Brush Biopsy and Mucosal Biopsy

ANDREW BUSH and PETR POHUNEK

Paediatric Respirology, Imperial School of Medicine, National Heart and Lung Institute, and Royal Brompton Hospital, London, United Kingdom; and Division of Pediatric Pulmonology, Second Pediatric Department, University Hospital Motol, Prague, Czech Republic

	WHAT DO WE KNOW?

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Fiberoptic bronchoscopy allows inspection and biopsy of superficial airway structures, typically including epithelium, basement membrane, and lamina propria. Thus, mucosal structures and events can be studied, but the technique is less applicable to studies of smooth muscle. Numerous bronchoscopic studies in adults have established the importance of helper T type 2 (Th2) lymphocyte-mediated, eosinophil-driven airway inflammation in adults with asthma. So commonplace is fiberoptic bronchoscopy in adult volunteers that serial bronchoscopies, for example, endobronchial segmental allergen challenges with repeat bronchoscopy, segmental lavage, and biopsy, are routinely performed. Bronchoscopy has enabled fundamental questions about airway biology and pathophysiological mechanism of inflammation to be addressed, not only in the context of asthma. A MEDLINE search combining [asthma and biopsy and (airway or endobronchial or bronchial or mucosal)] yielded 162 references. However, when this search was combined with [child or paediatric or pediatric or children] this was reduced to only three relevant hits, none of which were in the English language (1). One other study looked at changes after respiratory infection (4). Articles about adults have sometimes contained material about older children (5), and there are occasional informative case reports (6). The questions that arise are as follows.

1. Does this matter; are adults suitable models for pediatric asthma?

2. If it does matter, can we address this disparity through noninvasive testing?

3. If noninvasive testing is not adequate, how can we safely and ethically perform more bronchoscopic studies?

This article deals almost exclusively with asthma and related diseases. Other diseases are mentioned briefly.

Can We Learn about Asthma in Children from Studies of Adults?

Although eosinophilic inflammation is fundamental to asthma, there are clear-cut indications that other inflammatory pathways are also important. Even in adult studies, there is a marked dissociation between bronchial hyperreactivity and eosinophil count, both in induced sputum and in bronchial biopsy (7, 8). Subgroups of patients with asthma but without raised eosinophil counts and who are poorly responsive to inhaled steroids have been described (9). There is evidence that virus-associated wheeze may be noninflammatory and certainly noneosinophilic (10, 11) and that the pathology of severe asthma may not be the same as that of milder phenotypes (12). Finally, a bronchoscopic study of severe asthma, which included two older children, demonstrated that neutrophils but not eosinophils were elevated in bronchoalveolar lavage (BAL) fluid (5). Thus, there is already evidence that we have much more to learn about different patterns of asthmatic inflammation. Furthermore, studies in adults may only be telling us about cellular and molecular cycles that sustain inflammation, and nothing about initiating events. Thus, although we have learned much from the adult airway, it is inevitable that pediatric studies be pursued. Such comparisons as are available between pediatric and adult asthma have demonstrated at least quantitative differences in synthesis of proinflammatory cytokines in peripheral blood mononuclear cells (13).

In conclusion, adult studies, whether using bronchoscopy or less invasive means, cannot replace pediatric studies. There are important research questions that can be addressed only in children, and thus there is an ethical imperative to do those studies in children.

Are Noninvasive Studies Adequate?

Detailed consideration is given to many other techniques in separate parts of this workshop. In summary, methods available include sputum induction (only applicable to older children), breath analysis (gaseous and liquid/particulate phase; breath condensates), peripheral blood studies, and urinalysis. Many of these methods are either only an indirect reflection of events in the airway (blood, urine), or require subject cooperation (sputum induction, some breath tests). Furthermore, if it is reasonable to assume that direct inspection of airway biopsy is the "gold standard," there are few comparisons of bronchial histology and noninvasive markers in pediatric practice. In a small study, we have shown that elevated exhaled nitric oxide levels correlate with the presence of airway eosinophils but not lymphocytes in bronchial biopsy specimens (14). Until we know how and to what extent noninvasive markers correlate with bronchial histology, further invasive studies are inevitable. Furthermore, studies of airway remodeling are unlikely to be addressed by noninvasive means. The remainder of this article deals with pediatric bronchoscopy in clinical practice and for research.

