|Year : 2016 | Volume
| Issue : 2 | Page : 100-105
Artifacts in oral biopsy specimens: A comparison of scalpel, punch, and laser biopsies
Sarita Yanduri, Garima Pandey, Veerendra B Kumar, S Suma, MG Madhura
Department of Oral and Maxillofacial Pathology, D. A. Pandu Memorial R. V. Dental College and Hospital, Bengaluru, Karnataka, India
|Date of Web Publication||19-Dec-2016|
Department of Oral and Maxillofacial Pathology, D. A. Pandu Memorial R. V. Dental College and Hospital, No. CA 37, 24th Main Road, 1st Phase, J.P. Nagar, Bengaluru - 560 078, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Biopsy is an important tool which aids in the diagnosis of lesions ranging from simple non-neoplastic growths to malignancies. The choice of technique depends on factors such as anatomic site and morphology of the lesion. Oral biopsies can be performed using different techniques, in which scalpel biopsy is the conventional method. Punch biopsies are also quite popular because they are safe and can be rapidly performed. However, the above techniques cannot provide hemostasis which is required on highly vascular tissues. As a result, lasers have been gaining popularity. Nevertheless, laser-tissue interactions may produce some artifactual changes, especially at the margins, such as thermal damage and coagulation which may impair the histopathological diagnosis. Aim: The aim of this study was to compare artifacts produced in scalpel, punch, and laser biopsies and to determine the most effective tool of the three in terms of oral biopsies. Materials and Methods: Thirty hematoxylin and eosin stained sections, ten each of conventional scalpel, punch, and laser biopsies, were retrieved from the archives and analyzed for artifacts under a light microscope. Results: There was no statistically significant difference between the three techniques in terms of orientation, crush, and hemorrhage. Both horizontal and vertical splits were seen in scalpel biopsies (70%) while none were present in the other two techniques. Loss of epithelium was maximum with laser biopsies, and thermal damage was also exclusively seen in this technique. Conclusion: Punch biopsy revealed the least number of artifacts. Laser, compared to scalpel and punch, produces artifacts which may render the margins of the lesions uninterpretable.
Keywords: Artifacts, laser, oral biopsy, punch, scalpel
|How to cite this article:|
Yanduri S, Pandey G, Kumar VB, Suma S, Madhura M G. Artifacts in oral biopsy specimens: A comparison of scalpel, punch, and laser biopsies. Indian J Oral Health Res 2016;2:100-5
|How to cite this URL:|
Yanduri S, Pandey G, Kumar VB, Suma S, Madhura M G. Artifacts in oral biopsy specimens: A comparison of scalpel, punch, and laser biopsies. Indian J Oral Health Res [serial online] 2016 [cited 2019 Nov 20];2:100-5. Available from: http://www.ijohr.org/text.asp?2016/2/2/100/196147
| Introduction|| |
It is an accepted fact that biopsy is the gold standard procedure for the diagnosis of lesions ranging from simple non-neoplastic growths to malignancies. ,, Selection of the biopsy technique depends on multiple factors such as anatomic site and morphology of the lesion. , Scalpel biopsy is the conventional method of biopsy, but more often, the patient is left with a slow healing wound or with a wound breakdown. , The punch biopsies are also quite popular in oral units because they are safe and can be rapidly performed.  The use of laser has gained popularity and has advantages over a scalpel in that it can instantly disinfect the surgical wound and has a hemostatic effect.  Irrespective of the technique of biopsy, there is always a possibility of producing artifacts as the specimens removed from the oral cavity are often small.  These artifacts may result in alteration of normal morphologic and cytologic features thus interfering with arriving at a diagnosis.  It is essential to have adequate knowledge and an understanding of artifacts so that appropriate precautionary measures can be taken to avoid or minimize their occurrence. ,
Very few studies have compared the artifacts produced between scalpel and punch biopsies, and these have revealed that the punch biopsy produces the least number of artifacts. , Although artifacts can be produced in all the three techniques, studies have shown that there are few artifacts that are exclusively seen in laser biopsies and these are attributed to the heat produced during the procedure. 
