Molecular Protocols in Transfusion Medicine
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Table of contents
- Contentsv
- Prefacevii
- List of Abbreviationsix
- Part 1: Introduction1
- 1.1 Background1
- 1.2 Aim, Scope and Purpose of the Book2
- 1.3 Organization of the Book3
- 1.4 Terminology4
- 1.5 Regulatory Compliance5
- 1.6 References6
- Part 2: General Considerations9
- 2.1 Clinical Information9
- 2.2 Basic Principles of Molecular Analysis9
- 2.3 Troubleshooting16
- 2.4 References18
- Part 3: Protocols19
- 3.1 Sample Shipment and Storage19
- 3.2 Reagents19
- 3.3 Harvesting WBCs by RBC Sedimentation19
- 3.4 Short-term Cell Cultures from Amniotic Fluid21
- 3.5 Cultured Amniocyte Cell Harvesting23
- 3.6 Cell Preparation and DNA Extraction24
- 3.7 Assessment of DNA Quality, Quantity and Purity27
- 3.8 PCR Amplification29
- 3.9 Restriction Enzyme Digestion for PCR-RFLP Analysis34
- 3.10 Allele-specific PCR35
- 3.11 Agarose Gel Preparation and Electrophoresis38
- 3.12 Acrylamide Gel Preparation and Electrophoresis41
- 3.13 Single-strand Conformation Polymorphism (SSCP)43
- 3.14 Optimizing a PCR46
- 3.15 Gene Zygosity Assessment by Quantitative PCR Amplification50
- 3.16 VNTR and STR Analysis by PCR Amplification53
- 3.17 Multiplex PCR Analysis57
- 3.18 Southern Analysis60
- 3.19 References65
- Part 4: Red Cell Blood Groups67
- 4.1 Terminology67
- 4.2 Clinical Applications67
- 4.3 Situations where Genotype and Phenotype do not Agree71
- 4.4 References73
- 4.5 Facts Sheets, Gene Maps and Molecular Protocols75
- Part 5: Platelet Blood Groups141
- 5.1 Terminology141
- 5.2 Clinical Applications142
- 5.3 References145
- 5.4 Facts Sheets, Gene Maps and Molecular Protocols146
- Part 6: Neutrophil Blood Groups163
- 6.1 Terminology163
- 6.2 Clinical Applications164
- 6.3 References165
- 6.4 Facts Sheets, Gene Maps and Molecular Protocols166
- Part 7: Appendices171
- 7.1 Database Information171
- 7.2 Glossary172
- 7.3 References174
- 7.4 Works Sheets and Request Forms176
- Index181
- Useful Tables185
This essential methods manual for immunohematologists (or hematologists and immunohematologists) provides information on genes that encode antigens on red blood cells, platelets and neutrophils. The book begins by covering general concepts in molecular biology and specific protocols such as DNA preparation, PCR-RFLP and allele-specific PCR. Information on the erythrocyte, platelet and neutrophil antigen systems and the molecular basis of polymorphisms are presented clearly in a gene facts sheet format. Database accession numbers and useful adjuncts such as Request forms, worksheets for PCR/enzyme digests also serve to benefit the user. The information is clearly presented and easily accessible and is complemented by the excellent diagrams and tabular material. This book is invaluable for both new and experienced researchers in the field and other related disciplines.
Key Features
* Essential for hematologists and those involoved in tissue typing and the study of human genetic polymorphisms
* Presents clearly and concisely the information on a particular variant and the technique used to detect it
* Organized by antigen and provides sequences of polymorphisms and primers
* Details the general concepts and critical information on genes, their products, and sources of relevant nucleic acids
* Includes protocols that allow investigators to set up assays with minimal effort (protocols include primers, reagents, reaction conditions, sizes of amplified products, restriction fragment digests, and the relevant safety information)
* Provides information that helps interpret results in clinical settings
* Contains additional sources of information (e.g., key references, web site addresses, glossary, Database accession numbers, request
forms, and worksheets for PCR/enzyme digests)
Key Features
* Essential for hematologists and those involoved in tissue typing and the study of human genetic polymorphisms
* Presents clearly and concisely the information on a particular variant and the technique used to detect it
* Organized by antigen and provides sequences of polymorphisms and primers
* Details the general concepts and critical information on genes, their products, and sources of relevant nucleic acids
* Includes protocols that allow investigators to set up assays with minimal effort (protocols include primers, reagents, reaction conditions, sizes of amplified products, restriction fragment digests, and the relevant safety information)
* Provides information that helps interpret results in clinical settings
* Contains additional sources of information (e.g., key references, web site addresses, glossary, Database accession numbers, request
forms, and worksheets for PCR/enzyme digests)