1. Reoperation for Hirschsprung Disease:
Pathology of the Resected Problematic
Distal Pull-Through
ALEXANDER COE,1
MARGARET H. COLLINS,2*
TAIWO LAWAL,3
EMILY LOUDEN,3
MARC A. LEVITT,3
AND ALBERTO PEN˜ A
3
1
Department of Microbiology, Ohio State University, 643 Biological Sciences Building, 484 West 12th Avenue, Columbus, OH 43210,
USA
2
Division of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH
45229, USA
3
Colorectal Center for Children, Department of Surgery, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue,
Cincinnati, OH 45229, USA
Received February 9, 2011; accepted November 9, 2011; published online November 23, 2011.
ABSTRACT
Hirschsprung disease, which consists of aganglionosis of
the rectum and sometimes more proximal bowel, requires
surgical removal of the aganglionic bowel and creation
of ganglionated neorectum using proximal normally
innervated bowel. The border between aganglionic and
ganglionic bowel is irregular; the transition zone features
variable quantities of ganglion cells and numerous large
nerves. We report the histopathology of pull-through
bowel segments resected because of poor postoperative
outcome from 30 patients (22 boys, 8 girls). The most
common indication for reoperation was severe constipa-
tion/obstruction. Transition zone (bowel with at least two
nerves 40 mm diameter per 4003 high-power field, and
ganglion cells) or aganglionic bowel (bowel with at least
two nerves 40 mm per high-power field diameter,
but without ganglion cells) was found in 19/30 (63%)
resections. In colons resected because of familial
adenomatous polyposis, rare high-power fields showed
two enlarged nerves; the mean age of those patients (135
6 49.4 months) was significantly higher than that of the
patients undergoing redo pull-through surgery (67.9 6
42.8 months). Additional pathology included stricture and
enterocolitis. Although there are multiple causes for poor
outcomes following surgical therapy for Hirschsprung
disease, abnormal innervation of the bowel used for pull-
through is common. We recommend that intraoperative
consultation at primary pull-through procedure include
frozen section evaluation of the circumference of the
bowel to be used for pull-through to confirm histologi-
cally the presence of both ganglion cells and normal-
caliber nerves. The criteria used in this study are most
suitable for infants and young children.
Key words: Hirschsprung disease, pull-through surgery,
transition zone
INTRODUCTION
Hirschsprung disease is a complex genetic disorder that
occurs in approximately 1 in 5000 live births [1,2]. The most
common clinical presentation is a neonate who does not
pass meconium in the first few hours of life, but the disease
is also sometimes diagnosed in older children who have
chronic constipation with associated abdominal distension,
failure to thrive, and/or episodes of enterocolitis.
The main pathologic feature of Hirschsprung disease
is the total absence of ganglion cells from the rectum. In
addition to the lack of ganglion cells, hypertrophied and
hyperplastic extrinsic autonomic and sensory nerves are
found in the aganglionic bowel in most patients who have
Hirschsprung disease. The hyperplastic nerves in Hirsch-
sprung disease are cholinergic, and demonstration of
increased acetylcholinesterase activity is a useful diag-
nostic feature of Hirschsprung disease [3,4].
Both aganglionosis and nerve hypertrophy and
hyperplasia may extend various lengths proximally in the
bowel in Hirschsprung disease [5]. Rarely, aganglionosis
may extend to the entire length of the colon, involve the
small intestine, or even encompass the entire gastrointes-
tinal tract [6]. The transition zone is a segment of bowel
that varies in length and is situated between distal
aganglionic and proximal ganglionic bowel; it contains
variable quantities of ganglion cells and often exhibits
hypertrophied nerves.
Definitive therapy for Hirschsprung disease is surgical
removal of the abnormally innervated bowel with creation
of a neorectum that has normally innervated bowel tissue.
Several types of surgical procedures have been developed*Corresponding author, e-mail: Margaret.Collins@CCHMC.org
Pediatric and Developmental Pathology 15, 30–38, 2012
DOI: 10.2350/11-02-0977-OA.1
ª 2012 Society for Pediatric Pathology

2. to accomplish those goals. However, some patients continue
to experience significant signs and symptoms postopera-
tively. The Colorectal Center for Children (CCC) at the
Cincinnati Children’s Hospital Medical Center (CCHMC) is
a referral center for pediatric patients who have colorectal
diseases and includes a large cohort of patients who had
prior surgery for Hirschsprung disease but who continue to
experience significant clinical problems related to bowel
dysfunction [7,8]. Here, we expand and extend our prior
studies of the causes of suboptimal outcomes following
surgical therapy for Hirschsprung disease by documenting
and illustrating the pathology of specimens resected from
patients with poor postoperative outcomes, including
specimens that included retained transition zone.
