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

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A  HISTOCHEMICAL  GLYCOPROTEINS  ANALYSIS  OF T H E COLONIC  F R O M U L C E R A T I V E COLITIS, DIVERTICULAR  EPITHELIAL  CROHN'S  DISEASE  AND  DISEASE  by ELIZABETH  A THESIS SUBMITTED THE  ANN  ATKINS  IN P A R T I A L  REQUIREMENTS MASTER  F U L F I L M E N T OF  FOR T H E DEGREE OF OF SCIENCE  in THE  F A C U L T Y OF GRADUATE Department  of  Pathology  We accept this thesis as to the  THE  conforming  required standard  UNIVERSITY  O F BRITISH  Spring  °  STUDIES  COLUMBIA  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  ABSTRACT  The  aim  epithelial  of  the  glycoproteins  premalignant was  present  to  seen  markers  assess  study  or  in  was the  secondary  whether  to  assess  mucosa reactive  ulcerative  whether  adjacent  to  phenomena.  colitis  and  the  changes  tumors A  in  are  secondary  the  specific objective  Crohn's  Disease  could  epithelial  glycoproteins  be  distinguished from one another histochemically.  The  carbohydrate  prosthetic  characterized histologically 21  cases  of  histochemical the  Crohn's  using  a  specimen  were  addition,  the  inflammatory  - the  substitution  battery  of also  and relative  pattern  seven  colonic  of  of  19  cases  of  O-acetylated  sialic  O-acetylated  side-chain  acid  -  disease.  were  sections  hematoxylin sialic  colitis, Two  and sialomucin and  Serial  using  were  cases of ulcerative diverticular  techniques.  morphologically,  bowel diseases were  17  proportion of sulpho-  histochemical  evaluated  patterns  from  and histochemically from Disease  parameters  side-chain  groups  acid  assessed  from  and  from  each  eosin. the  In  three  compared to data previously acquired from  the  mucosa adjacent to colonic tumors.  Results are  indicate  specific  that  to  the  neither mucosa  histochemical parameter were histochemical  class  of  inflammatory  bowel  diseases  between ulcerative  colitis  focal  changes  adjacent  to  nor the  predominance of  tumors.  As  independent of changes  epithelial and,  glycoproteins  was  therefore,  was  and Crohn's Disease  ii  it  well,  in the  on the  changes  to  possible  basis  in  one  other parameter. No  specific not  sialomucins  of the  any to  of  the  distinguish  histochemical  techniques  used  in the  present  changes in ulcerative colitis, related  to  specimens  the  degree  showed  a  of  study.  It  was  also noted  that  the  histochemical  Crohn's Disease and diverticular specimens inflammation.  loss of  Finally,  as  a  group,  sulphomucin-sialomucin gradient  the crypts.  iii  were not  Crohn's  along the  Disease  length  of  TABLE  OF CONTENTS  Abstract  ii  L i s t o f Tables  vii  L i s t o f Figures  ix  L i s t o f Slides  x  Abbreviations  xi  Acknowledgements Figure  xii  1  xiii  1. Introduction 1.1. 1.2. 1.3.  1.4. 1.5. 1.6. 1.7. 1.8. 1.9. 1.10. 1.11. 1.12.  1  Objectives 1 G e n e r a l Macroscopic a n d Microscopic F e a t u r e s o f N o r m a l Colon 1 Pathologic Description of D i v e r t i c u l a r Disease, Ulcerative Colitis, Crohn's D i s e a s e , a n d T r a n s i t i o n a l M u c o s a 2 1.3.1. D i v e r t i c u l a r Disease 2 1.3.2. C r o h n ' s Disease a n d U l c e r a t i v e Colitis 3 1.3.3. T r a n s i t i o n a l M u c o s a 6 Histochemical Characterization of Normal Colonic Epithelial Glycoproteins 8 Characteristics o f Colonic E p i t h e l i a l Glycoproteins i n T u m o r s 11 Characteristics o f Colonic E p i t h e l i a l Glycoproteins Adjacent to T u m o r s (Transitional Mucosa) and Remote from T u m o r s 17 Characteristics of Colonic E p i t h e l i a l Glycoproteins i n Crohn's Disease .. 22 Characteristics o f E p i t h e l i a l Glycoproteins from U l c e r a t i v e Colitis .. 25 Characteristics o f E p i t h e l i a l Glycoproteins Associated with Benign Pathological Conditions 30 Premalignant M a r k e r s 33 Incidence of Colonic C a n c e r in U l c e r a t i v e Colitis, C r o h n ' s Disease a n d D i v e r t i c u l a r Disease 34 Thesis Rationale 35  2. M a t e r i a l s a n d Methods 2.1. Tissue 2.2. S t a i n i n g Procedures 2.2.1. H i s t o c h e m i c a l Procedures 2.2.1.1. Periodic acid oxidation (PA) 2.2.1.2. Borohydride reduction (Bh) 2.2.1.3. Saponification (KOH) 2.2.1.4. Phenylhydrazine Block Schiff (PAPS or PAPT) 43 2.2.1.5. Cationic Dye Methods with Alcian Blue 2.3. S t a i n i n g of S e r i a l Sections  iv  38 38 40 43 43 43 43 .... 44 44  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 K O H / A B 1 . 0 / P A P S 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  4.2. 4.3. 4.4. 4.5.  4.1.2. Patterns of O-acetylated sialic acid 80 Differential Diagnosis of Ulcerative Colitis and Crohn's Disease ... 82 Premalignant Markers or Nonspecific Changes? 83 The Usefulness of the Differential Diagnostic Scheme 86 Future Considerations for a Project of this Nature 88  5. Conclusions  90  REFERENCES  91  SLIDES  101  vi  LIST OF T A B L E S Page  Table  1:  Macroscopic differences between ulcerative colitis and Crohn's Disease of the Large Intestine  Table  2:  Microscopic differences between ulcerative colitis and Crohn's Disease of the Large Intestine  Table 3:  Comparison of normal hyperplastic colonic mucosa  Table 4:  Characteristics from tumors  and  nonpolypoid  epithelial  glycoproteins  14-16  Table 5:  Characteristics of the epithelial glycoproteins adjacent to tumors (transitional mucosa)  19-20  Table  6:  Characteristics of the epithelial from areas remote from tumors  glycoproteins  21  Table  7:  Characteristics of the epithelial from Crohn's Disease specimens  glycoproteins  23-24  Table  8:  Characteristics of the epithelial from ulcerative colitis specimens  glycoproteins  27-29  Table 9:  Characteristics of the from benign pathological  glycoproteins  31-32  Table  10:  Number of cases & specimens of ulcerative colitis, Crohn's Disease and diverticular disease from the descending and ascending colon  39  Table  11:  Serial sections and the histochemical procedure applied  41-42  Table  12:  Expected staining results from histochemical procedures used for given structural elements  47  Table  13:  Histologic and histochemical ulcerative colitis cases  characteristics  from  52  Table  14:  Histologic and histochemical Crohn's Disease cases  characteristics  from  53-54  Table  15:  Histologic and histochemical diverticular disease cases  characteristics  from  55-56  of  the  vii  epithelial conditions  corresponding  Table  16:  Relative proportion of sialo- and sulphomucins in the descending colon as assessed by the K O H / A B 1 . 0 / P A P S procedure  64  Table  17:  Relative proportion of sialoin the ascending colon as K O H / A B 1 . 0 / P A P S procedure  and sulphomucins assessed by the  67  Table  18:  Percentage and number of cases and specimens showing a predominance of sialomucins in the K O H / A B 1 . 0 / P A P S procedure  68  Table  19:  Assessment of the degree of inflammation versus histochemical class using the Chi Square test  70  Table  20:  Assessment of histochemical class verus using the Chi Square test  disease  71  Table 21:  Assessment of the degree of sulphation versus degree of side chain o-acetylation using the Spearman Rank Correlation Coefficient  73  Table  Analysis of O-acetylated (by specimen)  acid  74  Assessment of O-acetylated sialic acid with respect to disease using Chi Square analysis  76  22:  Table 23:  viii  side  chain  sialic  LIST OF FIGURES Page  Figure 1:  Quotation  xiii  Figure 2:  N-acetyl neuraminic acid and its side chain variants  Figure 3:  Differential diagnostic scheme colonic epithelial glycoproteins  ix  for  O-acetylated  10  classifying  58  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 - P B T / K O H / P A S  10.  Serial in which the focal change is not revealed by the K O H / A B 1 . 0 / P A P S procedure  11.  Serial in which the focal change is not revealed by the H & E procedure  12.  Normal pattern of O-acetylated sialic acid procedure  13.  Normal pattern of O-acetylated sialic acid - P B T / K O H / P A S procedure  14.  Moderate field change -  15.  Moderate field change - P B T / K O H / P A S  16.  Severe field change - P B T / K O H / P A S  17.  Crypts showing a predominance of sialomucins.  PAPT/KOH/Bh/PAS  PAPT/KOH/Bh/PAS  x  ABBREVIATIONS  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  ACKNOWLEDGEMENTS  I would like to thank and  patience  thesis. Drs.  I  am  this  also  grateful  review for his  much  appreciated  project  thesis  help  drafts.  and  to  W . K . Ovalle  Dr. P . E . Reid for his constant  the  and In  thesis  have  been  husband, the  David,  D.A.  Ainsley,  who  extremely  most of  Owen  particular, I  aid in photomicroscopy.  completed  while  without  for  his  than  grateful.  his  a I  a  sacrifices  would  provided  a  also  in  source  of  criticism  supervisory  their  would  like  terminology.  care  of  my  of  in my  that to  were thank  welcome,  children,  and  I would like to acknowledge  by my husband's  this  committee,  encouragement to  thank  and  Dr. D . A . his  expertise of L . Trueman,  help  completion  from  ability  my  would  family.  my and  finish  this  project  necessary  along  the  way,  my albeit  son,  Ryan,  constant,  and  assistance  on  and  with thanks the encouragement  I  am  daughter,  diversion.  syntax  not  To  to  grateful to my parents, Dr. and Mrs. Ainsley Atkins for their  support,  of  and Dr. D . C . Walker for  Calgary; such  deal  like  enthusiasm  appreciated.  unfailing faith  few  my  The technical  living great  helpful  for  in assessing tissue morphology  possible  more  especially  moral  was  for  members  Muelchen and C. Ramey is sincerely  This  for  very  of  Owen  B.  supervisor,  throughout  W . L . Dunn,  critical  my  I  am  constant medical provided  family. Finally, I would like to thank the University of British  Columbia for the award of a University Summer Graduate Fellowship.  xii  FIGURE  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 the  epithelial  glycoproteins  to characterize the carbohydrate prosthetic groups of in  cases  of  ulcerative  colitis,  Crohn's  Disease,  and  diverticular disease.  The overall objectives 1.  determine  were to:  whether  primary  the  changes  seen  premalignant phenomenon,  in  or  the  mucosa  alternatively,  close  an  to  effect  tumors  are  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  NORMAL  The  intestine  and  subdivisions colon.  AND  MICROSCOPIC  FEATURES  OF  COLON  large  (Morson  MACROSCOPIC  extends  Dawson, of  the  1979).  colon.  The proximal or  the  lower  aspect  of  the  Anatomic landmarks provide  The  cecum  ascending  flexure, while the transverse descending  from  marks  the  colon extends  most from  cecum the  the  splenic  flexure  to  the  cecum  to  anus  for further  the  the  hepatic  splenic flexure. The  "a point where  crosses the pelvis" (Morson and Dawson, 1979), and the  1  basis  the  proximal portion of  extends from the hepatic to the  portion extends from  to  the colon  sigmoid from that point  2 to the retrosigmoid junction.  As  in  all other  layers: The  a)  the  of the  mucosa,  mucosa  crypts  Morson,  at  the  co-exist.  an lie  exhibit  bases where  The surface  predominantly  the  normal colon consists  muscularis  component  propria, which  parallel and adjacent  consists  to one  are lined almost exclusively  undifferentiated  cells,  cells,  situated  with  the  at  APUD  the  of  another  serosa. straight,  and, in  1976;  except  and enterochromaffin of the  goblet cell  the  Chambers  by goblet cells,  opening  occasional  of four  and d) the  any branching (Filipe & Branfoot,  epithelium,  absorptive  c)  epithelial  1980). The crypts  very  digestive tract,  submucosa,  which  colon, do not  &  b)  possesses  perpendicular normal  areas  crypts,  cells  contains  interspersed  (Filipe  & Branfoot, 1976).  1.3.  PATHOLOGIC  ULCERATIVE  DESCRIPTION  COLITIS,  OF  CROHN'S  DIVERTICULAR  DISEASE,  AND  DISEASE,  TRANSITIONAL  MUCOSA  1.3.1. Diverticular Disease  Diverticular Dawson, The  disease,  1979)  gives  diverticulum  muscularis  the  mucosa  circular  rise  itself,  muscularis propria. in  which  arises to  the  consists  through  from  a  defect  inflammatory of  and  an  the  of  layer,  ie.  where  major  muscle,  known mucous  circular  The diverticulum, which invaginates  muscle  the  condition  inpouching  beyond  in  muscle at  blood  as  &  diverticulitis.  membrane layers  inherently vessels  (Morson  of  weak  cross,  and the spots  remains  3 surrounded by a thin layer of longitudinal muscle fat  region.  The  configuration  lies proximal to the the percolic fat.  the  develops  mucosal surface  diverticulum is  such  luminal aspect of the colon and the  As a consequence,  and subsequently the  of  and is situated that  in the pericolic its  narrow neck  pouch itself bulges into  fecal matter becomes entrapped in the pouch,  into a fecolith. The fecolith, in turn, constantly  and as  a consequence,  chronic, low-grade  abrades  inflammation  arises  in the area around the diverticulum.  1.3.2. Crohn's Disease and Ulcerative Colitis  The macroscopic and microscopic features are shown in Tables 1 and 2,  Although  the  in  cases  some  patients  features  show  cited  specimens a  with hematoxylin and eosin Fisher,  1967;  Shorter,  1982).  Filipe  &  respectively.  in Tables from  spectrum  of ulcerative colitis and Crohn's Disease  1 and 2 would  suspected  of changes  ulcerative  such  that  appear  colitis  Dawson,  1970;  Lev,  and  examination  does not provide a definitive 1970;  relatively  of  Crohn's disease sections  diagnosis  Filipe,  distinct,  1979;  stained  (Hellstrom and Kirsner  and  4 TABLE  1  MACROSCOPIC DIFFERENCES BETWEEN ULCERATIVE COLITIS AND C R O H N ' S D I S E A S E O F T H E L A R G E I N T E S T I N E (Morson & Dawson, 1979)  Ulcerative Colitis  Crohn's Disease  1. Disease in continuity.  1. Disease  2. Rectum almost always  involved.  3. Terminal ileum involved in  2. Rectum normal in 50%.  10%.  4. Granular and ulcerated mucosa. fissuring.  discontinuous.  3. Terminal ileum involved in 30%. No  4. Discretely ulcerated mucosa; cobblestone appearance; fissuring.  