BOOK
Dacie and Lewis Practical Haematology
Barbara J. Bain | Imelda Bates | Mike A Laffan | S. Mitchell Lewis
(2016)
Additional Information
Book Details
Abstract
Highly Commended at 2012 BMA awards in Internal Medicine Category.
Recognized worldwide as the standard reference work, Dacie & Lewis Practical Haematology is a must have reference for any haematology laboratory. It covers all of the techniques used in the investigation of patients with blood disorders, including the latest technologies as well as the tried and true manual methods of measurement. It discusses the principles of each test, possible causes of error, the rationale for choosing one method over another and the interpretation, significance and clinical relevance of these findings. Each chapter conforms to a template, providing quick access to key information
Table of Contents
| Section Title | Page | Action | Price |
|---|---|---|---|
| Front Cover | Cover | ||
| Dacie and Lewis Practical Haematology | iii | ||
| Copyright | iv | ||
| Contents | v | ||
| Preface | vii | ||
| Contributors | ix | ||
| Chapter 1: Collection and handling of blood | xiii | ||
| Biohazard precautions | xiii | ||
| Standardized procedure | xiii | ||
| Venous blood | 2 | ||
| Phlebotomy Tray | 2 | ||
| Disposable Plastic Syringes and Disposable Needles | 2 | ||
| Specimen Containers | 2 | ||
| Phlebotomy Procedure | 3 | ||
| Post-phlebotomy Procedure | 3 | ||
| Waste Disposal | 3 | ||
| Capillary blood | 4 | ||
| Collection of Capillary Blood | 4 | ||
| Blood Film Preparation | 4 | ||
| Differences between capillary and venous blood | 5 | ||
| Sample homogeneity | 5 | ||
| Serum | 5 | ||
| Defibrinating Whole Blood | 5 | ||
| Cold Agglutinins | 6 | ||
| Anticoagulants | 6 | ||
| Ethylenediaminetetra-acetic Acid (EDTA) | 6 | ||
| Trisodium Citrate | 7 | ||
| Heparin | 7 | ||
| Effects of storage on the blood count | 7 | ||
| Effects of storage on blood cell morphology | 7 | ||
| References | 9 | ||
| Chapter 2: Reference ranges and normal values | 11 | ||
| Reference ranges | 11 | ||
| Statistical procedures | 12 | ||
| Confidence Limits | 12 | ||
| Normal reference values | 13 | ||
| Physiological variations in the blood count | 15 | ||
| Red Cell Components | 15 | ||
| Pregnancy | 15 | ||
| The Elderly | 18 | ||
| Exercise | 18 | ||
| Posture | 18 | ||
| Diurnal and Seasonal Variation | 18 | ||
| Altitude | 18 | ||
| Smoking | 19 | ||
| Leucocyte Count | 19 | ||
| Platelet Count | 20 | ||
| Other Blood Constituents | 20 | ||
| Effects of Smoking | 20 | ||
| References | 20 | ||
| Chapter 3: Basic haematological techniques | 23 | ||
| Haemoglobinometry | 24 | ||
| Measurement of haemoglobin concentration using a spectrometer (spectrophotometer) or photoelectric colorimeter | 24 | ||
| Haemiglobincyanide (cyanmethaemoglobin) method | 25 | ||
| Diluent | 25 | ||
| Haemiglobincyanide Reference Standard | 25 | ||
| Method | 26 | ||
| Calculation of Haemoglobin Concentration | 26 | ||
| Preparation of Standard Graph and Standard Table | 26 | ||
| Oxyhaemoglobin method | 27 | ||
| Method | 27 | ||
| Standard | 27 | ||
| Direct spectrometry | 27 | ||
| Direct reading portable haemoglobinometers | 28 | ||
| Colour Comparators | 28 | ||
| Portable Haemoglobinometers | 28 | ||
| Non-invasive Screening Tests | 28 | ||
| Range of Haemoglobin Concentration in Health | 28 | ||
| Packed cell volume or haematocrit | 28 | ||
| Microhaematocrit Method | 29 | ||
| Accuracy of Microhaematocrit | 29 | ||
| Anticoagulant | 29 | ||
| Blood Sample | 29 | ||
| Capillary Tubes | 29 | ||
| Centrifuge | 29 | ||
| Reading | 29 | ||
| Plasma Trapping | 29 | ||
| International Council for Standardization in Haematology Reference Method | 30 | ||
| Surrogate Reference Method | 30 | ||
| Equipment | 30 | ||
| Method | 30 | ||
| Range of Packed Cell Volume in Health | 30 | ||
| Manual cell counts and red cell indices | 30 | ||
| Range of MCHC in Health | 30 | ||
| Manual differential leucocyte count | 30 | ||
| Method | 31 | ||
| Basophil and eosinophil counts | 32 | ||
| Range of Eosinophil Count in Health | 32 | ||
| Range of Basophil Count in Health | 32 | ||
| Reporting the Differential Leucocyte Count | 32 | ||
| Correcting the Count for Nucleated Red Blood Cells | 32 | ||
| Reference Differential White Cell Count | 32 | ||
| Range of Differential White Cells in Health | 33 | ||
| Platelet count | 33 | ||
| Range of Platelet Count in Health | 33 | ||
| Reticulocyte count | 33 | ||
| Reticulocyte Stains and Count | 33 | ||
| Staining Solution | 33 | ||
| Method | 33 | ||
| Counting Reticulocytes | 35 | ||
| Calculation | 35 | ||
| Differentiating between Reticulocytes and Other Red Cell Inclusions | 35 | ||
| Fluorescence Methods for Performing a Reticulocyte Count | 36 | ||
| Manual Reference Method | 36 | ||
| Range of Reticulocyte Count in Health | 36 | ||
| Automated blood count techniques | 36 | ||
| Haemoglobin concentration | 37 | ||
| Red blood cell count | 37 | ||
| Counting systems | 37 | ||
| Impedance Counting | 37 | ||
| Light Scattering | 38 | ||
| Reliability of electronic counters | 38 | ||
| Setting Discrimination Thresholds | 39 | ||
| Packed cell volume and mean cell volume | 39 | ||
| Red cell indices | 41 | ||
| Mean Cell Volume | 41 | ||
| Mean Cell Haemoglobin and Mean Cell Haemoglobin Concentration | 41 | ||
| Variations in red cell volumes: red cell distribution width | 41 | ||
| Percentage hypochromic red cells and variation in red cell haemoglobinization: haemoglobin distribution width | 42 | ||
| Total white blood cell count | 42 | ||
| Automated differential count | 43 | ||
| The automated immature granulocyte count | 44 | ||
| The automated nucleated red blood cell count | 44 | ||
| Automated digital imaging analysis of blood cells | 44 | ||
| New White Cell Parameters | 45 | ||
| Automated instrument graphics | 45 | ||
| Platelet count | 47 | ||
| Platelet Count in Health | 47 | ||
| Mean Platelet Volume | 47 | ||
| Reticulated Platelets and Immature Platelet Fraction | 48 | ||
| Reticulocyte count | 48 | ||
| Immature Reticulocyte Fraction | 48 | ||
| Reticulocyte Counts in Health | 49 | ||
| Measurement of Reticulocyte Haemoglobin | 49 | ||
| Point-of-care instruments | 49 | ||
| Calibration of automated blood cell counters | 49 | ||
| Flagging of automated blood counts | 50 | ||
| Microscopy | 51 | ||
| Microscope Components | 51 | ||
| Setting Up the Microscope Illumination | 52 | ||
| Examination of Slides | 52 | ||
| Routine Maintenance of the Microscope | 52 | ||
| References | 53 | ||
| Chapter 4: Preparation and staining methods for blood and bone marrow films | 57 | ||
| Preparation of blood films on slides | 57 | ||
