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140 Review Article

Hirschsprung’s Disease, One of the Most Difficult

Diagnoses in Pediatric Surgery: A Review of the
Problems from Clinical Practice to the Bench

Author G. Martucciello

Affiliation Paediatric Surgery Department, Scientific Inst. (IRCCS) Policlinico San Matteo, Pavia, Italy

Key words Abstract set of procedures and enzyme-histochemical re-

Downloaded by: Istituto Giannina Gaslini. Copyrighted material.

" Hirschsprung’s disease
l !
(HSCR) Purpose: The diagnosis of Hirschsprung’s disease
" enteric nervous system
l
(HSCR) should take place early in the neonatal pe-
(ENS)
" intestinal neuronal dysplasia
riod, because without an effective diagnosis and
l
(IND) appropriate treatment, a considerable proportion
" histochemistry
l of infants will go on to develop serious complica-
" lyophilization
l tions such as acute enterocolitis or toxic mega-
colon. Because no more than 10 % of HSCR cases
have a late presentation with classical chronic
constipation and megacolon, the clinician has to
make a difficult, early diagnosis, which is the crux
of the clinical problem. The aim of this review
paper is to present all tools currently available to
make a clear HSCR diagnosis and to discuss the
problems facing the clinician and the pediatric
surgeon in the correct identification of HSCR and
of other intestinal dysganglionoses.
Methods: Based on the current state of knowl-
edge and 24 years’ personal experience in clinical
practice and basic research in this field, I describe
an algorithmic approach that enables clinicians
and surgeons to rationalize and maximize the
clarity of diagnosis through a complementary
actions.
Results: Two innovative techniques, added to the
protocol in the last four years, are described: the
lyophilized HSCR diagnostic kit, and the one-tro-
car transumbilical laparoscopic intestinal full-
thickness biopsy technique (OTTLB).
Conclusion: The rational, algorithmic diagnostic
pathway proposed in this review paper aims to
optimize every diagnosis by the stepwise appli-
cation of a complementary set of procedures and
enzyme-histochemical reactions as they become
appropriate. In the interests of simplifying genet-
ic molecular diagnosis, I suggest the following
guidelines: 1) only in cases of total colonic agan-
glionosis (TCA) is it advisable to carry out full RET
mutation screening (the mutation rate is up to
70%); and 2) all HSCR patients should be tested
only for standard MEN2A and MTC mutations. If
these are present, the patients should be fol-
lowed up carefully with proper surveillance and
biochemical testing of other susceptible family
members as they are at risk of developing neuro-
endocrine tumors.

received March 3, 2008

accepted after revision
March 16, 2008
Bibliography
DOI 10.1055/s-2008-1038625
Eur J Pediatr Surg 2008; 18:
140 – 149 © Georg Thieme
Verlag KG Stuttgart • New York •
ISSN 0939-7248
Correspondence
Associate Prof.
Giuseppe Martucciello, M.D.,
Surgeon-in-Chief
Paediatric Surgery Department
Scientific Inst. (IRCCS)
Policlinico San Matteo
Via le Golgi 19
27100 Pavia
Italy
martucciello@yahoo.com
Introduction
! more than half of the colon is involved. This
Hirschsprung’s disease (HSCR) is actually a group
of congenital enteric nervous system (ENS) disor-
ders, all characterized by a lack of ganglia in the
myenteric and submucous plexuses [32, 41, 50,
68]. The absence of ganglia is associated with an
increased number of acetylcholinesterase posi-
tive nerve fibers in the rectal wall.
The aganglionic segment extends from the ano-
rectum for a variable colonic or intestinal dis-
tance. In 1982, Kaiser et al. [32] classified the dis-
orders into three groups: 1) classical HSCR, which
involves the rectum, the rectosigmoid, and ex-
tends to the splenic flexure; 2) ultra-short HSCR,
which is limited to the distal 2 – 3 cm of the rec-
tum [11, 27]; and 3) ultra-long HSCR, in which
group also includes those cases with small bowel
involvement [77].
Problems in clinical presentation
The clinical presentation of HSCR is dependent
not only on the length of the aganglionosis, but
also on the age of the patient. Ninety percent of
all cases of aganglionosis produce clinical signs
during the newborn period [37, 55, 69, 70]. More-
over, many series show that from 3.5 to 10% of
HSCR newborns are either premature or low
birth weight infants. The diagnosis of HSCR
should take place early in the neonatal period,
because without an effective diagnosis and ap-

