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UBC Theses and Dissertations

A histochemical analysis of the colonic epithelial glycoproteins from ulcerative colitus, Crohn's disease… Atkins, Elizabeth Ann 1987

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A H I S T O C H E M I C A L A N A L Y S I S O F T H E C O L O N I C E P I T H E L I A L G L Y C O P R O T E I N S F R O M U L C E R A T I V E COLITIS , C R O H N ' S D I S E A S E A N D D I V E R T I C U L A R D I S E A S E by E L I Z A B E T H A N N A T K I N S A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E O F M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Pathology We accept this thesis as conforming to the required standard T H E UNIVERSITY O F BRITISH C O L U M B I A Spring 1987 ° Elizabeth Ann Atkins, 1987 $ 2 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pathology The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V 6 T 1W5 Date: Spring 1987 A B S T R A C T The aim of the present study was to assess whether the changes in the epithelial glycoproteins seen in the mucosa adjacent to tumors are specific premalignant markers or secondary reactive phenomena. A secondary objective was to assess whether ulcerative colitis and Crohn's Disease could be distinguished from one another histochemically. The carbohydrate prosthetic groups from colonic epithelial glycoproteins were characterized histologically and histochemically from 17 cases of ulcerative colitis, 21 cases of Crohn's Disease and 19 cases of diverticular disease. Two histochemical parameters - the relative proportion of sulpho- and sialomucin and the side-chain substitution pattern of O-acetylated sialic acid - were assessed using a battery of seven histochemical techniques. Serial sections from each specimen were also evaluated morphologically, using hematoxylin and eosin. In addition, the patterns of O-acetylated side-chain sialic acid from the three inflammatory bowel diseases were compared to data previously acquired from the mucosa adjacent to colonic tumors. Results indicate that neither focal changes nor the predominance of sialomucins are specific to the mucosa adjacent to tumors. As well, changes in one histochemical parameter were independent of changes in the other parameter. No histochemical class of epithelial glycoproteins was specific to any of the inflammatory bowel diseases and, therefore, it was not possible to distinguish between ulcerative colitis and Crohn's Disease on the basis of the histochemical ii techniques used in the present study. It was also noted that the histochemical changes in ulcerative colitis, Crohn's Disease and diverticular specimens were not related to the degree of inflammation. Finally, as a group, Crohn's Disease specimens showed a loss of sulphomucin-sialomucin gradient along the length of the crypts. iii T A B L E O F C O N T E N T S Abs t rac t i i L i s t of Tables v i i L i s t of Figures ix L i s t of Slides x Abbreviat ions x i Acknowledgements x i i Figure 1 x i i i 1. Introduction 1 1.1. Objectives 1 1.2. General Macroscopic and Microscopic Features of N o r m a l Colon 1 1.3. Pathologic Descript ion of Diver t icular Disease, Ulcerat ive Colit is , Crohn's Disease, and Transi t ional Mucosa 2 1.3.1. Diver t icu la r Disease 2 1.3.2. Crohn 's Disease and Ulcerat ive Coli t is 3 1.3.3. Trans i t iona l Mucosa 6 1.4. Histochemical Character izat ion of N o r m a l Colonic Epi thel ia l Glycoproteins 8 1.5. Characterist ics of Colonic Epi the l ia l Glycoproteins in Tumors 11 1.6. Characteristics of Colonic Epi the l ia l Glycoproteins Adjacent to Tumors (Transit ional Mucosa) and Remote from Tumors 17 1.7. Characterist ics of Colonic Epi thel ia l Glycoproteins in Crohn's Disease .. 22 1.8. Characterist ics of Epi the l ia l Glycoproteins from Ulcera t ive Colitis .. 25 1.9. Characterist ics of Epi the l ia l Glycoproteins Associated wi th Benign Pathological Conditions 30 1.10. Premal ignant M a r k e r s 33 1.11. Incidence of Colonic Cancer in Ulcerat ive Col i t is , Crohn 's Disease and Diver t icular Disease 34 1.12. Thesis Rationale 35 2. Mate r ia l s and Methods 38 2.1. Tissue 38 2.2. Sta ining Procedures 40 2.2.1. His tochemical Procedures 43 2.2.1.1. Periodic acid oxidation (PA) 43 2.2.1.2. Borohydride reduction (Bh) 43 2.2.1.3. Saponification (KOH) 43 2.2.1.4. Phenylhydrazine Block Schiff (PAPS or PAPT) .... 43 2.2.1.5. Cationic Dye Methods with Alcian Blue 44 2.3. Sta ining of Ser ia l Sections 44 iv 2.3.1. Identification of Total Sialic Acid and Sulphate 44 2.3.1.1. KOHIAB1.0IPAPS 44 2.3.2. Identification of sulphate and side chain O-acetylated sialic acid 45 2.3.2.1. AB1.0IPAPS 45 2.3.2.2. PBT/KOHiABl.O/PAS 45 2.3.3. Identification of Total Sialic Acid, Sulphate, and Side-chain O-acetylated Sialic Acid 45 2.3.3.1. PBT/KOHIAB2.5/PAS 45 2.3.4. Identification of Side-chain O-acetylated Sialic Acids 45 2.3.4.1. PAPT/KOH/Bh/PAS 46 2.3.4.2. PATIKOHIBhIPAS 46 2.3.4.3. PBT/KOH/PAS 46 2.3.5. Procedure Controls 46 2.4. Assessment of Staining Patterns 48 2.4.1. Digitization 48 2.4.2. Classification of colonic epithelial glycoproteins 57 2.5. Morphologic Assessment 59 2.5.1. Morphologic Assessment of Focal Changes and Field Changes 59 2.5.2. Morphologic Criteria for Normal 60 2.5.2.1. Morphologic Assessment of Diverticular Disease Cases 61 2.6. Data Analysis 61 3. Results 62 3.1. Morphological Results 62 3.1.1. Relationship Between Inflammation and Mucosal Atrophy .... 62 3.2. Histochemical Results 62 3.2.1. Evaluation of the Relative Proportion of Sialomucin and Sulphomucin using the KOH/AB1.0/PAPS procedure .... 62 3.2.1.1. Assessment of the relative proportion of sialomucins and sulphomucins from the descending colon 63 3.2.1.2. Assessment of the relative proportion of sialomucins and sulphomucins from the ascending colon 65 3.2.1.3. Percentage and Number of Cases and Specimens Showing A Predominance of Sialomucins .... 65 3.2.2. Severity of Disease Versus Histochemical Class 69 3.2.3. Disease Versus Histochemical Class 69 3.2.4. Degree of Sulphation Versus Degree of Acetylation 72 3.2.5. Assessment of O-Acetylated Sialic Acids 72 4. Discussion 77 4.1. Histochemical Characteristics of Ulcerative Colitis, Crohn's Disease and Diverticular Disease 78 4.1.1. Assessment of the relative proportion of sialomucins and sulphomucins 78 v 4.1.2. Patterns of O-acetylated sialic acid 80 4.2. Differential Diagnosis of Ulcerative Colitis and Crohn's Disease ... 82 4.3. Premalignant Markers or Nonspecific Changes? 83 4.4. The Usefulness of the Differential Diagnostic Scheme 86 4.5. Future Considerations for a Project of this Nature 88 5. Conclusions 90 REFERENCES 91 SLIDES 101 vi LIST OF TABLES Page Table 1: Table 2: Macroscopic differences between ulcerative colitis and Crohn's Disease of the Large Intestine Microscopic differences between ulcerative colitis and Crohn's Disease of the Large Intestine Table 3: Comparison of normal hyperplastic colonic mucosa and nonpolypoid Table 4: Characteristics of the epithelial glycoproteins from tumors 14-16 Table 5: Table 6: Table 7: Table 8: Table 9: Table 10: Characteristics of the epithelial glycoproteins adjacent to tumors (transitional mucosa) Characteristics of the epithelial glycoproteins from areas remote from tumors Characteristics of the epithelial glycoproteins from Crohn's Disease specimens Characteristics of the epithelial glycoproteins from ulcerative colitis specimens Characteristics of the epithelial glycoproteins from benign pathological conditions Number of cases & specimens of ulcerative colitis, Crohn's Disease and diverticular disease from the descending and ascending colon 19-20 21 23-24 27-29 31-32 39 Table 11: Serial sections and the histochemical procedure applied corresponding 41-42 Table 12: Table 13: Expected staining results from histochemical procedures used for given structural elements Histologic and histochemical characteristics from ulcerative colitis cases 47 52 Table 14: Histologic and histochemical characteristics from Crohn's Disease cases 53-54 Table 15: Histologic and histochemical characteristics from diverticular disease cases 55-56 vii Table 16: Table 17: Table 18: Relative proportion of sialo- and sulphomucins 64 in the descending colon as assessed by the KOH/AB1.0/PAPS procedure Relative proportion of sialo- and sulphomucins 67 in the ascending colon as assessed by the KOH/AB1.0/PAPS procedure Percentage and number of cases and specimens 68 showing a predominance of sialomucins in the KOH/AB1.0/PAPS procedure Table 19: Assessment of the degree of inflammation versus histochemical class using the Chi Square test 70 Table 20: Table 21: Table 22: Table 23: Assessment of histochemical class verus disease 71 using the Chi Square test Assessment of the degree of sulphation versus 73 degree of side chain o-acetylation using the Spearman Rank Correlation Coefficient Analysis of O-acetylated side chain sialic acid 74 (by specimen) Assessment of O-acetylated sialic acid with 76 respect to disease using Chi Square analysis viii LIST OF FIGURES Page Figure 1: Quotation xiii Figure 2: N-acetyl neuraminic acid and its O-acetylated 10 side chain variants Figure 3: Differential diagnostic scheme for classifying 58 colonic epithelial glycoproteins ix LIST OF SLIDES 1. 'Scrambled' specimens 2. Crypts in which the bases and tops of the crypts are histochemically indistinguishable from each other (purple) 3. Crypts in which the bases and tops of the crypts histochemically distinct from each other 4. Focal change - PBT/KOH/AB1.07PAS 5. Focal change - PBT/KOH/AB2.5/PAS 6. Focal change - AB1.0VPAPS 7. Focal change - PAPT/KOH/Bh/PAS 8. Focal change - PAT/KOH/Bh/PAS 9. Focal change - PBT/KOH/PAS 10. Serial in which the focal change is not revealed by the KOH/AB1.0/PAPS procedure 11. Serial in which the focal change is not revealed by the H & E procedure 12. Normal pattern of O-acetylated sialic acid - PAPT/KOH/Bh/PAS procedure 13. Normal pattern of O-acetylated sialic acid - P B T / K O H / P A S procedure 14. Moderate field change - PAPT/KOH/Bh/PAS 15. Moderate field change - P B T / K O H / P A S 16. Severe field change - PBT/KOH/PAS 17. Crypts showing a predominance of sialomucins. x A B B R E V I A T I O N S CO - Sialic acid without O-acetyl side chain substituents C7 - Sialic acid with O-acetyl substituents at position C7 C8 - Sialic acid with O-acetyl substituents at position C8 C9 - Sialic acid with O-acetyl substituents at position C9 C4 - Sialic acid with O-acetyl substituents at position C4 xi A C K N O W L E D G E M E N T S I would like to thank my supervisor, Dr. P .E . Reid for his constant enthusiasm and patience throughout this project and for his most helpful criticism of this thesis. I am also very grateful to the members of my supervisory committee, Drs. W.L . Dunn, W.K. Ovalle and D.A. Owen for their encouragement and critical review of thesis drafts. In particular, I would like to thank Dr. D.A. Owen for his help in assessing tissue morphology and Dr. D.C. Walker for his much appreciated aid in photomicroscopy. The technical expertise of L . Trueman, B. Muelchen and C. Ramey is sincerely appreciated. This thesis was completed while living in Calgary; such completion would not have been possible without a great deal of help from my family. To my husband, David, for his unfailing faith in my ability to finish this project and for the more than a few sacrifices that were necessary along the way, I am especially grateful. I would also like to thank my son, Ryan, and daughter, Ainsley, who provided a source of welcome, albeit constant, diversion. I am extremely grateful to my parents, Dr. and Mrs. Ainsley Atkins for their constant moral support, care of my children, and assistance on syntax and medical terminology. I would like to acknowledge with thanks the encouragement provided by my husband's family. Finally, I would like to thank the University of British Columbia for the award of a University Summer Graduate Fellowship. xii F I G U R E 1 They are ill discoverers that think there is no land, when they can see nothing but sea. Francis Bacon, Advancement of Learning, Bk.I, vii. 5 xiii 1. INTRODUCTION 1.1. OBJECTIVES The aim of this project was to characterize the carbohydrate prosthetic groups of the epithelial glycoproteins in cases of ulcerative colitis, Crohn's Disease, and diverticular disease. The overall objectives were to: 1. determine whether the changes seen in the mucosa close to tumors are primary premalignant phenomenon, or alternatively, an effect secondary to an established disease process, and; 2. ascertain whether ulcerative colitis and Crohn's Disease can be distinguished from each other by histochemical methods. 1.2. GENERAL MACROSCOPIC AND MICROSCOPIC FEATURES OF NORMAL COLON The large intestine extends from the lower aspect of the cecum to the anus (Morson and Dawson, 1979). Anatomic landmarks provide the basis for further subdivisions of the colon. The cecum marks the most proximal portion of the colon. The proximal or ascending colon extends from the cecum to the hepatic flexure, while the transverse extends from the hepatic to the splenic flexure. The descending portion extends from the splenic flexure to "a point where the colon crosses the pelvis" (Morson and Dawson, 1979), and the sigmoid from that point 1 2 to the retrosigmoid junction. As in all other areas of the digestive tract, the normal colon consists of four layers: a) the mucosa, b) submucosa, c) muscularis propria, and d) the serosa. The mucosa possesses an epithelial component which consists of straight, perpendicular crypts which lie parallel and adjacent to one another and, in the normal colon, do not exhibit any branching (Filipe & Branfoot, 1976; Chambers & Morson, 1980). The crypts are lined almost exclusively by goblet cells, except at the very bases where undifferentiated cells, A P U D and enterochromaffin cells co-exist. The surface epithelium, situated at the opening of the crypts, contains predominantly absorptive cells, with the occasional goblet cell interspersed (Filipe & Branfoot, 1976). 1.3. PATHOLOGIC DESCRIPTION OF DIVERTICULAR DISEASE, ULCERATIVE COLITIS, CROHN'S DISEASE, AND TRANSITIONAL MUCOSA 1.3.1. Diverticular Disease Diverticular disease, which arises from a defect in the muscle, (Morson & Dawson, 1979) gives rise to the inflammatory condition known as diverticulitis. The diverticulum itself, consists of an inpouching of mucous membrane and muscularis mucosa through and beyond the circular muscle layers of the muscularis propria. The diverticulum, which invaginates at inherently weak spots in the circular muscle layer, ie. where major blood vessels cross, remains 3 surrounded by a thin layer of longitudinal muscle and is situated in the pericolic fat region. The configuration of the diverticulum is such that its narrow neck lies proximal to the luminal aspect of the colon and the pouch itself bulges into the percolic fat. As a consequence, fecal matter becomes entrapped in the pouch, and subsequently develops into a fecolith. The fecolith, in turn, constantly abrades the mucosal surface and as a consequence, chronic, low-grade inflammation arises in the area around the diverticulum. 1.3.2. Crohn's Disease and Ulcerative Colitis The macroscopic and microscopic features of ulcerative colitis and Crohn's Disease are shown in Tables 1 and 2, respectively. Although the features cited in Tables 1 and 2 would appear relatively distinct, in some cases specimens from suspected ulcerative colitis and Crohn's disease patients show a spectrum of changes such that examination of sections stained with hematoxylin and eosin does not provide a definitive diagnosis (Hellstrom and Fisher, 1967; Filipe & Dawson, 1970; Lev, 1970; Filipe, 1979; Kirsner and Shorter, 1982). 