Pediatric Bronchoscopy in a Clinical Context

Fiberoptic bronchoscopy is widely performed throughout the world even on very small children (15). The child requires either heavy sedation (16) or a general anesthetic, and techniques of anesthesia (face mask, laryngeal mask, intubation) depend on the questions being asked. Choice of anesthesia or sedation depends in part on the age of the child and the procedures planned. The procedure is generally safe and well tolerated. Endobronchial biopsy under direct vision and brush biopsy are routine and safe procedures for the diagnosis of tuberculosis and other airway granulomatous conditions (17). Transbronchial biopsy (TBB) can also be performed in children, and has an established place in lung transplant recipients (18), and a more controversial role in interstitial lung diseases (19). However, it is clear that TBB carries a greater risk than other procedures, and has only relatively recently been accepted as a research procedure in adult studies (20).

Retrospective analysis of data from Prague by one of the authors confirms the safety of bronchial biopsy. Over a 10-yr period, 278 endobronchial biopsies were performed (262 using the fiberscope). The mean age of the children was 11.6 yr (range, 8 mo to 19 yr). No more than three attempts were needed to obtain a macroscopically adequate sample; 71% of biopsies provided material adequate for histological diagnosis. There was a trend for biopsies to be more informative in older children, but differences between age groups did not reach significance. Only trivial and superficial bleeding was seen, and this lasted less than 2 min; there were no other complications. The best biopsies were obtained with new forceps, and the quality improved with practice. Oxygen saturation and electrocardiogram were monitored throughout and for 2 h after the procedure, and there were no significant decreases related to the biopsies. A chest radiograph was not routinely performed after endobronchial biopsy; it is mandatory after transbronchial biopsy.

Pediatric Bronchoscopy in a Research Context

Ethical guidelines for research in children conclude that only research procedures of no or minimal risk can be performed, unless the individual child will benefit from that research procedure. This would preclude children undergoing bronchoscopy purely for research; an adult, however, can legitimately consent to a research bronchoscopy being performed on him/ herself. However, the risk of the procedure lies not with the bronchoscopy but mainly with the anesthesia. Thus, if the child is being anesthetized for another purpose, then it is surely legitimate to seek consent for a bronchoalveolar lavage (BAL) or an endobronchial biopsy, provided that (1) they are performed by an experienced bronchoscopist and (2) there is no obvious contraindication such as a coagulopathy or respiratory compromise (if, e.g., the child is undergoing bronchoscopy for another purpose). The Royal Brompton Hospital Ethics Committee accepts that it is ethical to perform these additional procedures provided that prior consent is sought from the child and family. Indeed, there is an acknowledged ethical imperative to gain the maximum information from such procedures. In this context, it is disappointing to note that many bronchoscopic studies in cystic fibrosis in which lavage has been performed have not included bronchial biopsy (21). It is important that material be prepared for electron microscopy and immunohistochemistry as well as standard histopathology. Close collaboration between the laboratory and the clinician is essential.

Several groups have performed blind BAL for research purposes at the time of anesthesia for routine surgery, with great success. It would be ideal to do bronchoscopy under the same circumstances; the logistical difficulties are great, because the operating list may be delayed. In experienced hands, using prepared endotracheal access, the extra time required for this procedure would be small.

Methodological Problems in Biopsy Studies

The chief problems associated with biopsy studies relate to the size of the bronchoscopes available, the acquisition of normal data, and the obtaining of viable cells by brushing in the presence of airway inflammation.

Adults have big airways, children have small ones. Typically, adult studies use a 6.0-mm external diameter endoscope with a 2.8-mm biopsy channel. This instrument is too large for use in most children. The 4.9-mm endoscope has a 2.2-mm biopsy channel, and the 3.6-mm endoscope (and the prototype 2.7-mm instrument) have only a 1.2-mm channel. Thus, biopsies are much smaller and may be correspondingly more difficult to interpret. It may be advantageous to take the biopsy from more peripheral and thus sharper carinas, where the forceps usually grasp a better piece of tissue. The fiberscope is solid, and may occlude much of the airway, leading to the risks of carbon dioxide retention and inadvertent positive airway pressure (25, 26). Thus, the endoscope used must allow adequate ventilation around the instrument during the procedure, and the diameter of the instrument used must be much less than the narrowest diameter of the airway. The alternative is to perform rigid bronchoscopy; historically, most pediatricians have left this to thoracic surgeons. This allows larger biopsies to be obtained, but carries the corresponding risk of bronchial perforation. We recommend that if a rigid bronchoscope is used, then standard flexible forceps should be inserted along the optical rod, allowing the biopsy to be taken under direct vision.

A further difficulty is the acquisition of control data. By definition, children with healthy lungs will not undergo diagnostic bronchoscopy. However, older children with, for example, a suspected endobronchial foreign body, or undergoing surgery for congenital lung disease, could reasonably be asked to give consent for a bronchoscopic biopsy.

