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Built on a decade of experience with novel molecular diagnostics, this practice-oriented guide shows how to cope with validation issues during all stages of biomarker development, from the first clinical studies to the eventual commercialization of a new diagnostic test.

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List of Contributors XI

Preface XV

1 Biomarkers - Past and Future 1
Siegfried Neumann

1.1 Introduction 1

1.2 Definitions of Biomarkers 2

1.3 Biomarkers in the Past 3

1.4 Novel Molecules and Structural Classes of Biomarkers by New Technologies 7

1.5 Biomarkers in Drug Research 9

1.6 Current Development and Future Trends for Biomarkers in Laboratory Diagnostics 12

1.6.1 Biomarker Test Validation 12

1.6.2 Companion Diagnostics in Clinical Pharmacology 14

1.6.3 Biomarker Multivariate Index Assays 16

1.6.4 Regulatory Policies on Biomarker Tests 17

1.7 Summary and Outlook 19

References 20

2 Quantitative Proteomics Techniques in Biomarker Discovery 23
Thilo Bracht, Dominik AndreMegger,Wael Naboulsi, Corinna Henkel, and Barbara Sitek

2.1 Introduction 23

2.1.1 General Considerations 24

2.2 2D-Difference Gel Electrophoresis 27

2.3 Mass Spectrometry-Based Proteomics 29

2.3.1 Principles and Instrumentation 29

2.3.1.1 Ionization Methods 29

2.3.1.2 Mass Analyzers 30

2.3.2 Label-Free Protein Quantification 30

2.3.2.1 Area Under Curve (AUC) or Signal Intensity Measurement 30

2.3.2.2 Spectral Counting 31

2.3.3 Label-Based Proteome Analysis 31

2.4 MALDI Mass Spectrometry Imaging 33

2.5 Conclusion 36

References 36

3 Biomarker Qualification: A Company Point of View 39
Maximilian Breitner, Kaïdre Bendjama, and Hüseyin Firat

3.1 Introduction 39

3.2 Biomarker Uses 40

3.3 Biomarker Types 41

3.4 Validation vs. Qualification 43

3.5 Strategic Choices in Business Models 43

3.6 Validation of Analytical Methods 44

3.6.1 Currently Applicable Guidelines for the Validation of Analytical Methods 45

3.6.2 Laboratory Proficiency 46

3.6.3 Establishment of Reference Ranges for Candidate Biomarkers 46

3.7 Clinical Qualification of Candidate Biomarkers 47

3.7.1 Methodological Approaches 47

3.7.2 Study Size for Biomarker Performance Characterization 48

3.7.3 Sample Quality and Biobanking 50

3.7.3.1 Sample Collection 50

3.7.3.2 Storage of Sample 51

3.7.3.3 Clinical Data (Sample Annotation) 52

3.7.3.4 Ethical Considerations 53

3.8 Biomarker Qualification in the ´omics Era 53

3.9 An Example of a Biomarker Provider 54

3.10 Conclusion 55

References 55

4 Biomarker Discovery and Medical Diagnostic Imaging 59
Andreas P. Sakka and James R.Whiteside

4.1 Introduction 59

4.1.1 Imaging Modalities 59

4.1.1.1 Positron Emission Tomography (PET) 59

4.1.1.2 Single Photon Emission Computed Tomography (SPECT) 60

4.1.1.3 Computed Tomography (CT) 60

4.1.1.4 Magnetic Resonance Imaging (MRI) 60

4.1.1.5 Ultrasound (US) 61

4.2 Factors to Consider in Biomarker Selection for Imaging 61

4.3 Defining the Insertion Point of the Assay and Its Business Case 62

4.4 Practical In Vitro Methods Used to Identify Biomarkers 63

4.5 Preclinical Models 64

4.5.1 Model Species 64

4.5.2 Inducing Human Disease and Relevant Biomarker Expression 64

4.5.3 Genetic Manipulation 65

4.5.4 Pharmacological/Chemical Induction 65

4.5.5 Xenografts: Grafting Foreign Cells or Tissues 66

4.5.6 Physical Induction 66

4.6 Preclinical Analysis Techniques 67

4.7 Translational Considerations and Restrictions 67

4.8 Other Uses of Preclinical Models 68

4.9 Nuclear Imaging Infrastructure 69

4.10 Image Processing 70

4.11 Concluding Remarks 70

References 71

5 Breath: An Often Overlooked Medium in Biomarker Discovery 75
Jonathan D Beauchamp and Joachim D Pleil

5.1 Introduction 75

5.1.1 Breath Analysis: Past and Present 76

5.2 Breath Analysis Studies: Targets, Techniques, and Approaches 77

5.2.1 Exhaled Breath Gas, Condensate, and Aerosols 79

5.2.2 Sampling Techniques and Analytical Tools 80

5.2.3 Discovery Versus Targeted Study Approaches 81

5.3 Biomarker Confounders 83

5.3.1 Sampling Impact 83

5.3.1.1 Online Breath Sampling and Direct Analysis 84

5.3.1.2 Breath Sampling

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