5. Often intensely vascular.  5. Vascularity seldom pronounced.  6. Normal serosa (except in acute fulminating colitis.  6. Serositis  7. Muscular shortening of colon; fibrous structures very rare.  7. Shortening due to fibrosis; fibrous structure common.  8. Never spontaneous  8. Enterocutaneous in 10%.  fistulae.  9. Inflammatory polyposis extensive.  common and  10. Anal lesions in less than 25%; acute fissures, excoriation and rectovaginal fistula.  common.  or intestinal  fistulae  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 TABLE  2  MICROSCOPIC DIFFERENCES BETWEEN ULCERATIVE COLITIS AND C R O H N ' S D I S E A S E O F T H E L A R G E I N T E S T I N E (Morson & Dawson, 1979)  Ulcerative Colitis  Crohn's Disease  1. Mucosal and inflammation (except fulminating colitis). 2. Width reduced.  of  3. Often edema.  intense  submucosal in acute  submucosa  normal  vascularity.  or  Little  1. Transmural inflammation.  2. Width increased.  of  submucosa  3. Vascularity Edema marked.  seldom  normal  or  prominent.  4. Focal lymphoid hyperplasia restricted to the mucosa and superficial submucosa.  4. Focal lymphoid mucosa, submucosa, pericolic fat tissues.  5. 'Crypt abscesses' very common.  5. Crypt abscesses fewer in number.  6. Mucous secretion  6. Mucus secretion  grossly  7. Paneth cell metaplasia  impaired.  common.  8. Sarcoid-type granulomas bowel and lymph nodes.  absent from  10. Anal inflammation.  lesions  -  nonspecific  slightly impaired.  7. Paneth cell metaplasia rare. 8. Sarcoid-type granulomas in bowel and lymph nodes. 9.  9. 'Fissuring' absent.  hyperplasia in serosa and  in  60-70%  'Fissuring' very common.  10. Anal present.  lesions;  sarcoid  foci  often  6 1.3.3. T r a n s i t i o n a l M u c o s a  The of  'transitional mucosa' is transition  from  abnormal secretion 1976). Abnormal  frank was  a term coined by Filipe carcinoma  found in the  secretion,  in this  to  (1969) to describe  histochemically  absence of any  case, has  been  normal  "a zone  mucosa,  where  morphologic change" (Filipe,  defined  as  a predominance of  sialomucins with a reduction or absence of sulphated material.  Subsequent 1979; that some  studies  (Dawson  Rhatigan & Saffos, the  mucosa,  atypical  1979;  rather  features.  &  than The  Rhatigan (1977) represents  Filipe,  1976;  Greaves et al., being  data  absolutely shown  a composite  in  Filipe 1980;  &  Branfoot,  Lev et al.,  1976;  Filipe,  1985). revealed  morphologically normal, possessed Table  3,  definition of the  of normal mucosa and that adjacent to carcinoma.  taken  from  Saffos  light microscopic  &  features  7 TABLE  COMPARISON  3  O F N O R M A L A N D NONPOLYPOID M U C O S A (Saffos & Rhatigan,  Normal Mucosa  HYPERPLASTIC 1977)  COLONIC  Nonpolypoid Mucosa Adjacent to Carcinomas  Surface Epithelium More absorptive cells than goblet  cells.  More goblet cells than absorptive cells.  Crypts 0.5  - 0.8mm in length.  0.8  - 2.0mm in length.  Straight and parallel except for slight anthemal pattern.  Many crypts assume tortuous configuration and dilated crypts are seen.  Branching seldom or never seen.  Branching frequently seen.  Epithelium is mainly goblet cells except at base of crypts where A P U D and undifferentiated cells are present.  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.  HISTOCHEMICAL  EPITHELIAL  Chemical  CHARACTERIZATION  studies  (Filipe,  galactose,  galactosamine  1979;  Reid  small  are  (Reid et al.,  of  1973;  predominantly  Structural  studies  a complex  of  It has  indicate  The  amino  that  ester  1980;  1984a)  of  human  colonic  N-acetyl  glucosamine,  N-acetyl  been demonstrated that the majority of the at position C4, or in the polyhydroxyl side (Figure 2).  position  the  C7  In  normal  and/or  C8  colon,  (Reid  carbohydrate prosthetic  et  groups  sialic al.,  are  acids  1978b). composed  highly branched acidic and neutral oligosaccharide  linked O-glycosidically through N-acetyl galactosamine  residues  may  al.,  mannose,  1980)  at  mixture of often  or threonine  O-sulphate  1975;  substituted  chains of different lengths serine  COLONIC  that the carbohydrate prosthetic groups contain  quantities  and sialic acid.  et  sialic acids possess O-acyl substituents chain  NORMAL  GLYCOPROTEINS  epithelial glycoproteins demonstrate fucose,  OF  also  in the be  acid composition  polypeptide  present  of the  chain (Filipe & Branfoot,  but its  location  polypeptides  is  has  not been  typical of that  to  1976).  established.  of mucin-type  glycoproteins.  Histochemical  studies  indicate  predominantly  sulphomucins  Filipe,  1969,  1979;  Filipe  1985,  Habib  et al.,  1986)  that  the  colonic  epithelial  (sulphosialoglycoproteins) &  Branfoot,  However,  1976;  the  (Goldman  Culling  type  et  al,  colon (Gad, 1969a;  1969,  1979).  In  the  Filipe  & Branfoot,  descending  colon,  1976; the  &  Sheahan  lower  half  Ming,  1981;  of mucin secreted  colon varies according to both the level in the crypt, and the the  glycoproteins  1968;  Lev et  in the  al.,  normal  anatomic region of  & Jervis, of  are  the  1976;  crypt  Filipe  contains  9 predominantly contains in  the  sulphomucin,  the  upper  portion  and  variable proportions of sulphomucins and sialomucins same  goblet  (1970) and Filipe the lower little  whilst  to the  neutral  cell,  or  in  (1979) there  different  cells.  exist  in  normal  lower  the  predominant  in  remaining two-thirds  According  to  the  third  specimens.  in the  1976;  Sheahan & Jervis, 1976;  Culling  et al.  to  (1971), however,  predominant mucins  Branfoot,  which exist together  According  the  mucins  epithelium  Filipe  &  Dawson  is an increasing gradient of neutral mucins from  upper crypt. Subbuswamy  mucosubstances  surface  of the  Filipe,  (1979), O-acetyl  maintains  In  crypts are  the  are  that very  ascending  colon  sialomucins  while  sulphomucins  (Filipe &  1979).  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  the right colon than in the  left.  Developmental  shown  changes over little  suphate  studies have time is  (Lev, found  progressive increase noted  in the  adult pattern  12  the  however,  in  that  1970;  colonic  there  are  sulphate  right  fewer  goblet  cell  O-acetylated  variants  distribution in colonic  Lev & Orlic,  1974).  before  12  In the  weeks  week  human fetus  (Lev & Orlic,  intensely colon  established  for sulphomucins  stains  while  intermediately.  do not vary significantly with age  1974).  human fetus,  (Lev,  By early  such  that  in  epithelium  1970).  in sulphomucin staining from the right to left colon has  of sulphomucin distribution is  colon stains most and  1968;  that  the  A  been  infancy,  an  transverse  the left colon stains the least  Neutral  mucins  (Lev & Orlic,  1974).  and  sialomucins,  10 FIGURE  N-ACETYL-NEURAMINIC  ACID  2  AND ITS O-ACETYLATED SUBSTITUENTS  OH  SIDE  CHAIN  H  I ho s u l k i i c k i ( \ ' - . K c t y ! . K ' m a m m k - . K i d ) m o k v u k ' . -  •o  -OH -OH CH.OH  R = Ace t vi  -o  -O-OH CH.OH  -O  O  R  —OH -O R CH,OH  OH OH CH.O  11 1.5.  CHARACTERISTICS  OF COLONIC  EPITHELIAL  GLYCOPROTEINS  IN  TUMORS  As  shown  different  in Table 4, from  significant  normal  reduction  Subbuswamy,  the in  epithelial glycoproteins many  or  respects.  absence  1971a; Reid et al.,  of  Apart  mucins  1981;  general  the  epithelial  from  (Filipe,  Lev et al,  colonic tumors, several histochemical alterations  In  of colonic tumors are distinctly  glycoproteins  of  studies 1969,  1985;  which  1979;  report  Lev,  Reid et al.,  a  1970;  1985a), in  are evident.  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.,  1980,  1984a,  1977;  1985a).  1978;  molar al.,  1979;  Consequently,  neuraminidase al.,  Filipe,  the  (sialidase)  Reid  et  O-acyl/sialic  Montero & Sergura, sialic  labile  al.,  1980;  acid  ratio  than  acids normal  Lev et al., of  of  1980; tumor  1985).  et al.,  glycoproteins  (Korhonen et A  tumor glycoproteins  1984a), providing chemical evidence  Reid  al.,  significant has  been  1971;  are  more  Dawson  reduction reported  in  et the  (Reid et  that a decrease in O-acetylated sialic acid  is a characteristic feature of colonic tumor glycoproteins.  Although  the  relative  proportion of  O-acetylated  colonic tumors, acetyl esters are still present as reflected by their positive K O H / P A S  Gad  (1969b)  reported  that  the  higher  sialic  acid  reduced  in many  in many colonic tumor glycoproteins  effect (Culling et al,  the  is  degree  of  (1975).  differentiation,  the  more  12 closely  the mucosubstances  resemble  normal. Culling et al.  Segura (1980) confirmed this  finding, reporting that  the  and  degree  most  of  differentiation  poorly differentiated  the  pattern  tumors most often  of  (1977) and Montero &  a relationship exists between  O-acetyl  exhibiting the  substitution, loss of C8  with  the  substituted  sialic acid.  In addition to  decreases  in O-acetylated  sulphomucins  present  Subbuswamy,  1971a; Fenger  al.,  have  also  study uniform  exhibit  decreases  pattern  Subbuswamy,  of  in  been  & Filipe,  1985a). However, it is generally the  staining  sialic  in the  (Goldman  &  Listinsky & Riddell,  amount  Ming, 1981;  of  1968; Reid et  not the case that all tumors in a particular of  sulphomucins.  demonstrated  1971a; Montero & Segura,  in some cases the  reported  1981;  amount was  acid, decreases  1980;  (Goldman  Reid et al.,  tumors are predominantly sulphated while  sulphate or contain both sulphated and non-sulphated  More &  1981,  frequently,  no  Ming,  1968;  1985a).  Thus,  in others, they lack  glycoproteins.  LEGEND  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 P N A : Peanut agglutinin (specific for Gal-GalNAc) F P C : 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. P B T / K O H / P A S 3. P A P T / K O H / B h / P A S 4. P A S 5. P A P S 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 p H 3.0 16. Toluidine blue p H 5.0 17. AB1.0/PAS 18. Alcian Yellow  14 TABLE  CHARACTERISTICS  Findings  OF  4  COLONIC EPITHELIAL TUMORS  Comments  N A N A less substituted in side chain and C4; significant reduction in O A C - N A N A ratio Usually no secretory elements  Adenocarcinomas  In colorectal type mucosa of anal canal shift from sulpho to sialo with predominantly N-acyl derivative  Carcinoma near dentate line  Only tumors arising from cecum, colon or rectum give positive K O H / P A S reaction  GLYCOPROTEINS  Method  Reference  1,2,6  Reid et al., Reid et al.,  Filipe,  FROM  1984a; 1980  1969  1,2,11  Fenger & Filipe, 1981  2,4  Culling et al.,  1975  1977  Tumors from noncolonic sites & their metastases K O H / P A S negative decreased O-acetyl N A N A in poorly, moderately differentiated tumors  31 adenocarcinomas  4,6  Culling et al.,  Less resistant neuraminidase digestion  15 adenocarcinomas  2,6  Reid et al.,  to  Decreased mucin  7/8 colonic carcinomas  Lev  et al.,  1980  1985  15 P N A labelled goblet cell mucin in 100% cases  21 cases colonic cancer  64% cases sialo  25 cases colonic adenocarcinoma  52% cases sialo  33 cases rectal adenocarcinomas  44% colonic & 45% rectal adenocarcinomas decrease of O-acyl NANA  Abnormal pattern more frequent in less differentiated tumors  12/15 lesions associated with less than 15% sulpho  Colonic/rectal adenocarcinomas  Decreased neuraminidase resistance as compared to normal  IS cases colonic carcinomas  1,2,4,9  Dawson et al., 1978  Increased proportion sialo  24 cases of F P C  1,4,12  Filipe et al., 1980  36% Sulpho; 32% mixed sulpho/sialo; 28% sialo; 4% no mucin  25 cases colonic tumor  1,2,3,6  Reid et al., 1985a  Sialo  Dysplastic crypts  1,4  Dawson & Filipe, 1980  Decreased sulpho in over half of cases  31 colonic carcinomas  13-16  Goldman & Ming, 1968  Mucosubstances partially labile in 4/9 and totally labile in 5/9 cases  Mild sialidase digest without colon  1,2,6  Reid et al., 1984a  decreased [H3]-galactose  Boland et al.,  1,2,6  1982  Montero & Segura, 1980  Listinsky & Riddell, 1981  uptake  Increased differentiation of colonic carcinomas, more closely mucins resemble normal  Gad,  1969b  All decreased mucin: 3 no mucin; 1/29 neutral mucins only; 12/29 no sulpho; 16/29 sialo and sulpho  32 specimens from rectum and colon  10,17  Subbuswamy,  1971  Sialo  Dysplastic rat colon crypts  1  Dawson & Filipe, 1980  1.6.  CHARACTERISTICS  ADJACENT  OF  TO TUMORS  COLONIC  EPITHELIAL  (TRANSITIONAL  MUCOSA)  GLYCOPROTEINS  AND REMOTE FROM  TUMORS  The  definition of 'transitional mucosa' was initially based, not on its morphological  features,  but  rather,  surprising that, that  the  as  Branfoot, Isaacson  (Filipe,  1974, &  uptake  is  Dawson & Filipe,  1970,  Dawson 1979;  studies  reduced  mucosa  1969,  Attwood,  abnormal  or  &  Branfoot, affinities acids  1976;  et  confirmed  absent  in  the  most  to  the  resistance  of  their  Dawson  et  al.,  1983;  1980;  histochemical  transitional  (Montero & Segura,  mucosa  not found  exclusively Filipe al,  [35S]  crypts  &  1978;  Segura,  1980.  sulphate  (Filipe,  adjacent to colonic tumors include a sialic  1978;  1980),  1970;  acids Lev  (Filipe  et  al.,  and increased  is  directly  &  Cooke,  1985),  1974;  altered  decreased Filipe  lectin binding  of side-chain O-acetylated levels of total  &  sialic  hexosamines  and  1974). In addition, Greaves et al. (1980) noted that in  associated  of survival. Filipe  sialomucins  et  &  is  have  1971a;  results;  mucosa  it  almost  Dawson  Montero  with  adenocarcinomas  classified  inverse relationship existed between the length of the  in  are  Subbuswamy,  1982), a variable content  sialic acid (Filipe & Cook,  length  investigators  tumors  1976, al.,  Therefore,  1983).  (Boland et al.,  transitional  pattern.  1979;  Filipe,  Greaves  have  adjacent  1972,  Other changes in the glycoproteins neuraminidase  mucin  in Tables 5 and 6,  in the  1976:  Autoradiographic  its  illustrated  glycoproteins  non-sulphated  on  & Branfoot (1974) have related  to  the  extent  Duke's  B,  an  transitional mucosa and the  also noted of  as  invasion  that by  the a  "increase carcinoma,  18 according to Duke's classification".  