Abstract
Background:
Hirschsprung disease (HD) is an aganglionosis of variable length starting at the rectosigmoid colon with surgery as sole therapeutic option. The length of the resected bowel segment is a crucial information for the treating surgeons and influences the prognosis of the patient. It is often artificially altered due to post operative tissue shrinkage. The objective of this study is to quantify the extent tissue shrinkage of HD specimens.
Material and Methods:
Colorectal HD specimens were measured at the time of surgery and at the time of cut-up, either fresh or after formalin fixation and statistically analyzed.
Results:
Sixteen colorectal specimens were included. Following formalin fixation the specimen length decreased by 22.7% (P < .001). Without formalin fixation the specimens shrank by an average of 24.9% (P = .05). There was no significant difference in the extent of tissue shrinkage with or without formalin fixation (P = .76).
Conclusion:
This study showed that there is significant tissue shrinkage in HD specimens. The 2 different cohorts revealed that tissue shrinkage is mostly caused by tissue retraction/alteration after organ removal but also to a lesser extent by fixation with formalin. Surgeons and (neuro-)pathologists should be aware of the sizeable shrinking artifact to avoid unnecessary confusion.
Keywords: Hirschsprung, pediatric, PediPath, Surg Path, neuropathology, GI
Background
First described in 1888, Hirschsprung’s disease (HD) is an aganglionosis of the large bowel, characterized by failed migration of the ganglion cells of the plexus submucosus and myentericus. 1 Usually starting from the anus, varying lengths of the colon are affected, ranging from anorectal/rectosigmoid segment to affect the whole large bowel. 2 In rare cases, the whole intestine is affected. In about 70%, the disease appears isolated; in about 18% of patients, other malformations can be found without any underlying syndromic disease identified. Chromosomal alterations can be found in 12% of cases. 2 With the affected bowel being unable to relax, most of the infants (90%) fail to pass meconium in the first 24 hours. 1 Other symptoms include difficult bowel movements, poor feeding, emesis, and progressive abdominal distention during the first few months. Diagnostic procedures include anorectal manometry, contrast enema radiography, and histological evaluation of a rectal suction biopsy above the anorectal dentate line.1,2 Surgery is the only therapeutic option for HD with transanal endorectal pull-through (TERPT) being one of the most commonly performed procedures. 3 In TERPT, the affected colon part is resected transanally by pulling the aganglionic rectum/colon through the anus and resecting it while establishing a primary colo-anal anastomosis. 4 During this surgery, mapping biopsies are taken. These are examined by a pathologist with frozen sections to identify a normal ganglionated plexus of the bowel segment. Depending on the case, the proximal resection margin should also be examined intraoperatively by frozen section. 2 The length of the resected rectum/colon depends on the extent of HD and varies a lot. Upon formalin fixation, a (neuro-) pathologist determines the final length of the aganglionic segment and total resected specimens. In our routine practice, we have experienced misunderstandings because of the discrepancies in length at the time of surgery and after gross examination of the formalin-fixed specimens in the final pathological report. This information is crucial for quality assessment and communication between pathologist and surgeon. Since there is no literature available on shrinkage of HD bowel specimens in the process of workup, the objective of this study is to quantify the extent of HD specimens shrinkage from the time of the fresh specimen removal at the point of surgery up to formalin fixation.
Material and Methods
Colorectal HD specimens from TERPT operations (Figure 1) were measured directly at the time of surgery as well as at the time of gross examination. In total, 16 colorectal specimen from 2 institutions with slightly different gross examination techniques were included resulting in 2 separate cohorts. In the first cohort (Bristol, n = 8), the specimens were measured at the time of the surgery and after formalin fixation. To assess tissue shrinkage independent of formalin fixation, a second cohort (Frankfurt, n = 8) measured samples at the time of surgery and 25 minutes after organ removal without formalin fixation. Furthermore, one specimen was measured at the time of surgery, 25 minutes after surgery without fixation, and after 24 hours of formalin fixation. As a proof of principle, one specimen was pinned to see if specimen shrinkage can be avoided. All specimens were measured at room temperature.
Figure 1.
Transanal endorectal pull-through. (A) Pull-through surgery with specimen in situ. (B, C) Resection specimen before and after opening. The narrow part is the distal part devoid of muscularis propria. Opposite the narrow part is the healthy, fully ganglionic resection margin. Black arrows indicate biopsy sites.
For statistical significance of the specimen shrinkage, the data was analyzed with paired t-tests. All data was analyzed with SPSS 26 (IBM; Armonk, BY, USA) statistical software. The significance level was set to P < .05.