| Manual Method | 57 | ||
| Automated Methods | 58 | ||
| Labelling Blood Films | 58 | ||
| Fixing Blood Films | 58 | ||
| Bone Marrow Films | 59 | ||
| Staining blood and bone marrow films | 59 | ||
| Preparation of Solutions of Romanowsky Dyes | 59 | ||
| May-Grünwald Stain | 59 | ||
| Jenner's Stain | 59 | ||
| Giemsa's Stain | 59 | ||
| Azure B-Eosin Y Stock Solution | 60 | ||
| Chapter 5: Blood cell morphology in health and disease | 69 | ||
| Examination of blood films | 70 | ||
| Red cell morphology | 70 | ||
| Abnormal erythropoiesis | 71 | ||
| Anisocytosis (αiotaσzeta, unequal) and Poikilocytosis (πiotaκiotaλzeta, varied) | 71 | ||
| Macrocytes | 73 | ||
| Microcytes | 73 | ||
| Basophilic Stippling | 74 | ||
| Inadequate haemoglobin formation | 74 | ||
| Hypochromia (Hypochromasia) (πρ, under) | 74 | ||
| Anisochromasia (αiotaσzeta, unequal) and Dimorphic Red Cell Population | 76 | ||
| Damage to red cells after formation | 76 | ||
| Hyperchromasia (Hyperchromia) (περ, over) | 76 | ||
| Spherocytosis (σφαiotaρα, a sphere) | 76 | ||
| Irregularly Contracted Red Cells | 77 | ||
| Elliptocytosis and Ovalocytosis | 79 | ||
| Spiculated cells and red cell fragmentation | 80 | ||
| Schistocytosis (Fragmentation) (σχiotaστzeta, cleft) | 80 | ||
| Keratocytes (κεραzeta, horn) | 81 | ||
| Acanthocytosis (ακαnuθα, spine) | 81 | ||
| Echinocytosis (εχiotanuzeta, sea-urchin or hedgehog) | 82 | ||
| Miscellaneous erythrocyte abnormalities | 82 | ||
| Leptocytosis (λεπτzeta, thin) | 82 | ||
| Target Cells | 83 | ||
| Stomatocytosis (στμα, mouth) | 83 | ||
| Sickle Cells | 84 | ||
| Haemoglobin C Crystals and SC Poikilocytes | 85 | ||
| Erythrocyte Inclusions | 85 | ||
| Howell-Jolly Bodies | 85 | ||
| Pappenheimer Bodies | 85 | ||
| Rouleaux and Autoagglutination | 86 | ||
| Changes associated with a compensatory increase in erythropoiesis | 86 | ||
| Polychromasia (πλθzeta, many) | 86 | ||
| Erythroblastaemia | 87 | ||
| Effects of splenectomy and hyposplenism | 88 | ||
| Scanning electron microscopy | 88 | ||
| Morphology of leucocytes | 88 | ||
| Polymorphonuclear neutrophils | 89 | ||
| Granules | 90 | ||
| Vacuoles | 92 | ||
| Bacteria | 92 | ||
| Döhle Bodies | 92 | ||
| Nuclei | 93 | ||
| Hypersegmentation | 93 | ||
| Pelger-Huët Cells | 93 | ||
| Pyknotic Neutrophils (Apoptosis) | 94 | ||
| Eosinophils | 94 | ||
| Basophils | 94 | ||
| Monocytes | 95 | ||
| Lymphocytes | 95 | ||
| Platelet morphology | 97 | ||
| References | 99 | ||
| Chapter 6: Supplementary techniques including blood parasite diagnosis | 101 | ||
| Tests for the acute-phase response | 101 | ||
| Erythrocyte Sedimentation Rate | 102 | ||
| Conventional Westergren Method | 102 | ||
| Method | 102 | ||
| Range in health | 102 | ||
| Modified methods | 103 | ||
| Length of tube | 103 | ||
| Plastic glass tubes | 103 | ||
| Disposable glass tubes | 103 | ||
| Capillary method | 103 | ||
| Time | 103 | ||
| Sloping tube | 103 | ||
| Anticoagulant | 103 | ||
| Evaluation of a new routine method | 103 | ||
| Quality Control | 104 | ||
| Semiquantitative Slide Method | 104 | ||
| Mechanism of Erythrocyte Sedimentation | 104 | ||
| Plasma Viscosity | 105 | ||
| Reference Values | 105 | ||
| Whole Blood Viscosity | 105 | ||
| Heterophile antibodies in serum: diagnosis of infectious mononucleosis | 105 | ||
| Screening Tests for Infectious Mononucleosis | 106 | ||
| Clinical Value | 106 | ||
| Demonstration of Lupus Erythematosus Cells | 106 | ||
| Erythropoietin | 107 | ||
| Reference Range | 107 | ||
| Significance | 107 | ||
| Autonomous in vitro Erythropoiesis | 107 | ||
| Thrombopoietin | 107 | ||
| Haematological tests in sports medicine | 108 | ||
| Reticulocytes | 108 | ||
| Haemoglobin and Hct | 108 | ||
| Whole Blood Viscosity and ESR | 108 | ||
| Erythropoietin | 108 | ||
| Principles of parasite detection | 108 | ||
| Examination of blood films for parasites | 109 | ||
| General Principles | 109 | ||
| Staining Thin Films | 109 | ||
| Microscopic diagnosis of malaria | 109 | ||
| Fluorescence Microscopy | 110 | ||
| Quantitative Buffy Coat Method | 110 | ||
| Rapid diagnostic tests for malaria | 110 | ||
| Leishmaniasis | 116 | ||
| Diagnosis of Leishmaniasis in the Haematology Laboratory | 116 | ||
| Trypanosomiasis | 116 | ||
| African Trypanosomiasis | 116 | ||
| American Trypanosomiasis | 116 | ||
| Diagnosis of Trypanosomiasis in the Haematology Laboratory | 116 | ||
| Wet Preparations | 117 | ||
| Thick Blood Films or Chancre Aspirates | 117 | ||
| Concentration Techniques | 117 | ||
| Quantitative buffy coat method | 117 | ||
| Capillary tube method | 117 | ||
| Filariasis and loiasis | 118 | ||
| Diagnosis of Filariasis in the Haematology Laboratory | 118 | ||
| Wet Preparation | 118 | ||
| Concentration Techniques | 118 | ||
| Filtration method | 118 | ||
| Quantitative buffy coat and microhaematocrit methods | 118 | ||
| Lysed capillary blood | 118 | ||
| Babesiosis | 118 | ||
| Ehrlichiosis | 118 | ||
| References | 119 | ||
| Chapter 7: Bone marrow biopsy | 123 | ||
| Aspiration of the bone marrow | 124 | ||
| Consent and Safety | 124 | ||
| Performing a Bone Marrow Aspiration | 124 | ||
| Puncture of the Ilium | 125 | ||
| Puncture of the Sternum | 125 | ||
| Comparison of Different Sites for Marrow Puncture | 125 | ||
| Aspiration of the bone marrow in children | 125 | ||
| Marrow puncture needles | 126 | ||
| Processing of aspirated bone marrow | 126 | ||
| Preparing Films from Bone Marrow Aspirates | 126 | ||
| Concentration of Bone Marrow by Centrifugation | 128 | ||
| Preparation of Films of Post-mortem Bone Marrow | 128 | ||
| Examination of aspirated bone marrow | 128 | ||
| Principles of Marrow Aspirate Examination | 128 | ||
| Quantitative Cell Counts on Aspirated Bone Marrow | 129 | ||
| Differential Cell Counts on Aspirated Bone Marrow | 129 | ||
| Sources of Error and Physiological Variations | 129 | ||
| Cellular Ratios | 130 | ||
| Reporting bone marrow aspirate films | 130 | ||
| Systematic Scheme for Examining Bone Marrow Aspirate Films | 130 | ||
| Low Power (x10) | 130 | ||
| Higher Power (x40, x100 Oil-Immersion) | 130 | ||
| Reporting Results | 131 | ||
| Preparation of sections of aspirated bone marrow fragments | 131 | ||
| Percutaneous trephine biopsy of the bone marrow | 131 | ||
| Principles Behind Marrow Trephine Biopsy Examination | 133 | ||
| Imprints from Bone Marrow Trephine Biopsy Specimens | 133 | ||
| Processing of Bone Marrow Trephine Biopsy Specimens | 133 | ||
| Staining of Sections of Bone Marrow Trephine Biopsy Specimens | 135 | ||
| References | 137 | ||
| Chapter 8: Molecular and cytogenetic analysis | 139 | ||
| Introduction to the analysis of DNA | 139 | ||
