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A LABORATORY MANUAL
FOR HUMAN BLOOD ANALYSIS
M.K. BHASIN (
S.M.S. CHAHAL ( With a Foreword by: S. C. TIWARI First Printed: 1996 • Reprinted: 2013 • Pages: 351 • Size: 180 x 240 mm • ISBN 81-85264-12-0 •Binding : Hard • Price: US $ 45/- Rs. 1450/-
The “Laboratory Manual for Human Blood Analysis” is a unique textbook. It is “must be” for all laboratories, in which the genetic blood group systems of man are analysed. This “Laboratory Manual” does not offer only excellent possibilities to the beginner to become familiar with the multifaceted experimental and statistical methods employed in serological population genetics, but is also an excellent laboratory reference book for the advanced scholar. That is why one wish a broad distribution of this book. There is no doubt that in future the “Laboratory Manual” will become the Standard Text and Reference Book to all those who in research or in practice wish to work or are already working in the field of the genetic markers of the human blood.
—ANTHROPOLOGISCHER ANZEIGER (
Human blood studies,
both theoretical and practical, are an integral part of curricula of varied
disciplines - Medical Sciences, Biological Anthropology, Zoology, Forensic
Science, Genetics, Life Sciences, Human Biology, but there seems to be great
paucity of books covering laboratory techniques. The present book covers the
latest techniques, giving their description and methods of execution in lucid
terms. The book has thirteen chapters:
CONTENTS
Foreword
Preface
List of Figures
List of Tables
01. Blood
1.1. Characteristics of Blood 1.2. Collection of Blood (1.2.1. Equipment, 1.2.2. Collection of Blood from Finger-Tip, 1.2.3. Capillary Tube Method, 1.2.4. Venipuncture Method) 1.3. Preparation of Blood 1.4. Anticoagulants 1.5. Isotonic Solution Appendix 1.6. Basic Equipments and Apparatus 1.7 Abbreviations 1.8. Equivalents of Measures and Weights
02. Technique for Blood Cell Examinations
2.1 Complete Blood Count (2.1.1. White Cell Count, 2.1.2. Red Cell Count, 2.1.3. Haemoglobin Determination, 2.1.4. Differential White Cell Count and Stained Red Cell Examination, 2.1.4.1. Differential White Cell Count, 2.1.4.2. Stained Red Cell Examination) 2.2. Sedimentation Rate 2.3. Tests to Study Types of Anaemia (2.3.1. Haematocrit (Hct) Reading or Packed Cell Volume (PCV), 2.3.2. Reticulocyte Count, 2.3.3. Osmotic Fragility Test, 2.3.4. Mean Corpuscular Values, 2.3.5. Red Cell Indices) Appendix 2.4. Haemoglobin Nomogram
03. Haemoglobins
3.1. Normal Haemoglobins 3.2. Abnormal haemoglobins 3.3. Techniques (3.3.1. Haematological Studies, 3.3.2. Preparation of Red Blood Cell Haemolysate, 3.3.3. Electrophoretic Techniques, 3.3.4. Antenatal Diagnosis of Haemoglobinopathies, 3.3.5. Sickle Cell Examination, 3.3.6. Determination of Haemoglobin A2, 3.3.7. Determination of Foetal Haemoglobin (Hb F) Appendix 3. 4. Haemoglobins (3.4.1. Haemoglobin S, 3.4.2. Haemoglobin C, 3.4.3. Haemoglobin D, 3.4.4. Haemoglobin E, 3.4.5. Hereditary Persistence of Foetal haemoglobin, 3.4.6. Thalassaemia, 3.4.7. Other Haemoglobin Variants) 3.5. List of Haemoglobin Variants 3.6. Guidelines for Human Gene Nomenclature (ISGN, 1987) (3.6.1. Gene and Allele Terminology, 3.6.2. Genotype Terminology, 3.6.3. Phenotype Terminology, 3.6.4. Haemoglobin Nomenclature)
04. Glucose-6-Phosphate Dehydrogenase
