation, even if airway

5 Fig. 3 A typical flow volume curve (a) and a volume time curve (b). VC = vital capacity, FEV 1 = forced expired volume in one second obstruction is very moderate. In contrast, interstitial pulmonary diseases have normal FEV 1 /FVC and FEV 1 /VC volumes due to limitation of both lung function parameters. FEF : It is the average expired flow over the middle half of the FVC maneuver 25 75% and is regarded as a more sensitive measure of small airways narrowing than FEV 1. Unfortunately, FEF 25 75% has a wide range of normality, is less reproducible than FEV 1, and is difficult to interpret if the VC or FVC is reduced or increased.

6 106 K.F. Rabe et al. TLC IRV IC V T VC ERV FRC RV 0 Time Fig. 4 Normal spirogram showing the spirographic parameters: FEV 1 =forced expired volume in one second; FVC = forced vital capacity; VT = tidal volume or tidal breathing; ERV = expired residual volume; IRV = inspired residual volume; FRC = functional residual capacity; RV = residual volume; VC = vital capacity; TLC = total lung capacity Spirometry should be performed only when the spirometer is heated up to body temperature and pressure saturated with water vapor. Temperature differences between, for example, the warm exhaled air within a cold spirometer will cause an underestimation of the true lung volumes. Further, spirometers need to be calibrated prior to use. Technique How to Use It: Pitfalls and Problems To ensure an acceptable result, the spirometry maneuver must be performed with greatest effort starting with maximum expiration followed by a deep inspiration and an expiration phase lasting at least 6 s. The spirogram should be a smooth continuous curve. To achieve good results the procedure should be done only in a patient sitting erect with feet firmly on the ground (though standing gives a similar result) with a nose clip applied. The lips must seal firmly the mouthpiece and the patient must be urged to exhale forcibly. At least three technically acceptable blows should be obtained. Only the measurement with the largest VC or FVC is recorded. A good reproducibility means a difference of less than 200 ml between the highest and the second highest FVC [2, 3, 12]. Many patient-related problems may occur and result in inefficient flow volume curves (Fig. 5):

7 Fig. 5 Common pitfalls with the breathing technique of the patients Submaximal effort Leaks between the lips and the mouthpiece Incomplete inspiration or expiration prior to or during the forced maneuver Hesitation at the start of expiration, cough, and/or vocalization during the forced maneuver Obstruction of the mouthpiece by the tongue Poor posture Hazards and Quality Control Spirometry is a safe procedure and hazards have only been anecdotally reported. The quality of the spirometry can be just as good as the operator understands the principles underlying measurement and equipment operation. Various components

8 108 K.F. Rabe et al. of the spirometry system, including mouthpieces, nose clips, pneumotachs, valves, and tubing are potential vehicles for transmission of infection to subjects and staff. A tight infection control is therefore recommended. Interpretation of the Results Spirometry allows the distinction between obstructive and restrictive pulmonary diseases [13]. Further, reversibility of an airway obstruction can be tested easily (see bronchodilator [BD] test). In order to compare the individual results to healthy persons, reference tables or reference equations have been established and abnormalities are evaluated against predicted results. Predicted values are calculated based on age, height, and gender. Office spirometers are typically preprogrammed with prediction equations derived from the study of Caucasians, such as the European Community for Steel and Coal (ECSC) [14]. An obstructive ventilatory abnormality is defined as a disproportionate reduction in maximal airflow from the lung with respect to the maximal volume that can be displaced from the lung. Typically, reduction of FEV 1 /FVC (or FEV 1 /VC) FEV 1 is seen in asthma and COPD. The extent of lung function decline defines the disease stage of both diseases [8, 15]. The diagnosis of an obstruction should be followed up with a bronchodilator test (see below). Severity or airway obstruction is graded according to percentage of predicted FEV 1 [8]. A restrictive ventilatory defect is characterized physiologically by a reduction in total lung capacity (TLC) which, however, cannot be determined by spirometry. Regardless of this limitation, reduced FVC or VC together with reduced FEV 1 resulting in normal or high FEV1/FVC or FEV 1 /VC indicate a restrictive pattern. A range of conditions can reduce FEV 1 and FVC (or VC) such as diseases impeding the movement of the chest wall including pain, neuromuscular weakness, lung parenchymal diseases (interstitial lung diseases), or conditions causing reduced lung volume (lung resection). Bronchodilator Test The purpose of the bronchodilator (BD) test is to determine whether an airway obstruction, as measured by low FEV 1 % predicted and/or low FEV 1 /VC is reversible with an inhaled rapid acting β2-agonist. The test can be standardized as follows: Two puffs (100 μg) of salbutamol or equivalent are administered. A waiting period of at least 10 min is introduced. Two reproducible flow volume curves (FEV and/or FVC) are again obtained. 1 The best post-bronchodilator FEV is evaluated. 1 A significant improvement of at least 12% (or 15% depending on the society) and 200 ml from the best pre-bronchodilator FEV 1 is regarded as a positive reversibility,