| Extraction of DNA | 140 | ||
| DNA Extraction Kits | 140 | ||
| Polymerase chain reaction | 140 | ||
| Principle | 140 | ||
| Reagents | 141 | ||
| Method | 142 | ||
| Modifications and Developments | 143 | ||
| Problems and Interpretation | 143 | ||
| Analysis of polymerase chain reaction products | 143 | ||
| Presence or Absence of a Polymerase Chain Reaction Product | 143 | ||
| Amplification Refractory Mutation System | 143 | ||
| Principle | 143 | ||
| Interpretation | 143 | ||
| Gap-PCR | 144 | ||
| Size of the PCR Product | 144 | ||
| Chapter 9: Iron deficiency anaemia and iron overload | 175 | ||
| Iron metabolism | 176 | ||
| Dietary Iron Absorption | 176 | ||
| Dietary and Luminal Factors | 177 | ||
| Iron Absorption at the Molecular Level | 177 | ||
| Regulation of Iron Absorption | 177 | ||
| Cellular Iron Uptake and Release | 177 | ||
| Iron Storage | 178 | ||
| Regulation of Iron Metabolism | 178 | ||
| Plasma Iron Transport | 178 | ||
| Iron status | 179 | ||
| Disorders of iron metabolism | 179 | ||
| Methods for assessing iron status | 179 | ||
| Serum Ferritin Assay | 179 | ||
| Immunoassay for Ferritin | 180 | ||
| Preparation and Storage of Ferritin | 180 | ||
| Antibodies to Human Ferritin | 180 | ||
| Reagents and Materials | 180 | ||
| Conjugation of Antiferritin IgG Preparation to Horseradish Peroxidase36 | 180 | ||
| Buffer A | 182 | ||
| Buffer B | 182 | ||
| Buffer C | 182 | ||
| Buffer D | 183 | ||
| Substrate Solution | 183 | ||
| Sulphuric Acid | 183 | ||
| Preparation and Storage of a Standard Ferritin Solution | 183 | ||
| Coating of Plates | 183 | ||
| Preparation of Test Sera | 183 | ||
| Assay Procedure | 183 | ||
| Calculation of Results | 183 | ||
| Selecting an Assay Method | 183 | ||
| Interpretation | 184 | ||
| Estimation of serum iron concentration | 185 | ||
| Reagents and Materials | 185 | ||
| Preparation of Glassware | 185 | ||
| Protein Precipitant | 185 | ||
| Chromogen Solution (Ferrozine) | 185 | ||
| Iron-free Water | 185 | ||
| Iron Standard 80μmol/l | 185 | ||
| Method | 185 | ||
| Calculation | 185 | ||
| Alternative procedure: serum iron without protein precipitation | 186 | ||
| Reagents and Materials | 186 | ||
| Iron Standards 80 μmol/l and 40 μmol/l | 186 | ||
| Phosphate-Ascorbate Buffer (Stock) | 186 | ||
| Chromogen Solution | 186 | ||
| Microtitre Trays | 186 | ||
| Control Serum | 186 | ||
| Method | 186 | ||
| Calculations | 186 | ||
| Automated Methods for Serum Iron | 187 | ||
| Serum iron concentrations in health and disease | 187 | ||
| Iron-binding capacity, serum transferrin and transferrin saturation | 187 | ||
| Estimation of Total Iron-Binding Capacity | 187 | ||
| Principle | 187 | ||
| Reagents | 187 | ||
| Method | 187 | ||
| Determination of unsaturated iron-binding capacity | 188 | ||
| Reagents and Materials | 188 | ||
| Saturating Solution | 188 | ||
| Tris Buffer (Stock) | 188 | ||
| Tris-Ascorbate-Iron Buffer | 188 | ||
| Chromogen Solution | 188 | ||
| Microtitre Trays | 188 | ||
| Control Serum | 188 | ||
| Method | 188 | ||
| Calculations | 188 | ||
| Fully Automated Methods | 189 | ||
| Serum transferrin | 189 | ||
| Normal Ranges of Transferrin and Total Iron-Binding Capacity | 189 | ||
| Transferrin saturation | 189 | ||
| Transferrin Index | 189 | ||
| Serum transferrin receptor | 190 | ||
| Assays for the Serum Transferrin Receptor | 190 | ||
| Reference Ranges | 191 | ||
| Samples | 191 | ||
| Transferrin Receptor Concentrations in Diagnosis | 191 | ||
| Erythropoiesis | 191 | ||
| Iron Deficiency | 191 | ||
| Iron Overload | 191 | ||
| Erythrocyte protoporphyrin | 192 | ||
| Analysers | 192 | ||
| Diagnostic Applications | 193 | ||
| Units | 193 | ||
| Hepcidin | 193 | ||
| Methodological and biological variability of assays | 193 | ||
| Predictive value of blood tests for iron deficiency | 194 | ||
| Iron Deficiency Anaemia in Adults | 194 | ||
| Detection of Iron Deficiency in Acute or Chronic Disease | 194 | ||
| Functional Iron Deficiency | 195 | ||
| Iron Deficiency in Infancy and Childhood | 196 | ||
| Pregnancy | 196 | ||
| Conclusion | 196 | ||
| References | 196 | ||
| Chapter 10: Investigation of megaloblastic anaemia | 201 | ||
| Cobalamin absorption and metabolism | 202 | ||
| Folate absorption and metabolism | 202 | ||
| Rationale for Investigation of Cobalamin or Folate Status | 204 | ||
| Haematological features of megaloblastic anaemia | 204 | ||
| Differential Diagnosis of Macrocytic Anaemia | 205 | ||
| Testing strategy for suspected cobalamin or folate deficiency | 205 | ||
| Limitations of Cobalamin Assays | 210 | ||
| Sensitivity and Specificity of Cobalamin and Holotranscobalamin Assays | 210 | ||
| Utility of receiver operator characteristic curves | 210 | ||
| Utility of holotranscobalamin, methylmalonic acid and homocysteine assays | 211 | ||
| Clinical and Diagnostic Pitfalls of Folate Assays | 213 | ||
| Standards, Accuracy and Precision of Cobalamin and Folate Assays | 213 | ||
| Genetic Factors | 214 | ||
| Pre-analytical Sample Preparation | 214 | ||
| Analytical Factors | 215 | ||
| Limitations and Interference | 215 | ||
| Post-analytical Factors | 216 | ||
| Methods for cobalamin and folate analysis | 216 | ||
| General Principles of Competitive protein binding assays | 216 | ||
| Serum B12 assays | 216 | ||
| Release from Endogenous Binders and Conversion of Analyte to Appropriate Form | 216 | ||
| Binding of B12 to Kit Binder | 216 | ||
| Separation of Bound and Unbound B12 | 216 | ||
| Signal Generation | 217 | ||
| Electrochemiluminescence Immunoassay | 217 | ||
| Enzyme-linked Fluorescence Generation | 217 | ||
| Holotranscobalamin assays | 217 | ||
| Principle | 217 | ||
| Holotranscobalamin `Active B12´ Immunoassay | 217 | ||
| HoloTC Radioimmunoassay | 217 | ||
| Quantitation of Transcobalamin Saturation | 218 | ||
| Serum folate methods | 218 | ||
| Release from Endogenous Binders | 218 | ||
| Binding of Folate to Folate-Binding Protein | 218 | ||
| Separation of Bound and Unbound Folate | 218 | ||
| Signal Generation | 218 | ||
| Red cell folate methods | 218 | ||
| Haemolysate Preparation | 219 | ||
| Calculation of Red Blood Cell Folate from Haemolysate Folate Result | 219 | ||
| Serum B12 and Folate and Red Cell Folate Assay Calibration | 219 | ||
| Whole Blood Folate Standards | 220 | ||
| Primary Instrument Calibration | 220 | ||
| Internal Adjustment Calibration | 220 | ||
| Internal Quality Control | 220 | ||
| Methylmalonic acid measurement | 220 | ||
| Chapter 11: Laboratory methods used in the investigation of the haemolytic anaemias | 229 | ||
| Investigation of haemolytic anaemia | 230 | ||
