www.elsevier.com/locate/jpedsurg

Controversies concerning diagnostic guidelines foranomalies of the enteric nervous system:A report from the fourth International Symposium onHirschsprung’s disease and related neurocristopathies

Giuseppe Martuccielloa,*, Alessio Pini Pratob, Prem Puric,Alexander M. Holschneiderd, William Meier-Rugee, Vincenzo Jasonnib,Juan A. Tovarf, Jay L. Grosfeldg

aDepartment of Pediatric Surgery, IRCCS Policlinico San Matteo, Pavia I-27100, ItalybDepartment of Pediatric Surgery, G Gaslini Institute, Genova I-16148, ItalycDepartment of Pediatric Surgery, Our Lady’s Hospital, Crumlin 12, Dublin, IrelanddDepartment of Pediatric Surgery, Children’s Hospital of Cologne, Cologne D-50735, GermanyeDepartment of Pathology, University of Basel, Basel CH-4003, SwitzerlandfDepartment of Pediatric Surgery, University of La Paz, Madrid 28046, SpaingDepartment of Pediatric Surgery, Riley Children’s Hospital, Indianapolis, IN 46202-5200, USA

0022-3468/$ – see front matter D 2005

doi:10.1016/j.jpedsurg.2005.07.053

T Corresponding author.

Index words:Hirschsprung’s disease;

Aganglionosis;

Enteric nervous system;

ENS;

Intestinal neuronal

dysplasia

Abstract Intestinal Dysganglionoses (IDs) represent a heterogeneous group of Enteric Nervous System

anomalies including Hirschsprung’s 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

bHirschsprung’s disease and related neurochristopathies.QThe 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

Journal of Pediatric Surgery (2005) 40, 1527–1531

Elsevier Inc. All rights reserved.

G. Martucciello et al.1528

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.

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).

Intestinal dysganglionoses (IDs) represent a heteroge-

neous group of anomalies of the enteric nervous system

(ENS) including Hirschsprung’s 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 gene–related 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 bHirschsprung’s 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 bHirschsprung’s disease and

related neurocristopathies.Q The meeting’s 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.

Diagnostic guidelines for anomalies of the ENS 1529

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.

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.

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. 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).

G. Martucciello et al.1530

(AG Coran, JA Tovar) 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, bQuantitativemorphometric 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 we’ve 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

b

g

th

g

a

b

3. Co

Hi

whos

techn

imen

impor

of hy

adequ

diagn

trunks

from

Newl

istry w

of HD

the m

in the

OPTI

Al

diagn

level

surgic

ic, or

used

evalu

Anore

worku

Int

agang

onate

proce

also

proxim

Int

nized

cells i

activi

severe

that t

Myec

cases.

previo

sphin

the pr

of inc

Hy

iopsy. 4] Everyone agrees the diagnosis of hypogan-

lionosis is very difficult to make and some are not sure

is entity actually exists. (5) Finally, I think there is

eneral agreement that IND probably exists, whether it is

primary entity or a secondary phenomenon is yet to

e determined.Q

nclusions

rschsprung’s disease is a worldwide accepted entity

e diagnosis is based on rectal suction biopsy [13]. This

ique allows sampling of a mucosal-submucosal spec-

required for proper ENS evaluation. The most

tant diagnostic features of HD are the combination

pertrophic nerve trunks and aganglionosis in an

ate specimen. Acetylcholinesterase staining is the best

ostic technique to demonstrate hypertrophic nerve

in lamina propria mucosae, but many pathologists

different centers still use H&E staining effectively.

y developed diagnostic kits for enzymatic-histochem-

ill likely increase the use of AChE for the diagnosis

. The new industrial kits lyophilize the components of

edium that can be sent at room temperature anywhere

world (Hirschsprung’s disease diagnostic kit, BIO-

CA, Milan, www.bio-optica.it).

though barium enema is not essential to confirm the

osis of HD; in many cases, it is useful in evaluating the

of aganglionosis and aids in the decision regarding the

al approach (ie, transanal, transabdominal laparoscop-

open). Although anorectal manometry is frequently

in association with rectal biopsy and barium enema in

ating patients for HD, it is not routinely necessary.

ctal manometry is more useful in the diagnostic

p of IASNA rather than HD.

raoperative pathological evaluation of the extent of

lionosis is mandatory to be sure that normal gangli-

d bowel is used for a colostomy or pull through

dure. In some instances, intraoperative biopsies may

demonstrate the possible presence of associated

al hypoganglionosis or IND.

ernal anal sphincter neurogenic achalasia is a recog-

entity characterized by the presence of normal ganglion

n the submucosal plexus, slight reduction of NADPH-d

ty, and absence of rectoanal inhibitory reflex in a

ly constipated child. Treatment-wise, it seems clear

he patient should undergo initial anal dilatations.

tomy can be used with good results expected in selected

Attention should be paid to children who underwent

us anorectal operations because their internal anal

cter has presumably been damaged to some extent by

ior procedure. Further myectomy may increase the risk

ontinence.

poganglionosis is not yet considered as an isolated

entity worldwide. Moreover, it is not clear whether it

Diagnostic guidelines for anomalies of the ENS 1531

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.

