J Clin Pathol 1983;36:867-872

Changes in the Paneth cell population of human smallintestine assessed by image analysis of the secretorygranule area

MARGARET E ELMES, J GWYN JONES, MR STANTON

From the Department ofPathology, University Hospital of Wales, Heath Park, CardiffCF4 4XN

SUMMARY Estimates of the Paneth cell population in human jejunum and ileum were made usingmeasurement of the granule area in 'Um2 by image analysis in a defined number of crypts. Thisfigure was preferable to granule area per mm as there was a significant difference in crypts per

mm between biopsies and surgical samples.In the jejunum no significant difference was found between normal children and adults with

and without peptic ulcer. In adults with subtotal or partial villous atrophy the decrease in areawas not statistically significant and there was no decrease in area in children with partial villousatrophy and coeliac disease.There was a marked increase in granule area in the jejunum of patients who had had a previous

partial gastrectomy which was statistically significant.In the ileum patients with carcinoma of the caecum had higher values than patients with

non-inflammatory non-malignant conditions but this was not statistically significant and twopatients with Crohn's disease had an increased granule area.

Paneth cell populations are affected by alterations in the intestinal luminal environment due toprevious surgery or neoplastic or inflammatory disease.

The current techniques of estimating Paneth cellpopulations in sections of small intestine depend ongranule staining of cells either in a measured lengthof mucosa,' 2 or by calculating the number of Panethcells per crypt,34 or by measuring the proportion ofPaneth cell containing crypts in a section.56 In addi-tion to haematoxylin and eosin special stains such asMasson's trichrome, Lendrum' s phloxine tartrazineand immunostaining for lysozyme (muramidase)and IgA have been used.78 There are many potentialsources of error. The staining depends on a cell con-taining reactive granules and granule loss can occurduring fixation if acid fixatives such as Bouin's fluidare used.9 The nature of the marker used may causeproblems; phloxine tartrazine stains argentaffin cellsand lysozyme is found in goblet cells and somelamina propria cells as well as Paneth cells. Anyestimate of the number of cells present should takeinto account the average size of the cell and the factthat parts of any one cell will be present in adjacentsections. Even when all cells are accurately counted

Accepted for publication 28 March 1983

different degrees of tissue shrinkage during fixationand processing make comparisons difficult.The aim of this work was to devise a technique of

estimating Paneth cell populations in man in whichboth small biopsy specimens and large surgicalspecimens could be compared, and to minimiseerrors due to tissue shrinkage, subjective assessment,and difficulty in idenfifying Paneth cells. Imageanalysis was used to measure the area occupied bystained Paneth cell granules in a defined number ofcrypts, and changes in this area correlated with dif-ferent pathological conditions.

Material and methods

Samples of jejunal and ileal mucosa were obtainedfrom two sources-jejunal biopsy for diagnosticpurposes and intestinal resection for disease.A total of 77 specimens were examined, 56 adultsage 17-83 yr (mean 56) and 21 children age 6months-16 yr (mean 7).The patients were divided into the following groups:1 Biopsy samples (jejunum)(a) Non-coeliac disease This group consisted offive adults and 13 children. The adults were being

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investigated for anaemia or malabsorption and thechildren for short stature or unexplained diarrhoea.All mucosal samples were histologically normal.(b) Active coeliac disease The subjects were fouradults with untreated coeliac disease and eight chil-dren with subtotal or partial villous atrophy due tountreated coeliac disease in four cases and after glu-ten challenge in four cases. Gluten challenge con-sisted of a normal diet for 6 months to 2 yr afterprevious gluten withdrawal in three cases and agluten-free diet with either added gluten or breadfor three months in one case.

2 Surgical specimens. All adults(a) Jejunum was obtained during anastomoticoperations including partial gastrectomy for pepticulcer (n = 7), gastrointestinal anastomosis for recur-rent peptic ulcer after previous surgery (n = 7) andgastric carcinoma (n = 9).(b) Ileum from patients with non-inflammatory,non-malignant disease including Meckers diver-ticulum, angiodysplasia, polyposis coli withoutmalignant change and non-malignant obstruction (n= 7); ("Control"); carcinoma of right side of colon(n = 3); carcinoma of caecum (n = 8) and Crohn'sdisease (n = 6).

