ulcer-associated duodenitis correlation between area

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J Clin Pathol 1981 ;34:1181-1186 Measurements of intestinal villi in non-specific and ulcer-associated duodenitis correlation between area of microdissected villus and villus epithelial cell count

1 J Clin Pathol 1981 ;34: Measurements of intestinal villi in non-specific and ulcer-associated duodenitis correlation between area of microdissected villus and villus epithelial cell count MAHMUD HASAN, ANNE FERGUSON From the Gastro-Intestinal Unit, Western General Hospital and The University of Edinburgh, Crewe Road, Edinburgh EH4 2XU SUMMARY Measurements of villus height, villus area, together with counts of epithelial cells in individual villi, were performed on endoscopic duodenal biopsies from five groups of patients: controls, ulcer-associated duodenitis, mild and severe non-specific (non-ulcerative) duodenitis, cimetidine healed ulcer-associated duodenitis and cimetidine healed non-specific duodenitis The objectives of the study were two-fold: to establish if epithelial cell count correlated with simpler measurements of villus height or area; and to compare the findings in ulcer-associated and in nonspecific duodenitis Villus area correlated well with epithelial cell count per villus (r = 0O96); villus height correlated less well (r = 0O66) When compared with controls, there was a significant decrease in the epithelial cell count per villus in ulcer-associated and severe non-specific duodenitis, but this was confined to the visually inflamed area of the duodenal bulb After healing of inflammation with cimetidine villus height, area, and epithelial cell count returned to values similar to those in controls This study confirms that the effects of ulcer-associated and severe non-specific duodenitis on duodenal villi are identical Objective measurements of the sizes of small intestinal villi and crypts may be of value in clinical practice-for example, to assess the effects of gluten challenge in a coeliac patient, as well as in research Most measurements of intestinal villi have been conducted using histological sections, but the technique of microdissection of specimens stained in bulk, previously extensively used in animal work' has also been applied to material from human intestine2 Villus height may be measured as microns, or as the height of the villus epithelial cell column However, Wright and his colleagues3 have criticised the use of villus height as an index of damage to villi, for in a study of jejunal biopsies from coeliac patients they counted the total number of epithelial cells per villus and found that this correlated poorly with the villus length Unfortunately, it is impractical to use counts of villus epithelial cells in clinical research, for some 30 minutes per villus is necessary to perform this measurement Since normal human small intestinal villi are leaf or spade shaped, we reasoned that measurement of the area of a microdissected villus would be likely to Accepted for publication 2 April 1981 correlate well with total villus epithelial cell count We have therefore compared villus height, villus area, and epithelial cell count per villus in biopsies obtained from the duodenum at endoscopy We have applied these methods to specimens from patients with duodenitis, both ulcer-associated and nonspecific, and controls, in an attempt to detect minor deviations from normal in the duodenal villi of such patients A previous study has shown that villus height was abnormally low only in visually abnormal, severe duodenitis, ulcer-associated or non-specific4 Material and methods The protocol for this study was identical to that described in a previous paper on duodenal mucosal architecture in non-specific and ulcer-associated duodenitis4 Briefly, specimens of duodenal mucosa were obtained under direct vision from the first and second parts of the duodenum at upper gastrointestinal endoscopy Five groups of subjects were studied: controls, ulcer-associated duodenitis, nonspecific (non-ulcerative) duodenitis, healed ulcerassociated and non-specific duodenitis on treatment with cimetidine Specimens were taken from five 1181

1182 subjects in each group Controls were subjects in whom the only endoscopic abnormality was hiatus hernia Non-specific duodenitis patients were further subdivided into mild and severe categories

when associated with spontaneous bleeding and multiple erosions were classified as severe duodenitis Cimetidine treated cases were selected to include those in whom visual healing of inflammation had

any part of the bulb and distal second part of the duodenum In the ulcer-associated duodenitis, mild and severe non-specific duodenitis subjects two biopsies were taken from the duodenal bulb, one