| Is There Evidence of Increased Haemolysis? | 230 | ||
| What is the Type of Haemolytic Mechanism? | 231 | ||
| What is the Precise Diagnosis? | 231 | ||
| Plasma haemoglobin | 231 | ||
| Sample Collection | 231 | ||
| Peroxidase Method3 | 232 | ||
| Reagents | 232 | ||
| Benzidine compound | 232 | ||
| Hydrogen peroxide | 232 | ||
| Acetic acid | 232 | ||
| Standard | 232 | ||
| Method | 232 | ||
| Spectrophotometric Method | 232 | ||
| Normal Range | 232 | ||
| Significance of increased plasma haemoglobin | 232 | ||
| Serum haptoglobin | 233 | ||
| Electrophoresis Method | 233 | ||
| Principle | 233 | ||
| Reagents | 233 | ||
| Buffer (pH 7.0, ionic strength 0.05) | 233 | ||
| Haemolysates | 233 | ||
| Stain | 233 | ||
| Clearing solution | 233 | ||
| Acetic acid rinse | 233 | ||
| Method | 233 | ||
| Interpretation | 233 | ||
| Radial Immunodiffusion (RID) Method | 234 | ||
| Principle | 234 | ||
| Reagents | 234 | ||
| Single diffusion plates | 234 | ||
| Reference sera | 234 | ||
| Test serum | 234 | ||
| Method | 234 | ||
| Normal Ranges | 234 | ||
| Significance | 235 | ||
| Serum haemopexin | 235 | ||
| Examination of plasma (or serum) for methaemalbumin | 235 | ||
| Schumm´s Test | 236 | ||
| Chapter 12: Investigation of the hereditary haemolytic anaemias | 245 | ||
| Investigation of membrane defects | 246 | ||
| Osmotic fragility as measured by lysis in hypotonic saline | 246 | ||
| Principle | 246 | ||
| Reagents | 246 | ||
| Method | 246 | ||
| Notes | 246 | ||
| Osmotic Fragility after Incubating the Blood at 37C for 24 Hours | 247 | ||
| Method | 247 | ||
| Factors Affecting Osmotic Fragility Tests | 248 | ||
| Recording the Results of Osmotic Fragility Tests | 248 | ||
| Alternative methods of recording osmotic fragility | 249 | ||
| Interpretation of Results | 249 | ||
| Flow cytometric (dye-binding) test | 250 | ||
| Principle | 250 | ||
| Reagents | 250 | ||
| EMA | 250 | ||
| Bovine serum albumin (30%) solution (BSA) | 251 | ||
| PBS | 251 | ||
| Chapter 13: Acquired haemolytic anaemias | 273 | ||
| Assessing the likelihood of acquired haemolytic anaemia | 273 | ||
| Assessment of the blood film and count in suspected acquired haemolytic anaemia | 273 | ||
| Immune haemolytic anaemias | 274 | ||
| Types of Autoantibody | 275 | ||
| Warm Autoantibodies | 276 | ||
| Cold Autoantibodies | 277 | ||
| Combined Warm and Cold Autoantibodies | 277 | ||
| Methods of Investigation | 277 | ||
| Collection of Samples of Blood and Serum | 277 | ||
| Storage of Samples | 278 | ||
| Scheme for Serological Investigation of Haemolytic Anaemia Suspected to be of Immunological Origin | 278 | ||
| Detection of Incomplete Antibodies by Means of the Direct Antiglobulin (Coombs) Test | 279 | ||
| Principle | 279 | ||
| Precautions | 280 | ||
| Method | 280 | ||
| DAT Using Column Agglutination Technology | 280 | ||
| Significance of Positive Direct Antiglobulin Test | 280 | ||
| Positive DATs in Normal Subjects | 281 | ||
| Positive DATs in Hospital Patients | 281 | ||
| False-Negative Antiglobulin Test Results | 281 | ||
| DAT-Negative Autoimmune Haemolytic Anaemia | 281 | ||
| Preparing and Testing a Concentrated Eluate | 282 | ||
| Manual Direct Polybrene Test | 282 | ||
| Reagents | 282 | ||
| Method | 282 | ||
| Positive control | 282 | ||
| Negative control | 282 | ||
| Determination of the Blood Group of a Patient with AIHA | 283 | ||
| ABO Grouping | 283 | ||
| RhD Grouping | 283 | ||
| Demonstration of Free Antibodies in Serum | 283 | ||
| Identification by Adsorption Techniques of Coexisting Alloantibodies in the Presence of Warm Autoantibodies | 283 | ||
| Use of ZZAP Reagent in Autoadsorption Techniques | 283 | ||
| Reagents | 283 | ||
| Method | 283 | ||
| Notes | 283 | ||
| Alloadsorption Using Papainized R1R1, R2R2 and rr Cells | 284 | ||
| Method for Testing Alloadsorbed Sera | 284 | ||
| Example of alloantibody detection using the alloadsorption technique in a recently transfused patient with AIHA | 284 | ||
| Explanation of the results of testing alloadsorbed sera, A, B and C | 284 | ||
| Additional notes on adsorption techniques | 284 | ||
| Elution of Antibodies from Red Cells | 284 | ||
| Notes | 286 | ||
| Heat Elution | 286 | ||
| Freeze and Thaw Elution (Lui) | 286 | ||
| Screening Eluates | 286 | ||
| Determination of the Specificity of Warm Autoantibodies in Eluates and Sera | 286 | ||
| Titration of Warm Antibodies in Eluates or Sera | 286 | ||
| Determination of the Specificity of Cold Autoantibodies | 286 | ||
| Titration of Cold Antibodies | 286 | ||
| Normal range | 287 | ||
| Cold Agglutinin Titration Patterns | 287 | ||
| Determination of the Thermal Range of Cold Agglutinins | 287 | ||
| Detection and Titration of the Donath-Landsteiner Antibody | 287 | ||
| Direct Donath-Landsteiner Test | 287 | ||
| Indirect Donath-Landsteiner Test | 287 | ||
| Two-Stage Indirect Donath-Landsteiner Test | 288 | ||
| Titration of a Donath-Landsteiner Antibody | 288 | ||
| Detection of a Donath-Landsteiner Antibody by the Indirect Antiglobulin Test | 288 | ||
| Method | 288 | ||
| Thermal range of Donath-Landsteiner antibody | 288 | ||
| Specificity of the Donath-Landsteiner antibody | 288 | ||
| Treatment of Serum with 2-Mercaptoethanol or Dithiothreitol | 289 | ||
| Method | 289 | ||
| 2-Mercaptoethanol | 289 | ||
| Dithiothreitol | 289 | ||
| Drug-Induced Haemolytic Anaemias of Immunological Origin | 289 | ||
| Drug-Induced Autoimmune Haemolytic Anaemias | 289 | ||
| Detection of Antipenicillin Antibodies | 290 | ||
| Reagents | 290 | ||
| Penicillin-Coated Normal Red Cells | 290 | ||
| Control normal red cells | 290 | ||
| Method | 290 | ||
| Note | 290 | ||
| Detection of Antibodies against Drugs Other than Penicillin | 290 | ||
| Interpretation | 291 | ||
| Oxidant-induced haemolytic anaemia | 291 | ||
| Microangiopathic and mechanical haemolytic anaemias | 291 | ||
| Paroxysmal nocturnal haemoglobinuria | 291 | ||
| Acidified-Serum Lysis Test (Ham test) | 293 | ||
| Principle | 293 | ||
| Chapter 14: Investigation of abnormal haemoglobins and thalassaemia | 301 | ||
| The haemoglobin molecule | 302 | ||
| Structural variants of haemoglobin | 302 | ||
| Haemoglobins with Reduced Solubility | 303 | ||
| Hb S | 303 | ||
| Sickle Cell Disease | 303 | ||
| Other Forms of Sickle Cell Disease | 303 | ||
| Hb C | 303 | ||
| Other Sickling Haemoglobins | 303 | ||
| Unstable Haemoglobins | 304 | ||
| Haemoglobins with Altered Oxygen Affinity | 304 | ||
| Hb M | 304 | ||
| Thalassaemia syndromes | 304 | ||
| β Thalassaemia Syndromes | 305 | ||
| α Thalassaemia Syndromes | 306 | ||
| Thalassaemic Structural Variants | 306 | ||
| Increased Hb F in Adult Life | 307 | ||