Martucciello G. Hirschsprung’s Disease, One … Eur J Pediatr Surg 2008; 18: 140 – 149

propriate treatment a considerable proportion of infants will go
on to develop serious complications such as acute enterocolitis
or toxic megacolon [16, 23, 53, 55, 67, 75].
The early clinical signs of the disease are summarized by series
in l" Table 1. Although these provide useful pointers indicating a
possible HSCR diagnosis, they are not sufficient to obtain a defin-
itive and clear preoperative diagnosis. Because no more than
10% of HSCR cases have a late presentation with classical chronic
constipation and megacolon, the clinician has to make a difficult,
early diagnosis to avoid the development of serious complica-
tions such as acute enterocolitis or toxic megacolon. And this is
the crux of the clinical problem.
Problems of radiological studies
The supine radiological film can demonstrate a gaseous disten-
sion of bowel loops, while contrast enema at low insufflation
pressures (when used for diagnosis) shows a distal rectocolonic
segment with a proximal transition zone and a marked proximal
bowel distension [36, 39]. The choice of contrast Rx medium is
controversial; in my experience, fluid barium sulfate solution is
suitable only in older children, while an iso-osmolar iodate solu-
tion (Gastromiro/Iopamiro) is indicated in the neonatal period
and early infancy. I never suggest using Gastrografin or a barium
sulfate suspension in neonatal cases.
The number of false-negative radiological results is very high in
young children and newborns, because the transitional zone is
so easily distended and distorted. Like other authors, I do not
consider contrast enema findings sufficient for a diagnosis of
HSCR in most cases.
Problems in functional manometric diagnosis
The demonstration of genuine internal sphincter relaxation with
anorectal fluctuations at the beginning of the relaxation reflex
and after the return of the resting pressure level excludes a diag-
nosis of HSCR [28]. Unfortunately, the converse is not completely
true, i.e., the absence of internal sphincter relaxation is not al-
ways synonymous with a confirmation of the disease. The rate
of false positives is too high to consider manometry as a suffi-
cient diagnostic test for HSCR. When the patient is an infant of
less than 14 weeks of age [28], the internal sphincter reflex may
be physiologically rudimentary due an immaturity of its nerve
supply. Furthermore, in the first years of life there are technical
problems, and a good collaboration from the patient is necessary
to perform an appropriate functional anorectal manometric
evaluation. Nowadays, most patients are seen in the neonatal
period or during the first months of life, when diagnosis is diffi-
cult and rectal biopsy is always necessary. In 2007, Kawahara et
al. [35] introduced a specifically designed sleeve microassembly
for the manometric diagnosis of neonatal HSCR. However, few
institutions or pediatric surgeons currently rely on anorectal
manometry for HSCR diagnosis.
The era of histochemistry
It is well known that the characteristic feature of HSCR is the
congenital absence of ganglion cells in the distal hindgut. A reli-
able diagnosis using a hematoxylin-eosin morphological exami-
nation of mucosal biopsies is very difficult because the submu-
cosal ganglia are very small (3 – 5 cells per ganglion) and spread
inside the Meissner’s plexus. These anatomical findings explain
the low reliability and false positive results of classical histo-
morphological (H&E) studies of suction rectal biopsies in sus-
pected HSCR cases [46, 47, 71]. In 1972, Meier-Ruge [49] started
Martucciello G. Hirschsprung’s Disease, One … Eur J Pediatr Surg 2008; 18: 140 – 149
Review Article 141
to investigate the acetylcholinesterase (AChE) enzyme-histo-
chemical reaction and revealed a markedly increased AChE ac-
tivity in a large number of cholinergic nerve fibers and thick
nerve trunks present in HSCR. The main principle behind the
use of AChE as a diagnostic test for HSCR is based on pathophys-
iological studies of the disease. In association with rectal agan-
glionosis, there is a marked increase in extrinsic preganglionic
parasympathetic nerve fibers (of sacral origin), whose axons
continuously release acetylcholine, together with an excessive
production and accumulation of the enzyme acetylcholinester-
ase [60]. Today, AChE investigation in association with a comple-
mentary set of enzyme-histochemical reactions (which we will
discuss in this review) is routine for suction rectal biopsies, and
is considered the most reliable preoperative HSCR diagnostic
technique [47].
Diagnostic problems for pseudo-Hirschsprung
Complementary enzyme-histochemical staining of rectal muco-
sa biopsies has provided a reliable diagnostic tool for HSCR.
However, these also select for a number of other pathological
conditions which clinically resemble HSCR (pseudo-Hirsch-
Downloaded by: Istituto Giannina Gaslini. Copyrighted material.
sprung) despite the absence of aganglionosis [60]. These other
intestinal dysganglionoses (IDs) form a heterogeneous group of
ENS anomalies that include intestinal neuronal dysplasia (IND),
and hypoganglionosis [44, 59, 64, 65].
This means that in clinical practice, the pediatric surgeon needs
to follow a clear and complex algorithmic path of analysis (see
flow chart in l" Fig. 4) which can identify each type of dysgan-
glionosis.
Problems in molecular studies of HSCR
While the aim of my group in investigating the genetic basis of
HSCR [40] was to clarify its pathogenetic mechanisms, my per-
sonal hope was that we would obtain sufficient knowledge of
the disease to be able to make timely molecular diagnoses.
Some years later, in collaboration with Prof. Giovanni Romeo,
we set up a research project for the molecular genetic study of
HSCR based on the detection of an interstitial deletion of chro-
mosome 10 [40]. This and other studies showed that mutations
can affect the whole sequence of the RET gene, and therefore
that there are no critical mutations always detectable in the
same exon [43, 66]. This heterogeneity is reflected in the techni-
cal problems associated with narrowing down the diagnostic
choices in this multigenic and multifactorial disease. However,
general knowledge about HSCR has greatly increased over the
last decade following genetic studies, and some molecular in-
vestigations are now routinely taken into account in clinical
practice [38, 43].
What is the best approach for the diagnosis of HSCR
and other intestinal dysganglionoses (IDs)?
The aim of this review paper is to present all tools currently
available for a clear HSCR diagnosis and to discuss the problems
facing the clinician and the pediatric surgeon when attempting a
correct identification of HSCR and of other IDs. Based on our cur-
rent knowledge and 24 years’ personal experience in clinical
practice and basic research in this field [8, 9,17, 31, 40 – 45, 63,
73], I describe an algorithmic approach that will enable clini-
cians and surgeons to rationalize and maximize the clarity of di-
agnosis through a complementary set of procedures and en-
zyme-histochemical reactions.
142 Review Article