2 1182 subjects in each group Controls were subjects in whom the only endoscopic abnormality was hiatus hernia Non-specific duodenitis patients were further subdivided into mild and severe categories on the basis of endoscopic appearances Those with mild hyperaemia and swelling of the mucosa with slight contact bleeding were classified as mild duodenitis Intense hyperaemia and mucosal swelling, when associated with spontaneous bleeding and multiple erosions were classified as severe duodenitis Cimetidine treated cases were selected to include those in whom visual healing of inflammation had taken place after six to twelve weeks of treatment with conventional dosage of cimetidine In the control, healed ulcer-associated and healed non-specific duodenitis subjects biopsies were taken from any part of the bulb and distal second part of the duodenum In the ulcer-associated duodenitis, mild and severe non-specific duodenitis subjects two biopsies were taken from the duodenal bulb, one from visually inflamed and the other from visudlly normal areas; in addition biopsies were taken from the second part of the duodenum Specimens were fixed immediately after collection by immersion in Clarke's fixative (75% ethyl alcohol, 25% glacial acetic acid) After four hours at room temperature the specimen was transferred to 75 % ethyl alcohol in water and stored Specimens were stained in bulk by the Feulgen reaction These were kept in 50 % ethyl alcohol for 10 min; in tap water for 10 min with three changes; in molar hydrochloric acid for 6 min at 60 C in a waterbath; in tap water for 10 min with three changes; in Schiff's reagent at room temperature for 45 min; in tap water for 10 min with three changes; in Giemsa's stain (diluted 1/10 in water) at room temperature for 30 min The specimen was then rinsed in water to remove excess stain MICRODISSECTION AND MEASUREMENTS The specimen was placed in tap water under a dissecting microscope With fine forceps and a cataract knife, a number of strips (length 2 mm) and one or two villi thick were cut free Individual villi were separated from these strips with a fine needle, care being taken to cut the villus at its base A single villus was then put in a drop of 45% acetic acid on a slide and covered with a cover-slip (Fig 1) Its length was then measured with an eye-piece graticule under a light microscope Its image was then projected on a screen from a fixed distance and the outline of the villus mapped out on paper Afterwards, the area of the villus was measured by using a planimeter The same villus was then pressed gently under the cover-slip with the tip of a needle to spread the cells Hasan, Ferguson Fig I Microdissected single villus Schiff ard Giemsa x 160 out uniformly (Fig 2) The nuclei of the epithelial cells (oval shaped, paler stained) could be readily distinguished from the nuclei of plasma cells, lymphocytes, fibroblasts, polymorphonuclear cells (Fig 3) The number of epithelial cells was then counted field by field until all the fields on the slide had been examined Total cell counts were performed on 10 villi from each specimen Statistical methods Kruskal and Wallis' test and Wilcoxon's sum of ranks test were applied for comparison of values for the various measurements Results Preparations of duodenal biopsies from the different sites were adequate in every case, although it was Fig 2 Squashed villus cells Schiff and Giemsa x 125

Villus measurements and cell counts in duodenitis F ki * E :^4 :- _ a ttp 0 -W I: Or -1 I 4I 9 * u IS 0 4 ;P P 4* Fig 3 Squashed villus cells Schiff and Giemsa x 500 often necessary to examine two

