Cerebrospinal Fluid Biomarkers

Description

This volume covers the latest methods used in clinical neurochemistry laboratories for both clinical practice and research. Chapters in this book discuss topics such as techniques for cerebrospinal fluid (CSF) collection, pre-analytical processing, and basic CSF analysis; an examination of biomarkers including ELISA and automated immunochemical assays for amyloid and tau markers for Alzheimer’s disease; the analysis of neurofilaments by digital ELISA; and an example of successful novel immunoassay development. In the Neuromethods series style, chapters include the kind of detail and key advice from the specialists needed to get successful results in your laboratory.  Cutting-edge and thorough, Cerebrospinal Fluid Biomarkers is a valuable resource for clinicians and researchers to use in CSF labs and CSF courses. Series Preface Preface Contents Contributors Chapter 1: CSF Cells: Cell Count, Cytomorphology, Cytology, and Immunophenotyping 1 Introduction 2 CSF Cell Count 2.1 Methods 2.2 Interpretation 2.3 Pitfalls 3 Cytomorphology 3.1 Special Cases 3.2 Methods 3.3 Interpretation 3.4 Pitfalls 4 Immunophenotyping of CSF Cells by Flow Cytometry 4.1 Methods 4.2 Immunophenotyping of CSF Leukocytes 4.3 Pitfalls 4.4 Immunophenotyping for Lymphoma in the CNS 5 Clinical Significance of Complementary Cytological Methods 6 Summary References Chapter 2: Nephelometry and Turbidimetry: Methods to Quantify Albumin and Immunoglobulins Concentrations in Clinical Neurochem... 1 Introduction 2 Albumin Quotient (QAlb): A Biomakrer of the Blood-CSF Barrier Function 3 IgG, IgA, and IgM Quotients (QIgG/IgA/IgM). Intrathecal Fractions of the Blood-Derived Proteins. Hyperbolic Functions (Reibe... 4 A Couple of Examples of the Patterns of the CSF/Serum Quotients 5 Tips, Difficulties, and Pitfalls References Chapter 3: Oligoclonal Bands: Isoelectric Focusing and Immunoblotting, and Determination of κ Free Light Chains in the Cerebro... 1 Oligoclonal Bands 1.1 Introduction 1.1.1 Immunoglobulin G 1.1.2 History of Intrathecal IgG Detection 1.1.3 Principles of Isoelectric Focusing 1.1.4 Interpretation of IEF Patterns of Oligoclonal IgG Bands Definition of OCB Positivity 1.1.5 Application in Clinical Practice 1.2 Materials 1.2.1 Test Reagents 1.2.2 Samples 1.2.3 Storage 1.3 Methods 1.3.1 Gel 1.3.2 Isoelectric Focusing Preparation of Samples Preparation of Electrophoresis Chamber and Sample Application Run 1.3.3 Immunoblotting and -Staining Mechanical Transfer Immunolabeling Staining 1.4 Notes 1.4.1 Note 1 1.4.2 Note 2 1.4.3 Note 3 1.4.4 Note 4 1.4.5 Note 5 1.4.6 Note 6 2 κ Free Light Chains 2.1 Introduction 2.1.1 κ Free Light Chains 2.1.2 History of Free Light Chain Detection 2.1.3 Clinical Application 2.2 Materials 2.2.1 Assays 2.2.2 Samples 2.2.3 Storage 2.3 Methods 2.3.1 Principles Nephelometry κ-FLC Assays 2.4 Notes 2.4.1 Cross-Reactivity of Anti-κ-FLC Antibodies 2.4.2 Lot-to-Lot Variation 2.4.3 Interference 2.4.4 Antigen Excess 2.4.5 Nonlinearity 2.4.6 Non-reactivity References Chapter 4: Abeta CSF LC-MS 1 Introduction 2 Materials 3 Methods 3.1 Preparation of Solutions 3.2 Preparation of Calibrators 3.3 Preparation of Internal Standard 3.4 Preparation of Response