Comparative analysis of tissue shrinkage with different resection methods in porcine models

Introduction

Head and neck carcinomas rank as the sixth most common cancers worldwide, with surgical resection as the primary treatment modality (1-3). Surgeons are tasked with achieving clear surgical margins while preserving as much healthy tissue as possible, as involved margins are strongly linked to recurrence and mortality (2-5). The UK Royal College of Pathologists’ 1998 guidelines define margins as clear (>5 mm), close (1–5 mm), or involved (<1 mm) (6-8). A significant challenge in margin assessment is the discrepancy between in vivo and histopathological margins, largely due to tissue shrinkage (3-5).

Tissue shrinkage occurs rapidly within the first 5 minutes post-resection, driven by tissue dehydration and elastic recoil of collagen and elastin fibres, which are influenced by tissue composition (3,5,9,10). Contributing factors include tissue type, location, disease severity, fixation technique, and resection method (4,5,11,12). For instance, shrinkage varies across cancers, such as oesophageal (50%), melanoma (15–25%), breast (34%), and uterine (15.3%) (11). Within the same tissue type, variability is also evident: buccal tissues exhibit a wide range (21–72%), while cutaneous tissues show a narrower range (10–23%) (3,10,12-15).

Most research examines tissue shrinkage in two dimensions (length and width), yet it occurs three-dimensionally, highlighting the need for three-dimensional margin assessment. Furthermore, data on how resection methods influence shrinkage remains limited, and existing studies often lack three-dimensional measurements (11,16-18). This study addresses these gaps by evaluating the impact of various resection methods on tissue shrinkage across all dimensions and overall volume. Utilising porcine tissues, which closely resemble human tissues, this research provides a novel and extensive analysis of shrinkage patterns (19-23). Such insights are essential for improving margin assessment and mitigating the risks of false positives and negatives, which can lead to unnecessary treatment or increased recurrence and mortality (3,5,8).

Methods

Ethical statement

This study did not require Animal Ethics Committee (AEC) approval, as the tissues were sourced from animals culled for routine commercial food production. The study was prepared in accordance with the ARRIVE guidelines for animal research (available at https://www.theajo.com/article/view/10.21037/ajo-24-86/rc).

Specimen

Porcine tissues were sourced from a local butchery, refrigerated, and used within 7 days of collection. No previously frozen tissues were used. Porcine hocks and jowls were selected to represent the cutaneous and buccal tissue types, respectively.

Surgical devices

CO₂ laser: Lumenis AcuPulse^TM on SuperPulse^TM, continuous setting at 10 W. Monopolar diathermy: Covidien Valleylab^TM FT10 with SafeAir smoke evacuator and needle-point tip; power set to 20 W for both cutting and fulgurate modes. Scalpel: disposable size 10 blades.

Resection

A square plastic frame (2 cm × 2 cm) marked the resection margins for all tissue samples. All resections were performed in an operating theatre. Microscopy-assisted CO₂ laser resection was performed by the senior author (N.F.) with appropriate personal protective equipment and a laser safety officer present. The remaining resections were performed by the first author (J.L.). A size 10 scalpel was used for the resection of deep margins in CO₂ laser specimens. Cold steel resection used size 10 blades, replaced regularly to maintain sharpness. Monopolar diathermy for both cutting and fulgurate modes used a needle-point.

Measurements

Pre-resection dimensions were standardised at 2 cm × 2 cm using the square plastic frame, with an assumed depth of 0.5 cm, as both buccal and cutaneous tissues are approximately of this thickness (Figures 1,2). Length was marked with a dot using a permanent marker to avoid confusion in subsequent measurements. Post-resection dimensions were measured within 5 minutes of resection. Specimens were placed in 50 mL of 10% formalin solution, two samples per pot, maintaining a specimen-to-formalin ratio of 1:10. Measurements were repeated at 12, 24, and 48 hours in formalin, altogether grouped as post-fixation measurements. All measurements were performed by the first author (J.L.) using a ruler to the nearest millimetre. Volume was calculation by multiplying the length, width, and depth of each specimen. The mean values of the dimensions and volume, along with their standard deviations, were calculated after all measurements were collected.