MATERIALS AND METHODS
This study was performed with approval from the
Institutional Review Board at CCHMC. The database of
the CCC at CCHMC was searched for patients meeting
study entry criteria, specifically those who had clinically
significant signs and symptoms of obstruction follow-
ing a surgical procedure for Hirschsprung disease, with
reoperation and removal of the bowel used for the prior
pull-through at the CCC and slides available for review.
Clinical information, including follow-up treatment, was
obtained through review of the electronic medical records.
Specimens from the repeat surgery were received fresh
and in most cases oriented to the distal margin. Resection
specimens were opened longitudinally, and longitudinal
sections were obtained along the entire length. In most
cases, circumferential sections were obtained from each
resection margin.
All specimens were fixed in 10% formalin, routinely
processed and embedded in paraffin, cut into sections at
5 mm, and stained with hematoxylin and eosin. In addition,
distal samples of some resected bowels were snap frozen for
enzyme histochemistry using acetylcholinesterase stain, and
those sections were cut at 8 mm. Resections from prior
Duhamel procedures included posterior wall samples.
Hirschsprung disease was defined as the absence of
ganglion cells in the submucosa and myenteric plexus
with hypertrophied and hyperplastic nerves. A transition
zone was defined as the presence of ganglion cells in the
submucosa or myenteric plexus with hypertrophied
and hyperplastic nerves. Nerve hypertrophy was defined
as nerve trunks measuring 40 mm in maximum
diameter. Nerve hyperplasia was defined as the presence
of at least 2 hypertrophied nerves in one 4003 high-
power field (0.3 mm2
). Separate nerves were defined as
nerve bundles separated by submucosal collagen. Entero-
colitis was defined as increased inflammatory cells in the
mucosal lamina propria with crypt abscesses.
Nerve hypertrophy and hyperplasia were measured in
the submucosa only; hypertrophy or hyperplasia was not
measured in nerves in the myenteric plexus, extramural
nerves, or nerves whose location could not be identified
with certainty. Submucosal nerves oriented perpendicular
to the bowel lumen that were continuous with nerves in the
muscularis propria/myenteric plexus were not included.
Colons, along with the separately submitted proctectomy
specimen, resected for familial adenomatous polyposis
(FAP) were also examined, and the submucosal nerves
were measured in those specimens. Additional features
evaluated in all specimens were enlarged submucosal
ganglia (defined as 10 ganglion cells/ganglion cluster)
and the distribution of ganglion cells in the myenteric
plexus. Only ganglion cells containing nuclei in the plane
of section were counted.
The Student t test was performed to compare groups,
and significance was set at P 0.05. Group data are
presented as means 6 standard deviations.
RESULTS
Patient demographics
Thirty patients met inclusion criteria (Table 1); 28
patients had been referred from other institutions, and 5
of the 30 (17%) original pull-through surgeries had been
performed in other countries. Twenty-two of the 30 (73%)
patients were boys and 8 (27%) were girls. Mean age at
primary pull-through for 28 patients was 10.2 6 8.21 months
(range, 0.16–34 months); age at primary pull-through
operation was unknown for 2 patients. Mean age at
reoperation of all 30 patients at the CCC was 67.9 6
42.8 months (range, 12–190 months).
Six of 30 (20%) patients had total colonic Hirsch-
sprung disease diagnosed prior to the original pull-
through. Three patients (10%) had Down syndrome, 2
patients (7%) had urologic abnormalities, and 2 patients
(7%), including one with total colonic Hirschsprung
disease, had a family history of Hirschsprung disease.
Prior procedures and indications for reoperation
The primary operation had been Soave-type pull-through
in 17/30 (57%) patients, Duhamel in 11/30 (37%) patients,
and Swenson in 2/30 (7%) patients. Five of 30 (17%)
patients underwent redo pull-through procedures prior to
the third or fourth operation at the CCC; one patient had an
initial Duhamel pull-through followed by a Soave pull-
through, one had 2 prior Soave procedures, one had 3 prior
Soave procedures, and the type of redo procedure for one
further patient with a primary Duhamel and a primary
Swenson procedure is not known.
Prior to reoperation at the CCC, all patients had
obstructive symptoms, including severe intractable con-
stipation (many with abdominal distension) and/or fecal
impaction and failure to thrive. Half of the patients
displayed symptoms of enterocolitis.
