Lesson 4, Volume 16—Induced Sputum

By Ratko Djukanovic, MD, DM, FRCP

Objectives

1. Understand the methods and safety issues of sputum induction.
2. Understand the methods for processing sputum for cell and mediator analyses.
3. Recognize the need for validation of methods.
4. Appreciate the usefulness of induced sputum as a research tool.
5. Appreciate that there are some uses for induced sputum in clinical practice.

Key words

asthma; COPD; induced sputum; inflammatory cells; mediators; safety

Abbreviations

BHR = bronchial hyperresponsiveness; DTE = dithioerythritol; DTT = dithiotreitol; ECP = eosinophil cationic protein; PEF = peak expiratory flow

However, it was only in the early 1990s that the first attempt to standardize the method of induction and processing was made. Since then, numerous studies have been published attesting to the safety and relative feasibility of this method, and eventually leading to the establishment of the first task force on induced sputum methods sponsored by the European Respiratory Society.

Although sputum induction appears to be a safe and relatively simple method, it is important that the staff involved at all stages of this test be properly trained and, preferably, certified at one of the centers in the United States or Europe that has a reputation and considerable experience with this technique. Induction itself is not without hazard; therefore, a physician must be in the vicinity at all times if a nurse or technician is performing the induction. The induction procedure and analysis must adhere to approved standard operating procedure protocols, and all assays used to determine mediators and cells must be validated.³

Methods of Sputum Induction

Sputum can be obtained either spontaneously⁴ or using hypertonic saline solution,^5,6 depending on whether or not the patient has a productive cough. Induction with hypertonic saline is by far the most common, with analysis of spontaneously expectorated sputum possible only in patients who have a productive cough as a part of their disease (eg, chronic bronchitis or acute asthma exacerbation).

Hypertonic saline is delivered in the form of an aerosol produced by an ultrasonic nebulizer. There is at present no consensus as to the optimum concentration of NaCl that is needed for successful induction. Adequate samples have been obtained even with normal, ie, physiologic 0.9% saline,⁷ although the success rate is better with hypertonic saline.⁸ There are no differences in the cell composition between sputum samples induced by isotonic or hypertonic saline,^7,8 but it is unknown whether there are differential effects on mediator concentrations in the sputum fluid phase. Many studies to date have used incremental concentrations of NaCl, beginning with 3% and increasing to 5%.⁴ The reason for using isotonic saline or increasing concentrations of hypertonic saline has been the concern that hypertonicity might cause excessive bronchospasm. However, there have been no carefully conducted studies to show that there is any advantage in using incremental concentrations of NaCl as opposed to protocols using a single concentration of hypertonic saline.

So far, there has also been no consensus as to the type of nebulizer that should be used, although ultrasonic nebulizers have been reported to be more successful than jet nebulizers.⁸ Whether and to what extent the choice of ultrasonic nebulizer influences the success rate and the sputum cell and mediator contents is unknown. There are reports that induction using higher-output nebulizers is less acceptable to patients.⁸ Despite these uncertainties, there is a consensus that ultrasonic nebulizers should be used, with an output of 1 mL/min being sufficient for successful induction.

The duration of inhalation of saline should be kept constant whenever possible. Current recommendations state that the optimal time is 15 min; however, induction can be continued for another 5 min if an adequate sample has not been obtained. Problems may arise when hypertonic saline causes bronchoconstriction, which requires the procedure to be aborted before 15 min have elapsed. When repeat inductions are performed, as is the case in clinical trials with pre- and posttreatment inductions, the induction time should be the same to enable comparison. It has been clearly shown that the cell composition varies with the duration of inhalation, with neutrophils decreasing and macrophages increasing with more prolonged challenge, reflecting sampling of more distal airways where macrophages predominate.

Contamination of the sputum sample with saliva is a major concern and a common problem. Because of their size, squamous cells cover the inflammatory cells in cytospins, making counting of these imprecise. In an attempt to reduce squamous cell contamination various methods have been recommended: blowing the nose prior to induction and wearing a nose clip during induction, washing the mouth with water before each expectoration, and wiping the mouth with paper tissue. None of these methods has been clearly shown to be effective, ie, no comparative studies have been performed. Active encouragement of the patient to cough from deep within the chest as opposed to spitting from the mouth is recommended.