Methods and Criteria for Diagnosis

Table 1 Clinical presentation of HSCR in the first 6 months of life
!
Below, I analyze the diagnostic pathway, describing the phases I Main clinical presentations
Intestinal obstruction 55% Sieber WK, 1978
consider fundamental to building a rational, algorithmic frame-
Early mild constipation with 24% Sieber WK, 1978
work. The steps are presented as a consecutive sequence which
secondary abrupt obstruction
corresponds to the chronological process that is used in my per- Chronic severe constipation 3% Sieber WK, 1978
sonal protocol. Enterocolitis/ 18% Sieber WK, 1978
a) The clinical evaluation of the patient remains the most impor- Toxic megacolon 25% Werbeloff L, 1974
tant step in diagnosis. l
" Table 1 summarizes the main neona- 25% Nixon H, 1982
tal signs of HSCR, which must be carefully considered and an- 25% Deucher F, 1977
alyzed by clinicians and, in particular, by the pediatric sur- 10% Grand RJ, 1975
Clinical early signs
geon. Of full-term infants, 98 % pass meconium in the first 24
Prematurity 2.6 % Sieber WK, 1978
hours of life and the remainder will pass their first stool by 48
3.5 % Ehrenpreis T, 1971
hours [60, 74]. Sometimes partial spotting of meconium dur- 10% Swenson O, 1973
ing the first hours of life is erroneously reported in the case Delayed passage 94% Swenson O, 1973
history by the nurse as a complete emission. The distended of meconium 90% Dasgupta R and
infant who passes only a small amount of meconium should, Langer JC, 2004
in any case, be investigated for HSCR; however the passage of Distension of abdomen 55% Sieber WK, 1978
meconium during the first 48 hours does not completely ex- 87% Swenson O, 1973
Diarrhea 14% Langer B, 1959
clude HSCR. The severity of the constipation may vary from a
22% Lillie JG, 1971
complete inability to evacuate to one or two undersized daily

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movements. It is true that the explosive discharge of gas and
feces following a rectal examination or probing is highly sus-
picious for HSCR [67]. Generally speaking, all early onset
cases of constipation should be studied in order to exclude a
diagnosis of HSCR or other congenital ENS anomalies.
Schematically, four main types of HSCR clinical presentation [67]
have been described: 1) neonatal intestinal obstruction; 2) early
mild constipation followed by secondary abrupt intestinal ob-
struction; 3) chronic severe constipation without true intestinal
obstruction (older children with classical megacolon); and 4) en-
terocolitis or toxic megacolon [16, 53, 55] (l" Table 1).
Today the clinical aspects of HSCR are well known. However, I
decided to include these short notes on the clinical presentation,
because every diagnostic approach starts from a clinical suspi-
cion of the disease.
b) Starting from a clinical suspicion, diagnostic screening is car-
ried out on the basis of rectal suction biopsies (RSBs), studied
using a complementary set of enzyme-histochemical tech-
niques.
Since Noblett [56] described her innovative tool for RSBs, submu-
cosal and mucosal specimens can be obtained easily and pain-
lessly without anesthesia from patients with suspected HSCR.
Many variants have been developed over the last 30 years; I use
the Solo-RBT [57], which has some advantages such as the possi-
bility to adjust the tool (capsule and aspiration), easy disassem-
bly, and one-handed execution of the biopsy. In a recent study in
a series of 389 patients [58], Solo-RBT had a very low incidence
of complications: 2 rectal bleedings (0.5 %), and no rectal perfo-
rations. The inadequacy of RSB, i.e., an absence of submucosa,
was limited to 14.6 % of samples.
Meier-Ruge et al. [47] suggest performing multiple RSBs in a
geometric sequence (e.g., centimetres 1, 2, 4, 8, 16) proximally
to the dentate line. Of course, the pathologist will be happier as
this would be painful because of the very high number of sensi-
tive fibers at this point. However, I do agree that the lowest RSB
should be sampled at 1 cm from the pectinate line. I generally
perform no more than 3 or 4 biopsies (depending on the age of
the patient). In a newborn with a rectum which is only a few cen-
timeters long, the maximum number of biopsies that I perform
is 3 (at 1, 2, 3 cm). In older patients, I consider four levels (at
about 1, 2, 3, and 6 cm). If I suspect IND more than HSCR, RSBs
of the upper rectum are more useful, so I try to obtain an upper
biopsy at about 8 cm.
The RSBs have to be frozen and cut in the cryostat in sections of
15 µm [47]. The thickness is very important, because if the cryo-
static section is too thin, the nerve fibers cannot be correctly
stained along their whole length inside the mucosa.
Although acetylcholinesterase activity (AChE) [34] is diagnosti-
cally the most useful of the set of enzyme-histochemical reac-
tions, it is not sufficient. AChE stains the parasympathetic nerve
fibers and trunks of fibers that increase dramatically in the lam-
ina propria mucosae and submucous layer of HSCR patients, but
it is not a specific marker for ganglion cells. Other complementa-
ry enzyme-histochemical reactions are necessary – lactate dehy-
drogenase (LDH), alpha-naphthylesterase (ANE), and NADPH-di-
aphorase (NADPH-d) – which are enzymatic markers of the
soma of ganglion cells [14,17, 47]. In addition, succinic dehydro-
genase (SDH) may be used to evaluate the possible immaturity
of ganglion cells in the submucous plexus [47, 54]. The comple-
mentary association in the RSB examination protocol of a specif-
ic enzymatic marker for cholinergic fibers (AChE) with at least 3
other markers for ganglion cells (LDH, ANE, and NADPH-d) is a
safeguard against false positives or false negatives for HSCR
(l" Table 2).