Pathology of resected distal pull-through
specimens: abnormal innervation
The most common abnormality identified histopatholog-
ically was abnormal innervation of the bowel used for the
prior pull-through procedure: 19 of 30 (63%) resected
specimens had the histologic appearance of Hirschsprung
REOPERATION FOR HIRSCHSPRUNG DISEASE 31

3. disease (aganglionosis) (2 of 19, 11%) or transition zone
(17 of 19, 89%) (Figs. 1,2). Acetylcholinesterase stain
was performed on the distal margin of 2 resections with
abnormal innervation, and the pattern was abnormal in
both. Nerve hypertrophy and hyperplasia were most
prominent in the distal portions of the resections and
were less prevalent more proximally, forming a decreas-
ing gradient from distal to proximal and resembling the
pattern of transition zone histology in primary resections;
this pattern was also seen in an ileum in the resection
from a patient who had total colonic Hirschsprung disease
and prior Duhamel procedure. The mean age of the group
with nerve hypertrophy and hyperplasia in the redo pull-
through colon resection (n 5 19) did not differ from the
mean age of the group without abnormal nerves (n 5 11),
either at the time of primary pull-through surgery (8.48 6
6.09 vs 12.9 6 10.5 months, respectively; P 0.05) or at
the time of the redo surgery reported here (66.4 6 45.5 vs
70.4 6 39.5 months, respectively; P 0.05).
Nerve hypertrophy and hyperplasia were found in
resections following all types of primary pull-through.
Twelve of the 19 (63%) specimens that showed abnormal
innervation were from patients who had a Soave pull-
through as the primary procedure, 6 (32%) were from
patients who had Duhamel pull-through as the primary
procedure (the posterior segment was sampled in Duhamel
specimens), and 1 (5%) was from a patient who had a
Swenson pull-through as the primary procedure.
Table 1. Pathology of resected pull-through specimens
Patient
# Sex
Age at
PPT (mo) Type of PPT
Length/
location of
PPT bowel Enterocolitis Stricture
Age at
redo
PT (mo)
1 M 6 Swenson; additional Swenson Rectosigmoid No No 55
2 M 12 Soave Rectosigmoid No No 26
3 M 12 Soave Rectosigmoid No No 48
4 M 11 Duhamel 10.5 cm No Yes 124
5 M Unk Soave Unk Yes Yes 106
6 F 5 Soave; additional Soave Left colon No No 43
7 M 1 Soave Recto-sigmoid No No 23
8 M Unk Duhamel; subsequent redo,
type unk
Unk No No 56
9 F 2 Duhamel Splenic flexure No No 40
10 M 7 Soave 18 cm No No 55
11 M 0.16 Soave Above splenic flexure No No 27
12 M 24 Soave Rectosigmoid No No 39
13 M 5 Soave Rectosigmoid No No 33
14 M 18 Duhamel 7.5 cm No No 109
15 M 7 Soave 11.7 cm Yes No 47
16 M 6 Soave Unk No No 111
17 M 7 Soave Sigmoid Yes No 20
18 F 13 Duhamel IC for TCHD Yes No 190
19 M 8 Duhamel Rectosigmoid No No 110
20 M 25 Soave Unk No No 48
21 F 11 Swenson TC for TCHD No No 83
22 F 24 Duhamel Unk No No 46
23 F 34 Duhamel SC for TCHD No No 128
24 M 2 Duhamel TC for TCHD No No 96
25 F 2 Duhamel; subsequent Soave IC for TCHD Yes No 79
26 M 13 Soave; additional Soave 32 Rectosigmoid No Yes 137
27 F 4 Soave IC for TCHD Yes No 32
28 M 11 Soave Unk No Yes 39
29 M 6 Duhamel 20 cm No No 74
30 M 10 Soave Unk Yes No 12
DM, distal margin of resection; HD, Hirschsprung disease; IC, ileocolectomy; mo, months; N/A, not applicable; Nl, normal; PM, proximal margin of resection;
PPT, primary pull-through; PT, pull-through; SC, subtotal colectomy; S/P, status post; TC, total colectomy; TCHD, total colonic Hirschsprung disease;
TZ, transition zone; Unk, unknown; Q, decreased episodes.