4 T A B L E 1 MACROSCOPIC D I F F E R E N C E S B E T W E E N U L C E R A T I V E COLITIS A N D CROHN'S DISEASE OF T H E L A R G E INTESTINE (Morson & Dawson, 1979) Ulcerative Colitis 1. Disease in continuity. 2. Rectum almost always involved. 3. Terminal ileum involved in 10%. 4. Granular and ulcerated mucosa. No fissuring. 5. Often intensely vascular. 6. Normal serosa (except in acute fulminating colitis. 7. Muscular shortening of colon; fibrous structures very rare. 8. Never spontaneous fistulae. 9. Inflammatory polyposis common and extensive. 10. Anal lesions in less than 25%; acute fissures, excoriation and rectovaginal fistula. Crohn's Disease 1. Disease discontinuous. 2. Rectum normal in 50%. 3. Terminal ileum involved in 30%. 4. Discretely ulcerated mucosa; cobblestone appearance; fissuring. 5. Vascularity seldom pronounced. 6. Serositis common. 7. Shortening due to fibrosis; fibrous structure common. 8. Enterocutaneous or intestinal fistulae in 10%. 9. Inflammatory polyposis less prominant and less extensive. 10. Anal lesions in 75%; anal fistulae (often multiple); anal ulceration or chronic fissure; oedematous anal tags. 5 T A B L E 2 MICROSCOPIC D I F F E R E N C E S B E T W E E N U L C E R A T I V E COLITIS A N D CROHN'S DISEASE OF T H E L A R G E INTESTINE (Morson & Dawson, 1979) Ulcerative Colitis 1. Mucosal and submucosal inflammation (except in acute fulminating colitis). 2. Width of submucosa normal or reduced. 3. Often intense vascularity. Little edema. 4. Focal lymphoid hyperplasia restricted to the mucosa and superficial submucosa. 5. 'Crypt abscesses' very common. 6. Mucous secretion grossly impaired. 7. Paneth cell metaplasia common. 8. Sarcoid-type granulomas absent from bowel and lymph nodes. 9. 'Fissuring' absent. 10. Anal lesions - nonspecific inflammation. Crohn's Disease 1. Transmural inflammation. 2. Width of submucosa normal or increased. 3. Vascularity seldom prominent. Edema marked. 4. Focal lymphoid hyperplasia in mucosa, submucosa, serosa and pericolic fat tissues. 5. Crypt abscesses fewer in number. 6. Mucus secretion slightly impaired. 7. Paneth cell metaplasia rare. 8. Sarcoid-type granulomas in 60-70% in bowel and lymph nodes. 9. 'Fissuring' very common. 10. Anal lesions; sarcoid foci often present. 6 1.3.3. Transitional Mucosa The 'transitional mucosa' is a term coined by Filipe (1969) to describe "a zone of transition from frank carcinoma to histochemically normal mucosa, where abnormal secretion was found in the absence of any morphologic change" (Filipe, 1976). Abnormal secretion, in this case, has been defined as a predominance of sialomucins with a reduction or absence of sulphated material. Subsequent studies (Dawson & Filipe, 1976; Filipe & Branfoot, 1976; Filipe, 1979; Rhatigan & Saffos, 1979; Greaves et al., 1980; Lev et al., 1985). revealed that the mucosa, rather than being absolutely morphologically normal, possessed some atypical features. The data shown in Table 3, taken from Saffos & Rhatigan (1977) represents a composite definition of the light microscopic features of normal mucosa and that adjacent to carcinoma. 7 T A B L E 3 COMPARISON O F N O R M A L A N D NONPOLYPOID H Y P E R P L A S T I C COLONIC M U C O S A (Saffos & Rhatigan, 1977) Normal Mucosa  Surface Epithelium More absorptive cells than goblet cells. Nonpolypoid Mucosa Adjacent to Carcinomas More goblet cells than absorptive cells. Crypts 0.5 - 0.8mm in length. Straight and parallel except for slight anthemal pattern. Branching seldom or never seen. Epithelium is mainly goblet cells except at base of crypts where A P U D and undifferentiated cells are present. 0.8 - 2.0mm in length. Many crypts assume tortuous configuration and dilated crypts are seen. Branching frequently seen. Epithelium mainly goblet cells but strikingly increased in numbers. Mitoses confined to lower half of crypt. Mitoses confined to lower half of elongated crypt. 8 1.4. H I S T O C H E M I C A L C H A R A C T E R I Z A T I O N O F N O R M A L C O L O N I C E P I T H E L I A L G L Y C O P R O T E I N S Chemical studies (Filipe, 1979; Reid et al., 1980; 1984a) of human colonic epithelial glycoproteins demonstrate that the carbohydrate prosthetic groups contain fucose, galactose, small quantities of mannose, N-acetyl glucosamine, N-acetyl galactosamine and sialic acid. It has been demonstrated that the majority of the sialic acids possess O-acyl substituents at position C4, or in the polyhydroxyl side chain (Reid et al., 1973; 1975; 1980) (Figure 2). In normal colon, sialic acids are predominantly substituted at position C7 and/or C8 (Reid et al., 1978b). Structural studies indicate that the carbohydrate prosthetic groups are composed of a complex mixture of often highly branched acidic and neutral oligosaccharide chains of different lengths linked O-glycosidically through N-acetyl galactosamine to serine or threonine residues in the polypeptide chain (Filipe & Branfoot, 1976). O-sulphate ester may also be present but its location has not been established. The amino acid composition of the polypeptides is typical of that of mucin-type glycoproteins. Histochemical studies indicate that the colonic epithelial glycoproteins are predominantly sulphomucins (sulphosialoglycoproteins) (Goldman & Ming, 1968; Filipe, 1969, 1979; Filipe & Branfoot, 1976; Culling et al, 1981; Lev et al., 1985, Habib et al., 1986) However, the type of mucin secreted in the normal colon varies according to both the level in the crypt, and the anatomic region of the colon (Gad, 1969a; Filipe & Branfoot, 1976; Sheahan & Jervis, 1976; Filipe 1969, 1979). In the descending colon, the lower half of the crypt contains 9 predominantly sulphomucin, whilst the upper portion and surface epithelium contains variable proportions of sulphomucins and sialomucins which exist together in the same goblet cell, or in different cells. According to Filipe & Dawson (1970) and Filipe (1979) there is an increasing gradient of neutral mucins from the lower to the upper crypt. Subbuswamy (1971), however, maintains that very little neutral mucosubstances exist in normal specimens. In the ascending colon the predominant mucins in the lower third of the crypts are sialomucins while the predominant mucins in the remaining two-thirds are sulphomucins (Filipe & Branfoot, 1976; Sheahan & Jervis, 1976; Filipe, 1979). According to Culling et al. (1979), O-acetyl substituted side-chain sialic acids are evenly distributed along both the length of the crypt and the length of the colon. Filipe (1979), however, maintains that there are fewer O-acetylated variants in the right colon than in the left. Developmental studies have shown that sulphate distribution in colonic epithelium changes over time (Lev, 1968; 1970; Lev & Orlic, 1974). In the human fetus, little suphate is found in colonic goblet cell before 12 weeks (Lev, 1970). A progressive increase in sulphomucin staining from the right to left colon has been noted in the 12 week human fetus (Lev & Orlic, 1974). By early infancy, an adult pattern of sulphomucin distribution is established such that the transverse colon stains most intensely for sulphomucins while the left colon stains the least and the right colon stains intermediately. Neutral mucins and sialomucins, however, do not vary significantly with age (Lev & Orlic, 1974). 10 F I G U R E 2 N - A C E T Y L - N E U R A M I N I C ACID A N D ITS O - A C E T Y L A T E D SIDE CHAIN S U B S T I T U E N T S OH H I ho s u l k - i i ck i ( \ ' - .Kc t y ! . K ' m a m m k - .K id ) m o k v u k ' . •o -o -OH -OH -O--OH C H . O H C H . O H -O O H O H CH.O R O — O H -O R C H , O H R = Ace t vi 11 1.5. C H A R A C T E R I S T I C S O F C O L O N I C E P I T H E L I A L G L Y C O P R O T E I N S IN T U M O R S As shown in Table 4, the epithelial glycoproteins of colonic tumors are distinctly different from normal in many respects. Apart from studies which report a significant reduction or absence of mucins (Filipe, 1969, 1979; Lev, 1970; Subbuswamy, 1971a; Reid et al., 1981; Lev et al, 1985; Reid et al., 1985a), in colonic tumors, several histochemical alterations are evident. In general the epithelial glycoproteins of colonic and rectal adenocarcinomas contain a smaller proportion than normal of sialic acids with O-acetyl substituents at position C4 and in the polyhydroxyl side chain (Korhonen et al., 1971; Culling et al., 1977; Filipe, 1979; Montero & Sergura, 1980; Reid et al., 1980, 1984a, 1985a). Consequently, the sialic acids of tumor glycoproteins are more neuraminidase (sialidase) labile than normal (Korhonen et al., 1971; Dawson et al., 1978; Reid et al., 1980; Lev et al., 1985). A significant reduction in the molar O-acyl/sialic acid ratio of tumor glycoproteins has been reported (Reid et al., 1984a), providing chemical evidence that a decrease in O-acetylated sialic acid is a characteristic feature of colonic tumor glycoproteins. Although the relative proportion of O-acetylated sialic acid is reduced in many colonic tumors, acetyl esters are still present in many colonic tumor glycoproteins as reflected by their positive K O H / P A S effect (Culling et al, (1975). Gad (1969b) reported that the higher the degree of differentiation, the more 12 closely the mucosubstances resemble normal. Culling et al. (1977) and Montero & Segura (1980) confirmed this finding, reporting that a relationship exists between the degree of differentiation and the pattern of O-acetyl substitution, with the most poorly differentiated tumors most often exhibiting the loss of C8 substituted sialic acid. In addition to decreases in O-acetylated sialic acid, decreases in the amount of sulphomucins present have also been reported (Goldman & Ming, 1968; Subbuswamy, 1971a; Fenger & Filipe, 1981; Listinsky & Riddell, 1981; Reid et al., 1985a). However, it is generally not the case that all tumors in a particular study exhibit decreases in the amount of sulphomucins. More frequently, no uniform pattern of staining was demonstrated (Goldman & Ming, 1968; Subbuswamy, 1971a; Montero & Segura, 1980; Reid et al., 1981, 1985a). Thus, in some cases the tumors are predominantly sulphated while in others, they lack sulphate or contain both sulphated and non-sulphated glycoproteins. L E G E N D T O T A B L E S 4,5,6,7,8, A N D 9 Abbreviations Sialo: all or predominantly sialomucin Sulpho: all or predominantly sulphomucin PNA: Peanut agglutinin (specific for Gal-GalNAc) FPC: familial polyposis coli N A N A : N-acetyl neuraminic acid (sialic acid) gal: galactose rue: fucose GlcNAc: N-acetyl glucosamine GalNAc: N-acetyl galactosamine Methods 1. HID/AB2.5 2. PBT/KOH/PAS 3. PAPT/KOH/Bh/PAS 4. PAS 5. PAPS 6. P A T / K O H / P A S S. FITC-labelled lectin 9. PBT/KOH/neuraminidase/PAS 10. AB2.5/PAS 11. Diastase-PAS 12. HID 13. AB1.0 14. AB2.5 15. Toluidine blue pH 3.0 16. Toluidine blue pH 5.0 17. AB1.0/PAS 18. Alcian Yellow 14 T A B L E 4 CHARACTERISTICS OF COLONIC E P I T H E L I A L G L Y C O P R O T E I N S F R O M T U M O R S Findings Comments N A N A less substituted in side chain and C4; significant reduction in OAC-NANA ratio Method Reference 1,2,6 Reid et al., 1984a; Reid et al., 1980 Usually no secretory elements Adenocarcinomas Filipe, 1969 In colorectal type mucosa of anal canal shift from sulpho to sialo with predominantly N-acyl derivative Carcinoma near 1,2,11 dentate line Fenger & Filipe, 1981 Only tumors arising 2,4 Culling et al., 1975 from cecum, colon or rectum give positive K O H / P A S reaction Tumors from noncolonic sites & their metastases K O H / P A S negative decreased O-acetyl 31 adenocarcinomas 4,6 Culling et al., 1977 N A N A in poorly, moderately differentiated tumors Less resistant to 15 adenocarcinomas 2,6 Reid et al., 1980 neuraminidase digestion Decreased mucin 7/8 colonic carcinomas Lev et al., 1985 15 PNA labelled goblet cell mucin in 100% cases 21 cases colonic cancer Boland et al., 1982 64% cases sialo 25 cases colonic 1,2,6 adenocarcinoma Montero & Segura, 1980 52% cases sialo 33 cases rectal adenocarcinomas 44% colonic & 45% rectal adenocarcinomas decrease of O-acyl N A N A Abnormal pattern more frequent in less differentiated tumors 12/15 lesions associated with less than 15% sulpho Decreased neuraminidase resistance as compared to normal Increased proportion sialo 36% Sulpho; 32% mixed sulpho/sialo; 28% sialo; 4% no mucin Colonic/rectal adenocarcinomas IS cases colonic 1,2,4,9 carcinomas 24 cases of F P C 1,4,12 25 cases colonic 1,2,3,6 tumor Listinsky & Riddell, 1981 Dawson et al., 1978 Filipe et al., 1980 Reid et al., 1985a Sialo decreased [H3]-galactose uptake Decreased sulpho in over half of cases Mucosubstances partially labile in 4/9 and totally labile in 5/9 cases Dysplastic crypts 1,4 31 colonic 13-16 carcinomas Mild sialidase digest 1,2,6 without colon Dawson & Filipe, 1980 Goldman & Ming, 1968 Reid et al., 1984a Increased differentiation of colonic carcinomas, more closely mucins resemble normal All decreased mucin: 3 no mucin; 1/29 neutral mucins only; 12/29 no sulpho; 16/29 sialo and sulpho Gad, 1969b 32 specimens from 10,17 Subbuswamy, 1971 rectum and colon Sialo Dysplastic rat colon 1 crypts Dawson & Filipe, 1980 1.6. C H A R A C T E R I S T I C S O F C O L O N I C E P I T H E L I A L G L Y C O P R O T E I N S A D J A C E N T T O T U M O R S ( T R A N S I T I O N A L M U C O S A ) A N D R E M O T E F R O M T U M O R S The definition of 'transitional mucosa' was initially based, not on its morphological features, but rather, on its abnormal mucin pattern. Therefore, it is not surprising that, as illustrated in Tables 5 and 6, most investigators have found that the glycoproteins in the mucosa adjacent to tumors are almost exclusively non-sulphated (Filipe, 1969, 1970, 1972, 1979; Subbuswamy, 1971a; Filipe & Branfoot, 1974, 1976: Dawson & Filipe, 1976, 1983; Dawson et al, 1978; Isaacson & Attwood, 1979; Greaves et al., 1980; Montero & Segura, 1980. Autoradiographic studies have confirmed the histochemical results; [35S] sulphate uptake is reduced or absent in the transitional mucosa crypts (Filipe, 1970; Dawson & Filipe, 1983). Other changes in the glycoproteins adjacent to colonic tumors include a decreased neuraminidase resistance of their sialic acids (Filipe & Cooke, 1974; Filipe & Branfoot, 1976; Dawson et al., 1978; Lev et al., 1985), altered lectin binding affinities (Boland et al., 1982), a variable content of side-chain O-acetylated sialic acids (Montero & Segura, 1980), and increased levels of total hexosamines and sialic acid (Filipe & Cook, 1974). In addition, Greaves et al. (1980) noted that in transitional mucosa associated with adenocarcinomas classified as Duke's B, an inverse relationship existed between the length of the transitional mucosa and the length of survival. Filipe & Branfoot (1974) have also noted that the "increase in sialomucins is directly related to the extent of invasion by a carcinoma, 18 according to Duke's classification". Transitional mucosal features have also been identified remote from tumors (Filipe & Branfoot, 1974; Filipe et al., 1980), in the flat mucosa between polyps (Filipe et al., 1980), in dysplastic crypts (Ehsanullah et al., 1982b), and in resection margins (Listinsky & Riddell, 1981). Furthermore, Wood et al. (1985) has noted that a greater proportion of patients whose resection margins showed a marked increase in sialomucins at the time of initial surgery, subsequently developed a local recurrence as opposed to those patients whose resection margins contained predominantly sulphomucins. 