Abstract
Using a CO 2 laser beam for biopsy is compromised by thermal cytological artefacts that could be critical when assessing dysplastic changes, as they may simulate cytological atypia. This study examined wounds caused by CO 2 laser, evaluating their size and features, utilizing 25 Sprage rats randomly allocated to five groups: four glossectomized using a CO 2 laser at 3, 6, 9 and 12 W and a control group treated with a conventional scalpel. Samples were prepared in 4 μm sections, stained, and studied double-blind by two pathologists. The artefacts identified included cellular and nuclear polymorphism, nuclear hyperchromatism and loss of intercellular adherence; they were mainly located in the basal and suprabasal layers of the oral epithelium. Number of artefacts per specimen did not differ between experimental groups. The width of epithelial damage adjacent to the laser incisions revealed 298.7 ± 150.7 μm of damaged tissue (range 100–750 μm), with no differences between low (3 W) and high wattages (6, 9 and 12 W) ( X i − X j = 41.6; 95% CI = −125.1 to 208.4). No changes were observed in the control group. CO 2 laser (3–12 W) generates epithelial damage that can simulate light dysplasia with atypias mainly affecting basal and suprabasal layers that may lead to erroneous therapy.
An oral biopsy specimen can be affected by a number of artefacts resulting from crush, fulguration, injection or improper fixation and freezing . The selection of technique and surgical instruments to avoid artefacts is controversial. Punch biopsy has revealed some benefits , but its advantages could not be confirmed under controlled experimental conditions .
Using a carbon dioxide (CO 2 ) laser beam to procure diagnostic biopsy specimens is compromised by thermal cytological artefacts that include vacuolation of the superficial layer, detachment and shredding of keratin, basal cell degeneration, and separation from the lamina propia . These artefacts could be critical when assessing dysplastic changes, as thermal damage induced along the laser-treated margins would simulate cytological atypia . Similar events are experienced with electrocautery .
To the best of the authors’ knowledge, there are no studies systematically analyzing cytological atypias or structural changes in the oral epithelium (epithelial pseudodysplastic artefacts), nor their association with different CO 2 laser wattage.
The aims of this study are to examine the influence of thermal wounds caused by different CO 2 laser wattages by assessing cytological and epithelial architectural changes and to evaluate the width of the damage lateral to the incision.
Material and methods
An experimental study with 25 Sprage rats (250 g) was designed. The specimens were randomly allocated to five groups: four experimental groups in which they underwent glossectomy using a CO 2 laser beam at different wattages (3, 6, 9 and 12 W) and a control group glossectomized using a number 15 blade (B/BRAUN, Aesculap AG, Tuttlingen, Germany).
The laser beam (Opmilas CO2-L, Carl Zeiss AG, Oberkochen, Germany) emitted light at a wavelength of 10,600 nm via an articulated arm with a focusing hand piece (f-125) and was set to the pulsated wave mode (0.05 s). The surgical technique was performed by a single surgeon directing the laser beam perpendicular to the dorsum of the tongue while stabilizing the specimen with non-toothed Adson forceps applied to the tongue tip. The rats were killed immediately after the intervention by anaesthetic overdosage (intraperitoneal injection of 30% chloral hydrate) according to EU ethical protocols . Specimens were immediately placed in a wide-mouthed container and fixed in a generous amount of 10% formol buffered saline for 24 h.
A single pathologist cut all the specimens longitudinally, using a new disposable scalpel for every section, and orientated them before they were embedded in paraffin. Samples were prepared in 4 μm sections, stained with haematoxylin and eosin and processed by the same technician. The specimens were coded and all 25 samples were studied double-blind by two pathologists until a consensus was reached for each case. All specimens were examined using an Optiphot-2 microscope (Nikon, Tokyo, Japan) equipped with a millimetre-calibrated eyepiece graticle (Graticules Town Bridge, Kent, UK) to measure thermal damage.
The histological epithelial features evaluated included loss of polarity of the basal cells, the presence of more than one layer with basaloid appearance, increased nuclear-cytoplasmatic ratio, drop-shaped rete ridges, irregular epithelial stratification, increased number of mitotic figures, abnormal mitotic figures, presence of mitotic figures in the superficial half of the epithelium, cellular and nuclear polymorphism, nuclear hyperchromatism, enlarged nucleoli, loss of intercellular adherence, and keratinization of single cell groups in the prickle cell layer . Histological examination assessed the prevalence and location of thermal artefacts within the epithelium.
Statistical analysis was performed using a SPSS + 11.0 statistical package. χ 2 (Fisher’s exact test) was used to compare proportions and the ANOVA test to compare means. The level of significance chosen was 5%.
Results
During histopathological examination, several artefacts were identified including the presence of fusiform cells with marked nuclear elongation, cellular and nuclear polymorphism, nuclear hyperchromatism and loss of intercellular adherence. Their distribution according to the wattage used is given in Table 1 and illustrated in Figs. 1–5 .
| Histological alterations | 3 W laser n (%) | 6 W laser n (%) | 9 W laser n (%) | 12 W laser n (%) |
|---|---|---|---|---|
| Cellular and nuclear polymorphism | 5 (100%) | 5 (100%) | 4 (80%) | 5 (100%) |
| Nuclear hyperchromatism | 5 (100%) | 5 (100%) | 5 (100%) | 4 (80%) |
| Loss of intercellular adherence | 2 (40%) | 4 (80%) | 5 (100%) | 4 (80%) |
| Width of side thermal damage (μm). Mean ± SD | 330 ± 237.7 | 300 ± 79.0 | 320 ± 119.1 | 245 ± 158.5 |
| Number of artefacts by specimen. Mean ± SD | 2.4 ± 0.54 | 2.8 ± 0.44 | 2.8 ± 0.44 | 2.6 ± 0.89 |
These artefacts were mainly located in the basal and suprabasal layers of the oral epithelium. No significant differences could be identified in terms of prevalence of artefacts per specimen among the four experimental groups. There was no statistical significance between the alterations related to the use of low (3 W) and high power (6–12 W). None of the following were found in the samples analyzed: loss of polarity of the basal cells, presence of more than one layer with basaloid appearance, drop-shaped rete ridges, irregular epithelial stratification, increased number of mitotic figures, abnormal mitotic figures, presence of mitotic figures in the superficial half of the epithelium, and keratinization of single cell groups in the prickle cell layer.
The mean width of epithelial damage adjacent to the laser incisions for all wattages was 298.7 ± 150.7 μm (range 100–750 μm). The extent of the thermal damage at the basal cell layer for the low power (3 W) group was 330 ± 237.7 μm and 288.3 ± 118.7 μm for the high wattage group (6, 9 and 12 W); this difference was not statistically significant ( X i − X j = 41.6; 95% CI = −125.1 to 208.4), as occurred when variations among the four groups ( Fig. 6 ) were investigated ( p > 0.05).
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