Transitional mucosal features & Branfoot, 1974; et  al.,  1980),  margins that  local  Filipe et al.,  in dysplastic  1980), in the flat mucosa between  crypts  (Listinsky & Riddell,  a greater  increase  have also been identified remote from tumors (Filipe  (Ehsanullah et  1981). Furthermore,  proportion of patients  in sialomucins  recurrence as  at  opposed  predominantly sulphomucins.  the to  al.,  Wood et al.  whose resection  time  of initial  those  patients  1982b),  and  polyps (Filipe in  (1985) has  margins showed  surgery, subsequently whose resection  resection  a  noted  marked  developed  a  margins contained  19 TABLE  5  CHARACTERISTICS O F COLONIC EPITHELIAL GLYCOPROTEINS 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 ('TM')  Findings  Comments  Sialo  10 surgical specimens of T M  Method  Reference Filipe & Branfoot, 1976 Greaves et al., 1980; Dawson & Filipe, 1980  Gradual decrease sulphated material T M present in 93/95 adenocarcinoma; increase length of T M , decrease survival  ADJACENT  Filipe,  According to Duke's classification  1969  Greaves et al., 1980.  Sulpho-sialo gradient is lost in crypts  1, 4, 5  Lev  TM  8  Boland et al.,  1, 2, 6  Montero & Segura, 1980  binds P N A  et al,  1985  1982  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.  80/80 sialo  80 specimens adjacent to adenocarcinoma  Isaacson & Attwood, 1979  8/9  Cases adjacent to noncolorectal carcinomas  Isaacson & Attwood, 1979  cases sialo  Decreased neuriminidase resistance  18 cases T M  1, 2, 4, 9  Dawson et al,  1978  Sialo, decrease or absence of sulpho  25  Loss of sulpho-sialo gradient  No difference between 'diseased' (from nonmalignant diseases) and T M specimens noted  specimens  Filipe & Branfoot, 1974 Lev et al.,  1985  21 TABLE  CHARACTERISTICS  OF COLONIC EPITHELIAL GLYCOPROTEINS AREAS REMOTE FROM TUMORS  Findings  Comments  8/10 2/10  "Normal" remote from tumors  cases sulpho, cases sialo  6  Method  Wood et al.,  Resection  Increased proportion sialo  Flat mucosa between polyps from patients with F P C  Increased  11/25 cases remote from tumors  sialo  Reference 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 4/15 cases less than 25% sulpho  FROM  1985  Listinsky & Riddell, 1981  margins  Filipe et al.,  1  1980  Filipe & Branfoot, 1974  22 1.7.  CHARACTERISTICS  CROHN'S  OF COLONIC  EPITHELIAL  GLYCOPROTEINS  DISEASE  Investigations  of the  reveal  uniform  any  mucins  in Crohn's  pattern  of  Disease  change.  of the  Some  colon  (Table  investigators  sulphomucins to be the predominant mucins for all (Habib et al., (Filipe,  1969)  Franzin  et  of the specimens examined, while others  al.,  1983a)  have  found  sialomucins  to  7)  do not  have  found  1986), or some  (Ehsanullah et al.,  be  the  also  shown  (Culling et al., disease. Fisher  there  1979)  to  be  a  reduction in  and that these changes  1982b;  predominant mucin  present in all or the majority of specimens. Studies of Crohn's Disease have  IN  specimens  O-acetylated side-chain sialic  acid  are related to the severity of the  No decrease in the amount of mucin secreted was noted by Hellstrom & (1967),  marked (Dawson,  Filipe  (1970),  and  mucin secretion has been observed in rectal biopsies from  some  1972).  Other  degrading glycosidases activity to normal  (1969,  1979)  investigations  or  Filipe  have  &  Dawson  revealed that  sulphatases  in  fact,  patients  and mucin  from the faeces of Crohn's Disease patients have a similar  (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  from normal individuals.  desialating effects  than glycoproteins  Shamsuddin & Phillips (1981) also have reported that a  large proportion of Crohn's Disease as transitional mucosa.  of fecal extracts  specimens  share the  same  histologic  features  23 TABLE  CHARACTERISTICS  OF  COLONIC CROHN'S  7  EPITHELIAL D I S E A S E (CD)  GLYCOPROTEINS  FROM  Findings  Comments  Method  Reference  Normal, sialo or intermediate mucin pattern  9 cases rectal C D ; 8 cases colonic C D  1,10  Filipe,  No decrease in amount mucin secreted  29 cases  1,4,10  Filipe & Dawson, 1970  More resistant to disialating effects from normal fecal extracts than controls or U C specimens  18 rectal biopsies CD  Rhodes et al.,  1985c  Normal fecal sulphatase activity  17 cases  Rhodes et al.,  1985b  Mucus degrading glycosidases have similar fecal activities to normal  19 cases  Rhodes et al.,  1985a  Sulpho  10 cases  1  Habib et al.,  20/42 - sialo  Rectal biopsies  1,4  Ehsanullah et al., 1982b  8/20  - sialo  Moderate to marked mucus secretion; pattern is normal or sialo  1969  Franzin et al., 1983a Dawson,  1972  1986  24 Reduction in sidechain O-acetylated N A N A as compared to normal ; these changes related to severity of disease  14 cases C D  2,6  Culling et al., 1979.  25 1.8.  CHARACTERISTICS  ULCERATIVE  COLITIS  Studies  epithelial  8)  of the  show  a  (Hellstrom was  reduction  the  1967;  which was  1979,  Dawson,  1972;  of  Culling et al,  related  to  C7  1979;  with  ulcerative  and  C8  Reid  et al,  colitis  substituted 1981,  FROM  (Table  sialic  acid  1984a)  that  of disease; a decrease in the amount of mucin  the  Franzin  GLYCOPROTEINS  from patients  proportion  larger with increased severity  secreted  EPITHELIAL  glycoproteins  in  & Fisher,  OF  intensity et  of inflammation  al,  1983a);  a  (Filipe,  decrease  1969,  1970,  the  molar  in  galactose-fucose and GlcNAc-GalNac ratios in the descending colon as compared to normal  (Reid et al,  chronic  active  al,  1984);  groups  (Allan et al,  a  subgroup of the  a  than  1984);  crypt in  the  1985),  cell  decrease  descending  production  remission  in the  rate  groups,  and a significant  molar fucose-NANA ratio in  colon as that  compared to  was  which  in  45%  turn  faster  was  normal (Reid et in  faster  colitis  like Crohn's Disease  pattern  of  change  the  Habib  activity, et  al  uncomplicated of  duration, (1986)  by  specimens,  (Ehsanullah  reported that when abnormal patterns to  relapse  than  normal  1967).  to the relative proportion of sialomucins and sulphomucins, ulcerative  specimens,  uniform  the  reduction of neutral mucins in all regions  of the colon in cases showing severe histologic changes (Greco et al,  With respect  the  extent of  reported  that  the  al,  1982b).  do occur, the  and  dysplasia,  et  do not  in  the  appear  to  However,  possess it  abnormal secretion  disease  ulcerative  majority of specimens  (Ehsanullah et colitis  cases  possessed  a  has  any been  is related  al, which  1982b). were  predominance  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  associated with 1986)  dysplasia  or carcinoma  distinquish  between  appear  two.  predominate  (Boland et al.,  (Filipe,  1979).  uncomplicated  complicated by carcinoma on the that dysplastic  to  foci secreted either  1984;  However,  in  basis of their sulphomucins,  of  Filipe et al.,  Jass  ulcerative  cases  et al., colitis  ulcerative  1985;  (1986) and  respective sialomucins  colitis  Habib et who  could  ulcerative  mucin patterns,  al, not  colitis found  or a mixture of the  27 TABLE  8  CHARACTERISTICS O F COLONIC E P I T H E L I A L GLYCOPROTEINS F R O M U L C E R A T I V E COLITIS (UC)  Findings  Comments  Decreased C7C8 side chain N A N A  Increase severity, increase reduction of side chain N A N A  2,6  Culling et al,  N A N A less substituted in side chain and C4  U C of the descending colon  1,2,6  Reid et al,  Molar gal-fuc; GlcNAc-GalNAc ratio smaller than normal  Descending colon  Molar fuc-NANA and g a l - N A N A ratios smaller than normal  Descending colon: chronic active subgroup  Secretory activity absent in surface epith, predominant mucin present is sialo  9/15  Reduction in amount of mucin, related to degree of inflammation  62  C C P R 45% faster in relapse group than in remission group which was 19% faster than normal  Ulcerative proctocolitis  Allan et al,  Normal fecal suphatase activity  39  Rhodes et al,  Method  cases  biopsies  patients  References  Filipe,  1,4,10  1979  1984a  1969  Filipe & Dawson, 1970  1985  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  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  10,14,18  Greco et al,  1967  8 cases treated with total colectomy - all developed cancer or precancer  Filipe et al,  1985  U C uncomplicated by dysplasia  Habib et al,  1986  4/8 cases - normal mucins, no dysplasia 1/20  10/20  - sialo  - sialo  U C complicated by dysplasia  13/18 abnormal goblet cell mucins dysplasia later developed in 6 and carcinoma in 1  Rectal biopsies from 18 patients with stable U C  35/38 biopsies sialo  UC  54/120 sialo; 19/120 - mixed; 47/120 normal  UC  with dysplasia  1,4  Boland et al,  1984  Ehsanullah et 1985  al,  Ehsanullah et 1982b  al,  29 Uninvolved mucosa irregular mucin distribution; active U C - mucus depletion, sialo; quiescent U C - sialo.  40 cases U C  1  Franzin et al., 1983a  No difference between 11 U C patients with cancer and 11 U C patients without cancer  Colonoscopic biopsies  1, P N A  Jass et al.,  i  1986  30 1.9.  CHARACTERISTICS  WITH  BENIGN  GLYCOPROTEINS  ASSOCIATED  P A T H O L O G I C A L CONDITIONS  Table 9 describes conditions,  OF EPITHELIAL  the mucin characteristics  including  parameter investigated  diverticular  disease.  of a number of benign pathological In  general,  the  only  histochemical  was the relative proportion of sialo- and sulphomucins. As  is evident, no uniformly consistent pattern was demonstrated.  31 TABLE  9  CHARACTERISTICS  OF EPITHELIAL PATHOLOGICAL  Findings  Comments  3/5 lesions less than 25% sulpho  Endometriosis  Listinsky & Riddell, 1981  16/27  Solitary ulcer syndrome  Franzin et al.,  4/4 cases sialo  Colostomy specimens  Isaacson & Attwood, 1979  14/21  Solitary ulcer syndrome  cases sialo  cases sialo  GLYCOPROTEINS CONDITIONS  Method  1, 4  76/78 normal mucin pattern  Nonspecific  Irregular mucin pattern with abnormal morphology  40 juvenile & inflammatory polyps  2/5 sialo & abnormal morphology 3/5 irregular mucins & abnormal morphology  5 cases ischemic colitis  2/5 sialo & abnormal morphology 1/5 sulpho & normal morphology 2/5 sulpho & abnormal morphology  Polypectomy  64/65 cases normal mucins  Nonspecific  Sulpho  Metaplastic mucosa  Sialo & abnormal morphology  Pedunculated hyperplastic polyps  FROM  BENIGN  Reference  1981  Ehsanullah et al., 1982a  proctitis  Franzin et al., 1983a  scars  proctitis  Ehsanullah et al., 1982b Franzin et al., 1983a  32 12/45  7/28  sialo  sialo  Sessile hyperplastic polyps Anastomosis  sites  Sunter et al.,  1985  Increase in sialo in mid & lower crypts  Ureterosigmoidostomy sites of 9 patients  Marcheggiano et al., 1984  17/17 normal mucin pattern  Villous adenomas and adenomatous polyps  Goldman & Ming, 1968  Histologic features of TM  8 cases diverticulosis  Rhatigan & Saffos, 1979  Sialo  12 benign tumors  Subbuswamy,  Normal mucin pattern  12 cases diverticulitis  Filipe,  Normal pattern  Diverticular disease  Reid et al.,  staining  1971a  1969  1984a  33 1.10.  The  PREMALIGNANT  majority of the  MARKERS  investigations  the histochemical patterns to  address  studies  the  have  provide only  seen in various diseases; few  significance  attempted  previously cited  of  to  the  answer  changes  were specifically  observed.  whether  the  a description of  However,  changes  seen  a in  designed  number the  of  mucosa  adjacent to tumors are specific.  Several  investigators  Rhatigan & Saffos, 19S3b; Lev et al.,  (Saffos 1979;  &  Rhatigan,  1977;  Listinsky & Riddell,  1985)  Isaacson  1981;  &  Attwood,  Franzin et al.,  adopt the position that the changes  1981,  changes  is to  based those  on that  demonstrating occur  in  the  that  identical  transitional  histochemical  mucosa  1983a,  seen in the mucosa  adjacent to tumors are nonspecific. Almost without exception, the evidence contention  1979;  can  be  or  for this histologic  observed  in  diseases with no malignant potential.  Similarly compelling arguments have been proffered by those who believe  that the  changes  markers  (Filipe  in  the  transitional  & Branfoot,  1979; Filipe et al,  1974; 1980;  mucosa  Filipe  specifically  & Cooke, 1974;  Ehsanullah et al.,  represent Filipe,  preneoplastic 1979;  Filipe & Fenger,  1982b). The problem of showing that  transitional mucosa changes  are specific has been approached from three  perspectives.  and  Firstly,  Filipe  Branfoot (1976)  demonstrated the existence of isolated patches  and Filipe  et  al.,  different  (1980)  have  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  Ehsanullah et al. of  transitional  (1982b) has  mucosa  are  in male Wistar rats (Lazosky, 1986). Finally,  shown that  associated  the histochemical changes  with  dysplasia  or  with  characteristic  other  potentially  ULCERATIVE  COLITIS,  malignant conditions.  1.11.  INCIDENCE  CROHN'S  Of  the  OF  DISEASE  three  COLONIC  CANCER  AND DIVERTICULAR  inflammatory  bowel  diseases  colitis is the one most universally accepted risk  of colonic  1985b;  cancer  Heimann et  (Morson & Pang,  al.,  1985)  According  IN  DISEASE  investigated  in  this  as being associated  1967; to  (Filipe,  1969,  Lennard-Jones et  study,  ulcerative  with an increased 1979; al.  Allen et (1977),  al.,  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  study  - from  times  the  increased "very  normal  risk  cited by different  investigators  low" in Hendriksen's et al.  risk  in  Lennard-Jones' study  (1985)  varies  study  to  (Lennard-Jones et  from  study  to  a high of  23  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  that there  are subgroups in this population who have  developing  colonic  cancer.  studies conducted studies  it  was  in the  Shorter Mayo  concluded  with longstanding  (Shamsuddin  as  1981;  (1983) reviewed  Clinic,  that,  et al.,  New  to  1983)  from  three  large-scale  and Birmingham. In the  contend  a greatly increased risk of  results  York  compared  Shorter,  general  all  population,  three  patients  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 patients  increased  who  risk  in  the  New  York  study).  As  in  are at the greatest risk for developing cancer  had extensive disease of longstanding  duration and who  ulcerative  are  colitis,  those who  developed  the  have  disease in  childhood (Shorter, 1983).  