Histochemical diagnosis Hirschsprung's disease · Fig. 2 Rectalmucosain Hirschsprung's disease with...

J Clin Pathol 1980; 33: 336-343

Histochemical diagnosis of Hirschsprung's diseaseand a comparison of the histochemical andbiochemical activity of acetyicholinesterase in rectalmucosal biopsiesWJA PATRICK, GTN BESLEY, AND II SMITH

From the Department ofPaediatric Pathology, Royal Hospital for Sick Children, Edinburgh, UK

SUMMARY Three hundred and seventy-two rectal mucosal biopsies, taken from 150 children andyoung adults with chronic constipation, were subjected to histochemical and biochemical analysis ofacetylcholinesterase to exclude Hirschsprung's disease. The relative merits of the procedures were

compared. The histochemical method was considered to be the most practical for laboratorieshandling small numbers of biopsies but the biochemical estimation of acetylcholinesterase activitywas found to be a useful complementary procedure and an accurate quantitative assessment ofenzyme activity.

The introduction of acetylcholinesterase histo-chemistry' has resulted in a reliable means ofexcluding Hirschsprung's disease in rectal mucosalbiopsies.2

Previously, the diagnosis of Hirschsprung's diseasedepended on standard histological techniques,3-7although some workers have used histochemicalmethods to confirm the diagnosis on resectedspecimens.8-'2

Acetylcholinesterase histochemistry is now usedin preference to routine histological methods in manycentres. Its diagnostic reliability in rectal mucosalbiopsies has been emphasised by Meier-Ruge andhis colleagues3 and confirmed by other workers14-'9and ourselves.20The present paper is an account of our experience

over a two-year period of the use of acetylcholin-esterase histochemistry as a screening procedure toexclude Hirschsprung's disease in rectal mucosalbiopsies and compares the histochemical findingswith the biochemical activity of the enzyme.2'

Material and methods

Over a two-year period, which ended in January1979, 372 rectal mucosal biopsies were performed on150 children and young adults aged between 6 daysand 28 years (Table 1) and the specimens wereprocessed for acetylcholinesterase histochemistry.

Received for publication 8 October 1979

Table 1 Age and sex incidence ofpatients biopsied:numbers of cases of Hirschsprung's disease in parentheses

Age group Males Females

0-4 weeks 7 (4) 0 (0)1-12 months I I (0) 7 (1)1-4 years 30 (4) 18 (1)5-12 years 52 (6) 18 (0)13-20 years 3 (1) 1 (0)> 21 years 1 (0) 2 (0)Totals 104 (15) 46 (2)

The biopsies were taken by means of laryngealpunch biopsy forceps at several levels from the analmargin (Table 2). The patients attended hospital asday cases on prearranged days of the week, and theprocedure was carried out with sedation or undermild general anaesthesia. The biopsy specimens weretransported to the laboratory on ice and orientatedunder a dissecting microscope; frozen sections weretaken for histochemistry within an hour of removal.

Table 2 Levels of individual biopsies: number ofbiopsies from cases of Hirschsprung's disease inparentheses.

Level (cm) Number %

1-5 180 (23) 506-10 35 (18) 1011-15 9 (4) 3> 15 3 (3) 1Not stated 130 (27) 36Totals 357 (75) 100

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Histochemical diagnosis of Hirschsprung's disease

Fifteen specimens were subsequently excludedbecause they were either too badly traumatised orbecause they were of anal as opposed to rectalmucosa.One hundred and sixteen of the original 372

biopsy specimens were divided longitudinally so thathalf of the specimen went for histochemical and theother half for biochemical analysis of acetylcholin-esterase. The piece of tissue for biochemistry waswrapped in aluminium foil, snap frozen, and storedfor up to one year in the vapour phase of liquidnitrogen before biochemical analysis.

