Controversies concerning diagnostic guidelines for

anomalies of the enteric nervous system:

A report from the fourth International Symposium on
Hirschsprungs disease and related neurocristopathies
Giuseppe Martucciello
a,
*
, Alessio Pini Prato
b
, Prem Puri
c
,
Alexander M. Holschneider
d
, William Meier-Ruge
e
, Vincenzo Jasonni
b
,
Juan A. Tovar
f
, Jay L. Grosfeld
g
a
Department of Pediatric Surgery, IRCCS Policlinico San Matteo, Pavia I-27100, Italy
b
Department of Pediatric Surgery, G Gaslini Institute, Genova I-16148, Italy
c
Department of Pediatric Surgery, Our Ladys Hospital, Crumlin 12, Dublin, Ireland
d
Department of Pediatric Surgery, Childrens Hospital of Cologne, Cologne D-50735, Germany
e
Department of Pathology, University of Basel, Basel CH-4003, Switzerland
f
Department of Pediatric Surgery, University of La Paz, Madrid 28046, Spain
g
Department of Pediatric Surgery, Riley Childrens Hospital, Indianapolis, IN 46202-5200, USA
Abstract Intestinal Dysganglionoses (IDs) represent a heterogeneous group of Enteric Nervous System
anomalies including Hirschsprungs disease (HD), Intestinal Neuronal Dysplasia (IND), Internal Anal
Sphincter Neurogenic Achalasia (IASNA) and Hypoganglionosis. At present HD is the only recognised
clinico-pathological entity, whereas the others are not yet worldwide accepted and diagnosed. This
report describes the areas of agreement and disagreement regarding definition, diagnosis, and
management of IDs as discussed at the workshop of the fourth International Meeting on
bHirschsprungs disease and related neurochristopathies.Q
The gold standards in the preoperative diagnosis of IDs are described, enlighting the importance of
rectal suction biopsy in the diagnostic workup. The most important diagnostic features of HD are the
combination of hypertrophic nerve trunks and aganglionosis in adequate specimens. Acetylcholines-
terase staining is the best diagnostic technique to demonstrate hypertrophic nerve trunks in lamina
propia mucosae, but many pathologist from different centers still use H&E staining effectively.
Moreover, the importance of an adequate intraoperative pathological evaluation of the extent of IDs
to avoid postoperative complications is stressed. Although it is not clear whether IND is a separate entity
or some sort of secondary acquired condition, it is concluded that both IND and IASNA do exist. Other
interesting conclusions are provided as well as detailed results of the discussion. Further investigation is
0022-3468/$ see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.jpedsurg.2005.07.053
T Corresponding author.
Index words:
Hirschsprungs disease;
Aganglionosis;
Enteric nervous system;
ENS;
Intestinal neuronal
dysplasia
Journal of Pediatric Surgery (2005) 40, 15271531
www.elsevier.com/locate/jpedsurg
needed to resolve the many controversies concerning IDs. The fourth International Conference in Sestri
Levante stimulated discussion regarding these entities and led to the International guidelines to serve
the best interest of our patients.
D 2005 Elsevier Inc. All rights reserved.
Intestinal dysganglionoses (IDs) represent a heteroge-
neous group of anomalies of the enteric nervous system
(ENS) including Hirschsprungs disease (HD), intestinal
neuronal dysplasia (IND), internal anal sphincter neurogenic
achalasia (IASNA), and hypoganglionosis.
At present, internationally, the only widely recognized
and accepted clinical-pathological entity is HD, whereas
IND, IASNA, and hypoganglionosis are not yet accepted
conditions, nor are these diagnosed by clinicians in many
countries. bAre they true congenital malformations or
acquired phenomena?Q This is the major debate regarding
the origins of these anomalies. Some authors firmly believe
in the independent existence of all forms of IDs, for which
they defined diagnostic criteria and proposed therapeutic
protocols [1- 4]. Conversely, others maintain a critical
position regarding these controversial conditions as they
consider ENS anomalies as secondary acquired phenomena
sometimes associated with other diseases such as bowel
atresias, HD, or simple constipation [5-7].
