ISSN : 2146-3123
E-ISSN : 2146-3131

Shrinkage of Nasal Mucosa and Cartilage during Formalin Fixation
Leyla Kansu1, Erdinç Aydın1, Hampar Akkaya2, Suat Avcı1, Nalan Akalın3
1Departments of Otolaryngology-Head and Neck Surgery, Başkent University School of Medicine, Ankara, Turkey
2Departments of Pathology, Başkent University School of Medicine, Ankara, Turkey
3Departments of Biochemistry, Başkent University School of Medicine, Ankara, Turkey
DOI : 10.4274/balkanmedj.2015.1470

Background:After resection, specimens are subjected to formalin fixation during histologic processing. This procedure can result in tissue shrinkage, with amount of shrinkage related to tissue composition and tissue type.
Aims: In this study,we aimed to evaluated shrinkage of nasal mucosa and cartilage tissue and compared differences in shrinkage after resection, after formalin fixation, and during microscopic examination to understand differences in rate of shrinkage with different tissue types.
Study Design:Animal experimentation.
Methods:Fresh sheep nasal septa were excised (10 mm diameter in 40 sheep and 20 mm diameter in 40 sheep). The mucosa was separated from 1 side of the cartilage, with the contralateral mucosa remaining attached to the cartilage. Specimen diameters were measured in situ, after resection, after fixation for 6 or 24 hours (10% formalin), and during microscopic examination.
Results:We found no significant changes in specimen diameters (both 10 mm and 20 mm) between in situ and resected tissue. Shrinkage was noted in all fixated specimens, and further shrinkage was noted after microscopic examination. We found p=0.004 for free mucosa, p=0.005 for mucosa attached to cartilage, and p=0.008 for cartilage in 10 mm, and p=0.007 for free mucosa, p=0.004 for mucosa attached to cartilage, and p=0.01 for cartilage in 20 mm specimen diameter. Tissue shrinkage was greatest in free mucosal tissue and least in cartilage.
Conclusion: We found that tissue shrinkage was greatest in free mucosal tissue, less in mucosa attached to cartilage, and least in cartilage. These results should be considered when evaluating patients undergoing surgical procedures fornasal cavity and paranasal sinus malignancies. Surgical margins should be measured before fixation or evaluated if possible before fixation and shrinkage.

Carcinomas of the nasal cavity and paranasal sinuses (NCPS) account for 1% of all malignancies and 3% of head and neck malignancies. These neoplasms may be epithelial, mesenchymal, neural, neuroectodermal, or haematopoietic. Squamous cell carcinoma (SCC) is the most common malignancy of the mucosal surfaces of NCPS (1,2). The primary treatment of carcinoma of NCPS is complete surgical resection, which is usually followed by postoperative radiotherapy (3). The goal of surgical treatment is complete eradication of the primary tumour with a safe margin (4). Despite improvements in surgical techniques and radiotherapy, patients with SCC of NCPS have a poor prognosis, and the 5-year survival rate is 50% (1).

The most important prognostic factor for SCC of NCPS is complete surgical removal of the neoplasm. Failure to eradicate the primary tumour is the leading cause of local recurrence of this type of cancer. Local recurrence is likely when gross tumour remains, and this can lead to patient death. The presence of microscopic cancer at the margin of resection is associated with local recurrence and poor survival. Local recurrence occurs in approximately 50% of patients, even when surgical margins are microscopically negative for residual tumour (3).

Surgical and pathological margins may differ. The head and neck surgeon may determine the margin of normal tissue around the resected tumour 1 to 2 cm wide, but the margin measured by the pathologist may be smaller. This difference may be related to tissue shrinkage after resection and during specimen preparation (5). Most tissues shrink when placed in a formalin fixative solution (4,6). A formaldehyde solution is the most commonly used fixative during histopathological examination. However, this fixative may cause marked deformation of tissue dimensions and shape (5); therefore, pathologists typically report that resection margins closer than those measured by the surgeon during surgery (7).