HISTOCHEMICAL METHOD

Reagents(1) Substrate: Acetylthiocholine iodide(AThCh) 5mg,dissolved in 0-1 M-acetate buffer at pH 5-5, with theaddition of 0 5 ml 01 M-sodium citrate, 10 ml30mM-cupric sulphate, 10 ml distilled water,10 ml 5 mM-potassium ferricyanide, and 0-2ml0-01 % (w/v) tetramonoisopropylpyrophosphoramide(iso-OMPA).(2) DAB/Hanker- Yates solution: 3.3-diaminobenzi-dine tetrahydrochloride (DAB) 5 mg, dissolved in10 ml 0O1 M-sodium phosphate buffer at pH 6-8;subsequently Hanker-Yates reagent, 5 mg, dissolvedin 10 ml 0-1 M-sodium phosphate buffer at pH 6-8.22

ProcedureThe histochemical method used was based on themodification by Karnovsky and Roots23 of thatoriginally described by Koelle and Friedenwald'using acetylthiocholine iodide as substrate.

Cryostat sections, cut at 10 ,tm, were fixed instandard formol calcium for 1 minute,24 washed indistilled water, and incubated with the substrate for1 hour at 37°C in the presence of iso-OMPA as aselective irreversible inhibitor of non-specific chol-inesterase activity.10 After incubation the sectionswere washed in distilled water, and the reactionwas enhanced by treatment with DAB for 45 minutesand osmium tetroxide for 1 minute.25 The sectionswere counterstained with haematoxylin and mounted.The histochemical procedure was carried out

routinely on three sections; a fourth control sectionwas incubated in the absence of substrate, and a fifthwas stained with haematoxylin and eosin.

BIOCHEMICAL METHOD

Reagents(1) Substrate (0-015 M A ThCh): Acetylthiocholineiodide (AThCh), 21 67 mg, added to 4 95 ml dis-tilled water followed by 0-05 ml 1 M-hydrochloricacid.

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(2) Colour reagent (0 01 M DTNB): 5.5.dithio-bis-2-nitrobenzoic acid (DTNB), 39 5 mg, and sodiumbicarbonate 15 mg, dissolved in 10 ml 0-1 M-phosphate buffer pH 7T0.(3) Inhibitor (8 52 x 10-4 M Lysivane): 10-(2-diethyl-aminopropyl)phenothiazine hydrochloride (etho-propazine HCI, Lysivane), 27-1 mg, dissolved in30 ml 2 M-hydrochloric acid and made up to 100 mlwith 0-1 M-phosphate buffer pH 7 0.

ProcedureThe biochemical method used was a modification byDale and his colleagues26 of a photometric methodoriginally described by Ellman et al.27The assays were carried out without prior knowl-

edge of the histochemical findings as single deter-minations as there was usually insufficient materialto do otherwise. The biopsies were trimmed to givea final specimen weighing less than 10 mg. The tissuewas hand-homogenised in 0-1 M-phosphate bufferat pH 8-0 in a glass Potter-Elvehjem type homo-geniser using 15 strokes. The volume of buffer wassufficient to give a final concentration of 10 mgtissue per ml. The homogenate was centrifuged at12 000 g for 4 minutes to remove cellular debris.Each stage of the procedure was carried out atapproximately 5°C.Two enzyme determinations were made on each

sample, corresponding to (1) total acetylcholin-esterase activity and (2) true acetylcholinesteraseactivity. The total (AChE + ChE) activity wasdetermined at 25°C in microcuvettes by adding80p, of tissue extract to 480,u1 0 1 M-phosphatebuffer at pH 8-0 and 20 ,ul colour reagent (DTNB).After allowing the solutions to stabilise for 5 minutes,20 ,ul of substrate (AThCh) was added. The rate ofchange of absorbance per minute was measured at412 nm with a recording spectrophotometer. Follow-ing determinations of total activity, specific acetyl-cholinesterase (AChE) activity was measured by theaddition of 10 pl of the non-specific cholinesterase(ChE) inhibitor, Lysivane.

Results

The product of the histochemical reaction appears asa dark brown precipitate at the site of nerve fibresand ganglia.