Factor Sample 3.5 Sample Preparation 3.5.1 Note: Thaw Samples to Be Measured at Room Temperature on a Roller 3.6 Solid Phase Extraction 3.7 Liquid Chromatography 3.8 Mass Spectrometric Analysis 3.9 Data Processing 4 Notes References Chapter 5: Immunoassay and Mass Spectrometry Methods for Tau Protein Quantification in the Cerebrospinal Fluid 1 Introduction 2 Materials 2.1 ELISA Tau 2.2 CLEIA Tau 2.3 Mass Spectrometry 2.3.1 Tau Standard Preparation 2.3.2 Protein Precipitation 2.3.3 Solid Phase Extraction 2.3.4 Sample Drying 2.3.5 Protein Digestion 2.3.6 LC-MS Analysis 2.3.7 Data Analysis 2.4 Other Supplies 2.5 Other Equipment 3 Method 3.1 Samples 3.2 Preparation of Immunoassay Internal Quality Controls (iQC) 3.3 Immunoassays (ELISA, CLEIA) 3.4 Quality Control (QC) 3.5 Mass Spectrometry 3.5.1 Tau Standards Preparation 3.5.2 Protein Precipitation 3.5.3 Solid Phase Extraction 3.5.4 Sample Drying 3.5.5 Protein Digestion 3.5.6 LC-MS Analysis 3.5.7 Data Analysis 4 Notes References Chapter 6: CSF RT-QuIC and the Diagnosis of Creutzfeldt-Jakob Disease Abbreviations 1 Introduction 1.1 A Brief RT-QuIC Work Plan 2 Materials 2.1 Safe Handling and Disposal of sCJD Brain Homogenates Important! 3 Methods 3.1 Preparing the Brain Homogenate Controls 3.2 Example Unseeded and Brain Homogenate Controls (Fig. 6.7) 3.2.1 Alternate Positive Control for Category 2 Laboratories 3.3 Example of the Alternate Positive Control in RT-QuIC (Fig. 6.8) 3.3.1 Preparation of Master Mix 3.3.2 Preparation of Master Mix Components 3.3.3 RT-QuIC Buffer Recipes 3.3.4 Example Calculation 3.4 Assembling the RT-QuIC Plate 3.5 Running the RT-QuIC Plate 3.5.1 Summary of RT-QuIC Instrument Protocol 3.6 RT-QuIC Data Analysis 4 Notes 4.1 Troubleshooting RT-QuIC 5 Introduction 5.1 Making Recombinant PrP-Overview 6 Materials 7 Methods 7.1 Making Recombinant PrP-Molecular Biology 7.2 Disclaimer 7.3 Waste Handling 7.4 Gene Sequence 7.5 Making Recombinant PrP- Protein Purification 7.6 ``8DB´´ Denaturing Buffer 7.7 ``6DB´´ Denaturing Buffer 7.8 ``RB´´ Refolding Buffer 7.9 ``EB´´ Elution Buffer 7.10 Cleaning Resin-``DEB´´ Denaturing Elution Buffer 7.11 ``20x DIB´´ (20x Dialysis Buffer) 7.12 NaOH Solution 8 Notes 8.1 Troubleshooting PrP Production References Chapter 7: Lumbar Puncture: Consensus Guidelines 1 Introduction 2 Contraindications 3 LP Procedure 3.1 Equipment 3.2 Needle Selection 3.3 Patient Positioning (Lateral Recumbent Position or Seated) 3.4 Needle Insertion 3.5 Measuring CSF Pressure 3.6 Volume of CSF to Be Collected 4 Conclusions: Recommendations References Chapter 8: Pre-Analytical Processing and Biobanking Protocol for CSF Samples 1 Introduction 2 Materials 3 Methods 3.1 Biobanking Protocol 4 Notes References Chapter 9: Communicating Complex Results of Cerebrospinal Fluid Analysis Abbreviations 1 Introduction 2 CSF Contaminated with Blood 3 Correction of CSF Leucocytes in Artificially Bloody CSF 4 Correction of CSF Cells Contaminated with Peripheral Blood Cells 4.1 Simple Correction: Based on CSF Erythrocyte Count 4.2 CBC Leuco: Leucocytes Corrected Using the Blood Differential 4.3 CBC Diff. Leucocytes, Polymorphic and Mononuclear Cells Corrected Using the Blood Differential with WBC Differential Count 5 Correction of CSF Protein Concentration in Blood Contaminated Samples 6 Interpretation of the Antibody Index 7 Biomarkers of Dementia 8 Is CSF Analysis Worth the Effort? 