4.1. Introduction 4.2. Quantitative Determination 4.3. Molecular Forms 4.4. Techniques (4.4.1. Screening Procedures, 4.4.2. Quantitative Techniques Reflecting G6PD Activity, 4.4.3. Electrophoretic Characterisation of G6PD Variants, 4.4.4. Partial Purification of Human Red Cell (G6PD), 4.4.5. Determination of Substrate Affinity or Michaelis Constant (Km), 4.4.6. Utilization of Substrate Analogues, 4.4.7. Thermostability, 4.4.8. Other Kinetic Measurements, 4.4.9. Other Means of Characterising G6PD Appendix 4.5. List of G6PD Variants
05. Techniques in Blood Grouping
5.1. Apparatus and Chemicals 5.2. Precautions (5.2.1. Cleaning of Dirty Glassware) 5.3. Designation of Agglutination Reaction 5.4. Blood Group Terminology 5.5. The ABO Blood Group System (5.5.1. The ABO Blood Groups, 5.5.2. Method for Detecting Subgroups of A and AB, 5.5.3. The Bombay (Oh) Phenotype) 5.6. The MNSs Blood Group System (5.6.1. The MN Blood Groups, 5.6.2. The MNS Blood Groups) 5.7 The P Blood Group System 5.8. The Rhesus Blood Group System [(5.8.1. Anti-D (Anti-Rh 0) Test Method), 5.8.2. Du Test Method, 5.8.3. Rh Complete Sera (IgM) (Saline Agglutinating), 5.8.4. Rh Incomplete Sera (IgG) (Albumin Agglutinating)] 5.9. Secretors and Non-Secretors (The ABH Secretion System) 5.10. The Lewis System 5.11 Blood Grouping By Indirect Antiglobulin Test (Coombs Test) (5.11.1. The Lutheran Blood Group System, 5.11.2. The Kell Blood Group System, 511.2.1. The Sutter Groups, 5.11.3. The Duffy Blood Group System, 5.11.4. The Kidd Blood Group System, 5.11.5. The Diego Blood Group System, 5.11.6. The Colton Blood Group System, 5.11.7. The Xg Blood Group System) 5.12 Anti-Human Globulin Serum (For Direct or Indirect Antiglobulin/Coombs Tests) 5.13. Blood Grouping from Bloodstains, Fragments of Muscle, Skin etc. and Lysed Blood Samples (5.13.1. The ABO Blood Groups, 5.13.2. The MN Blood Groups, 5.13.3. The Rh Blood Groups, 5.13.4. Grouping Tests on Saliva) Appendix 5.14. Blood as a Forensic Indicator
06. Techniques in Human Leukocyte Grouping
6.1. The HLA System 6.2. Techniques (6.2.1. Serological Methods, 6.2.2. Data Analysis, 6.2.3. Reasons for False Reactions, 6.2.4. Quality Control Appendix
07. Techniques in Immunoglobulin Typing 7.1 Immunoglobulin Plasma Protein Allotypes: The Gm, Km (Inv) and Am Systems 7.2. Techniques in Immunoglobulin Typing: Gamma Globulin Factors (Gm) and Kappa Markers (Km) [7.2.1. Agglutination-Inhibition Tests]
08. Techniques in Electrophoresis
8.1. Electrophoresis 8.1.1. Apparatus for Electrophoresis, 8.1.2. Applications of Electrophoresis, 8.1.3. Starch Gel Electrophoresis, 8.1.4. Agarose Gel Electrophoresis, 8.1.5. Cellulose Acetate Electrophoresis, 8.1.6. Polyacrylamide Gel Electrophoresis, 8.1.7. Block Electrophoresis, 8.1.8. Isolectric Focusing, 8.1.9. Disc Electrophoresis, 8.1.10. Protein Blotting, 8.1.11. Pulsed Field Gel Electrophoresis 8.2. Immunological Techniques (8.2.1. Immunoelectrophoresis, 8.2.2. Immunodiffusion Appendix 8.3. Guidelines for Human Gene Nomenclature (ISGN, 1987) [8.3.1 Enzyme and Protein Nomenclature]
09. Serum Protein Polymorphisms
9.1. Introduction 9.2. Protein Polymorphisms [9.2.1 Haptoglobin (Hp) System, 9.2.2. Serum Transferrin (Tf) System, 9.2.3. Group Specific Component (Gc) System, 9.2.4. Complement Component 3 (C3) System, 9.2.5. Properdin Factor B (Bf) System]
10. Red Cell Enzyme Polymorphisms