In  contrast  (Rhatigan  to  &  ulcerative  Saffos,  colitis  1979)  and  that  Crohn's Disease,  diverticular  disease  is  it  is  not  generally associated  accepted with  an  increased risk of developing malignancy.  1.12.  THESIS  RATIONALE  Changes in the glycoproteins  composition of the carbohydrate prosthetic groups of the epithelial  are  mucosa adjacent  associated to tumors  with  tumors  of  the  ('transitional mucosa')  ah,  1980;  Ehsanullah et  changes  observed  markers  while  in  others  the  al.,  1982b;  transitional  (Saffos  &  Reid mucosa  Rhatigan,  intestine  (Table  4),  (Table 5), and with the  remote from tumors (Table 6). Some investigators et  large  mucosa  (Filipe & Branfoot, 1976;  et  al.,  1985a)  represent  1977;  Isaacson  contend  specific &  the  Filipe  that  the  premalignant  Attwood,  1979;  36 Rhatigan & Saffos,  1979; Listinsky & Riddell,  1983b),  maintain that  clearly,  if  it  were  the  changes  the  case  transitional  mucosa  are  of  with  high risk  patients  diagnostic  a  are  that  a  1981; Franzin et al.,  nonspecific  the  reactive  histochemical  premalignant markers, then for developing  seen  histochemical  colorectal cancer  1983a,  phenomena.  changes  early  1981,  Quite in  the  screening  might be  of some  value.  Changes of a similar, but not identical nature have inflammatory  bowel  (Table  7).  bowel  disease,  diseases  -  ulcerative  colitis  However, in diverticulitis, which is the  epithelial  glycoproteins  (Table  also  do  also been 8)  and  classified  not  differ  described for two Crohn's  as  Disease  an inflammatory  radically  from  normal  (Table 9).  Although  the  figures  (Lennard-Jones with  an  1977)  risk  alterations  associated  with  suggesting  that  changes  et al.,  increased  histochemical  vary  an  of  from  study  and Crohn's cancer,  may  be  increased  of  In  are  that  colitis  associated  cancer  diverticulitis, which is  has is  ulcerative  1983)  possibility  contrast,  inflammation, in and of itself,  both  (Shorter,  the  cancer  a  normal  not responsible  mucin  and not  profile  for the mucin  seen in the mucosa adjacent to and remote from tumors.  The significance of the histochemical changes colitis  study,  Disease  suggesting  related. risk  to  and  Crohn's  Disease  is  unknown.  in the transitional mucosa, ulcerative 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  sialomucin  and  from  one  histochemical  sulphomucin  -  a  parameter  parameter  which  using the high iron diamine-Alcian Blue p H 2.5 recent  studies  interpretation 1985;  In  of  the  led  results  Rhodes et al.,  present  histochemical the  have  some with  HID/AB2.5  1985c; Reid et al.,  study,  parameters  new  new  techniques  would  that  the  of  generally  side chain sialic  whether  or  not  the  has  proportion been  suggest et  al.,  more  1985;  of  assessed However,  caution  in  the  et  al.,  McFadden  1985b).  methodology  characterize  relative  (HID/AB2.5) technique.  to  (Lev  the  was  used  which  - the relative proportion of sialo-  pattern of O-acetylated  issue  investigators  -  acid. It  epithelial  changes  in  represent premalignant markers could be resolved.  was  two  and sulphomucins, and anticipated  glycoproteins the  investigated  mucosa  more adjacent  that  these  fully,  such  to  tumors  2. M A T E R I A L S  A N D METHODS  TISSUE  2.1.  Thirty-five  specimens  from  cases of Crohn's Disease diverticular disease Hospitals (Table  17  cases of ulcerative colitis, 51  of the colon and 37  were  obtained from  the  specimens  specimens from  Vancouver  from  21  nineteen cases of  General  and Shaughnessy  10).  All specimens had been fixed in 10% formalin-calcium and subsequently embedded in  paraplast. In most  paraplast.  Following  were  taken  bath  (46 °C)  from  cases,  the  specimens  re-embedding, 20  each  block  containing  serial  of tissue.  0.1%  obtained were sections,  cut  Serials were  chrome  alum  in  re-embedded in fresh at  floated  1.0%  intervals on a  gelatin  as  of 5Mm,  warm an  water  adhesive,  mounted onto clean glass slides, and subsequently exposed to formalin vapour at 6 0 ° 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  specimen was assigned a letter (a-t) to denote from where in the serial it  was  taken.  Table  11  shows  the  order  stained with the methods described below.  38  in  which  the  serial  each  sequence  sections  were  39 TABLE  NUMBER DISEASE  10  O F C A S E S A N D S P E C I M E N S O F U L C E R A T I V E COLITIS, CROHN'S A N D DIVERTICULAR DISEASE FROM T H E DESCENDING AND ASCENDING COLON  ULCERATIVE COLITIS  CROHN'S DISEASE  DIVERTICULAR DISEASE  Ascending only  4  8  1  Descending only  11  9  18  Both  2  4  0  17  21  19  Ascending  10  24  2  Descending  25  27  35  Total #  35  51  37  Cases  Total #  Cases  Specimens  Specimens  40  2.2. STAINING PROCEDURES  Sections  were  morphological Table  assessment  11. The  discussed  stained  with and  mechanisms  (Reid et al,  1981,  hematoxylin w i t h the  and  and  seven  eosin  histochemical  specificities of these stains  1984b,  1985a,  1985b).  (Culling,  1974)  stains  outlined  have  as been  for in  extensively  41 TABLE  SERIAL  Serial Section  SECTIONS  Histochemical  11  AND THE CORRESPONDING PROCEDURE APPLIED  Procedure  HISTOCHEMICAL  Acronym  Reference  Potassium hydroxide/ Alcian Blue p H 1.0/ Periodic acid phenylhydrazine - water - peridic acid - Schiff.  KOH/AB1.0/PAPS  Reid et al., 1985b  Periodic acid borohydride treatment/ potassium hydroxide/ Alcian Blue ph 1.0/ Periodic acid - Schiff.  PBT/KOH/AB1.0/PAS  Reid et al., 1985b  Alcian Blue pH 1.0/ Periodic acid phenylhydrazine blockade - water periodic acid Schiff.  AB1.0/PAPS  Reid et al., 1985b  Periodic acid borohydride treatment/ potassium hydroxide/ Alcian Blue pH 2.5/ Periodic acid - Schiff.  P B T / K O H / A B 2.5/P A S  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 Schiff/ Potassium hydroxide/ Borohydride treatment/ Periodic acid Schiff.  PAT/KOH/Bh/PAS  Reid et 1984b  43  2.2.1. Histochemical  The  histochemical  standard  Procedures  stains used  in this project were constructed  from the  following  techniques:  2.2.1.1. Periodic acid oxidation (PA) 1% aqueous  periodic acid at room temperature oxidizes  produce Schiff-reactive was  aldehydes.  performed, sections were  steps, the  performed for two  are  reduced  2.2.1.3. Saponification  hours and the  to  Schiff  esters  are  unreactive  primary  alcohols  1% (W/V) N a H P O „  for  2  oxidation subsequent  removed  engendered  in  30  application  minutes  of  at room  (KOH)  2.2.1.4. Phenylhydrazine  phenylhydrazine  by  1972).  from  glycoproteins  by  application  potassium hydroxide (KOH) in 70% alcohol for 15 minutes  overnight  two  step  reduction (Bh)  temperature (Lillie and Pizzolato,  Periodate  oxidation  oxidized for 2 hours; in those with  0.1%(W/V) sodium borohydride in  O-acetyl  one  minutes.  2.2.1.2. Borohydride Aldehydes  In procedures in which only  initial oxidation step was  one for 30  pre-existing vicinol diols to  Block Schiff (PAPS aldehydes  hydrochloride distilled  for  water.  are 2  or  at  0.5%  (W/V)  at room temperature.  PAPT)  treated  hours  of  with  room  Phenylhydrazine  0.5%  temperature  hydrochloride  (W/V) and  aqueous then  held  selectively  and  44 irreversibly blocks tissue diols. However, the blockade of sialic acid is  reversed  specific  by application  of pararosaniline  Schiff staining of such  or Thionin  Schiff,  monoaldehydes  resulting  in the  residues.  2.2.1.5. Cationic Dye Methods with Alcian Blue Manipulation electrostatic  of  the  binding  to  pH  of  both  0.3%  carboxyl  (W/V) aqueous and sulphate  Alcian groups  Blue  at  results  p H 2.5  in  and to  sulphate esters only at p H 1.0.  2.3. STAINING OF SERIAL SECTIONS  The  histochemical  procedures  identify the following specific  shown  in Table  11  were  employed  in order  to  chemical groups:-  2.3.1. Identification of Total Sialic Acid and Sulphate  2.3.1.1.  KOHIAB1.0IPAPS  In  procedure,  this  then  treated  sialic  acids  with  the tissues Alcian  are stained  (PAPS) procedure.  are saponified  to remove  Blue p H 1.0 to visualize magenta  with  the periodic  O-acyl  sulphate  esters  esters.  and are  Subsequently,  acid phenylhydrazine  - Schiff  2.3.2. Identification of sulphate and side chain O-acetylated sialic acid  2.3.2.1. This  AB1.0/PAPS  method  stains  sulphate  esters  unsubstituted or are substituted  2.3.2.2. This  acids  those  sialic  acids  which  are  sulphate  esters  and  at C7 and/or C9, magenta.  allows  with  for  the  side-chain  simultaneous  O-acyl  which have two or three side chain  2.3.3.  and  PBTIKOHIAB1.0IPAS  procedure  sialic  aqua  Identification  of  Total  visualization  substituents  located  of  at  position  C7 pr C8 or  substituents.  Sialic  Acid,  Sulphate,  and  Side-chain  O-acetylated Sialic Acid  2.3.3.1.  PBTIKOHIAB2.5IPAS  In  technique,  this  substituents  or  Those  acids  sialic  substituted)  sulphate  which  are  with  esters  or  substituted  O-acetyl esters  sialic at at  C9 C7  acids and or  which  have  O-sulphate C8  (or  esters  which  stain purple.  2.3.4. Identification of Side-chain O-acetylated Sialic Acids  no  are  side  chain  stain  aqua.  di- or tri-  46 2.3.4.1.  Those  PAPT/KOH/Bh/PAS  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 TABLE  EXPECTED  12  STAINING RESULTS FROM HISTOCHEMICAL USED FOR GIVEN STRUCTURAL E L E M E N T S  STAIN  PROCEDURES  Structural Element  Sialic  Acid  CO  C9  C8  C7  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  PBT/KOH/PAS  0  0  0  R  R  0  KOH/AB.O/PAPS  A  =  Aqua; R =  OSulphate Ester  Red; B  =  Blue; P =  Purple; 0 =  No  Tissue Diols  staining  48  2.4. ASSESSMENT OF STAINING PATTERNS  All  histochemical  before  carrying  simultaneously range data  and out  any  colours  from  each was  present. case  noted,  the  Results  from  the  AB1.0/PAPS  (R),  Red-purple (RP),  (DA),  or  Aqua  was  (A).  Purple Data  Red-purple  (RP), Purple  staining), 1,2,3  entire  assessed  graded  Initially,  were  then  Where  any  specimen  was  on  all  assessments  specimens  were  made  microscope to establish  carried  out  discrepancy  examined  the  individually, and between  again by both  PBT/KOH/AB1.0/PAS,  the  two  observers  PBT/KOH/AB2.5/PAS,  assessed on a seven point color scale as  was  was  completed  microscope.  procedures  procedure  compared.  KOH/AB1.0/PAPS,  and  was  using a double-headed  Assessments  was  using a double-headed  staining  assessment.  by two observers  of  observers  morphological  (P), from  on (P),  on  a  Blue-purple the  five  five  Royal-blue  PAPT/KOH/Bh/PAS point  color  Blue-purple (BP) a  (BP),  point  or 4 (maximum staining  scale  as  or Blue  intensity  being either Red  and  (RB), Dark  aqua  PAT/KOH/Bh/PAS  being  either  Red (R),  (BP). The P B T / K O H / P A S  scale  as  being  either  0  (no  intensity).  2.4.1. Digitization  Tables Disease  13,  14  and  and  15  represent  Diverticular  KOH/AB1.0/PAPS,  the  Disease  PBT/KOH/AB1.0/PAS  results cases,  from  ulcerative  respectively.  Data  and P B T / K O H / A B 2 . 5 / P A S  converted to a 13 point numerical scale as  follows:  colitis,  Crohn's  from  procedures  the was  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  The  results  was  converted  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  from  the  from  color  the  PBT/KOH/PAS  cases the  predominated  co-existed in the the  dominant  magenta than  the  than  the  PAT/KOH/Bh/PAS  procedures  follows:-  Red Red to red-purple Red-purple Red-purple to purple Purple Purple to blue-purple Blue-purple Blue-purple to blue Blue  numerical value was  In many  and  to a 9 point numerical scale as  4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0  Results  PAPT/KOH/Bh/PAS  entered  field  color's the  dominant  another field.  which  less  significant  color,  these  specimens,  numerical  score  if  color,  a  initially  recorded  as  a  not homogeneous. Frequently, one particular  In  dominant  was  as that value.  viewed was  while same  procedure,  color. value  If of  the 0.5  the less was  or  a value  less  a  spectrum  of  0.5  significant  dominant substracted  color from  of  was  colors,  added  to  color  was  more  was  more  aqua  the  latter  color  50 score.  In  some  specimens  distinct from other  homogeneous adjacent areas.  areas  were  seen  In these cases,  that  data  were  was  histochemically  recorded separately  for each area of the slide.  Lastly,  a  'scrambled'  few (Slide  cases  possessed  1). Scrambled  specimens specimens  which showed  which no pattern could be discerned. In these cases, as having several major classes of glycoprotein.  were a  described  vast the  range  of  as  being  colors  in  specimen was recorded  LEGEND  TO TABLES  13,  14 A N D 15  Column Number 1. 2. 3. 4.  Specimen code number Disease: U C = ulcerative colitis, CD = Crohn's Disease, D=Diverticular disease Anatomic site: D = descending colon, A = ascending colon 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: I V A , I V C , IVD, V A , V C , V D 14. Digitized data - K O H / A B 1 . 0 / P A P S 15. Digitized data - P B T / K O H / A B 1 . 0 / P A S 16. Digitized data - P B T / K O H / A B 2 . 