| Inherited Abnormalities That Increase Hb F Concentration | 307 | ||
| Investigation of patients with a suspected haemoglobinopathy | 307 | ||
| Laboratory detection of haemoglobin variants | 308 | ||
| Blood Count and Film | 308 | ||
| Collection of Blood and Preparation of Haemolysates | 309 | ||
| Preparation of Haemolysate for Qualitative Haemoglobin Electrophoresis | 309 | ||
| Preparation of Haemolysate for the Quantification of Haemoglobins and Stability Tests | 309 | ||
| Control Samples | 310 | ||
| Quality Assurance | 310 | ||
| Cellulose Acetate Electrophoresis at Alkaline pH | 310 | ||
| Principle | 310 | ||
| Equipment | 310 | ||
| Reagents | 311 | ||
| Method | 311 | ||
| Interpretation and Comments | 311 | ||
| Citrate Agar Electrophoresis at pH 6.0 | 312 | ||
| Equipment | 312 | ||
| Reagents | 313 | ||
| Buffer | 313 | ||
| Method | 313 | ||
| Interpretation and Comments | 313 | ||
| Agarose Gel Electrophoresis | 313 | ||
| Reagents and Method | 313 | ||
| Interpretation | 314 | ||
| Automated High-Performance Liquid Chromatography | 314 | ||
| Principle | 314 | ||
| Chapter 15: Erythrocyte and leucocyte cytochemistry | 333 | ||
| Erythrocyte cytochemistry | 333 | ||
| Siderocytes and Sideroblasts | 333 | ||
| Method of Staining Siderotic Granules | 334 | ||
| Significance of siderocytes | 335 | ||
| Haemoglobin Derivatives | 336 | ||
| Heinz Bodies in Red Cells | 336 | ||
| Demonstration of Heinz Bodies | 336 | ||
| Unstained preparations | 336 | ||
| Stained preparations | 337 | ||
| Demonstration of Haemoglobin H Inclusions | 337 | ||
| Method | 337 | ||
| Carboxyhaemoglobin and Methaemoglobin | 338 | ||
| Fetal Haemoglobin | 338 | ||
| Reagents | 338 | ||
| Method | 338 | ||
| Haemoglobin S and Other Haemoglobin Variants | 339 | ||
| Leucocyte cytochemistry | 339 | ||
| Myeloperoxidase | 339 | ||
| Method with 3,3-Diaminobenzidine | 340 | ||
| Reagents | 340 | ||
| Method | 340 | ||
| Technical considerations | 340 | ||
| Results and interpretation | 340 | ||
| Pathological variations | 341 | ||
| Sudan Black B | 341 | ||
| Chapter 16: Immunophenotyping | 353 | ||
| Introduction | 353 | ||
| Methods for the study of immunological markers | 354 | ||
| Preparation of the Specimens and Cell Separation | 354 | ||
| Ficoll-Gradient Method of Separation | 354 | ||
| Red Blood Cell Lysing Methods | 354 | ||
| Multicolour Flow Cytometry Methods | 354 | ||
| Detection of membrane antigens | 354 | ||
| Rationale for choosing antibody panels | 356 | ||
| Detection of Surface Immunoglobulin | 356 | ||
| Method 1: Wash-Stain-Lyse-Wash | 356 | ||
| Method 2: Lyse-Stain-Wash | 357 | ||
| Detection of Intracellular Antigens | 357 | ||
| Chapter 17: Diagnostic radioisotopes in haematology | 373 | ||
| Sources of radioisotopes | 374 | ||
| Radiation protection | 374 | ||
| Apparatus for Measuring Radioactivity in Vitro | 375 | ||
| Apparatus for Measuring Radioactivity in Vivo | 376 | ||
| Surface Counting | 376 | ||
| Imaging | 376 | ||
| Measurement of Radioactivity with a Scintillation Counter | 376 | ||
| Standardization of Working Conditions | 376 | ||
| Counting Technique | 377 | ||
| Measurement of radioactivity | 377 | ||
| Correction for Physical Decay | 377 | ||
| Double Radioisotope Measurements | 377 | ||
| Differential decay | 377 | ||
| Physical separation | 377 | ||
| Blood volume | 377 | ||
| Measurement of Blood Volume | 377 | ||
| Principle | 377 | ||
| Red Cell Volume | 378 | ||
| Radioactive Chromium Method | 378 | ||
| Technetium Method | 378 | ||
| Chapter 18: Investigation of haemostasis | 393 | ||
| Components of normal haemostasis | 394 | ||
| The Blood Vessel | 394 | ||
| General Structure of the Blood Vessel | 394 | ||
| Endothelial Cell Function | 394 | ||
| Vasoconstriction | 395 | ||
| Platelets | 395 | ||
| Platelet Function in the Haemostatic Process | 395 | ||
| Platelet Aggregation | 396 | ||
| Blood Coagulation | 396 | ||
| The Contact Activation System | 397 | ||
| Tissue Factor | 397 | ||
| The Vitamin K-Dependent Factors | 398 | ||
| Cofactors | 398 | ||
| Fibrinogen | 399 | ||
| Factor XIII | 399 | ||
| Inhibitors of Coagulation | 399 | ||
| The Fibrinolytic System | 399 | ||
| General approach to investigation of haemostasis | 400 | ||
| Clinical Approach | 400 | ||
| Principles of Laboratory Analysis | 400 | ||
| Immunological | 400 | ||
| Assays using chromogenic peptide substrates (amidolytic assays) | 401 | ||
| Coagulation assays | 401 | ||
| Other Assays | 401 | ||
| Notes on equipment | 401 | ||
| Waterbaths | 401 | ||
| Refrigerators and Freezers | 402 | ||
| Centrifuges | 402 | ||
| Reagents and Buffers | 402 | ||
| Plastic and Glass Tubes | 402 | ||
| Pipettes | 402 | ||
| Stopwatches and Stopclocks | 402 | ||
| Automated Coagulation Analysers | 402 | ||
| Evaluating and choosing an automated analyser | 402 | ||
| Mandatory requirements | 402 | ||
| Desirable additional requirements | 402 | ||
| Safety | 403 | ||
| Pre-analytical variables including sample collection | 403 | ||
| Collection of Venous Blood | 403 | ||
| Blood Sample Anticoagulation | 403 | ||
| Time of Sample Collection | 404 | ||
| Transportation to the Laboratory | 404 | ||
| Centrifugation: Preparation of Platelet-Poor Plasma | 404 | ||
| Storage of Plasma and Sample Thawing | 404 | ||
| Some Common `Technical´ Errors | 404 | ||
| Calibration and Quality Control | 405 | ||
| Reference Standard (Calibrator) | 405 | ||
| Calibration of Standard Pools and Suggested Calibration Procedure | 405 | ||
| Control Plasma | 405 | ||
| Variability of Coagulation Assays | 405 | ||
| Performance of Coagulation Tests | 405 | ||
| Handling of Samples and Reagents | 405 | ||
| Eliminating a Time Trend | 406 | ||
| Assay Monitoring and Endpoint Detection | 406 | ||
| Manual Methods | 406 | ||
| Electromechanical | 406 | ||
| Impedance, steel ball | 406 | ||
| Photo-Optical Analysis | 406 | ||
| Scattered light detection for clotting assays (660nm) | 406 | ||
| Transmitted light detection for chromogenic assays (405nm, 575nm, 800nm) | 406 | ||
| Transmitted light detection for immunoassays (405nm, 575nm, 800nm) | 406 | ||
| Nephelometry | 406 | ||
| Photo-optical endpoint determination and analyses | 406 | ||
| Percentage Detection Method | 406 | ||
| Rate Method | 407 | ||
| VLin Integral Method | 407 | ||
| Analysis Time Over | 407 | ||
| Turbidity Level Over | 408 | ||
| Clot Signatures: Normal and Abnormal APTT Clot Waveforms | 408 | ||
| Molecular Mechanism of the Biphasic Waveform: LC-CRP | 408 | ||
| Commonly Used Reagents | 409 | ||
| CaCl2 | 409 | ||
| Barbitone buffer | 409 | ||
| Barbitone buffered saline, pH 7.4 | 409 | ||
| Glyoxaline buffer | 409 | ||
| Owren's veronal buffer | 409 | ||