In normal mucosal biopsies, a few clearly definedslender nerve fibres are present in the connectivetissue of the lamina propria and between musclebundles in the muscularis mucosae (Fig. 1). Thenerve fibres in the submucosa occur in bundles, andganglion cells are usually prominent, the reactionappearing as fine particulate deposit in the cyto-plasm. The supportive Schwann cells are also con-



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Fig. 1 Normal rectal mucosa with slender AChEpositive nerve fibres (single arrows) in the connectivetissue of the lamina propria (lp) and muscularismucosae (mm), and prominent ganglia (double arrow) inthe submucosa (sin). x 25.

spicuous, and the reaction usually outlines smoothmuscle elements.The biopsies in cases of Hirschsprung's disease

show a marked increase in the number and size ofpositively staining nerve fibres in the muscularismucosae and lamina propria (Figs 2 and 3), anincrease in the size of the nerves in the submucosa,and an absence of submucosal ganglia. The abnormalnerve plexuses in the muscularis mucosae andlamina propria are not readily seen in haematoxylinand eosin stained sections.The biochemical activity of acetylcholinesterase

was expressed according to Dale and his colleagues26as units of specific (AChE) and non-specific (ChE)activity. The specificAChE activity was also expressedas a percentage of total (AChE + ChE) activity.

In 101 biopsies showing a normal histochemicalreaction, AChE activity (Fig. 4) was in the range0-3-7l9 units (mean 2-7, SD 1-4) and ChE activity(Fig. 5) in the range 0-3-5-8 units (mean 2-6, SD 1-0).

Patrick, Besley, and Smith

The range of AChE and ChE activity in nine biopsiesshowing the histochemical features of Hirschsprung'sdisease was 5 3-30 2 units (mean 15-2, SD 9 0) and2 1-7 9 units (mean 3-8, SD 2 0) respectively (Figs 4and 5). In six biopsies which were histochemicallyequivocal, that is to say, biopsies showing someincrease in the number of nerve fibres in themuscularis mucosae and, to a lesser extent, the laminapropria, AChE activity (Fig. 4) was within thenormal range 1-7-5-7 units (mean 3 7, SD 1 7).AChE activity expressed as a percentage of total

activity in the 101 normal biopsies (Fig. 6) was in therange 27-79O% (mean 54-6, SD 105), and, for thenine biopsies showing the histochemical features ofHirschsprung's disease, in the range 70-84% (mean78 9, SD 6 8).

Discussion

Over 75% of the patients investigated in our serieswere aged between I and 12 years (Table 1). Ahigher proportion of the cases presenting in infancywere found to have Hirschsprung's disease. Malesoutnumbered females both in the number of casespresenting with a history of chronic constipation andin the number who subsequently were shown tohave Hirschsprung's disease. This remarkable pre-ponderance of males with Hirschsprung's disease hasbeen noted by others,28-30 and our male to femaleratio is close to the 8: 1 ratio reported in negroinfants by Leenders and his colleagues.29The histochemical criteria for the diagnosis of

Hirschsprung's disease as stated above are thosegenerally accepted by other workers.13-17 19 Theincrease in the number and size of nerve fibres in themuscularis mucosae and lamina propria is a par-ticularly striking feature, and a diagnosis can be madeconfidently without reference to the ganglia in thesubmucosa,13 19 a view that conflicts with that of atleast two other groups who consider that the presenceof submucosa is essential to accurate diagnosis.15 16The absence of abnormal nerve fibres in the normalhypoganglionic zone immediately above the pectinateline described by Aldridge and Campbell31 wouldexclude the disease.

In the histochemical assessment of the sections,care must be taken to be certain that structuresresembling ganglia in the submucosa do actuallycontain ganglion cells and are not just a cluster ofsupportive cells. As Naik and Cauna32 pointed out,both types of cell show moderate AChE activity.Difficulty may also be encountered if cellular detailin the lamina propria is obscured by haemor-rhage.17 19

Accurate definition of the aganglionic segment inHirschsprung's disease would require biopsies to be

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Fig. 2 Rectal mucosa in Hirschsprung's disease with an increase in the number and sizeofAChE positive nerve fibres (single arrows) in the connective tissue of the lamina propria(lp) and muscularis mucosae (mm), and aggregates of Schwann cells in the submucosa (sm)that could be mistaken for ganglia (short arrow). x 38.

__L _Fig. 3 Superficial rectal mucosa in Hirschsprung's disease with a marked increase in thenumber and size of AChE positive nerve fibres (single arrow) in the lamina propria:L = lumen. x 47.

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Patrick, Besley, and Smith

EQUIVOCAL: NUMBER

RANGE 1.

MEAN

S.D.