3 Villus measurements and cell counts in duodenitis F ki * E :^4 :- _ a ttp 0 -W I: Or -1 I 4I 9 * u IS 0 4 ;P P 4* Fig 3 Squashed villus cells Schiff and Giemsa x 500 often necessary to examine two biopsies from a site in order to obtain 10 villi suitable for measurement and epithelial cell count The villi showed considerable variation in shapes and sizes in biopsies from both normal and abnormal areas It was not possible to differentiate normal and inflamed areas on the basis of appearances of the villi Correlation between villus cell count, villus height and villus area The relations between measurements of villus size by cell count, projected villus area and villus height are here illustrated by analysis of results for three biopsies of duodenum from control subjects For purposes of simplicity, the term villus area will be used to denote the projected villus area in the results and discussion sections Total number of epithelial cells per villus was 2138 ± 90 in the duodenal bulb and 2118 ± 79 in the second part of the duodenum (these values and those given in the rest of the results section are all expressed as mean and standard error of the mean of the group concerned) Coefficient of correlation (r) was 0O66 between height of the villus and the number of epithelial cells per villus in control specimens from the duodenal bulb (p < 0001) The value of (r) between the area of villi and the number of epithelial cells per villus was 0-96 in the control duodenal bulb specimens (p < 0 001) Calculation of coefficient of correlation in the other groups of specimens between villus height and number of epithelial cells per villus and villus area and number of epithelial cells per villus gave results which were similar to those obtained in the control duodenal bulb specimens Thus in each group the epithelial cell population in a villus correlated well with villus area but the correlation with villus height was less satisfactory Figures 4, 5, and 6 shows the correlation between the three parameters, villus height, villus area and E 900 r 800 * 0 r= ,J 1* , Number of epitheliol cells per vil lus (from ttree control bulb speciimens ) Fig 4 Correlation betveen villus height and number of epithelial cells per villus 601 r (A E,, 40-0 o 30 a) 201~ 1J - 0, * *0/ 00-0/ * */0 * 0 II/9 r= 9 1T _- 11a,1o Number of epithelial cells per villus (From three control bulb specimens ) 3000 Fig 5 Correlation between villus area and number of epithelial cells per villus * 1183

11848Hasan, Ferguson number of epithelial cells per villus in three control duodenal bulb specimens 60 e7 50' E -=4 0-0 0 3 < 30 20 Fig 6 00 * * * : 0 /; 'a -06-1 i 300 400 500 600 700 800 900

4 11848Hasan, Ferguson number of epithelial cells per villus in three control duodenal bulb specimens 60 e7 50' E -= < Fig 6 00 * * * : 0 /; 'a i Height of viilus (,um J (from three control bulb specimens) Correlation between villus height and villus area Duodenum Site Duodenal bulb seadnd part Clinical C DU NSD DPC C DU NSD category 1000 Boo Visually normal Mean *tse i-mild duodenitis * p<oo 200 Severe duodenitis Fig 7 Measurement ofvillus height in pm C = controls, DU = ulcer-associated duodenitis, NSD = non-specific duodenitis, DUPC = healed ulcerassociated duodenitis after cimetidine treatment, NSDPC healed non-specific duodenitis after cimetidine = treatment Measurements of villi in duodenal biopsiesfrom control subjects andfrom patients with duodenitis Figure 7 shows the differences in villus height in the various categories of specimens studied The control group (C) had villus height 687 ± 20 ptm in the duodenal bulb and 720 ± 14,um in the second part of the duodenum Values for specimens from inflamed areas of ulcer-associated duodenitis (DU) and severe non-specific duodenitis (NSD) were 527 ± 19 and 552 ± 9,um respectively, both being significantly shorter (p < 0-01) than values in control duodenal bulb specimens Specimens from visually normal areas of bulb in ulcer-associated duodenitis (DU) and non-specific duodenitis (NSD) had villus height of 691 ± 12 and 702 ± 13 Mum respectively Areas of mild NSD had villus height of 691 ± 14,um These values were not significantly different from the controls Cimetidine healed ulcer-associated duodenitis (DUPC) and severe non-specific duodenitis (NSDPC) had villus height not significantly different from the controls, the values being 671 ± 13 and 684 ± 14,m respectively In the second part of the duodenum, villus height in biopsies from DU and NSD cases were similar to the control values, the figuresbeing722 ± 13,715 ± 14, and 720 ± 14,um respectively Similar differences emerged in comparison of villus area between the different groups (Fig 8) The control value for duodenal bulb 33 ± 2-1 (in cm2) was significantly greater than values for inflamed areas of DU (17-3 ± 1P6 cm2) and severe NSD (18-8 ± 09 cm2), the value for p being less than 0-01 in both cases Values for uninvolved bulb in DU and NSD, mild NSD, DUPC and NSDPC were 31P , , , 29-5 ± 1-9, and Site Duodenal bulb Duodenum second part Clinical category C DU NSD DIPNSDPC C DU NSD 40- E 30- (A 20 e 201 a i < 10 'Visually normal Mean *SE 10o' Mild duodenitis * P<0-01 i Severe duodenitis Fig 8 Measurements of villus area Abbreviations as in Fig 7

