THE SIMULTANEOUS IMMUNOCYTOCHEMICAL STUDY OF CDla AND S100 ANTIGENS IN CERVIX AND SKIN The Development and Use of an Immunocytochemical Technique. A thesis submitted for the degree of Doctor of Philosophy in the Faculty of Medicine University College London by PETER HENRY MADDOX Dept, of Histopathology St Marys Wing Whittington Hospital London. 1992 ProQuest Number: 10609757 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10609757 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 2 ABSTRACT. A technique using formalin-calcium pre-fixed frozen sections has been developed which enabled the simultaneous demonstration of CDla and S100 antigens using the Dako antibodies, monoclonal Nal/34 and polyclonal S100 and an avidin-biotin complex peroxidase label. A reproducible counting method has been described which made a direct comparison of these two antigens in the epithelium of human skin and cervix. The Wilcoxon non-parametric test indicated no significant difference between the CDla and S100 counts of normal cervix (Z=1.02) but a difference between the seven counts showing minimal change papilloma virus infection (Z=2.63). A significant difference was shown in biopsies of normal skin (Z=3.37). The same test performed upon biopsies of cervix and skin of patients infected with Human Immune Deficiency Virus gave Z-values of 5.14 and 4.17 respectively. There was no significant difference between the CDla counts of these biopsies and those of normal biopsies (cervix, Z=0.01; skin, Z=0.62), but a significant difference when the same comparison was made for the S100 counts (cervix, Z=5.17; skin, Z=2.89). 3 The ratio of CDla/SlOO positivity in normal epithelium was found to approach unity, rising in value in cases of minimal change Human Papilloma Virus infection and rising still higher in cases of Human Immune Deficiency Virus infection. The effect of subcutaneous malignant breast disease upon the expression of the two antigens in the overlying epithelia indicated a lower CDla positive count in epithelia distant from the tumour and a normal count above the tumour (Z=2.86). There was no significant difference in the S100 counts of the two sites (Z=0.26). The final study used morphometric and colorimetric methods to support an observation previously reported in cytological material that the two antigens are expressed upon an individual Langerhans cell. ACKNOWLEDGEMENTS The initiation, management and completion of this project would not have been possible without the permission and support of Dr. D. Jenkins MRCPath. (Director of Clinial Research) and Dr. J. Dyson FRCPath. (Head of Histopathology and Cytology Department) both of the Whittington Hospital. I am indebted to the Technical Head of the Histopathology and Cytology Department, Mr. Ron Yabsley, for his invaluable support and kindness in negotiating finance and allowing me laboratory time in persuit of my studies. My thanks are due to Mr. A. Singer FRCOG (Consultant Gynaecologist), for allowing me access to his cases and to the theatre staff of the Royal Northern for informing me of the availability of "normal" cervical material. I gratefully acknowledge the contribution of Doctors, S. Barton and J. Langtry of the Westminster Hospital, London, and JR. Smith, P. French and SM. Forster of St. Mary's Hospital, London, for taking the cervical and skin biopsies used for the Human Immune Deficiency study. I also acknowledge the cooperation of Consultant Mr. AW. Wilson FRCS, of the Whittington Hospital Surgical Unit in allowing me access to the mastectomy resections cases. My thanks go, to members of the Whittington and University College photographic department, Mr. Simon Clarke, Miss. Carol Buckman, and Mr. Noel Cadet, for their help and advice with the illustrations; and to the Chief Librarian of the Islington District Library, Miss Jane Stephen, for helping to obtain the numerous journal references, many of which proved to be extremely difficult. In performing the statistical analysis of the results I wish to acknowledge the advice of Dr. Jack Cuzick, Head of the Imperial Cancer Research Fund department of Mathematics, Statistics and Epidemiology and the help of Miss Ah Mun Kuan, statistician to the Whittington Hospital. There are two people to whom I owe so much for the completion of this project. The first is Dr. Anne Szarewski, Research Fellow of the Imperial Cancer research Fund, who acted as my mentor and reader. The second is Miss Margaret Hills, Research Assistant to the Clinical Research Department, who gave me invaluable support and encouragement and so frequently deputized for me in the routine immunocytochemical department to allow me to study. Finally, thanks to my wife Muriel, for her understanding and encouragement at my undertaking this project, and particularly for her tolerance at my withdrawal to an office and word-processor whilst the domestic scene moved into disrepair. 