STAINING TECHNIQUEAll samples were fixed in formol saline and proces-sed routinely. Paraffin sections (4-5 ,um) were cutand stained with haematoxylin and eosin and alsowith a modification of Lendrum's phloxine tar-trazine omitting the nuclear stain, the procedurebeing as follows:The dewaxed sections were stained for 5 min in

0-5% phloxine in 0-5% CaCl2, rinsed in water andthen differentiated in a saturated solution of tar-trazine in Cellosolve until the Paneth cell granulesstood out clearly against a yellow background. Thistook between 10 and 60 min and was controlled foreach slide. When complete the slide was rinsed inCellosolve, put in 95% ethanol and dehydrated andmounted in Canada balsam or DPX. The Paneth cellgranules, enterochromaffin cell granules and eryth-rocytes appeared bright red against a yellow back-ground.Long periods of differentiation were needed for

tissue from some cases of coeliac disease andCrohn's disease and in a few cases it was impossibleto remove all red staining from the enterocyte cyto-plasm.

ASSESMENT OF TISSUE SHRINKAGEThe number of crypts per mm was measured in 74specimens stained with haematoxylin and eosinusing an eyepiece graticule with a total scale length

Elmes, Jones, Stanton

of 0-6 mm at a magnification of x 160. At least twoconsecutive fields with a linear crypt base measure-ment of 1-2 mm were used but in larger specimensfive consecutive fields with a crypt base measure-ment of 3 mm were observed and the number ofcrypts per mm calculated.

PANETH CELL GRANULE AREA MEASUREMENTCell granule area measurements were made on 75slides stained with phloxine tartrazine using a com-puter controlled quantitative image analyser.'0 Thisinstrument comprises a closed circuit television sys-tem connected via a beam splitter attachment to amicroscope. The image received from the micro-scope is processed by the TV system and a mini-computer so that component parts of the image canbe selected according to the degree of light intensitywhich they contain.

Microscope specimens are stained so that items ofinterest, when viewed on a television monitor,assume a specific degree of light intensity and cantherefore be discriminated from the background.To aid the process of discrimination a

pseudocolour binary image is superimposed overthose parts of the television picture which share thesame values of light intensity.'0 Once this has beendone the selected regions can be processed by thecomputer to extract specific parameters relating toarea, count etc. (Fig. 1).To measure Paneth cell area the slide was

examined using an objective power of x 40 at anarrow bandwidth light wavelength of 546 nm. Thisensured that the Paneth cells granules appeared onthe television screen as dense black objects setagainst a relatively light background (Fig. 2)The image analyser was calibrated with a Zeiss

stage micrometer so that an area of 100 ,um2 at themicroscope specimen plane was equivalent to a tele-vision picture area of 134 x 134 picture points(pixels).

MONITORCAMERA

1j\L~~~~~~~~~~~~P~~

Fig. 1 Diagram ofImage Analyser showing microscope,camera, monitor and diagram ofappearances oftwo Panethcells on the monitor to right offigure.

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Changes in the Paneth cell population ofhuman small intestine

a.

Fig. 2 Appearances ofImage Analyser monitor withpseudocolour laid over Paneth ceUl granule area. Originalslide magnification x 400.

Each Paneth cell group together with a smallamount of background were isolated from surround-ing artefact by enclosing the granules in a variablerectangular "window" which could be positionedanywhere on the screen. Only items within the win-dow were included in the measurements.The black Paneth cell granules were isolated from

the relatively light background by a computer pro-gramme which examined the bimodal distribution ofintensity levels within the image and then selectedan optimum "threshold" at which to separate one

from the other.A pseudocolour was then superimposed over the

detected Paneth cell area and the numerical datarelating to this area was obtained from the printer.The discrimination of the Paneth cells by the compu-ter was successful in approximately 95% of cases.On the other occasions it was necessary to set thethreshold level by hand. If artefactual regions couldnot be avoided and came within the measuring win-dow, then these regions were eliminated by using a

special screen light pen.11 Artefacts consisted oferythrocytes, enterochromaffin cells and persistentphloxine stain in enterocytes.The area occupied by Paneth cell granules in a