A

NORMAL: NUMBER 101

RAMIE 0.3 - 7.9

MmAN 2.7

S.D. 1.4 Fig.4 AChEactivity in biopsyspecimens show-ing a normalhistochemicalreaction (A), and

12 16 20 24 28 32 in those with thehistochemicalfeatures ofHirschsprung'sdisease (B). The

B specimens thatwere histochemi-cally equivocal

6 ANORMAL: NUMBER 9 are represented.7 - 5.7 RANWE 5.3 - 30.2 by interrupted3.7 MmAN 15.2 lines.1.7 S.D. 9.0

I0 2 4 6 8 12 16 20 24 28 3:

AChE ACTIVITY

NORMAL NUMBER 101

RANGE 0.3 - 5.8MEAN 2.6S.D. 1.0

2 4 6 8 12 16 20 24 28 32

EQUIVOCAL NUMER 6 ABWRML : NUME 9RANGE 1.3 - 2.8 RA^NGE 2.1 - 7.9MEAN 2.0 t{lea 3.8S.D. 0.5 S.D. 2.0

I *SI1lll1 1 1 1 N

2

A

Fig. 5 ChEactivity in biopsyspecimens show-ing a normalhistochemicalreaction (A), andin those with thehistochemicalfeatures ofHirschsprung'sdisease (B). The

B specimens thatwere histochemi-cally equivocalare representedby interruptedlines.

ChE ACTIVITY

6

4

2 U'M1115n2

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I

0 2 4 6 8

6

4

2

11

6

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% AChE ACTIVITY

Fig. 6 AChE activity expressed as a percentage of totalactivity in biopsy specimens showing a normal histo-chemical reaction (top) and in those with thehistochemical features of Hirschsprung's disease (bottom).The specimens that were histochemically equivocal are

represented by interrupted lines.

taken at several levels from the anal margin. Weaimed to make these levels below 5 cm, between5 and 10 cm, and above 10 cm to correspond with ourdefinition of ultrashort, short, and long segmentHirschsprung's disease respectively.33 The levelswere proportionately less in younger children. Inpractice, eight of the 17 cases of Hirschsprung'sdisease in our series required repeat biopsies todefine the aganglionic segment, 80% of all our cases

had biopsies taken from only one or two levels, and52% of the biopsies were from levels less than 5 cmfrom the anal margin. For 36% of the biopsies thelevel was not stated.

Biopsies were obtained routinely from more thanone level in three of the previously reported series.Chow and his colleagues6 obtained a minimum ofthree biopsies 3, 4, and 5 cm from the anal verge in

younger children and 5, 6, and 7 cm from the analverge in older children; Martinez-Almoyna and hiscolleagues'8 had biopsies taken at 3, 5, and 10cmfrom the pectinate line; and in the series of Lake andhis colleagues'9 the biopsy specimens were taken at2, 4, and 5 cm from the mucocutaneous junction inmost cases. The levels chosen by Chow and hiscolleagues6 in younger children and by Martinez-Almoyna and his colleagues'8 in older children arein line with our recommendations and diagnosticapproach to children with chronic constipation20and would be more likely to define accurately thelength of any aganglionic segment. In our opinion,this would directly influence the surgical managementand obvitate the need for costly radiographic andmonometric investigations.34Up to 150 sections may have to be examined to

exclude Hirschsprung's disease using standardhistological techniques,4 7 whereas with the histo-chemical method a maximum of five sections is allthat is required for accurate diagnosis. In 96% ofour cases the diagnosis was unequivocal, and in noinstance did we have a false-positive result. Thiscompares favourably with histochemical resultsreported by other authors and is as good as theresults obtained using routine histologicalmethods.7 15 30

Established techniques were used in this study forboth the histochemical and biochemical methods.The essential difference between the two methodswas in the inhibitors of non-specific cholinesterase(ChE) activity. The inhibitors used (iso-OMPA andLysivane, respectively) were not the same for eachmethod, but both have been shown to be selectiveinhibitors of ChE activity'0 35 and therefore wouldnot be expected in any way to complicate the results.The results we obtained biochemically are in almost

all instances directly comparable with the histo-chemical findings, and we agree with Dale and hiscolleagues21 that the biochemical estimation ofAChEactivity is a useful complementary procedure, par-ticularly if the histochemical diagnosis is equivocal.Overall the levels of activity are lower in our seriesthan in Dale's-something that may have been dueto the fact that the tissue was stored before theanalyses were carried out. The figures for AChEactivity expressed as a percentage of total activitysuggest that the increased activity in Hirschsprung'sdisease is a true increase in specific (AChE) activity.