| Factor-Deficient Plasmas | 409 | ||
| The `clotting screen´ | 409 | ||
| Prothrombin Time | 409 | ||
| Principle | 409 | ||
| Reagents | 409 | ||
| Patient and control plasma samples | 409 | ||
| Thromboplastin | 409 | ||
| CaCl2 | 410 | ||
| Method | 410 | ||
| Expression of Results | 410 | ||
| Normal Values | 410 | ||
| Interpretation | 410 | ||
| Activated Partial Thromboplastin Time | 410 | ||
| Principle | 410 | ||
| Reagents | 410 | ||
| Chapter 19: Investigation of a thrombotic tendency | 447 | ||
| Introduction to thrombophilia | 447 | ||
| Tests for the presence of a lupus anticoagulant | 448 | ||
| Sample Preparation | 448 | ||
| Dilute Russell's Viper Venom Time | 449 | ||
| Principle | 449 | ||
| Reagents | 449 | ||
| Reagent Preparation | 449 | ||
| Method | 449 | ||
| Interpretation | 449 | ||
| Platelet Neutralization Test | 450 | ||
| Principle | 450 | ||
| Reagents for preparation of platelet neutralization reagent | 450 | ||
| Method | 450 | ||
| Interpretation | 450 | ||
| Interpretation of Tests for Lupus Anticoagulant | 450 | ||
| Kaolin Clotting Time | 451 | ||
| Chapter 20: Laboratory control of anticoagulant, thrombolytic and antiplatelet therapy | 467 | ||
| Oral anticoagulant treatment using vitamin K antagonists | 467 | ||
| Selection of Patients | 468 | ||
| Methods Used for the Laboratory Control of Oral Anticoagulant Treatment | 468 | ||
| Standardization of Oral Anticoagulant Treatment | 468 | ||
| Calibration of Thromboplastins | 468 | ||
| Principle | 468 | ||
| Reagents | 468 | ||
| Method | 468 | ||
| Calibration | 469 | ||
| Calculation of International Sensitivity Index | 469 | ||
| Local Calibration of Thromboplastins | 470 | ||
| Geometric Mean Normal Prothrombin Time | 470 | ||
| Calibration Audits | 470 | ||
| Determination of the International Normalized Ratio | 470 | ||
| Capillary Reagent | 470 | ||
| Therapeutic Range and Choice of Thromboplastin | 470 | ||
| Management of Overanticoagulation | 470 | ||
| Point-of-Care Testing | 471 | ||
| Heparin treatment | 472 | ||
| Selection of Patients | 472 | ||
| Laboratory Control of Heparin Treatment | 472 | ||
| Activated Partial Thromboplastin Time for Heparin Monitoring | 473 | ||
| Principle | 473 | ||
| Reagents and Method | 473 | ||
| Therapeutic Range | 473 | ||
| Near-Patient Heparin Monitoring | 474 | ||
| Chapter 21: Blood cell antigens and antibodies | 483 | ||
| Erythrocytes | 483 | ||
| Red Cell Antigens | 483 | ||
| ABO System | 485 | ||
| ABO Antigens and Encoding Genes | 486 | ||
| Secretors and Non-Secretors | 487 | ||
| ABO Antigens and Disease | 488 | ||
| ABO Antibodies | 488 | ||
| Anti-A and anti-B | 488 | ||
| Anti-A1 and anti-H | 488 | ||
| Lewis System | 488 | ||
| Lewis Antigens and Encoding Genes | 488 | ||
| Lewis Antibodies | 489 | ||
| The P System and Globoside Collection | 489 | ||
| Antigens | 489 | ||
| Antibodies | 489 | ||
| Rh System | 489 | ||
| Rh Antigens and Encoding Genes | 489 | ||
| Antibodies | 490 | ||
| Kell and Kx Systems | 491 | ||
| Antigens and Encoding Genes | 491 | ||
| Kell Antibodies | 491 | ||
| Duffy System | 491 | ||
| Duffy antigens and encoding genes | 491 | ||
| Duffy antibodies | 491 | ||
| Kidd (JK) System | 491 | ||
| Kidd antigens and encoding genes | 491 | ||
| Kidd antibodies | 491 | ||
| MNSs System | 491 | ||
| MNSs antigens and encoding genes | 491 | ||
| MNSs antibodies | 492 | ||
| Other Blood Group Systems | 492 | ||
| Lutheran system | 492 | ||
| Yt (Cartwright) system | 492 | ||
| Colton system | 492 | ||
| Dombrock system | 492 | ||
| Clinical Significance of Red Cell Alloantibodies | 492 | ||
| Mechanisms of Immune Destruction of Red Cells | 494 | ||
| Antigen-Antibody Reactions | 495 | ||
| Quality Assurance within the Laboratory | 495 | ||
| General Points of Serological Technique | 496 | ||
| Serum versus Plasma | 496 | ||
| Collection and Storage of Blood Samples | 497 | ||
| Storage of Sera or Plasma | 497 | ||
| Red Cell Suspensions | 497 | ||
| Normal ionic strength saline | 497 | ||
| Low ionic strength saline | 497 | ||
| Reagent Red Cells | 497 | ||
| Use of Enzyme-Treated Cells | 498 | ||
| Agglutination of Red Cells by Antibody: A Basic Method | 498 | ||
| Tube Tests | 498 | ||
| Tubes | 498 | ||
| Temperature and Time of Exposure of Red Cells to Antibody | 498 | ||
| Slide Tests | 498 | ||
| Reading Results of Tube Tests | 498 | ||
| Microscopic reading | 498 | ||
| Macroscopic reading | 498 | ||
| Demonstration of Lysis | 499 | ||
| Controls | 500 | ||
| Antiglobulin Test | 500 | ||
| Antiglobulin Reagents | 500 | ||
| Polyspecific (broad-spectrum) reagents | 500 | ||
| Monospecific reagents | 500 | ||
| Quality Control of Antiglobulin Reagents | 500 | ||
| Recommended Antiglobulin Test Procedure | 501 | ||
| Alternative Technology for Antibody Detection by the Antiglobulin Test | 502 | ||
| Assessment of Individual Worker Performance | 502 | ||
| Titration of Antibodies | 503 | ||
| Preparation of serial dilutions of patient's or other sera | 503 | ||
| Addition of red cell suspensions to dilutions of serum | 503 | ||
| Test for ABH Substance Secretion | 504 | ||
| Method | 504 | ||
| Red Cell Genotyping | 504 | ||
| Platelet and Neutrophils | 504 | ||
| Platelet and Neutrophil Alloantigen Systems | 504 | ||
| Clinical Significance of Platelet and Neutrophil Antibodies | 504 | ||
| Alloantibodies | 504 | ||
| Isoantibodies | 506 | ||
| Autoantibodies | 506 | ||
| Drug-Induced Antibodies | 507 | ||
| Demonstration of Platelet and Neutrophil Antibodies | 507 | ||
| Alloantibodies | 507 | ||
| Autoantibodies | 508 | ||
| Drug-Induced Antibodies | 508 | ||
| Methods of Demonstrating Antibodies | 508 | ||
| The Immunofluorescent Antiglobulin Methods | 509 | ||
| Patient's and Screening Panel Cells | 509 | ||
| Patient's Serum | 509 | ||
| Control Sera | 509 | ||
| Eluate from Patient's Sensitized Cells | 509 | ||
| Heat Eluate | 509 | ||
| Platelet Preparation | 509 | ||
| Granulocyte Preparation | 509 | ||
| Platelet and Granulocyte Immunofluorescence Tests | 510 | ||
| Indirect Test | 510 | ||
| Scoring Results | 511 | ||
| Use of Flow Cytometry | 511 | ||
| Chloroquine Treatment of Platelets and Granulocytes | 511 | ||
| Interpretation of Results with Chloroquine-Treated Cells | 512 | ||
| MAIPA Assay | 512 | ||
| Other Methods | 513 | ||
| Molecular Genotyping of Platelet Alloantigens | 513 | ||
| References | 514 | ||
| Chapter 34: Laboratory aspects of blood transfusion | 519 | ||
| Technology and automation in blood transfusion laboratories | 520 | ||
| Pre-transfusion compatibility systems | 522 | ||
| Documentation of the Transfusion Process | 523 | ||