All the pathological features of ENS have been described
in depth, and more than 70 diagnostic, enzymatic-histo-
chemical, and immunohistochemical staining techniques
have been proposed for ENS evaluation. These include
H&E, acetylcholinesterase (AChE), rapid AChE, lactate
dehydrogenase (LDH), succinic dehydrogenase, alphanaph-
tylesterase, and nicotinamide adenine dinucleotide phos-
phate diaphorase (NADPH-d) [8-15]. Peripherin, cathepsin
D, PGP 9.5, synaptophysin, chromogranins, S-100 protein,
2 nerve markers-erythrocyte-type glucose transporter
(GLUT-1), choline acetyltransferase, nerve growth factor
receptor, neuron-specific enolase, neurofilaments, vasoac-
tive intestinal peptide, neuropeptide galantine, substance P,
neuropeptide Y, calcitonin generelated peptide, gastrin
releasing peptide, enkephalin (Met-ENK), and acridine
orange comprise the numerous histochemical stains cur-
rently in use [8,16-21].
Most of the hospital facilities dealing with IDs cannot
afford the adoption of expensive and sophisticated staining
techniques on a daily routine basis for ENS examination [22]
and, therefore, frequently use nonspecific histomorpholog-
ical staining techniques such as H&E to diagnose HD.
This report describes the areas of agreement and
disagreement regarding definition, diagnosis, and manage-
ment of IDs, discussed at a workshop of the fourth
International Meeting on bHirschsprungs disease and
related neurocristopathies.Q The aim is to focus on aspects
of IDs for which some consensus among specialists in the
field was achieved and some general guidelines devel-
oped. It is fully recognized, however, that this only
represents a starting point for future important clinical and
research studies.
1. Materials and methods
The workshop entitled bCriteria for Classification and
Diagnosis of DysganglionosesQ was part of the fourth
International Meeting on bHirschsprungs disease and
related neurocristopathies.Q The meetings participants
included basic scientists, geneticists, pathologists, and
pediatric surgeons. The meeting was held in Sestri
Levante, Genoa, Italy, on April 22 to 24, 2004, and was
organized as follows:
Some of the most recognized authors in this field
(W Meier-Ruge, P Puri, G Martucciello, and AM Holsch-
neider) participated in a round table. The chairmen of the
session (JL Grosfeld and JA Tovar) presented 10 items for
debate regarding IDs to stimulate open discussion from the
roundtable members and the audience. Approximately
150 participants joined in-depth discussion on each debated
point. Among them were important investigators in the field
such as K Georgeson, A Coran, R Kapur, H Kobayashi, A
Chakravarti, P Tam, and MD Gershon (Fig. 1). The dis-
cussion was tape recorded (Figs. 2 and 3).
The 10 debated topics included the following:
1. Acetylcholinesterase is necessary for the diagnosis
of HD.
Fig. 1 Participants from every country and field were present to
discuss different aspects on research and clinical features of IDs.
From left to right, Stanislav Lyonnet and Aravinda Chakravarti
(world famous geneticists).
G. Martucciello et al. 1528
2. Rectal suction biopsy is adequate for the diagnosis
of HD.
3. Acetylcholinesterase should be associated with at
least one other histochemical technique.
4. Anorectal manometry is not a useful tool in the
diagnosis of HD. It is much more useful when
biopsies are negative for HD in order to exclude the
possible diagnosis of IASNA.
5. Barium enema is useful in deciding on the surgical
approach rather than to confirm the diagnosis.
6. Adequate intraoperative biopsy for histologic stain-
ing is mandatory during surgery of HD.
7. At least one enzymatic-histochemical staining tech-
nique among LDH, nonspecific esterase, and rapid
AChE should be used for intraoperative assessment.
8. The diagnosis of IASNA is the result of a clinical,
manometric, and histochemical studies.
9. The most difficult diagnosis is hypoganglionosis.
Without laparoscopic/or open seromuscular biopsies,
it is very difficult to obtain a correct diagnosis.
10. Does IND exist?
A list of agreements/disagreements will be presented
with important comments made during the discussion.