Hirschsprung's disease was confirmed in 16 of our17 cases (Table 1) by histological and histochemicalexamination of specimens obtained at myectomy orafter resection. The remaining case was a micro-cephalic infant who died before definitive treatmentcould be undertaken. Of the 16 confirmed cases, sixhad long segment disease, eight had short segment

, *X * * E sX 1 """_11 S __ 341

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342 Patrick, Besley, and Smith

disease, and two had ultrashort segment disease byour definition. Four of the six histochemicallyequivocal biopsies with biochemical AChE activitywithin the normal range were from this singleunconfirmed case of Hirschsprung's disease in theseries.For most laboratories handling small numbers of

biopsies the histochemical method is probably themore practical. Both methods are straightforwardand inexpensive, but the biochemical assay has theadditional advantage of offering an accuratequantitative assessment of enzyme activity.

We thank Mr A R Cobb and Mr T McLaren fortechnical assistance; Mr F Coleman for help with thepreparation of the illustrations; and Mrs RhonaAnderson for typing the manuscript. We are alsograteful to Dr G Dale for supplying us with a sampleof Lysivane.

References

Koelle GB, Friedenwald JS. A histochemical methodfor localising cholinesterase activity. Proc Soc ExpBiol Med 1949;70:617-22.

2 Meier-Ruge W. Das Megacolon-Seine Diagnose undPathophysiologie. Virchows Arch (Pathol Anat)1968 ;344:67-85.

3 Swenson 0, Fisher JH, MacMahon HE. Rectal biopsyas an aid in the diagnosis of Hirschsprung's disease.New EnglJ Med 1955;253:632-5.

4 Dobbins WO, Bill AH Jr. Diagnosis of Hirschsprung'sdisease excluded by rectal suction biopsy. New EnglJMed 1965 ;272:990-3.

Campbell PE, Noblett Helen R. Experience withrectal suction biopsy in the diagnosis of Hirsch-sprung's disease. J Pediatr Surg 1969;4:410-15.

6 Shandling B, Auldist AW. Punch biopsy of the rectumfor the diagnosis of Hirschsprung's disease. JPediatr Surg 1972;7:546-52.

7 Pease PWB, Corkery JJ, Cameron AH. Diagnosis ofHirschsprung's disease by punch biopsy of rectum.Arch Dis Child 1976;51:541-3.

8 Kamijo K, Hiatt RB, Koelle GB. Congenital mega-colon. A comparison of the spastic and hyper-trophied segments with respect to cholinesteraseactivities and sensitivities to acetylcholine, DFP, andthe barium ion. Gastroenterology 1953 ;24:173-85.

'Adams CWM, Marples EA, Trounce JR. Achalasia ofthe cardia and Hirschsprung's disease. The amountand distribution of cholinesterases. Clin Sci 1960;19 :473-81.

1Niemi M, Kouvalainen K, Hjelt L. Cholinesterasesand monoamine oxidase in congenital megacolon.J Path Bact 1961 ;82:363-6.

Garrett JR, Howard ER, Nixon HH. Autonomicnerves in rectum and colon in Hirschsprung's disease.A cholinesterase and catecholamine histochemicalstudy. Arch Dis Child 1969;44:406-17.

12 Ludvikovsky J, Lukas Z. Bioptische Diagnostik desMorbus Hirschsprung unter Verwendung derMembrantechnik zur histochemischen Sichtbar-machung der Cholinesterasen. Zentralbl Chir 1976;101:1607-10.

13 Meier-Ruge W, Lutterbeck PM, Herzog B, Morger R,Moser R, Scharli A. Acetylcholinesterase activity insuction biopsies of the rectum in the diagnosis ofHirschsprung's disease. J Pediatr Surg 1972;7:11-7.

14 Elema JD, De Vries JA, Vos LJM. Intensity and proxi-mal extension of acetylcholinesterase activity in themucosa of the rectosigmoid in Hirschsprung'sdisease. JPediatr Surg 1973;8:361-8.

15 Trigg, PH, Belin R, Haberkorn S et al. Experience witha cholinesterase histochemical technique for rectalsuction biopsies in the diagnosis of Hirschsprung'sdisease. J Clin Path 1974;27:207-13.