Figure 1 Pre-resection porcine tissue specimen dimensions.

Figure 2 Tools used in the study—Stamp dye, 2 cm × 2 cm plastic mould, ruler (from left to right).

Groups

CO₂ laser resection was performed on buccal tissues only, as CO₂ lasers are not typically used for cutaneous excisions in practice. Both cold steel and monopolar diathermies were used on buccal and cutaneous tissues. Twenty specimens were assigned to each type of resection, with a total of 80 buccal specimens and 60 cutaneous specimens. Figure 3 represents a diagrammatic view of the study design.

Figure 3 Diagrammatic view of the study design.

Statistical analysis

Sample size calculations for CO₂ laser specimens with cold steel as a control were based on t-test parameters, with effect size of 0.5, 90–95% power, and P<0.05 significance level, based on the effect sizes from previous studies ranging from 0.502 to 0.585. For comparing the three resection methods (cold steel, cutting monopolar, fulgurate monopolar) across the two tissue types (buccal, cutaneous), sample sizes were determined using F-test parameters, with similar effect size power, significance settings. The sample size was standardised to 20 per group, achieving approximately 92% power. Data distribution was assessed using the Mann-Whitney U test and Kruskal-Wallis test, and subsequent pairwise comparisons with Dunn-Bonferroni correction were conducted. The effect of formalin over multiple time points was evaluated using the Friedman test and Wilcoxon Signed-Rank test, with Dunn-Bonferroni correction applied. Effect sizes were calculated using rank-biserial correlation. Calculations were performed on G*Power software (RRID:SCR_013726), and data analysis was performed on IBM SPSS Statistics (RRID:SCR_016479).

Results

Buccal tissues and CO₂ laser

Table 1 and Figure 4 show tissue shrinkage of 4% (P=0.158), 15% (P<0.001), 13% (P=0.002), and 27% (P<0.001) post resection, and further 3% (P=0.164), 6% (P=0.040), 17% (P=0.003), and 24% (P<0.001) post-fixation in length, width, depth, and volume, respectively. Width decreased mainly post-resection (15%, P<0.001), whereas depth continued to shrink during fixation, at 13% and 17% respectively (P<0.05). Overall, depth experienced the greatest reduction of 28% (P<0.001), compared to 6.75% in length and 19.75% in width (P<0.05). While length did not undergo substantial changes post-resection or during fixation, the overall reduction was statistically significant. Due to the non-normal distribution of the measurements, as indicated by the Shapiro-Wilk test, the Mann-Whitney U test was used for comparisons, with cold steel as the control. This analysis showed that CO₂ laser specimens had significantly greater reductions in length (4% vs. +9%) and width (15% vs. 2%) at post-resection and post-fixation stages versus the control. In contrast, cold steel specimens exhibited greater depth shrinkage post-resection (41% vs. 13%), even though the depth margins for CO₂ laser specimens were also resected using cold steel. Overall, there were no significant differences in tissue volumes between the two groups (P>0.05). Detailed values are provided in Tables S1,S2.

Figure 4 Changes in porcine buccal tissue specimen dimensions and volume with CO₂ laser.