Safety of Sputum Induction

Safety of any research procedure is paramount; in respect to sputum induction, certain measures have to be taken before and during induction. Lung function measurement, preferably FEV₁ rather than peak expiratory flow (PEF), should be performed before induction. When working outside the laboratory (eg, at the workplace), PEF can be measured as an alternative. In cases of poor lung function, induction should be commenced with isotonic saline, especially in patients with very hyperreactive airways as is the case in severe asthma or during exacerbation. Lung function should also be monitored at regular intervals during induction as an indicator of tolerance, preferably using FEV₁ (see Table 1).

Premedication should be given as standard using a b₂-antagonist bronchodilator administered via metered-dose inhaler. Although the full effects of this treatment on sputum parameters are not known, premedication is necessary in order to avoid bronchoconstriction caused by exposure of the airways to hypertonic fluid. Studies have reported no effect of albuterol on inflammatory cell counts⁸ and levels of eosinophil cationic protein (ECP), but there may be possible effects on histamine levels. The effects on other mediators are unknown. There is a consensus that a standard dose of albuterol (200 mg) should be given. Pretreatment with higher doses is not recommended; this does not prevent excessive bronchospasm induced by hypertonic saline and increases the risk that subsequent doses of albuterol may be ineffective.

Analysis of Induced Sputum

As stated above, analysis of sputum, although simple at first glance, demands trained staff who adhere to standard operating procedure protocols that have been validated and for which the technicians have been trained and preferably certified in another laboratory with experience with this technique. This is particularly important if samples are collected as part of multicenter trials, as is increasingly the case. In addition, each assay used to analyze the cells and mediators must be validated.

There are two validated and widely used methods for processing induced sputum: the first consists of selecting the visibly viscous (mucoid) parts of sputum using an inverted microscope,⁴ and the second consists of processing the entire expectorated sample. At present, there are no recommendations/guidelines as to which method should be used because not enough comparative studies have been conducted. The advantage of selected sputum is slightly lower squamous cell contamination. Unselected sputum can contain saliva, although this can be avoided by giving clear instructions to the patient, and removing the fluid that looks obviously like saliva. The disadvantage of the sputum selection method is that it needs an inverted microscope, which is not always readily available, especially if the induction is conducted outside the laboratory. In addition, it takes a little longer. Finally, the accuracy of cell counts is better if squamous contamination is low, which is the case with selected sputum, but it is unclear whether differential cell counts are different in the two methods.

Before analysis, the sputum sample must be solubilized. This should be done as soon as possible, or at least within 2 h, as there is a possibility of deterioration, not only of cells but also of soluble mediators. Samples should be kept cold (on ice). Sputum is usually expectorated into a Petri dish and then removed into a conical tube, where it is solubilized with either dithiotreitol (DTT) or dithioerythritol (DTE). Both act by breaking up disulfide bonds in the large mucin molecules. Sputum can also be solubilized with phosphate-buffered saline. Both methods enable the release of cells and mediators from the mucus. However, DTE and DTT have been shown to be far superior for making high-quality cytospins, but both can interfere with the detection of soluble mediators by way of their effects on disulfide bonds, as is the case with chemokines. DTT and DTE appear not to affect detection of interleukin-5 and fibrinogen, but clearly raise levels of ECP and IgA,⁹ not because of cell activation but because of increased release from mucins.

As already stated, sputum should be processed rapidly in order to avoid cell and mediator degradation. There is probably a role for protease inhibitors, which have been used in some studies,¹⁰ but this has not been fully elucidated. For the same reasons, samples should be stored on ice prior to processing, and centrifugation should be conducted at 4ºC using speeds of 400 g, as higher speeds may activate cells.

