Advanced Techniques in Diagnostic Cellular Pathology Advanced Techniques in Diagnostic Cellular Pathology Edited by Mary Hannon–Fletcher and Perry Maxwell © 2009 John Wiley & Sons, Ltd. ISBN: 978-0-470-51597-6 Advanced Techniques in Diagnostic Cellular Pathology Edited by Mary Hannon-Fletcher The University of Ulster, Coleraine, UK and Perry Maxwell Belfast Health & Social Care Trust, Belfast, UK This edition first published 2009, # 2009 by John Wiley & Sons Ltd Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical and Medical business with Blackwell Publishing. 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I. Hannon-Fletcher, Mary. II. Maxwell, Perry, 1958- [DNLM: 1. Cytodiagnosis–methods. 2. Cytodiagnosis–trends. 3. Cytological Techniques– methods. 4. Cytological Techniques–trends. 5. Pathology, Clinical–methods. 6. Pathology, Clinical–trends. QY 95 A2435 2009] RB43.A38 2009 611’.01815–dc22 2008055188 ISBN: 9780470515976 A catalogue record for this book is available from the British Library. Set in 11/13 Minion by Thomson Digital, Noida, India. Printed in Spain by Grafos SA, Barcelona. First Impression 2009 Cover design adapted from an ISH detection of HER-2 gene amplification in breast cancer. Contents List of Contributors vii Preface ix List of Tables xi List of Figures xiii Supplementary Materials xvii 1 Virtual Microscopy 1 Jim Diamond and David McCleary 1.1 Introduction 1 1.2 Digital (virtual) microscopy: equipment for implementation 2 1.3 The virtual slide format 11 1.4 Image serving and viewing 13 1.5 Applications of virtual microscopy 14 1.6 Virtual microscopy in education 23 1.7 Computational aspects of virtual microscopy 30 1.8 Conclusions 35 2 Cytopathology 37 Mary Hannon-Fletcher 2.1 Introduction 37 2.2 Basic principles 38 2.3 Cytodiagnosis 44 2.4 Gynaecological cytopathology 49 2.5 Conclusions 64 3 Flow Cytometry 69 Ian Dimmick 3.1 Introduction 69 3.2 Sample preparation 72 3.3 Principles of the flow cytometer 73 3.4 Clinical applications 83 3.5 The future for flow cytometry 95 3.6 Conclusions 97 4 Immunocytochemistry 99 Perry Maxwell and Merdol Ibrahim 4.1 Introduction 99 4.2 Basic principles 100 4.3 Clinical immunocytochemistry 110 4.4 Conclusions 130 5 Microarray-based Comparative Genomic Hybridization 135 David S.P. Tan, Rachael Natrajan and Jorge S. Reis-Filho 5.1 Introduction 135 5.2 Principles of array CGH 138 5.3 aCGH platforms 140 5.4 Choosing the right platform 144 5.5 Analysis and validation 146 5.6 Finding the target 147 5.7 Data integration 152 5.8 Clinical applications 152 5.9 Conclusions 153 6 Tissue In Situ Hybridization 163 Anthony O’ Grady, John O’ Loughlin and Hilary Magee 6.1 Introduction 163 6.2 ISH probes 164 6.3 Probe labels 166 6.4 ISH detection systems 168 6.5 Tissue preparation and the ISH procedure 168 6.6 Signal amplification 170 6.7 ISH controls 171 6.8 Clinical applications of tissue ISH 172 6.9 ISH automation 179 6.10 Image capture and analysis 180 6.11 Recent developments and future directions in tissue ISH 181 6.12 Conclusions 183 Index 189 vi CONTENTS List of Contributors Jim Diamond Lecturer Queen’s University Belfast Centre for Cancer Research & Cell Biology 97 Lisburn Road Belfast, UK Ian Dimmick Flow Cytometry Core Facility Manager Institute of Human Genetics Bioscience Centre International Centre for Life Newcastle Upon Tyne, UK Mary Hannon-Fletcher Lecturer University of Ulster Coleraine, UK Merdol Ibrahim Manager UKNEQAS ICC & ISH London, UK Hilary Magee Senior Medical Scientist Department of Cellular Pathology Adelaide & Meath Hospital, Dublin incorporating the National Children0s Hospital Tallaght Dublin, Ireland Perry Maxwell Principal Clinical Scientist Belfast Health & Social Care Trust Centre for Cancer Research & Cell Biology Belfast, UK David McCleary Doctoral Student Queen’s University Belfast Centre for Cancer Research & Cell Biology Belfast, UK Rachael Natrajan Molecular Pathology The Breakthrough Breast Cancer Research Centre Institute of Cancer Research London, UK Anthony O’ Grady Chief Medical Scientist Department of Pathology Royal College of Surgeons in Ireland Education & Research Centre Beaumont Hospital Dublin, Ireland John O’ Loughlin Chief Medical Scientist Department of Cellular Pathology Adelaide & Meath Hospital, Dublin incorporating the National Children’s Hospital Tallaght Dublin, Ireland Jorge S. Reis-Filho Molecular Pathology The Breakthrough Breast Cancer Research Centre Institute of Cancer Research London, UK David S.P. Tan Molecular Pathology The Breakthrough Breast Cancer Research Centre Institute of Cancer Research London, UK viii LIST OF CONTRIBUTORS Preface Cellular Pathology in recent years has become more closely involved in the direct management of patients with the introduction of molecular tech- nologies and targeted therapies. Through this, we have seen the introduc- tion of specialist pathology. It is the aim of this book to introduce these concepts and the key technologies that are influencing clinical practice today. Throughout this book we show how clinical practice