more material is available. However, I think that in the interest of
reducing the invasiveness, the pediatric surgeon should choose a
reasonable compromise which will make the procedure less
dangerous but at the same time supply sufficient material for
the histochemical diagnosis.
Most of our cases are newborns or infants aged only a few
months, so their rectum is short. The surgeon must be careful
not to perform the biopsy at the same level as the dentate line:
Rectal suction biopsy is agreed to be the current gold standard in
the diagnosis of HSCR [44], and European investigators routinely
use AChE to diagnose HSCR. Unfortunately, it seems that only
very specialized centers can rely on this enzyme-histochemical
technique [44] with many pathologists still using H&E staining.
Acetylcholinesterase staining is the best diagnostic technique to
demonstrate hypertrophic nerve trunks in the lamina propria
mucosae of the aganglionic rectum.

Martucciello G. Hirschsprung’s Disease, One … Eur J Pediatr Surg 2008; 18: 140 – 149
 Review Article 143

Table 2 Histochemical stainings used in the diagnosis of HSCR and pseudo-HSCR

Technique Type of technique Staining Diagnostic usefulness Commercial production

AChE Enzymo- " HSCR big increase of brown " Preoperative diagnosis of HSCR Hirschsprung diagnostic kit,
histochemistry nervous fibers in the mucosa and IND on RSBs Bio-Optica, Milan, Italy
" Use in full-thickness biopsies Code: 30-50110
for IND
ANE Enzymo- " Incubation at room tempera- " Preoperative diagnosis of HSCR Hirschsprung diagnostic kit,
histochemistry ture for 5 – 10 min and IND on RSBs Bio-Optica, Milan, Italy
" Submucosal ganglia brown " Very fast reaction used in intra- Code: 30-50111
" Myoenteric ganglia brown operative diagnosis
" Use in full-thickness biopsies
for IND and hypoganglionosis
NADPH-d Enzymo- " Incubation at 37 8C for 10 – " Preoperative diagnosis of HSCR Hirschsprung diagnostic kit,
histochemistry 30 min and IND on RSBs Bio-Optica, Milan, Italy
" Submucosal ganglia blue " Intraoperative diagnosis Code: 30-50112
" Myenteric ganglia blue " Use in full-thickness biopsies
for IND and hypoganglionosis
SDH Enzymo- " Incubation at 37 8C for 45 min " Immaturity of ganglion cells on Hirschsprung diagnostic kit,
histochemistry " Submucosal ganglia granular RSBs Bio-Optica, Milan, Italy
blue only if mature Code: 30-50113
LDH Enzymo- " Incubation at 37 8C for 10 min " Preoperative diagnosis of HSCR Hirschsprung diagnostic kit,
histochemistry " Submucosal ganglia blue and IND on RSBs Bio-Optica, Milan, Italy

Downloaded by: Istituto Giannina Gaslini. Copyrighted material.

" Myenteric ganglia blue " Intraoperative diagnosis
" Use in full-thickness biopsies
for IND and hypoganglionosis
C-KIT Immuno- " c-kit immunoreactivity of in- " Decrease of ICC (pacemaker
histochemistry terstitial cells of Cajal (ICC) cells in smooth muscle layer)
" Associated with hypoganglio-
nosis
Code: 30-50114
" Monoclonal anti c-kit,
Novocastra Laboratory,
Newcastle, UK
" Policlonal anti c-kit CD117

antihuman; DAKO, Glos-

trup, Denmark
Picrosirius red staining Histology " Incubation in picrosirius red " Atrophy of connective tissue EMS, Hatfield, PA, USA
or other tricromic solution for 60 min net. Colonic desmosis Code EMS catalog #26357
stainings (Van Gieson; " Collagen stains red smooth
Azan-Mallory, etc…) muscle stain yellow