Sputum is very rich in mediators, much more so than BAL fluid. The presence of fluid-phase mediators in sputum can be assessed using immunoassays (enzyme-linked immunosorbent assay or radioimmunoassay), enzyme assays, or bioassays. Most studies to date have used immunoassays to detect mast cell and eosinophil products such as tryptase, ECP, and myeloperoxidase. Before accepting the results obtained by immunoassay, it is essential that each assay, ie, each antibody used as part of the detection system, be validated. Different antibodies raised against the same mediator may be affected differently by DTT/DTE. Sputum is a complex medium rich in proteins and particularly in mucins, with mucins being the greatest component of sputum. It also contains degradation products such as DNA and actin released from dead cells, which can interfere with cytokine function.¹¹ Mediators are also often associated with other molecules present in sputum, such as autoantibodies (eg, against interleukin-8) and a₂-macroglobulin.⁹ Association of mediators with these may hide the epitope for which the antibody in the enzyme-linked immunosorbent assay is specific. Methods are currently being validated to increase extraction of mediators, including the use of acidification to precipitate large mucins, and the application of N-cetyl-NNN-trimethyl ammonium bromide, a cationic detergent, which lyses cells and thus enables extraction of total (released and intracellular) mediators such as ECP and myeloperoxidase.

Because of all the possible interactions between mediators and other components present in sputum, validation of the assay must include what are referred to as "spiking" experiments in which a known quantity of mediator standard is added. Some studies have gone further in detecting recovery by using radiolabeled mediators, which does not depend on good recognition by antibody and which may point to loss of mediator sticking to containers used during processing.¹⁰

Enzyme assays have also been used successfully to analyze sputum. Sputum is rich in such enzymes as neutrophil-derived elastase and cathepsin G, which can be detected using specific chromogenic substrates that produce a colored product that can be quantified by spectrophotometry. The activity of some proteases, such as matrix metalloproteinases, can be quantified using substrate gel zymography.

The fluid phase of induced sputum is increasingly being used for the detection of functions attributable to cytokines such as chemokines.⁵ There are also attempts to grow cells out of sputum. At present these methods have not been fully validated and should be reserved for centers with considerable expertise.

The Use of Induced Sputum in Clinical Research

The apparent feasibility and so far excellent safety of induced sputum has resulted in a large number of publications in which this method has been used both to provide more insight into the pathology of asthma and its determinants of severity and to study the effects of established and novel asthma drugs. Ideally, one would wish to use this noninvasive tool to either confirm or exclude a clinical diagnosis (such as asthma), and to enable better monitoring of disease activity and response to treatment. In addition, one would hope that effects of occupational and environmental hazards might be amenable to study with induced sputum.¹² Whenever considering the use of induced sputum, one has to compare the merits of this technique with those of other noninvasive tools, including measurement of volatile gases in exhaled air and analysis of mediators or their metabolites excreted into urine. The ability to measure mediators in condensates of exhaled air is looking attractive, but it is far from being standardized, and the sensitivity of methods used in studies of urine is inferior to that of sputum.

Induced sputum has most widely been used in the study of asthma. The presence of elevated eosinophil counts above the upper limit of 3% of nonsquamous cells can help diagnose this condition in the absence of typical findings on examination and lung function testing. The main feature reported so far is sputum eosinophilia, which appears to be broadly correlated with disease severity and BHR.^5,13,14 Eosinophil counts also rise after exposure to allergen and are reduced after treatment with a number of anti-inflammatory drugs. This has suggested that induced sputum can be used to monitor and adjust asthma treatment. Thus, studies have shown that apparently stable asthmatics with higher sputum eosinophil counts are more likely to develop an exacerbation on reduction of their regular corticosteroid therapy. The presence of eosinophilia has been shown to predict response to corticosteroids in patients with airways disease, including both asthma¹⁵ and COPD.² Persistently elevated eosinophil counts in patients who are taking corticosteroids would indicate either poor compliance or a degree of relative corticosteroid resistance.¹⁴ It is not as yet entirely clear whether further increases in corticosteroid dosages in such patients would necessarily have a beneficial effect in terms of reducing eosinophils or achieving better clinical control. It is, however, known that reduction of the corticosteroid dosage leads to increased sputum eosinophilia, which precedes clinical deterioration¹⁶; this could be used to decide on the minimum dose of corticosteroids needed to control disease.

The presence of eosinophilia has also been used successfully in the context of occupational asthma.¹² This could be used as a diagnostic tool because eosinophilia can be seen during exposure to the sensitizing agent, and decreases when the patient has been removed from work.¹⁷ The features of occupational asthma are similar to those of the more common atopic forms of the disease.