9 Lung Function and Bronchial Challenge Testing for the Allergist 109 a strong indication for asthma. Typically, COPD patients lack a positive test result due to fixed airway obstruction [3, 16, 17]. Percentage improvement in FEV 1 can be calculated using the following formula: [FEV 1 pre-bd 2 FEV 1 post-bd /FEV 1 pre-bd ] 100 For an accurate interpretation of a negative response, patients must have been weaned from bronchodilators for at least 12 h, if medically possible. Limitations Spirometry is an effort-dependent test that requires careful instructions and the cooperation of the test subject. Inability to perform acceptable maneuvers may be due to poor subject motivation or failure to understand instructions. Physical impairment and young age (e.g., children over 5 years of age) may also limit the subject s ability to perform spirometric tests. These limitations do not preclude attempting spirometry, but should be noted and taken into consideration when the results are interpreted. Bodyplethysmography The measurement of absolute lung volumes, residual volume (RV), functional residual capacity (FRC), and total lung capacity (TLC) are technically more challenging procedures, which has limited the use of bodyplethysmography in clinical practice. Also, the role of lung volume measurements in the assessment of disease severity, functional disability, course of disease, and response to treatment remains to be determined for all age groups, although sometimes measurements of lung volume are necessary for a correct physiological diagnosis [3]. Definitions of Lung Volumes The term lung volume usually refers to the volume of gas within the lungs, as measured by body plethysmography and the measurements obtained are graphically illustrated in Fig. 6. Measurement of FRC Using Body Plethysmography Plethysmographic measurements are based on Boyle s law, which states that, under isothermal conditions, when a constant mass of gas is compressed or decompressed, the gas volume decreases or increases and gas pressure changes such that the product of volume and pressure at any given moment is constant [18, 19].

10 110 K.F. Rabe et al. Fig. 6 The functional residual capacity (FRC) is the volume of gas present in the lung at endexpiration during tidal breathing. The expiratory reserve volume (ERV) is the volume of gas that can be maximally exhaled from the end-expiratory level during tidal breathing (i.e., from the FRC). The maximum volume of gas that can be inspired from FRC is referred to as the inspiratory capacity (IC). The inspiratory reserve volume is the maximum volume of gas that can be inhaled from the end-inspiratory level during tidal breathing. The residual volume (RV) refers to the volume of gas remaining in the lung after maximal exhalation. The volume of gas inhaled or exhaled during the respiratory cycle is called the tidal volume (TV or VT). The thoracic gas volume (TGV or VTG) is the absolute volume of gas in the thorax at any point in time and any level of alveolar pressure. The total lung capacity (TLC) refers to the volume of gas in the lungs after maximal inspiration, or the sum of all volume compartments The changes in thoracic volume that accompany a compression or decompression of the gas in the lungs during respiratory maneuvers can be obtained using a body plethysmograph by measuring the changes either as: (1) pressure within a constant-volume chamber (variable-pressure plethysmograph); (2) volume within a constant-pressure chamber (volume-displacement plethysmograph); or (3) airflow in and out of a constant-pressure chamber (flow plethysmograph). Regardless of the type, a transducer capable of measuring mouth pressure ±5 kpa ( ±50 cm H 2 O), with a flat frequency response in excess of 8 Hz, is essential for measurements (Fig. 7). The transducer measuring changes in the chamber pressure must be capable of accurately measuring a range of ±0.02 kpa (±0.2 cm H 2 O) [20]. All components of plethysmographs that are used for the measurement of lung volumes should meet published standards for the accuracy and frequency response of spirometric devices [20, 21].