Results
In total, 16 colorectal specimens were included. Specimen length at the time of surgery ranged from 100 to 480 mm. The mean length at the time of the surgery was 235.2 mm (±6.9 mm); the mean length at the time of the sectioning, was 181.1 mm (±5.8 mm), resulting in an average shrinkage of 23.8% (P < .001). The lengths of the individual specimen are displayed in Table 1.
Table 1.
Postoperative Tissue Shrinkage in the Individual HD Specimen. (A) Individual Lengths and Shrinkage of the Pull Through Specimen Direct After Surgery and After Fixation With Formalin. (B) Individual Lengths and Shrinkage of the Pull Through Specimen Direct After Surgery and After 25 minutes Without Formalin Fixation.
| A. Patients | T = 0 min (mm) | Post fixation (mm) | Shrinkage (%) |
|---|---|---|---|
| P1 | 180 | 132 | 26.7 |
| P2 | 230 | 190 | 17.4 |
| P3 | 100 | 80 | 20.0 |
| P4 | 130 | 80 | 38.5 |
| P5 | 155 | 115 | 25.8 |
| P6 | 198 | 170 | 14.1 |
| P7 | 250 | 190 | 24.0 |
| P8 | 130 | 110 | 15.4 |
| Mean | 171.6 | 133.3 | 22.7 |
| B. Patients | T = 0 min (mm) | After 25 min (mm) | Shrinkage (%) |
| P9 | 300 | 220 | 26.7 |
| P10 | 180 | 115 | 36.1 |
| P11 | 220 | 150 | 31.8 |
| P12 | 480 | 420 | 12.5 |
| P13 | 290 | 280 | 3.4 |
| P14 | 450 | 270 | 40.0 |
| P15 | 350 | 295 | 15.7 |
| P16 | 120 | 80 | 33.3 |
| Mean | 298.7 | 228.7 | 24.9 |
The 8 colorectal specimens from the cohort which underwent formalin fixation (Bristol) measured from 100 to 250 mm with a mean length of 171.6 mm (±6.1 mm) directly after resection. Following formalin fixation in 4% formalin, the mean length was 133.9 mm (±5.3 mm) ranging from 80 to 190 mm. The specimen length decreased significantly by 22.7% (Figure 2(A); P < .001).
Figure 2.
Postoperative tissue shrinkage. (A) Mean length of the pull-through specimen direct after surgery and after fixation with formalin (**P > .001). (B) Mean length of the pull-through specimen direct after surgery and after 25 minutes without formalin fixation (*P > .05). (C) Mean tissue shrinkage with and without fixation (P = .76). Values are shown as means with standard deviation.
In a second cohort without formalin fixation (Frankfurt), 8 other specimens were measured at the time of the surgery and 25 minutes after organ removal in order to assess the effect of shrinkage unrelated to formalin fixation. In this group the mean length after organ removal was 298.8 mm (±14.7 mm) with specimen length ranging from 120 to 480 mm. 25 minutes after organ removal the mean length was 228.8 mm (±13.0 mm) with a minimal length of 80 mm and a maximal length of 420 mm, showing a significant tissue shrinkage of 24.9% (Figure 2(B); P = .005). There was no significant difference in the extent of tissue shrinkage with or without formalin fixation (Figure 2(C); P = .76).
One specimen was measured at the time of the surgery, left for 25 minutes before formalin fixation, and then measured a third time after formalin fixation. In the first 25 minutes, the specimen shrank by 19% (155–125 mm), then shrank an additional 8% (125–115 mm) upon formalin fixation, implying that most of the shrinkage (78%) occurs directly after resection and is unrelated to formalin fixation. As a proof of principle, one specimen was pinned up after resection. This specimen did not show any shrinkage and stayed at the length of 125 cm.
Discussion
Although tissue shrinkage after organ removal is a common phenomenon, literature about the topic is surprisingly sparse. In the past, few studies investigated the effect of specimen shrinkage in different organs (Table 2). In order to properly examine operative specimens, it is common practice to fix them in 4% formalin immediately after the resection to avoid autolysis. During the fixation process, formalin diffuses into the tissue, generating crosslinks between proteins and proteins or proteins and nucleic acids. This hardens and preserves the tissue but can also lead to shrinkage and distortion of the tissue as well as changes in antigen epitopes.5,6 Furthermore, the resected specimen can shrink due to retraction forces that occur in the organ after being removed out of its integrity in the human body.
Table 2.
Postoperative Tissue Shrinkage of Different Specimen After Formalin Fixation Reported in Literature.
| Author | Specimen | Shrinkage (%) |
|---|---|---|
| Goldstein et al 7 | Colon | 57 |
| Weese et al 8 | Colon | 48 |
| Lam et al 9 | Colorectal | 14.99 |
| Siu et al 10 | Esophagus | 50 |
| Dauendorffer et al 11 | Skin | 16–18 a |
| Chen et al 12 | Head and neck cancer | 4.1–6.4 |
| Tran et al 6 | Renal tumors | 4.6 |
| Park et al 13 | Lung cancer | 4.06 |
The 16–18% in skin tissue shrinkage appeared after resection and before formalin fixation.