Villus measurements and cell counts in duodenitis 31 6 ± 1 8 (in cm2) respectively None of these values was significantly different from the control value In the second part of duodenum, values for

5 Villus measurements and cell counts in duodenitis 31 6 ± 1 8 (in cm2) respectively None of these values was significantly different from the control value In the second part of duodenum, values for control, DU and NSD were , 31P , and 30 4 ± l9 (in cm2) respectively, showing no significant difference from each other The number of epithelial cells per villus was 1318 ± 77 in the specimens from inflamed areas of DU and 1373 ± 40 in inflamed areas of NSD (Fig 9) These were significantly differerent (p < 0-01) Site Duodenal bulb Duodenum second part Clinrcal C DU NSD NSDPC C DU NSD In o V 1iooo Visually normal Mean ±SE n0 imild duodenitis * p<o0 E i Severe duodenitis Fig 9 Fig 7 Yillus epithelial cell counts Abbreviations as in from control values of duodenal bulb ( ) The number of epithelial cells per villus in visually normal areas of DU, NSD and inflamed areas of mild NSD were 2043 ± 74, , and 2106 ± 81 respectively None of these was significantly different from the control value No significant difference from controls was found in the numbers of epithelial cells per villus in DUPC and NSDPC, the values being 2045 ± 81 and 2024 ± 67 respectively In the second part of the duodenum, the number of epithelial cells per villus was 2078 ± 68 for DU, 2082 ± 82 for NSD These values were similar to the control value from second part of duodenum, this being 2118 ± 79 Discussion Microdissection of endoscopically obtained biopsies of the duodenal mucosa provides a simple and inexpensive method for performing measurements of 1185 intestinal mucosal villi and crypts We have now shown that although biopsies are small it is possible to separate individual villi and count the number of epithelial cells per villus We have confirmed the previous report3 that in disease, the reduction in the number of epithelial cells per villus is of greater magnitude than the corresponding reduction in villus height In circumstances where there is considerable variation in shape and size of villi, as in the normal duodenum, direct enumeration of the epithelial cell population of a single villus is one way to obtain precise information on the villus size However, since measurement of the villus area correlates very well with the total number of epithelial cells in the villus, villus area is likely to be a more practicable measurement for clinical investigation and research We have also confirmed that the villus height correlates less well, although significantly, with villus cell count Duodenitis may be defined as a clinical entity on the basis of radiological appearances of duodenum, by visual examination at endoscopy, and by changes at histological examination of duodenal biopsies On the basis of subjective assessment at endoscopy and histopathology, duodenitis has often been classified as mild or severe For the purposes of this study, we have chosen to define duodenitis on the basis of visual appearances at endoscopy (see methods) That this definition is valid has been demonstrated by objective measurements of villi and crypts, counting of mitotic figures in crypts4 and of intraepithelial and lamina propria inflammatory cellular infiltrate These studies have shown that increased cellular infiltrate and mucosal architecture abnormalities are found in biopsies from visually inflamed areas of duodenitis and are normal in visually normal areas Now by using direct enumeration of epithelial cells per villus we have confirmed our previous observation, based on measurement of villus height, that there is reduction in villus size in non-specific duodenitis as well as in ulcer-associated duodenitis and that the changes are identical in the two diseases4 The reduction in villus cell count was confined to visually abnormal areas of the duodenal bulb and was not present in the second part of the duodenum Furthermore, villus cell population returned to normal after healing of inflammation in non-specific or ulcer-associated duodenitis with cimetidine We are grateful to Professor Nicholas Wright for his advice on the method of epithelial cell counting and to Miss Frances Allan for her help with the setting up of the technique We acknowledge the help of the endoscopy theatre staff for their co-operation with the collection of the biopsies and of the Association