References

[1] Puri P, Wester T. Intestinal neuronal dysplasia. Semin Pediatr Surg

1988;7:181 -6.

[2] Meier-Ruge W, Gambazzi F, Kaufeler RE, et al. The neuropatholog-

ical diagnosis of neuronal intestinal dysplasia (NID B). Eur J Pediatr

Surg 1994;4:267 -73.

[3] Martucciello G, Torre M, Pini Prato A, et al. Associated anomalies in

intestinal neuronal dysplasia. J Pediatr Surg 2002;37:219-23.

[4] Gillick J, Tazawa H, Puri P. Intestinal neuronal dysplasia: results of

treatment in 33 patients. J Pediatr Surg 2001;36:777 -9.

[5] Sacher P, Briner J, Hanimann B. Is neuronal intestinal dysplasia (NID)

a primary disease or a secondary phenomenon? Eur J Pediatr Surg

1993;3:228 -30.

[6] Huang CC, Ko SF, Chuang JH, et al. Lipoblastomatosis combined with

intestinal neuronal dysplasia. Arch Pathol Lab Med 1998;122:191 -3.

[7] Galvez Y, Skaba R, Vajtrova R, et al. Evidence of secondary neuronal

intestinal dysplasia in a rat model of chronic intestinal obstruction.

J Invest Surg 2004;17:31 -9.

[8] Nogueira AM, Barbosa AJ, Carvalho AA, et al. Usefulness of

immunocytochemical demonstration of neuron-specific enolase in the

[1

[1

[1

[1

[1

[1

[1

[1

[1

[1

[2

[2

[2

diagnosis of Hirschsprung’s disease. J Pediatr Gastroenterol Nutr

1990;11(4):496 -502.

[9] Park WH, Choi SO, Kwon KY, et al. Acetylcholinesterase histo-

chemistry of rectal suction biopsies in the diagnosis of Hirschsprung’s

disease. J Korean Med Sci 1992;7(4):353 -9.

0] Kobayashi H, O’Briain DS, Hirakawa H, et al. A rapid technique of

acetylcholinesterase staining. Arch Pathol Lab Med 1994;118(11):

1127-9.

1] Karnovsky MJ, Roots L. A bdirect coloringQ thiocholine method for

cholinesterase. J Histochem Cytochem 1964;12:219.

2] Martucciello G, Favre A, Torre M, et al. A new rapid acetylcholin-

esterase histochemical method for the intraoperative diagnosis of

Hirschsprung’s disease and intestinal neuronal dysplasia. Eur J Pediatr

Surg 2001;11(5):300 -4.

3] Pini Prato A, Martucciello G, Jasonni V. Solo-RBT: a new instrument

for rectal suction biopsies in the diagnosis of Hirschsprung’s disease.

J Pediatr Surg 2001;36(9):1364 -6.

4] Martucciello G, Mazzola C, Favre A, et al. Preoperative enzymo–

histochemical diagnosis of dysganglionoses associated with anorectal

malformations (ARM) with recto–vestibular and recto–perineal

fistula. Eur J Pediatr Surg 1999;9(2):96-100.

5] Hirakawa H, Kobayashi H, O’Briain DS, et al. Absence of NADPH-

diaphorase activity in internal anal sphincter (IAS) achalasia. J Pediatr

Gastroenterol Nutr 1995;20(1):54 -8.

6] Petchasuwan C, Pintong J. Immunohistochemistry for intestinal

ganglion cells and nerve fibers: aid in the diagnosis of Hirschsprung’s

disease. J Med Assoc Thai 2000;83(11):1402-9.

7] Kakita Y, Oshiro K, O’Briain DS, et al. Selective demonstration of mural

nerves in ganglionic and aganglionic colon by immunohistochemistry for

glucose transporter-1: prominent extrinsic nerve pattern staining in

Hirschsprung disease. Arch Pathol Lab Med 2000;124(9):1314-9.

8] Ratcliffe EM, deSa DJ, Dixon MF, et al. Choline acetyltransferase

(ChAT) immunoreactivity in paraffin sections of normal and diseased

intestines. J Histochem Cytochem 1998;46(11):1223-31.

9] Kobayashi H, Hirakawa H, O’Briain DS, et al. Nerve growth factor

receptor staining of suction biopsies in the diagnosis of Hirsch-

sprung’s disease. J Pediatr Surg 1994;29(9):1224 -7.

0] Romanska HM, Bishop AE, Brereton RJ, et al. Immunocytochemistry

for neuronal markers shows deficiencies in conventional histology in

the treatment of Hirschsprung’s disease. J Pediatr Surg 1993;28(8):

1059-62.

1] Sarnat HB, Seagram GF, Trevenen CL, et al. A fluorochromic stain for

nucleic acids to demonstrate submucosal and myenteric neurons in

Hirschsprung’s disease. Am J Clin Pathol 1985;83(6):722 -5.

2] Meier-Ruge WA, Bruder E. Pathology of chronic constipation in

pediatric and adult coloproctology. Basel: Karger; 2005.