19 T A B L E 5 CHARACTERISTICS O F COLONIC E P I T H E L I A L G L Y C O P R O T E I N S A D J A C E N T T O T U M O R S - TRANSITIONAL M U C O S A ('TM') Findings Comments Method Reference Sialo Gradual decrease sulphated material T M present in 93/95 adenocarcinoma; increase length of T M , decrease survival 10 surgical specimens of T M According to Duke's classification Filipe & Branfoot, 1976 Greaves et al., 1980; Dawson & Filipe, 1980 Filipe, 1969 Greaves et al., 1980. Sulpho-sialo gradient is lost in crypts T M binds PNA Variable content of O-acetylated N A N A ; 67% specimens from colonic adenocarcinoma and 37% from rectal adenocarcinoma sialo. T M adjacent to 21 cases colonic & 24 cases rectal adenocarcinoma. 1, 4, 5 8 1, 2, 6 Lev et al, 1985 Boland et al., 1982 Montero & Segura, 1980 80/80 sialo 80 specimens adjacent to adenocarcinoma Isaacson & Attwood, 1979 8/9 cases sialo Cases adjacent to noncolorectal carcinomas Isaacson & Attwood, 1979 Decreased neuriminidase resistance 18 cases T M 1, 2, 4, 9 Dawson et al, 1978 Sialo, decrease or absence of sulpho Loss of sulpho-sialo gradient 25 specimens No difference between 'diseased' (from nonmalignant diseases) and T M specimens noted Filipe & Branfoot, 1974 Lev et al., 1985 21 T A B L E 6 CHARACTERISTICS O F COLONIC E P I T H E L I A L G L Y C O P R O T E I N S F R O M A R E A S R E M O T E F R O M T U M O R S Findings Comments Method Reference 8/10 cases sulpho, 2/10 cases sialo "Normal" remote from tumors Dawson & Filipe, 1976 16/130 proximal and 21/130 distal resection margins sialo; 3/4 patients who later developed local recurrence had sialo at resection margin Wood et al., 1985 4/15 cases less than 25% sulpho Increased proportion sialo Resection margins Flat mucosa between polyps from patients with F P C Listinsky & Riddell, 1981 Filipe et al., 1980 Increased sialo 11/25 cases remote 1 from tumors Filipe & Branfoot, 1974 22 1.7. C H A R A C T E R I S T I C S O F C O L O N I C E P I T H E L I A L G L Y C O P R O T E I N S IN C R O H N ' S D I S E A S E Investigations of the mucins in Crohn's Disease of the colon (Table 7) do not reveal any uniform pattern of change. Some investigators have found sulphomucins to be the predominant mucins for all (Habib et al., 1986), or some (Filipe, 1969) of the specimens examined, while others (Ehsanullah et al., 1982b; Franzin et al., 1983a) have found sialomucins to be the predominant mucin present in all or the majority of specimens. Studies of Crohn's Disease specimens have also shown there to be a reduction in O-acetylated side-chain sialic acid (Culling et al., 1979) and that these changes are related to the severity of the disease. No decrease in the amount of mucin secreted was noted by Hellstrom & Fisher (1967), Filipe (1969, 1979) or Filipe & Dawson (1970), and in fact, marked mucin secretion has been observed in rectal biopsies from some patients (Dawson, 1972). Other investigations have revealed that sulphatases and mucin degrading glycosidases from the faeces of Crohn's Disease patients have a similar activity to normal (Rhodes et al., 1985a, 1985b). Further, Rhodes et al., (1985c) have shown that the epithelial glycoproteins in rectal biopsies from these patients are more resistant to the desialating effects of fecal extracts than glycoproteins from normal individuals. Shamsuddin & Phillips (1981) also have reported that a large proportion of Crohn's Disease specimens share the same histologic features as transitional mucosa. 23 T A B L E 7 CHARACTERISTICS OF COLONIC E P I T H E L I A L G L Y C O P R O T E I N S F R O M CROHN'S DISEASE (CD) Findings Comments Method Reference Normal, sialo or intermediate mucin pattern No decrease in amount mucin secreted More resistant to disialating effects from normal fecal extracts than controls or U C specimens Normal fecal sulphatase activity Mucus degrading glycosidases have similar fecal activities to normal Sulpho 20/42 - sialo 8/20 - sialo Moderate to marked mucus secretion; pattern is normal or sialo 9 cases rectal CD; 8 cases colonic CD 29 cases 18 rectal biopsies CD 17 cases 19 cases 10 cases Rectal biopsies 1,10 1,4,10 1 1,4 Filipe, 1969 Filipe & Dawson, 1970 Rhodes et al., 1985c Rhodes et al., 1985b Rhodes et al., 1985a Habib et al., 1986 Ehsanullah et al., 1982b Franzin et al., 1983a Dawson, 1972 24 Reduction in 14 cases CD 2,6 sidechain O-acetylated N A N A as compared to normal ; these changes related to severity of disease Culling et al., 1979. 25 1.8. C H A R A C T E R I S T I C S O F E P I T H E L I A L G L Y C O P R O T E I N S F R O M U L C E R A T I V E COLITIS Studies of the epithelial glycoproteins from patients with ulcerative colitis (Table 8) show a reduction in the proportion of C7 and C8 substituted sialic acid (Hellstrom & Fisher, 1967; Culling et al, 1979; Reid et al, 1981, 1984a) that was larger with increased severity of disease; a decrease in the amount of mucin secreted which was related to the intensity of inflammation (Filipe, 1969, 1970, 1979, Dawson, 1972; Franzin et al, 1983a); a decrease in the molar galactose-fucose and GlcNAc-GalNac ratios in the descending colon as compared to normal (Reid et al, 1984); a decrease in the molar fucose-NANA ratio in the chronic active subgroup of the descending colon as compared to normal (Reid et al, 1984); a crypt cell production rate that was 45% faster in the relapse groups than in the remission groups, which in turn was faster than normal (Allan et al, 1985), and a significant reduction of neutral mucins in all regions of the colon in cases showing severe histologic changes (Greco et al, 1967). With respect to the relative proportion of sialomucins and sulphomucins, ulcerative colitis specimens, like Crohn's Disease specimens, do not appear to possess any uniform pattern of change (Ehsanullah et al, 1982b). However, it has been reported that when abnormal patterns do occur, the abnormal secretion is related to the activity, duration, and extent of the disease (Ehsanullah et al, 1982b). Habib et al (1986) reported that in ulcerative colitis cases which were uncomplicated by dysplasia, the majority of specimens possessed a predominance of sulphomucins, while the majority with dysplastic changes possessed sialomucins. 26 In contrast, Franzin et al. (1983a) reported that uncomplicated cases of ulcerative colitis possessed a predominance of sialomucins. As a rule, sialomucins appear to predominate in cases of ulcerative colitis associated with dysplasia (Boland et al., 1984; Filipe et al., 1985; Habib et al, 1986) or carcinoma (Filipe, 1979). However, Jass et al., (1986) who could not distinquish between uncomplicated ulcerative colitis and ulcerative colitis complicated by carcinoma on the basis of their respective mucin patterns, found that dysplastic foci secreted either sulphomucins, sialomucins or a mixture of the two. 27 T A B L E 8 CHARACTERISTICS O F COLONIC E P I T H E L I A L G L Y C O P R O T E I N S F R O M U L C E R A T I V E COLITIS (UC) Findings Decreased C7C8 side chain N A N A Comments Method Increase severity, 2,6 increase reduction of side chain N A N A References Culling et al, 1979 N A N A less substituted in side chain and C4 U C of the 1,2,6 descending colon Reid et al, 1984a Molar gal-fuc; GlcNAc-GalNAc ratio smaller than normal Descending colon Molar fuc-NANA and gal-NANA ratios smaller than normal Secretory activity absent in surface epith, predominant mucin present is sialo Descending colon: chronic active subgroup 9/15 cases Filipe, 1969 Reduction in amount of mucin, related to degree of inflammation 62 biopsies 1,4,10 Filipe & Dawson, 1970 CCPR 45% faster in relapse group than in remission group which was 19% faster than normal Ulcerative proctocolitis Allan et al, 1985 Normal fecal suphatase activity 39 patients Rhodes et al, 1985b 28 Mucus degrading glycosidases, including neuraminidase, had normal fecal activities 35 patients Rhodes et al, 1985a No association between H L A phenotype and presence or absence or U C 56 patients Cottone et al, 1985 Large quantities neutral mucin, normally absent from sigmoid Those showing mild histologic change in sigmoid 10,14,18 Greco et al, 1967 Significant reduction of neutral mucins Severe histologic change in all regions of the colon 4/8 cases - sialo even in absence of inflammation or dysplasia 8 cases treated with total colectomy - all developed cancer or precancer Filipe et al, 1985 4/8 cases - normal mucins, no dysplasia 1/20 - sialo 10/20 - sialo U C uncomplicated by dysplasia U C complicated by dysplasia Habib et al, 1986 13/18 abnormal goblet cell mucins -dysplasia later developed in 6 and carcinoma in 1 Rectal biopsies from 18 patients with stable U C Boland et al, 1984 35/38 biopsies -sialo U C with dysplasia 1,4 Ehsanullah et al, 1985 54/120 sialo; 19/120 - mixed; 47/120 -normal U C Ehsanullah et al, 1982b Uninvolved mucosa -irregular mucin distribution; active U C - mucus depletion, sialo; quiescent U C - sialo. No difference between 11 U C patients with cancer and 11 U C patients without cancer 29 40 cases U C 1 Franzin et al., 1983a Colonoscopic biopsies 1, P N A Jass et al., 1986 i 30 1.9. C H A R A C T E R I S T I C S O F E P I T H E L I A L G L Y C O P R O T E I N S A S S O C I A T E D W I T H B E N I G N P A T H O L O G I C A L C O N D I T I O N S Table 9 describes the mucin characteristics of a number of benign pathological conditions, including diverticular disease. In general, the only histochemical parameter investigated was the relative proportion of sialo- and sulphomucins. As is evident, no uniformly consistent pattern was demonstrated. 31 T A B L E 9 CHARACTERISTICS OF E P I T H E L I A L G L Y C O P R O T E I N S F R O M BENIGN P A T H O L O G I C A L CONDITIONS Findings Comments Method Reference 3/5 lesions less than Endometriosis 25% sulpho 16/27 cases sialo 4/4 cases sialo Solitary ulcer syndrome Colostomy specimens Listinsky & Riddell, 1981 Franzin et al., 1981 Isaacson & Attwood, 1979 14/21 cases sialo 76/78 normal mucin pattern Irregular mucin pattern with abnormal morphology 2/5 sialo & abnormal morphology 3/5 irregular mucins & abnormal morphology 2/5 sialo & abnormal morphology 1/5 sulpho & normal morphology 2/5 sulpho & abnormal morphology 64/65 cases normal mucins Solitary ulcer syndrome Nonspecific proctitis 40 juvenile & inflammatory polyps 5 cases ischemic colitis Polypectomy scars 1, 4 Ehsanullah et al., 1982a Franzin et al., 1983a Nonspecific proctitis Ehsanullah et al., 1982b Sulpho Sialo & abnormal morphology Metaplastic mucosa Pedunculated hyperplastic polyps Franzin et al., 1983a 32 12/45 sialo 7/28 sialo Increase in sialo in mid & lower crypts 17/17 normal mucin pattern Sessile hyperplastic polyps Anastomosis sites Ureterosigmoid-ostomy sites of 9 patients Villous adenomas and adenomatous polyps Histologic features of 8 cases diverticulosis T M Sialo Normal mucin pattern 12 benign tumors 12 cases diverticulitis Sunter et al., 1985 Marcheggiano et al., 1984 Goldman & Ming, 1968 Rhatigan & Saffos, 1979 Subbuswamy, 1971a Filipe, 1969 Normal staining pattern Diverticular disease Reid et al., 1984a 33 1.10. P R E M A L I G N A N T M A R K E R S The majority of the investigations previously cited provide only a description of the histochemical patterns seen in various diseases; few were specifically designed to address the significance of the changes observed. However, a number of studies have attempted to answer whether the changes seen in the mucosa adjacent to tumors are specific. Several investigators (Saffos & Rhatigan, 1977; Isaacson & Attwood, 1979; Rhatigan & Saffos, 1979; Listinsky & Riddell, 1981; Franzin et al., 1981, 1983a, 19S3b; Lev et al., 1985) adopt the position that the changes seen in the mucosa adjacent to tumors are nonspecific. Almost without exception, the evidence for this contention is based on demonstrating that identical histochemical or histologic changes to those that occur in the transitional mucosa can be observed in diseases with no malignant potential. Similarly compelling arguments have been proffered by those who believe that the changes in the transitional mucosa specifically represent preneoplastic markers (Filipe & Branfoot, 1974; Filipe & Cooke, 1974; Filipe, 1979; Filipe & Fenger, 1979; Filipe et al, 1980; Ehsanullah et al., 1982b). The problem of showing that transitional mucosa changes are specific has been approached from three different perspectives. Firstly, Filipe and Branfoot (1976) and Filipe et al., (1980) have demonstrated the existence of isolated patches of transitional mucosa remote from tumors. This suggests that transitional mucosa does not arise as a result of local malignant transformation. Second, one study has demonstrated that histochemical 34 changes precede morphologic change in male Wistar rats (Lazosky, 1986). Finally, Ehsanullah et al. (1982b) has shown that the histochemical changes characteristic of transitional mucosa are associated with dysplasia or with other potentially malignant conditions. 1.11. I N C I D E N C E O F C O L O N I C C A N C E R IN U L C E R A T I V E COLITIS , C R O H N ' S D I S E A S E A N D D I V E R T I C U L A R D I S E A S E Of the three inflammatory bowel diseases investigated in this study, ulcerative colitis is the one most universally accepted as being associated with an increased risk of colonic cancer (Morson & Pang, 1967; (Filipe, 1969, 1979; Allen et al., 1985b; Heimann et al., 1985) According to Lennard-Jones et al. (1977), those most predisposed to developing cancer are those: 1. who have had the disease for 10 or more years, 2. in which the colitis involves the entire colon, 3. who developed the disease in childhood, and finally, 4. who show evidence of epithelial dysplasia. Although the increased risk cited by different investigators varies from study to study - from "very low" in Hendriksen's et al. (1985) study to a high of 23 times the normal risk in Lennard-Jones' study (Lennard-Jones et al., 1977) -there is probably little doubt that there is a high risk group of colitics who are more likely than the general population to develop colonic cancer. In the last ten years, the widely held belief that Crohn's Disease patients were not at an increased risk of developing colonic cancer has, in many quarters, been 35 abandoned. Some investigators (Shamsuddin et al., 1981; Shorter, 1983) contend that there are subgroups in this population who have a greatly increased risk of developing colonic cancer. Shorter (1983) reviewed results from three large-scale studies conducted in the Mayo Clinic, New York and Birmingham. In all three studies it was concluded that, as compared to the general population, patients with longstanding Crohn's Disease were at an increased risk of developing colonic cancer (fourfold increased risk in the Mayo Clinic and Birmingham studies and a twentyfold increased risk in the New York study). As in ulcerative colitis, patients who are at the greatest risk for developing cancer are those who have had extensive disease of longstanding duration and who developed the disease in childhood (Shorter, 1983). In contrast to ulcerative colitis and Crohn's Disease, it is generally accepted (Rhatigan & Saffos, 1979) that diverticular disease is not associated with an increased risk of developing malignancy. 