16 Chow CW, Chan WC, Yue PCK. Histochemicalcriteria for the diagnosis of Hirschsprung's disease inrectal suction biopsies by acetylcholinesteraseactivity. J Pediatr Surg 1977;12:675-80.

17 Toorman J, Bots GTAM, Vio PMA. Acetylcholin-esterase-activity in rectal mucosa of children withobstipation. Virchows Arch (Pathol Anat) 1977;376:159-64.

18 Martinez-Almoyna C, Claver Criado MC, MonereoGonzilez JM, Contreras F. Diagnostico histo-quimico en biopsias rectales por succi6n pediatricas.An Es Pediatr 1977;10:341-50.

19 Lake BD, Puri P, Nixon HH, Claireaux AE. Hirsch-sprung's disease. An appraisal of histochemicallydemonstrated acetylcholinesterase activity in suctionrectal biopsy specimens as an aid to diagnosis.Arch Path Lab Med 1978;102:244-7.

20 Hamdy MH, Scobie WG, Smith II, Patrick WJA. Thechanging position of punch biopsy of the rectumcombined with acetylcholinesterase study in themanagement of chronic constipation in childhood.Z Kinderchir 1979;26:124-32.

21 Dale G, Bonham JR, Lowdon P, Wagget J, RangecroftL, Scott DJ. Diagnostic value of rectal mucosalacetylcholinesterase levels in Hirschsprung's disease.Lancet 1979;1:347-9.

22 Hanker JS, Yates PE, Metz CB, Rustioni A. A newspecific, sensitive and non-carcinogenic reagent forthe demonstration of horseradish peroxidase. Histo-chem J 1977;9:789-92. (Letter.)

23 Karnovsky MJ, Roots L. A 'direct coloring' thio-choline method for cholinesterases. J HistochemCytochem 1964;12:219-21.

24 El-Badawi A, Schenk EA. Histochemical methods forseparate, consecutive and simultaneous demon-stration of acetylcholinesterase and norepinephrinein cryostat sections. J Histochem Cytochem 1967;15 :580-8.

25 Hanker JS, Thomburg LP, Yates PE, Moore HG. Thedemonstration of cholinesterases by the formation ofosmium blacks at the sites of Hatchett's brown.Histochemie 1973;37:223-42.

26 Dale G, Bonham JR, Riley Katherine WA, Wagget J.An improved method for the determination ofacetylcholinesterase activity in rectal biopsy tissue



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Histochemical diagnosis of Hirschsprung's disease 343

from patients with Hirschsprung's disease. ClinChim Acta 1977;77:407-13.

2 Ellman GL, Courtney KD, Andres V, Jr, FeatherstoneRM. A new and rapid colorimetric determination ofacetylcholinesterase activity. Biochem Pharmacol1961 ;7:88-95.

28 Bodian M, Carter CO. A family study of Hirschsprung'sdisease. Ann Hum Genet 1963;26:261-77.

29 Leenders E, Sieber WK, Girdany BR, KiesewetterWB. Aganglionic megacolon in infancy. SurgGynecolObstet 1970;131:424-30.

30 Swenson 0, Sherman JO, Fisher JH. Diagnosis ofcongenital megacolon: an analysis of 501 patients.J Pediatr Surg 1973;8:587-94.

31 Aldridge RT, Campbell PE. Ganglion cell distributionin the normal rectum and anal canal. A basis for thediagnosis of Hirschsprung's disease by anorectal

biopsy. J Pediatr Surg 1968 ;3 :475-90.32 Naik NT, Cauna N. Histochemical observations on

cholinesterase activity in the autonomic ganglia ofthe human sympathetic trunk and vagus nerve.Histochem J 1971 ;3:47-53.

33 Scobie WG. Personal communication, 1979.34 Morikawa Y, Donahoe Patricia K, Hendren WH.

Manometry and histochemistry in the diagnosis ofHirschsprung's disease. Pediatrics 1979;63 :865-71.

35 Naik NT. Technical variations in Koelle's histo-chemical method for demonstrating cholinesteraseactivity. Q J Micro Sci 1963;104:89-100.

Requests for reprints to: Dr WJA Patrick Department ofPaediatric Pathology, Royal Hospital for Sick Children,Glasgow, UK.



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