The obstacles to a widespread use of enzyme-histochemical re-
actions for HSCR diagnosis are linked to technical difficulties in
some pathology laboratories: a) the fresh preparation of the me-
dium is time-consuming; b) potentially toxic reagents are used
during preparation; c) technicians are reluctant to work on me-
dium preparation; d) the medium has to be stored at – 25 8C and
cannot be transported from lab to lab. However, a commercial
diagnostic kit that makes use of the lyophilized modified com-
ponents of the medium is now available, and it is likely that this
will increase the use of AChE in the diagnosis of HSCR. The kit
(Hirschsprung’s disease diagnostic kit, Bio-Optica, Milan, Italy;
www.bio-optica.it) can be sent anywhere in the world at room
temperature.
We have compared the performance of the new diagnostic kit
with conventional laboratory-prepared incubation media, and
found it to be excellent. One hundred and sixty-five rectal and
colon biopsies were studied comparatively. Cryostat sections of
15 µm were obtained for incubation and enzyme-histochemical
staining from all the frozen (– 80 8C) specimens. Half of the sec-
tions were conventionally stained with self-prepared media (fro-
zen and stored at – 25 8C in plastic tubes) of acetylcholinesterase
age of 5 on an index of 1 – 5, vs. an average of 2 for those obtained
using the frozen, conventionally obtained media.
Whereas it is often very difficult to self-prepare the frozen incu-
bation media in pathology laboratories, the new Bio-Optica
lyophilized industrial kit is ready for use and can guarantee a
standard high quality for the preoperative and intraoperative
histochemical diagnosis of HSCR. Thus, the pathologist no longer
has an alibi not to perform acetylcholinesterase and enzyme-
histochemistry in HSCR diagnosis.
The new commercial enzyme-histochemical diagnostic kits
could open a new era in the routine study of Hirschsprung pa-
tients, which may lead to a dramatic improvement in the diag-
nosis and radical treatment of patients.
The diagnostic criteria for HSCR and other IDs I have used for the
last 4 years are as follows:
HSCR shows: 1) an increase in AChE activity in nerve fiber nets in
the lamina propria mucosae, which is absent in normally inner-
vated mucosa; 2) trunks of AChE-positive nerve fibers in the
submucosa; 3) an absence of ganglion cells in the submucosa,
with a negative result using enzyme-histochemical markers
LDH, ANE, NADPH-d [47] (l " Fig. 1).

(AChE) according to Karnovsky [34] and alpha-naphthylesterase
(ANE) according Davis [14]; the other half of the sections were
stained using Bio-Optica’s new lyophilized Enzyme-Histochemi-
cal Diagnostic Kit (AChE, LDH, ANE, NADPH-d).
In a blind randomized analysis of the AChE and ANE stainings,
those obtained with the lyophilized kit were ranked at an aver-
However, it is important to observe that there are two different
patterns of AChE positivity in the lamina propria mucosae, fol-
lowing the criteria of Huntley et al. for HSCR diagnosis [29]. In
pattern A, prominent nerve fibers staining for AChE are seen
throughout the lamina propria (classical positivity for HSCR). In
pattern B, similar fibers are seen only in the muscularis mucosae

Martucciello G. Hirschsprung’s Disease, One … Eur J Pediatr Surg 2008; 18: 140 – 149
144 Review Article
and immediately adjacent areas of the lamina propria. AChE pat-
tern B is characteristic of the newborn period.
Experience suggests that if positivity type B is encountered, RSBs
obtained from the same neonate 1 – 2 weeks later can yield clas-
sical type A positivity, because increased AChE activity is sec-
ondary to aganglionosis, and is age related. This must be taken
into account in order to avoid false negative results for HSCR
during the neonatal period.
Total Colonic Aganglionosis (TCA) has the same pattern as other
forms of HSCR, but shows a lower density of AChE fibers [47].
Ultra-short HSCR (UHD) shows: 1) a reduced net of AChE nerve
fibers in the lamina propria mucosae, which is, however, positive
in the deeper part proximal to the muscularis mucosae; 2) strong
AChE positivity in the muscularis mucosae; 3) trunks of fibers in
the submucosa; 4) negativity for LDH, ANE and NADPH-d gan-
glion cell staining (absence of ganglia) [11].
Hypoganglionosis [44] is difficult and often misdiagnosed in
RSBs, because the best approach for diagnosis is the study of my-
oenteric biopsies and the evaluation of the myenteric plexus
with LDH, ANE, and NADPH-d enzyme-histochemical reactions,
which show small myenteric ganglia with a reduced number of
ganglion cells per ganglion (see paragraph on one-trocar tran-
sumbilical laparoscopic intestinal full-thickness biopsies).
Intestinal Neuronal Dysplasia (IND) type B [10, 20, 47] meets the
following criteria: 1) giant ganglia in the submucosa with more
than 8 ganglion cells positive to the enzyme-histochemical
markers LDH, ANE and NADPH-d; 2) twenty percent of submu-
cosal ganglia are giant; 3) AChE positivity around submucous
vessels; 4) increase in AChE positive fibers in the lamina propria
mucosae (additional, age-dependent criterion); 5) heterotopic
ganglion cells inside lamina propria mucosae (additional criteri-
on) [6, 47].
Martucciello G. Hirschsprung’s Disease, One … Eur J Pediatr Surg 2008; 18: 140 – 149
Fig. 1 a to d Lyophilized Hirschsprung diagnostic
kit (Bio-Optica). a RSB in normal mucosa, a sub-
mucosal ganglion is present. b Classical Hirsch-
sprung acetylcholinesterae (AChE) pattern of posi-
tivity. c and d Are slides of AChE in a 1-month-old
Hirschsprung patient.
Downloaded by: Istituto Giannina Gaslini. Copyrighted material.
c) Radiological studies are carried out after RSB screening. The
usefulness of contrast enema in HSCR is more related to the
choice of surgical approach (laparoscopic, transanal, open
technique, etc.) than to understanding of the type of agan-
glionosis.
The choice of contrast Rx medium is controversial: I personally
use fluid barium sulfate solution only in older children, with
iso-osmolar iodate solution (Gastromiro/Iopamiro) being more
indicated for the neonatal period and early infancy. I never sug-
gest Gastrografin or barium sulfate suspension in neonatal cases.
d) Anorectal manometric study has only a limited application in
my protocol for HSCR and other IDs. However, if histochemis-
try has excluded HSCR – the main difference between UHD
and ISA is that the first disorder presents a specific increase
in AChE activity (as previously described), while in the latter
AChE is negative (functional and myogenic ISA) – manometry
is the next step in order to obtain information on the patient’s
anorectal pressure and the absence/presence of internal anal
sphincter relaxation. Manometry enables a relatively nonin-
vasive diagnosis of internal anal sphincter achalasia (ISA)
(the inability of the internal anal sphincter to relax) [27, 28].
Manometry, then, is useful to plan the treatment of non-HSCR
cases (ISA and some IND patients) with an absence of internal
sphincter relaxation and high anorectal pressure profile, in
whom a sphincteromyotomy or sphincter dilatation under
anesthesia is sometime indicated.
e) In the last 4 years, I have on rare occasions included a special
procedure in my protocol for pseudo-HSCR ENS abnormal-
ities requiring further diagnostic work: the one-trocar trans-
umbilical laparoscopic intestinal full-thickness biopsy (OTTLB).
Our results suggest that this technique, which combines the
advantages of both open and laparoscopic procedures, is the