10.1 Introduction 10.2. Enzyme Polymorphisms [10.2.1. 6-Phosphogluconate Dehydrogenase (6-PGD) System (E.C. 1.1.1.44), 10.2.2. Adenylate Kinase (AK) system (E.C. 2.7.4.3), 10.2.3. Phosphoglucomutase Locus 1 (PGM1) System (E.C. 5.4.2.8 earlier E.C. 2.7.5.1), 10.2.4. Acid Phosphatase (ACP) System (E.C. 3.1.3.2), 10.2.5. Adenosine Deaminase (ADA) System (E.C. 3.5.4.4), 10.2.6. Esterase D (EsD) System (E.C. 3.1.1.1.), 10.2.7. Glyoxalase I (GLO I) System (E.C. 4.4.1.5), 10.2.8. Phosphohexose Isomerase (PHI) System (E.C. 5.3.1.9), 10.2.9. Glutamate Pyruvate Transaminase (GPT) System (E.C. 2.6.1.2.) Appendix 10.3. Scheme of Classification and Numbering of Eyzymes
11. DNA Polymorphisms
11.1. Introduction 11.2. Reagents, Apparatus and Equipments 11.3. Techniques [11.3.1. Isolating Pure DNA from Human Cell (Specimen Collection and Preparation for Analysis), 11.3.1.1. Cell Preparation, 11.3.1.2. Extraction of DNA 11.3.1.3. Determination of DNA Concentration by Spectrophotometry, 11.3.2. Restriction Enzyme Digestion, 11.3.3. Agarose Gel Electrophoresis, 11.3.4. Southern Transfer, 11.3.5. DNA Hybridization, 11.3.6. Colour Development and Autoradiography, 11.3.6.1. Colour Development, 11.3.6.2. Autoradiography, 11.3.6.3. Interpretation of Photograph or Autorads, 11.3.7. Cell Preparation and Extraction of DNA From Forensic Exhibits] Appendix
12. OTHER GENETIC TRAITS
12.1. Colour Blindness (12.1.1. Technique) 12.2. Taste Testing with Phenylthiocarbamide (PTC) [12.2.1. Technique, 12.2.2. Allele Frequency Estimations] 12.3. Ability to Smell Hydrogen Cyanide 12.4. Human Cerumen Type (12.4.1. Technique, 12.4.2. Allele Frequency Estimations)
13. Statistical Analysis
13.1. Allele Frequency Calculations (13.1.1. The ABO System, 13.1.1.1. Allele Frequencies when Anti-A and Anti-B have been used, 13.1.1.2. Allele Frequencies when Anti-A, Anti-B and Anti-A1 have been used, 13.1.2. The MNSs System, 13.1.2.1. Allele Frequencies when Anti-M and Anti-N have been used, 13.1.2.2. Haplotype Frequencies when Anti-M, Anti-N, and Anti-S have been used, 13.1.2.3. Haplotype Frequencies when Anti-M, Anti-N, Anti-S and Anti-s have been used, 13.1.3. The Rh System, 13.1.3.1. Allele Frequencies when Anti-d has been used, 13.1.3.2. Haplotype Frequencies when Anti-C (rh’), Anti-D (Rh0), anti-E (rh”), Anti-c (hr’) and Anti-E (hr”) have been used, 13.1.4. The Kidd (Jk) System, 13.1.4.1. Allele Frequencies when only Anti-Jka has been used, 13.1.4.2. Allele Frequencies when both Anti-Jka and Anti-Jkb have been used, 13.1.5. Acid Phosphatase (ACP) System, 13.1.6.The Gm (1,2,5) System, 13.1.7. X-Linked Systems, 13.1.7.1. The Xg System, 13.1.7.2. Glucose-6 Phosphate Dehydrogenase (G6PD) System 13.2. Estimation of Measures of Genetic Variation and Genetic Distance (13.2.1. Heterozygosity, 13.2.2.Genetic Differentiation, 13.2.2.1. Wahlund’s Variance, 13.2.2.2. Nei’s Gene Diversity Analysis, 13.2.3. Genetic Distance, 13.2.3.1. Steps of Computation of Nei’s Standard Genetic Distance 13.3. Calculations Pertaining to Forensic Applications (13.3.1. Measures of Individualization and Discrimination Potential, 13.3.2. Measures of Paternity Exclusion, 13.3.2.1. Measure of Paternity Exclusion Based on Single System, 13.3.2.2. Measure of Paternity Exclusion Based on More than One System)
Glossary
APPENDIX
1. List of Chemicals, Their Abbreviations, Molecular Weights and Specifications 2. Sources of Reagents and Equipments
Bibliography
Index
Almost all recent researches in serology and haemotology have been consulted for preparing this manual.
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