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Ann Thorac Surg 1995;60:1605-1607
© 1995 The Society of Thoracic Surgeons

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Original Articles: General Thoracic

Influence of Sample Number and Biopsy Site on the Histologic Diagnosis of Diffuse Lung Disease

Departments of Pathology, Internal Medicine, Biostatistics, and Surgery, School of Medicine, and School of Public Health, The University of Michigan, Ann Arbor, Michigan

Accepted for publication July 20, 1995.

	Abstract

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Background. Although open biopsy is considered the optimal method for obtaining lung tissue for the diagnosis of diffuse infiltrative pulmonary disorders, there are no universally established guidelines concerning biopsy site selection and the ideal number of tissue samples. Relatively few investigations have been devoted to the influence exerted by the site and number of biopsy samples on the histologic diagnosis.

Methods. Seventy-seven open biopsy samples obtained from different lobes of 28 patients with idiopathic pulmonary fibrosis were analyzed. The histopathologic features were evaluated semiquantitatively and the results from each sample compared with those of the other samples obtained from each patient.

Results. Statistically significant differences in histopathologic features were not observed between samples.

Conclusions. A single generous (2 cm or greater diameter) sample, obtained from a representative region of the radiographically most involved lobe, will suffice for diagnostic and evaluation purposes.

	Introduction

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See also page 1607.

Open lung biopsy is generally regarded as the optimal method for obtaining lung tissue for the diagnosis of diffuse interstitial lung disease [1], and thoracoscopic biopsy has become increasingly popular in this regard [2, 3]. The site of biopsy is primarily determined by the extent and location of the radiographic abnormalities, and by physical inspection at the time of operation. Although several studies have evaluated the influence of biopsy site on the histologic diagnosis, relatively little attention has been paid to determining the optimal number of biopsy samples necessary for accurate diagnosis [4–9]. In this communication, we hypothesize that the number and site of biopsy samples has little influence on the evaluation of histologic features present in open lung biopsy samples obtained from patients with idiopathic pulmonary fibrosis.

	Material and Methods

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Patients enrolled in the University of Michigan Idiopathic Pulmonary Fibrosis Specialized Center of Research program formed the basis for the study. Participating in the prospective project were 15 male and 13 female patients whose mean age was 55.7 years (range, 28 to 74 years). As part of their evaluation, all patients underwent open lung biopsy by formal thoracotomy (n = 10) or video-assisted thoracoscopy (n = 18). Similar-sized biopsy samples were obtained by both techniques. In the case of open thoracotomy, areas manifesting radiographic abnormalities were sampled. Direct palpation helped localize the biopsy site. With regard to thoracoscopy, peripheral portions of radiographically abnormal parenchyma were sampled. All three lobes on the right side or the upper and lower lobes of the left lung were sampled. Obviously fibrotic areas as well as the dependent tips of the lingular segment or middle lobe were not sampled. The diagnosis of usual interstitial pneumonitis (the pathologic counterpart of idiopathic pulmonary fibrosis) was based on previously published pathologic criteria [10] and the clinical and radiographic findings.

Each specimen was processed in a routine fashion, and four histologic sections were prepared from each tissue block. Individual slides were stained with hematoxylin and eosin, pentachrome stain (which demonstrates elastic tissue, collagen, and mucopolysaccharide-rich stroma), Prussian blue (iron stain), and a trichrome stain (which demonstrates collagenized connective tissue and muscle).

The alveolar septa, alveolar spaces, blood vessels, and airways were examined for specific histopathologic features. Thirteen histopathologic features were evaluated where applicable: cellular infiltrates (extent and severity), bronchiolar metaplasia, pneumocyte hyperplasia, intraalveolar granulation tissue, intraalveolar cellularity, interstitial fibrosis, interstitial young connective tissue, honeycombing, vessel myointimal changes, smooth muscle metaplasia, airway (terminal and respiratory bronchioles) inflammation, fibrosis, and luminal granulation tissue. The pleura was not included for evaluation as few pathologic alterations of the pleura are encountered in cases of idiopathic pulmonary fibrosis. Evaluation and scoring were carried out by a single observer (A.F.), and the extent and severity of the histologic changes were graded in a semiquantitative fashion (on a scale of 0 to 5) according to a previously published grading protocol [11]. A score of 0 to 5 (absent, occasional, <25%, 25% to 49%, 50% to 75%, and >75%), roughly approximating the percentage of parenchyma involved, was assigned to most of the histologic features. A similar score (0 to 5) was assigned to the severity of the histologic features. Intraalveolar granulation tissue and airway inflammation, fibrosis, and luminal granulation tissue were graded as absent, present, or marked. The scores were tabulated separately and incorporated into inflammatory/exudative changes (alveolar wall cell infiltrate, bronchiolar metaplasia, pneumocyte hyperplasia, alveolar space cellularity, and granulation tissue), fibrotic/reparative changes (interstitial young connective tissue, interstitial fibrosis, honeycombing, smooth muscle metaplasia, vessel myointimal changes), and airway changes (mural inflammation, mural fibrosis, luminal granulation tissue). Clinical outcomes were not included in the study, and prognosis was not evaluated.

Scores were grouped into two categories for comparison: 0 to 3 and 4 to 5. For each patient, comparisons of the lung biopsy sample evaluation scores for each variable of both the inflammatory/exudative and fibrosis/reparative sections were computed for first sample versus second sample, in the case of left lung biopsy; and for first sample versus second sample, first sample versus third sample, and second sample versus third sample in the case of right lung biopsy, depending on whether two or three samples were obtained. Two by two frequency tables (three for each variable) were created, and the percentage of score categories that matched exactly from sample to sample was calculated. Other variables were coded as either absent, present, or marked. Present and marked were grouped together, and two by two frequency tables were also created for these variables. McNamar ² statistics were calculated for all of the two by two tables.