2. Results
These are the results of the discussion for each point
raised during the workshop:
1. European and Asian investigators (W Meier-Ruge,
G Martucciello, P Puri, JA Tovar, A Holschneider
and H Kobayashi) routinely use AChE to diagnose
HD, whereas the American contingent (JL Grosfeld,
K Georgeson, AG Coran, and R Kapur) think that
H&E is more user friendly, cheaper, and more
reliable for the diagnosis of HD. Although AChE
is an extremely useful tool to diagnose HD, it seems
that only very specialized centers can rely on this
staining technique to diagnose HD.
2. The unanimous opinion of the group was that rectal
suction biopsy is the current gold standard in the
diagnosis of HD.
3. This point overlaps the first question concerning
enzymatic-histochemistry and histomorphology.
European and Asian participants were divided in
opinion regarding the best other technique to use
along with AChE (ie, LDH, NADPH-d, or others),
whereas some American participants still believe that
H&E is the only technique required to diagnose HD.
4. Although there is broad agreement that anorectal
manometry is unnecessary to diagnose HD, some
still use manometry as an adjunct to help diagnose
HD or as a study complement (JA Tovar). The
discussion focused on the debated treatment options
for IASNA, namely, botulinum toxin injection, anal
dilatation, and sphincteromyectomy. Few partici-
pants were convinced that Botulinum toxin injec-
tion was an enduring treatment. There was general
agreement on the importance of anal dilatation as
first step in treatment. Sphincteromyectomy was the
most debated option: some pediatric surgeons
(A Holschneider, V Jasonni) avoid it in most cases
because of the risk of incontinence. Others,
however, (AG Coran, P Puri, JL Grosfeld) consider
this procedure safe and effective in selected
pediatric patients. Sphincteromyectomy may in-
crease the risk of fecal incontinence in patients
who have previously undergone anorectal surgery
(A Holschneider, AG Coran).
5. Everybody agreed that the barium enema was often
useful in selecting the operative approach, but some
think that the barium enema can sometimes be
misleading in evaluating the proximal extent of
aganglionic bowel (K Georgeson and AG Coran).
6. Everybody agreed that an intraoperative biopsy was
necessary, although controversies remain regarding
which staining technique should be used (H&E or
histochemical staining like LDH, NADPH-d, and
nonspecific esterase).
7. This point was not discussed at length as it overlaps
the previous item.
8. Everybody agreed on this point. Moreover, none
excluded manometry as enzymatic-histochemistry,
and histology alone cannot provide the diagnosis that
is achieved by demonstrating the absence of the anal
inhibitory reflex.
9. There was broad agreement regarding the difficulty
in diagnosing hypoganglionosis (A Holschneider,
W Meier-Ruge, G Martucciello). Some participants
did not think that hypoganglionosis really exists
Fig. 2 The meeting was opened, with a keynote lecture
describing the historical background of HD, by JL Grosfeld
(pediatric surgeon) who also chaired the workshop on criteria for
classification and diagnosis of intestinal dysganglionoses.
Diagnostic guidelines for anomalies of the ENS 1529
(AG Coran, JATovar) because they have never seen
a case in their extensive experience.
10. Almost all the participants believe that IND does
exist. Some believe in presently defined diagnostic
criteria (G Martucciello, W Meier-Ruge), whereas
others suggest that these diagnostic criteria are not
reliable enough (A Holschneider), referring to a
report by Dr Coerdt that showed the presence of
ENS anomalies similar to those of IND in perfectly
healthy children (abstract entitled, bQuantitative
morphometric analysis of the submucous plexus in
age-related control groupsQ). Some participants ques-
tion if IND is a truly separate entity or an acquired
secondary phenomenon related to long-standing
constipation or chronic obstruction (A Coran,
K Georgeson, JL Grosfeld).
The following comments were made by the chairman, JL
Grosfeld, at the end of the session:
bWell, the hour is late. We could ask other questions
about IND and staining but I think this would be
redundant since weve heard all about the different
staining techniques before. There is a consensus about
some of the issues we discussed today including: (1) You
can make the diagnosis of HD with a submucosal biopsy,
I think everybody would agree with that. There is a slight
disagreement whether you need H&E alone or whether
you need H&E plus other stains but that varies in
different countries. (2) In regard to anorectal achalasia
(IASNA), I believe there is consensus that you need the
presence of ganglion cells and the absence of the
anorectal inhibitory reflex to make that diagnosis. There
are some controversies regarding treatment but at least to
achieve the diagnosis we have consensus.