Buccal tissues and the three resection methods

Table 1 and Figures 5-7 summarise the mean dimensions and overall volume of buccal specimens following each resection method. Cold steel resection resulted in a 37% (P<0.001) reduction in tissue volume, occurring predominantly post-resection, with minimal shrinkage during fixation. Overall, length increased by 9% (P=0.004), which was offset by greater reductions in width (6%, P=0.006) and depth (39%, P<0.001). In contrast, cutting monopolar diathermy led to a 58.5% (P<0.001) volume reduction, with substantial shrinkage of 52% (P<0.001) and 14% (P=0.026) at both post-resection and during fixation, respectively. However, the change in length did not reach statistical significance (P=0.069). Fulgurate monopolar diathermy caused a 52% (P<0.001) volume reduction, displaying similar patterns of 39% and 20% (P<0.001) shrinkage post-resection and during fixation, respectively. All dimensions reached statistical significance post-fixation (P<0.05). The Shapiro-Wilk test indicated mixed normality in the measurements, so Kruskal-Wallis H test was utilised. This revealed significant differences among resection methods in terms of dimensions and volume at both post-resection and post-fixation stages (P<0.05). Pairwise comparisons with the Dunn-Bonferroni correction showed significant differences between cold steel and monopolar diathermies for lengths, widths, and volumes at both stages (P<0.05), with less shrinkage observed in cold steel specimens. Fulgurate monopolar diathermy demonstrated the least shrinkage in depth post-resection (25%, P<0.001). However, this difference was not significant in pairwise comparison and was evened out by post-fixation. No significant differences were found between the two monopolar techniques. Detailed values are available in Tables S1-S4.

Figure 5 Changes in porcine buccal tissue specimen dimensions and volumes with cold steel.

Figure 6 Changes in porcine buccal tissue specimen dimensions and volumes with cutting monopolar diathermy.

Figure 7 Changes in porcine buccal tissue specimen dimensions and volumes with fulgurate monopolar diathermy.

Cutaneous tissues and the three resection methods

Table 2 and Figures 8-10 summarise the mean dimensions and overall volume of cutaneous specimens following each resection method. Cold steel resection resulted in a 47% (P<0.001) reduction in tissue volume, occurring primarily post-resection. Overall, length increased by 1% (P=0.042), which was offset by greater reductions in width (3%, P=0.024) and depth (46%, P<0.001), similar to the pattern observed in buccal specimens. Cutting and fulgurate monopolar diathermies resulted in similar volume reductions (46% and 51%, respectively, P<0.001), also predominantly post-resection. In the fulgurate monopolar group, length increased (4%, P<0.05), while all dimensions decreased in the cutting monopolar group (P<0.001). Depth exhibited the greatest shrinkage across all resection methods, by between 42% and 46% (P<0.001). Except for the length in cold steel and width in cutting monopolar specimens, there were no substantial changes in dimensions or volume during fixation (P<0.05). The Shapiro-Wilk test indicated mixed normality in the data, necessitating the use of the Kruskal-Wallis H test. This test revealed significant differences among resection methods in post-resection width and post-fixation length and width (P<0.001), but not in overall volume. Pairwise comparisons with the Dunn-Bonferroni correction showed significant differences in width between cold steel and both monopolar diathermies as well as between the two diathermy techniques (P=0.003), but no significant differences in other dimensions or in overall volume. Fulgurate monopolar specimens showed the greatest shrinkage in width (13.5%, P<0.001), while cutting monopolar specimens had the most shrinkage in length post-fixation (2%, P<0.001). Depth and overall volume shrinkage were comparable across all resection methods. Detailed values are outlined in Tables S5-S8.

Figure 8 Changes in porcine cutaneous tissue specimen dimensions and volumes with cold steel.

Figure 9 Changes in porcine cutaneous tissue specimen dimensions and volumes with cutting monopolar diathermy.

Figure 10 Changes in porcine cutaneous tissue specimen dimensions and volumes with fulgurate monopolar diathermy.