Since there are no studies to compare the artifacts produced by scalpel, punch, and laser biopsies, this study aimed to do the same and to determine which is the most effective tool of the three in terms of oral biopsies.
| Materials and methods|| |
A retrospective study was conducted wherein a total of thirty hematoxylin and eosin stained slides (ten scalpel biopsies, ten punch biopsies, ten laser biopsies) were randomly selected from the archives. All the biopsies had been carried out as routine procedures for the purpose of diagnosis of various lesions [Table 1]. Sections were evaluated by two oral pathologists blinded to the type of biopsy procedure used. After establishing the diagnosis, the sections were analyzed for the presence of various artifacts under a light microscope under different magnifications (×4, ×10, ×40) and scoring was done according to the histological pro forma provided. Artifacts such as orientation, loss of epithelium, curling, crush, hemorrhage, split, fragmentation, pseudocyst, and vacuolation were analyzed for their presence or absence. In addition, artifacts produced by laser biopsy such as thermal damage, inter- and intra-epithelial edema, trichocariosis, and hyperchromatic nuclei were also analyzed. To assess the thermal damage at both the epithelial and connective tissue levels, photomicrographs of the representative sites were taken using ProgRes CapturePro 2.5 (Jenoptik, Germany) and depth of the maximum thermal damage was measured using Image-Pro Express 6.0 software (Media Cybernetics Inc., USA). The results were statistically analyzed using the Chi-square test and Kolmogorov-Smirnov test. P < 0.05 was considered to be statistically significant.
|Table 1: List of histopathologic diagnosis of lesions evaluated in the study|
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| Results|| |
When all thirty samples were considered, statistically significant findings were found in relation to artifacts such as loss of epithelium (P = 0.02) and split (P = 0.001). Loss of epithelium was maximum with the laser biopsies (70%) followed by scalpel (40%) and then punch biopsy (10%) [Figure 1]. Both horizontal and vertical splits were seen in scalpel biopsies (70%) while none were present in the other two techniques [Figure 2].
|Figure 1: Histopathologic section showing partial loss of epithelium (H and E, ×10)|
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No statistically significant differences in terms of orientation, crush, hemorrhage, pseudocyst formation, and vacuolation were identified [Figure 3]. Curling was maximum (50%) in punch biopsies and fragmentation was observed in a majority of all sections (scalpel biopsy - 100%, punch biopsy - 80%, and laser - 70%) [Figure 4]. However, these results were not statistically significant. The artifacts encountered in the histopathological examination are listed in detail in [Table 2].
|Figure 4: Histopathologic section showing curling artifact (H and E, ×10)|
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Few artifacts were found to be exclusively seen in only laser biopsies. Thermal damage was evident in the connective tissue of all cases and ranged from 65.59 to 374.62 μm with a mean value of 153.02 μm [Figure 5]. However, it could be assessed in the epithelium in only one case (41.05 μm). Other artifacts such as trichocariosis were found in all sections of laser biopsies; intercellular edema (10%), intracellular edema (3%), and hyperchromatic nuclei (60%) were also observed [Figure 6] and [Table 3].
|Figure 5: Histopathologic section showing depth of thermal damage seen in the lamina propria of specimen removed by laser biopsy (H and E, ×10)|
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|Figure 6: Histopathologic section showing intracellular edema (black arrow), hyperchromatism (green arrow) and trichocariosis (flattened epithelial cells with elongated nuclei) (red arrow) in specimen of laser biopsy (H and E, ×20)|
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|Table 3: List of additional artifacts found exclusively in laser biopsies|
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| Discussion|| |
An appropriate biopsy needs to be representative of the lesion. To achieve this, three major factors, namely, selection of the biopsy site, the type of biopsy, and finally adequate submission of the specimen to the laboratory will determine the quality of the biopsy specimen. , High vascularity and limited access to the oral cavity do not allow adequate biopsies. Thus, the possibility of producing artifacts is enhanced in such cases. ,,
Oral biopsies by virtue of often being small may be subjected to tissue distortion by even the most minimal compression. This may lead to the production of squeeze artifacts such as crush, hemorrhage, splits, fragmentation, and pseudocysts. ,
In the present study, split artifacts were present only in scalpel biopsy. Horizontal splits were found in 40% of cases, and vertical splits were found in 30% of sections of scalpel biopsy (P = 0.001). Split artifacts have often been attributed to multiple cuts caused by improper use of the blade. Other reasons could be due to excessive force used during traction by sutures. , As suture traction was not used in any of the present cases, this could explain the absence of this artifact in punch and laser biopsies.
Areas of fragmentation were found to be maximum in scalpel biopsies (100%), followed by punch (80%) and laser biopsies (70%). However, results were not statistically significant. Fragmentation might be due to excessive stretching of tissue during a biopsy procedure or may be attributed to the use of scissors at the base of tissue for releasing the core of the tissue. ,,
Sometimes, due to the inappropriate utilization of a toothed forceps, the surface epithelium may be forced through the connective tissue and form a small pseudocyst. However, the total absence of pseudocysts in all cases of the present study can likewise be explained due to the use of blunt forceps during handling. , Use of a blunt forceps instead of a toothed forceps and grasping the tissue away from the main site of interest may help avoid most of the compression zones and perforation. 