(3) An intraoperative biopsy is necessary in patients
with HD to make sure that you bring down bowel with
normal ganglia. There is slight disagreement whether
you evaluate the biopsy with an H&E stain alone and/or
other special histochemical and enzymatic studies.
However, everyone agrees you need an intraoperative
Fig. 3 Experts from different countries participated at the
meeting and discussed many topics during the workshop. From
left to right, Juan Alberto Tovar, Prem Puri, and Vincenzo Jasonni
(pediatric surgeons).
biopsy. 4] Everyone agrees the diagnosis of hypogan-
glionosis is very difficult to make and some are not sure
this entity actually exists. (5) Finally, I think there is
general agreement that IND probably exists, whether it is
a primary entity or a secondary phenomenon is yet to
be determined.Q
3. Conclusions
Hirschsprungs disease is a worldwide accepted entity
whose diagnosis is based on rectal suction biopsy [13]. This
technique allows sampling of a mucosal-submucosal spec-
imen required for proper ENS evaluation. The most
important diagnostic features of HD are the combination
of hypertrophic nerve trunks and aganglionosis in an
adequate specimen. Acetylcholinesterase staining is the best
diagnostic technique to demonstrate hypertrophic nerve
trunks in lamina propria mucosae, but many pathologists
from different centers still use H&E staining effectively.
Newly developed diagnostic kits for enzymatic-histochem-
istry will likely increase the use of AChE for the diagnosis
of HD. The new industrial kits lyophilize the components of
the medium that can be sent at room temperature anywhere
in the world (Hirschsprungs disease diagnostic kit, BIO-
OPTICA, Milan, www.bio-optica.it).
Although barium enema is not essential to confirm the
diagnosis of HD; in many cases, it is useful in evaluating the
level of aganglionosis and aids in the decision regarding the
surgical approach (ie, transanal, transabdominal laparoscop-
ic, or open). Although anorectal manometry is frequently
used in association with rectal biopsy and barium enema in
evaluating patients for HD, it is not routinely necessary.
Anorectal manometry is more useful in the diagnostic
workup of IASNA rather than HD.
Intraoperative pathological evaluation of the extent of
aganglionosis is mandatory to be sure that normal gangli-
onated bowel is used for a colostomy or pull through
procedure. In some instances, intraoperative biopsies may
also demonstrate the possible presence of associated
proximal hypoganglionosis or IND.
Internal anal sphincter neurogenic achalasia is a recog-
nized entity characterized by the presence of normal ganglion
cells in the submucosal plexus, slight reduction of NADPH-d
activity, and absence of rectoanal inhibitory reflex in a
severely constipated child. Treatment-wise, it seems clear
that the patient should undergo initial anal dilatations.
Myectomy can be used with good results expected in selected
cases. Attention should be paid to children who underwent
previous anorectal operations because their internal anal
sphincter has presumably been damaged to some extent by
the prior procedure. Further myectomy may increase the risk
of incontinence.
Hypoganglionosis is not yet considered as an isolated
entity worldwide. Moreover, it is not clear whether it
G. Martucciello et al. 1530
represents a severe form of ID or just an ENS abnormality
that leads to constipation. There are divergent views
concerning this condition in Asia (H Kobayashi) and
Europe (G Martucciello), and further studies are necessary
to reach a consensus on definition and diagnosis.
Finally, IND likely does exist, although it is not clear as
yet whether it is a separate primary entity or some sort of
secondary acquired condition. Definition and diagnosis of
IND are yet to be determined because more than one
criterion has been proposed in recent years. Most authors
suggest nonoperative conservative treatment in most cases.
Treatment depends on clinical presentation of the patients:
conservative treatment is sufficient in 90% of cases, but
enterostomy may be necessary in about 10% of patients.
Further investigation is needed to resolve the many
controversies concerning IDs. The fourth International
Conference in Sestri Levante stimulated discussion regarding
these entities and led to the International Guidelines noted
above as an effort serve the best interest of our patients.
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Diagnostic guidelines for anomalies of the ENS 1531