Abstract
A pilot study was performed to examine the presence of nerve fibres in minor salivary gland tissue samples obtained by two procedures: punch and linear incisional techniques. The study was undertaken on a convenience sample of five cryopreserved corpses (mean age 74 ± 3.5 years; three males and two females). Biopsies were performed on the buccal side of the lower lip, between the mid-line and the corner of the mouth. Each corpse had one side of the lower lip biopsied by punch and the contralateral side using a linear incision. All punch samples (100%) displayed severed nerve fibres, whereas no nerve fibres (0%) could be identified in the samples obtained by means of the linear incision technique. While the linear incision approach permitted retrieval of at least five glands, punch biopsies did not provide enough material for the diagnosis of Sjögren’s syndrome. Within the limitations of the study, our results strongly discourage the punch technique for minor salivary gland lip biopsy and provide information on the superiority of the linear incisional biopsy in terms of neural damage. These results may also promote the undertaking of clinical trials on patients in whom Sjögren’s syndrome is suspected, comparing the morbidity associated with the linear incisional technique vs. minimally invasive biopsies.
The minor salivary gland biopsy (MSGB) has been used for the diagnosis of systemic disorders, such as amyloidosis, sarcoidosis, and Sjögren’s syndrome (SS), and for the confirmation of neonatal haemochromatosis.
The presence of focal lymphocytic sialadenitis with a focus score >1 per 4 mm 2 of glandular tissue is an objective criterion for consideration when diagnosing Sjögren’s syndrome. MSGB has proven validity and diagnostic confidence, with a high specificity and positive predictive value, and an average sensitivity of 79%. These features make MSGB particularly useful for patients with inconclusive clinical findings, incipient forms of the syndrome, SS with negative anti-Ro/la serology, and extra-glandular involvement.
A wide range of surgical approaches have been described for harvesting at least five accessory glands from the lower lip using different instruments (scalpel, punch, or cup forceps) and for producing different incisions (circular, linear, or elliptical) in a variety of sizes (from 2 mm to 3 cm) and orientations (parallel to the lip, oblique, or even vertical). The use of a forceps with a fenestrated active end (chalazion forceps) to stabilize the lip has also been suggested.
The selection of the best surgical approach in terms of related morbidity is hampered by the absence of comparative studies and the proliferation of descriptive papers that do not state negative outcomes associated with the technique performed. Moreover, those reports describing percentages of surgical complications have a limited validity due to the lack of standardization when defining and categorizing the complications according to their severity. Nonetheless, persistent lip numbness is the most frequently published surgical complication, occurring in up to 6% of MSGBs performed in the lower lip.
Despite the existing investigations discouraging the removal of labial mucosa with attached glands when performing MSGB because of the potential for neurological damage, punch use has been widely recommended because of safety and handling simplicity reasons, as this procedure is not technically demanding and can be undertaken in an outpatient setting. However, and to the best of our knowledge, there are no quality comparative studies assessing neurological damage induced by the different techniques for MSGB. Thus, the aim of this pilot study was to examine the presence of nerve fibres in minor salivary gland tissue samples obtained by means of two different procedures: punch technique and linear incisional technique.
Materials and methods
On the basis of the feasibility of the investigation and to minimize potential ethical conflicts, an observational, descriptive pilot study designed to replicate the techniques for minor salivary gland biopsy for SS diagnosis was undertaken on a convenience sample of five non-formolized, frozen corpses (mean age 74 ± 3.5 years; three males and two females). All subjects had bequeathed their bodies for medical – scientific research and training purposes and all procedures were undertaken in accordance with the university ethics committee recommendations (14/2007).
Biopsies were performed at the inner side of the lower lip, between the mid-line and the corner of the mouth. Each corpse had one side of the lower lip biopsied by punch and the contralateral side using a linear incision. The biopsy site was randomly allocated to each technique using a computer-generated list of random numbers.
The punch biopsy technique was undertaken following previously established protocols by everting the lip, perpendicularly positioning a 4-mm diameter punch (Stiefel Laboratories, Madrid, Spain), and performing simultaneous rotational movements under gentle pressure to reach 8 mm deep into the lip. The cylinder of tissue was removed from its base using a scalpel with a No. 15 blade and placed onto a filter paper to avoid curling or twisting artefacts.
For the incisional biopsy, the lip was stabilized with a forceps (OEPM No. 201200158) and the incision performed away from the mid-line using a No. 15 scalpel blade. This incision was directed horizontally for about 1.5 cm, just penetrating the epithelium and combined with a blunt dissection of the borders of the wound. Five minor salivary glands were harvested from each corpse.
All specimens were immediately placed in a wide-mouthed container, coded, and fixed in a generous amount of 10% formalin buffered saline for 24 h.
A single pathologist cut all specimens longitudinally with a new disposable scalpel for every section to obtain three slices 200 μm apart from each specimen and orientated them before paraffin embedding. Samples were prepared in 4-μm sections, stained with haematoxylin and eosin, and processed by the same technician. All specimens were examined using an Optiphot-2 microscope (Nikon, Tokyo, Japan) equipped with a millimetre-calibrated eyepiece graticule (Graticules Ltd, Tonbridge, Kent, UK) in order to measure the length of the core of tissue obtained by punch procedure. Pathological analysis also assessed the presence of severed nerve fibres within the tissue samples and the number of glands obtained by each technique.
The scores obtained for each variable were recorded and the confidence intervals for the differences between techniques calculated using Epidat 3.1 statistical package (Xunta de Galicia, Santiago de Compostela, Spain).
Results
The punch technique for minor salivary gland biopsy produced specimens of 7.2 ± 1.1 mm length. The procedure harvested one minor salivary gland per sample in three cases; another case showed two glands located at the same depth in the tissue sample and the last case showed no glandular tissue in the specimen.
All punch biopsy samples (100%) displayed severed nerve fibres, located deeper in the tissue than the minor salivary glands. Only one sample showed nerve fibres close to the glandular tissue, at a more superficial level ( Fig. 1 ). No nerve fibres (0%) could be identified in the samples obtained by means of the linear incision technique ( Fig. 2 ). While the linear incision approach permitted retrieval of at least five glands, punch biopsies did not provide enough material for the diagnosis of Sjögren’s syndrome. The results are summarized in Table 1 .
| Case | Age, years | Gender | Punch specimen length (mm) | No. of glands in each sample | Presence of nerve tissue | ||
|---|---|---|---|---|---|---|---|
| Punch | Linear incision | Punch | Linear incision | ||||
| 1 | 76 | F | 5.5 | – | 5 | + | − |
| 2 | 72 | M | 7.0 | 1 | 5 | + | − |
| 3 | 79 | M | 7.0 | 1 | 5 | + | − |
| 4 | 70 | F | 8.5 | 1 | 5 | + | − |
| 5 | 73 | M | 8.2 | 2 | 5 | + | − |
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