best approach to study selected, very complex cases of hypo-
ganglionosis, intestinal neuronal dysplasia, decrease of inter-
stitial cells of Cajal [62, 72, 76], and desmosis of the colon [47,
48]. HSCR is the most common (1 : 5000 live births) ENS
anomaly. However, other complex ENS conditions such as
hypoganglionosis, intestinal neuronal dysplasia (IND), and
colonic desmosis can cause severe chronic constipation and
motility disorders of the gut, and their diagnosis can require
full-thickness intestinal biopsies and other specific histo-
chemical investigations.
At the time of surgery, patients receive antibiotic prophylaxis
and a preoperative bowel preparation. Under general anesthesia,
the periumbilical region is infiltrated with bupivacaine. The op-
eration is performed with one infraumbilical sagittal incision, an
11-mm port is inserted using the Hasson technique and pneu-
moperitoneum is established at 8 to 12 mmHg (depending on
the age of the patient).
In the procedures, a 10-mm operative telescope (Karl Stortz op-
erative canal) (l " Fig. 2) is used, with a 5-mm atraumatic grasper
introduced through the operative channel. Using one-trocar
transumbilical laparoscopic exploration, it is possible to identify
the different intestinal segments and to study and pull critical
regions outside the abdominal cavity through the umbilical inci-
sion. The technique is the best approach to obtain several full-
thickness biopsies without peritoneal contamination (l " Fig. 3).
The OTTLB technique could also be applied to appendectomy
and to the study of appendix innervation. However, I would like
to stress very strongly that the vermiform appendix can present
specific intrinsic innervation abnormalities without other intesti-
nal abnormalities [47], and that it is thus not a suitable sample
for the diagnosis of ENS disorders.
From 2004 to the present, the author has performed this proce-
dure on 12 selected patients, obtaining biopsies from sigma co-
lon, descending colon, transverse colon and ascending colon. The
histochemical studies of these biopsies have enabled diagnoses
of intestinal hypoganglionosis of the myenteric plexus in 8 cases
and diffuse IND in 4 cases of chronic pseudo-obstruction. We
have had no postoperative complications.
f) Intraoperative enzyme-histochemical diagnosis is the last step
in focusing on the evaluation of HSCR, because it permits the
length of the aganglionic and proximal hyopoganglionic seg-
ment to be identified. Different seromuscular biopsies are
performed laparoscopically during a Georgeson-Soave proce-
dure [22] or open seromuscular biopsies are sampled during
an open classical Soave technique [31]. The enzyme-histo-
Martucciello G. Hirschsprung’s Disease, One … Eur J Pediatr Surg 2008; 18: 140 – 149
Review Article 145
Fig. 2 a and b A 10-mm operative telescope (Karl
Storz operative canal) is used, with a 5-mm atrau-
matic grasper introduced through the operative
channel. During one-trocar transumbilical laparo-
scopic exploration, it is possible to identify the
different intestinal segments and to study and pull
critical regions outside the abdominal cavity
through the umbilical incision.
Downloaded by: Istituto Giannina Gaslini. Copyrighted material.
Fig. 3 The one-trocar transumbilical laparoscopic intestinal full-thickness
biopsy technique (OTTLB) in cases where there is a suspicion of complex
pseudo-HSCR. OTTLB is used to obtain full-thickness intestinal biopsies
which can then be studied to reach differential diagnoses. The technique is
the best approach to obtain several full-thickness biopsies without perito-
neal contamination.
chemical markers used during surgery are ANE and NADPH-d
(Bio-Optica Hirschsprung diagnostic kit).
g) Molecular studies complete the HSCR diagnostic process. The
RET gene has been found to be mutated in about 35% of
sporadic and 49% of familial cases of HSCR and in a higher
percentage of long than of short type HSCR (76% vs. 32%)
[12, 52]. RET mutations that lead to HSCR can occur through-
out the 21 exons of the gene [50] and more than 100 different
mutations have been identified, including nonsense muta-
tions, missense mutations, small deletions and insertions
[12]. Only 5 – 10% of patients show mutations in other genes
such as glial cell lined derived neurotrophic factor (GDNF),