has been affected by these respective technologies and how further development will influence the practice and delivery of Cellular Pathology, which will impact on the patient through targeted therapeutics and diagnostics. In Virtual Microscopy, we deal with the changing face of the most traditional aspect of Cellular Pathology: that of microscopy itself. For centuries, the glass microscope slide has been the sole method of visualising cells and tissues, but with the addition of computing, imaging and communications technologies, it is now possible to digitise the glass slide and deliver it via the World Wide Web, transforming the ability of the practitioner to deliver a diagnosis, consultation or high-throughput image analysis for biomarker research. Cytopathology and the introduction of Liquid-based Cytology have greatly improved the quality and thus the sensitivity of the traditional Papnicolaou smear and non-gynaecological cytology specimens. An added bonus of this technology is the surplus of well-preserved material that can be used for additional diagnostic procedures and for research. The intro- duction of the Human Papilloma Virus vaccination programme may well change the future of the cervical screening programme; only time will tell how effective it will be. Flow Cytometry has expanded over recent years with the introduction of multicolour cell and protein labelling. Yet it is through understanding the components of the flow cytometer and the properties of the labels that the precise identification of elements important to the practitioner will be enabled. Immunocytochemistry has seen the continued expansion of the anti- body repertoire with diagnostic, prognostic and therapeutic demands. The experience of peer groups and external quality assurance is a vital part in delivering on the promise of identifying patients suitable for targeted therapies. The search for new biomarkers and therapeutic targets continues and we highlight a strategic means of identifying these key genes and proteins through array Comparative Genomic Hybridization, and outline how this is used in association with companion diagnostic technologies. In Situ Hybridization has shown promise of being used in the routine laboratory for more than a decade but it is the introduction of the technology for Her2/Neu gene amplification that has realised this potential. Its extension to other biomarkers has followed rapidly. The content of this book is particularly suitable for students with a basic working knowledge of Cellular Pathology, although each author has given space to providing some basic principles and key references for students less familiar with thesenew technologies. The book is a suitable text forstudents to Masters Level in Cellular Pathology but it is hoped that it will support both students and practising scientists who wish to understand more fully the principles and clinical value of the technologies described within. Mary Hannon-Fletcher, Coleraine Perry Maxwell, Belfast October 2008 x PREFACE List of Tables 2.1 Changes associated with a) the cytoplasm and b) the nucleus 52 2.2 Follow-up procedure following an abnormal Pap smear result 54 2.3 European age-standardized mortality from cervical cancer by age group, England and Wales . . . . . . . . . . . . . . . . . . . 56 2.4 Sampling errors using traditional Pap smear collection . . . . 58 2.5 Some examples of the advantages of using LBC for gynaecological sample collection and processing . . . . . . . . . 61 4.1 A scoring system which accounts for both the staining intensity and the number of cells staining positive . . . . . . . 109 4.2 Commonly-used cytokeratin monoclonal antibodies and their CK targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 4.3 UK NEQAS ICC cytology module data for CD45 markers over a two-year period . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 4.4 UK NEQAS ICC & ISH general module data CD45 (1.5 years) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 4.5 General module data CD45 (1.5 years) showing the performance of the participants by clone, based on pass, borderline or fail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 4.6 Optimal staining patterns to be expected on sections of appendix by angiogenic markers CD31, CD34 and von Willebrand factor VIII (vWF) . . . . . . . . . . . . . . . . . . . . . . 120 4.7 Results from UK NEQAS ICC & ISH breast hormone module ER 124 4.8 Results from the UK NEQAS ICC & ISH alimentary tract module where participants used HMER prior to staining . . 129 4.9 Results from the UK NEQAS ICC & ISH Alimentary tract module, stained at the same time as in Table 4.8 but without the use of HMER prior to staining . . . . . . . . . . . . 129 5.1 Comparison of aCGH and other techniques for genetic analysis 137 5.2 Parameters to be considered in the design of microarray-based comparative genomic hybridization studies 138 6.1 Examples of chromosomal and genetic targets for ISH testing of solid tumours . . . . . . . . . . . . . . . . . . . . . . . . . . 177