11 Lung Function and Bronchial Challenge Testing for the Allergist 111 Fig. 7 Body plethysmorgraphy (MasterScreen, CardinaHealth) consists of a body box (right) and an analyzing unit (left) Measurement Technique Measurements should practically adhere to the following steps: 1. The equipment should be turned on and allowed an adequate warm-up time. 2. The equipment is set up for testing, including calibration, according to manufacturer s instructions. 3. The equipment is adjusted so that the patient can sit comfortably in the chamber and reach the mouthpiece without having to flex or extend the neck. 4. The patient is seated comfortably, with no need to remove dentures. The procedure is explained in detail, including that the door will be closed, the patient s cheeks are to be supported by both hands, and a nose clip is to be used.

12 112 K.F. Rabe et al. 5. The plethysmograph door is closed, and time is allowed for the thermal transients to stabilize and the patient to relax. 6. The patient is instructed to attach to the mouthpiece and breathe quietly until a stable end-expiratory level is achieved (usually three to ten tidal breaths). 7. When the patient is at or near FRC, the shutter is shortly closed at end- expiration and the patient is instructed to perform a series of gentle pants (~±1 kpa [~±10 cm H 2 O]) at a frequency between 0.5 and 1.0 Hz [22, 23]. 8. A series of three to five technically satisfactory panting maneuvers should be recorded after which the shutter is opened and the patient performs an ERV maneuver, followed by a slow IVC maneuver. If needed, the patient can come off the mouthpiece and rest between TGV/VC maneuvers. 9. For those unable to perform appropriate panting maneuvers (e.g., young children), an alternative is to perform a rapid inspiratory maneuver against the closed shutter. In this situation, it is essential that the complete rather than the simplified version of the TGV computation equation [18] be used in the calculation of TGV. 10. With regard to repeatability, at least three FRCpleth values that agree within 5% should be obtained and the mean value reported. Quality Control The accuracy of the flow and volume output of the mouth flow-measuring device should comply with published recommendations [2]. The mouth pressure transducer should be physically calibrated daily. The plethysmograph signal should also be calibrated daily, using a volume signal of similar magnitude and frequency as the respiratory maneuvers during testing. A validation of accuracy using a known volume should be performed periodically using a container of known volume [18, 24]. Care should be taken to adjust the calculated volumes to ambient temperature and saturated conditions (BTPS) during the calculations. The accuracy of adult plethysmographs in measuring the gas volume of the container should be ±50 ml or 3%, whichever is greater, based on a mean of five determinations [18]. Monthly, or when errors are suspected, two reference subjects should have their FRCpleth and related RV and TLC measured. Values that differ significantly (e.g., 0.10% for FRC and TLC, or 0.20% for RV) from the previously established measurements suggest errors that need to be followed up [25]. Reference Values Interpretation of lung volumes critically depends on reference values which are related to body size, with standing height being the most important factor. In children and adolescents, lung growth appears to lag behind the increase in standing

13 Lung Function and Bronchial Challenge Testing for the Allergist 113 height during accelerated growth, shifting the relationship between lung volume and height during this period [26, 27]. Pitfalls and Problems A number of factors need to be considered when selecting predictive values for absolute lung volumes. The reference populations need to match the patient populations and this information is not always easily available. Furthermore, the appropriate extrapolation of regression equations needs to be exerted considering the size and age range of subjects actually studied. Finally, the differences in testing methodology between clinical laboratories and studies from which predicted reference values are derived need to be considered before clinical interpretation of plethysmographic measurements. Bronchoprovocation Tests Direct and Indirect Bronchial Challenges Airway hyperresponsiveness (AHR) is a key characteristic of asthma and can be subdivided into a transient (relatively reversible) and more persistent (relatively irreversible) component [28]. Measurements of airway responsiveness are particularly helpful when lung function tests are normal despite symptoms or history of respiratory allergies. In general, the pathophysiology of AHR is complex and not entirely clarified. Transient AHR can be induced by indirect stimuli (e.g., exercise, cold dry air, hypertonic saline, adenosine monophosphate (AMP), and mannitol) and is generally well-responsive to inhaled corticosteroids. Hence, there is evidence that this component of AHR is linked to airway inflammation [28]. Indeed, some indirect stimuli are applied as exacerbation models of airway inflammation [29]. Alternatively, persistent AHR is more marked to direct stimuli, including cholinergic agents and histamine, appears relatively refractory to inhaled corticosteroids, and reflects the structural changes within the airways, referred to as airway remodeling (Table 1) [28]. Direct Challenges Direct stimuli or agonists, exemplified by methacholine and histamine, induce airway narrowing through interaction with specific receptors on airway smooth muscle cells [28]. Bronchial challenge tests with these agonists are standardized, usually performed by the 2-min tidal breathing method, and amply used for the assessment and quantification of the AHR in clinical and research settings [28, 30]. The severity