neurturin (NTN), endothelin3 (EDN3), endothelin B receptor
(EDNRB), endothelin converting enzyme 1 (ECE1), the tran-
scriptional factor SOX10, Smad interacting protein-1 (SIP1)
and the PHOX2B gene [1, 4, 5,12, 21].
RET is a proto-oncogene that is primarily expressed during em-
bryonic life in the neural crest, urogenital precursors, adrenal
medulla and thyroid and later on throughout postnatal life in
the central and peripheral nervous systems and the endocrine
system [50].
146 Review Article
Fig. 4 Algorithmic diagnostic pathway, aimed at optimizing the diagnosis using a complementary set of procedures and enzyme-histochemical reactions.
Mutations in the RET gene play an essential role in two common
neurocristopathies, MEN2 (OMIM 171400 and 162300), an auto-
somal dominant disorder caused by activating mutations and
HSCR (OMIM 142623) which is believed to be caused by a loss
of function mutations. HSCR and MEN2 are usually observed in
isolated cases and probably result from different molecular and
cellular mechanisms due to different mutation types. Thus, the
identification of RET mutations (C618 and C620) giving rise to
both HSCR and MEN2 (FMTC and MEN2A) in the same families
and even in the same patients was surprising. The underlying
mechanism that leads to both diseases is unknown [52] and has
been reported in a number of families who have HSCR but carry
the mutation that leads to MEN2 [30]. In order to explain how the
same mutations can produce such diverse phenotypes, we can
hypothesize that they are the result of molecular mechanisms
occurring in different periods of embryonic and postnatal life.
Another explanation for the complex inheritance pattern of
HSCR and the low detection rate of RET mutations is the pres-
ence of several common polymorphisms of the RET gene that
are associated with a variable risk of causing HSCR. Specific RET
haplotypes have been found to act as protective or predisposing
factors or to modulate the severity of the disease [7, 34, 35].
Since HSCR represents a part of many multigenic or multifacto-
rial conditions, the determination of the recurrence risk and of
proper genetic counseling faces certain difficulties. HSCR can be
defined as a sex-modified multifactorial disorder with an overall
risk of 4% for siblings and first degree relatives of the proband. It
is more difficult to estimate the recurrence risk in isolated cases
but it can be done by taking into account certain data such as the
sex of the proband and consultand, and the length of the agan-
Martucciello G. Hirschsprung’s Disease, One … Eur J Pediatr Surg 2008; 18: 140 – 149
Downloaded by: Istituto Giannina Gaslini. Copyrighted material.
glionic segment [4]. Counseling seems to be easier in familial
cases with known RET mutations: the pattern of inheritance is
autosomal dominant and carries a 50 % risk for the offspring of
having the mutation. Neither the penetrance nor the expressiv-
ity of the trait can be predicted, although a study of 17 HSCR
families showed that RET penetrance is sex dependent (72%
and 51% in males and females, respectively) [3]. All these factors
weigh against performing prenatal diagnosis for HSCR.
In general, HSCR exhibits marked sex differences which depend
on the length of the aganglionic segment, with both the inci-
dence and the penetrance being almost 2-fold to 4-fold higher
in males than in females [12]. Finally, genetic counseling usually
takes into account the great improvements in surgical manage-
ment of HSCR over the last decades.
In the interests of simplifying genetic molecular diagnosis, I sug-
gest the following guidelines: 1) only in cases of total colonic
aganglionosis (TCA) is it advisable to carry out full RET mutation
screening (the mutation rate is up to 70%); and 2) all HSCR pa-
tients should be tested only for standard MEN2A and MTC muta-
tions. If these are present, patients should be followed up care-
fully with proper surveillance and biochemical testing of other
susceptible family members as they are at risk of developing
neuroendocrine tumors.
Results and Discussion
!
The steps described here for the diagnosis for HSCR and IDs have
been applied and analyzed over a span of 24 years of personal
experience in clinical practice (more than 400 IDs cases) and in