Endobronchial Brush Biopsy

Brush biopsy is a well-established clinical technique, for example, in the diagnosis of tuberculosis in children and measurement of ciliary beat frequency to diagnose ciliary dyskinesia. A protected (sheathed) brush can be used with the 4.9-mm bronchoscope. The only brush available for the 3.6-mm endoscope is unsheathed and there is a risk of losing cells if the brush is pulled back through the channel. The best yields are obtained if after brushing the airway wall, the brush is left protruding from the bronchoscope while the instrument is withdrawn, taking care not to touch the brush onto the upper airway. The tip of the brush is then cut off, and the cells displaced by agitating it in appropriate medium. Even with these precautions, if the airway is heavily inflamed, it may be difficult to obtain viable cells; often, only necrotic material is obtained (27).

Alternatively, a direct smear can be made onto glass slides, with immediate fixation to preserve cell morphology. However, the advantage of this method over lavage and bronchial biopsy is the potential to access fresh single epithelial cells, albeit only those cells that are poorly adherent and therefore easily shed.

Safety of Bronchoscopy in Older Children with Difficult Asthma

There has been no systematic study of the safety of bronchoscopy in young children with asthma. Therefore, we must rely on (1) reports of clinical bronchoscopy, which is a well-established procedure even in the youngest children and (2) experiences in older children with asthma. The Brompton group has collated information gathered from bronchoscopies performed in 34 children with severe asthma. There were 21 males, and the mean age of the group was 11.7 yr (range, 6-16 yr). The mean dose of inhaled fluticasone propionate or equivalent was 2.9 mg/d (range, 1.6-4.0 mg/d), and 26 patients were also taking a long-acting ₂-agonist. The FEV₁ before bronchoscopy was on average 78% of the predicted value (range, 41-109% pred). Ventilation was performed via an endotracheal tube (n = 27), laryngeal mask (n = 6), or face mask (n = 1). Various anesthetic techniques were used, usually induction with sevoflurane and maintenance of anesthesia with a mixture of nitrous oxide and oxygen. All children underwent BAL (two aliquots of 40 ml, bronchoscope wedged in right middle lobe), and at least six endobronchial biopsies, always from the same (right) side, were taken. Other than minor bleeding at the time of the biopsy, which stopped spontaneously in all cases, there were no acute complications. After the procedure, one child was coughing and unwell and kept in overnight; all others went home on the day of the procedure. Two children required extra bronchodilators for 2 d after the procedure; one child was readmitted to his local hospital with a fever and infiltrate on the same side as his BAL. He was given intravenous antibiotics and recovered rapidly. Interestingly, his BAL subsequently grew Streptococcus pneumoniae. No other complications were encountered (D. Payne, unpublished data).

Bronchoscopy in Children with Undiagnosed Respiratory Symptoms

Although wheezing is common in children and usually improves without treatment, delayed treatment of the inflamed airway may prejudice the development of normal lung function. At the moment, there is no test predictive of persistent wheeze. One study, as yet published only in abstract form, examined the predictive value of endobronchial biopsy in 27 children between the ages of 1.2 and 11.7 yr (28). Children experiencing recurrent symptoms underwent diagnostic bronchoscopy, and were then reevaluated 22-80 mo later. Patients who were subsequently diagnosed as asthmatic had a significantly greater number of activated eosinophils in their biopsies (Figure 1) than those not diagnosed with asthma. In addition, there was an increase in mast cell numbers and thickness of the subepithelial collagen (Figure 2) in the patients subsequently diagnosed with asthma. However, there was overlap between the two groups, and it is not known whether such measurements can be refined to make them of diagnostic use in individuals. Nevertheless, they raise important questions as to the time of onset of inflammation with respect to symptoms and the relationship between inflammation and airway remodeling. In contrast, in a study of 16 wheezy children, Teig and coworkers (29) found that subepithelial collagen and fibronectin deposition on bronchial biopsy could not be used to discriminate between infants who would and would not outgrow their wheezing.

View larger version (7K): [in this window] [in a new window]   Figure 1.   Numbers of activated (EG2+) eosinophils in children who subsequently did or did not develop asthma (28).

View larger version (86K): [in this window] [in a new window]   Figure 2.   Bronchial biopsy from a child with a 2-yr history of clinical asthma. Note the epithelial shedding and marked thickening of the basement membrane. The biopsy was prepared as follows: formol fixed, paraffin embedded, and stained (hematoxylin-eosin). Original magnification: ×150.