All studies of induced sputum in asthma show a great deal of heterogeneity within the asthmatic population. This is seen even in well-characterized patients classified according to symptoms and air flow limitation.¹⁴ This would suggest that there are phenotypes of asthma that are not apparent on the basis of the clinical picture alone, and further research is needed to identify factors in sputum that help to better define these phenotypes. Sputum studies have also identified neutrophilia as a feature of more severe asthma¹⁴ and exacerbations,¹⁸ especially if caused by viral infection.

The study of sputum has also been very helpful in COPD, but it should not be used to diagnose this syndrome. In view of the chronic nature of COPD, spontaneous sputum production analysis is much easier in COPD. Sputum has been used to study the relationship between bacterial colonization and inflammation and to predict with high sensitivity, but not as high specificity, the presence of bacteria in discolored sputum when antibiotic treatment may be most needed. Sputum is also increasingly used to study cytokines in this disorder. However, it is unclear whether there are clear advantages of using induced as opposed to spontaneously expectorated sputum. Induction with saline is safe in COPD and that should not be a factor when choosing between the two methods. Sputum expectorates can be expected to contain more cellular debris. To what extent this may influence soluble mediators is not clear, and it should be borne in mind that induction by hypertonic saline does probably cause hypersecretion and should therefore be seen as a form of challenge.

The Value of Induced Sputum in Clinical Practice

It is increasingly believed that there is a place for induced sputum in the diagnosis and monitoring of asthma, but its exact place in routine practice remains to be determined. The presence of sputum eosinophil counts above the upper limit of normal has been shown to be better than PEF variation and bronchodilator response at making the diagnosis of asthma. Initial studies suggest that corticosteroids are ineffective in individuals with airways disease in whom sputum eosinophil counts are < 3% of total nonsquamous cells.¹⁵ However, it remains to be seen how widely this can be applied in practice and whether it might guide the practitioner to use other asthma drugs. At least two studies are currently underway comparing therapeutic regiments that aim to reduce eosinophil counts to within the normal range with standard protocols based on lung function and symptoms alone. Further studies are also needed to compare the effects on sputum markers of inflammation of doubling the dose of inhaled corticosteroid as compared with adding long-acting b₂-antagonists to the current dose of inhaled corticosteroid. One study has suggested that reduction of the dose of corticosteroid in the presence of good disease control by a long-acting b₂-agonist may mask untreated eosinophilic inflammation,¹⁹ but whether this is necessarily associated with poor overall control such as more frequent exacerbations is not clear. Many studies to date have shown the latter approach to be superior in terms of clinical control; what is now needed is to clearly show that this is not at the expense of increased inflammation masked by potent bronchodilatation.

Induced sputum has enabled the identification of a new respiratory syndrome consisting of chronic cough with sputum eosinophilia but the absence of BHR. A significant proportion of patients with cough attending clinics have sputum eosinophilia; some of them are eventually identified as having asthma, but a significant proportion also have eosinophilic bronchitis without asthma.¹ Importantly, the latter condition responds favorably to inhaled corticosteroids. Therefore, protocols have been designed that incorporate induced sputum as part of the assessment of patients with chronic cough of unknown origin.²⁰ Cough is occasionally associated with gastroesophageal reflux. One recent study has noted increased macrophages that are laden with lipid in induced sputum from such patients.²¹

The Use of Induced Sputum To Study Inflammation in Children

Probably the greatest benefit of induced sputum has been in its application to the study of airways inflammation in children, in whom the use of bronchoscopy has been very limited for ethical and safety reasons.²² Studies to date have reported on successful and safe sputum induction in children as young as 6 years. Protocols similar to those used in adults have been applied to children, with the premedication and monitoring issues being similar for adults and children. Similar success rates have been achieved and it has been suggested that entertainment during induction, such as the use of a cartoon video, helps improve the success rate. As with adults, there has been some variety in the processing methods used for sputum samples from children. There is great potential for induced sputum to be used in further long-term monitoring of children. It would be particularly interesting to see whether there are any changes during spontaneous remissions of asthma during puberty and whether there are any markers in sputum that predict which children will subsequently have a recurrence of the disease.

References

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