Molecular Biology in Cellular Pathology

The latest edition of this highly successful text, covers the major advances in the methods used in cellular and molecular pathology. In recent years, knowledge of the molecular organization of the cell has led to the development of powerful new techniques that bring greater accuracy and objectives to the diagnosis, prognosis and management of many diseases and to the study of pathological states. This book describes the latest molecular techniques available for the analysis of diseases. In particular it includes new techniques using fluorescent dyes, DNA microarrays, protein chemistry, and mass spectrometry. It also incorporates information from the Human Genome Project, and the new disciplines of genomics and proteomics, where relevant to pathology. Color plates are a new feature of this edition, illustrating the advances in fluorescence labeling of cells.

John Crocker is the editor of Molecular Biology in Cellular Pathology, published by Wiley.

Paul G. Murray is the editor of Molecular Biology in Cellular Pathology, published by Wiley.

Preface

xiii

Preface to Molecular Biology in Histopathology

xv

List of Contributors

xvii

1 Blotting Techniques: Methodology and Applications

1

(18)

Fiona Watson and C. Simon Herrington

1.1 Introduction

1

(1)

1.2 Blotting techniques

1

(14)

1.3 References

15

(4)

2 In-situ Hybridisation in Histopathology

19

(30)

Gerald Niedobitek and Hermann Herbst

2.1 Introduction

19

(1)

2.2 Experimental conditions

20

(3)

2.3 Probes and labels

23

(4)

2.4 Controls and pitfalls

27

(2)

2.5 Double-labelling

29

(2)

2.6 Increasing the sensitivity of ISH

31

(2)

2.7 What we do in our laboratories

33

(2)

2.8 Applications of ISH: examples

35

(4)

2.9 Perspective

39

(1)

2.10 References

40

(9)

3 DNA Flow Cytometry

49

(12)

M. G. Ormerod

3.1 Introduction

49

(1)

3.2 Definitions and terms

49

(1)

3.3 Dye used for DNA analysis

50

(2)

3.4 Sample preparation for DNA analysis

52

(1)

3.5 Analysis of the DNA histogram

53

(1)

3.6 Quality control

53

(2)

3.7 Computer analysis of the DNA histogram

55

(2)

3.8 Multiparametric measurement

57

(2)

3.9 Acknowledgements

59

(1)

3.10 References

59

(2)

4 Interphase Cytogenetics

61

(18)

Sara A. Dyer and Jonathan J. Waters

4.1 Introduction

61

(1)

4.2 Interphase cytogenetics

62

(5)

4.3 Applications

67

(9)

4.4 Conclusion

76

(1)

4.5 References

77

(2)

5 Oncogenes

79

(26)

Fiona Macdonald

5.1 Introduction

79

(1)

5.2 Identification of the oncogenes

79

(1)

5.3 Functions of the proto-oncogenes

80

(9)

5.4 Mechanism of oncogene activation

89

(2)

5.5 Oncogenes in colorectal cancer

91

(3)

5.6 Oncogenes in breast cancer

94

(1)

5.7 Oncogenes in lung cancer

95

(1)

5.8 Oncogenes in haematological malignancies

96

(3)

5.9 Other cancers

99

(1)

5.10 Conclusion

100

(1)

5.11 References

100

(5)

6 Molecular and Immunological Aspects of Cell Proliferation

105

(32)

Karl Baumforth and John Crocker

6.1 The cell cycle and its importance in clinical pathology

105

(3)

6.2 Molecular control of the cell cycle

108

(3)

6.3 Cell cycle control

111

(1)

6.4 The cell cycle and cancer

112

(3)

6.5 Immunocytochemical markers of proliferating cells

115

(18)

6.6 References

133

(2)

6.7 Further Reading

135

(2)

7 Interphase Nucleolar Organiser Regions in Tumour Pathology

137

(16)

Massimo Derenzini, Davide Treré, Marie-Françoise O'Donohue and Dominique Ploton

7.1 Introduction

137

(1)

7.2 The AgNORs

138

(4)

7.3 NOR silver-staining

142

(3)

7.4 Quantitative AgNOR analysis

145

(1)

7.5 AgNORs as a parameter of the level of cell proliferation

146

(1)

7.6 Application of the AgNOR technique to tumour pathology

147

(4)

7.7 What future for AgNORs in tumour pathology?

151

(1)

7.8 References

152

(1)

8 Apoptosis and Cell senescence

153

(40)

Lee B. Jordan and David J. Harrison

8.1 Introduction

153

(1)

8.2 Apoptosis

153

(21)

8.3 Cell senescence

174

(4)

8.4 Summary

178

(1)

8.5 References

179

(14)

9 The Polymerase Chain Reaction

193

(20)

Timothy Diss

9.1 Introduction

193

(1)

9.2 Principles

194

(3)

9.3 Analysis of products

197

(2)

9.4 RT-PCR

199

(1)

9.5 Quantitative PCR

200

(1)

9.6 DNA and RNA extraction

200

(1)

9.7 Correlation of the PCR with morphology

201

(1)

9.8 Problems

202

(1)