| Identification and Storage of Blood Samples | 523 | ||
| ABO and D grouping | 524 | ||
| ABO Grouping | 524 | ||
| Reagents for ABO Grouping | 524 | ||
| D Grouping | 524 | ||
| Reagents for D Grouping | 524 | ||
| Methods | 524 | ||
| Tube and slide tests | 525 | ||
| EDTA for diluents | 525 | ||
| Slide method | 525 | ||
| Liquid-phase microplate methods | 525 | ||
| Column agglutination techniques | 525 | ||
| Solid-phase techniques | 526 | ||
| Controls | 526 | ||
| Causes of Discrepancies in ABO/D Grouping | 526 | ||
| False-Positive Reactions | 526 | ||
| Rouleaux | 526 | ||
| Cold autoagglutination and cold reacting alloantibodies | 526 | ||
| T-activation/polyagglutination | 526 | ||
| Acquired B | 527 | ||
| Potentiators | 527 | ||
| In vitro bacterial contamination | 527 | ||
| False-Negative Reactions | 527 | ||
| Failure to add reagents | 527 | ||
| Loss of potency | 527 | ||
| Failure to identify lysis | 527 | ||
| Mixed-field appearance | 527 | ||
| D variant phenotypes | 527 | ||
| Antibody screening | 528 | ||
| Red Cell Reagents | 528 | ||
| Methods | 528 | ||
| Indirect Antiglobulin Techniques | 529 | ||
| Column Agglutination | 529 | ||
| Solid-Phase Systems | 529 | ||
| Liquid-Phase Techniques - Tubes and Microplates | 529 | ||
| Controls | 529 | ||
| Antibody identification | 530 | ||
| Principles | 530 | ||
| Phenotyping | 530 | ||
| Additional Panels/Techniques | 531 | ||
| Reagents | 531 | ||
| Antibody Cards | 531 | ||
| Selection and transfusion of red cells | 531 | ||
| Crossmatching | 532 | ||
| Choice of Test | 532 | ||
| Indirect Antiglobulin Crossmatch | 532 | ||
| Immediate Spin Crossmatch | 533 | ||
| False-negative results (in immediate spin crossmatch) | 533 | ||
| False-positive results (in immediate spin crossmatch) | 533 | ||
| Electronic Issue | 533 | ||
| Emergency blood issue | 534 | ||
| Rapid ABO and D Typing | 534 | ||
| Confirmation | 534 | ||
| Selection of Units | 534 | ||
| Compatibility Testing | 534 | ||
| Antibody Screening | 534 | ||
| Massive Transfusion | 534 | ||
| Selection of Platelets and Plasma | 535 | ||
| Potential Errors | 535 | ||
| Antenatal serology and haemolytic disease of the newborn | 535 | ||
| Haemolytic Disease of the Fetus and Newborn | 535 | ||
| Antenatal Serology | 535 | ||
| ABO and D Grouping and Antibody Screening | 535 | ||
| Follow-Up Antibody Screening | 535 | ||
| Prediction of Fetal Blood Group | 536 | ||
| Partner Testing | 536 | ||
| Testing Fetal DNA in the Maternal Circulation | 536 | ||
| Fetal Blood Sampling | 536 | ||
| Antenatal Assessment of the Severity of Haemolytic Disease of the Fetus and Newborn | 536 | ||
| Antibody Titrations during Pregnancy | 536 | ||
| Antibody Quantitation | 536 | ||
| Assessment of Fetal Anaemia | 536 | ||
| Tests on Maternal and Cord Blood at Delivery | 537 | ||
| Prevention of Haemolytic Disease of the Fetus and Newborn as a Result of Anti-D | 538 | ||
| Anti-D Prophylaxis | 538 | ||
| Measurement of Fetomaternal Haemorrhage | 539 | ||
| Recommended Action at Delivery (or Potentially Sensitizing Event) | 539 | ||
| ABO Haemolytic Disease of the Newborn | 540 | ||
| Serological Investigation | 540 | ||
| Compatibility testing in special transfusion situations | 540 | ||
| Neonates and Infants within First 4 Months of Life | 540 | ||
| Investigations on the Maternal Sample | 540 | ||
| Investigations on the Infant Sample | 540 | ||
| Selection of Blood and Other Components | 540 | ||
| Intrauterine (Fetal) Transfusion | 541 | ||
| Patients Receiving Transfusions at Close Intervals | 541 | ||
| Chronic Transfusion Programmes | 541 | ||
| Allogeneic Haemopoietic Stem Cell Transplantation | 542 | ||
| Investigation of a transfusion reaction | 542 | ||
| Acute Transfusion Reactions | 543 | ||
| Acute Intravascular Haemolysis | 543 | ||
| Documentation check | 544 | ||
| Serological investigations | 544 | ||
| Tests for haemolysis | 544 | ||
| Microbiological tests | 544 | ||
| Delayed Haemolytic Transfusion Reaction | 544 | ||
| Haematological Investigation | 544 | ||
| Serological Investigation | 544 | ||
| References | 545 | ||
| Chapter 23: Approach to the diagnosis and classification of blood diseases | 549 | ||
| Common presentations of haematological diseases | 549 | ||
| Initial screening tests | 549 | ||
| Interpretation of Screening Tests | 550 | ||
| Quantitative Abnormalities of Blood Cells | 550 | ||
| Increased Numbers of Cells | 550 | ||
| Increases affecting more than one cell line | 550 | ||
| Erythrocytosis | 550 | ||
| Leucocytosis | 550 | ||
| Neutrophilia | 550 | ||
| Lymphocytosis | 550 | ||
| Monocytosis | 550 | ||
| Eosinophilia | 551 | ||
| Basophilia | 551 | ||
| Thrombocytosis | 551 | ||
| Reduced Numbers of Cells | 551 | ||
| Reductions in more than one cell line | 551 | ||
| Anaemia | 551 | ||
| Microcytic Anaemia | 551 | ||
| Macrocytic Anaemia | 553 | ||
| Normocytic Anaemia | 554 | ||
| Leucopenia | 554 | ||
| Neutropenia | 554 | ||
| Reduced numbers of lymphocytes, monocytes, eosinophils and basophils | 554 | ||
| Thrombocytopenia | 554 | ||
| Pancytopenia | 554 | ||
| Qualitative Abnormalities of Blood Cells | 554 | ||
| Abnormalities of All Cell Lines | 554 | ||
| Abnormalities of Individual Cell Lines | 555 | ||
| Red cells | 555 | ||
| White cells | 555 | ||
| Platelets | 555 | ||
| Specific tests for common haematological disorders | 555 | ||
| Red Cell Disorders | 555 | ||
| Microcytic Hypochromic Anaemias | 555 | ||
| Macrocytic Anaemias | 556 | ||
| Aplastic Anaemia11 | 556 | ||
| Haemolytic Anaemias | 556 | ||
| White Cell Disorders | 556 | ||
| Acute Leukaemia | 556 | ||
| Neutropenia | 556 | ||
| Chronic Myelogenous Leukaemia | 556 | ||
| Chronic Lymphoproliferative Disorders/Lymphadenopathy | 556 | ||
| Myelomatosis (Plasma Cell Myeloma) | 557 | ||
| Other Disorders | 557 | ||
| Myeloproliferative Neoplasms | 557 | ||
| Myelodysplastic syndromes | 557 | ||
| Pancytopenia with Splenomegaly | 557 | ||
| Classification of haematological neoplasms | 557 | ||
| Classification of Acute Myeloid Leukaemia | 558 | ||
| Classification of the Myelodysplastic Syndromes | 558 | ||
| Classification of Acute Lymphoblastic Leukaemia | 559 | ||
| Classification of Myeloproliferative Neoplasms and Related Conditions | 559 | ||
| References | 562 | ||
| Chapter 24: Laboratory organization and management | 563 | ||
| Management structure and function | 564 | ||
| Staff Appraisal | 564 | ||
| Continuing Professional Development | 565 | ||
| Strategic and Business Planning | 565 | ||
| Workload Assessment and Costing of Tests | 565 | ||
| Financial Control | 566 | ||
| Calculation of Test Costs | 566 | ||
| Test reliability | 567 | ||
| Test selection | 567 | ||