5 / P A S 17. Digitized data - AB1.0/PAPS 18. Digitized data - P A P T / K O H / B h / P A S 19. Digitized data - P A T / K O H / B h / P A S 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  COLITIS DISEASE  UC uc uc UC uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc UC uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc uc  • 0 • 0 0 0 A  3ea u c 2 isl uc 2 tal u c 4oal u c 4081 u c 3 3 8 | UC 35Bt UC 358 u c 338 uc 428 u c 428 u c 42B u c 7 1* u c 7 1* u c 43* uc 43* uc soe uc soa uc 438 uc 458 u c 4 SB 438 uc 498 u c 498 u c 498 u c 498 u c 478 u c 4 7B u c 488 uc 4 SB u c 48* uc 48* uc 30* uc SO* u c 67* uc 67* uc  18  IB ia IB S4B 648 SA 5* SA SA 23A 23* SB 38 IOA IO» 3 IB 318 94 9A 7 IB 7 IB 12* 12* 55* 3 5 *  37* S74 138 138  sea 36B S68 sea 198 I9B 30B 308 178 I7B 468 468 46* 46* 20B  20a 36a  uc  • -• N H  F F  H  r  N  A A A A 0 • • 0 D 0 D 0 0 0 A  N N N N N N N M  M  N N N N N N N  A  M  F F 3 S F F A A  A  N  M  A • D 0 • 0 0 0 • 0 0 0 0 • A  N N N N N N  M M H M M M M M H  S3 S3 53  • • •  • •  * *  A 0 • 0 0 0 D 0 0 0 0 D 0 0 0 0 0 A A  H  N N N N N N H  N N N H M  N N N N N N N N  U  N N N N N N N M N N N N N N N  ti N N M  N N M U  N N M N N N  • •  M M M M M M M M M M M M M M M M M M M M F F  A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A  A A A A . A A A A A p p p p p A A A A A A A A P p p p p p A A p p A A A  *  4  A  A  « A A 0 0 0 0 0 0 • 0 0 • 0 0 A A 0 0 A A A  N N  A A A A A A A A A A A 4 A A A A A A A A A A A A A A A  A  15 IS IS  F F F F F F F F F M M H H M M F F F F F F F F F F F F F F F F H M M  N N M  A A A A A A A A A A A A A A A A A A A A A A p P p p p P p p p p p p A A A A A A A A A A A A A  15  13 13  30 30 30 30 30 30 26 26 26 26 26 26 31 31 31 31 79 79 79 79 79 79 19 19 19 19 19 19  34 34 34 34 37 37  37 S7 37 37  26 26 26 26 40 40 40 40 S3  • •  •  64 64 64 64 sa sa ss sa sa ss ss ss S3 53 53 S3 57 37 37 37 39 39  A  A A A A A A A A A A A A A A A A A  2 "J 2 2 2  :  2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 f U u 2 2 U U U U u u u u 1 1 2 2 0 0 1 1 0 0 1 t  1 1 3 3 3 3 2 3 2 2 I t 1 u u ! 1 2 2 2 2 3 2 t 1 t l  U  u  3 3 u  u  3 3 3 3  OF  ULCERATIVE  SPECIMENS  VO vc VO vc VO vc VO ivo 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 ivo VO VO VO VO VO VO VO VO VO VO VO VO VO VO VO IVA VA IVA  IVA IVA vc VO vc VO lvc IVC VO VD VO vo VO vo IVC ivc 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  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 too 10.0 3.5 3 5 12 0 12 O 9 s 9 3 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 0 9 .0 9 .0 9 .0 6 .0 7 o 7 0 7 o 4.0 4.0 7.0 7.0 3.0 3.0 7.3 7.3 8.0 a.o 3.3  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  O 0  o o  0 o 10. 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 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  2.0 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 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 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  2.0 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 3.5  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 99.9 99.9 99.9 3.5 3.5 3. 3. 2. 2.  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 2 2 2 2 2 2 2.3 2.S 1 2 2 2 2 1.5 1.5 2  53 TABLE 14 HISTOLOGIC  AND  HISTOCHEMICAL DISEASE  (~  IA CO IA CO IA CD IA CO :a CO CO 29 368 CO 268 CO 48 CD CO 48 CO '8 48 CO 108 CO 108IC0 • SB CO 168 CO 20A CO 20A CO 20A CO 20A CO 6A CO SA CO ISA CO I6A CO 33B CO 338 CO ISA CO ISA CO 37B CO 378 CO 378 CO 378 CD 228 CO 228 CD 228 CO 228 CO 388 CO 388 CO 3SB CO 388 CO 238 CO 238 CO 4 18 CO 4 18 CO 43A CO 43A CO 258 CD 258 CO 258 CD 258 CO 3SA CO 3SA CD 3SA CO 36A CD 298 CO 298 CO 59A CD 59A CO 398 CO 398 CO 54A CO 54A CO 5SA CO SSA CO 548 CO 548 CO 548 CD 548 CO 588 CO 588 CO 588 CD 588 CO 588 CO 588 CO S<A CO S IA CO s a a CO S88 CD 6SA CO SSA CO S9A CO 69A|C0 288 CO 288 CO 288lC0 288 CO  A A A A 0 0 0 0 A A A A A A 0 0 0 0 0 0 0 0 A A A A 0 0 0 0 0 D A A A A A A A A 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 A A A A A A 0 0 D 0 0 0 o 0 0 0 A A 0 0 0 0 0 0 0 0 0 0  N  M N N N N N N M N N N M N N N N M M N M N N N N N N M N N N M N N N N N N N N H H  N N  N N N N H H H H  N H  N N N N N N N N N N N N N M N .N N N N N M N  N N N H  M N  n  M N M  H H M H F F F F H M H M M M M H M M M F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F F P F F F F F f  22 22 23 22 70 70 70 70 14 14 14 14 14 14 14 14 14 14 14 14 S3 S3 30 30 30 30 7S 7S 76 7S 76 76 71 7 1 71 7 1 7 1 71 71 7 1 17 17 17 17 17 17 24 24 34 24 24 24 24 24 14 14 14 14 S3 S3 18 ta 18 18 35 35 35 35 35 35 35 35 35 35 38 38 38 38 83 83 83 S3 63 S3 63 S3  I  1 1 1 2 2 2 2 2 2 1 1 1 1 1 1 3 3 2 2 2 2 1 t 2 2 1 1 1 1 1 1 1 1 2 2 1 1 1 1 3 3 2 2 2 2 2 2 2 2 1 1 1 1 U  u u u u  2 2 1 1  1 1 2 2 2 2 2 3 1 1 3 2 1 1 U u u u i i  2 2  U u u u  3 3 2 2 2 2 2 2 2 2 1 1 1 1 U u  2 2 1 1 1 1 1 1 3 2 2 2 2 2 2 2 3 3 3 3 1 1 3 3 U  2 2 1 1 1 1 1 1 U  2 7 2  u tj u u  2 2 2 2 1 1 1 1 2 2 0 0 1 1 1 1 0 0 0 0 3 •3  1 1 I I 1 1 1 2 22 2 0 0 0 0 1 1 1 1 1 1 t 1 0 2 0 2 o 0 0 0 2 2 2 2 O 0 0 t 0 0 0 2 1 1 1 1 1 2 1 1 1 1 1 1 1 u1 1 1 2 u 2 1 2 2 li 1 2 2 u t 2 2 1 1 o X ! X 1 0 2 2 2 32 3 2 1 1 1 l* I 1 1 1 t 1 1 1 1 3 3 3 3 2 33 33 33 2 3 3 3 2 3 3 3 2 2 3 3 3 2 2 2 2 22 2 2 2 2 2 2 2 1 t 1 1 1 1 1 1 1 1 1 1 1 1 l 1 3 3 U U  1 1  U  U l 1 1  2 2 2 2 2 1 1 2 1 1 2 2 1 1 2 2 2 2 2 I  * 2  3  2 2 1 1 1 t 1 1 2 2  u u u  t  U 3 3  0 0 0 O 0 0 1 1 1 f  U 3 3  0 0 0 0 0 0 U u u u  3 3 3  0 0 0 o 0 0 1 1 1 1 0 0 0 0 3 2 3 2 1  2 2 oo 0 2 0 2 2 0 0 2 0 0 3 33 2 2 3 3 2 2 3 3 2 2 3 2 2 2 1 1 2 1 2 t 1 2 3 2 1 1 1 1 t 2 2 u 1 2 1 u 2 1  , U .  1  1  ' ' ' 1 ! ' 1  1  1  «0  u  0 0 0 0 0 0 0 0 0 0 o  CHARACTERISTICS  OF  CROHN'S  SPECIMENS MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA  r 8  1  8 T 8 r a r a T a T  3 T a T  a T a  r  a r a r a r a T T a a T a T 8 T a T a T a T a r a r T  a  8 r a r a T a r  a  r a T a i a  r  T 8 a T B T a T a  r  a r a r 8 T a T a T a  VO vo vo vo vo vo vo IVO VO VO vo IVO VA VA VA VA VO VO VA VA VO VO VA IVA VO VO VA iva vo VA vo VA VO VO IVC IVC IVC IVC vc IVC vo VO VO VO IVC IVC vo ivo vo IVO IVO IVO vo VD vo vo vo vo vo VO vo vo vo vo IVO vo IVO VO IVO ivc vo VO IVD ivc VO vo IVD ivo ivc IVC IVC IVC VO VO VA VA  0 10 0 5 0 7 5 3 0 9 0 a 5 5 0 7 5 33 0 11 0 11 0 S6 0 11 0 0 11 0 11 0 0 11 0 20 10 5 to 5 9 0 1 0 s 2s 11 0 11 0 9 0 13 0 2 55 a 0 a 0 4 0 1 00 1 12 0 12 0 9 0 13a 0 13 8 0 S 0 5 0 0 15 a 0 6 0 5 0 a 0 13 10 0 9 0 a5 0 139 0 1S 182 n 12 0 0 0 13 0 3 5 2 0 10 0 0 a8 5 9 9 0 2 0 70 0 8 0 3 0 10 6 5 03 8 0 2 0 10 6 5 03 a 0 90 S 0 1 0 1 20 a 0 1 s 05 0 10 1 20 9 s 10 10 1 0 1 13 9 05 , 1 0 510 1 0 1 315 9 9 0 0 9 0 3 a 0 a 0 5 0 7 0 23 a 0 2 0 10 7 0 0 3 1 2 0 0 0 510 13 0 0 5 to 0 1 0 0 0 9 0 13 10 0 10 0 5 0 9 0 1a 9 0 132 0 3 0 1 00 0 5 11 0 0 9 0 13 0 0 S 9 00 a 0 53 0 10 0 0 5 90 2 0 0 10 0 0 3 9 a 0 5 0 10 0 0 5 9 0 2 0 a3 0 10 0 0 5 70 9 0 5 70 35 70 9 0 8 5 7 0 3 3 12 00 12 0 sa 55 0 0 3 3 12 12 0 0 0 33 12 0 12 0 7 0 0 0 3 5 1•J2 0 12 0 7 0 0 0 33 0 S 5 70 3 0 3 3 12 0 t 10 7 0 0 0 3 S 9 5 1 o0 1 70 90 3 0 95 1 1 70 90 3 0 0 10 o0 to 9 0 9 0 9 9 9 10 5 IO 0 9 0 9 0 99 9 11 1 0 5 9 0 0 0 3 0 1a1 5 10a 5 9 0 0 0 3 0 8 0 a 0 23 0 0 8 0 1 0 1 0 12 2 10 25 a 0 a a 0 a 0 0 2 S3 12 00 13 00 a 0 11 0 2 3 0 0 12 0 12 7 0 1 2 3 0 12 13 0 7 0 138 0 5 5 3 0 75 7 5 70 a 5 3 0 7 7 70 5 a 5 90 a 0 70 95 3 0 3 0 9 0 0 7 0 9 a 0 a 0 70 3 3 9 0 a 0 8 0 7 0 9 0 2 S3 a 5 45 S 5 90 1 s 10 1 0 1 65 1 0 1l 3 t 5 0 5 0 20 0 3 0 7 o 1 0 3 o3 s5 o 2o 5 0 10 3 5 0 2 0 6 0 10 3 5 0 2 0 12 0 t 155 88 0 t 10 2 3 10 0 10 5 8 0 9 0 3 3 12 0 1 51 a 0 1 0 1 23 10 0 1 0 0 90 3 3 12 0 13 0 55 0 12 0 3 3 12 0 12 0 5 0 0 0 3 3 10 0 10 0 5 0 10 0 3 S a 0 95 0 70 3 3 12 0 13 0 55 0 13 0 3 3 12 0 1 2 0 0 00 33 10 55 10.5 aa 0 1 0 12 5 s 1 0 1 0 0 1 0 5 25 13 0 1 2 0 6 0 1 0 1 20 12 0 12 0 6 0 1 0 1 20 a 0 33 1 0 0 1 0 . 0 9 0 to 0 1 0 0 9 0 0 0 25 13 0 1 2 0 70 0 0 33 13 0 13 0 0 0 0 3» 8 0 a o 5 0 8 0 1 0 S 5 s 5 0 0 S 0 10 8 0 a 0 3 0 8 0 10 s S 5 0 2 0 S 0 10 10  1  1  2.0 2 3 I.S 3 1 2.0 3 5 1.» 2 ! 2.0 2 5 1.5 2 2.0 2 3 1.3 2 2.0 4 03 1 2.0 4 0 3 1 1.0 3 0 1.5 1 l . 0 3 0 1.5 1 1.3 2 3,2 3 1.3 2 3 3 2 1.3 2 3 1t 0 1.3 2 83 0 0.0 0 8 I.S 1 0.0 0 3 I.S 1 0.0 0 3 1 1 1 0.0 0 1i 1.0 2 35 2 1. 1. i 2 1. 0 3 0.0 1 0 2 0 0.0 410 2 0 2.0 0 3 2. s 2 0 4 0 3 25 0 3 03 1 0 0.3 0 5 1 0 1-0 2 3 2 t 1.0 2 5 2 1 0.0 0 5 1 1 0.0 3 0 1 1 0.3 10 1 2 0.3 10 11 2 2 0.3 10 0.3 10 11 2 1.8 35 03 5 1 .83 5 1 0 1.8 3 3 2 22 1 .83 S 2 2.0 3 3 1 ll 2.0 3 5 1 1 2.0 3 5 1 1 2.0 3 3 1 2 0 3 3 3 33 2.0 3 5 3 3 2.0 3 S 3 3 2.0 3 3 3 2.0 3 3 3 33 2.0 3 a3 3 3 1.0 3 2 t . 02 8 2 2 1. 0 2a 2 2 t . 02 a 2 2 2.0 3 55 1 11 2.0 3 5 1 1 2.0 3 5 11 1 2.0 3 4 0 3 3 1. 0 4 0 3 3 1. 0 2.0 3 0 3 33 2.0 3 0 3 3 5 1. 0 1 0 1.0 3 5 1 0 2.0 1o0 1 0 3.0 1 1 0 2.0 11 0 1t 0 2.0 0 0 2.0 3 0 2 11 2.0 3 0 2 1 2.0 3 0 2 1 2.0 3 0 2 2.0 2 55 2 0 2.0 2 3 0 2.0 3 0 2 0 2.0 3 0 3 0 2.0 3 8 3 3 a 2.0 3 3 2 2.0 4 0 3 2 2.0 4 0 3 2 .S I S 3.0 4 0 II.S 1 5 2.0 4 0 3.0 3 0 2 11 2.0 3 0 2 2.0 3 5 3 11 2.0 3 » 1.0  0 3  a.i 1.0 a. 5 1.0  1.0  0 a  1 t  0 0 0 0  | i  TABLE 14 (CONT.)  6B SB SB 23A 23A 33A 33A 7A 7A 1 IA 1 1A 1 IA 1 IA 4 1A 4 IA 39A 39A 39A 38A 38A JOA 4 OA 32A 22A 7B 7B 7B 7B SA 8A SB 9B 3SA 35A 3SA 3SA 9B 9B 9B 98 26A 2SA 13A 13A  I3A 134 I3A I3A I4A !4A SSB 558 I4B 14B 44A 44A  CD CO CD CO CO CO CD CO CD CO CO CO CD CO CO CO CD CO CO CO CO CO CD CD CO CD CO CD CO CD CO CO CO CO CD CO CD CO CO CO CO CO CO CO CO CO CO CD CO CO CO CD CO CD CO CO  A A A  N N  A  N N N N N N N N M N N M N N N N N N N  A A A A A A A A A 0 D 0 0 0 0 0 0 0 A A A A A A A A A A A A A A 0 D 0 D D D A A A A A A A A D 0 A A A A  N  U  N N N N N N N N M N N N N  M M M M M M M M H W M M M M M M H M M M M M M M F F F f  F F F F F F F F  N H  N N N N N N N N H  N  N N N N N N N N  M M H F F F F F F F F F F F F f  82 83 82 82 82 82 82 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 37 37 37 37 37 37 37 37 37 37 37 37 44 44 44 44 44 44 25 25 25 23 25 23 25 23 25 25 29 29 29 29  P P p A A A A P P A A A A P P P P P P P P P P P P P P P A A A A A A A A A A A A A A P P P P P P P P P P A A A A  A A A A A A A P P A A A A A A A A A P P A  ' A A A A A A A A A A A A A A A P P P P A A P P P P P P A A P P A A A A  0 0 0 2 2 2 2 1 1 1 0 1 0 2 2 2 2 2 2 2 t 1 1 1 t I 1 t 1 1 1 1 1 1 2 2 2 2 1 1  2 2 3 2 2 2 2 2 2 2 1 1 1 1  0 0 0 2 2 2 2 t 1 1 0 1 0 2 2 2 2 2 U U 1 1  2 2 1 1 t 1 0  o  t 1 1 1 1 1 2 3 2 2 1 1  u u  u u  u  0 0 0 2 2 2 2 1 1 2 0 2 0 2 2 2 2 2 2 2 1 1 •  2 2 2 2 0 O I 1 1 \  t 2 2 2 3  1  0 0 0 2 2 2 2 2 2 1 0 1 0 1 1 2 2 2 U U 1  • 1 1 1 I 0 0 2 2 2 2 2 2 2 2 2 2 1  «  0 0 0 2 2 2 2 0 0 1 0 1 0 1 1 2 2 2 1 1 1 1  .  2 2 2 2 0 O 2 2 2 2 2 2 2 2 2 2  ,  1  <  1 t  1 U U  !  t  It  i  u  \  )  1  u 1 1  1 t 1 1 I 0 0 0 0  t  u 2 2 u  2 3  1 t 1 1  2 t 1 1 1  u  0 0 0 2 2 2 2 2 2 I 0 1 0 2 2 2 2 2 1 1 1 1 0 0 2 2 2 2 0 0 2 2 2 2 2 2 2 2 2 2  t  «  t  0 0 0 0  u u u  u 0 0 0 0  i  <  MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA MA  T B B T T 8 8 7 B 7 T B B T B T T B  r  B r B T B T T B B T B T B T T •  a r B T  a  T B T T T B B B T B T B T B T 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 VD  VO  vo vo vo  VD VD  8.0 8.0 10.0 10.0 12.0 0 0 7.0 O 12.0 O 7.0 O O 3 0 5 O 4.0 3 2.0 5 5.0 5 2.0 5 9.0 3 5.0 0 0 10.0 I 1 .0 11.0 S.O 7.0 5.0 9.5 5.0 5.0 10.0 1 0 . 0 7.0 0 7.5 0 7.5 0 7.0 5 1 1 .0 0 7.0 5 1 1 .0 0 7.0 0 I I .O o 9.0 o 9.0 0 9.0 9. 0 1 1 .011.0 1 1 0 11.0 10 5 1 0 . 0 9.0 9 0 9.0 9.0 12.0 11.5 12.0 M S 12.0 12.0 12.0 12.0 9.0 9.0 9.0 9.0 12.0 12.0 12.0 12.0 9.0 9.0 9.0 9.0 7.0 7.0 10.0 10.0 8.3 8.0 8.5 8.0 5.0 7.0 9.0 10.0 5.0 5.0 9.0 9.0  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  8.5 10.3 12.0 3.0 0.0 3.0 0.0  3.0 3.0 3.0 2.0 2.0 2.0 2.0 3.0 3.0  10.0 9.5 9.3 3 4.S 3 9.0 3 0.0 3 1 .0 3 8 . 0 99 7.0 99.9 9.0 3.0 4.0 3.0 10.5 3.3 7.0 3.3 1.0 3.S 0.0 7 .0 3.5 I .0 7.0 3.3 0.0 7.0 1 .0 S.O 3. 0.0 9.0 3. 3.0 8.0 3. 3.0 8.0 3 1.0 O 0.0 3. O 0.0 3 0 11.0 3. o 10.5 3 o 10. 5 2. 2 0 11.0 2. 0 11.0 0 0.0 3 0 0.0 4.0 3. 0 3. o 3. 3.0 9.0 0 . 0 3 . 9.0 0.0 3. 9.0 3 . 0 3 . 9.0 4.0 3. 9.0 8 . 0 10.0 3.0 8 .0 10.0 3.0 8.0 9.0 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  DISEASE  2A 2A 2A 2A €78 S78 878 S78 3A 3A 3A 3A  0  0 0 0 0 0 0 0 0 0 0 0  S28  D 0  328  S28 328  0 0 0 0  4A 4A 4A 4A 43B 438 438 43B  0  1 1 8  IB 1 1 IB t IB  11 8 I IB 118 I 1 8 128 12B 128 12B  27A  27A 27A 27A 708 70S 708 708 27B 278 278 27B 278 27B 23A 28A 28A 28A 32A 32A 32A 32A 37A  10 0  ID 10 10 10  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  1 8 318 318 334 334 3  0 0 0 0 0  s sp p  s3 p p  s s p p  s sp p  *  s5  M M  p p p  s s s s p  *  p p p  0 0 0 0 0 0 0 0 0 0 0 0 D •  0 0 •  0 0 0 0 0 0 0 0 0 0 0 D  0 0 0  0 0  0  0  0  0 0 0 D  0 0  0 0 0 0 0  0 0 0  0  F P  s s p  4 4 4 4 4  4 4 4 4 4 4  r F F F F F F F F F F F F F  s. sp p  s sp p  s s p p  s s s sp p  s s p p  s s p p  ss p p  M  *  H  * F F F F F  P P  F F  P P P  F F F F  P  F  P  M M M M M M M M H M F F  P P P  J  s s  p p N N N N N p p  s sp p  s s sp p  s sp p  s s  ** **  M  * M  P  F  P P P P P P P P F  P P P f  48  48 48 48 4S 48 48 48 79 79 79 79 79 79 79  79 30 30 30 90 90 80  ao ao 78  78 78 78  78 78 78 79 78 78  78  73  36 36 38  36  76 78 76 76 64 64 64 64 64 64 64 64 64 64 66 66 66  66 66  4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4  66 66 66  J  64 64 64 64 64  4 4 4 4 4 4  64  *  64 32 52 32 52  7a 78 78 78 78 78 78 78 78 71  4 A 4 A 4 A 4 A 4 A 4 A 4 A 4 4 4 4  1 1 71 71 71 * 7 7  4 4 4 4 4 4 4 4 4 4 A 4 A 4 4 A A 4 4 4 4 4 4 4 4 4 A 4 A 4 A 4 A 4 A 4 A 4 4 4 4 4 4 4 A 4 A 4 A A A A A A A A A A A  *  0 0  0 0  0 o  0 0  0  0  1 t  1 1 0 0  1 1 0  *  1 1 0 0  1 1 0  0  0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0  1 1 0 1 1 0 1 1  1 1  1 1  1 1  0  0  0 0  0 0  0 0  0 0  0 0  0 0  0 0  0  0  0  0 0 o  0 0 0  0 0 0 0 0 0 0 0 0 0 o 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0  0 0 0 0 0 0 0  1 1  1 1  • 11 1 01 0 1 1 0 1 1 1 1 0  1 1 0 1 1 1 1 0  1 1  1 1 0 1 1  0 0 o 0  1 1 1 t  0  0 0  0 0  0 0  0 0  0  0  0  0 2 2  1 1 0 1 1 1 1 1 1 2 2  0 0 2 2  0 0  1 1  0 0  1 r  A A A A A A A A A A A A A A A A A A A A A 4 4 4 4  1 1 0  0 0 o o 0  0 0 2 2  2 2  1 1 o 1 1 T 1 1 1 2 2  2 2  1 1 0 1 1 1 1 1 1 2 2  0 0  0 o  0  0 0  0 0  0 0  2 2 O  1 1 1 1  0 o o o 0 0 0 2 2  2 2  1 1 1 1  0 0 0 o 0 0 0 2 2  1 1 1 1 1 1 1 1 1 1 1 0  1 1 1 1 1 1 1 1 1 1 1 0  0  0  0  0  0 0  1 1 0 0  1 1 0  1 1 I 1 1 1 1 1 1 1 1 0 1 1  1 1  1 1 0  1 1 0 0  1 1  1 1  1 1 0  1 1  1 1  0 0 0  0 0 0 0 0  1 1 1 1 0 0  1 1  0 0  1 1  o 0 0 0 0 0 0 0 0 0 o 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0  0  0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o  0  1 1  0  1 1  1 1  OF  DIVERTICULAR  SPECIMENS  M4 M4 M4 MA M4 MA M4 M4 M4 M4 M4 M4 M4 M4 M4 MA M4 M4 M4 M4 M4 M4 M4 M4 M4 M4 M4 M4 M4 M4 M4 M4 M4 M4 M4 MA M4 MA M4 MA M4 MA M4 MA M4 MA MA MA MA MA MA MA MA MA MA MA M4 M4 M4 M4 M4 M4 MA M4 MA M4 MA M4 MA M4 M4 M4 M4 MA M4 M4 M4 M4 M4 M4 M4 M4 M4 MA M4 M4 M4 MA  T  8 T  3 T 8 T 8  T 8  7 3 T a T a T a T B  T 3 T 8 T  VO VO VO VO VO VO VO  vo vo VO VO  vo vo vc vo vc  [VD  vo ivo vo  IVO  vo vo vo  VO  3 T  vo  B  T 3  VC VC  T  vo vo vc  a T a T a T 3 T  a  T  3 r 8 T  a  VC VO VO  vo vo  IVA [VA IVA IVA IVC IVC IVC IVC IVC IVC  3 r  vc ivc  a  ivo vo ivo  a7  vo ivo  T 8 T  T B T  3 T  a  T  s T  a  IVC IVC  IVO IVO  VO IVO VO VO VO VO VC  T  vo  T  vo ivo  a r 8 7 B T  8 7 a T a T r a T a r a r 8 7 a  VC  VC  ivo vo IVC  vc vo  VC  vo vc  IVO VO  vo vo vc vo vo vo vo vo vo  10 0 10 3' 7"0 9 0 3.3 2 0 3.3 0 a 0 9 0 7 0 9 . 