14 114 K.F. Rabe et al. Table 1 Tests identifying pathophysiologic characteristics of asthma eno EBC Sputum BAL Biopsy AMP BPT Airway inflammation Airway remodeling Transbronchial biopsy Imaging Methacholine BPT (?) Airway hyperresponsiveness BPT: Methacholine, Histamine and Exercise Asthma symptoms Key features of asthma and possible sampling methods. eno = exhaled NO, EBC = exhaled breath condensate, BAL = bronchoalveolar lavage, AMP = Adenosine 5' monophosphate, BPT = bronchoprovocation test. (mild to severe) of AHR is expressed by the provocative dose or concentration causing a 20% fall in FEV 1 from baseline (PD 20 and PC 20, respectively) [30]. Direct challenges are highly sensitive, although not specific for asthma, and can be used as diagnostic tools in subjects with a history of asthma with normal spirometry and no reversibility to inhaled β2-agonists [28, 30]. Indirect Challenges Indirect stimuli contract airway smooth muscle causing subsequent bronchoconstriction through pathways mediated by the release of mediators from inflammatory cells and sensory nerves [31]. Therefore, when performed serially, an adequate washout period should be allowed between consecutive challenges to correct for these pro-inflammatory carry-over effects [31, 32]. Similarly to direct provocation tests, most indirect challenges are standardized procedures [30, 33]. In 2003, an ERS Task Force document has been published on the pathophysiology, methodology, and applicability of indirect challenges with physical or pharmacological stimuli [33]. Physical stimuli with exercise challenge and the related isocapnic hyperventilation of cold, dry air are validated exacerbation models of exercise-induced bronchoconstriction (EIB) [31]. Bronchoprovocation with the pharmacological stimulus hypertonic saline (mostly NaCl 4.5%), aerosolized by an ultrasonic nebulizer, shares similar pathophysiological mechanisms with EIB and cold, dry air hyperventilation [33]. This simple, inexpensive method is an attractive tool for both diagnostic (EIB, risk assessment for scuba divers with asthma) and research

15 Lung Function and Bronchial Challenge Testing for the Allergist 115 purposes (EIB-modeling, airway sampling by sputum induction) [31, 34]. Mannitol is another hyperosmolar pharmacological stimulus, comprising of a dry powder inhaler and capsules [35]. Sharing a similar pathophysiological mechanism, i.e., increasing airway osmolarity through dehydration, both hypertonic saline and mannitol have been shown to be well-related to exercise and cold, dry air in steroid-naive asthmatics [35, 36]. Despite mechanistic similarities, there are marked differences in the methodology, equipment, and costs [30, 33, 35]. Making mannitol possesses superior properties: being a simple, reproducible, and relatively inexpensive method [35, 37]. Apart from its diagnostic potential, mannitol appeared as a sensitive predictor of a drug s anti-inflammatory efficacy and thus may be applicable for monitoring of asthma control both in clinical practice and in clinical trials [33, 37]. Adenosine monophosphate (AMP) is another related, pharmacological stimulus that could act as a marker of disease activity [31, 33]. Exacerbation Models Asthma exacerbations can be modeled by indirect stimuli that can trigger more or less specific immunological pathways and, consequently, mimic various pathophysiological features of asthma (Table 2) [30, 38 45]. In early clinical trials, validated exacerbation models can be applied for proof of mechanism (POM), as they only require short-term pretreatment in a (relatively) small number of patients [38, 39]. For obvious reasons, the choice of an adequate exacerbation model should Table 2 Most commonly known direct stimuli and indirect stimuli/exacerbation models Direct stimuli Indirect stimuli Cholinergic agonists: Physical stimuli: Acetylcholine Exercise Carbachol Cold, dry air Methacholine Histamine Pharmacological stimuli: Hypertonic saline Mannitol Adenosine monophosphate (AMP) Exacerbation models/cellular mechanism: Exercise (mast cells) Endotoxin (LPS) (neutrophils) Ozone (neutrophils) Virus (eosinophils/neutrophils) Aspirin (eosinophils) Allergen (mast cells/eosinophils) Steroid tapering (eosinophils)