9.9 Applications

202

(1)

9.10 Diagnostic applications

203

(6)

9.11 Infectious diseases

209

(1)

9.12 Identity

209

(1)

9.13 The future

210

(1)

9.14 References

210

(2)

9.15 Online information

212

(1)

10 Laser Capture Microdissection: Techniques and Applications in the Molecular Analysis of the Cancer Cell

213

(20)

Amanda Dutton, Victor Lopes and Paul G. Murray

10.1 Introduction

213

(1)

10.2 The principle of LCM

214

(2)

10.3 Technical considerations

216

(1)

10.4 Advantages and disadvantages of LCM

217

(5)

10.5 Applications of LCM

222

(7)

10.6 Future perspectives

229

(1)

10.7 Acknowledgements

229

(1)

10.8 References

229

(4)

11 The In-situ Polymerase Chain Reaction

233

(18)

John J. O'Leary, Cara Martin and Orla Sheils

11.1 Introduction

233

(1)

11.2 Overview of the methodology

234

(1)

11.3 In-cell PCR technologies

235

(3)

11.4 In-cell amplification of DNA

238

(4)

11.5 Detection of amplicons

242

(1)

11.6 Reaction, tissue and detection controls for use with in-cell DNA PCR assays

243

(1)

11.7 In-cell RNA amplification

244

(2)

11.8 Problems encountered with in-cell PCR amplification

246

(1)

11.9 Amplicon diffusion and back diffusion

247

(1)

11.10 Future work with in-cell PCR-based assays

247

(2)

11.11 References

249

(2)

12 TagMan® Technology and Real-Time Polymerase Chain Reaction

251

(18)

John J. O'Leary, Orla Sheils, Cara Martin, and Aoife Crowley

12.1 Introduction

251

(1)

12.2 Probe technologies

252

(2)

12.3 TagMan® probe and chemistry (first generation)

254

(2)

12.4 Second generation TagMan® probes

256

(2)

12.5 Hybridisation

258

(1)

12.6 TagMan® PCR conditions

259

(1)

12.7 Standards for quantitative PCR

260

(1)

12.8 Interpretation of results

261

(1)

12.9 End-point detection

262

(1)

12.10 Real-time detection

263

(1)

12.11 Relative quantitation

263

(1)

12.12 Reference genes

264

(1)

12.13 Specific TagMan® PCR applications

265

(3)

12.14 References

268

(1)

13 Gene Expression Analysis Using Microarrays

269

(18)

Sophie E. Wildsmith and Fiona J. Spence

13.1 Introduction

269

(1)

13.2 Microanay experiments

269

(4)

13.3 Data analysis

273

(11)

13.4 Recent examples of microarray applications

284

(1)

13.5 Conclusions

284

(1)

13.6 Acknowledgements

284

(1)

13.7 References

284

(2)

13.8 Further Reading

286

(1)

13.9 Useful websites

286

(1)

14 Comparative Genomic Hybridisation in Pathology

287

(20)

Marjan M. Weiss, Mario A.J.A. Herensen, Antoine Snijders, Horst Buerger, Werner Boecker, Ernst J. Kuipers, Paul J. van Diest and Gerrit A. Meijer

14.1 Introduction

287

(2)

14.2 Technique

289

(3)

14.3 Data analysis

292

(1)

14.4 Applications

293

(6)

14.5 Clinical applications

299

(1)

14.6 Screening for chromosomal abnormalities in fetal and neonatal genomes

299

(1)

14.7 Future perspectives

300

(1)

14.8 Acknowledgements

301

(1)

14.9 References

301

(6)

15 DNA Sequencing and the Human Genome Project

307

(22)

Philip Bennett

15.1 Introduction

307

(1)

15.2 DNA sequencing: the basics

308

(10)

15.3 Applications of DNA sequencing

318

(2)

15.4 The Human Genome Project

320

(7)

15.5 References

327

(1)

15.6 Further Reading

327

(1)

15.7 Useful websites

328

(1)

16 Monoclonal Antibodies: The Generation and Application of 'Tools of the Trade' Within Biomedical Science

329

(22)

Paul N. Nelson, S. Jane Astley and Philip Warren

16.1 Introduction

329

(2)

16.2 Antibodies and antigens

331

(1)

16.3 Polyclonal antibodies

332

(1)

16.4 Monoclonal antibody development

333

(5)

16.5 Monoclonal antibody variants

338

(3)

16.6 Monoclonal antibody applications

341

(4)

16.7 Therapy

345

(1)

16.8 Specific applications

346

(1)

16.9 Conclusions

347

(1)

16.10 Acknowledgements

347

(1)

16.11 References

347

(4)

17 Proteomics

351

(20)

Kathryn Lilley, Azam Razzaq and Michael J. Deery

17.1 Introduction

351

(1)

17.2 Definitions and applications

352

(1)

17.3 Stages in proteome analysis

352

(16)

17.4 Future directions

368

(1)

17.5 References

368

(3)

Index

371

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Molecular Biology in Cellular Pathology

Summary

Author Biography

Table of Contents

Supplemental Materials