32 A. COE ET AL.

4. Five colon resections from 5 female patients with a
mean age of 135 6 49.4 months who had total colectomy
because of FAP were examined to determine whether
submucosal nerve hypertrophy and/or hyperplasia were
present. The mean age of the FAP patients was signi-
ficantly greater than the mean age of the patients
undergoing redo surgery, both at the time of primary
pull-through (P , 0.0001) and at the time of the redo
surgery reported here (P , 0.003); it was also greater than
the age of the patients who had transition zone histology in
the resected bowel (P , 0.007) as well as those who did not
(P , 0.01). One of 2 FAP patients for whom nonsurgical
medical records were available reportedly had constipation
alternating with diarrhea prior to the colectomy. In the
submucosa of the FAP bowels, nerves in the deep
submucosa near the muscularis propria were usually larger
than nerves in the more superficial submucosa near the
muscularis mucosa. In contrast to the bowels resected at
redo operations, none of the FAP colons showed
aganglionosis. However, there were rare instances of
submucosal nerve hypertrophy and hyperplasia at high-
power magnification (two or more nerves 40 mm in
diameter in a field at 4003) in the rectum and colon of the
two oldest FAP patients (192 months and 163 months,
respectively, at the time of colectomy).
Submucosal ganglion cell clusters containing 10
ganglion cells were found in both redo pull-through resections
and in FAP resections. Enlarged submucosal ganglion cell
clusters were found in 12 of 30 (40%) of the redo pull-through
resections (ie, 9 of 19 [47%] of resections with abnormal
Length of
redo PT
bowel (cm)
Length of
TZ (cm)
Distance
from TZ to
PM (cm) PM histology
DM
histology
Enlarged
submucosal
ganglia Follow-up age and status
17 11 6 Nl TZ Absent 105 mo; improved. Ileostomy;
recurrent stricture
14.4 3.5 11 Nl TZ Present 81 mo; improved
4.5 4.5 0 TZ in 1 of 3
sections
TZ Present 105 mo; improved; Q enterocolitis.
Neoappendicostomy for incontinence
43 20 23 Nl TZ Absent 171 mo; improved
3.4 3.4 0 TZ in 2 of 5
sections
HD Absent 109 mo; improved
18 8.7 9.3 Nl TZ Present 44 mo; improved
9.5 2.5 7 Nl TZ Present 35 mo; improved. Requires irrigations.
8.5 8.5 Unk Nl (additional
proximal biopsy)
TZ Absent 78 mo; improved. Soiling.
5 5 Unk Nl (additional
proximal biopsy)
TZ Absent 67 mo; improved
5.2 3.4 1.8 Nl TZ Present 60 mo; improved
18.5 4 14.5 Nl TZ Present 59 mo; improved
14.5 10 4.5 Nl TZ Present 64 mo; improved
6.5 4 2.5 Nl HD Absent 48 mo; improved
7.5 7.5 Unk Nl (additional
proximal biopsy)
TZ Absent 112 mo; improved. Colostomy
33 32 1 Nl TZ Present 70 mo; improved. Constipation, soiling.
23.5 5.1 18.4 Nl TZ Present 128 mo; improved. Periodic enterocolitis
9.5 2.8 6.7 Nl TZ Absent 43 mo; improved
33.5 24 9.5 Inflamed TZ Absent 192 mo; improved
9.5 6.6 2.9 Nl TZ Absent 146 mo; improved
18.5 N/A N/A Nl Nl Present 92 mo; S/P total colectomy; small
bowel dysmotility
7.2 N/A N/A Nl Nl Absent 126 mo; improved. Ileostomy.
25 N/A N/A Nl Nl Absent 103 mo; improved
7.1 N/A N/A Nl Nl Absent 173 mo; improved. Ileostomy
9 N/A N/A Nl Nl Present 101 mo; improved
32 N/A N/A Nl Nl Absent 93 mo; improved. Periodic enterocolitis
6.5 N/A N/A Nl Nl Present 153 mo; improved
8 N/A N/A Nl Nl Absent 60 mo; improved. Streaking
11 N/A N/A Nl Nl Absent 44 mo; improved. Soiling
8.5 N/A N/A Nl Nl Absent 95 mo; improved. Streaking
10.1 N/A N/A Nl Nl Absent 22 mo; improved. Episodic diarrhea
Table 1. Extended.
REOPERATION FOR HIRSCHSPRUNG DISEASE 33

5. innervation and 3 of 11 [27%] of the remaining resections).
The maximum number of enlarged submucosal clusters in a
slide was 5 and that was found in just 1 patient; a maximum of
2 enlarged ganglia in a slide was found in 4 cases, and only 1
enlarged submucosal ganglion cell cluster in a slide was
found in 7 cases. The average number of slides reviewed per
case was 17 (range, 9–36), the average number of slides per
case that exhibited enlarged submucosal ganglia was 2 (range,
1–3), and the average percentage of slides that exhibited
enlarged submucosal clusters was 15% (range, 7%–29%).