crypt was measured five times and a mean for eachcrypt obtained. A crypt was defined as any part of acrypt that was within 30 ,Am of the muscularismucosa and this included sections across the outeredge of a crypt that had no identifiable Paneth cells

in them. Slides with longitudinally cut crypts werepreferred but if a small badly orientated region wasencountered it was ignored and the count continuedin the next satisfactory area. Fifty consecutive cryptsper slide were counted and if the sample was toosmall to have 50 crypts per slide a second slide sepa-rated by more than 5 ,um was used to complete thecount to avoid counting the same cells twice.By this means a Paneth cell granule area in pixels

per crypt for each tissue was obtained but statisticaltransformation was necessary as the values obtainedincluded many zeros and a normal distribution wasobtained by using log,0 (x + 20) for each observa-i h i t°g~~~~log1(X +20)idtion.The geometric mean,lg x+20

was determined50

for each patient and all statistical analyses per-formed using transformed data. The final resultswere converted into ,tm2 by converting back intopicture points (antilog10 - 20) and multiplying bythe calibration factor 0.557 (1342 picture points =100 ILm2).

Results

ASSESSMENT OF TISSUE SHRINKAGETable 1 shows the mean crypts per mm length ofsmall intestine in the jejunum and ileum in bothbiopsy and surgical specimens. The group of controlpatients in the results from the ileum includednon-malignant, non-inflammatory conditions requir-ing surgery such as Meckel's diverticulum,angiodysplasia of the ileum, polyposis coli andnon-malignant stricture.The overall pooled data was tested using a paired t

test and a significant difference (p < 0-05) was

Table 1 Arithmetc means and standard deviations ofcryptsper mm length ofsection

JejunumSurgical specimensPeptic ulcerGastric carcinomaOverall pooled mean

BiopsiesNormal adultsNormal childrenVillous atrophy, dcildrenOverall pooed mean

IleumSurgical specimens (adult)ControlCarcinoma colonCarcinoma caecumInactive Crohn'sActive Crohn'sOverall pooled mean

15.9+2-9 n=1018-7 + 3-0 n = 917.2± 3-2 n= 19

13-4 _ 2-79 3 2.4

10.0 + 2*710.3 + 2-8

13-1 _ 2-711-7 _ 1-211-8 2-314 0 2-912-5 6.412-7 2-8

n= 5n = 13n= 8n = 26

n= 8n= 3n= 9n= 7n= 2n = 29

Sinificant difference between surgical and biopsy speamens injejunum (p < 0.05) and between jejunal and ileal surcalspecimens (p < 0)001) paired t test.

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Table 2 Jejunal Paneth cell granule area in iom2 per crypt in biopsy specimens from normals and patients with partialvillous atrophy (PVA)

Group I Group 2 Group 3 Group 4Normal adult PVA adult Normd chldren PVA chidren

120.3 98*3 117.5 29.651.2 13-9 36-0 44.634-1 50.8 12.3 36.329.0 111.7 9-6 46.751*9 5-7 31*7

40.9 25.019.0 37-3

101.4 33.840-533.249-962-920-4

Mean51.1 34.3 30-9 35-1

No statistically significant differences between groups.

found between surgical specimens and biopsies inthe jejunum. Fewer crypts/mm were found in biopsyspecimens, probably due to lack of a muscle coat,and therefore less shrinkage during fixation. The

smaller number of crypts/mm in children's biopsiesmay in part have been due to different techniques inhandling the tissue before fixation, the children'sbiopsies being spread out more than the adult ones.There was no difference between children withnormal biopsies and those with subtotal or partialvillous atrophy.

In adults when surgical specimens from thejejunum and ileum were compared more crypts permm were found in the jejunum than in the ileum (p< 0-001), probably anatomical variation rather thantissue shrinkage.

PANETH CELL GRANULE AREA

'I;

Fig. 3 Same slide as Figure 2 with Paneth cels arrowed.Post gastrectomy jejunum, phloxine tartrazine x 400.