1.12. T H E S I S R A T I O N A L E Changes in the composition of the carbohydrate prosthetic groups of the epithelial glycoproteins are associated with tumors of the large intestine (Table 4), the mucosa adjacent to tumors ('transitional mucosa') (Table 5), and with the mucosa remote from tumors (Table 6). Some investigators (Filipe & Branfoot, 1976; Filipe et ah, 1980; Ehsanullah et al., 1982b; Reid et al., 1985a) contend that the changes observed in the transitional mucosa represent specific premalignant markers while others (Saffos & Rhatigan, 1977; Isaacson & Attwood, 1979; 36 Rhatigan & Saffos, 1979; Listinsky & Riddell, 1981; Franzin et al., 1981, 1983a, 1983b), maintain that the changes are a nonspecific reactive phenomena. Quite clearly, if it were the case that the histochemical changes seen in the transitional mucosa are premalignant markers, then early histochemical screening of patients with a high risk for developing colorectal cancer might be of some diagnostic value. Changes of a similar, but not identical nature have also been described for two inflammatory bowel diseases - ulcerative colitis (Table 8) and Crohn's Disease (Table 7). However, in diverticulitis, which is also classified as an inflammatory bowel disease, the epithelial glycoproteins do not differ radically from normal (Table 9). Although the figures vary from study to study, both ulcerative colitis (Lennard-Jones et al., 1977) and Crohn's Disease (Shorter, 1983) are associated with an increased risk of cancer, suggesting the possibility that cancer and histochemical alterations may be related. In contrast, diverticulitis, which is not associated with an increased risk of cancer has a normal mucin profile suggesting that inflammation, in and of itself, is not responsible for the mucin changes seen in the mucosa adjacent to and remote from tumors. The significance of the histochemical changes in the transitional mucosa, ulcerative colitis and Crohn's Disease is unknown. Perhaps the absence of a definitive answer with respect to this issue is due to the reliance of most investigators on the HID/AB2.5 technique. Most conclusions from previous studies have been based 37 on the results from one histochemical parameter - the relative proportion of sialomucin and sulphomucin - a parameter which generally has been assessed using the high iron diamine-Alcian Blue pH 2.5 (HID/AB2.5) technique. However, recent studies have led some investigators to suggest more caution in the interpretation of results with HID/AB2.5 (Lev et al., 1985; McFadden et al., 1985; Rhodes et al., 1985c; Reid et al., 1985b). In the present study, new methodology was used which investigated two histochemical parameters - the relative proportion of sialo- and sulphomucins, and the pattern of O-acetylated side chain sialic acid. It was anticipated that these new techniques would characterize the epithelial glycoproteins more fully, such that the issue of whether or not changes in the mucosa adjacent to tumors represent premalignant markers could be resolved. 2. M A T E R I A L S A N D M E T H O D S 2.1. T I S S U E Thirty-five specimens from 17 cases of ulcerative colitis, 51 specimens from 21 cases of Crohn's Disease of the colon and 37 specimens from nineteen cases of diverticular disease were obtained from the Vancouver General and Shaughnessy Hospitals (Table 10). All specimens had been fixed in 10% formalin-calcium and subsequently embedded in paraplast. In most cases, the specimens obtained were re-embedded in fresh paraplast. Following re-embedding, 20 serial sections, cut at intervals of 5Mm, were taken from each block of tissue. Serials were floated on a warm water bath (46 °C) containing 0.1% chrome alum in 1.0% gelatin as an adhesive, mounted onto clean glass slides, and subsequently exposed to formalin vapour at 60° C for 24 hours. Of the twenty serial obtained, the first five sections and the last seven were stored for future use. Each 5um section obtained from each specimen was assigned a letter (a-t) to denote from where in the serial sequence it was taken. Table 11 shows the order in which the serial sections were stained with the methods described below. 38 39 T A B L E 10 N U M B E R O F CASES A N D SPECIMENS OF U L C E R A T I V E COLITIS, CROHN'S DISEASE A N D D I V E R T I C U L A R DISEASE F R O M T H E D E S C E N D I N G A N D A S C E N D I N G C O L O N Cases Ascending only Descending only Both Total # Cases U L C E R A T I V E COLITIS 4 11 2 17 CROHN'S DISEASE 8 9 4 21 D I V E R T I C U L A R DISEASE 1 18 0 19 Specimens Ascending Descending Total # Specimens 10 25 35 24 27 51 2 35 37 40 2.2. STAINING PROCEDURES Sections were stained wi th hematoxyl in and eosin (Cull ing, 1974) for morphological assessment and wi th the seven histochemical stains as outlined in Table 11. The mechanisms and specificities of these stains have been extensively discussed (Reid et al, 1981, 1984b, 1985a, 1985b). 41 T A B L E 11 SERIAL SECTIONS A N D T H E CORRESPONDING H I S T O C H E M I C A L P R O C E D U R E APPLIED Serial Section Histochemical Procedure Acronym Reference Potassium hydroxide/ Alcian Blue pH 1.0/ Periodic acid -phenylhydrazine - water - peridic acid - Schiff. Periodic acid -borohydride treatment/ potassium hydroxide/ Alcian Blue ph 1.0/ Periodic acid - Schiff. KOH/AB1.0/PAPS Reid et al., 1985b PBT/KOH/AB1.0/PAS Reid et al., 1985b Alcian Blue pH 1.0/ Periodic acid -phenylhydrazine blockade - water -periodic acid Schiff. Periodic acid -borohydride treatment/ potassium hydroxide/ Alcian Blue pH 2.5/ Periodic acid - Schiff. AB1.0/PAPS Reid et al., 1985b P B T / K O H / A B 2.5/P AS Reid et al., 1985d Periodic acid -phenylhydrazine blockage - water -thionin Schiff/ Potassium hydroxide/ Borohydride treatment/ Periodic acid - Schiff PAPT/KOH/Bh/PAS Reid et al., 1984b Periodic acid -borohydride treatment/ potassium hydroxide/ Periodic acid - Schiff. PBT/KOH/PAS Reid et al., 1973 Periodic acid - thionin PAT/KOH/Bh/PAS Reid et Schiff/ Potassium 1984b hydroxide/ Borohydride treatment/ Periodic acid Schiff. 43 2.2.1. Histochemical Procedures The histochemical stains used in this project were constructed from the following standard techniques: 2.2.1.1. Periodic acid oxidation (PA) 1% aqueous periodic acid at room temperature oxidizes pre-existing vicinol diols to produce Schiff-reactive aldehydes. In procedures in which only one oxidation step was performed, sections were oxidized for 2 hours; in those with two oxidation steps, the initial oxidation step was performed for two hours and the subsequent one for 30 minutes. 2.2.1.2. Borohydride reduction (Bh) Aldehydes are reduced to Schiff unreactive primary alcohols by application of 0.1%(W/V) sodium borohydride in 1% (W/V) N a 2 H P O „ for 30 minutes at room temperature (Lillie and Pizzolato, 1972). 2.2.1.3. Saponification (KOH) O-acetyl esters are removed from glycoproteins by application of 0.5% (W/V) potassium hydroxide (KOH) in 70% alcohol for 15 minutes at room temperature. 2.2.1.4. Phenylhydrazine Block Schiff (PAPS or PAPT) Periodate engendered aldehydes are treated with 0.5% (W/V) aqueous phenylhydrazine hydrochloride for 2 hours at room temperature and then held overnight in distilled water. Phenylhydrazine hydrochloride selectively and 44 irreversibly blocks tissue diols. However, the blockade of sialic acid monoaldehydes is reversed by application of pararosaniline or Thionin Schiff, resulting in the specific Schiff staining of such residues. 2.2.1.5. Cationic Dye Methods with Alcian Blue Manipulation of the pH of 0.3% (W/V) aqueous Alcian Blue results in electrostatic binding to both carboxyl and sulphate groups at pH 2.5 and to sulphate esters only at pH 1.0. 2.3. STAINING OF SERIAL SECTIONS The histochemical procedures shown in Table 11 were employed in order to identify the following specific chemical groups:-2.3.1. Identification of Total Sialic Acid and Sulphate 2.3.1.1. KOHIAB1.0IPAPS In this procedure, the tissues are saponified to remove O-acyl esters and are then treated with Alcian Blue pH 1.0 to visualize sulphate esters. Subsequently, sialic acids are stained magenta with the periodic acid phenylhydrazine - Schiff (PAPS) procedure. 2.3.2. Identification of sulphate and side chain O-acetylated sialic acid 2.3.2.1. AB1.0/PAPS This method stains sulphate esters aqua and those sialic acids which are unsubstituted or are substituted at C7 and/or C9, magenta. 2.3.2.2. PBTIKOHIAB1.0IPAS This procedure allows for the simultaneous visualization of sulphate esters and sialic acids with side-chain O-acyl substituents located at position C7 pr C8 or which have two or three side chain substituents. 2.3.3. Identification of Total Sialic Acid, Sulphate, and Side-chain O-acetylated Sialic Acid 2.3.3.1. PBTIKOHIAB2.5IPAS In this technique, sulphate esters or sialic acids which have no side chain substituents or which are substituted at C9 and O-sulphate esters stain aqua. Those sialic acids with O-acetyl esters at C7 or C8 (or which are di- or tri-substituted) stain purple. 2.3.4. Identification of Side-chain O-acetylated Sialic Acids 46 2.3.4.1. PAPT/KOH/Bh/PAS Those forms of sialic acid that have no side-chain substituents or which are substituted at position C7 or C9 stain blue while C8 substituted sialic acids stain magenta. Mixtures of unsubstituted and substituted sialic acids stain purple. 2.3.4.2. PAT/KOH/Bh/PAS The PAT/KOH/Bh/PAS procedure is identical to the PAPT/KOH/Bh/PAS technique, with the exception that the phenylhydrazine blockade is omitted. Thus, the blue staining in the PAT procedure can be attributed to tissue diols in addition to CO, C7 and C9 substituted sialic acids. 2.3.4.3. PBT/KOH/PAS This procedure stains only those sialic acids that are substituted at C7 or C8 (or those which are di- or tri-substituted). Table 12 summarizes the results expected following the application of these procedures. 2.3.5. Procedure Controls A periodic acid-borohydride/Schiff control (PBT/S) and a periodic acid borohydride/ periodic acid-Schiff control (PBT/PAS) were used where appropriate. 47 T A B L E 12 E X P E C T E D STAINING R E S U L T S F R O M H I S T O C H E M I C A L PROCEDURES U S E D FOR G I V E N S T R U C T U R A L E L E M E N T S STAIN Structural Element O- Sialic Acid Tissue Sulphate Diols Ester CO C9 C8 C7 KOH/AB.O/PAPS A R R R R 0 PBT/KOH/AB1.0/PAS A 0 0 R R 0 PBT/KOH/AB2.5 /PAS A A A P P 0 AB1.0/PAPS A R R 0 R 0 PAPT/KOH/Bh/PAS 0 B B R B 0 PAT/KOH/Bh/PAS 0 B B R B B P B T / K O H / P A S 0 0 0 R R 0 A = Aqua; R = Red; B = Blue; P = Purple; 0 = No staining 2.4. ASSESSMENT OF STAINING PATTERNS 48 All histochemical and morphological staining was completed on all specimens before carrying out any assessment. Initially, assessments were made simultaneously by two observers using a double-headed microscope to establish the range of colours present. Assessments were then carried out individually, and data from each case was compared. Where any discrepancy between the two observers was noted, the entire specimen was examined again by both observers using a double-headed microscope. Results from the KOH/AB1.0/PAPS, PBT/KOH/AB1.0/PAS, PBT/KOH/AB2.5/PAS, and AB1.0/PAPS was assessed on a seven point color scale as being either Red (R), Red-purple (RP), Purple (P), Blue-purple (BP), Royal-blue (RB), Dark aqua (DA), or Aqua (A). Data from the PAPT/KOH/Bh/PAS and PAT/KOH/Bh/PAS procedures was assessed on a five point color scale as being either Red (R), Red-purple (RP), Purple (P), Blue-purple (BP) or Blue (BP). The PBT/KOH/PAS procedure was graded on a five point intensity scale as being either 0 (no staining), 1,2,3 or 4 (maximum staining intensity). 2.4.1. Digitization Tables 13, 14 and 15 represent the results from ulcerative colitis, Crohn's Disease and Diverticular Disease cases, respectively. Data from the KOH/AB1.0/PAPS, PBT/KOH/AB1.0 /PAS and PBT/KOH/AB2.5 /PAS procedures was converted to a 13 point numerical scale as follows: 49 13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 Red Red to red-purple Red-purple Red-purple to purple Purple Purple to blue-purple Blue-purple Blue-purple to royal-blue Royal-blue Royal-blue to dark aqua Dark aqua Dark aqua to aqua Aqua The results from the PAPT/KOH/Bh/PAS and the PAT/KOH/Bh/PAS procedures was converted to a 9 point numerical scale as follows:-Results from the P B T / K O H / P A S procedure, which was initially recorded as a numerical value was entered as that value. In many cases the field viewed was not homogeneous. Frequently, one particular color predominated while another less significant color, or a spectrum of colors, co-existed in the same field. In these specimens, a value of 0.5 was added to the dominant color's numerical score if the less significant color was more magenta than the dominant color. If the less dominant color was more aqua than the dominant color, a value of 0.5 was substracted from the latter color 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Red Red to red-purple Red-purple Red-purple to purple Purple Purple to blue-purple Blue-purple Blue-purple to blue Blue 50 score. In some specimens homogeneous areas were seen that were histochemically distinct from other adjacent areas. In these cases, data was recorded separately for each area of the slide. Lastly, a few cases possessed specimens which were described as being 'scrambled' (Slide 1). Scrambled specimens showed a vast range of colors in which no pattern could be discerned. In these cases, the specimen was recorded as having several major classes of glycoprotein. L E G E N D T O T A B L E S 13, 14 A N D 15 Column  Number 1. Specimen code number 2. Disease: U C = ulcerative colitis, CD = Crohn's Disease, D=Diverticular disease 3. Anatomic site: D = descending colon, A = ascending colon 4. Only applicable to diverticular disease cases: N = not applicable to disease S = Surface mucosa adjacent to diverticula P = Diverticulum 5. Sex: M = male, F = female 6. Age 7. Presence or absence of granulomas: A = absent, P = present 8. Presence or absence of ulcers: A = absent, P = present 9. Degree of inflammation for each third (a,b,c) of specimen 10. Degree of mucosal atrophy for each third (a,b,c) of specimen 11. Major or minor class of glycoprotein: M A = major, MI = minor 12. Crypt position: T = top, B=bottom 13. Glycoprotein class: IVA, IVC, IVD, V A , V C , V D 14. Digitized data - KOH/AB1.0/PAPS 15. Digitized data - PBT/KOH/AB1.0/PAS 16. Digitized data - PBT/KOH/AB2.5/PAS 17. Digitized data - AB1.0/PAPS 18. Digitized data - PAPT/KOH/Bh/PAS 19. Digitized data - PAT/KOH/Bh/PAS 20. Digitized data - P B T / K O H / P A S 21. Averaged value of degree of inflammation 22. Averaged value of degree of mucosal atrophy 52 TABLE 13 HISTOLOGIC AND HISTOCHEMICAL CHARACTERISTICS OF ULCERATIVE COLITIS DISEASE SPECIMENS • - • 1 8 UC • N F 15 A A 2 IB u c 0 H F 15 A A "J i a uc • H r IS A A 2 IB UC 0 N F IS A A 2 S4B u c 0 N F 13 A A 2 648 u c 0 M F 13 A A : SA u c A N F 30 A A 2 5* u c « N F 30 A A 2 SA u c A H F 30 A A 2 SA u c A N F 30 A A 2 23A u c 0 N F 30 A A 2 23* u c 0 N F 30 A A 2 SB u c 0 N M 26 A A 2 38 u c 0 N M 26 A A 2 IOA u c 0 N H 26 A A 2 IO» u c 0 H H 26 A A 2 3 IB u c • N M 26 A A 2 318 u c 0 N M 26 A A 2 94 u c 0 N F 31 A A 2 9A u c • H F 31 A A 2 7 IB u c 0 M F 31 A A 1 7 IB u c 0 N F 31 A A f 12* UC A N F 79 A p U 12* u c A N F 79 A P u 5 5 * u c 0 N F 79 A p 2 3 5 * u c 0 N F 79 A p 2 3 7 * u c A N F 79 A p U S74 u c A N F 79 A P U 1 3 8 u c A N F 19 A p U 1 3 8 u c A N F 19 A p U s e a u c A N F 19 A p u 36B u c A N F 19 4 p u S68 u c A N F 19 A p u s e a uc A N F 1 9 A p u 198 u c 0 N H 34 A A 1 I9B u c • N M 34 A A 1 30B uc • N M 34 A A 2 308 u c 0 M M 34 A A 2 178 u c D N F 37 A A 0 I7B u c 0 N F 3 7 A A 0 468 u c D N 3 37 A A 1 468 u c 0 N S S7 A A 1 46* u c 0 N F 37 A A 0 46* u c 0 N F 3 7 A A 0 20B uc A N A 26 A A 1 20a u c A M A 26 A A t 36a u c A N M 26 A A 1 3ea u c A N M 26 A A 1 2 i s l u c • N M 40 A A 3 2 tal u c D N H 40 A A 3 4oal u c 0 N M 40 A A 3 4081 u c • N M 40 A . A 3 338| UC 0 N M S 3 A A 2 35Bt UC 0 U M S3 A A 3 358 u c 0 N M S3 A A 2 338 u c • N H 53 A A 2 428 u c 0 N • • A p I 428 u c 0 N • • A p t 42B uc 0 N • • A p 1 7 1* u c 0 N • • A p u 7 1* u c • N • • A p u 43* uc A M M 64 A A ! 