Enlarged submucosal ganglion cell clusters were also found
in 2 of the 5 (40%) FAP resections, who were not the oldest
FAP patients, and one had a history of alternating constipation
and diarrhea. Neither of the resections exhibiting enlarged
submucosal ganglia had 2 or more enlarged nerves in any
high-power field. The maximum number of submucosal
clusters in a slide was 4 in one case and 2 in the other. The
maximum number of slides exhibiting any enlarged submu-
cosal ganglia were 6 of 12 (50%) from one patient and 2 of 15
(13%) from the other. The mean age of Hirschsprung disease
patients with enlarged submucosal ganglia in the redo bowel
(58.3 6 36.5 months) did not differ significantly from those
without enlarged ganglia (74.2 6 46.4 months) (P 0.05).
The mean age of patients whose resection showed nerve
hypertrophy and hyperplasia and who had enlarged submu-
cosal ganglia (n 5 9/19, 47%) (46.6 6 26.6 months) did not
differ from the mean age of patients whose bowel showed
abnormal nerves but not enlarged submucosal ganglia (n 5
10/19, 53%) (84.3 6 52.6) (P . 0.05). Similarly, the mean
age of the patients whose resection did not show nerve
hypertrophy or hyperplasia but who had enlarged ganglion
cell clusters (n 5 3/11, 27%) (93.7 6 44.5 months) did
not differ significantly from the mean age of patients who did
not have either (n 5 8/11, 73%) (61.6 6 36.7 months) (P .
0.05).
There were no instances of partial aganglionosis of
the myenteric plexus in any of the resection specimens.
Enterocolitis
Enterocolitis was diagnosed clinically in 16 of the 30
(53%) patients. Inflammation consistent with enterocolitis
was found in 7 of 30 (23%) resections, which included 6 of
the 16 (38%) patients who had the clinical diagnosis. Also,
10 of the 16 (63%) patients with a clinical diagnosis of
enterocolitis had transition zone in their resected bowel.
Additional pathology in resected pull-
through specimens
Of the 30 cases, strictures were found at the anastomotic
sites in 4 (13%) resection specimens (3 Soave, 1 Duhamel)
(Fig. 1). In 2 of the 4 (50%) specimens that demonstrated
stricture, nerve hypertrophy and hyperplasia were identi-
fied in the distal portion of the bowel used for prior pull-
through, and histologic evidence of enterocolitis was found
in one of those. An additional specimen was reported
to have a stricture that was not recognized in the resect-
Figure 1. Bar graph indicating the number of cases with the
major findings in this study.
Figure 2. A. Enlarged submucosal nerves, some exceeding 50 mm are seen in the submucosa of a resected specimen.
Ganglion cells (arrows) are seen in the enlarged nerve at the right (arrows); HE, 3100. B. Enlarged nerve exceeding 80 mm
is seen in the myenteric plexus. Ganglion cells are seen in the smaller nerve in lower right (arrows); HE, 3200.
34 A. COE ET AL.

6. ed bowel that had been opened prior to submission to
pathology.
An inflamed channel consistent with a rectovaginal
fistula was found in a specimen from a patient who had
2 prior Soave procedures and had a rectovaginal fistula
repaired during a prior reoperation. A suture abscess was
present in a specimen resected 15 months after the initial
surgery, and focal ischemic changes were seen in the
muscularis propria of another specimen resected 14 years
after the initial procedure.
Resections without histopathological correlation
with clinical dysfunction
There were 5 cases that did not display stricture, nerve
hypertrophy or hyperplasia, or enterocolitis. Clinical
findings that correlated with bowel dysfunction in those
cases were dilated Duhamel pouches in 4 cases and chronic
bowel dilation in 1 case, as evidenced on contrast enema
studies.
Follow-up
Follow-up information, including age and symptoms at last
encounter recorded in the electronic medical record, is
provided in Table 1. The mean length of follow-up was 24.8
6 18 months (range, 1–57 months). All patients showed
clinical improvement after reoperation with resolution of
presenting obstructive symptoms.