JejunumFig. 3 shows Paneth cell granules in a post gastrec-tomy patient with Paneth cell increase including onehigh up in the crypt.Table 2 gives the result found in biopsies from

adults and children and there is no statisticallysignificant difference between normal adults andchildren. The children have a lower mean value andsome biopsies yielded very low counts due to appar-ent loss of Paneth cell granules as non-granulatedcells in the correct position resembling Paneth cellswere seen. This is probably due to biopsy trauma ormedication as these cells were not found in surgicalspecimens. In a few children poorly stained Panethcell granules were seen but the image analysis wasusually sufficiently sensitive to measure granuleswith any tinge of red. Children with villous atrophyappeared to have fewer Paneth cells but due to thewide range of values in normal children this was notstatistically significant. No child with villous atrophyhad a high value in contrast to two of the normalchildren.The results of both groups had a wide range of

870 Elmes, Jones, Stanton

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Changes in the Paneth cell population ofhuman small intestine

values. The mean value in villous atrophy was againlower than normal but did not attain statisticalsignificance.The remaining estimations of Paneth cell area

were done on surgical specimens and in every casesome pathology was present in either stomach orintestine so no group can be considered as normal.Table 3 compares Paneth cell area in patients in

group 1 (peptic ulcer, first operation), group 2 (pre-vious partial gastrectomy requiring revision or arecurrent peptic ulcer) and group 3 (gastric car-cinoma).

It is evident that the reoperated patients in group2 had a mean Paneth cell granule area significantlyhigher than the other two groups (p < 0-05).

IleumAs in the assessment of tissue shrinkage results thepatients were classified into groups. (Table 4)Group I included non-inflammatory, non-malignantconditions such as Meckel's diverticulum, angiodys-plasia, polyposis coli and non-malignant stricture("Control"). n = 7.Group 2 Carcinoma of the caecum. n = 7.Group 3 Crohn's disease. n = 6.Group 1 had the lowest overall mean (71-2 4m2)compared with 113-3 ,Um2 in group 2 and 91-4 ,um2in group 3, but it is apparent that there is no markeddifference between groups 1 and 3. Group 2appeared to have a considerably higher value thangroup 1 but on statistical analysis (paired t test) the pvalue was not significant in spite of the high levels in4 of 8 patients. This is due to the small number ofcases and one very low level. No significant increasein area over group 1 was found in the cases ofCrohn's disease in group 3-the first four cases hadno lesions in the portion of mucosa examined andhad values comparable with group 1. Two patients

Table 3 Jejunal Paneth cell granule area per crypt in ,.un2in surgical specimens

Group I Group 2 Group 3Peptc ulcer Reoperated Gastric carcinoma

peptic ulcer

54-9 192-1 76-134-5 95-2 33-239-8 60-1 68-334-8 123-0 44-327-6 25-4 49-541-8 48-8 16-860-6 136-4 36-4

83-966-6115-9

Mean40-0 82-2 52-7

A paired t test on the geometric means of the above figures showeda significant difference between groups 1 and 2; p < 0-05.

Table 4 Ileal Paneth cell granule area per crypt in on' insurgical specimens

Group I Group 2 Group 3"Control" Carcinoma caecum Crohn's disease

83-9 44-4 92-486-3 193-9 87-241-4 148.4 83-9119-9 355-9 40-579-3 58-5 152-591-8 58-0 131-032-6 158-0

104-8Mean71-2 113-3 91-4

No statisticaily significant difference between groups.

with characteristic lesions in the mucosa examineddid have high values but statistical significance wasnot attained.

Discussion

The choice of a quantifiable marker for any cell typeis always difficult. In this case it was decided to usethe acidophilic secretory granule area as an estimateof the Paneth cell population although this value canbe lowered by either unstained granules, as wasfound in a few children, or by granule discharge.This was particularly apparent in jejunal biopsiesfrom normal children where degranulated Panethcells were seen resulting in low granule area values.In studies using a combined muramidase/phloxine/tartrazine stain, (B Gormley, personal communica-tion, 1982) the muramidase reaction productoccupies a larger part of the cell than the phloxinestained granules and appears to surround the redgranules. This results in an increase in the number ofcells identified as Paneth cells by the muramidasetechnique as Scott and Brandtzaeg7 point out. Inspite of this we confirmed Scott and Brandtzaeg'sobservation of no significant decrease in Paneth cellgranule area in the jejunum in adults or childrenwith villous atrophy.The study on the effect of tissue preparation on

crypts per mm demonstrates that techniques ofestimating Paneth cell populations using linearmeasurements are more subject to error than thoseusing a defined number of crypts. If both biopsy andsurgical material is to be used the crypt countingmethod is essential.