0 3.3 2.0 3.3 0 9 0 9 0 7 0 9. 0 2 .3 2 0 3.3 1 a 0 9 0 7 0 9 . 0 2.3 2 0 3.3 1 9 0 2 10 0 1 0 0 7 0 .3 2 0 3.0 0 6 0 a 0 70 4 0 2 .3 2.0 3.0 0 to 3 a 0 7 0 4 0 2 .3 2 0 3.0 1 6 0 a 0 70 4 0 2 .3 2 0 3.0 1 9 0 9 0 70 9 0 2 . 3 2 0 4.0 0 9 4 30 5 0 0 0 2 3 2 0 4.0 0 a 0 9 0 9 0 9 0 3.3 2 0 4.0 1 9 0 9 0 9 0 9 0 3 3 2 0 4.0 1 10 00 10 0 7 0 10 0 3 0 2 0 4.0 0 3 7 0 7 0 3 0 3.0 2 0 4.0 0 10 0 10 0 7 0 10 0 3 0 2 0 4.0 1 3 0 7 0 7 0 3 0 3 0 2 0 4.0 1 12 0 12 0 3 0 12 0 3 5 2 0 3.5 0 7 0 7 0 3 0 7 0 3.5 2 0 3.3 0 12 0 12 0 3 0 12 0 3 5 2 0 3.3 1 7 0 7 0 3 0 7 0 3 5 2 0 3.5 1 12 0 12 0 9999 99 12 0 2 0 2 0 3.3 0 a 0 a 0 a 0 2 0 2 0 3.3 0 io 0 IO 0 5 0 to 0 2.5 2 0 3 .3 1 a 0 6 0 5 0 7 0 2 5 2 0 3.3 1 8 3 a 3 7 0 4 0 3 5 2 0 J.3 0 8 0 6 5 70 4 0 3 5 20 3 .3 0 a 3 6 3 7 0 3 0 3 3 2 0 3.3 0 a 0 S 5 7 0 3 0 3 5 2 0 3.3 0 8 3 6 3 7 0 4 0 3 3 20 3 .3 1 a 0 6 3 7 0 4 0 3 3 2 0 3.3 1 a 3 6 3 7 0 3 0 3 3 2 0 3.3 1 a 0 6 3 7 0 3 0 3 3 2 0 3.3 1 a 3 7 0 7 0 4 0 3 0 2 0 3.0 0 3 3 7 0 7 0 4 0 3 0 2 0 3.0 0 a 5 7 0 7 0 4 0 3 0 2 0 3.0 2 8 3 7 0 7 0 4 0 3 0 20 3 .0 2 12 0 0 0 2 0 t2 0 0 3 0 3 t . O 0 12 0 0 0 2 0 12 0 0 3 0 3 1.0 0t 13 1 0 0 2 0 11 5 0 3 0 3 2.3 1 51 0 0 2 0 1 3 1 0 3 0 3 2.3 1 12 0 12 0 5 0 0 0 3 3 10 3.3 0 12 00 12 0 5 0 0 0 3 3 10 3.3 0 12 0 12 0 5 0 0 0 3 3 10 3.3 1 0 0 3 3 10 3 12 0 12 0 5 0 .3 1 12 12 0 5 o0 00 00 3 3 2 0 3.0 1 12 0 1 2 0 3 3 3 2 0 3 .0 1 a 0 a 0 5 0 1 0 3 3 2 0 3.3 1 0 12 0 1 2 0 3 0 0 3 3 2 0 3 . 5 t 12 0 12 0 3 0 0 0 3 3 2 0 3.0 2 12 0 12 0 3 0 0 00 3 3 2 0 3.0 2 12 0 10 1 3 0 12 2 0 10 2.5 0 6 0 4 0 5 0 7 0 2 0 10 2 .5 0 12 0 12 0 3 0 0 0 992 0 10 1.5 2 9 12 0 12 0 5 0 0 0 10 1.3 2 12 0 13 0 3 0 12 0 10 10 3.0 0 6 0 6 0 3 0 6 0 t 0 10 3 .0 0 12 0 12 0 5 0 12 0 2 5 2 0 2.5 1 6 04 6 0 3 0 6 0 2 5 2 0 2 .3 1 12 0 12 0 3 0 1 0 1 2 0 2 0 3.0 0 a . 0 7. 0 3.0 a.0 2.0 2.0 3.0 0 to . 0 IO . 0 7.0 9 ..00 2. S i 2 .0 2.0 1 a . S a . 37. 0 3 2.5 3.0 2.0 1 10.5 to4 .0 5.0 9 . 5 2.0 1.0 2.0 0 3 . 0 . 0 3 . 0 3.0 3.0 l1. 0 2.0 0 IO . 3to .o 5.o 9 . 3 3.0 .o 3.0 o 3.0 4 .0 3.0 3.0 3.0 l .o 3.0 o 10.0 10.0 3.0 10.0 3.5 2.0 3.3 0 12.0 12.0 5.0 12.0 3.3 2.0 3.3 0 7.0 7.0 3.0 3.0 3.5 2.0 3.3 0 12.0 12 .0 3.0 1 .01 3.0 2.0 3.5 2 8 . 55. 0 9 .3 3.0 2.0 3.3 2 a.3 11 5 1 . 0 1 3 . 0 0 .0 3.5 1.3 3.0 1 9. 0 toa .0 5.0 to2.0 3.5 1.3 3.0 1 10 . 0 0 3 . 0 .0 3 3 2.0 4.0 1 6. 0 6 .0 3 .0 2.0 3.5 2.0 4.0 1 S. 10 0 1 0 . 0 0 1 0 .0 3.5 2.0 4.0 1 6 0 6. 0 3.0 2.0 3.5 2.0 4.0 1 1 2 . 0 1 2 . 0 3 . 0 1 2 . 0 3.3 1.3 4.0 1 IO 0 to 0 3. 0 11.0 3.3 1.3 4.0 1 8 0 7 0 3. 0 5 0 3.3 1.3 4.0 1 10 0 to .0 3 0 9 .0 3.5 2.0 4.0 1 0 0 5 0 2 . 0 3.3 2.0 4.0 1 a a 10 0 to4 .0 3.0 to .o0 3.0 2.0 2.0 0 a 0 0 5 0 7 3 0 2 0 2.0 0 io 0 1 0.0 3 0 to .0 2.0 2.0 2.0 1 a 0 4 0 3 0 7.0 3 0 2.0 1 2.0 1 10 0 10 0 3.0 9 .0 2.0 2.0 2.0 0 a 0 8 .0 3.0 4 . 0 2.0 2.0 I 2.00  0 0 0 0 0 0 0 0 0 0 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 ' 0 1 1 0 0 1 1 0 0 0 0 0 0 o 0 0 o o  0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  56 TABLE 15 (CONT.)  53A 53A 578 578 578 578 3BA 58A 58A S8A «OA 60A 60A SOA SOA 60A 60A 60S 608 60S 608 608 &oe 608 638 638 638 63B 618 6 IB 6 <B  0 0 0 0 0 • 0 0 0 0 0 0 0 0 D 0 0 0 D 0 0 0 0 D 0 • 0 0 0 0 0 0  62A 62A 62A 62A 62A 62A 64A 64 A 64A 64A 64A 64A 64A 64A 628 628 628 62B 62B 68A 68A 684 68A 66A 66A 66A 66A 66A 66A 668 668 66B 66B S98 69B 698 69B 698 69B 72A 72A 72A 72A 72B 728 72B 728 72B 72B  0 0 0 0 0 0 0 0 • 0 0 D 0 D 0 0 0 0 D 0 0 0 0 D 0 0 D D 0 0 0 D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 D 0 0  eoA  sialo  0 0 0 0 0 0 0 D 0 D 0 0 0 0 0 D 0 D 0 0 0 0 0 D 0 0 D 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 D 0 0 0 • D 0 0 0 0 0 0 0 D 0 0 0 D 0 D 0 0 0 0 D D 0 0 D 0 0 0 0 D 0 0 0 0 0  P P  r  s s  M H M M H H M H M M H H H M M M M M H H M H H H M M M M H H M F F F F F F F F F F F F F F M H M M M M M M H M M M M M H M M M M F F F F F F M H H M M M M M M M  p p  s 5 P P  s s s s  P p p p  s s s  »  p p p  s s p p  s s  p p S  s  p p p p  s s 5  s  p p p p  s s s  p p  s s  p p  s s s s p p  s s  p p  s s 5 5 0  P S  s  P p 5 S 5 5 P P  f  71 71 66 66 66 66 66 66 66 66 70 79 79 79 79 79 79 79 79 79 79 79 79 79 79 69 69 69 69 69 69 69 C9 43 43 43 43 43 43 43 43 43 43 43 43 43 43 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 83 82 82 82 82 82 82 82 82 82  k  A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A  A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A  A A A A A A A A A A A A  *  i t 0 0 t i 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 2 2 0 0 2 2 0 0 2 2 2 2 0 0 0 0 2 2 2 2 0 0 0 2 2 0 0 1 1 0 0 0 0 1 1 0 0 1 1 0 0 0 0 2 2 1  1  « 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  10 O 5.0 8 0 5.0 12.0 3.0 8.3 S.O 1 1 0 5.0 8.5 3.0 12.0 7.0 12.0 7.0 12.0 7.0 12.0 7.0 0.0 3.0 2.0 2.0 2.0 2.3 0.0 2.3 0.0 2.0 1 .0 2.0 1 .0 2.0 0.0 2.0 0.0 2 .0 2.0 2 .0 2.0 2 .0 0.0 2 .0 2.0 2 .0 0.0 2 0 3.0 2 .0 10.0 6 .0 S.O 6.0 10.0 6.0 S.O 6.0 9.0 6.0 3.0 6.0 9.0 3.0 5 5.0 5 3.0 5 S.O 8 3.0 S.O 3.0  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 7.0 7 7.0 9.0 9.0 9.0 9 7 7.0 7 7.0 9 10.0  a a a  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  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  sO a .0 a .0 8 .0 8 .0 .0 .0  a a 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  2.0 2.0  .o .o .o o .o  7.0 9.3 11.0 11 .0 9.3 9.3 11 0 12 0 10 8 0 12 10 0 12 0 0 9 0 2 0 9 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  a  o o o  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  0.0 0.0 0 0 O 0 .0 0 .0 0 .0 .0  o o o0 0  o o  0 2.0 2.0 2.0 2.0 2.0 2.0  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  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  absence  and  of both  sialic  acid  sulphate  using  this  esters.  method, indicating the  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  glycoproteins.  - permits  the identification  of eleven  distinct subclasses of  58  FIGURE DIFFERENTIAL  DIAGNOSTIC  SCHEME  EPITHELIAL  STEP  3 FOR  GLYCOPROT EIN  KOH-AB10-PAPS  "Vd M P M  P B T - K O H - A B 1 0 -PAS PBT-K0H-AB2-5 -PAS AB10-PAPS  >v 2  COLONIC  GLYCOPROTEINS  STAIN  PAP-H 0-T-KOH-Bh-PAS PAT-KOH-Bh-PAS PBT-KOH-PAS  CLASSIFYING  P P M  M P 0  I  II  III  V  M  0  A  A  P  'Va  1  1 1  !1 1 V  0 A M  0 0 0  A A A  A A A  1  1 1  1 1 1v  0 B 0  0 0 0  0 B 0  'V  b  B B M  IV  M P M  B B 0  a  a  A A P  v  c  P P A  a  B B 0  v  c  M P M  P P P  v  b  B B M  v  d  P P 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,  throughout  the  the  length  predominant  of the  glycoprotein  crypt (Slide  2),  appeared  to  whereas in others,  be  the  the  glycoprotein  in the tops of the crypts were distinct from that in the bases (Slide  2.5.  MORPHOLOGIC  This portion of the of  the  each  to  project was  separately intensity  of  for  was its  scale was  degree  of to  divided  mucosal  atrophy,  into  assess both the 0,0,  respectively,  thirds and  with  assessment  The hematoxylin  inflammation  atrophy; this scale varied from and  Morphologic  assessment.  arbitrarily  used  carried out in conjunction  Pathology.  histochemical  specimen  3).  ASSESSMENT  Department  subsequent  and  Dr. David was  was  and  eosin  section from  each  third  was  mucosal  atrophy.  A  asessed four-point  degree of inflammation and mucosal  indicating both  the  to a maximum of 3,3,  considered to have intermediate  Owen  performed  absence of  inflammation  indicating the  of severe inflammation and mucosal atrophy. Hence, a tissue assigned 2,0  same  presence  a value of  inflammation but no mucosal atrophy.  2.5.1. Morphologic Assessment of F o c a l Changes and F i e l d  Changes  Reid et al. (1985a) defined focal changes as the "existence of one or more crypts which and  stained  0-1  blue  with the  to  blue  purple  PBT/KOH/PAS  the normal range"(Slides  (0-1)  with  the  PAPT/KOH/Bh/PAS  procedure  procedure in sections which otherwise stained in  4-10). In H & E slides, focals are not distinct from the  60 surrounding field (Slide  11).  Therefore, the  closest histochemical section  & E slide which demonstrated focal change, slide,  and a morphologic evaluation  of the  was matched exactly focal and its  to the  H  to the H & E  surrounding crypts  was  carried out according to the above criteria.  A  'field change' was  the  staining  the  PBT/KOH/PAS  (Slides  12  of  and  respectively). severe.  Sections  2  (Slides  the  epithelial  glycoproteins  and  PAPT/KOH/Bh/PAS  the  show  a normal  changes  were  such  and 14  the  subdivided  PAPT/KOH/Bh/PAS  and  15,  combined score  as  a "generalized alteration in  that  the  sum  procedures  of the  was  scores of  less  than  5.  pattern of staining for these two procedures,  with moderate field change  (Slides  changes,  As  13  Field  PBT/KOH/PAS than  the  defined by Reid et aZ.(1985a)  respectively). for the  two  into  two  categories:  moderate  and  are those in which the  sum of the  scores  but  In  is  sections  procedures is  less  than  5  possessing less  greater  "severe"  field  than or equal to  2  16).  field  changes  histochemical  are,  slides  by to  definition, the  H  a  &  generalized  E  morphologic assessment of specimens  slides  phenomenon,  was  not  matching of  necessary.  demonstrating field changes  was  the  Therefore, carried out  according to the above criteria.  2.5.2. Morphologic Criteria for  In  the  dysplasia)  absence the  of  gross  morphologic  Normal  pathologic criteria  for  features normal  (ie. was  frank based  carcinoma, on  the  atypia  or  absence  of  61 mucosal atrophy or inflammation. Hence, when a tissue was zero  for both inflammation  and mucosal atrophy, it was  assigned  considered  a value of  to be normal  by morphologic standards.  2.5.2.1.  The  Morphologic Assessment of Diverticular  diverticulum  assessed  and  pathologic surface  the  recorded  process  difficult  to  and obtain,  compared  to  adjacent  separately.  found  in  area. The surface  inflammation  often  and  then,  mucosal the  the  ulcerative  surface In  most  diverticulum was  atrophy.  surface  Disease Cases  area colitis  mucosa cases did  from of  not  each  diverticular extend  generally  classified  Because  morphologically  from the  specimen  as  into  disease, the  the  adjacent  0 for both degree of 'normal'  tissue  diverticular disease specimens  and Crohn's Disease  was  specimens,  and in  is was this  respect served as a control.  2.6.  DATA  Data was  ANALYSIS  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 between  the  Therefore,  degree  further  the severity  of  a statistically  significant  inflammation  assessment  of the  and  relation  the of  correlation (p<.001, df = degree  any  of  mucosal  histochemical  9)  atrophy.  parameter  to  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 and and  14  in Tables  13,  sulphomucin in the descending  colon  14  relative  upper and lower portions of the for  each  diverticular disease from the was  and 15 shows the  disease.  ascending  omitted from the statistical  analysis  As  colon  will was  be  proportion of sialomucin crypts of the seen,  examined.  only  case  of  Therefore, this case  of the ascending colon.  62  one  ascending  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,  diverticular disease  3.2.1.1.  the  diverticula  and  the  uninvolved  surface  mucosa  of  the  specimens were considered separately.  Assessment of the relative proportion of sialomucins and  sulphomucins from  the descending colon  The of  results obtained (Table the  crypts  from  diverticular  more sulphated than those between  the  bases  specimens (Table  No  differences  the  tops  and  sulphated  from than  indicated that the glycoproteins in the bases  disease  and  tops  of  the  crypts  colitis  were  significantly  classes  were  were  detected  in  Crohn's  Disease  16a).  between  disease  ulcerative those  from  colitis the  detected  16b). However, the  in  the  glycoproteins  from  glycoproteins in the bases in  and  Crohn's  Disease  were  surface  mucosa  adjacent  to  glycoproteins in the diverticulum were  Disease  ulcerative  in the tops of the crypts. In contrast, no differences  of the crypts (Table  specimens  the  16 a-c)  significantly  diverticula.  more sulphated than those  less  Further,  in Crohn's  specimens but did not differ from those in ulcerative colitis specimens.  64 TABLE  16  RELATIVE PROPORTION OF SIALOAND SULPHOMUCINS IN T H E DESCENDING C O L O N AS ASSESSED B Y T H E KOH/AB1.0/PAPS PROCEDURE  A. Tops Versus Bases of Crypts for each Disease Disease  P Value  Results  a. DI(S)  0.0001  Significant  b. DI(D)  0.0001  Significant  c. U C  0.0212  Significant  d. C D  0.2033  Not Significant  B. Between-disease Comparison of Tops of Crypts a. DI(S) vs. C D  0.8389  Not  significant  b. DI(S) vs. U C  0.7673  Not  Significant  c. DI(D) vs. C D  0.2451  Not  Significant  d. DI(D) vs. U C  0.9990  Not  Significant  e. DI(S) vs. DI(D)  0.7091  Not  Significant  f. U C vs. C D  0.6331  Not Significant  C. Between-disease Comparison of Bases of Crypts a. DI(S) vs. C D  0.0002  Significant  b. DI(S) vs. U C  0.0239  Significant  c. DI(D) vs. C D  0.0059  Significant  d. DI(D) vs. U C  0.0901  Not  Significant  e. DI(S) vs. DI(D)  0.1635  Not  Significant  f. U C vs. C D  0.1314  Not  Significant  DI(S)=Diverticular disease surface mucosa; DI(D) = Diverticular diverticulum; U C = Ulcerative Colitis; C D = Crohn's Disease  disease  65 3.2.1.2. Assessment of the relative proportion of sialomucins and  sulphomucins from  the ascending colon  As  shown  in Table  sulphation existed  17(a-c),  between  tops  of the  sulphated  than the  bases  the  of  Disease  specimens  crypts revealed  and Crohn's Disease  specimens, latter.  