Studies in Other Diseases

There are few data in the published literature on the use of biopsy techniques in other diseases. Durieu and coworkers (30) studied structural changes in the bronchial wall in patients with cystic fibrosis and found evidence of airway fibrosis and matrix degradation, presumably due to proteolytic activity. The relationship between early disease of the lower respiratory tract and chronic cough was determined in a bronchoscopic study of seven children (4). Early infection was related to subsequent chronic epithelial inflammation. However, the data are based on small numbers of patients and so no firm conclusions can be made.

	WHAT DO WE NEED TO KNOW?

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There is nothing that we need to know about the techniques of bronchoscopy, the safety of the procedure, and the ethical constraints to the procedure. What we do need to know is how we can ethically use this technique to greater advantage, and what areas are likely to be most fruitful for study.

1. First, we need to know far more about the early proinflammatory events in the pediatric airway if we are to devise therapeutic interventions to prevent progression to established disease.

2. We need to try to understand if there is a presymptomatic phase of inflammation, seeking every opportunity to obtain biopsies of the airways of high-risk, presymptomatic infants. The only bronchoscopic study performed when children were symptomatic, but without an established diagnosis, showed that in those children who went on to develop clinical asthma, inflammation and airway remodeling were present before the occurrence symptoms of sufficient severity to merit a diagnosis of asthma (28).

3. A further issue is the site of biopsy. We can sample the central airways and one or two branching generations under direct visual control. Smaller airways can only be biopsied blind, which is not considered to be safe and is therefore unethical as a research procedure. We need to know whether and to what extent central airway biopsy is representative of distal airways in children.

4. We need to know more about the different molecular and cellular phenotypes of asthma so that treatment can be rationalized, in particular for those patients with severe asthma. Preliminary analysis suggests that there are several phenotypes of severe asthma; these include persistent eosinophilic inflammation despite high-dose oral prednisolone; persistent symptoms with no evidence of airway inflammation; and eosinophilic inflammation that responds only to high-dose steroids (31). Bronchoscopy is fully justified in these patients if it can be shown that treatment (e.g., steroid-sparing agents, subcutaneous terbutaline) is influenced by the results of biopsy.

5. We also need to know more about the early stages of airway remodeling so that appropriate prevention strategies can be devised. Although the traditional view of remodeling is that it is the result solely of unchecked airway inflammation, more recent studies have challenged this view. We need to know what initiates and also who sustains remodeling and how to prevent it.

6. We need to become more aware of the risks of not performing a bronchoscopy. For example, failing to treat eosinophilic inflammation in an infant wheezer may risk irreversible airway remodelling (32, 33), while overtreatment with inhaled steroids risks growth suppression (34).

	HOW CAN WE ACHIEVE THIS?

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1. We need to devise noninvasive means of monitoring inflammation and remodeling, and validate them against invasive studies; bronchoscopy can never be a routine clinic monitoring tool, but can be used to validate other methods based on (for example) breath analysis.

2. We need to seek opportunities for doing further bronchoscopic studies at the time of anesthesia for other procedures, or as part of a clinically indicated bronchoscopy, where bronchial biopsy and brushing should be considered as a routine part of the procedure. These studies must have a clearly defined hypothesis and plan of investigation that are clinically and scientifically valid, and should not merely degenerate into haphazard specimen collection.

3. Consideration needs to be given to obtaining control data from normal children, for example, those undergoing bronchoscopy for removal of an endobronchial foreign body or prior to lobectomy for cystic adenomatoid malformation. For example, perhaps some children with atopic dermatitis who need an anesthetic for an unrelated procedure should have an airway biopsy at the same time to determine whether atopy in the absence of respiratory symptoms is associated with histological changes in the airways. These studies will require resources; at the least, a biopsy study will take 10 min and this may significantly impinge on operating time and the tolerance of our colleagues. Those who edit and referee for journals should question the publication of bronchoscopic studies using lavage but not biopsy.

4. We need to improve bronchoscope technology to allow us to obtain larger biopsies from small children without sacrificing safety. Advances in optics and loss of some of the maneuverability of modern bronchoscopes may achieve this (as, e.g., with the 2.7-mm endoscope with a 1.2-mm channel). This will provide pathologists and immunologists with better samples, and enhance our knowledge of fundamental molecular mechanisms. Only in this way can we challenge the many widely held and probably erroneous assumptions surrounding asthma.

	Footnotes

Correspondence and requests for reprints should be addressed to Andrew Bush, M.D., Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. E-mail: a.bush{at}rbh.nthames.nhs.uk

	References

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