1186 of Commonwealth Universities who financed the study of Dr Mahmud Hasan References Clarke RM Control of intestinal epithelial replacement: lack of evidence for tissue-specific blood-borne factor

6 1186 of Commonwealth Universities who financed the study of Dr Mahmud Hasan References Clarke RM Control of intestinal epithelial replacement: lack of evidence for tissue-specific blood-borne factor Cell Tissue Kinet 1974;7: Ferguson A, Sutherland A, Macdonald TT, Allan F Technique for microdissection and measurement in biopsies of human small intestine J Clin Pathol 1977 ;30: Hasan, Ferguson Wright N, Zucoloto S, Bramble M, Record C Assessment of villus and crypt population sizes in human small intestinal biopsies Gut 1979;20:A 921 4Hasan M, Sircus W, Ferguson A Duodenal mucosal architecture in non-specific and ulcer-associated duodenitis Gut (in press) Requests for reprints to: Dr M Hasan, Gastro-Intestinal Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Scotland Reports and Bulletins prepared by the Association of Clinical Biochemists The following reports and bulletins are published by the Association of Clinical Biochemists They may be obtained from The Publishing Department, British Medical Journal (ACB Technical Bulletins), BMA House, Tavistock Square, London WC1H 9JR Overseas readers should remit by British Postal or Money Order SCIENTIFIC REVIEWS (price 1E00/f$200 each) 1 The assessment of thyroid function March 1971 FV FLYJNN and 1R HOBBS 2 Renal function tests suitable for clinical practice January 1972 FL MITCHELL, N VEALL, and RWE WATTS 3 Biochemical tests for the assessment of fetoplacental function May 1975 CE WILDE and RE OAKEY 4 Test of exocrine pancreatic function March 1977 AH GOWENLOCK 5 Assay of cholinesterase in clinical chemistry March 1979 ELSIE SILK, J KING, and MARY WHITIAKER TECHNICAL BULLETINS (price 1-00/$200 each) 22 Bilirubin standards and the determination of bilirubin by manual and technicon AutoAnalyzer methods January 1971 BARBARA BILLING, RUTH HASLAM, and N WALD 23 Interchangeable cells for spectrophotometers and fluorimeters September 1971 ss BROWN and AH GOWENLOCK 24 Simple tests to detect poisons March 1972 BW MEADE et al 25 Blood gas analysers May 1972 K DIXON 26 Kits for enzyme activity determination September 1972 SB ROSALKI and D TARLOW 27 Assessment of pumps suitable for incorporation into existing continuous flow analytical systems November 1972 A FLECK et al 28 Routine clinical measurements of transferrin in human serum September 1973 K DIXON 29 Control materials for clinical biochemistry (5th edition) September 1973 JF STEVENS 30 Notes on the quality of performance of serum cholesterol assays September 1973 ss BROWN 31 Determination of uric acid in blood and in urine July 1974 RWE WATTS 32 A survey of amino acid analysers readily available in the United Kingdom September 1974 JE CARLYLE and P PURKISS 33 Definitions of some words and terms used in automated analysis November 1974 A FLECK, R ROBINSON, SS BROWN, and JR HOBBS 34 Measurement of albumin in the sera of patients January 1975 LINDA SLATER, PM CARTER, and JR HOBBS 35 Investigation of the validity of temperature correction factors for serum aspartate and alanine transaminases March 1975 SB ROSALKI et al 36 Factors influencing the assay of creatinine November 1975 JGH COOK 37 A survey of enzyme reaction rate analysers readily availabl in the United Kingdom July 1977 RA SAUNDERS and RF BURNS 38 Transport of specimens for clinical chemistry analysis November 1977 P WILDING, JF ZILVA, and CE WILDE 39 A scheme for the evaluation of diagnostic kits May 1978 PH LLOYD 40 A practical guide to gamma-counting in radioimmunoassay January 1980 CE WILDE and D OTTEWELL 41 The use of biochemical tests in the diagnosis of disorders of calcium metabolism July 1980 ANGELA FAIRNEY

Gonadal infiltration in children with leukaemia and lymphoma

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