crypts  results  obtained  the bases and tops  colitis or Crohn's Disease the  the  was  indicated that  no difference in  of the crypts for either ulcerative  (Table 17a). A between-disease comparison of  a significant difference indicating that  the  However, no difference demonstrated  between  former were in the  between  ulcerative  significantly less  glycoproteins  ulcerative  colitis  colitis  from  and  the  Crohn's  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  were  red to red-purple (R-RP) or Red (R) in the  KOH/AB1.07PAPS  tops  and  specimen  corresponding  evaluation,  some  of  bottoms  the  of  a  specimens  particular showed  a  (Slide  predominance  when  they  in both the 17).  of  In  this  sialomucins  throughout the entire tissue sample, while in others, only a part of the specimen fulfilled  this  criterion.  The  results,  colon  no  distinction  was  made  -  as  shown  between  the  in  Table  ascending  18,  are  and  for  the  entire  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 TABLE  17  RELATIVE PROPORTION OF SIALOA N D SULPHOMUCINS IN T H E ASCENDING COLON AS ASSESSED B Y T H E KOH/AB1.0/PAPS PROCEDURE  A.  Tops vs. Bases of Crypts for each Disease  Disease  Results  a. C D  Not Significant  b.  Not Significant  UC  B. Between-disease Comparison of Tops of Crypts  a. U C vs. CD  C. Between-disease  a. U C vs. CD  Significant  (p<.05)  Comparison of Bases of Crypts  Not Significant  U C = Ulcerative Colitis; CD = Crohn's disease.  68 TABLE  18  P E R C E N T A G E A N D N U M B E R O F CASES A N D SPECIMENS SHOWING A P R E D O M I N A N C E O F S I A L O M U C I N S IN T H E K O H / A B 1 . 0 / P A P S P R O C E D U R E  Ulcerative Colitis  Specimens Cases  l:  values  22.8%  (8)*  41.0% (7)  Crohn's Disease  Diverticular Disease  15.7% (8)  16.2%  (6)  28.6%  21.1%  (4)  (6)  in brackets are actual numbers of cases or specimens.  69  3.2.2. Severity of Disease Versus Histochemical Class  Statistical  analyses  were  existed between the of disease. top  and  Without exception, bottom  two  of  a  (ascending/descending)  class  are  associated  the  particular  determine  whether  or  not  in a specimen  degree of inflammation was crypt.  However,  the  was  performed  for  each  relationship  and the the  severity  same for the  epithelial  disease,  and each level in the crypt  each  glycoproteins  of  one  another,  ie)  anatomic  area  (top/bottom).  summarized in Table 19 show that disease severity independent  a  differed from those in the bottom. Therefore, analysis of  parameters  The results  to  type of glycoprotein present  present in the top often these  performed  the  degree  of  and histochemical  inflammation  is  not  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  the  crypt  As  for  each  anatomic  area  (ascending/descending)  and  each  level  in  (top/bottom).  shown  disease, disease.  ie.  in Table no  20,  no  relationships  particular class  exists  of glycoprotein  between is  histochemical  associated  with  class and  any particular  70 TABLE  ASSESSMENT  19  OF T H E DEGREE OF INFLAMMATION VS. CLASS USING T H E CHI S Q U A R E T E S T  HISTOCHEMICAL  Disease  Crypt/ Anatomic Location  df  Results  UC  Ascending-top Ascending-bottom Descending-top Descending-bottom  6 6 15 12  NS NS NS NS  CD  Ascending-top Ascending-bottom Descending-top Descending-bottom  8 15 6 8  NS NS NS NS  DI(S)  Descending-top Descending-bottom  5 5  NS NS  DI(D)  Descending-top Descending-bottom  10 10  NS NS  All diseases  Ascending-top Ascending-bottom Descending-top Descending-bottom  15 15 15 15  NS NS NS NS  U C = Ulcerative Colitis; CD = Crohn's Disease; DI(S) Diverticular disease, mucosa: DI(D) = Diverticular Disease, diverticulum; NS = not significant.  surface  71 TABLE  ASSESSMENT  20  OF HISTOCHEMICAL CLASS VERSUS SQUARE ANALYSIS  DISEASE  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.  USING  CHI  72  3.2.4. Degree of Sulphation Versus Degree of Acetylation  Previous  work  (Reid  et  al.,  1985a)  on  the  mucosa  adjacent  to  colonic  suggests than an abnormal staining pattern for side chain O-acetylated is  not  necessarily  sulphomucins. assess  the  Therefore, the of  associated  However, presence  analysis  in that or  was  disease to evaluate O-acetylation,  as  with  a  study,  absence  revealed  by  statistical  of  carried out whether  concomitant  abnormal analysis  association for  each  was  between  disease  staining  these  and  for  results,  O-acetylated and  as sialic  summarized acid  are  in  sialic acid pattern  for  performed  to  two  each  variables.  area  or not a relationship exists between the the  PBT/KOH/PAS  stain,  and  sialo- and sulphomucins, as revealed by the K O H / A B 1 . 0 / P A P S  The  not  Table  independent  21,  show  that  of changes  in  the  within pattern  pattern  of  stain.  changes the  tumors  in  side  proportion of  chain sulpho-  sialomucins.  3.2.5. Assessment of O-Acetylated Sialic Acids  The as  pattern being:  change,  of side-chain  O-acetylated  1. normal 2. focal  change  sialic  acid was  3. moderate  assessed  field change,  according to the criteria of Reid et a/.(1985a).  for  each  section  or 4. severe field  73 TABLE  21  ASSESSMENT OF T H E DEGREE OF SULFATION VERSUS DEGREE 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 CORRELATION COEFFICIENT  Disease  Anatomic/Crypt Location  Results  Crohn's disease  Ascending/Top  NS  Ascending/Bottom  NS  Descending/Top  NS  Descending/Bottom  NS  Ascending/Top  NS  Ascending/Bottom  NS  Descending/Top  NS  Descending/Bottom  NS  Diverticular disease  Descending/Top  NS  (Surface  Descending/Bottom  NS  Diverticular disease  Descending/Top  NS  (Diverticulum)  Descending/Bottom  NS  Ulcerative Colitis  NS = not  mucosa)  significant  74 TABLE  ANALYSIS  OF O-ACETYLATED  22  SIDE-CHAIN  SIALIC A C I D (BY SPECIMEN)  Disease  Normal  Moderate  Severe  Focal  TOTAL  DI(S)  22  4.5  3  6.5  36*  DI(D)  28  5  2  0  35**  UC  23  3  9  0  35  CD  30  11.5  6  3.5  51  MCT  15  7  14  26  62  TOTAL  118  31  34  36  219  DI(S) = Diverticular disease, surface mucosa; DI(D) = Diverticular disease, diverticulum; U C = Ulcerative colitis; CD = Crohn's disease; M C T = M u c o s a close to tumors. * 1/37 specimens - no mucosal surface component ** 2/37 specimens - no diverticulum component  75 The  actual  each  number of  disease  obtained  are  from  normal,  shown  in  a previous  focal Table  and 22.  field  changes  These  were  (Reid et al.,  study  for  each  specimen  compared  1985a) on the  to  the  mucosa  from results  adjacent  to  patterns  of  tumors.  Statistical  analysis  of  the  data  in  Table  22  suggests  O-acetylated sialic acid are more closely associated others.  The  results,  as  summarized  in  Table  23  that  some  with a particular disease than show  that,  as  a  group  glycoproteins from the mucosa adjacent to tumors (MCT) are statistically  the  different  from any of the three inflammatory bowel diseases. Further, comparisons between inflammatory adjacent  to  bowel  diseases  diverticula  revealed that  differed  only ulcerative colitis  significantly  from  each  other  and the in  their  mucosa 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  the  was  due  presence  in the  observed  between  Disease,  the  to  higher  the  to  latter the  absence  disease mucosa  of  focal  changes  (.005<p<.01). adjacent  diverticula and the number  of  focal  to  in  Similarly,  tumors  and  the  the  former  significant  ulcerative  mucosa adjacent to diverticula is changes  (p<.001), (p<.001), (p<.001), (.01<p<.025),  in  the  mucosa  respectively.  and  colitis,  their  difference Crohn's  apparently due  adjacent  to  tumors,  76 TABLE  ASSESSMENT  23  OF O-ACETYLATED SIALIC ACID WITH D I S E A S E U S I N G CHI S Q U A R E A N A L Y S I S  Disease  .  RESPECT  df  _P_ Value  3  NS  DI(S)  vs. DI(D)  DI(S)  vs. U C  3  .01<p<.025  DI(S)  vs.  CD  3  NS  DI(S)  vs.  MCT  3  .001<p<.005  DI(D) vs.  UC  3  NS  DI(D) vs.  CD  3  NS  DI(D) vs. M C T  3  p<.001  UC  vs.  CD  3  NS  UC  vs.  MCT  3  p<.001  CD  vs.  MCT  3  p<.001  TO  DI(S) = Diverticular disease, surface mucosa; DI(D) = Diverticular disease, diverticula; U C = Ulcerative colitis; C D = Crohn's disease ;MCT=Mucosa close to tumors; N S = not significant.  4. DISCUSSION  In general, results  from  have  on  been  based  HID/AB2.5 this  procedure.  previous a  studies of human colonic epithelial  relatively  Several  simple  investigators  histochemical are  of the  procedure should be interpreted more cautiously  et al.,  1985,  Rhodes et al.,  more  fully  characterize  1985c). It was  the  histochemical  the  glycoproteins  technique,  namely  opinion that  (Lev et al.,  intent of this  changes  seen  results  1985,  a  more  diseases,  complete  and therefore  histochemical primary study  characterization  or  was  it was  phenomena  to assess whether  epithelial  anticipated  changes seen in the secondary  of  stains used  in  mucosa could be  that  the  McFadden  ulcerative  to  colitis,  sophisticated  project allowed for  glycoproteins  adjacent  in  these  various  issue of whether  or not  to  represented  resolved.  or not ulcerative  in this  from  study, therefore,  Crohn's Disease and diverticular disease with a wider array of more histochemical procedures. The histochemical  the  colonic A  colitis  tumors  further  objective  of  the  this  and Crohn's Disease could  be distinguished from each other.  In this study the results obtained from the surface were  used  for  associated  with  specimens  were  comparative an  increased  histologically  purposes cancer normal,  as risk the  inflammation.  77  controls and  the  mucosa adjacent to diverticula because very  remainder  the  great  showing  disease  is  not  majority  of  the  only  very  mild  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  Filipe  &  Branfoot,  shown  that  in  from the 1976;  the  H1D/AB2.5 Sheahan  normal  procedure, previous  &  Jervis,  descending  proportionately more sulphate  colon  1976; the  studies  Filipe,  bases  (Gad,  1969,  of  the  1969a;  1979)  crypts  than the tops of the crypts, while in the  have contain  ascending  colon the lower portions of the crypts contain relatively less sulphate than to the upper  portions.  diverticula  were  The  results  consistent  obtained  with  sulphomucin gradient along the  that  from expected  length  of the  being more sulphated than the upper halves comparison  between  between the two,  the  diverticula  the  and  surface  from  mucosa  normal  crypts, the  indicating that histochemically,  mucosa the  they  showed  bases and lower  in the descending  surface  as  adjacent  to a  halves  colon. A statistical  revealed  no  difference  diverticula possess a normal  pattern of sulphomucins and sialomucins.  In  the  descending  significant more  difference  heavily  descriptions &  Branfoot,  colon,  specimens  between the  sulphated  than  the  from  ulcerative  colitis  bases and tops of the tops.  This  finding is  showed  a  crypts, the in keeping  statistically bases being  with  previous  of normal sulphomucin and sialomucin distribution (Gad, 1969a; Filipe 1976;  Sheahan  & Jervis,  1976;  between-disease comparison of the bases of the  Filipe,  1969,  1979).  crypts from the  However,  descending  a  colon  79 showed  that  between  ulcerative  colitis  and  the  surface  mucosa  not  ulcerative  colitis  and  the  diverticula. This finding implies that ulcerative colitis deviates somewhat  from  the  normal  a  statistical adjacent  histochemical  (Ehsanullah et al,  A  significant  difference to  existed  diverticula  pattern.  This  but  finding  1982b; Franzin et al,  finding  in  this  study  is  in  1983a;  was  for  the  accord  with  Filipe et al.,  absence  of  previous  1985).  a  statistical  between the bases and tops of the crypts from Crohn's Disease portions absence This the  of  the  colon.  This  finding  Crohn's  Disease  difference  specimens  specimens  in all  reflects  the  of the normal pattern of sulphomucin distribution in the descending colon.  deviation crypts  diverticular difference  as  from  normal  there  disease between  was  probably did not originate no  statistical  specimens. the  bases  the  abnormalities  sialomucins  in  sulphation.  This  the  exist  bases  was,  the  crypts  of  difference  in the  between  however, in  a  Crohn's  upper portions of  Crohn's  Disease  statistically Disease  and  significant  specimens  and  surface mucosa adjacent to diverticula, indicating  in  of  difference  There  those in the diverticula and the that  in  studies  the  the from  relative  crypts,  the  normal  has  proportion  of  alteration  being  not  been  sulphomucins a  reported  reduction  and in  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 bases  and  tops  of  the  crypts  indicating the loss of a sulphate ascending colon was  no statistically significant difference between the in  either  ulcerative  gradient. The absence  colitis  or  Crohn's  Disease,  of such a gradient in the  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  obtained  from  between  the  than  the  the  bases.  ascending  normal  As  no  it  was  colon,  pattern  of  sulpho-  diverticular not  and  disease  possible  to  sialomucin  specimens  make  were  comparisons  distribution  and  those  patterns found in either ulcerative colitis or Crohn's Disease.  In  summary,  in  the  between  the  bases  disease  specimens.  descending  and  tops  These  colon,  of  the  results  a  sulphate  crypts  for  in  the  gradient  ulcerative  distribution  was  colitis  of  demonstrated  and diverticular  sulphomucins  keeping with previous descriptions of diverticular disease (Filipe, 1969; 1984a;  Habib  (Habib  et  et al.,  al.,  specimens  the  and  1986).  The  all  portions  from  However,  1986)  use  may be responsible  of the  for  absence of  ulcerative of  a  the  sulphate  colon  has  KOH/AB1.0/PAPS  for this  colitis  not  procedure  in  been  dysplasia  Crohn's Disease  reported  previously.  of the  HID/AB2.5  instead  finding, as McFadden et al.  in  Reid et al.,  uncomplicated by  gradient  are  (1985) note that former  technique is a more sensitive indicator of sulphomucin distribution.  4.1.2. Patterns of O-acetylated  Culling  et  al.  proportion (Reid  et  greater  of al.,  in  (1979) C7C8 1984a)  the  between  the  previously  substituted and  former.  