43* u c * N M 64 A A 1 s o e u c * N M 64 A A 2 s o a u c A N M 64 A A 2 438 u c 0 N M sa A A 2 458 u c • N M sa A A 2 4 SB uc 0 N M s s A A 3 438 uc 0 N M sa A A 2 498 u c 0 ti M s a A P t 498 u c D N M s s A p 1 498 u c 0 N M s s A p t 498 u c 0 M M s s A p l 478 u c 0 N M S3 A p U 4 7B u c 0 N M 53 A p u 488 u c D M M 53 A A 3 4 SB u c 0 U M S3 A A 3 48* u c 0 N M 57 A p u 48* u c 0 N M 37 A p u 30* u c 0 M M 37 A A 3 SO* u c 0 N M 37 A A 3 6 7 * u c A N F 39 A A 3 6 7 * u c A N F 39 * 4 3 VO vc VO vc VO vc VO i v o VO IVO IVA IV* VA VA VA VA VO VO VO VO IVC IVC VO VO IVC IVC IVC IVC IVC VO IVC VO IVO VO i vo VO VO VO VO VO VO VO VO VO VO VO VO VO VO VO IVA VA IVA IVA IVA v c VO vc VO l v c IVC VO VD VO vo VO vo IVC i v c VO VO IVA IVA 0 0 O 5 .0 o 3 .0 7 .S 13.0 12.0 12.0 7 .0 7 .0 7 .0 7 .0 10.0 6 .0 10. 10. to. to. 12. 12. to. 10. 12. t2. 12. 12. 1 I S. I I . s . 12 3. 12. 6. 9. 3. a . o a . o 9 . 3 10.3 3 .0 8 0 7 .5 12 .0 n o 12.0 t 1 .0 13.0 10.0 9 . 0 9 . 3 9 . 3 11.0 11.0 12 .0 12 .0 9 . 3 3 5 3 O O 3 3 O 0 0 o o 0 I t .0 n o 12.0 12.0 9 . 0 5 . 0 IO. 0 7 .5 9 . 0 3 . 0 a . o 12.0 9 . 0 12.0 0 . 0 0 . 0 2 . 0 2 . 0 4 . 5 4.S 1.0 1.0 t o o 10.0 3 . 5 5 0 O s 3 3 12 12 9 9 12.0 12 .0 12 .0 12.0 1 1 6 1 1 6 12 3 I 1 .0 4 . 0 9 . 0 3 . 3 a . o a . o o 3 0 0 3 7 0 7 .0 7 .0 7 .0 7 . 0 7.01 3 . 0 3 . 0 3 . 0 3 . 0 ' 3 . 0 3 . 0 4 0 4 . 0 3 . 3 3 . 3 1 .0 l .0 5 .0 5 .0 3 . 0 3 . 0 9 . 0 9 . 0 9 . 0 9 . 0 S.O 8 . 0 a . o 8 . 0 9 9 9 9 6 7 7 7 0 .0 .0 .0 .0 o 0 o 4 . 0 4 . 0 7 .0 7 .0 3 .0 3 .0 7 .3 7 .3 8 . 0 a . o 3 .3 10. O 0 o o 0 o 0 11.0 10.0 n o 12.0 12.0 7 . 0 7 . 0 9 . 0 9 . 0 10.0 S.O 11.0 I 1.0 9 . 0 9 . 0 0 . 0 0 . 0 10.0 IO.0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 9 . 0 0 . 0 9 . 0 13.0 9 . 0 12.0 4 . 0 a . s 3 . 0 8 . 0 0 o o 0 .0 o .0 3 3 3 5 0 0 .0 .0 .0 o .0 o o .0 o .0 .0 0 .0 .0 o 0 0 o .0 o .0 5 3 0 o 11 .0 n o it.o n o 3 .0 3 . 0 3 .0 3 .0 3 .0 3 .0 I . 3 1.3 I .5 1.3 0 .5 0 . 5 0 . 5 0 . 3 1.0 t .0 0 . 3 0 . 5 2 .0 2 .0 2 .3 2 .3 3 3 2 2 3 3 3. 3. 2 2 . a 2 .3 2 .3 2.3 2 .0 2 .0 2 .0 2 .0 2 .0 2 .0 3 S 3 3 0 . 5 0 . 5 0 . 5 0 . 3 0 . 0 0 . 0 0 . 0 0 . 0 .5 .3 .0 .0 .0 .0 .0 3 .3 3 .5 3 0 O 3 3 3 3 3 3 3 3 O .0 .3 .3 .3 .3 .0 .0 .0 .0 .9 .9 .9 . 3 .3 3 2 .3 0 . 3 0 . 3 3 .3 3 .3 3 .3 3.3 3 . 0 3 . 0 3 . 0 3 . 0 3.3 2.S 2 . 0 2 . 0 2.3 2 .3 3 .5 1.5 0 . 3 0 . 5 2 2 2 2 2 2 . 0 2 . 0 2 . 0 2 .0 2 . 0 2 . 0 2 . 0 2 . 0 2 .0 2 .0 2 .0 1.0 1 .0 1.5 1.3 2 .0 2 .0 2 .0 2 .0 1 .a a 2 .0 . 2 .0 2 .0 2 .0 2 .0 2 .0 2 2 2 2 . 3 o . a o . s 0 . 3 o . s 0 . 8 0 . 3 0 . o . a 4 . 0 4 . 0 3 . 3 3 . 3 3 . 0 3 .0 2.S 2 . 5 2.S 2.3 3 . 3 3 . 3 19.9 9 9 . 9 9 9 . 9 9 9 . 9 3 . 5 3 . 5 3. 3. 2. 2. 2 2 2 2 2 2 2 2 2 2 2.3 2 .S 1 2 .0 2 .0 0 . 0 0 . 0 2 .0 2 . 0 2 .0 2 .0 2 . 0 2 . 0 2 .0 2 . 0 2 .0 2 .0 2 . 0 3 .0 2 .0 2 .0 2 .0 2 .0 0 .0 0 . 0 3 .3 3 .S 2.8 4 . 0 4 . 0 0 . 3 O.S 0 .3 O.S 0 . 3 0 . 3 0 .5 O.S 0 . 5 3 .0 3 .0 1 .0 1.0 4 . 0 4 . 0 4 . 0 4 . 0 2 2 2 2 3 3 3 3 3 .3 3 .3 3 .3 3 .3 3 .3 0 . 3 0 . 3 2 2 2 2 1.5 1.5 2 53 HISTOLOGIC AND TABLE 14 HISTOCHEMICAL CHARACTERISTICS DISEASE SPECIMENS OF CROHN'S (~ IA CO A N H 22 IA CO A M H 22 IA CD A N M 23 IA CO A N H 22 :a CO 0 N F 70 29 CO 0 N F 70 368 CO 0 N F 70 268 CO 0 N F 70 48 CD A M H 14 48 CO A N M 14 ' 8 CO A N H 14 48 CO A N M 14 108 CO A M M 14 108IC0 A N M 14 • SB CO 0 N M 14 168 CO 0 N H 14 20A CO 0 N M 14 20A CO 0 M 14 20A CO 0 M M 14 20A CO 0 N M 14 6A CO 0 M F S3 SA CO 0 N F S3 ISA CO A N F 30 I6A CO A N F 30 33B CO A N F 30 338 CO A N F 30 ISA CO 0 N F 7S ISA CO 0 M F 7S 37B CO 0 N F 76 378 CO 0 N F 7S 378 CO 0 N F 76 378 CD D M F 76 228 CO A N F 71 228 CD A N F 7 1 228 CO A N F 7 1 228 CO A N F 7 1 388 CO A N F 7 1 388 CO A N F 7 1 3SB CO A N F 71 388 CO A N F 7 1 238 CO 0 H F 17 238 CO 0 H F 17 4 18 CO 0 N F 17 4 18 CO 0 N F 17 43A CO 0 N F 17 43A CO 0 N F 17 258 CD 0 N F 24 258 CO 0 N F 24 258 CD 0 H F 34 258 CO 0 H F 24 3SA CO 0 H F 24 3SA CD 0 H F 24 3SA CO 0 N F 24 36A CD 0 H F 24 298 CO 0 N F 14 298 CO 0 N F 14 59A CD 0 N F 14 59A CO 0 N F 14 398 CO A N F S3 398 CO A N F S3 54A CO A N F 18 54A CO A N F ta 5SA CO A N F 18 SSA CO A N F 18 548 CO 0 N F 35 548 CO 0 N F 35 548 CD D N F 35 548 CO 0 M F 35 588 CO 0 N F 35 588 CO 0 . N F 35 588 CD o N F 35 588 CO 0 N F 35 588 CO 0 N F 35 588 CO 0 N F 35 S<A CO A M F 38 S IA CO A N F 38 s a a CO 0 N F 38 S88 CD 0 N F 38 6SA CO 0 N P 83 SSA CO 0 H F 83 S9A CO 0 M F 83 69A|C0 0 N S3 288 CO 0 n F 63 288 CO 0 M F S3 2 8 8 l C 0 0 N F 63 288 CO 0 M f S3 I u * 2 u 2 MA r VO 10 0 10 0 5 0 7 5 3 0 2.0 2 3 I.S 3 1 1 1 u 2 2 tj 2 MA 8 vo 9 0 a 5 5 0 7 5 3 0 2.0 3 5 1.» 2 ! 1 u 2 7 u 2 MA 1 vo 11 0 11 0 6 0 11 0 3 0 2.0 2 5 1.5 2 1 u 2 2 u 2 MA 8 vo 11 0 11 0 S 0 11 0 2 0 2.0 2 3 1.3 2 2 2 2 t 1 1 MA T vo 10 5 to 5 9 0 10 s 2 s 2.0 4 0 3 1 2 2 2 1 I 1 MA 8 vo 11 0 11 0 9 0 13 0 2 5 2.0 4 0 3 1 2 1 1 I 1 1 MA r vo a 0 a 0 4 0 10 0 1 5 1.0 3 0 1.5 1 2 1 1 1 1 1 MA a IVO 12 0 12 0 9 0 13 0 1 3 l .0 3 0 1.5 1 2 2 2 2 2 MA r VO 8 0 S 0 5 0 a 0 1 5 1.3 2 3, 2 3 2 2 2 2 MA a VO a 0 6 0 5 0 a 0 1 3 1.3 2 3 3 2 1 1 1 0 0 0 MA T vo 10 0 9 0 a 0 9 0 1 S 1.3 2 3 1 0 1 1 1 0 0 0 MA a IVO 12 n 12 0 5 0 13 0 1 3 1.3 2 3 t 0 1 2 1 1 1 MA T VA 8 0 3 5 2 0 10 0 0 a 0.0 0 8 I.S 1 1 2 1 1 1 MA 3 VA 9 5 9 0 2 0 7 0 0 8 0.0 0 8 I.S 1 1 1 1 1 1 1 MA T VA 8 0 3 0 1 0 6 5 0 3 0.0 0 3 1 1 1 1 1 t 1 1 MA a VA 8 0 2 0 1 0 6 5 0 3 0.0 0 3 1 1 3 2 0 2 0 MA T VO a 0 9 0 S 0 1 1 0 2 0 1.0 2 3 2 1. i 3 2 2 0 2 0 MA a VO a 0 10 5 s 0 1 1 0 2 0 1 .0 35 2 1. i 2 2 2 o 0 0 MA T VA 9 s 1 0 1 0 1 1 0 1 3 0.0 1 0 2 0 2 2 2 0 0 0 MA a VA 9 5 , 1 0 1 0 1 1 0 1 5 0.0 1 0 2 0 2 2 2 2 2 3 MA r VO 9 0 9 0 5 0 9 0 3 3 2.0 4 0 3 2. s 2 3 2 2 2 •3 MA a VO a 0 a 0 5 0 7 0 2 3 2 0 4 0 3 2 5 1 1 I O 0 0 MA r VA a 0 2 0 1 0 7 0 0 3 0 3 0 3 1 0 t 1 t 0 0 0 MA a IVA 12 0 0 0 1 0 13 0 0 5 0.3 0 5 1 0 2 3 2 1 1 1 MA r VO to 0 10 0 5 0 9 0 1 3 1-0 2 3 2 t 2 2 2 1 1 1 MA a VO 10 0 10 0 5 0 9 0 1 a 1.0 2 5 2 1 1 1 1 1 1 1 MA r VA 9 0 2 0 3 0 10 0 0 5 0.0 0 5 1 1 1 1 1 1 1 1 MA a iv a 11 0 13 0 9 0 13 0 0 S 0.0 3 0 1 1 1 U 1 2 u 2 MA T vo 9 0 a 0 5 0 10 0 0 5 0.3 1 0 1 2 1 u 1 2 u 2 MA T VA 9 0 2 0 3 0 10 0 0 3 0.3 1 0 1 2 1 u 1 2 li 2 MA a vo 9 0 a 0 5 0 10 0 0 5 0.3 1 0 1 2 1 u t 2 u 2 MA a VA 9 0 2 0 3 0 10 0 0 5 0.3 1 0 1 2 1 i 1 1 X o MA T VO 7 0 9 0 a 5 7 0 3 5 1.8 3 5 1 0 3 1 i 1 ! X 0 MA a VO 7 0 9 0 8 5 7 0 3 3 1 .8 3 5 1 0 5 2 2 2 2 2 2 MA T IVC 12 0 12 0 a 5 0 0 3 3 1.8 3 3 2 2 2 2 3 2 3 MA 8 IVC 12 0 12 0 s 5 0 0 3 3 1 .8 3 S 2 2 1 U 1 1 1 MA T IVC 12 0 12 0 7 0 0 0 3 5 2.0 3 3 1 l 1 u 1 I l * 1 MA a IVC 12 0 12 0 7 0 0 0 3 3 2.0 3 5 1 l 1 u t 1 1 MA T vc •J 0 S 5 7 0 3 0 3 3 2.0 3 5 1 1 1 u 1 1 1 MA a IVC 12 0 t 1 0 7 0 0 0 3 S 2.0 3 3 1 1 3 3 3 3 3 3 MA T vo 9 5 1 1 0 7 0 9 0 3 0 2 0 3 3 3 3 3 3 2 3 3 3 MA a VO 9 5 1 1 o 7 0 9 0 3 0 2.0 3 5 3 3 2 2 2 3 3 3 MA T VO 10 0 to 0 9 0 9 0 99 9 2.0 3 S 3 3 2 2 2 3 3 3 MA a VO 10 o IO 0 9 0 9 0 99 9 2.0 3 3 3 3 2 2 2 3 3 3 MA r IVC 11 5 10 5 9 0 0 0 3 0 2.0 3 3 3 3 2 2 2 3 3 3 MA a IVC 11 5 10 5 9 0 0 0 3 0 2.0 3 3 3 3 2 2 2 2 2 MA r vo a 0 a 0 8 0 a 0 2 3 1.0 3 a 2 3 2 2 2 2 2 2 MA T ivo 12 0 12 0 8 0 11 0 2 5 t .0 2 8 2 2 2 2 2 2 2 2 MA a vo a 0 a 0 a 0 a 0 2 S 1 .0 2a 2 2 2 2 2 2 MA 8 IVO 12 0 13 0 a 0 11 0 2 3 t .0 2 a 2 2 1 1 t 1 1 1 MA r IVO 12 0 12 0 7 0 12 0 3 0 2.0 3 5 1 1 1 1 1 1 1 1 MA a IVO 12 0 13 0 7 0 13 0 3 0 2.0 3 5 1 1 1 1 1 1 1 1 MA r vo 7 5 7 5 7 0 8 5 3 0 2.0 3 5 1 1 1 1 1 1 l 1 MA a VD 7 5 7 5 7 0 a 5 3 0 2.0 3 5 1 1 U U 3 U U 3 MA T vo 9 0 a 0 7 0 9 5 3 0 1 .0 4 0 3 3 U u 3 U U 3 MA a vo 9 0 a 0 7 0 9 5 3 0 1 .0 4 0 3 3 2 2 2 3 3 3 MA r vo a 0 a 0 7 0 9 0 3 3 2.0 3 0 3 3 2 2 2 3 3 3 MA a vo a 0 8 0 7 0 9 0 2 3 2.0 3 0 3 3 1 1 1 0 0 0 MA r vo a 5 4 5 S 5 9 0 1 S 1 .0 3 5 1 0 1 1 1 0 0 0 MA a VO 10 s 1 1 0 6 5 1 1 0 l 3 1.0 3 5 1 0 1 1 1 0 0 0 MA T vo 5 0 t 0 3 0 5 0 2 0 2.0 1 0 1 0 1 1 t O 0 o MA a vo 7 o 1 0 3 o s o 2 o 3.0 1 o 1 0 1 1 1 0 0 0 MA i vo 5 0 1 0 3 3 5 0 2 0 2.0 1 0 1 0 1 1 1 0 0 0 MA a vo 6 0 1 0 3 5 5 0 2 0 2.0 1 0 t 0 3 U 2 1 U 1 MA r IVO 12 0 t 1 5 8 0 t 1 0 2 3 2.0 3 0 2 1 2 u 2 1 u 1 MA T vo 10 0 10 5 8 0 9 0 3 3 2.0 3 0 2 1 2 u 1 u 1 MA 8 IVO 12 0 1 1 5 8 0 1 1 0 2 3 2.0 3 0 2 1 2 u f u 1 MA a VO 10 0 10 5 a 0 9 0 3 3 2.0 3 0 2 1 2 2 2 o 0 0 MA T IVO 12 0 13 0 5 0 12 0 3 3 2.0 2 5 2 0 2 2 o 0 0 MA B i v c 12 0 12 0 5 0 0 0 3 3 2.0 2 5 3 0 2 2 2 0 0 0 MA T vo 10 0 10 0 5 0 10 0 3 S 2.0 3 0 2 0 2 2 0 0 0 MA a VO a 0 9 5 5 0 7 0 3 3 2.0 3 0 3 0 3 3 3 2 2 3 MA T IVD 12 0 13 0 5 0 13 0 3 3 2.0 3 8 3 3 3 3 3 2 2 2 MA a i v c 12 0 12 0 5 0 0 0 3 3 2.0 3 a 3 2 3 3 3 2 2 3 MA r VO 10 5 10.5 a 0 1 1 0 2 5 2.0 4 0 3 2 3 3 3 2 2 2 MA a vo 10 5 10 s a 0 10 5 2 5 2.0 4 0 3 2 1 2 1 1 2 1 MA r IVD 13 0 12 0 6 0 1 1 0 2 0 3.0 4 0 I . S I S 1 2 1 t 2 1 MA a i v o 12 0 12 0 6 0 1 1 0 2 0 2.0 4 0 I.S 1 5 3 3 2 1 1 1 MA r i v c 10 0 10.0 9 0 a 0 3 3 3.0 3 0 2 1 3 2 2 1 t 1 MA 8 IVC to 0 10 0 9 0 0 0 2 5 2.0 3 0 2 1 U 2 u 1 u MA T IVC 13 0 12 0 7 0 0 0 3 3 2.0 3 5 3 1 U 2 , U . « u MA a IVC 13 0 13 0 1 0 0 0 3 » 2.0 3 » 1 l 1 1 1 1 0 o 0 MA T VO 8 0 a o 5 0 8 0 1 0 1.0 0 3 0 1 1 ' 0 0 0 MA a VO S 5 s 0 5 0 S 0 1 0 1.0 a . i 1 0 1 ' ' 0 0 0 MA T VA 8 0 a 0 3 0 8 0 1 0 1.0 a. 5 0 | 1 ! 1 ' 0 0 0 MA a VA S 5 s 0 2 0 S 0 1 0 1.0 0 a t 0 i TABLE 14 (CONT.) 6B CD A N M 82 P A 0 0 0 0 0 0 MA T SB CO A N M 83 P A 0 0 0 0 0 0 MA B SB CD A N M 82 p A 0 0 0 0 0 0 MA B 23A CO A N M 82 A A 2 2 2 2 2 2 MA T 23A CO A N M 82 A A 2 2 2 2 2 2 MA T 33A CO A N M 82 A A 2 2 2 2 2 2 MA 8 33A CD A N M 82 A A 2 2 2 2 2 2 MA 8 7A CO A N M 49 P P 1 t 1 2 2 0 MA 7 7A CD A N H 49 P P 1 1 1 2 2 0 MA B 1 IA CO A N W 49 A A 1 1 2 I 1 1 MA 7 1 1A CO A N M 49 A A 0 0 0 0 0 0 MA T 1 IA CO A M M 49 A A 1 1 2 1 1 1 MA B 1 IA CD A N M 49 A A 0 0 0 0 0 0 MA B 4 1A CO 0 N M 49 P A 2 2 2 2 1 1 MA T 4 IA CO D M M 49 P A 2 2 2 2 1 1 MA B 39A CO 0 N M 49 P A 2 2 2 2 2 2 MA T 39A CD 0 N H 49 P A 2 2 2 2 2 2 MA T 39A CO 0 N M 49 P A 2 2 2 2 2 2 MA B 38A CO 0 N M 49 P P 2 U 2 1 U 1 MA r 38A CO 0 N M 49 P P 2 U 2 1 U 1 MA B JOA CO 0 N M 49 P A t 1 1 1 1 1 MA r 4 OA CO 0 N M 49 P ' A 1 1 1 1 1 MA B 32A CD A U M 49 P A 1 2 • 0 • MA T 22A CD A N M 49 P A 1 2 0 1 . MA B 7B CO A N F 37 P A t 1 2 2 1 2 MA T 7B CD A N F 37 P A I 1 2 2 1 2 MA T 7B CO A N F 37 P A 1 t 2 2 I 2 MA B 7B CD A N f 37 P A t 1 2 2 2 MA B SA CO A N F 37 A A 0 0 0 0 0 MA T 8A CD A N F 37 A A o O 0 0 O MA B SB CO A N F 37 A A 1 t I 2 2 2 MA T 9B CO A M F 37 A A 1 1 2 2 2 MA B 3SA CO A N F 37 A A 1 1 1 2 2 2 MA T 35A CO A N F 37 A A 1 1 1 2 2 2 MA T 3SA CD A N F 37 A A 1 1 \ 2 2 2 MA • 3SA CO A N F 37 A A 1 1 t 2 2 2 MA a 9B CD 0 N 44 A P 2 2 2 2 2 2 MA r 9B CO D H M 44 A P 2 3 2 2 2 2 MA B 9B CO 0 N 44 A P 2 2 2 2 2 2 MA T 98 CO D N 44 A P 2 2 3 2 2 2 MA a 26A CO D N M 44 A A 1 1 1 1 MA T 2SA CO D N H 44 A A 1 1 t « 1 , MA B 13A CO A N F 25 P P 2 u U i MA T 1 3 A CO A N F 25 P P 2 u 1 < U ! MA T I3A CO A N F 25 P P 3 u 1 u t MA T 134 CO A N F 23 P P 2 u t t u I MA B I3A CO A H F 25 P P 2 u \ i u < MA B I3A CD A N F 23 P P 2 u ) 1 u 1 MA B I4A CO A N F 25 P A 2 2 2 t 1 t MA T !4A CO A N F 23 P A 2 2 3 1 1 MA B SSB CO D N F 25 P P 2 u « u 1 MA T 558 CD 0 N F 25 P P 2 u 2 t u I MA B I4B CO A N F 29 A A 1 1 t 0 0 0 MA T 14B CD A N F 29 A A 1 t 1 0 0 0 MA B 44A CO A N f 29 A A 1 1 1 0 0 0 MA T 44A CO A N 29 A A 1 1 1 0 0 0 MA B VD VO IVO vo IVO VO IVO vo vo VD VA VO VA VO vo VD ivc vc VO vo vo vo VD VO VC IVC vc IVC vc IVC vc vc vc IVC IVC vo VO vo IVO IVO IVC ivc VO VC IVC IVC VC VD VO V D VO vo vo vo VD VD 8 . 0 10.0 12.0 7 . 0 12.0 7 . 0 O 0 O 3 5 5 5 3 0 0 I 1 .0 7 .0 9 .5 5 .0 10.0 7 .0 7 .5 7 .5 7 .0 1 1 .0 7 .0 1 1 .0 7 .0 I I . O 9 . 0 9 . 0 9 . 0 1 1 .0 0 5 0 1 1 10 9 9 . 0 12.0 12.0 12.0 12.0 9 . 0 9 . 0 12.0 12.0 9 . 0 9 . 0 7 . 0 10.0 8 . 0 8 . 0 7 . 0 10.0 5 . 0 9 . 0 8 . 0 10 .0 0 0 O O O 3 5 4 . 0 2 . 0 5 .0 2 . 0 9 . 0 5 .0 10.0 11.0 S.O 5 . 0 5 .0 10.0 0 0 0 5 0 5 0 0 o o 0 0 11.0 11.0 10.0 9 . 0 9 . 0 11.5 M S 12.0 12.0 9 . 0 9 . 0 12.0 12 .0 9 . 0 9 . 0 7 . 0 10.0 8 . 3 8 . 5 5 . 0 9 . 0 5 . 0 9 . 0 9. O 0 o 0 o 0 o 0 o 0 0 0 4 . 0 S.O 5 . 0 7 . 0 7 . 0 7 . 0 7 .0 7 . 0 S . O 6 . 0 9 . 0 9 . 0 7 . 0 7 .0 7 . 0 7 . 0 S .O 9 . 0 8 . 0 8 . 0 O O 0 o o 0 0 0 0 0 o 9 . 0 9 . 0 9 . 0 9 . 0 9 . 0 8 . 0 8 .0 9 . 0 8 . 5 10.3 12.0 3 . 0 0 . 0 3 . 0 0 . 0 10.0 9 . 5 9 . 3 4 . S 9 . 0 0 . 0 1 . 0 8 . 0 7 . 0 9 . 0 4 . 0 10.5 7 . 0 1.0 0 . 0 I . 0 0 . 0 1 . 0 0 . 0 3 . 0 3 . 0 1.0 0 . 0 0 . 0 11 .0 10.5 10. 5 11.0 11.0 0 . 0 0 . 0 4 . 0 3 . 0 0 . 0 0 . 0 3 . 0 4 . 0 10.0 10 .0 8 . 0 3 3 3 3 3 99 9 9 . 9 3 .0 3 .0 3 .3 3 .3 3 .S 3.5 3 .3 3 . 0 3 . 0 3 . 0 2 .0 2 . 0 2 .0 2 .0 3 . 0 3 . 0 3. 3. 3. 3 3. 3 3. 3 2. 2 2. 3 3. 3. 3. 3 . 3. 3 . 3. 3 . 0 3 . 0 2.5 2 .5 2 .5 2.3 1.0 1 . 0 2 . 0 2 .0 2 .0 1 .0 1.0 1.0 1 .0 2 .0 2 .0 1.0 1 .0 1.0 1 . 0 2 .0 2 .0 2 .0 2 .0 2 .0 2 .0 2 . 2. 2. 2. 2 . 2. 2. 2. 2. 2. 2 .0 2 .0 2 .0 2 . 0 2 .0 2 .0 2 .0 1.5 1 .5 1.5 1.5 2 .0 2 .0 V9.9 99 .9 49 .9 ! 9 . 9 : i9.9 : '9.9 2 .0 2 .0 2 .0 2 .0 2 .0 2 .0 1.0 1 .0 55 TABLE 15 HISTOLOGIC AND HISTOCHEMICAL CHARACTERISTICS OF DIVERTICULAR DISEASE SPECIMENS 2A 2A 2A 2A €78 S78 878 S78 3A 3A 3A 3A S28 328 S28 328 4A 4A 4A 4A 43B 438 438 43B 1 IB 1 18 1 IB t IB 1 18 I IB 118 I 18 128 12B 128 12B 27A 27A 27A 27A 708 70S 708 708 10 27B 0 278 ID 278 10 27B 10 278 10 27B 23A 28A 28A 28A 32A 32A 32A 32A 37A 37A 37A 374 348 348 348 348 538 ssa 658 448 448 338 338 474 474 474 474 494 494 4 94 494 494 SIS 3 18 318 318 334 334 0 s r 48 4 4 0 0 0 0 0 0 M4 T VO 0 s F 48 4 4 0 0 o 0 0 0 M4 8 VO 0 p F 48 4 4 1 1 1 0 0 0 M4 T VO 0 p F 48 4 4 t 1 1 0 0 0 MA 3 VO 0 s F 4S 4 4 0 0 0 0 0 0 M4 T VO 0 3 F 48 4 4 0 0 0 0 0 0 MA 8 VO 0 p F 48 4 4 1 1 1 0 0 0 M4 T VO 0 p F 48 4 4 1 1 1 0 0 0 M4 8 vo 0 s F 79 4 4 0 0 0 0 0 0 M4 T vo 0 s F 79 4 4 0 0 0 0 0 0 M4 8 VO 0 p F 79 4 A 1 1 1 1 1 1 M4 7 VO 0 p F 79 4 4 1 1 1 1 1 1 M4 3 vo D s F 79 4 A 0 0 0 0 0 0 M4 T vo 0 s F 79 4 4 0 0 0 0 0 0 M4 a vc 0 p F 79 4 4 1 1 1 1 1 1 M4 T vo 0 p P 79 4 A 1 1 1 1 1 1 MA a vc 0 s 30 4 A 0 0 0 0 0 0 M4 T [VD 0 s * 30 4 4 0 0 0 0 0 0 M4 a vo 0 p M 30 4 4 • 1 1 1 0 0 0 M4 T ivo 0 p * 90 4 4 1 1 1 0 0 0 M4 B vo 0 s M 90 4 4 0 0 0 0 0 0 M4 T IVO 0 5 M 80 4 4 0 0 0 0 0 0 M4 3 vo 0 p H ao 4 4 1 1 1 0 0 0 M4 T vo 0 p * ao 4 4 1 1 1 0 0 0 M4 8 vo 4 s F 78 4 4 0 0 0 0 0 0 M4 T VO 4 s F 78 4 4 0 0 0 0 0 0 M4 3 vo 4 s F 78 4 A 0 0 o o 0 0 M4 T VC 4 s F 78 4 4 o 0 0 0 0 0 M4 B VC 4 p F 78 4 A 1 1 1 0 0 0 M4 T vo * p P 78 4 4 1 1 1 0 0 0 M4 3 vo 4 p P 78 4 A 1 1 1 0 0 0 M4 T vc 4 p F 79 4 4 1 1 t 0 0 0 M4 a VC 4 s. F 78 4 A 0 0 0 0 0 0 M4 T VO 4 s P 78 4 4 0 0 0 0 0 0 M4 a VO 4 p P 78 4 A 2 2 2 0 0 0 M4 T vo 4 p 73 4 4 2 2 2 0 0 0 MA a vo 0 s P 36 4 A 0 0 0 0 0 0 M4 T IVA 0 s F 36 4 4 0 0 0 0 0 0 MA 3 [VA 0 p F 38 4 4 1 1 1 1 1 1 M4 T IVA 0 p F 36 4 4 1 1 1 1 1 1 MA a IVA 0 s F 76 4 4 0 o 0 0 0 0 M4 T IVC 0 s P 78 4 4 0 0 0 0 0 0 MA 3 IVC 0 p F 76 4 4 1 1 1 0 0 0 M4 r IVC 0 p P 76 4 4 1 1 1 0 0 0 MA 8 IVC 0 s M 64 4 A 1 T 1 0 0 0 M4 T IVC 0 s M 64 4 4 1 1 1 0 0 0 MA a IVC 0 s M 64 4 A 1 1 1 0 0 0 MA 3 vc 0 s M 64 4 4 1 1 1 0 0 0 MA r ivc D p M 64 4 A 2 2 2 1 1 1 MA T IVC • p M 64 4 A 2 2 2 1 1 1 MA 8 IVC 0 s M 64 4 A 0 0 0 0 0 0 MA T ivo 0 s M 64 4 A 0 0 o 0 0 0 MA a vo • p H 64 4 A 2 2 2 1 1 1 MA T ivo 0 p M 64 4 A 2 2 2 1 1 1 MA B IVO 0 s F 66 4 A 0 O 0 o 0 0 MA T IVO 0 s F 66 4 A 0 0 0 0 0 0 MA a vo 0 p P 66 4 A 1 1 1 0 0 0 M4 7 ivo 0 p P 66 4 A 1 1 1 0 0 0 M4 3 VO 0 s P 66 4 A 0 0 0 0 0 0 M4 T IVO 0 0 0 s p p J 66 66 66 J * 0 1 r 0 1 1 0 1 1 0 0 0 0 0 0 0 0 0 M4 M4 M4 a T s VO VO VO 0 s 64 4 A 0 0 0 0 0 0 MA T VO 0 s 64 4 A 0 o 0 0 0 0 M4 a VC D p 64 4 A o o 0 o 0 0 MA T vo 0 p ** 64 4 A o o o 0 0 0 M4 a VC 0 N ** 64 4 A 0 0 0 0 0 0 MA r vo 0 N 64 4 A 0 0 0 0 0 0 M4 T ivo 0 N 64 * A 0 0 0 0 0 0 MA 8 VC 0 N 32 4 A 2 2 2 1 1 1 M4 7 ivo 0 N M 52 A A 2 2 2 1 1 1 M4 B vo 0 p * 32 4 A 1 1 1 0 0 0 M4 T IVC 0 p M 52 A A 1 1 1 0 0 0 M4 8 vc 0 s P 7a 4 A 1 1 I 0 0 0 MA 7 vo 0 s F 78 A A 1 1 1 0 0 0 M4 a VC 0 p P 78 4 A 1 1 1 0 0 0 M4 T vo D p P 78 A A 1 1 1 0 0 0 M4 a vc 0 s P 78 4 A 1 1 1 0 0 0 M4 T IVO 0 s P 78 A A 1 1 1 0 0 0 M4 r VO 0 s P 78 4 A 1 1 1 0 0 0 M4 a vo 0 p P 78 A A 1 1 1 0 0 0 M4 T vo 0 p P 78 4 A 1 1 1 0 0 0 M4 a vc 0 s P 71 A A 0 0 0 0 0 0 M4 r vo 0 s F 7 1 4 4 0 0 0 0 0 0 MA a vo 0 p P 7 1 4 4 1 1 1 0 0 0 M4 r vo 0 p P 7 1 4 4 1 1 1 0 0 0 M4 8 vo 0 s P 7 1 4 4 0 0 0 0 0 0 M4 7 vo 0 s f 7 1 * * 0 0 0 0 o o MA a vo 10 0 10 3' 7" 0 9 0 3. 