16 116 K.F. Rabe et al. fit the asthma phenotype and the drug s target. In this chapter, we will address the most commonly applied exacerbation models. Mast Cell-Triggered Challenges Exercise and Related Challenges Exercise challenge and isocapnic hyperventilation of cold, dry air have been shown to induce bronchoconstriction in over 70% of the asthmatics [32]. Although the underlying mechanisms mediated by these stimuli are not fully understood, there is convincing evidence that airway cooling and drying cause the release of bronchoactive mediators from mast cells and stimulation of sensory nerves [31, 33]. Other indirect challenges acting through similar pathways, comprise adenosine monophosphate (AMP), hypertonic saline, and mannitol [33, 35]. When performed serially in a clinical trial, these indirect challenges require washout periods ranging from hours up to several days to correct for refractoriness and carry-over effects [31, 33, 46]. Models of Eosinophilic Airway Inflammation Allergen Challenge Allergy is a key mechanism inducing the development of persisting inflammation and structural changes within the airways that may result in symptomatic asthma. Several novel treatment modalities are presently being developed targeting the allergic mechanisms. Therefore, methods and tools that can rapidly predict a new drug s clinical efficacy are pivotal for the development of novel therapies. Allergen challenge meets most of these criteria and is a useful model for POM studies with anti-allergic therapies [44]. Presently, there are three allergen challenge techniques. First, nasal allergen challenge is a validated and reproducible method in which relevant allergens are administered intranasally to sensitized subjects [47, 48]. The subsequent upper airway response mimics symptoms and signs of allergic rhinitis and upper airway inflammation. Consequently, this model enables to study the (relationship between) kinetics of the clinical symptoms and the allergic upper airway response [49]. The allergen-induced nasal early response occurs within 10 min of the provocative allergen concentration and is mainly characterized by nasal congestion, itching, sneezing, and rhinorrhea, caused by mast cell-derived pro-inflammatory mediators. These symptoms can be scored and quantified by a validated questionnaire according to Lebel and colleagues [50]. Alternatively, the nasal late response is dominated by a prolonged nasal congestion and airway eosinophilia [49]. Rhinomanometry can be used for an objective assessment of the changes in nasal patency and several sampling techniques can be used to quantify

17 Lung Function and Bronchial Challenge Testing for the Allergist 117 the inflammatory nasal response [51]. Although nasal biopsies are the golden standard to study the cellular inflammatory response, serial samplings are limited due to its invasive nature [52]. Being less invasive techniques, nasal brushings, nasal lavages, and possibly nasal NO seem to be alternatives for repetitive nasal samplings of airway inflammation [53 55]. In patients with combined allergic rhinitis and asthma syndrome (CARAS), nasal allergen challenge may induce airway inflammation and hyperresponsiveness within the lower airways, thus allowing studying the link between both airway compartments [56 58]. The second technique comprises segmental allergen challenge, an invasive method requiring bronchoscopy for direct airway sampling (biopsy, bronchoalveolar lavage) following locally instilled allergen [59]. A safe and effective allergen dose is determined by prior inhaled allergen bronchoprovocation [60]. However, serial applicability of this method is limited due to its invasive nature, providing histopathological/cytological information on a restricted part of the lower airways. The third method includes inhaled allergen challenge, a highly reproducible exacerbation model mimicking several acute and some chronic features of (allergic) asthma [29, 30, 61]. Traditionally, the allergen dose is individually determined by a diluted skin prick test and/or methacholine PC 20 by Cockcroft s formula or a derived method [62, 63]. Within min of inhalation, this provocative allergen dose induces an early asthmatic response (EAR) in sensitized asthmatics that usually subsides at 3 h post-allergen. The EAR is an acute inflammatory airway narrowing, characterized by a self-limiting 15 20% fall in FEV 1 from baseline, in approximately 50% followed by a late asthmatic response (LAR) [29, 32]. In the patho physiology of the EAR, IgE-triggered mast cells have been shown to play the key role and convincing evidence points to their involvement in the LAR and the subsequent AHR [64]. The LAR represents an episode of progressive and longer-lasting airway narrowing, characterized by a fall in FEV 1 of at least 15% from baseline, usually occurring between 3 and 7 h post-allergen [30, 62]. In parallel, there is a more persistent airway inflammation in which activated eosinophils and their mediators play a key effector role causing tissue damage with subsequent AHR [65]. Consequently, to avoid carry-over effects in clinical trials, a washout period of at least 3 weeks is recommended between two consecutive allergen challenges [66]. The allergen-induced airway responses are defined as maximal percentage fall from baseline FEV 1, or, more precisely, as the area under the time response curve post-allergen (AUC), during the EAR (0 3 h), and the LAR (3 7 h), respectively [30]. Especially when combined with noninvasive sampling techniques enabling to study several aspects of the allergen-induced airway responses and their relation to the inflammatory events within the airways, the allergen challenge is a useful tool in POM studies with novel drugs targeting allergen-induced mechanisms [29, 44, 67]. And if properly conducted, allergen challenge can predict a drug s clinical efficacy [44]. Indeed, agents inhibiting the sequelae of the LAR have generally shown efficacy in clinical asthma (e.g., inhaled corticosteroids, leukotriene modifiers, and anti-ige), while those that did not affect the LAR have not. Based on previous studies and although not all drugs that did not show efficacy against the LAR have been tested in a clinical setting, this model seems to have a moderate positive predictive value, but an excellent negative predictive value [44].