A correction factor may be used to compensate for tissue shrinkage during specimen processing. The purpose of using a correction factor is to estimate the actual length of unfixed tumour in vivo and to obtain data that are comparable between laboratories (8). The composition and type of tissue may affect the amount of tumour shrinkage. Therefore, studies on changes in tissue size due to formalin fixation should be organ specific (9).

Studies on the shrinkage behaviour of tissue have been performed in liver, muscle, spleen, kidney, lingual mucosa, prostate, lung, cornea, colon, oesophagus, and brain tissue (6,7,10-12). For skin tissue, the degree of shrinkage caused by formalin fixation is controversial (13,14). A literature review revealed no previous reports on shrinkage of the nasal mucosa and cartilage of the nasal septum after excision and histological processing.

In the present study, our aim was to evaluate and quantify the shrinkage of specimens taken from the nasal mucosa and cartilage of sheep and to document whether inconsistencies exist between measurements of in situ margins before excision and histological margins.

MATERIALS AND METHODS

Animals
This experimental study included 80 heads from freshly killed sheep obtained from a local abattoir. The study was approved by our Institutional Review Board (Başkent University project no: DA10/22) and supported by research funds from our university.

Procedure
The nasal septa were removed from all sheep heads by the same surgeon (L.K.). Nasal septa that had lacerations or abnormal colour were excluded. Nasal septa (full layer: mucosa, cartilage, and contralateral mucosa) were excised in round diameters that were measured in situ and marked with a surgical marker (Devon surgical skin marker; Covidien, Minneapolis, MN, USA). Two in situ diameters were obtained: 10 mm (40 sheep) and 20 mm (40 sheep) (Figure 1). After excision, the mucosa from one side of the septum was dissected free, with the contralateral mucosa remaining attached to the cartilage. The diameters of the free mucosa, mucosa remaining attached to the cartilage, and cartilage were measured with a millimetre ruler ("after resection" diameters) (Figure 2). The free mucosa became contracted because it was very thin, but it was spread out on a hard, smooth surface before measurement.

All specimens were completely immersed in 10% neutral-buffered formalin (formaldehyde 37-40%; Merck, Darmstadt, Germany) immediately after excision, with measurements of length and calculations of percent differences also made immediately. After fixation for 6 or 24 hours, the specimens were removed from the formalin. Specimen diameters were measured by a pathologist (H.A.) ("after fixation" diameters). The tissue was marked, and sections were cut for paraffin embedding and histological preparation. One slide was prepared from each specimen, which was stained with haematoxylin and eosin. The diameters on the stained slide were measured with an ocular micrometer ("microscopic" diameters) (Axioscop 2; Carl Zeiss, Oberkochen, Germany).

The specimens were grouped with specimens of similar initial diameter (10 mm or 20 mm) and fixation time (6 hours or 24 hours), and the components of each specimen group were grouped separately (free mucosa, mucosa attached to cartilage, and cartilage).

Statistical analysis
Data were analysed using statistical software (Statistical Package for the Social Sciences, version 22.0, SPSS Inc., Armonk, IBM Corp., NY, USA). Data are expressed as number (percent) or mean ± standard deviation. Comparisons were made by the Friedman test and Wilcoxon signed rank test with Bonferroni correction. The mean difference was significant at the p<0.017 level (p/n=0.05/3).

RESULT

There were no differences between the in situ and after-resection diameters of any tissue components (free mucosa, mucosa attached to cartilage, and cartilage) of all nasal specimens (10- or 20-mm diameter and 6- or 24-hour fixation). Therefore, no shrinkage had occurred. However, significant shrinkage occurred between resection and after-fixation (Table 1). Regarding tissue specimens that were fixed for different durations (6 or 24 hours), we observed a significantly smaller mean tissue diameter in specimens fixed for 24 hours versus those fixed for 6 hours for mucosa attached to cartilage (in the 10-mm diameter after-fixation samples), free mucosa (in the 20-mm diameter after-fixation samples), mucosa attached to cartilage (in the 20-mm diameter after-fixation and microscopic measurement samples), and cartilage (in