because no statistical  sialic acid  sialic  finding  difference and  was  that,  acid was  Crohn's Disease  This  diverticulum  reported  was  compared  not  to  reduced in both  specimens,  observed  either  as  with  confirmed  in the  ulcercative  the  in  the  normal,  the  ulcerative  colitis  reduction  being  present  study  O-acetyl substitution  pattern  colitis  Disease  or  Crohn's  81 specimens  or between  Disease  the mucosa adjacent to diverticulum specimens  specimens.  comparisons,  it  is  As  these  possible  that  conclusions the  are  reason  based  why  on  ulcerative  and Crohn's  between-disease  colitis  and  Crohn's  Disease  specimens do not differ significantly from diverticular disease specimens  because  the  acid.  latter  However,  possess  an  certainly  do not truly  Reid  O-acyl  not .the  diverticular  et  al.,  (1984a)  staining  pattern  case that  disease  reflect  the  specimens  the  normal  report  that  very  results  pattern of O-acetylated sialic  similar  from  this  is  diverticular disease  specimens  to  is  normal  study  are  and due  which do not possess a normal  it to  almost  selection  O-acyl  of  substitution  pattern. Further, the lack of statistical difference between  the mucosa adjacent to  diverticula  acid  and  the  diverticula indicated  that  the  sialic  O-acyl  substitution  pattern was uniform throughout this condition.  A  statistically  mucosa 6.5  adjacent  focal  significant to  changes  disease was  difference  (.01<p<.025)  diverticula and ulcerative in  the  former  apparently responsible  (Table for this  22)  was  colitis  observed  specimens.  and  their  between  the  The presence  absence  finding (.005<p<.01)  in  the  of  latter  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  instances,  from  that  these differences the  mucosa  arose  from  adjacent  to  changes  in  Disease  or diverticular disease  changes  responsible  for  found in the  the  mucosa  adjacent  to  a proportionately greater tumors  specimens.  significant  In  than  in  tumors. In all number of focal  ulcerative  no  instances,  difference  observed  were  colitis,  Crohn's  moderate  between  the  field  mucosa  82 adjacent field  to  tumors  changes  responsible  and the  three  (.025<p<.05),  for the  inflammatory bowel  in combination  significant  difference  with  focal  diseases.  However,  changes,  were  observed between the  severe  apparently  mucosa adjacent to  tumors and the diverticulum specimens.  It was  found that of the  proportion acid,  a  with  an  of sulphomucin  histochemically alteration  two  histochemical parameters examined, ie) the  and  sialomucin  and the  abnormal pattern  in the  other  (Table  for 21).  pattern  one  of  parameter  These  results  are  (1985a), in which, for example,  specimens  showed  adjacent  the presence  4.2.  to  tumors  a  normal  O-acetylated was  those reported by Reid et al.  distribution  relative  not  in  of  associated  keeping  25.8%  sialic  with  of mucosal  sulphomucins  in  of an abnormal pattern of O-acetylated sialic acid.  DIFFERENTIAL DIAGNOSIS  O F U L C E R A T I V E COLITIS  A N D CROHN'S  DISEASE From  a diagnostic  differentiates found  any  one disease from another.  between  parameters  point of view,  ulcerative  examined,  no  colitis useful  and  technique  is  only  useful  Although some statistical Crohn's  diagnostic  Disease  patterns  by  could be  if it  consistently  differences  both  were  histochemical  identified  for either  disease.  The  data shown in Table 22  indicate that focal changes are exclusively  to Crohn's disease. However, the disease is of little value 1.  use of focal changes as  confined  an indicator of Crohn's  as:  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  would be of any value to the diagnostic  4.3. PREMALIGNANT MARKERS  It has  been demonstrated  a distance ulcerative have cancer  developed  although cancer  dysplasia  it  is  (Morson  possibility dilemma  of  &  Pang,  with  no  has  believed  that  dysplasia  1967),  whether  cancer  is  prompted  investigators  still to  of  a  association  is not  been  the its  question.  search  that  dysplasia.  previously  or  a significant indicator  (1985) noted  a weaker  than  (1985)  al (1985) found dysplasia  evidence  exist  concurrent  has  et al.,  to suggest that  might  generally  Ransohoff et  and Filipe  cancer  prompted investigators  degree of dysplasia is  Recently,  from colonic cancer  and  pathologist.  OR NONSPECIFIC CHANGES?  that the  transformation.  colitis  study  by Morson and Pang, (1967); Ehsanullah et al.,  and Ransohoff et al.,(1985) of malignant  employed in this  These  more  marker  should  findings  reported. Therefore,  absence  for  cases of  between colonic  histologic  As  4/8  at  a  precursor rules  out  consequence, diagnostically  to the this  ideal  premalignant markers.  It  would  seem  that  the  ideal  premalignant  meet  the  criteria:1.  It must precede morphologically identifiable malignant transformation;  following  84 2.  It must be specific  3.  Its  sensitivity  for malignancy;  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  not  the  of  specificitj'  changes  nonspecific.  In  nonspecific  rely  the  mucosa  associated  noted  general,  is  of  in  the  those  mucosa who  on demonstrating  adjacent with  to  an  tumors  & Attwood, 1979;  Franzin  et  1981,  adjacent  favor that  are  increased  Isaacson  al.,  particular importance  the  the  also  malignant  1983b).  In  to  respect  tumors  view  that  predominance  are  the of  array  potential  (Saffos  &  the  present  whether  or  premalignant or seen  sialomucins  wide  1979;  to  changes  seen in a  Rhatigan & Saffos,  1983a,  with  are  seen in  of diseases not Rhatigan,  1977;  Listinsky & Riddell,  1981;  study,  the  acceptance  or  rejection of the premalignant marker hypothesis was based on a similar rationale.  The  results  sialomucins not  from from  directly  the  assessment  ulcerative  compared  to  colitis, those  However, inspection of Table portion of some specimens, entire  crypt.  As  Table  18  of  the  Crohn's  obtained  18 showed  showed clearly  relative Disease  from  the  proportion  of  sulpho-  and diverticular disease mucosa  adjacent  that in some instances,  to  and were  tumors.  all, or a major  a predominance of sialomucins throughout the demonstrates,  the  percentage  of  cases  and  specimens all  which demonstrated  these cases  that  the  later  presence  therefore,  this characteristic was  developed  colonic  cancer,  relatively  these  results  of a predominance of sialomucins  whether  further investigations  of this  high. Barring that  favor the  is nonspecific.  conclusion  It is doubtful,  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 tumors  indicated that,  without  diseases differed significantly to  tumors  (Table 23).  O-acyl  pattern  whole,  this  was  from the  However,  not  group  exception,  as  an  O-acetyl substitution  patterns  Table 22  found in the  showed  the  mucosa  O-acyl  mucosa  observed shows,  from all these mucosa  adjacent  a unique alteration in  adjacent  substitution  in the  adjacent to  to  profile  tumors. that  the  Rather, as  was  a  significantly  different from that seen in the other diseases. The most probable explanation for this  significant  mucosa  difference  adjacent  to  between  tumors  is  the  that  a  inflammatory smaller  bowel  proportion of  diseases  and  focal  changes  that  focal  the were  present in the former than in the latter.  The  results  represent  a  from  study  disease  with  Although the  study  premalignant  present  associated  this  were no  against  marker. Over  identified  known  in  the  cancer  with  the  greatest  there  was  no  necessary  argue  risk  unique  criteria for  a  risk. of  O-acyl  useful  the  hypothesis  50% of the  surface  mucosa  Conversely, cancer,  focal  was  substitution  premalignant  changes found in  adjacent  ulcerative totally  changes  to  diverticula,  colitis,  without  pattern  such  marker, it  is  the a  the  disease  focal  change.  that quite  it fulfilled clear  that  86 some  change  alterations  is  in  occurring  the  these changes,  O-acyl  however,  some  et al.  respects,  the  (1985d) was  results  results  pattern  diagnostic  to  sialic  tumors acid.  which  produce  The  significance  DIAGNOSTIC  SCHEME  scheme,  originally described by  redundancy which is built into this  going  undetected.  Thus,  serial sections from  procedure  and  red  such a chemically inconsistent  of the  of  OF T H E DIFFERENTIAL  differential  from  KOH/AB1.0/PAPS  one  adjacent  of some value in analyzing the data presented  in which two  whereas  mucosa  substitution  There is no doubt that the erroneous  the  of  remains to be elucidated.  4.4. T H E U S E F U L N E S S  In  in  techniques  was  used.  the in  for example,  same the  in this  scheme  one  specimen  question  purple in the  PBT/KOH/ABl.O/PAS  procedure  finding would failed to be detected  From  this  point of view  study.  prevents  would  were  Reid  then,  the  if only  scheme  is  highly useful.  Within  the  whether  context  or  histochemical  not  that  their  (Class  V),  glycoproteins  one  degree  Franzin  occurrence  acid but  project,  et  may  be  distinguishes  no  O-sulphate  would  associated  expect  the  diagnostic  of  inflammation  al.  (1983a,  that the presence  diagnostic scheme sialic  this  the  class.  support the view  of  a  scheme  was  1983b),  was  related  and  Lev  used to  et  al.  of sialomucins is a nonspecific  response  to  ischemia  or  a  to  assess  particular (1985)  who  event, suggest  inflammation. As  the  between those epithelial glycoproteins which contain esters to  (Class see  a  with a high degree  IV)  and  significant  those  which  proportion  possess both of  Class  IV  of inflammation, if such changes  are  87 due  to  inflammation.  However,  as  no  association  histochemical  class and inflammation, it was  the  of  mucosa  inflammatory  bowel  concluded that  diseases  are  assessing the effect of inflammation then, the enabled  one  to  Culling et al.  eliminate  (1979) showed  pronounced in more the present  The of  glycoproteins. of  severely  of  the  Because  epithelial  changes,  considered number  of  cases  of  inflamed tissues.  This  between  the  changes  to  inflammation.  scheme was  seen in  useful  histochemical  that decreases in O-acetylated  procedure does not  further advantage  focal  due  diagnostic  possible  this  distinguish  scheme  glycoproteins  it  does  In  as it  change.  sialic acid were more  finding was  was  when  artifactual serials,  of this viewed in the  scheme was in  nature. chances  only  useful  not confirmed in  of  not  in  histochemical  between  showing  eleven  that  classes different  none  of  with a particular histochemical  in its detection one  When  between any  differentiate  inflammatory bowel diseases were associated  A  not  observed  study.  HID/AB2.5  classes  one  was  serial  these  could  changes  detecting  them  class.  of focal changes. either are  be  seen  are  the  These  missed  throughout  reduced  and  or a the  possibility of the change being an artifact of one method is eliminated.  Despite the benefits, 1.  The procedures  there are disadvantages used  are extremely  to using this scheme.  time-consuming.  From  They are:-  a technical  point  of view, this consideration cannot be underestimated. 2.  The  IVd  structures.  and  Vd  classes  of  glycoproteins  contain  too  many  possible  88 3.  The scheme does not distinguish between the various gradations of colors ie. blue-purple, purple, royal-blue, but rather classifies  4.  all under the  'Purple. Thus, information on the relative proportion of components  is lost.  In  class  sections  which  are  not  homogeneous  glycoprotein, interpretation  of results  time-consuming.  than  'scrambled'  In  such  more  that  some  red-purple or aqua, etc.  a  for  a  can be extremely few  crypts  specimens  were  particular  tedious  and  sections  were  quite  the  purple,  Correct  classification  of  while  use of all seven stains. which are necessary information their  outweighed  epithelial  in  glycoproteins  However, some stains,  to correctly classify  in and of themselves.  inclusion  the  others  Thus in order to correctly classify  the  scheme  often  the  red,  glycoproteins, necessary.  necessitated  the  yield little  useful  whether  or not  therefore,  purposes  were  PBT/KOH7AB2.5/PAS,  the glycoproteins,  One questions,  for  the  usually  ie. the  of  difficult,  exact matching of crypts from one serial to another was 5.  heading of  of  classification  is  not  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  mentioned  project  histochemical  previously,  in  the  assessment  morphologic  divided into thirds and each third was the  degree  of  mucosal  atrophy.  such division of the specimen  In  preceded  morphologic  assessment,  the  tissue  assessment. was  As  arbitrarily  scored for the degree of inflammation and the  histochemical  was made. In specimens  assessment, where  however,  no  each or one of the  89  thirds was be  scored  reviewed  morphology.  again  differently so  as  from  to  Had all the  the  other,  match each  specimens  all the  third  stained  one  precisely  the  case that  of the histochemistry more time-efficient 2.  With  the  the  specimen  was  was  with  corresponding  However, more often  not homogeneous.  very time-consuming  the  slides had to  color for a particular stain,  review, of course, would not have been necessary. it was  histochemical  Thus,  this  and it would therefore  this  than not "re-review" have  been  to have assessed the morphology first.  exception  epithelial glycoproteins,  of  allowing  in this  for  particular  the  histochemical  study very little  gained from carrying out the  PBT/KOH/AB2.5/PAS,  AB1.0/PAPS  both  procedures.  time-consuming,  it  is  designs such as  this.  As  questionable  the  techniques  whether  they  the  classification useful  of  the  information  was  P A T / K O H / B h / P A S or the  and should  the be  analysis included  were in  very future  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.  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