| Likelihood Ratio | 568 | ||
| Receiver-Operator Characteristic Analysis | 568 | ||
| Test Utility | 568 | ||
| Instrumentation | 569 | ||
| Equipment Evaluation | 569 | ||
| Principles of Evaluation | 569 | ||
| Precision | 569 | ||
| Linearity | 569 | ||
| Carryover | 570 | ||
| Accuracy and Comparability | 570 | ||
| Maintenance logs | 570 | ||
| Data processing | 570 | ||
| Laboratory Computers | 571 | ||
| Pre-analytical and post-analytical stages of testing | 572 | ||
| Test Requesting | 572 | ||
| Specimen Collection and Delivery | 572 | ||
| Pre-Analytical Phase | 572 | ||
| Post-Analytical Phase | 572 | ||
| Test Turnaround Time | 574 | ||
| Point-of-Care Testing | 574 | ||
| Point-of-Care Testing Beyond the Laboratory | 575 | ||
| Patient Self-Testing | 575 | ||
| Laboratory Services for General Practitioners | 575 | ||
| Pre-Analytical Service | 575 | ||
| Post-Analytical Service | 575 | ||
| Standard Operating Procedures | 575 | ||
| Laboratory audit and accreditation | 576 | ||
| Audit | 576 | ||
| Accreditation | 577 | ||
| International standards of practice | 578 | ||
| Benchmarking | 579 | ||
| Laboratory Safety | 579 | ||
| Principles of Safety Policy | 579 | ||
| Design of Laboratory | 580 | ||
| Electrical and Radiation Safety | 580 | ||
| Fire Hazard | 581 | ||
| Chemical Safety | 581 | ||
| Eyewash Facilities | 581 | ||
| Biohazardous Specimens | 581 | ||
| Universal Precautions | 581 | ||
| Disinfectants | 582 | ||
| Sodium hypochlorite (chlorine) | 582 | ||
| Alcohols | 582 | ||
| Applications of Disinfectants | 583 | ||
| Automated equipment | 583 | ||
| Centrifuges | 583 | ||
| Syringes and needles | 583 | ||
| Gloves | 583 | ||
| Laundry | 583 | ||
| Waste Disposal | 583 | ||
| Specimen Shipping | 584 | ||
| References | 585 | ||
| Chapter 25: Quality assurance | 587 | ||
| Standardization | 587 | ||
| Control materials and reference standards | 588 | ||
| Reference Standards | 589 | ||
| Assigning Values to Reference Materials | 590 | ||
| Quality assurance procedures | 590 | ||
| Internal Quality Control | 592 | ||
| Control Charts | 592 | ||
| Duplicate Tests on Patients' Specimens | 593 | ||
| Correlation Check | 593 | ||
| External Quality Assessment | 594 | ||
| Standardization of EQA Schemes | 595 | ||
| Assessment of Participant Performance | 596 | ||
| Quantitative Tests | 596 | ||
| Deviation index | 596 | ||
| Consecutive monitoring | 597 | ||
| Out of consensus method | 597 | ||
| Target values and bias | 597 | ||
| Youden (xy) plot | 597 | ||
| Methodology check | 598 | ||
| Clinical significance | 598 | ||
| Semi-Quantitative Tests | 598 | ||
| Interpretive Tests | 598 | ||
| Internal audit for total quality management | 598 | ||
| Internal Audit of Examination Processes | 599 | ||
| Continuous Quality Improvement | 599 | ||
| Control of Non-Compliance | 599 | ||
| Preparation of extended-life material for use in quality assessment | 599 | ||
| Preparation of Preserved Whole Blood | 599 | ||
| Method4,15 | 599 | ||
| Preparation of Haemolysate | 599 | ||
| Method | 599 | ||
| Preparation of Stabilized Whole Blood Control Material | 600 | ||
| Chapter 26: Haematology in under-resourced laboratories | 603 | ||
| Introduction: types of laboratories | 603 | ||
| Organization of clinical laboratory services | 604 | ||
| Level A: Sub-District Facilities Including Health Centres | 604 | ||
| Level B: District Hospitals | 604 | ||
| Level C: Central and Teaching Hospitals | 605 | ||
| Availability of tests at each level | 605 | ||
| Level A | 605 | ||
| Level B | 605 | ||
| Level C | 605 | ||
| Microscopes | 606 | ||
| Care of the Microscope | 606 | ||
| `Essential´ haematology tests | 606 | ||
| Cost per Test | 606 | ||
| Diagnostic Reliability | 606 | ||
| Clinical Usefulness | 607 | ||
| Maintaining quality and reliability of tests | 607 | ||
| Quality Control of a Test Method (Technical Quality) | 607 | ||
| Internal Quality Control | 607 | ||
| External Quality Assessment | 607 | ||
| Basic haematology tests | 607 | ||
| Haemoglobinometry | 607 | ||
| Direct Reading Haemoglobinometers | 608 | ||
| HemoCue Blood Hemoglobin System**Available from HemoCue AB, Angelholm, Sweden; www.hemocue.com (Accessed 25 January 2010). | 608 | ||
| Haemoglobin Colour ScaleAvailable from Teaching Aids at Low Cost; www.talcuk.org/accessories/haemoglobin-colour-scale.htm (Acce | 608 | ||
| Packed Cell Volume | 609 | ||
| Manual Cell Counts Using Counting Chambers | 609 | ||
| Counting Chambers | 609 | ||
| Total White Blood Cell Count | 610 | ||
| Diluent | 610 | ||
| Appendix | 619 | ||
| Preparation of commonly used reagents | 619 | ||
| Water | 619 | ||
| Anticoagulants and Preservative Solutions | 619 | ||
| Acid-Citrate-Dextrose (ACD) Solution - NIH-A | 619 | ||
| Acid-Citrate-Dextrose (Alsever's) Solution | 619 | ||
| Citrate-Phosphate-Dextrose (CPD) Solution, pH 6.9 | 620 | ||
| Citrate-Phosphate-Dextrose (CPD) Solution, pH 5.6-5.8 | 620 | ||
| Citrate-Phosphate-Dextrose-Adenine (CPD-A) Solution, pH 5.6-5.8 | 620 | ||
| Low Ionic Strength Solution (LISS)1 | 620 | ||
| EDTA | 620 | ||
| Neutral EDTA, pH 7.0, 110mmol/l | 620 | ||
| Neutral Buffered EDTA, pH 7.0 | 620 | ||
| Saline (Normal Ionic Strength) | 621 | ||
| Trisodium Citrate (Na3C6H5O7.2H2O), 109mmol/l | 621 | ||
| Heparin | 621 | ||
| Buffers | 621 | ||
| Barbitone Buffer, pH 7.4 | 621 | ||
| Barbitone Buffered Saline, pH 7.4 | 621 | ||
| Barbitone Buffered Saline, pH 9.5 | 621 | ||
| Barbitone-Bovine Serum Albumin Buffer, pH 9.8 | 621 | ||
| Citrate-Saline Buffer | 621 | ||
| Glycine Buffer, pH 3.0 | 621 | ||
| HEPES Buffer, pH 6.6 | 621 | ||
| HEPES-Saline Buffer, pH 7.6 | 622 | ||
| Imidazole Buffered Saline, pH 7.4 | 622 | ||
| Phosphate Buffer, Iso-Osmotic | 622 | ||
| Phosphate Buffered Saline | 622 | ||
| Phosphate Buffer, Sörensen's | 622 | ||
| Tris-HCl Buffer (200mmol/l) | 623 | ||
| Tris-HCl Bovine Serum Albumin (BSA) Buffer, pH 7.6, 20mmol/l | 623 | ||
| Buffered Formal Acetone | 623 | ||
| Preparation of glassware | 623 | ||
| Siliconized Glassware | 623 | ||
| Cleaning Slides | 623 | ||
| New Slides | 623 | ||
| Dirty Slides | 623 | ||
| Cleaning Glassware | 623 | ||
| Iron-free Glassware | 623 | ||
| Sizes of tubes | 623 | ||
| Speed of centrifuging | 624 | ||
| Statistical procedures | 624 | ||
| Calculations | 625 | ||
| Variance (s2) | 625 | ||
| Standard deviation (SD) | 625 | ||
| Coefficient of variation (CV) as percentage | 625 | ||
| Standard error mean (SEM) | 625 | ||
| Standard deviation of paired results | 625 | ||
| Standard deviation of median | 625 | ||
| Confidence interval | 625 | ||
| Analysis of Differences by t-Test | 625 | ||
| Index | 633 |