Effect of formalin on tissue shrinkage

Formalin fixation led to significant shrinkage in buccal tissues with monopolar diathermies, showing reductions across all dimensions and overall volume, as shown in Tables 1,2. Cutting monopolar diathermy displayed significant reduction in length (8%, P<0.001) and width (4%, P=0.006), while fulgurate monopolar diathermy resulted in greater reductions in length (7%, P<0.001), width (5%, P=0.002), and depth (11%, P=0.002). These changes corresponded to a significant reduction in overall volume, 14% (P=0.026) for cutting and 20% (P<0.001) for fulgurate monopolar diathermy. In contrast, cold steel specimens showed a significant reduction only in width (5%, P=0.012), with no significant changes in other dimensions or in overall volume. Shrinkage was less pronounced in cutaneous tissues. Cutting monopolar diathermy had minor width reduction (2%, P=0.01) and fulgurate monopolar showed no significant changes in any dimension. Notably, cold steel specimens exhibited a slight increase in length (1%, P=0.042) without affecting other dimensions or volume. Overall, tissue shrinkage in formalin was less than observed post-resection, regardless of tissue type or resection method. To assess the effect of formalin on tissue shrinkage over 48 hours, the Friedman test was performed. This revealed significant changes in all dimensions and overall volume for buccal tissues (P<0.05), while only depth and volume exhibited changes in cutaneous specimens (P<0.001). The Wilcoxon Signed-Rank test with Dunn-Bonferroni correction indicated that in buccal tissues, significant reductions in length and width occurred within the first 12 hours of formalin fixation (P=0.006), with significant changes in depth and overall volume between 12 to 48 hours (P<0.05). For cutaneous specimens, significant changes were only noted in depth (P=0.006). Significant shrinkage was detected from post-resection to 24 hours of formalin fixation, but not between post-resection to 12 or 12 to 24 hours. Similarly, changes were significant between 24 to 48 hours, but no difference was observed from post-resection to 48 hours. Detailed values are listed in Tables S8,S9.

Resection methods and tissue types

The Shapiro-Wilk test indicated a non-normal data distribution, leading to the use of the Mann-Whitney U test to evaluate differences in each dimension and overall volume at both post-resection and post-fixation stages for each resection method. Rank-biserial correlations were calculated from the U values to determine effect sizes. Cold steel resection showed significant differences only in post-resection and post-fixation lengths (P<0.05), with buccal tissues demonstrating greater increase with moderate effect sizes (r=0.48–0.55). No significant differences were observed in other dimensions or in overall tissue volume. Cutting monopolar diathermy revealed a significant reduction in both post-resection and post-fixation widths for buccal tissues (P<0.001) compared to cutaneous tissues, with substantial effect sizes (r=0.86–0.93). While this difference did not extend to other dimensions, it was reflected in overall tissue volume with moderate effect sizes (P<0.001, r=0.655). Fulgurate monopolar diathermy showed significant differences in depth (P<0.05), with cutaneous tissues showing greater shrinkage of moderate effect sizes (r=0.44). In contrast, post-fixation length and width exhibited significant reduction in buccal tissues, also with moderate effect sizes (P<0.001, r=0.60–0.66). Detailed values are provided in Table S10.

Discussion

Buccal tissues exhibit a wide range of shrinkage, reported between 21% and 72% in the literature, often resulting in substantial discrepancies between in vivo and histopathological margins (4,8,12). This variability likely arises from differences in resection and measurement techniques across studies, many of which assessed shrinkage only in a single dimension. The lack of comprehensive three-dimensional data in previous studies complicates direct comparisons. CO₂ laser resection, commonly used in laryngeal surgery for its precision and simultaneous cauterisation, has been reported to cause 35–45% shrinkage post-fixation when measured in one dimension (18). Our study similarly showed an overall volume reduction of 45% with significant variability by dimension (7–28%), with width exhibiting the greatest reduction post-resection, closely followed by depth. Although a scalpel was used for deep margins in both the cold steel and CO₂ laser groups, cold steel specimens displayed significantly greater shrinkage in depth (39%). Monopolar diathermy specimens showed the greatest shrinkage in depth (33–45%), followed by width (22–24%), regardless of the technique (cutting or fulgurate). All three resection methods resulted in varying degrees of shrinkage across dimensions, with cold steel showing a unique pattern where length increased, and width shrank disproportionately less than depth. This dimension-specific shrinkage highlights the anisotropic pattern of tissue response to resection, where different dimensions change at different rates. The warping of rectangular prism-shaped specimens over the 48-hour period of observation further supports this pattern. These findings emphasise the importance of a three-dimensional approach to margin assessment, as relying solely on two-dimensional margins may miss areas of concern.