3 2 0 3.3 0 0 a 0 9 0 7 0 9 . 0 3. 3 2. 0 3.3 0 0 9 0 9 0 7 0 9. 0 2. 3 2 0 3.3 1 0 a 0 9 0 7 0 9 . 0 2. 3 2 0 3.3 1 0 10 0 10 0 7 0 9 0 2. 3 2 0 3.0 0 0 6 0 a 0 7 0 4 0 2. 3 2. 0 3.0 0 0 to 3 a 0 7 0 4 0 2. 3 2 0 3.0 1 0 6 0 a 0 7 0 4 0 2. 3 2 0 3.0 1 0 9 0 9 0 7 0 9 0 2. 3 2 0 4.0 0 0 3 0 5 0 9 0 4 0 2 3 2 0 4.0 0 0 a 0 9 0 9 0 9 0 3. 3 2 0 4.0 1 1 9 0 9 0 9 0 9 0 3 3 2 0 4.0 1 1 10 0 10 0 7 0 10 0 3 0 2 0 4.0 0 0 3 0 7 0 7 0 3 0 3. 0 2 0 4.0 0 0 10 0 10 0 7 0 10 0 3 0 2 0 4.0 1 1 3 0 7 0 7 0 3 0 3 0 2 0 4.0 1 1 12 0 12 0 3 0 12 0 3 5 2 0 3.5 0 0 7 0 7 0 3 0 7 0 3. 5 2 0 3.3 0 0 12 0 12 0 3 0 12 0 3 5 2 0 3.3 1 0 7 0 7 0 3 0 7 0 3 5 2 0 3.5 1 0 12 0 12 0 99 9 12 0 2 0 2 0 3.3 0 0 a 0 a 0 99 9 a 0 2 0 2 0 3.3 0 0 io 0 IO 0 5 0 to 0 2. 5 2 0 3.3 1 0 a 0 6 0 5 0 7 0 2 5 2 0 3.3 1 0 8 3 a 3 7 0 4 0 3 5 2 0 J.3 0 0 8 0 6 5 7 0 4 0 3 5 2 0 3.3 0 0 a 3 6 3 7 0 3 0 3 3 2 0 3.3 0 0 a 0 S 5 7 0 3 0 3 5 2 0 3.3 0 0 8 3 6 3 7 0 4 0 3 3 2 0 3.3 1 0 a 0 6 3 7 0 4 0 3 3 2 0 3.3 1 0 a 3 6 3 7 0 3 0 3 3 2 0 3.3 1 0 a 0 6 3 7 0 3 0 3 3 2 0 3.3 1 0 a 3 7 0 7 0 4 0 3 0 2 0 3.0 0 0 3 3 7 0 7 0 4 0 3 0 2 0 3.0 0 0 a 5 7 0 7 0 4 0 3 0 2 0 3.0 2 0 8 3 7 0 7 0 4 0 3 0 2 0 3.0 2 0 12 0 0 0 2 0 t2 0 0 3 0 3 t . O 0 0 12 0 0 0 2 0 12 0 0 3 0 3 1.0 0 0 1 1 3 0 0 2 0 11 5 0 3 0 3 2.3 t 1 1 1 5 0 0 2 0 1 1 3 0 3 0 3 2.3 1 1 12 0 12 0 5 0 0 0 3 3 1 0 3.3 0 0 12 0 12 0 5 0 0 0 3 3 1 0 3.3 0 0 12 0 12 0 5 0 0 0 3 3 1 0 3.3 1 0 12 0 12 0 5 0 0 0 3 3 1 0 3.3 1 0 12 0 12 0 5 o 0 0 3 3 2 0 3.0 1 0 12 0 12 0 3 0 0 0 3 3 2 0 3.0 1 0 a 0 a 0 5 0 1 0 3 3 2 0 3.3 1 ' 012 0 12 0 3 0 0 0 3 3 2 0 3.5 t 0 12 0 12 0 3 0 0 0 3 3 2 0 3.0 2 1 12 0 12 0 3 0 0 0 3 3 2 0 3.0 2 1 12 0 1 1 0 3 0 12 0 2 0 1 0 2.5 0 0 6 0 4 0 5 0 7 0 2 0 1 0 2.5 0 0 12 0 12 0 3 0 0 0 2 0 1 0 1.5 2 1 12 0 12 0 5 0 0 0 99 9 1 0 1.3 2 1 12 0 13 0 3 0 12 0 1 0 1 0 3.0 0 0 6 0 6 0 3 0 6 0 t 0 1 0 3.0 0 0 12 0 12 0 5 0 12 0 2 5 2 0 2.5 1 0 6 04 6 0 3 0 6 0 2 5 2 0 2.3 1 0 12 0 12 0 3 0 1 1 0 2 0 2 0 3.0 0 0 a . 0 7 .0 3 .0 a .0 2 .0 2 .0 3.0 0 0 to . 0 IO .0 7 .0 9 .0 2 . S i 2 .0 2.0 1 o a . S a . 3 7 .0 3 .0 2 .5 3 .0 2.0 1 0 10 .5 to .0 5 .0 9 . 5 2 .0 1 .0 2.0 0 0 3 .0 4 .0 3 .0 3 .0 3 .0 1 .0 2.0 0 o IO . 3 to .o 5 .o 9 .3 3 .0 l .o 3.0 o o 3 .0 4 .0 3 .0 3 .0 3 .0 l .o 3.0 o 0 10.0 10 .0 3 .0 10 .0 3 .5 2 .0 3.3 0 0 12 .0 12 .0 5 .0 12 .0 3 .3 2 .0 3.3 0 0 7 .0 7 .0 3 .0 3 .0 3 .5 2 .0 3.3 0 0 12 .0 12 .0 3 .0 1 1 .0 3 .0 2 .0 3.5 2 1 a .3 8 . 5 5 .0 9 .3 3 .0 2 .0 3.3 2 1 11 5 1 1 .0 3 .0 0 .0 3 .5 1 .3 3.0 1 0 9 .0 a .0 5 .0 2 .0 3 .5 1 .3 3.0 1 0 10 .0 to 0 3 .0 to .0 3 3 2 .0 4.0 1 0 6 .0 6 .0 3 .0 2 .0 3 .5 2 .0 4.0 1 0 10 0 10 .0 S .0 10 .0 3 .5 2 .0 4.0 1 0 6 0 6 .0 3 .0 2 .0 3 .5 2 .0 4.0 1 0 12 .0 12 .0 3 .0 12 .0 3 .3 1 .3 4.0 1 0 IO 0 to 0 3 .0 11 .0 3 .3 1 .3 4.0 1 0 8 0 7 0 3 .0 5 0 3 .3 1 .3 4.0 1 0 10 0 to .0 3 0 9 .0 3 .5 2 .0 4.0 1 0 a 0 a 0 5 0 2 .0 3 .3 2 .0 4.0 1 0 10 0 to .0 3 .0 to .0 3 .0 2 .0 2.0 0 0 a 0 4 0 5 0 7 o 3 0 2 0 2.0 0 0 io 0 10 .0 3 0 to .0 2 .0 2 .0 2.0 1 0 a 0 4 0 3 0 7 .0 3 0 2 .0 1 2.0 1 0 10 0 10 0 3 .0 9 .0 2 .0 2 .0 2.0 0 0 a 0 8 .0 3 .0 4 .0 2 .0 2 .0 I 2.0 0 0 TABLE 15 (CONT.) 56 53A 0 0 P r 71 k A i 53A 0 0 P f 71 A A t 578 0 0 s M 66 A A 0 578 0 0 s H 66 A A 0 578 0 0 p M 66 A A t 578 • 0 p M 66 A A i 3BA 0 0 s H 66 A A 0 58A 0 D 5 H 66 A A 0 58A 0 0 P M 66 A A 0 S8A 0 D P H 66 A A 0 «OA 0 0 s M 70 A A 0 eoA 0 0 s M 79 A A 0 60A 0 0 s H 79 A A 0 60A 0 0 s H 79 A A 0 SOA D 0 P H 79 A A 0 SOA 0 D p M 79 A A 0 60A 0 0 p M 79 A A 0 60A 0 D p M 79 A A 0 60S D 0 s M 79 A A 0 608 0 0 s M 79 A A 0 60S 0 0 s H 79 A A 0 608 0 0 » H 79 A A 1 608 0 0 p M 79 A A 1 &oe D D p H 79 A A 1 608 0 0 p H 79 A A 1 638 • 0 s H 69 A A 0 638 0 D s M 69 A A 0 638 0 0 p M 69 A A 2 63B 0 0 p M 69 A A 2 618 0 0 s M 69 A A 0 6 IB 0 0 s H 69 A A 0 6 <B 0 0 p H 69 A A 2 sialo 0 p M C9 A A 2 62A 0 0 S F 43 A A 0 62A 0 0 s F 43 A A 0 62A 0 0 p F 43 A A 2 62A 0 0 p F 43 A A 2 62A 0 0 p F 43 A A 2 62A 0 0 p F 43 A A 2 64A 0 0 s F 43 A A 0 64 A 0 0 s F 43 A A 0 64A • 0 5 F 43 A A 0 64A 0 0 s F 43 A A 0 64A 0 D p F 43 A A 2 64A D 0 p F 43 A A 2 64A 0 0 p F 43 A A 2 64A D 0 p F 43 A A 2 628 0 • s M 61 A A 0 628 0 D s H 61 A A 0 628 0 0 s M 61 A A 0 62B 0 0 p M 61 A A 2 62B D 0 p M 61 A A 2 68A 0 0 s M 61 A A 0 68A 0 0 s M 61 A A 0 684 0 0 p M 61 A A 1 68A 0 0 p H 61 A A 1 66A D D s M 61 A A 0 66A 0 0 s M 61 A A 0 66A 0 0 s M 61 A A 0 66A D 0 s M 61 A A 0 66A D D p M 61 A A 1 66A 0 0 p H 61 A A 1 668 0 D s M 61 A A 0 668 0 0 s M 61 A A 0 66B D 0 p M 61 A A 1 66B 0 0 p M 61 A A 1 S98 0 0 s F 61 A A 0 69B 0 D s F 61 A A 0 698 0 D 5 F 61 A 0 69B 0 0 5 F 61 A A 0 698 0 0 0 F 61 A A 2 69B 0 D P F 61 A A 2 72A 0 0 S M 83 A A 72A 0 0 s H 82 A A 1 72A 0 0 P H 82 A A 72A 0 0 p M 82 A A 72B 0 D 5 M 82 A A 1 728 0 0 S M 82 A A 72B 0 0 5 M 82 A A 728 D 0 5 M 82 A A 72B 0 0 P M 82 A A « 72B 0 0 P M 82 * 1 VO vo IVC VO vo vo ivo IVO IVO IVO IVA VA VA IVA IVA IVA IVA VA VA IVA VA IVA VA VO vc vo vc vo vc VO vc VC vc vc vo vc vo VO vo VO vo vo vo vo VO VO vc vo vo vo IVC vc IVC vc vo vo vo vo vo vo vo vo vo VO vc vo vc vo vo vu vo vo vo vo vo vo IVD IVO IVO IVO 8 10 9 12 10 7 12 10 12 7 8 4.0 a.s 4.0 9.0 4.0 9.0 4.0 5.0 5.0 8.0 8.0 S.O 8.0 9.0 9.0 7.0 7.0 9.0 9 7 7 9 a a 9 9 12 a 12.0 8.0 7.0 3.0 10.0 1 1 .0 3.5 5.5 7.5 7.0 7.5 7.0 8.5 8.5 5.3 10 O 8 0 12.0 8.3 1 1 0 8.5 12.0 12.0 12.0 12.0 0.0 2.0 2.0 0.0 0.0 1 .0 1 .0 0.0 0.0 2.0 2.0 0.0 2.0 0.0 3.0 10.0 S.O 10.0 S.O 9.0 3.0 9.0 5 5 5 8 a a a 7 7 9.0 9.0 7.0 7.0 10.0 a.o a.o 9.0 9.0 12.0 7.0 12 7 9 3 1 1 5 5 7 7.0 7.5 7.0 a.o a.o 8.0 a.o a.o a.o 9.0 9.0 9.0 9.0 8.0 8.0 12.0 12.0 12.0 12.0 5.0 5.0 3.0 S.O 5.0 3.0 7.0 7.0 7.0 7.0 3.0 2.0 2.3 2.3 2.0 2.0 2.0 2.0 2 2 2 2 2 2 2 6 .0 .0 .0 .0 .0 0 .0 .0 6.0 6.0 6.0 6.0 6.0 3.0 5.0 3.0 S.O 3.0 S.O 3.0 s a a 8 8 a a .0 .o .o .o o .o 7.0 9.3 11.0 11 .0 9.3 9.3 11 12 10 8 12 10 12 a 9 2 9 O .0 .0 .0 .0 .0 .0 s.o a.o 7.0 7.0 7.0 7.0 7.0 6.0 6.0 6.0 6.0 3.0 5 5 S 5 S.O a.o s.o s.o 5 3 S 3. 5 5 5 5 3 5 5 3 5 3 S. 9.0 4.0 0.0 9.0 O 0 0 o 0 o 0 o 0 0 0 0 2.0 9.0 1.0! 9.0 1.0 2.0 2.0 1.0 9.0 1 .0 9.0 9.0 9.0 6.0 6.0 9.0 9.0 6.0 6.0 9.0 3.0 9.0 8.5 9.5 0.0 3.0 0.0 3.0 5.5 5.0 9.0 9.0 4.0 4.0 9.0 9.0 9.0 9.0 2.0 9.0 2.0 9.0 7.0 7.0 9.0 9.0 9.0 2.0 2.0 0.5 0 5 0.5 O.S O.S 0.5 0.5 0.5 0.5 0.3 0.5 O.S O.S 3.3 3.5 3.3 3.3 3.3 3.3 3.3 3.3 3.5 3.S 2.5 2.3 2.5 2.3 2.5 2.3 2.5 2.3 2.5 2.5 3.5 0.0 0.0 0 0 O 0 .0 0 .0 0 .0 .0 o o o-0 0 o o 0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0 O o o o o 0 0 0 o o o o 1.5 1.5 1.5 1.3 1.5 1.3 1.5 1.3 1.5 ' •» 57 2.4.2. Classification of colonic epithelial glycoproteins The epithelial glycoproteins were classified according to the diagnostic scheme of Reid et al. (1985d)(Figure 2). As is evident from Figure 2, the histochemical stains used in this study produce a unique staining pattern for eleven different classes of glycoproteins. In total, five general classes (designated I-V) can be identified using the KOH/AB1.0/PAPS method: Class I glycoproteins do not stain using this method, indicating the absence of both sialic acid and sulphate esters. Both Class II and III glycoproteins stain Aqua (A), reflecting the absence of sialic acid residues but the presence of sulphate esters. Conversely, Class IV glycoproteins stain magenta (M), indicating the absence of sulphate esters, but the presence of sialic acids. Finally, Class V glycoproteins stain purple (P), indicating the presence of both sialic acid residues and sulphate esters. Subdivision of the glycoproteins into nine subclasses is established by the staining of additional sections with the PBT/KOH/AB1.0/PAS, the PBT/KOH/AB2.5/PAS and the AB1.0/PAPS procedures. For example, Class Va stains Purple (P), Aqua (A), Aqua (A), and Purple (P) in the KOH/AB1.0/PAPS, PBT/KOH7AB1.0/PAS, PBT/KOH/AB2.5/PAS and the AB1.G7PAPS procedures, respectively. Using the last three procedures - the PAPT/KOH/Bh/PAS, PAT/KOH/Bh/PAS and the PBT/KOH/PAS - permits the identification of eleven distinct subclasses of glycoproteins. 58 FIGURE 3 D I F F E R E N T I A L DIAGNOSTIC S C H E M E FOR C L A S S I F Y I N G COLONIC E P I T H E L I A L G L Y C O P R O T E I N S S T E P S T A I N GLYCOPROT E I N K O H - A B 1 0 - P A P S IV I M 0 II III A A V P "Vd 'Va 1 1 1 ! 1 1 V a v c P B T - K O H - A B 1 0 - P A S M M 0 0 A A A P P P B T - K 0 H - A B 2 - 5 - P A S P P A 0 A A A P P A B 1 0 - P A P S M 0 M 0 A A P A P >vb ' V a 1 1 1 1 1 1 v a v c v b v d P A P - H 2 0 - T - K O H - B h - P A S P B M B 0 0 0 B M B P P A T - K O H - B h - P A S P B P B B 0 B B P B P P B T - K O H - P A S M M M 0 0 0 0 0 M M M 59 After having assessed the staining pattern, two observers assigned a histochemical class to the predominant glycoprotein in the tops and bottoms of the crypts. In some specimens, the predominant glycoprotein appeared to be the same throughout the length of the crypt (Slide 2), whereas in others, the glycoprotein in the tops of the crypts were distinct from that in the bases (Slide 3). 2.5. M O R P H O L O G I C A S S E S S M E N T This portion of the project was carried out in conjunction with Dr. David Owen of the Department of Pathology. Morphologic assessment was performed subsequent to histochemical assessment. The hematoxylin and eosin section from each specimen was arbitrarily divided into thirds and each third was asessed separately for its degree of inflammation and mucosal atrophy. A four-point intensity scale was used to assess both the degree of inflammation and mucosal atrophy; this scale varied from 0,0, indicating both the absence of inflammation and mucosal atrophy, respectively, to a maximum of 3,3, indicating the presence of severe inflammation and mucosal atrophy. Hence, a tissue assigned a value of 2,0 was considered to have intermediate inflammation but no mucosal atrophy. 2.5.1. Morphologic Assessment of Focal Changes and Field Changes Reid et al. (1985a) defined focal changes as the "existence of one or more crypts which stained blue to blue purple (0-1) with the PAPT/KOH/Bh/PAS procedure and 0-1 with the P B T / K O H / P A S procedure in sections which otherwise stained in the normal range"(Slides 4-10). In H & E slides, focals are not distinct from the 60 surrounding field (Slide 11). Therefore, the closest histochemical section to the H & E slide which demonstrated focal change, was matched exactly to the H & E slide, and a morphologic evaluation of the focal and its surrounding crypts was carried out according to the above criteria. A 'field change' was defined by Reid et aZ.(1985a) as a "generalized alteration in the staining of the epithelial glycoproteins such that the sum of the scores of the PBT/KOH/PAS and the PAPT/KOH/Bh/PAS procedures was less than 5. (Slides 12 and 13 show a normal pattern of staining for these two procedures, respectively). Field changes were subdivided into two categories: moderate and severe. Sections with moderate field change are those in which the sum of the PBT/KOH/PAS and the PAPT/KOH/Bh/PAS scores is less than 5 but greater than 2 (Slides 14 and 15, respectively). In sections possessing "severe" field changes, the combined score for the two procedures is less than or equal to 2 (Slides 16). As field changes are, by definition, a generalized phenomenon, matching of the histochemical slides to the H & E slides was not necessary. Therefore, morphologic assessment of specimens demonstrating field changes was carried out according to the above criteria. 2.5.2. Morphologic Criteria for Normal In the absence of gross pathologic features (ie. frank carcinoma, atypia or dysplasia) the morphologic criteria for normal was based on the absence of 61 mucosal atrophy or inflammation. Hence, when a tissue was assigned a value of zero for both inflammation and mucosal atrophy, it was considered to be normal by morphologic standards. 2.5.2.1. Morphologic Assessment of Diverticular Disease Cases The diverticulum and the adjacent surface mucosa from each specimen was assessed and recorded separately. In most cases of diverticular disease, the pathologic process found in the diverticulum did not extend into the adjacent surface area. The surface then, was generally classified as 0 for both degree of inflammation and mucosal atrophy. Because morphologically 'normal' tissue is difficult to obtain, the surface area from the diverticular disease specimens was often compared to ulcerative colitis and Crohn's Disease specimens, and in this respect served as a control. 2.6. D A T A A N A L Y S I S Data was analyzed using nonparametric statistics (Chi-Square Analysis, Spearman Rank Correlation Coefficient and Mann-Whitney U Test) (Zar, 1984). 3. RESULTS 3.1. MORPHOLOGICAL RESULTS 3.1.1. Relationship Between Inflammation and Mucosal Atrophy Analysis showed there was a statistically significant correlation (p<.001, df = 9) between the degree of inflammation and the degree of mucosal atrophy. Therefore, further assessment of the relation of any histochemical parameter to the severity of disease, was based solely on the degree of inflammation. 3.2. HISTOCHEMICAL RESULTS 3.2.1. Evaluation of the Relative Proportion of Sialomucin and Sulphomucin using the KOH/AB1.07PAPS procedure Column 14 in Tables 13, 14 and 15 shows the relative proportion of sialomucin and sulphomucin in the upper and lower portions of the crypts of the ascending and descending colon for each disease. As will be seen, only one case of diverticular disease from the ascending colon was examined. Therefore, this case was omitted from the statistical analysis of the ascending colon. 62 63 The data presented in Tables 16 and 17 consists of a statistical comparison between:-1. the bottoms and tops of the crypts for each disease; 2. diseases for the tops of the crypts; 3. diseases for the bases of the crypts. In these analyses, the diverticula and the uninvolved surface mucosa of the diverticular disease specimens were considered separately. 3.2.1.1. Assessment of the relative proportion of sialomucins and sulphomucins from the descending colon The results obtained (Table 16 a-c) indicated that the glycoproteins in the bases of the crypts from diverticular disease and ulcerative colitis were significantly more sulphated than those in the tops of the crypts. In contrast, no differences between the bases and tops of the crypts were detected in Crohn's Disease specimens (Table 16a). No differences between disease classes were detected in the glycoproteins from the tops of the crypts (Table 16b). However, the glycoproteins in the bases in specimens from ulcerative colitis and Crohn's Disease were significantly less sulphated than those from the surface mucosa adjacent to diverticula. Further, the glycoproteins in the diverticulum were more sulphated than those in Crohn's Disease specimens but did not differ from those in ulcerative colitis specimens. 