9 Closing Statement References Chapter 10: Method and Clinical Validation of Biomarkers for Neurodegenerative Diseases 1 Introduction 2 Method Validation 2.1 Robustness 2.2 Precision 2.3 Trueness 2.4 Selectivity 2.5 Measurement Range 2.6 Dilution Linearity and Parallelism 2.7 Stability 2.8 Quality Controls 2.9 Validation Report 3 Clinical Validation 3.1 Biological Variation 3.2 Sensitivity 3.3 Specificity 3.4 Accuracy 3.5 Predictive Values 3.6 The Receiver Operating Characteristic (ROC) Curve 3.7 Likelihood Ratios 3.8 Cut-Point Determination 3.9 Clinical Robustness 4 Closing Remark References Chapter 11: Sample Preparation for Proteomic Analysis of Cerebrospinal Fluid 1 Introduction 2 Materials 3 Methods 4 Notes References Chapter 12: Brain Biomarkers: Follow-Up of RNA Expression Discovery Approach: CSF Assays for Neurogranin, SNAP-25, and VILIP-1 Abbreviations 1 Introduction 2 Glutamate Dehydrogenase, MW 67 (GAD 67, GAD 1) 3 Myelin-Associated Oligodendrocyte Basic Protein (MOBP) 4 Neurogranin (Ng) 5 Neuroserpin (SerpinI1) 6 Synaptosomal-Associated Protein, MW 25,000 (SNAP-25) 7 (Syndapin-1) Protein Kinase C and Casein Kinase Substrate in Neurons 1 8 Visinin-Like Protein-1 (VILIP-1) 9 Zygin 10 CSF Immunoassay Procedures for Neurogranin, SNAP-25, and VILIP-1 10.1 Assay Methods Overview 10.1.1 Assay Flowchart (Fig. 12.3) 10.2 Materials 10.2.1 Equipment 10.2.2 Supplies 10.2.3 Reagents 10.2.4 Chemicals 10.2.5 Buffers Used for All Assays 10.2.6 Buffers Used for Specific Assays 10.2.7 Capture Antibody Magnetic Microparticle Reagent 10.2.8 Labeled Detection Antibody Reagent Preparation 10.2.9 Quality Control Samples 10.2.10 Assay Standardization 10.3 Assay Preparation 10.3.1 Technical Guidelines 10.3.2 CSF Sample Preparation 10.3.3 Capture Antibody Reagent Preparation 10.3.4 Standard Curve Preparation 10.4 Assay Procedure (All Assays) 10.4.1 Capture Step Setup Protocols 10.4.2 Post-Capture Wash Protocol 10.4.3 Detection Step Protocols 10.4.4 Post-Detection Wash Protocol 10.4.5 Manual Plate 1 to Plate 2 Transfer 10.4.6 Final Aspiration 10.4.7 Detection Antibody Elution Protocol 10.5 Data Analysis 10.5.1 Calculation of Results 10.5.2 Acceptance of Results 10.6 Notes 10.6.1 Evaluating Assay Failure 10.6.2 Adverse Interactions 10.6.3 Biotin Labeling of Capture Antibodies 10.6.4 Degree of Labeling for Alexa Fluor Dye 10.6.5 Strategies for Improving Precision 10.6.6 Assay Throughput 10.7 Summary References Chapter 13: Quantification of the Neurofilament Light Chain Protein by Single Molecule Array (Simoa) Assay 1 Introduction 2 Materials 2.1 Preparation of Calibrators 2.2 Coating of Beads with Capture Antibody 2.3 Measuring Protocol 3 Method 3.1 Preparation of Calibrators 3.2 Coating of Beads with Capture Antibody (Note 5) 3.3 Measuring Protocol 4 Notes 4.1 Note 1. Intra- and Inter-Assay Precision 4.2 Note 2. Analytical Sensitivity 4.3 Note 3. Parallelism and Selectivity 4.4 Note 4. Assay Stability over Time 4.5 Note 5. Reducing Variability within a Project 4.6 Note 6. Use of EDC 4.7 Note 7. Diluents Preparation References Index