basic research in this field. The last four years have seen the
completion of the protocol with the introduction of two innova-
tive procedures: the Bio-Optica HSCR diagnostic kit, and the
one-trocar transumbilical laparoscopic intestinal full-thickness
biopsy technique (OTTLB).
The rational, algorithmic diagnostic pathway (l " Fig. 4) proposed
here aims to optimize every diagnosis by the stepwise applica-
tion of a complementary set of procedures and enzyme-histo-
chemical reactions, as they become appropriate.
l" Fig. 4 shows that if a clinical evaluation suggests a diagnosis of
HSCR (step 1), the next step is RBS with the use of complemen-
tary histochemical staining techniques. This will lead to a result
of HSCR positivity (P) or HSCR negativity (N). If the result was P,
Rx with contrast enema can give useful additional information
for surgical treatment but manometry will not be necessary. Mo-
lecular studies should be performed to identify whether MEN2A
or MTC mutations are present [2, 51, 66]. If there was an N result,
manometry is indicated, as is a one-trocar transumbilical laparo-
scopic intestinal full-thickness biopsy (OTTLB) in cases where
complex pseudo-HSCR is suspected. The full-thickness intestinal
biopsies can then be studied to reach differential diagnoses.
It is evident that this protocol requires many enzyme-histo-
chemical techniques. AChE, LDH, ANE and SDH are used (Bio-Op-
tica kit) during the preoperative diagnostic study of RSBs. During
intraoperative histochemical study, ANE and NADPH-d enzyme-
histochemical techniques allow clear and rapid evaluation of the
length of the aganglionic and proximal hypoganglionic segments
requiring resection.
In complex disorders, where OTTLB is performed, I suggest:
1) picrosirius red staining and other trichromic morphological
stainings in order to identify desmosis of the colon [47, 48];
2) AChE, ANE, LDH and NADPH-d, and c-kit immunohistochemi-
cal technique (decrease in cells of Cajal) (Novocastra Laboratory,
Newcastle, UK) [62, 76] in hypoganglionosis; 3) AChE, ANE, LDH
and NADPH-d in order to demonstrate an IND type B (see l " Table
2).
The fluorescent glyoxylic acid technique, which is a catechol-
amine fluorescence reaction suggested by Meier-Ruge [47] to
identify the decrease in sympathetic innervation in IND type A
[20, 47], has been excluded for the reason that I have never en-
countered a diagnosis of IND type A, although I have studied
more than 400 IDs. I believe that IND type A does not exist, or
that it is extremely rare.
RSB in conjunction with a complementary preoperative set of
enzyme-histochemical markers has been recognized by some of
the most preeminent authors in the field as the gold standard for
HSCR [44]; and Asian and European investigators routinely use
AChE in RSB analysis. Some experienced investigators report a
99 % sensitivity and no false-positive results using AChE [42,
62]. In another recent study, de Lorijn et al. [15] compared differ-
ent diagnostic tests for HSCR, and established that RSB stained
with AChE is the most accurate test, having both the highest
mean sensitivity (93 %) and mean specificity (98 %).
In spite of all the evidence, some American pathologists [33] still
consider H&E staining more user-friendly and reliable for HSCR
diagnosis. It is paradoxical that in a country which invests mil-
lions of dollars every year in the development of molecular diag-
nostic techniques and in biotechnological research, the patholo-
gists should be reluctant to introduce AChE and other enzyme-
histochemical techniques in routine investigations for HSCR.
Another diagnostic trend has involved the application of an
enormous number of expensive immunohistochemical markers
Martucciello G. Hirschsprung’s Disease, One … Eur J Pediatr Surg 2008; 18: 140 – 149
Review Article 147
to paraffin-embedded samples. Many papers [18, 26, 61, 62] have
been published that document differences in the distributions of
various antigens in aganglionic vs. normal bowel wall. Such
claims seem unjustified, as appropriately designed studies to
evaluate the diagnostic utility of these reagents in the context
of RSBs are almost entirely lacking [33]. I also completely agree
with William Meier-Ruge and Elisabeth Bruder [47], who stress
the fact that while immunohistochemichemistry offers funda-
mental insights into the protein chemistry of tissues, it is a “stat-
ic” method. In contrast, enzyme-histochemistry is a “functional”
technique, because each reaction permits the evaluation, through
the intensity of acetylcholinesterase activity, of the parasym-
pathicotonus of colonic tissue [47].
Another example is that of dehydrogenase and esterase activities
(LDH, SDH, and ANE), which give information about the effec-
tiveness of ganglion cell performance, and can be applied intra-
operatively [17].
With the introduction of the OTTLB technique, this protocol ef-
fectively covers diagnoses of other disorders within the pseudo-
HSCR presentation in addition to that of HSCR. In my experience,
it has enabled the diagnosis of 8 cases of hypoganglionosis in 4
Downloaded by: Istituto Giannina Gaslini. Copyrighted material.
years, i.e., more than 20 % of all the IDs I saw in that period. As
a result, I no longer consider hypoganglionosis a rare ENS dis-
order but rather the most difficult to diagnose. When a patient
presents with severe constipation but an RSB negative for classi-
cal HSCR and is thus not suitable for conservative treatment, hy-
poganglionosis should be suspected, and the clinician should
consider the option of studying full-thickness biopsies from dif-
ferent portions of the gut using the OTTLB technique and a com-
bined set of stainings.
To conclude, my hope is that future research will continue to de-
velop better and better technologies for functional studies to re-
fine and optimize the diagnosis of intestinal motility disorders.
This will give us the best possible head start in the treatment of
this complex group of diseases.
Conflict of Interest: None
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