64 T A B L E 16 R E L A T I V E PROPORTION O F SIALO- A N D S U L P H O M U C I N S IN T H E D E S C E N D I N G C O L O N AS A SSESSED B Y T H E KOH/AB1.0/PAPS P R O C E D U R E A. Tops Versus Bases of Crypts for each Disease Disease P Value a. DI(S) b. DI(D) c. U C d. CD 0.0001 0.0001 0.0212 0.2033 B. Between-disease Comparison of Tops of Crypts a. DI(S) vs. CD b. DI(S) vs. U C c. DI(D) vs. CD d. DI(D) vs. U C e. DI(S) vs. DI(D) f. U C vs. CD 0.8389 0.7673 0.2451 0.9990 0.7091 0.6331 C. Between-disease Comparison of Bases of Crypts a. DI(S) vs. CD b. DI(S) vs. U C c. DI(D) vs. CD d. DI(D) vs. U C e. DI(S) vs. DI(D) f. U C vs. CD 0.0002 0.0239 0.0059 0.0901 0.1635 0.1314 Results Significant Significant Significant Not Significant Not significant Not Significant Not Significant Not Significant Not Significant Not Significant Significant Significant Significant Not Significant Not Significant Not Significant DI(S)=Diverticular disease - surface mucosa; DI(D) = Diverticular disease diverticulum; U C = Ulcerative Colitis; CD = Crohn's Disease 65 3.2.1.2. Assessment of the relative proportion of sialomucins and sulphomucins from the ascending colon As shown in Table 17(a-c), the results obtained indicated that no difference in sulphation existed between the bases and tops of the crypts for either ulcerative colitis or Crohn's Disease specimens (Table 17a). A between-disease comparison of the tops of the crypts revealed a significant difference between ulcerative colitis and Crohn's Disease specimens, indicating that the former were significantly less sulphated than the latter. However, no difference in the glycoproteins from the bases of the crypts was demonstrated between ulcerative colitis and Crohn's Disease specimens. 3.2.1.3. Percentage and Number of Cases and Specimens Showing A Predominance of Sialomucins Sections were defined as containing a predominance of sialomucins when they were red to red-purple (R-RP) or Red (R) in the KOH/AB1.07PAPS in both the tops and corresponding bottoms of a particular specimen (Slide 17). In this evaluation, some of the specimens showed a predominance of sialomucins throughout the entire tissue sample, while in others, only a part of the specimen fulfilled this criterion. The results, as shown in Table 18, are for the entire colon - no distinction was made between the ascending and descending colon. Furthermore, results obtained from the surface mucosa adjacent to diverticula and diverticula were combined and considered as one group, diverticular disease. 66 As will be seen, at least 15% of all specimens and at least 20% of all cases from all disease categories showed a predominance of sialomucins. 67 T A B L E 17 R E L A T I V E PROPORTION O F SIALO- A N D S U L P H O M U C I N S IN T H E A S C E N D I N G C O L O N AS ASSESSED B Y T H E KOH/AB1.0/PAPS P R O C E D U R E A. Tops vs. Bases of Crypts for each Disease  Disease Results a. CD Not Significant b. U C Not Significant B. Between-disease Comparison of Tops of Crypts a. U C vs. CD Significant (p<.05) C. Between-disease Comparison of Bases of Crypts a. U C vs. CD Not Significant U C = Ulcerative Colitis; CD = Crohn's disease. 68 T A B L E 18 P E R C E N T A G E A N D N U M B E R OF CASES A N D SPECIMENS SHOWING A P R E D O M I N A N C E O F SIALOMUCINS IN T H E KOH/AB1.0 /PAPS P R O C E D U R E Ulcerative Colitis Crohn's Disease Diverticular Disease Specimens 22.8% (8)* 15.7% (8) 16.2% (6) Cases 41.0% (7) 28.6% (6) 21.1% (4) l:values in brackets are actual numbers of cases or specimens. 3.2.2. Severity of Disease Versus Histochemical Class 69 Statistical analyses were performed to determine whether or not a relationship existed between the type of glycoprotein present in a specimen and the severity of disease. Without exception, the degree of inflammation was the same for the top and bottom of a particular crypt. However, the epithelial glycoproteins present in the top often differed from those in the bottom. Therefore, analysis of these two parameters was performed for each disease, each anatomic area (ascending/descending) and each level in the crypt (top/bottom). The results summarized in Table 19 show that disease severity and histochemical class are independent of one another, ie) the degree of inflammation is not associated with any particular class of glycoprotein. 3.2.3. Disease Versus Histochemical Class Statistical analysis was performed to ascertain if any disease was associated with a particular class of epithelial glycoprotein. Analysis of these two parameters was performed for each anatomic area (ascending/descending) and each level in the crypt (top/bottom). As shown in Table 20, no relationships exists between histochemical class and disease, ie. no particular class of glycoprotein is associated with any particular disease. 70 T A B L E 19 A S S E S S M E N T OF T H E D E G R E E OF I N F L A M M A T I O N VS. C L A S S USING T H E CHI S Q U A R E T E S T H I S T O C H E M I C A L Disease Crypt/ Anatomic Location df Results U C CD DI(S) DI(D) All diseases Ascending-top 6 Ascending-bottom 6 Descending-top 15 Descending-bottom 12 Ascending-top 8 Ascending-bottom 15 Descending-top 6 Descending-bottom 8 Descending-top 5 Descending-bottom 5 Descending-top 10 Descending-bottom 10 Ascending-top 15 Ascending-bottom 15 Descending-top 15 Descending-bottom 15 NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS U C = Ulcerative Colitis; CD = Crohn's Disease; DI(S) Diverticular disease, surface mucosa: DI(D) = Diverticular Disease, diverticulum; NS = not significant. 71 T A B L E 20 A S S E S S M E N T OF H I S T O C H E M I C A L CLASS V E R S U S DISEASE USING CHI S Q U A R E A N A L Y S I S Anatomic/Crypt Location df Results a. Ascending/Top 15 NS* b. Ascending/Bottom 15 NS c. Descending/Top 15 NS d. Descending/Bottom 15 NS NS = Not significant. 3.2.4. Degree of Sulphation Versus Degree of Acetylation 72 Previous work (Reid et al., 1985a) on the mucosa adjacent to colonic tumors suggests than an abnormal staining pattern for side chain O-acetylated sialic acid is not necessarily associated with a concomitant abnormal staining pattern for sulphomucins. However, in that study, statistical analysis was not performed to assess the presence or absence of association between these two variables. Therefore, analysis was carried out for each disease and for each area within the disease to evaluate whether or not a relationship exists between the pattern of O-acetylation, as revealed by the P B T / K O H / P A S stain, and the pattern of sialo- and sulphomucins, as revealed by the KOH/AB1.0 /PAPS stain. The results, as summarized in Table 21, show that changes in side chain O-acetylated sialic acid are independent of changes in the proportion of sulpho-and sialomucins. 3.2.5. Assessment of O-Acetylated Sialic Acids The pattern of side-chain O-acetylated sialic acid was assessed for each section as being: 1. normal 2. focal change 3. moderate field change, or 4. severe field change, according to the criteria of Reid et a/.(1985a). 73 T A B L E 21 A S S E S S M E N T OF T H E D E G R E E O F S U L F A T I O N V E R S U S D E G R E E OF SIDE-CHAIN O - A C E T Y L A T I O N USING T H E S P E A R M A N R A N K C O R R E L A T I O N C O E F F I C I E N T Disease Anatomic/Crypt Results Location Crohn's disease Ascending/Top NS Ascending/Bottom NS Descending/Top NS Descending/Bottom NS Ulcerative Colitis Ascending/Top NS Ascending/Bottom NS Descending/Top NS Descending/Bottom NS Diverticular disease Descending/Top NS (Surface mucosa) Descending/Bottom NS Diverticular disease Descending/Top NS (Diverticulum) Descending/Bottom NS NS = not significant 74 T A B L E 22 A N A L Y S I S OF O - A C E T Y L A T E D SIDE-CHAIN SIALIC ACID (BY SPECIMEN) Disease Normal Moderate Severe Focal T O T A L DI(S) 22 4.5 3 6.5 36* DI(D) 28 5 2 0 35** U C 23 3 9 0 35 CD 30 11.5 6 3.5 51 M C T 15 7 14 26 62 T O T A L 118 31 34 36 219 DI(S) = Diverticular disease, surface mucosa; DI(D) = Diverticular disease, diverticulum; U C = Ulcerative colitis; CD = Crohn's disease; MCT=Mucosa close to tumors. * 1/37 specimens - no mucosal surface component ** 2/37 specimens - no diverticulum component 75 The actual number of normal, focal and field changes for each specimen from each disease are shown in Table 22. These were compared to the results obtained from a previous study (Reid et al., 1985a) on the mucosa adjacent to tumors. Statistical analysis of the data in Table 22 suggests that some patterns of O-acetylated sialic acid are more closely associated with a particular disease than others. The results, as summarized in Table 23 show that, as a group the glycoproteins from the mucosa adjacent to tumors (MCT) are statistically different from any of the three inflammatory bowel diseases. Further, comparisons between inflammatory bowel diseases revealed that only ulcerative colitis and the mucosa adjacent to diverticula differed significantly from each other in their O-acetyl substitution pattern (.01<p<.025). Subdivision of the contingency tables for between-disease comparisons showed that the significant difference between ulcerative colitis and the the mucosa adjacent to diverticula was due to the absence of focal changes in the former and their presence in the latter disease (.005<p<.01). Similarly, the significant difference observed between the mucosa adjacent to tumors and ulcerative colitis, Crohn's Disease, the diverticula and the mucosa adjacent to diverticula is apparently due to the higher number of focal changes in the mucosa adjacent to tumors, (p<.001), (p<.001), (p<.001), (.01<p<.025), respectively. 76 T A B L E 23 A S S E S S M E N T O F O - A C E T Y L A T E D SIALIC ACID WITH R E S P E C T TO DISEASE USING CHI S Q U A R E A N A L Y S I S Disease df _P_ Value DI(S) vs. DI(D) . 3 NS DI(S) vs. U C 3 .01<p<.025 DI(S) vs. CD 3 NS DI(S) vs. M C T 3 .001<p<.005 DI(D) vs. U C 3 NS DI(D) vs. CD 3 NS DI(D) vs. M C T 3 p<.001 U C vs. CD 3 NS U C vs. M C T 3 p<.001 CD vs. M C T 3 p<.001 DI(S) = Diverticular disease, surface mucosa; DI(D) = Diverticular disease, diverticula; U C = Ulcerative colitis; CD = Crohn's disease ;MCT=Mucosa close to tumors; NS = not significant. 4. DISCUSSION In general, results from previous studies of human colonic epithelial glycoproteins have been based on a relatively simple histochemical technique, namely the HID/AB2.5 procedure. Several investigators are of the opinion that results from this procedure should be interpreted more cautiously (Lev et al., 1985, McFadden et al., 1985, Rhodes et al., 1985c). It was the intent of this study, therefore, to more fully characterize the histochemical changes seen in ulcerative colitis, Crohn's Disease and diverticular disease with a wider array of more sophisticated histochemical procedures. The histochemical stains used in this project allowed for a more complete characterization of epithelial glycoproteins in these various diseases, and therefore it was anticipated that the issue of whether or not the histochemical changes seen in the mucosa adjacent to colonic tumors represented primary or secondary phenomena could be resolved. A further objective of this study was to assess whether or not ulcerative colitis and Crohn's Disease could be distinguished from each other. In this study the results obtained from the surface mucosa adjacent to diverticula were used for comparative purposes as controls because the disease is not associated with an increased cancer risk and the very great majority of the specimens were histologically normal, the remainder showing only very mild inflammation. 77 78 4.1. HISTOCHEMICAL CHARACTERISTICS OF ULCERATIVE COLITIS, CROHN'S DISEASE AND DIVERTICULAR DISEASE 4.1.1. Assessment of the relative proportion of sialomucins and sulphomucins Based on results from the H1D/AB2.5 procedure, previous studies (Gad, 1969a; Filipe & Branfoot, 1976; Sheahan & Jervis, 1976; Filipe, 1969, 1979) have shown that in the normal descending colon the bases of the crypts contain proportionately more sulphate than the tops of the crypts, while in the ascending colon the lower portions of the crypts contain relatively less sulphate than to the upper portions. The results obtained from the surface mucosa adjacent to diverticula were consistent with that expected from normal as they showed a sulphomucin gradient along the length of the crypts, the bases and lower halves being more sulphated than the upper halves in the descending colon. A statistical comparison between the diverticula and surface mucosa revealed no difference between the two, indicating that histochemically, the diverticula possess a normal pattern of sulphomucins and sialomucins. In the descending colon, specimens from ulcerative colitis showed a statistically significant difference between the bases and tops of the crypts, the bases being more heavily sulphated than the tops. This finding is in keeping with previous descriptions of normal sulphomucin and sialomucin distribution (Gad, 1969a; Filipe & Branfoot, 1976; Sheahan & Jervis, 1976; Filipe, 1969, 1979). However, a between-disease comparison of the bases of the crypts from the descending colon 79 showed that a statistical difference existed between ulcerative colitis and the surface mucosa adjacent to diverticula but not for ulcerative colitis and the diverticula. This finding implies that ulcerative colitis deviates somewhat from the normal histochemical pattern. This finding is in accord with previous studies (Ehsanullah et al, 1982b; Franzin et al, 1983a; Filipe et al., 1985). A significant finding in this study was the absence of a statistical difference between the bases and tops of the crypts from Crohn's Disease specimens in all portions of the colon. This finding in Crohn's Disease specimens reflects the absence of the normal pattern of sulphomucin distribution in the descending colon. This deviation from normal probably did not originate in the upper portions of the crypts as there was no statistical difference between Crohn's Disease and diverticular disease specimens. There was, however, a statistically significant difference between the bases of the crypts in Crohn's Disease specimens and those in the diverticula and the surface mucosa adjacent to diverticula, indicating that the abnormalities exist in the relative proportion of sulphomucins and sialomucins in the bases of the crypts, the alteration being a reduction in sulphation. This difference from normal has not been reported previously. However, it is interesting that Filipe (1969) noted that in transitional mucosa the decrease in sulphate begins in the lower crypts. In the ascending colon there was no statistically significant difference between the bases and tops of the crypts in either ulcerative colitis or Crohn's Disease, indicating the loss of a sulphate gradient. The absence of such a gradient in the ascending colon was presumably due to the loss of sulphate in the upper portion 80 of the crypts, as in the normal ascending colon the tops of the crypts are more heavily sulphated than the bases. As no diverticular disease specimens were obtained from the ascending colon, it was not possible to make comparisons between the normal pattern of sulpho- and sialomucin distribution and those patterns found in either ulcerative colitis or Crohn's Disease. In summary, in the descending colon, a sulphate gradient was demonstrated between the bases and tops of the crypts in ulcerative colitis and diverticular disease specimens. These results for the distribution of sulphomucins are in keeping with previous descriptions of diverticular disease (Filipe, 1969; Reid et al., 1984a; Habib et al., 1986) and for ulcerative colitis uncomplicated by dysplasia (Habib et al., 1986). The absence of a sulphate gradient in Crohn's Disease specimens from all portions of the colon has not been reported previously. However, the use of the KOH/AB1.0/PAPS procedure instead of the HID/AB2.5 may be responsible for this finding, as McFadden et al. (1985) note that former technique is a more sensitive indicator of sulphomucin distribution. 