In the jejunum the most marked increase wasfound in patients who required a second operationafter a partial gastrectomy for peptic ulcer. Severalof these patients had a very obvious increase inPaneth cells on microscopy with Paneth cells seen inunusual positions in the crypt (Fig. 3). It has beensuggested that Paneth cells are concerned in the

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872maintenance of a normal bacterial flora in the smallintestine.'2

Preliminary work in rats demonstrated an

increase in Paneth cells in intestinal stasis due tomecamylamine or a self-emptying blind loop but a

decrease in Paneth cells in self-filling blind loops andself-emptying blind loops that showed a markedincrease in the bacterial flora of the lumen.13 Furtherwork has demonstrated an increase in the Panethcell population in the ileum of rats reared in a lowbacterial environment and in isolated Thiry-Vellaloops but a decrease in self-filling blind loops.8The aim of partial gastrectomy is to reduce acidity

and this combined with altered intestinal mobilitypredisposes to gastrointestinal bacterial over-

growth.'4 We suggest that the Paneth cell prolifera-tion observed is an attempt to prevent bacterialovergrowth in an abnormal hypochlorhydric stateand individual patients who do not show thisresponse are liable to have bacterial overgrowth inthe intestinal lumen. Preliminary results in furtherwork to be reported later support this view.

Dr J Gwyn Jones was supported by the WelshScheme for the Development of Health and SocialResearch.We wish to thank the physicians and surgeons of

the University Hospital of Wales and LlandoughHospital, Penarth for allowing us to study theirpatients.

Referenes

Mols G. Recherches cytologiques et experimentales sur les cel-lules de Paneth. Arch Biol (Pans) 1930;40:111-50.

Elmes, Jones, Stanton2Malan GI. Direct quantitative etm of Paneth and toial

celi populations in the jejunal glands of Ueberkhn. Am JAnat 1975;142:201-4.

3Gibbs NM. Incidence and sificance of argentaffin and Panethcells in some tumours of the large intatine. J Clin Pa*dol1967;20:826-31.

4Elmes ME. The Paneth cell population of the small intestine ofthe rat.J Padol 1976:113:183-91.

s Hertzog AJ. The Paneth cegl. Am J Pahdo 1937;13:351-60.6 Lewin K. The Paneth cel in disease. Gut 1969;1'.804-811.7Scott H, Brandtzaeg P. Enumeration of Paneth cells in coeliac

disease. Gut 1981;22:812-6.' Rodning CB, Erlandsen SL, Dodd Wilson I, Carpenter AM.

Iight microscopic morphometric analysis of rat ileal mucosa.U Component quantitation of Paneth cells. Anat Rec1982;204:33-8.

9 Sandow MJ, Whitehead R. The Paneth cell. Gut 1979;20:420-31

Coyne MB. Quntitative image analyser using a colour televisiondisplay for medical applications. Medical Biological Engineer-ing 1974;12:295-302.

"Stanton MR, Garrahan NJ. The use of a simple light-pen systemwith a colur television image analyser. Medical BiologicalEngineering 1975;13:311-4.

12 Erlandsen SL, Parsons JA, Taylor TD. Ultastrualimmunocytochemical locastion of lysozyme in the Panethcels of man. J Histochm Cytochem 1974;22:401-13.

3 Elmes ME, Love AHG. Paneth cels and the intestinalmicroflora. Proceedings European Society for Clinical Inves-tigation, 9th Meeting. Abstract No. 191, 1975.

4 Enander LK, Nilson R, Ryden AC, Schwan A. The aerobic andanaerobic microflora of the gastric remnant more than 15years after Billroth II resection. Scand J Gastroenterol1982;17:715-20.

Requests for reprints to: Dr Margaret E Elmes, Depart-ment of Pathology, University Hospital of Wales, HeathPark, Cardiff CF4 4XN, Wales.

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