DISCUSSION
Pathologists whose practice includes surgical and/or
gastrointestinal pathology may be involved in the care of
patients who have received surgical therapy for Hirsch-
sprung disease and who may manifest significant bowel
dysfunction after their pull-through. Poor outcomes are
explainable by gross or microscopic pathologic findings
[8]. Signs and symptoms that define poor outcome
continue until surgically corrected, and therefore patholo-
gists who care for adults as well as those who care for
children need to know the causes of bowel dysfunction
after surgery for Hirschsprung disease. Our study identifies
gross and microscopic pathologies that correlate with poor
outcomes. These data demonstrate that the use of
abnormally innervated bowel for pull-through procedure
is not dependent on the type of pull-through. These data
also suggest that abnormally innervated pull-through tissue
increases the risk of postoperative enterocolitis.
The processing of specimens removed during a
second or subsequent operation to correct Hirschsprung
disease poses challenges. Pathologists must know the
type of pull-through that was performed previously, and
the specimens must be oriented correctly to ensure proper
examination and interpretation of findings. For example,
a Duhamel pouch by definition is partly aganglionic,
since part of the pouch (the anterior portion) consists
of the native aganglionic rectum; therefore, samples for
histology must be taken specifically from the bowel that
was used in the pull-through procedure (namely, the
posterior portion of the Duhamel pouch). Our study is a
retrospective review of existing slides and gross descrip-
tions from specimens handled by multiple pathologists
using customary lab procedures for evaluating primary
bowel resections removed for Hirschsprung disease. A
standard protocol for processing resections of previous
pull-through operations was not used, but despite this
limitation, in all cases except one, we ascertained
histologic sections that included the distal margin of
the bowel that had been used as the pull-through bowel.
In this series of patients who required reoperation
because of persistent clinically significant signs and
symptoms of bowel obstruction following primary
surgery for Hirschsprung disease, the most prevalent
abnormality was evidence of abnormal innervation in
the pull-through bowel. The use of aganglionic bowel or
transition zone for pull-through procedures in Hirsch-
sprung disease can be prevented by the use and proper
interpretation of intraoperative frozen sections. Although
the interpretation error rate of intraoperative frozen
sections may be low, the consequences of misinterpreta-
tion may be so severe as to require reoperation [9–11]. In
a large series, frozen section diagnosis was compared
to the final diagnosis in leveling procedures for Hirsch-
sprung disease. Reoperation was not required in cases in
which more extensive bowel resection was performed
because ganglion cells were said to be absent on frozen
sections but were present on permanent sections, whereas
reoperation was required for cases in which ganglion cells
were said to be present on frozen sections but were not
actually present on permanent sections [10]. In other
words, cases in which ganglionic bowel was used for pull-
through had superior outcomes compared to those in
which aganglionic bowel was used for the pull-through.
These data, and the data reported herein, emphasize the
importance of accurate intraoperative recognition and
reporting of abnormally innervated bowel during primary
operative procedures for Hirschsprung disease.
Multiple abnormalities reported in the transition
zone strongly suggest that such bowel does not function
normally. Although the transition zone in Hirschsprung
disease contains ganglion cells focally, it is not normally
innervated. Structural alterations in the transition zone
include isolated ganglia that are not connected with nerve
bundles (which explains why they are not functional),
as well as ganglia that are displaced into the inner or
outer layers of the muscularis propria [12]. Absent and
decreased neuropeptides (substance P and met-enkepha-
lin, respectively) that are essential for normal bowel
function have been found in bowel corresponding to the
transition zone [13]. Interstitial cells of Cajal, required for
normal intestinal motility, are decreased in the transition
zone in Hirschsprung disease [14]. Suboptimal postoper-
ative function could occur following pull-through proce-
dures utilizing such bowel.
Histologic recognition of the transition zone is
confounded by numerous factors. The transition zone is
REOPERATION FOR HIRSCHSPRUNG DISEASE 35

7. not histologically homogeneous, ie, ganglion cells are not
found circumferentially in bowel comprising the transi-
tion zone; instead, ganglion cells are interspersed with
aganglionic areas [15]. This configuration is a result of
the fact that there may be one or multiple ‘‘leading
edges’’ of ganglion cells extending caudally into the
aganglionic segment in Hirschsprung disease in both
submucosa and myenteric plexus. Unfortunately, myen-
teric plexus ganglion cells may appear to be present in
normal numbers at the leading edge, masking contigu-
ous as well as more proximal hypoganglionosis [15].
Furthermore, the leading edges of ganglion cells in the
submucosa and myenteric plexus are not always present
at the same level [15,16]. Therefore, the discovery of
ganglion cells in the myenteric plexus in an intraoperative
seromuscular biopsy submitted for frozen section does not
necessarily indicate that ganglion cells are present in the
corresponding submucosa.