6 TABLE OF CONTENTS. PAGE. ABSTRACT. 2 ACKNOWLEDGEMENTS. 4 TABLE OF CONTENTS. 6 FIGURES. 14 TABLES. 18 LIST OF ABBREVIATIONS. 20 CHAPTER 1. General Introduction. 23 The Langerhans cell and Epidermal Surveillance. 23 The Melanocyte Theory. 24 The Keratinocyte Theory. 25 The Silberberg Theory. 25 Skin Associated Lymphoid Tissue. 26 The Skin Immune System. 28 Ultrastructure of Langerhans cells. 29 Methods of Identifying Langerhans cells. 32 Metal Impregnation. 32 Supravital Staining. 33 Enzyme Histochemistry. 34 7 PAGE. Immunocytochemistry. 3 5 Cluster of Differentiation. 37 CD1 Antigens. 38 S100 Protein Antigens. 42 Behavioural Studies on S100 Protein. 47 Less Specific Antigens used for the 47 Demonstration of Langerhans cells. 47 Vimentin. 47 Class II Antigens. 48 Fc and C3 Antigens. 50 CD4 antigens. 50 General Principles of Morphometry. 51 Selection of Test Groups. 52 Collection and Handling of the Specimen. 53 Collection of Numerical Data. 53 8 PAGE. CHAPTER 2. Introduction to Studies. 55 Formaldehyde as a fixative. 56 Unfrozen Unembedded Sections. 58 Pre-unfixed Frozen Sections. 58 Pre-fixed Frozen Sections. 59 Fixed Processed EmbeddedSections. 59 Other Embedding Media. 61 Sections Adhesives. 63 Water Soluble Proteins. 63 Cationic Polymerised Amino-acids. 63 of Poly-L-lysine. Organic Silanes. 64 Light Microscopy Immunocytochemical Labelling Methods. 65 Peroxidase Labelling Systems. 65 Other Enzyme Labels. 67 Colloidal Gold Markers. 68 The Quantitative Analysis of Langerhans Cells in Various Dermatological Diseases. 70 The Quantitative Analysis of Langerhans Cells on the Epidermis of Patients with Human Immunodeficiency Virus (HIV). 73 The Quantitative Analysis of Langerhans cells Overlying Primary Human Tumours. 7 6 Problems in Langerhans cell Enumeration in Tissue Sections. Investigations of the CDla and S100 Phenotypic Expression On Langerhans cells. CHAPTER 3. Aims of Thesis. Problems to be Overcome. Project Studies. CHAPTER 4. Materials. Subjects (Main Study and Study Id). Subjects for Statistical Reproducibility of Counting (Study la). Subjects for Assessment of "Normal Counts" (Study la). Subjects for Assessment of CDla and S100 positivity in Skin and Cervix of Patients with HIV Infection (Study lb). Subjects for Assessment of CDla and S100 positivity in Skin of Patients with Breast Cancer (Study lc). 10 PAGE. Reagents for Paraffin Processing. 87 Fixatives. 87 Dehydration Solutions. 87 Clearing Solutions. 88 Embedding Reagents. 88 Processing Equipment. 88 Reagents for Cryo-processing. 89 Fixatives. 89 Post-fixed Cryosections. 89 Pre-fixed Cryosections. 89 Immunocytochemical Reagents. 89 Buffers. 90 Trypsin. 90 Endogenous Peroxidase Inhibitor. 91 Chromagens. 91 Equipment. 91 Antibody Suppliers. 92 Adhesives. 94 CHAPTER 5. Methods. Main study.(Study 1). The Demonstration of CDla and S100 Antigens in Frozen and Paraffin Sections. 95 General collection and processing of Cervix. 95 Basic Paraffin Technique for The Demonstration of S100. 95 11 PAGE. Frozen technique for the demonstration of CDla. 96 Immunocytochemical Staining of Sections for S100 and CDla antigens. 97 Nursing" of the CDla Antigen during "Wax" Processing. 98 The "Nursing" of S100 Antigens in Frozen Sections. 98 Post-Fixed Cryosections. 99 Prefixed Frozen Sections. 99 Experimental comparison of adhesives. 100 Preparation of Formal-calcium fixative. 101 Compression effect of section. 101 Morphometric assessment of the four peroxidase labelling methods. 102 Staining and Morphometric Analysis of Sections. 103 Study la. Statistical Reproducibility of Counting. 106 Count Analysis of Normal Skin and Cervix. 106 Study lb. A Quantitative Assessment of CDla and S100 positivity in Skin and Cervix of Patients with HIV infection. 108 Study lc. A Quantitative Assesssment of CDla and S100 positivityin Skin of Patients with Breast Cancer. 109 12 PAGE. Study Id. A Numerical Analysis of Single and Mixed Counts. 110 Colorimetric Method of Analysis. 112 Immunogold Labelling of CDla. 112 Fluorochrome Labelling of S100 Antigens. 113 Phosphatase Labelling of S100 Antigens. 113 CHAPTER 6. Results (Study 1).