	Results

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Seventy-seven samples were obtained from 28 patients (average of three samples per patient): 22 samples were obtained from the right upper lobe, 16 from the right middle lobe, 22 from the right lower lobe, 11 from the left upper lobe, and 6 from the left lower lobe. Some patients underwent multiple biopsies of the same lobe rather than of different lobes. Thorascopy samples were slightly larger (2.1 cm) in average diameter than thoracotomy specimens (1.7 cm). The pathologic diagnosis of usual interstitial pneumonitis was rendered for each patient, based on the features manifested by the biopsy samples. End-stage fibrosis was not observed in any of the samples. After thorascopic biopsy, 1 patient experienced a persistent air leak and another patient an infection; all other patients had uncomplicated postoperative recoveries.

For the inflammatory and fibrous variables, although the original scoring used a six-point scale (0 to 5), the agreement measures were based on dichotomous scaling (0 to 3 versus 4 to 5). The average agreement for the inflammatory variables was 79%, and for the fibrosis variables, 86%. Placing all the variables together, the overall agreement average was 82% between samples from each patient. The average agreement for the other variables that were scored as absent/present (vascular and airway changes) was 56%. McNamar-type ² statistics for correlated data ranged from 0 to 2.57. There were no statistically significant differences between the various samples obtained from each patient. The p values ranged from 0.10 to 0.99.

	Comment

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Presently, open biopsy remains the recommended form of obtaining lung tissue for the diagnosis of many of the diffuse infiltrative lung disorders [1]. Although there are no universally established guidelines concerning biopsy site selection, most authorities agree that those regions of the lung most involved by disease of long-standing duration may manifest end-stage fibrosis of unrecognizable etiology [12]. Radiographic findings often serve as a guide to the most appropriate area for biopsy. However, approximately 10% of patients with histologically confirmed chronic diffuse infiltrative lung disease may have a normal chest roentgenogram [6], and a small number of patients may also have normal high-resolution computed tomographic findings [13, 14]. In general, it has been recommended that more than one biopsy sample be obtained to assure adequate sampling of the disease process [15]. Biopsy samples obtained from less involved areas of the lung will generally show an active and diagnosable process [12, 16].

Newman and colleagues [4] evaluated 50 consecutive postmortem specimens obtained from the left upper, left lower, and lingular segments, and concluded that the lingula overrepresented fibrosis and pulmonary vascular changes compared with the other sampled areas of the lung. Studying a different population of patients with bilateral diffuse disease, Wetstein [7] came to the opposite conclusion that histopathologic results of lingular biopsy samples correlated with those results based on samples obtained from other segments of the lung. Wetstein's conclusions were supported by the observations of Miller and associates [8], who also concluded that lingular and right middle lobe biopsy were diagnostically useful. Other investigators have noted other effects of biopsy site on histologic features. In patients with mitral stenosis, Gough [17] noted that hemosiderin was more widely distributed in the upper than in the lower lobes. In a related study, Heath and Whitaker [18] observed that hypertensive pulmonary vascular changes were more prevalent in the lower, dependent segments of the lung.

In a study of 20 patients with diffuse interstitial pulmonary disease from whom multiple biopsy samples were obtained, Winterbauer and colleagues [9] noted considerable intralobar and interlobar variation in diagnosis when using Liebow's classification of interstitial pneumonia. Furthermore, analysis of the histologic variation within a lobe frequently showed a wide discrepancy, in a semiquantitative analysis (0 to 4+), of a variety of histopathologic features. Winterbauer and colleagues did not report whether there were similar interlobar discrepancies. In contrast, Miller and associates [5] demonstrated interlobar uniformity of bronchoalveolar lavage findings in patients with systemic sclerosis. In a recent study, Chechani and co-workers [6] studied 10 patients with various chronic infiltrative lung diseases to determine the benefits of obtaining multiple open biopsy samples. Two lobes were sampled from each patient: a representative region of the radiographically most involved lobe, and a second sample from an adjacent lobe accessible through the thoracotomy incision. The same histologic diagnosis was reached for each of the two biopsy samples in all patients. Chechani and co-workers concluded that there was no need for multiple open biopsy specimens when a representative region of the radiographically most involved lobe was sampled.

Patients with idiopathic pulmonary fibrosis usually undergo open lung biopsy not only to confirm the diagnosis, but also to evaluate the intensity or stage of the disease to predict prognosis or determine therapy [19]. The histologic variability of usual interstitial pneumonitis is conspicuous, and it might be assumed that multiple sites should be sampled to compensate for this variability [20]. As expected, histologic variability was observed in the multiple samples obtained from our patients. However, this variability was observed within each specimen, and there were no statistically significant differences in the severity and extent of the histologic features between samples. Additionally, an unequivocal diagnosis of usual interstitial pneumonitis could be established from each specimen. From our study, the pathologic diagnosis was independent of biopsy site and similar, though not identical, histologic features were observed in all samples removed from each patient. We conclude that a single generous sample (2 cm or greater diameter) obtained from a representative region of the radiographically most involved lobe will be sufficient for diagnostic and evaluation purposes.

	Acknowledgments

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Supported in part by National Institutes of Health NHLBI grant P50HL46487.

We thank Michael A Schork, MPH, PhD, Professor of Biostatistics, School of Public Health, for his guidance concerning statistical analysis.

	References

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Richard I. Whyte Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Flint, A. Articles by Lynch, J. P. Search for Related Content PubMed PubMed Citation Articles by Flint, A. Articles by Lynch, J. P., III Related Collections Related Article