The increased shrinkage in the CO₂ laser and diathermy groups aligns with literature suggesting that thermal injury from hot devices causes greater mucosal damage and subsequent tissue shrinkage compared to cold steel (17,18,24,25). This is further evidenced by the continued shrinkage in formalin for specimens resected with hot devices, in contrast to minimal changes in cold steel specimens. The increased mean rank differences between hot devices and cold steel from post-resection to post-fixation further support this observation (Tables S3-S6). While significant differences were noted in post-resection depth and borderline differences in post-fixation depths among the resection methods, pairwise comparisons did not reveal meaningful disparities. This suggests that temperature, rather than specific resection technique, is the primary factor influencing tissue shrinkage in formalin. It is hypothesised that tissues exposed to high temperatures become more susceptible to formalin, resulting in prolonged shrinkage.

Cutaneous tissues also show variable shrinkage depending on tissue location, though it is less pronounced than in buccal tissues. Current literature reports only two-dimensional measurements, with reductions in length (16–23%) and width (9.5–18%) occurring within minutes post-resection (10,14,15). The arrangement of collagen and elastic fibres along skin tension lines is thought to significantly influence shrinkage in cutaneous tissues (10). Our study found more modest changes, with length varying from a 4% increase to a 2% decrease, and width decreasing by 3–13.5%, indicating a narrower range of shrinkage across resection methods compared to buccal tissues. However, depth showed significant reduction (42–46%), greater than observed in buccal specimens. This anisotropic pattern, with substantial depth reduction likely compensating for lesser changes in other dimensions, mirrors that of buccal tissues. The overall volume reduction of 46–51% in our study differs from previous findings; however, prior studies did not account for depth, suggesting that shrinkage in cutaneous tissues may be under-reported. Notably, minimal changes were observed in formalin across all methods, in contrast to buccal specimens, which showed greater shrinkage with hot devices. This indicates that the tissue’s response to temperature and susceptibility to formalin is not universal but tissue-specific. Cutaneous tissues may be more resistant to thermal effects and less affected by the resection method compared to buccal tissues.

Tissue fixation is essential in histopathology for preserving tissue integrity, with 10% formalin being the standard fixative (26). The extent of formalin-induced shrinkage varies across studies and tissue types (27). Buccal tissues appear more susceptible, with shrinkage ranging from 4–22% in length and 6–24% in width, whereas cutaneous tissues experience less shrinkage, with 3.5–8% in length and 0.8–3% in width (1,27-29). Depth measurements are rarely reported. In our study, buccal tissues demonstrated relatively modest reduction: 7–8% in length, 4–5% in width, and 4–11% in depth, compared to the current literature. Shrinkage in length and width occurred mainly within the first 12 hours of fixation, with minimal changes observed thereafter up to 48 hours. In contrast, depth and overall volume showed a gradual reduction from 12 to 48 hours, indicating a more prolonged shrinkage pattern. For cutaneous tissues, significant changes were observed only in depth and volume, occurring within the first 24 hours and again between 24 to 48 hours, with no notable changes during the intermediate timeframes. This suggests that formalin’s effect on cutaneous tissues is cumulative over time, rather than isolated changes at specific intervals. Formalin exerts its effect by penetrating the tissue through diffusion and accumulating within them (27). The temporal difference in shrinkage between buccal and cutaneous tissues may be explained partly by differences in tissue composition, and partly by their varying susceptibility to thermal effects. Our results show that two-dimensional margins (length and width) stabilise after the initial 12 hours of fixation for both tissue types. Notably, previous studies did not control for resection methods, and hot devices can cause continued shrinkage in formalin due to residual thermal effects, potentially leading to misconception about formalin causing the shrinkage. In our study, only the diathermy groups showed continued shrinkage in formalin in buccal tissues, whereas cold steel resection showed negligible differences. By contrast, cutaneous tissues showed minimal changes across resection methods. We hypothesise that buccal tissues are more susceptible to thermal effects compared to cutaneous tissues, and that true formalin-induced shrinkage is unlikely to significantly contribute to discrepancies between in vivo and histopathological margins.

