Cerebrospinal Fluid Biomarkers

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