Another histologic feature that should be evaluated
during intraoperative consultation at primary pull-through
procedure is nerve hypertrophy and hyperplasia, which
are well described as histologic features aiding in the
recognition of both aganglionosis and transition zone in
rectal biopsy specimens obtained for diagnosis of
Hirschsprung disease [4,5]. However, this histologic
feature is much less emphasized in intraoperative frozen
sections to guide selection of bowel for definitive surgical
therapy. In transition zones, hypertrophied submucosal
nerves are not always found in fields with submucosal
ganglion cells [15]. Therefore, identifying submucosal
ganglion cells in an intraoperative frozen section obtained
from a single site in the bowel circumference does not
rule out the possibility that hypertrophied nerves are
present at a different site at the same level.
The criterion for nerve hypertrophy used in this
study (diameter 40 mm) was developed in a study of
rectal biopsy specimens of young patients who had
Hirschsprung disease [5]. A criterion for nerve hyperpla-
sia was not provided in that study. In this study, the
presence of 2 or more hypertrophied nerves in a 4003
(0.3 mm2
) high-power field was used as a minimum
criterion to define nerve hyperplasia and to define the
proximal extent of the transition zone. Nerve hyperplasia
in the transition zones in the resected pull-through bowels
reported in this study was not subtle; the criteria we used
for nerve hypertrophy and hyperplasia were minimum
requirements to determine abnormal nerves. They are
intended to define the proximal extent of the ganglionated
transition zone by distinguishing transition zone from
normal bowel and may be useful during intraoperative
consultations to determine the ideal location of pull-
through. Our study does not permit recommendations
concerning the number of sections that should be
examined during intraoperative consultations, but multi-
ple sections are virtually always more revealing than a
single section.
Although all patients in this series had primary
surgery for Hirschsprung disease, followed by reoperation
because of signs and symptoms of obstruction, nerve
hypertrophy and hyperplasia as defined in this study were
not found in all resections, supporting the application of
these criteria to identify transition zone at either primary
surgery or reoperation. However, our data also suggest
that these criteria for nerve hypertrophy and hyperplasia
may be age-specific, since bowels from children without
Hirschsprung disease who were older than the patients
with Hirschsprung disease in this series exhibited few
examples of nerve hypertrophy and hyperplasia, suggest-
ing that nerve diameter and density may change with
age. Age-appropriate criteria that suitably define nerve
hypertrophy and hyperplasia for older children and
adolescents undergoing primary pull-through should be
developed.
Enlarged submucosal ganglia were found in resec-
tions from patients in all groups in this study. The
relationship between submucosal nerve hypertrophy and
hyperplasia and enlarged submucosal ganglia was incon-
sistent. Enlarged ganglia were found in more than one
third of bowels in the redo pull-through and FAP groups
that did not exhibit nerve hypertrophy and hyperplasia
according to our criteria, and they were found in fewer
than half of the resections exhibiting nerve hypertrophy
and hyperplasia. Therefore, enlarged submucosal ganglia
cannot be considered as surrogate markers for submucosal
nerve hypertrophy and hyperplasia and should not be used
as the sole criterion to identify transition zone.
In the bowels that exhibited nerve hypertrophy and
hyperplasia, the changes were most apparent in the distal
bowel and decreased in frequency and severity with
increasing distance from the distal margin. Two of our
cases had foci of nerve hypertrophy and hyperplasia at
the proximal margin of the bowel resected at our redo
operation. In 1 case, the decision to perform the redo
surgery transanally without entering the abdomen was
made preoperatively with the patient’s family, with the
knowledge that the resection might not remove the entire
transition zone. All of the patients improved postopera-
tively, which included fewer episodes of enterocolitis.
These observations suggest that the concentration or
prevalence of changes at the proximal margin might be
important for future bowel function.
Other studies that included histopathologic analyses
of pull-through bowels that were removed because of
poor clinical outcomes following surgical therapy for
Hirschsprung disease have identified abnormal innerva-
tion in the resected specimens [9,17–23]. Those studies
and our own lead us to make several observations and
recommendations concerning the selection of bowel to be
used for the primary pull-through surgery.
First, site selection should include intraoperative
frozen section examination of the level initially identified
during surgery as potentially normal. If this site is deemed
abnormal by the pathologist, then additional specimens
for frozen section evaluation should be submitted from
bowel proximal to the initial biopsy area.
36 A. COE ET AL.

8. Second, seromuscular biopsy for intraoperative local-
ization of normal bowel is not adequate for several reasons.