4.1.2. Patterns of O-acetylated sialic acid Culling et al. (1979) previously reported that, as compared to normal, the proportion of C7C8 substituted sialic acid was reduced in both ulcerative colitis (Reid et al., 1984a) and Crohn's Disease specimens, with the reduction being greater in the former. This finding was not confirmed in the present study because no statistical difference was observed in the O-acetyl substitution pattern between the diverticulum and either ulcercative colitis or Crohn's Disease 81 specimens or between the mucosa adjacent to diverticulum specimens and Crohn's Disease specimens. As these conclusions are based on between-disease comparisons, it is possible that the reason why ulcerative colitis and Crohn's Disease specimens do not differ significantly from diverticular disease specimens is because the latter do not truly reflect the normal pattern of O-acetylated sialic acid. However, Reid et al., (1984a) report that diverticular disease specimens possess an O-acyl staining pattern very similar to normal and it is almost certainly not .the case that the results from this study are due to selection of diverticular disease specimens which do not possess a normal O-acyl substitution pattern. Further, the lack of statistical difference between the mucosa adjacent to diverticula and the diverticula indicated that the sialic acid O-acyl substitution pattern was uniform throughout this condition. A statistically significant difference (.01<p<.025) was observed between the mucosa adjacent to diverticula and ulcerative colitis specimens. The presence of 6.5 focal changes in the former (Table 22) and their absence in the latter disease was apparently responsible for this finding (.005<p<.01) rather than an overall field reduction of C7C8 substituted sialic acid. The O-acyl substitution pattern in each of the three inflammatory bowel diseases differed significantly from that found in the mucosa adjacent to tumors. In all instances, these differences arose from a proportionately greater number of focal changes in the mucosa adjacent to tumors than in ulcerative colitis, Crohn's Disease or diverticular disease specimens. In no instances, were moderate field changes responsible for the significant difference observed between the mucosa 82 adjacent to tumors and the three inflammatory bowel diseases. However, severe field changes (.025<p<.05), in combination with focal changes, were apparently responsible for the significant difference observed between the mucosa adjacent to tumors and the diverticulum specimens. It was found that of the two histochemical parameters examined, ie) the relative proportion of sulphomucin and sialomucin and the pattern of O-acetylated sialic acid, a histochemically abnormal pattern for one parameter was not associated with an alteration in the other (Table 21). These results are in keeping with those reported by Reid et al. (1985a), in which, for example, 25.8% of mucosal specimens adjacent to tumors showed a normal distribution of sulphomucins in the presence of an abnormal pattern of O-acetylated sialic acid. 4.2. D I F F E R E N T I A L D I A G N O S I S O F U L C E R A T I V E COLITIS A N D C R O H N ' S D I S E A S E From a diagnostic point of view, any technique is only useful if it consistently differentiates one disease from another. Although some statistical differences were found between ulcerative colitis and Crohn's Disease by both histochemical parameters examined, no useful diagnostic patterns could be identified for either disease. The data shown in Table 22 indicate that focal changes are exclusively confined to Crohn's disease. However, the use of focal changes as an indicator of Crohn's disease is of little value as: 1. the incidence of focals is so low (8%) that only a very small percentage of 83 Crohn's Disease cases can be distinguished by this criterion, 2. focal changes also appear in the surface mucosa adjacent to diverticula and in the mucosa adjacent to tumors (Reid et al., 1985a), indicating that these changes are not specific for Crohn's Disease. It is doubtful, therefore, that the histochemical techniques employed in this study would be of any value to the diagnostic pathologist. 4.3. PREMALIGNANT MARKERS OR NONSPECIFIC CHANGES? It has been demonstrated by Morson and Pang, (1967); Ehsanullah et al., (1985) and Ransohoff et al.,(1985) that the degree of dysplasia is a significant indicator of malignant transformation. Recently, Ransohoff et al (1985) found dysplasia at a distance from colonic cancer and Filipe et al., (1985) noted that 4/8 cases of ulcerative colitis developed cancer with no evidence of dysplasia. These findings have prompted investigators to suggest that a weaker association between colonic cancer and dysplasia might exist than has previously been reported. Therefore, although it is generally believed that dysplasia is the histologic precursor to cancer (Morson & Pang, 1967), whether or not its absence rules out the possibility of concurrent cancer is still a question. As a consequence, this dilemma has prompted investigators to search for more diagnostically ideal premalignant markers. It would seem that the ideal premalignant marker should meet the following criteria:-1. It must precede morphologically identifiable malignant transformation; 84 2. It must be specific for malignancy; 3. Its sensitivity must be high ie) it should detect very early manifestations of malignant transformation; 4. It must be easily recognized; 5. Its absence must exclude the possibility of cancer ie. it must be present in all cases of premalignanc}'. In this study the design of the project was such that only the second criterion ie. specificity, could be addressed. The issue of specificitj' is of particular importance with respect to whether or not the changes noted in the mucosa adjacent to tumors are premalignant or nonspecific. In general, those who favor the view that the changes seen are nonspecific rely on demonstrating that the predominance of sialomucins seen in the mucosa adjacent to tumors are also seen in a wide array of diseases not associated with an increased malignant potential (Saffos & Rhatigan, 1977; Isaacson & Attwood, 1979; Rhatigan & Saffos, 1979; Listinsky & Riddell, 1981; Franzin et al., 1981, 1983a, 1983b). In the present study, the acceptance or rejection of the premalignant marker hypothesis was based on a similar rationale. The results from the assessment of the relative proportion of sulpho- and sialomucins from ulcerative colitis, Crohn's Disease and diverticular disease were not directly compared to those obtained from the mucosa adjacent to tumors. However, inspection of Table 18 showed that in some instances, all, or a major portion of some specimens, showed a predominance of sialomucins throughout the entire crypt. As Table 18 clearly demonstrates, the percentage of cases and specimens which demonstrated this characteristic was relatively high. Barring that all these cases later developed colonic cancer, these results favor the conclusion that the presence of a predominance of sialomucins is nonspecific. It is doubtful, therefore, whether further investigations of this particular histochemical parameter as an indicator of premalignancy would serve any useful purpose. A statistical comparison of the O-acetyl substitution pattern from ulcerative colitis, Crohn's Disease and diverticular disease with those from the mucosa adjacent to tumors indicated that, without exception, the O-acetyl substitution from all these diseases differed significantly from the patterns observed in the mucosa adjacent to tumors (Table 23). However, as Table 22 shows, a unique alteration in the O-acyl pattern was not found in the mucosa adjacent to tumors. Rather, as a whole, this group showed an O-acyl substitution profile that was significantly different from that seen in the other diseases. The most probable explanation for this significant difference between the inflammatory bowel diseases and the mucosa adjacent to tumors is that a smaller proportion of focal changes were present in the former than in the latter. The results from this study argue against the hypothesis that focal changes represent a premalignant marker. Over 50% of the focal changes found in the present study were identified in the surface mucosa adjacent to diverticula, a disease with no known cancer risk. Conversely, ulcerative colitis, the disease associated with the greatest risk of cancer, was totally without focal change. Although there was no unique O-acyl substitution pattern such that it fulfilled the necessary criteria for a useful premalignant marker, it is quite clear that 86 some change is occurring in the mucosa adjacent to tumors which produce alterations in the O-acyl substitution pattern of sialic acid. The significance of these changes, however, remains to be elucidated. 4.4. T H E U S E F U L N E S S O F T H E D I F F E R E N T I A L D I A G N O S T I C S C H E M E In some respects, the differential diagnostic scheme, originally described by Reid et al. (1985d) was of some value in analyzing the data presented in this study. There is no doubt that the redundancy which is built into this scheme prevents erroneous results from going undetected. Thus, for example, one would question results in which two serial sections from the same specimen were purple in the KOH/AB1.0/PAPS procedure and red in the P B T / K O H / A B l . O / P A S procedure whereas such a chemically inconsistent finding would failed to be detected if only one of the techniques was used. From this point of view then, the scheme is highly useful. Within the context of this project, the diagnostic scheme was used to assess whether or not the degree of inflammation was related to a particular histochemical class. Franzin et al. (1983a, 1983b), and Lev et al. (1985) who support the view that the presence of sialomucins is a nonspecific event, suggest that their occurrence may be a response to ischemia or inflammation. As the diagnostic scheme distinguishes between those epithelial glycoproteins which contain sialic acid but no O-sulphate esters (Class IV) and those which possess both (Class V), one would expect to see a significant proportion of Class IV glycoproteins associated with a high degree of inflammation, if such changes are 87 due to inflammation. However, as no association was observed between histochemical class and inflammation, it was concluded that the changes seen in the mucosa of inflammatory bowel diseases are not due to inflammation. In assessing the effect of inflammation then, the diagnostic scheme was useful as it enabled one to eliminate one of the possible cases of histochemical change. Culling et al. (1979) showed that decreases in O-acetylated sialic acid were more pronounced in more severely inflamed tissues. This finding was not confirmed in the present study. The HID/AB2.5 procedure does not distinguish between any histochemical classes of glycoproteins. Because this scheme does differentiate between eleven different classes of epithelial glycoproteins it was useful in showing that none of the inflammatory bowel diseases were associated with a particular histochemical class. A further advantage of this scheme was in its detection of focal changes. These focal changes, when viewed in only one serial could either be missed or considered artifactual in nature. When these changes are seen throughout a number of serials, the chances of not detecting them are reduced and the possibility of the change being an artifact of one method is eliminated. Despite the benefits, there are disadvantages to using this scheme. They are:-1. The procedures used are extremely time-consuming. From a technical point of view, this consideration cannot be underestimated. 2. The IVd and Vd classes of glycoproteins contain too many possible structures. 88 3. The scheme does not distinguish between the various gradations of colors ie. blue-purple, purple, royal-blue, but rather classifies all under the heading of 'Purple. Thus, information on the relative proportion of components is lost. 4. In sections which are not homogeneous for a particular class of glycoprotein, interpretation of results can be extremely difficult, tedious and time-consuming. In more than a few specimens the sections were quite 'scrambled' such that some crypts were purple, while others were red, red-purple or aqua, etc. Thus in order to correctly classify the glycoproteins, exact matching of crypts from one serial to another was often necessary. 5. Correct classification of the epithelial glycoproteins usually necessitated the use of all seven stains. However, some stains, ie. the PBT/KOH7AB2.5/PAS, which are necessary to correctly classify the glycoproteins, yield little useful information in and of themselves. One questions, therefore, whether or not their inclusion in the scheme for the purposes of classification is not outweighed by the fact that the procedure is relatively time-consuming. 4.5. FUTURE CONSIDERATIONS FOR A PROJECT OF THIS NATURE Two possible alterations in the design of this project would have undoubtedly reduced the time it took to interpret the data. They are:-1. In this project histochemical assessment preceded morphologic assessment. As mentioned previously, in the morphologic assessment, the tissue was arbitrarily divided into thirds and each third was scored for the degree of inflammation and the degree of mucosal atrophy. In the histochemical assessment, however, no such division of the specimen was made. In specimens where each or one of the 89 thirds was scored differently from the other, all the histochemical slides had to be reviewed again so as to match each third precisely with the corresponding morphology. Had all the specimens stained one color for a particular stain, this review, of course, would not have been necessary. However, more often than not it was the case that the specimen was not homogeneous. Thus, this "re-review" of the histochemistry was very time-consuming and it would therefore have been more time-efficient to have assessed the morphology first. 2. With the exception of allowing for the histochemical classification of the epithelial glycoproteins, in this particular study very little useful information was gained from carrying out the PBT/KOH/AB2.5/PAS, the PAT/KOH/Bh/PAS or the AB1.0/PAPS procedures. As both the techniques and the analysis were very time-consuming, it is questionable whether they should be included in future designs such as this. 5. CONCLUSIONS In summary, the results from this study indicate that: 1. It is not possible to differentiate between ulcerative colitis and Crohn's Disease on the basis of the histochemical techniques used in this study. 2. The presence of sialomucins, or conversely, the absence of sulphomucins, does not appear to be specific to the mucosa adjacent to tumors as such a phenomenon was noted in all three disease entities studied. 3. The presence of focal change, moderate field change or severe field change does not appear to be specific to the mucosa adjacent to tumors. 4. There is a quantitative but not a qualitative difference in the alterations in O-acetylated sialic acid between inflammatory bowel diseases and the mucosa adjacent to tumors. 5. The histochemical changes observed in ulcerative colitis, Crohn's Disease and diverticular disease are not related to inflammation. 6 . Changes in O-acetylated sialic acid are independent of changes in the relative proportion of sulphomucin and sialomucins. 7. Crohn's Disease specimens from both the ascending and descending colon showed an absence of a sulphomucin gradient in the crypts. 90 REFERENCES Allan A, Bristol JB , Williamson R C N (1985c). 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