18 118 K.F. Rabe et al. Aspirin Challenge Aspirin challenge is a more or less specific provocation test that can be used to confirm the diagnosis of aspirin-exacerbated respiratory disease better known as aspirin-induced asthma (AIA). The incidence of this asthma phenotype ranges from 3% to 21% among adult asthmatics and usually starts after the age of 30 years [68]. Upper respiratory tract symptoms are usually the first manifestations of AIA (rhinorrhea, nasal congestion, and polyps), while asthma and aspirin hypersensitivity develop some years later, persisting throughout life. Despite avoidance of aspirin and related nonsteroidal anti-inflammatory drugs (NSAIDs), approximately 50% of patients have severe persistent asthma, characterized by profound blood and airway eosinophilia [68]. Aspirin challenge is a specific and sensitive tool for both diagnostic and research purposes. This challenge can be performed with oral aspirin (ASA) or local (intranasal, endobronchial, or inhaled) solutions of lysine-aspirin (L-ASA) [43, 69]. In sensitized patients, aspirin challenge provokes an acute bronchoconstrictor response, often accompanied by extra-bronchial symptoms (conjunctivitis, rhinorrhea, nasal congestion, gastro-intestinal symptoms, and flushing of head and neck). The aspirin-induced airway inflammatory response is characterized by profound eosinophilia releasing large amounts of cysteinyl leukotrienes: bronchoactive mediators that can be quantified by urinary leukotriene E 4 [43, 69]. Despite a timeconsuming procedure requiring close monitoring for potential anaphylaxis, aspirin challenge is a useful tool for intervention studies with drugs targeting the aspirininduced airway responses, e.g., anti-leukotriene therapy [70]. Noninvasive Airway Sampling Methods and Biomarkers In contrast with the classical concepts of asthma, symptoms and lung function adequately reflect neither the activity of underlying airway inflammation nor the severity of AHR [71]. Hence, the efficacy of novel drugs should be evaluated by multiple (surrogate) biomarkers instead of symptom scores and lung function measurements only [38]. Consequently, an increasing number of noninvasive sampling methods have been developed to study the components of the asthmatic airway inflammation in search of responsive and reproducible biomarkers [32]. Exhaled Nitric Oxide Measurement of the fraction of nitric oxide in exhaled breath (FENO) is a simple and versatile method of noninvasive inflammometry. Nitric oxide (NO) is implicated in several biological processes, including the regulation of vascular and bronchial tone, neurotransmission, and inflammation. This volatile molecule