Early studies on tissue alteration focused on adult colonic cancer specimen and reported a great extent of specimen shrinkage. In 26 colorectal specimens, Goldstein et al 7 reported a total shrinkage of 57%. Weese et al 8 found a reduction of 48% in length. In contrast to that, Lam et al 9 recently described a much lower effect of 15% specimen shrinkage in a group of 46 patients. This result is much closer to our results with a median reduction of 26.3% after formalin fixation. At least part of the reason might be the fact that Goldstein used higher concentrated formalin (10%) for the fixation process. In esophageal specimens, Siu et al 10 reported an average shrinkage of 50%. In contrast to gut specimens, the effect of formalin fixation appears to be lower in parenchymatous organs and solid tumors. Tumors in the kidney, the lung and head and neck cancer showed an average shrinkage of 4–6%.6,12,13 Interestingly, skin appears to react differently to formalin fixation. Along others, Dauendorffer et al 11 reported a shrinkage of 16–18% upon resection of the skin specimens, with no further changes after formalin fixation. While this is most striking in skin specimens, this is a phenomenon which other studies, including our own, observed as well. The largest part of tissue shrinkage happens directly after removing the specimens from the body, before formalin fixation. In skin, almost 100% of shrinkage happens before formalin fixation. Siu et al reported that 80% of esophageal shrinkage occurred before formalin fixation. In colonic specimens ~70% of shrinkage happens in the first 10–20 minutes, while only 30% of shrinkage happens due to formalin fixation.7,8 These observations are in line with our results. In the Bristol cohort with formalin fixation, a shrinkage of 22.7% was seen. In the Frankfurt cohort, in which the HD specimens were measured without formalin fixation, a length reduction of 24.9% was observed, showing no significant difference to the Bristol cohort. The fact that there is no difference in tissue shrinkage with the addition of formalin fixation implies that most of tissue shrinkage happens independent of formalin fixation, immediately after organ removal by its intrinsic tissue retraction. This effect might be slightly increased because at the time of the surgery the specimens are usually measured under slight tension. The different extent of shrinkage fresh versus formalin fixated tissue was also shown in one specimen, which was measured at the time of the surgery, 25 minutes later and then after fixation with formalin, where most of the shrinkage (78%) happened before formalin fixation.
The observed tissue shrinkage implies direct consequences for surgeons and pathologists. Kapur et al showed that in short-segment HD the transition zone, a segment of ganglionic, but neuroanatomically abnormal bowel, is confined to a 5 cm region proximal of the aganglionic segment. Therefore, they suggested a resection with a minimum of additional 5 cm proximal to the site with the first identified ganglionic cells. 14 Our study implies that additional 1–2 cm should be resected to ensure an anastomosis of completely ganglionated bowl segments. Our study showed that the measurements in HD specimens can vary significantly. Therefore, an intraoperative assessment of full circumference sections of the proximal margins should be considered, to reliably identify fully ganglionic resection margins and to avoid a resection within the transition zone in TERPT. 15
Our study presents the first data on HD specimen shrinkage. It revealed there is significant tissue shrinkage comparable to what is seen in adult large bowel specimens. As in most studies, our data imply that post surgical tissue shrinkage is a multifactorial process with tissue retraction immediately after organ removal being a more relevant factor for tissue shrinkage than tissue alteration due to formalin fixation.
Conclusion
A diagnosis of HD causes a degree of anxiety for parents. Therefore, precise communication is key to a successful treatment. To avoid unnecessary confusion, surgeons as well as pathologists and clinicians should be aware that the specimen length can vary from the time when the surgeon removes the organ after resection to when the specimen is examined histologically. This phenomenon appears to happen primarily due to tissue retraction/alteration but also, to a minor extent, due to fixation artifacts. This is the first study which shows the extent of HD specimen shrinkage which should be expected by the surgeon and which should always be communicated to avoid unnecessary misunderstandings.
Footnotes
Author Contributions: Conceptualization, S. G. and E. G.; methodology, E. G. and P. N. H.; data curation and analysis, S. G., K. J. W., Y. B., and J. M.; writing—original draft preparation, S. G. and E. G.; writing—review and editing, K. J. W., Y. B., P. N. H., U. R., and J. M. All authors have read and agreed to the published version of the manuscript.
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding: The author(s) received no financial support for the research, authorship, and/or publication of this article.
ORCID iDs: Steffen Gretser 
https://orcid.org/0000-0002-4875-3808
Elise Gradhand https://orcid.org/0000-0003-3143-0487
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