The leading edge of ganglion cells in the myenteric plexus
in the transition zone may appear normally ganglionated,
masking transition zone proximal to the leading edge, and
the corresponding submucosa may be aganglionic. In a
separate analysis of the patients examined here, we found
that 3 patients with transition zone pathology in their pull-
through bowel resected because of persistent bowel
obstruction had an intraoperative frozen section evaluation
of a seromuscular biopsy at the primary pull-through that
showed ganglion cells [24]. This practice of a seromuscular
biopsy is common since the advent of laparoscopic pull-
through procedures. We recommend a full-thickness
biopsy instead.
Third, an intraoperative transmural biopsy for frozen
section is highly preferable to a seromuscular biopsy
because it provides the ability to identify submucosal
aganglionosis and submucosal nerve hyperplasia. Even
so, the transition zone can display deceptively normal-
looking histology intermixed with more ominous histol-
ogy that may be missed with a single-site transmural
biopsy.
Finally, therefore, the optimum intraoperative eval-
uation is a frozen section of a ‘‘donut’’ of the site planned
for pull-through. This can be sent for confirmation after a
preliminary laparoscopic biopsy prior to commencing the
anastomosis. A recent analysis of myenteric ganglion cell
density and distribution documented that an examination
that does not include the full circumference of bowel wall
yields results that vary significantly from counts obtained
by examining the entire circumference [25]. Furthermore,
the percent of the circumference of the proximal margin
of the resected bowel that is occupied by ganglionated
myenteric plexus correlates with immediate and long-
term outcomes following surgery for Hirschsprung
disease [11]. We reiterate the recommendation made a
decade ago [23] that the optimal method to choose bowel
for pull-through is to examine the entire circumference of
the bowel, a method that has become standard practice
in some labs [11]. If it is not possible to obtain a frozen
section on a donut, the sample should be submitted for
permanent sections and evaluated for both ganglion cells
and nerve hypertrophy, as this information will help to
guide postoperative management if there is a poor clinical
outcome. If a separate sample is not submitted by the
surgeon, the donut should be obtained by the pathologist
from a circumferential section of the proximal margin of
the resected bowel.
We recognize that the significance of nerve hyper-
trophy and hyperplasia is determined by the clinical
context. The admonition to remember that enlarged
nerves may be found at sites of enterostomy or biopsy
specimens from patients with imperforate anus is
appropriate [5]. If, however, enlarged and numerous
nerves are found in an enterostomy site in the setting
of Hirschsprung disease in a young child, the possibility
of transition zone at that site should be considered.
Similarly, enlarged nerves in specimens from patients
who have imperforate anus or other forms of anorectal
malformation could conceivably be part of the malfor-
mation, but the possibility of coexistent Hirschsprung
disease (and transition zone) should also be considered,
particularly for biopsies obtained because of constipation
following repair of the malformation [26].
There are several limitations to our study and
therefore to our recommendations. Since most of our
cases were referrals, we did not have the opportunity to
re-review the proximal margin of the bowel resected at
primary pull-through; however, the distal margin of the
bowel resected at redo is likely to have been immediately
adjacent to the proximal margin of the primary resection
and therefore provide a reasonable facsimile of that
margin. Also, this is a retrospective study of a select
group of patients who experienced unacceptable out-
comes after surgical therapy for Hirschsprung disease.
We do not know how many, or whether, patients who
have good outcomes after surgical therapy have abnor-
mally innervated bowel in the neorectum; those data
likely will not ever be known because those patients
do not have postoperative rectal biopsies. It is highly
improbable that there will ever be a prospective
randomized study comparing outcomes following surgery
using transition zone for primary pull-through with
outcomes following surgery using histologically normal
bowel (bowel that contains ganglion cells and does not
contain hypertrophied nerves) for primary pull-through.
We hope that our plea to consider more than ganglion
cells to determine the suitability of bowel to be used for
pull-through will prevent the inadvertent incorporation of
transition zone in the neorectum, which we truly believe
is an action that would not be taken deliberately. We
recognize that there are other causes, including surgical
technique, of poor outcomes following primary pull-
through that do not involve abnormal innervation, such as
strictures (which were also found among our cases).
However, we have now seen 16 cases in which
microscopic abnormalities were the only abnormalities
identified, and all of those patients improved following
redo operations [24]. We recognize that Hirschsprung
disease is a complex disease that may include abnormal-
ities in the bowel that appear normal on routine stains.
Nevertheless, the collective data suggest that incomplete
resection of transition zone as defined in this or other
studies of Hirschsprung disease may contribute to
postoperative morbidity requiring reoperation in some
cases, and that a conservative approach to avoid such
complications might be to follow the intraoperative
recommendations provided.
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