Poor orientation was found in only one case, and none of the cases showed vacuolation. Some authors have suggested that by placing the epithelial surface down on a piece of card (usually held with suture) before immersion in fixative reduces the chance of poor orientation. ,, Epithelial vacuolation may be generated by intralesional injection of anesthetic solution. Therefore, the anesthetic solution should be injected 3-4 mm away from the lesional tissue at four regions, i.e., top, bottom, left, and right of the lesion. ,,
In the present study, it was noticed that the least number of artifacts were associated with punch biopsies. This is similar to the findings of Moule et al. and Meghana et al., who compared artifacts in specimens of punch and scalpel biopsy. , It was also seen that loss of epithelium was found in only 10% of cases of punch biopsy while split artifact was completely absent. The minimal number of artifacts seen in this technique along with the fact that it is a simple and quick procedure, gives high-quality samples, causes minimal discomfort to patients without remarkable esthetic sequelae making it a popular choice in dental practice. However, it is well known that punch biopsy has its own limitations such as limited area of application, especially in the region of the soft palate, maxillary tuberosity, or floor of the mouth, due to the lack of firm tissue fixation or support. It also cannot be used for nodules or growths and is not applicable for deep lesions. ,
Scalpel biopsy, on the other hand, is a gold standard procedure and can be used in all the above conditions where a punch biopsy is not applicable. It also has further advantages of ease to use, accuracy, and minimal damage to the surrounding tissue. However, scalpels cannot provide the hemostasis that is helpful for use on the highly vascular tissue. 
Hemostasis is best provided by laser biopsy which has several other advantages such as minimal postoperative swelling, scarring, improved wound healing, and less postoperative pain. , It has a sealing effect on vessels smaller than 500 μm in diameter so prevents occult micrometastasis. Laser has been recommended to treat benign oral lesions, such as fibromas, papillomas, gingival hyperplasia, hemangiomas, aphthous ulcers, mucosal frenula or ankyloglossia, and even premalignant lesions such as oral leukoplakias.  Factors that determine the initial tissue effect include the laser wavelength, laser power, the available laser waveform (continuous wave, chopped, and pulsed beams), and tissue thermal properties. ,
One major disadvantage of the use of laser is with respect to the damage which occurs at the margins of the lesion.  The damage is thermal in nature and occurs due to tissue protein coagulation, which microscopically appears as a broad band of basophilic coagulum giving an amorphous appearance to the epithelium and connective tissue. Epithelial cells, as seen in the present study, may also appear detached, fusiform, hyperchromatic, and undergo vacuolar degeneration, making it useless for diagnosis, especially if the specimen is small. ,,,
Assessment of margins for the presence or absence of epithelial dysplasia and invasion is extremely important in the histopathological evaluation of premalignant and malignant lesions.  In laser biopsies, the thermal damage to the epithelium may make the margins uninterpretable. In addition, the marginal artifacts such as crush, trichocariosis, and hyperchromatic nuclei may actually simulate dysplasia and may lead to a misdiagnosis.  It has also been noticed that the heat emitted from the laser may cause a separation and subsequent loss of epithelium, thus further affecting margin interpretation. ,, In the present study, 70% of cases showed loss of epithelium.
Thermal damage was noticed in the lamina propria as well, but the depth (mean = 153.02 μm) of thermal damage was far lesser and within the tolerable limits of the margin clearance allowed for oral squamous cell carcinoma specimens. Thus, we agreed with the findings of Makki et al. that laser biopsies preserve the ability to interpret invasive malignancy but make an assessment of the presence or absence of dysplasia difficult. 
Suter et al. recommended that the use of a CO 2 laser statistically reduced the amount of thermal damage as compared to a diode laser and thus stated that the CO 2 laser is used for potentially malignant and malignant disorders.  Further, the use of low power settings of the laser may help to a certain extent reduce the possibility of separation of the epithelium and its subsequent loss during tissue processing. 
| Conclusion|| |
The findings of the present study highlight that the choice of the instrument for biopsy depends not only on the ease with which the biopsy can be performed but also on the number of artifacts that may be produced as a result of the instrument usage. To the best of our knowledge, no study to date has compared artifacts produced by scalpel, punch, and laser in oral biopsies.
The study shows that all the three techniques can produce artifacts which can be greatly reduced by proper handling of the tissue during the biopsy procedure. Punch biopsy reveals the least number of artifacts but it has its own limitations in usage. Laser provides the best hemostasis compared to scalpel and punch but produces artifacts which may render the margins of the lesions uninterpretable. Thus, we suggest that scalpel or punch biopsies be used for incisional biopsies of malignant and premalignant lesions and the use of lasers be restricted to excisional biopsies and relatively large specimens where sufficient margins can be obtained.
We also recommend that more studies with larger sample size be carried out to confirm these findings. Additional parameters such as determining the artifacts produced by different types of lasers and addition of immunohistochemical markers may help further enhance our knowledge on this topic.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]