Cold steel resection exhibited the least variation in dimensions and volume across tissue types, showing moderate differences in length and more pronounced shrinkage in cutaneous tissues. The unexpected increase in specimen length for both buccal and cutaneous tissues likely resulted from an anisotropic pattern of tissue shrinkage, with disproportionately greater reduction in depth affecting the changes in the other dimensions. While some dimensional changes during fixation reached statistical significance, they were relatively minor compared to post-resection changes and had little impact on overall volume. In contrast, cutting monopolar diathermy consistently caused greater shrinkage in the width of buccal tissues, with corresponding volume reductions showing moderate effect sizes. This aligns with previous findings that buccal tissues are more responsive to heat-induced shrinkage than cutaneous tissues. Fulgurate monopolar diathermy produced mixed results: it led to more depth shrinkage in cutaneous tissues, but greater reduction in post-fixation length and width in buccal tissues. These changes in length and width may be attributed to the thermal effect of the resection method. The relatively greater depth shrinkage in cutaneous tissues may account for the minimal changes observed in post-fixation length and width. The lack of significant difference in post-fixation volume between buccal and cutaneous tissues, despite significant dimensional variations, suggests that anisotropic shrinkage patterns contribute to minimal overall volume changes.

This study has several limitations that should be acknowledged. Firstly, while porcine tissues provide valuable insights, they do not perfectly mimic the complex tissue responses observed in human specimens. The use of cadaveric tissues, rather than live samples, also fails to accurately capture the dynamic physiological responses of live tissues. Refrigeration similarly does not stop processes such as protein denaturation that occurs in ex vivo tissues and will likely affect the water content and thus tissue hydration and elasticity. Additionally, disease-free tissues limit the applicability of the findings to clinical scenarios where disease presence and severity affect tissue shrinkage and margin assessment. The dimensions used may not fully reflect clinical practice, and equal measurement of length and width, though helpful in illustrating anisotropic shrinkage, could introduce inaccuracies. Increasing specimen depth might better represent real-life conditions but risks involving multiple tissue types. More precise measurement tools and a larger sample size would also improve accuracy and robustness of the findings.

Conclusions

In conclusion, the choice of resection method has a profound impact on tissue shrinkage, contributing to discrepancies between in vivo and histopathological margins. Cold steel resection demonstrated the most consistent shrinkage patterns across tissue types, suggesting its superiority for margin assessment accuracy. In contrast, thermal methods caused substantial and persistent shrinkage, complicating margin evaluation. Depth changes were the most pronounced across all methods and tissue types, highlighting a critical yet underexplored area in current research. Formalin fixation exerted its primary effect within the first 12 hours, with minimal further impact on length and width up to 48 hours. Given the high risk of recurrence and mortality associated with involved margins, careful selection of resection methods is essential to ensure accurate margin assessment. Further studies are required to confirm these findings in human tissues.

Acknowledgments

The authors would like to thank Dr. J Francois Malan for the provision of clinical space and in-kind consumables, Ms Tamara Allwood for serving as the laser safety officer and facilitating theatre access, and Dr. Ahmed Mehdi for his assistance with biostatistics.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://www.theajo.com/article/view/10.21037/ajo-24-86/rc

Data Sharing Statement: Available at https://www.theajo.com/article/view/10.21037/ajo-24-86/dss

Peer Review File: Available at https://www.theajo.com/article/view/10.21037/ajo-24-86/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://www.theajo.com/article/view/10.21037/ajo-24-86/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study did not require Animal Ethics Committee (AEC) approval, as the tissues were sourced from animals culled for routine commercial food production.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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