Mucosal biopsy specimens were immediately transported to the laboratory on a slide in a humidified, ice-cooled container, and subsequently embedded (in TissueTek; Sakura Finetek USA; Torrance, Calif) and snap-frozen by immersing in precooled isopentane at —80°C. Sections were cut at 4 μm and stored at —80°C until use. To evaluate the morphologic architecture of the bronchial tissue, the sections were stained in intervals of 50 μm with Mayer’s hematoxylin-eosin. The largest section of a series of 20 serial sections was selected for size estimation. Size of the tissue was estimated using an eyepiece graticule (double square lattice with cross-points each 100 μm at X80 magnification), counting the number of points covering morphologically intact tissue. Morphology was assessed by the aspect of the epithelium, the BM, and the intactness of the submucosa. Crush artifacts and disruption of tissue are defined as any disruption of tissue leading to inaccurate determination of cell counts per area. These were considered relevant when causing >10% false increase or decrease of tissue area in which cell counting is to be performed (false increase in surface area is observed in case of edema and diffuse disruption, whereas crush artifacts are mainly responsible for false decrease of real biopsy area).
The biopsy specimens were assessed in a blinded fashion. The Mann-Whitney U test was used to compare among the three groups the number of grid crossings covering morphologically intact tissue. The x2-test was used to compare the intactness of the submucosa and the integrity of the epithelium. Statistical analysis was performed with a statistical package (SPSS/PC + v 4.0.1; SPSS Inc; Chicago); p values <0.05 were considered statistically significant.
Biopsy specimens from five patients could not be evaluated. They contained only mucus or blood without tissue (two with forceps type FB-19C, two with FB-35C); biopsy specimens of another patient could not be used due to technical artifacts (FB-19C). Thus, bronchial biopsy specimens of 25 patients were available for evaluation.
The biopsy specimens taken with the FB-35C and FB-21C forceps were equal in size, and the biopsy specimens taken with the FB-19C were the smallest, when measured by the number of grid crossings. The difference in size between the FB-21C and FB-19C biopsy specimens was significant (p<0.05, Table 1).
In the biopsy specimens taken with the FB-21C forceps, the epithelium had the best preserved morphology; the biopsy specimens taken with the FB-19C and FB-35C showed damage of the epithelium in a larger proportion of the tissue sections (Figs 2 and 3).
The submucosal layer in the biopsy specimens taken with the FB-35C forceps was relatively superficial (Table 1), which means that <100 |Jim of submucosa beneath the BM was available for evaluation. All sections taken with FB-21C forceps showed submucosa with intact morphologic architecture. The depth of the submucosal layer in the biopsy specimens obtained with the forceps FB-21C was larger than in the sections of the biopsy specimens taken with the FB-19C and FB-35C, allowing evaluation of superficial and deep parts for inflammatory infiltrates. In the biopsy specimens taken with the FB-19C, specimens from five of the seven evaluable patients had submucosa that showed intact morphology. In the other two, the submucosa showed a more detached structure.
Table 1-—Qualitative Aspects and Size of Bronchial Biopsy Specimens Using Different Forceps Sizes
|Type of Forceps||No. of Patients||Median (Range) Grid Crossings||Intactness of Submucosa||Classification of Epithelium|
|FB-19C||10||29 ‘||5/10||4/7: + + + 2/7: + + 1/7: +|
|FB-21C||10||50||9/10||7/10: + + + 3/10: + + 0/10: +|
|FB-35C||10||53||4/10||4/8: + + + 3/8: + + 1/8: +|
Figure 2. Top: section of a bronchial biopsy specimen taken with forceps FB-21C (hematoxylin-eosin, X64). Note integrity of tissue (in this biopsy specimen with not-representative relative loss of epithelium). Bottom: higher magnification (X160).
FIGURE 3. Top: section of a bronchial biopsy specimen taken with forceps FB-35C (hematoxylin-eosin, X64). Note mechanical damage in submucosa, better observed in bottom, which is higher magnification (X160).