19 Lung Function and Bronchial Challenge Testing for the Allergist 119 is nonspecific for asthma and affected by several exogenous and endogenous factors including food, sex, atopic status, and smoking [72]. In patients with (allergic) asthma uncontrolled by ICS, FENO has been found to correlate with eosinophils, key components of (allergic) airway inflammation, and thus may be indicative of asthma control [73 76]. In agreement with this finding, ICS and other anti- eosinophil therapies, including leukotriene modifiers and anti-ige, dose- dependently reduced FENO [77 80]. Similarly, several tapering studies have shown that loss of asthma control is associated with an increase in FENO [81 83]. Hence, FENO has been proposed as a biomarker for the diagnosis of asthma and treatment monitoring. To this end, the American Thoracic Society (ATS) and the European Respiratory Society (ERS) issued recommendations for standardized exhaled NO measurements from upper and lower respiratory tracts in adults [84]. A separate document for children was approved by ATS and ERS [85]. The currently recommended FENO sampling technique is performed during a singlebreath exhalation against a fixed resistance allowing online measurements by validated chemoluminescence analyzers [84]. More recently, a two-compartment model has been proposed to discriminate between alveolar and conducting airways NO fractions [86, 87]. Using this model in patients with severe refractory asthma, alveolar NO appeared as a potential biomarker for distal airway inflammation that could not be reached by ICS [88]. The introduction of the less expensive, handheld NIOX MINO, yielding comparable measurements to the stationary, costly chemoluminescence NO-analyzers, will enable further implementation of NO measurements into daily practice [89]. Sputum Induction Induced sputum is another validated sampling method of the airways. This relatively simple, though demanding, method requires specific equipment and expertise including a dedicated lab and a certified (cyto)pathologist. The introduction of this reproducible technique helped to identify several components of the airway inflammation and their response to treatment, resulting in the definition of inflammatory asthma phenotypes [90]. In 2002, the ERS Task Force formulated guidelines for standardization of the different induction and processing techniques [34]. The commonly recommended method comprises inhalations of hypertonic saline (NaCl 4.5%) for three to four times 5 7 min through a mouthpiece the aerosols being generated by an ultrasonic nebulizer [34]. Subsequently, the sputum is processed within 2 h according to guidelines and divided into a cell pellet and supernatant [84, 91]. The pellet is resuspended, cytospined, stained, and the viability of the cells is assessed. Subsequently, cells are counted and the differentials are expressed as percentage of 500 nucleated, non-squamous cells. In analyzable sputum samples the percentage of squamous cells should not exceed 80% [84]. Depending on the availability of validated bioassays, several soluble biomarkers can be quantified in the sputum supernatant [91].

20 120 K.F. Rabe et al. Sputum eosinophil and neutrophil counts are reproducible biomarkers of airway inflammation and well-correlated with asthma severity [81, 90, 92, 93]. Sputum eosinophils are increased in models of eosinophilic exacerbations (e.g., allergen- induced LAR) and in eosinophil-dominated asthma phenotypes with an overall good response to anti-inflammatory interventions, especially inhaled cortico steroids [94 96]. In line with phenotype-directed treatment, Green and colleagues demonstrated that sputum eosinophils are superior guides of asthma control in patients with moderate to severe persistent asthma than symptom scores and lung function [90, 97]. Alternatively, the neutrophilic asthma phenotype, reflecting more severe asthma, is relatively refractory to inhaled corticosteroids and hence requires different targeted therapy [90]. In general, sputum eosinophil and neutrophil counts can serve as biomarkers for diagnostic purposes and early drug development [38]. Presently, several novel detection techniques are being tested to optimize quantification of soluble markers in the sputum supernatant, including peptidomics and metabolomics [98, 99]. Exhaled Breath Condensate Exhaled breath condensate (EBC) is another, relatively novel, noninvasive technology, allowing collection and measurement of various volatile compounds potentially implicated in disease processes [91]. An ATS/ERS Task Force document has been published on EBC addressing methodology and yet unresolved issues [91]. Exhaled breath consists of two components: the gaseous phase, containing volatile organic compounds (VOCs) such as NO and carbon dioxide (CO 2 ), and a liquid phase containing nonvolatile components including water-soluble inflammatory markers (Fig. 8). Fig. 8 CO-diffusion capacity, rebreathing functional residual capacity (FRC) but also NO-CO-diffusion capacity can easily be measured in daily routine (MasterScreen, PFT series, Cardinal Health)

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