BOOK
Dacie and Lewis Practical Haematology E-Book
Barbara J. Bain | Imelda Bates | Mike A Laffan
(2016)
Additional Information
Book Details
Abstract
For more than 65 years, this best-selling text by Drs. Barbara J. Bain, Imelda Bates, and Mike A. Laffan has been the worldwide standard in laboratory haematology. The 12th Edition of Dacie and Lewis Practical Haematology continues the tradition of excellence with thorough coverage of all of the techniques used in the investigation of patients with blood disorders, including the latest technologies as well as traditional manual methods of measurement. You’ll find expert discussions of the principles of each test, possible causes of error, and the interpretation and clinical significance of the findings.
- A unique section on haematology in under-resourced laboratories.
- Ideal as a laboratory reference or as a comprehensive exam study tool.
- Each templated, easy-to-follow chapter has been completely updated, featuring new information on haematological diagnosis, molecular testing, blood transfusion- and much more.
- Complete coverage of the latest advances in the field.
- An expanded section on coagulation now covers testing for new anticoagulants and includes clinical applications of the tests.
Table of Contents
| Section Title | Page | Action | Price |
|---|---|---|---|
| Front Cover | Cover | ||
| Inside Front Cover | ES2 | ||
| Dacie and Lewis Practical Haematology | iii | ||
| Copyright | iv | ||
| Contents | v | ||
| Preface | vi | ||
| Contributors | vii | ||
| Chapter 1 Collection and Handling of Blood | 1 | ||
| Biohazard precautions | 1 | ||
| Procurement of venous blood | 1 | ||
| Equipment | 1 | ||
| Specimen containers | 1 | ||
| Phlebotomy procedure | 2 | ||
| Postphlebotomy procedure | 3 | ||
| Capillary blood | 3 | ||
| Collection of capillary blood | 3 | ||
| Blood film preparation | 3 | ||
| Differences between capillary and venous blood | 3 | ||
| Sample homogeneity | 4 | ||
| Serum | 4 | ||
| Cold agglutinins | 4 | ||
| Anticoagulants | 4 | ||
| Ethylenediaminetetra-acetic acid (EDTA) | 4 | ||
| Trisodium citrate | 4 | ||
| Heparin | 5 | ||
| Effects of storage on the blood count | 5 | ||
| Effects of storage on blood cell morphology | 5 | ||
| Acknowledgement | 6 | ||
| References | 6 | ||
| Chapter 2 Reference Ranges and Normal Values | 8 | ||
| Reference ranges | 8 | ||
| Statistical procedures | 9 | ||
| Confidence limits | 9 | ||
| Normal reference values | 10 | ||
| Physiological variations in the blood count | 13 | ||
| Red cell components | 13 | ||
| Age and gender | 13 | ||
| Pregnancy | 14 | ||
| The elderly | 14 | ||
| Exercise | 14 | ||
| Posture | 15 | ||
| Diurnal and seasonal variation | 15 | ||
| Altitude | 15 | ||
| Smoking | 15 | ||
| Further information | 16 | ||
| Leucocyte count | 15 | ||
| Platelet count | 16 | ||
| Other blood constituents | 16 | ||
| Effects of smoking on haematological normal reference values | 16 | ||
| References | 16 | ||
| Chapter 3 Basic Haematological Techniques | 18 | ||
| Haemoglobinometry | 19 | ||
| Measurement of haemoglobin concentration using a spectrometer (spectrophotometer) or photoelectric colorimeter | 19 | ||
| Haemiglobincyanide (cyanmethaemoglobin) method | 20 | ||
| Diluent | 20 | ||
| Haemiglobincyanide reference standard | 20 | ||
| Method | 21 | ||
| Calculation of haemoglobin concentration | 21 | ||
| Preparation of standard graph and standard table | 21 | ||
| Direct spectrometry | 22 | ||
| Direct reading portable haemoglobinometers | 22 | ||
| Colour comparators | 22 | ||
| Portable haemoglobinometers | 22 | ||
| Noninvasive screening tests | 23 | ||
| Range of haemoglobin concentration in health | 23 | ||
| Packed cell volume or microhaematocrit | 23 | ||
| Accuracy of microhaematocrit | 23 | ||
| Anticoagulant | 23 | ||
| Blood sample | 23 | ||
| Capillary tubes | 23 | ||
| Centrifuge | 23 | ||
| Reading | 23 | ||
| Plasma trapping | 24 | ||
| International Council for Standardisation in Haematology reference method | 24 | ||
| Surrogate reference method | 24 | ||
| Equipment | 24 | ||
| Method | 24 | ||
| Range of packed cell volume in health | 24 | ||
| Manual cell counts and red cell indices | 24 | ||
| Range of MCHC in health | 25 | ||
| Manual differential leucocyte count | 25 | ||
| Method | 25 | ||
| Basophil and eosinophil counts | 26 | ||
| Range of eosinophil count in health | 26 | ||
| Range of basophil count in health | 26 | ||
| Reporting the differential leucocyte count | 26 | ||
| Correcting the count for nucleated red blood cells | 26 | ||
| Reference differential white cell count | 27 | ||
| Range of differential white cells in health | 27 | ||
| Platelet count | 27 | ||
| Range of platelet count in health | 27 | ||
| Reticulocyte count | 27 | ||
| Reticulocyte stains and count | 27 | ||
| Staining Solution | 29 | ||
| Method | 29 | ||
| Counting reticulocytes | 29 | ||
| Calculation | 29 | ||
| Differentiating between reticulocytes and other red cell inclusions | 30 | ||
| Fluorescence methods for performing a reticulocyte count | 30 | ||
| Manual reference method | 30 | ||
| Range of reticulocyte count in health | 30 | ||
| Automated blood count techniques | 30 | ||
| Haemoglobin concentration | 31 | ||
| Red blood cell count | 31 | ||
| Counting systems | 31 | ||
| Impedance counting | 31 | ||
| Light scattering | 32 | ||
| Reliability of electronic counters | 32 | ||
| Setting discrimination thresholds | 33 | ||
| Packed cell volume and mean cell volume | 33 | ||
| Red cell indices | 35 | ||
| Mean cell volume | 35 | ||
| Mean cell haemoglobin and mean cell haemoglobin concentration | 35 | ||
| Variations in red cell volumes: red cell distribution width | 35 | ||
| Percentage hypochromic red cells and variation in red cell haemoglobinisation: haemoglobin distribution width | 36 | ||
| White blood cell count | 36 | ||
| Automated differential count | 37 | ||
| The automated immature granulocyte count | 38 | ||
| The automated nucleated red blood cell count | 38 | ||
| Automated digital imaging analysis of blood cells | 39 | ||
| New white cell parameters | 39 | ||
| Automated instrument graphics | 40 | ||
| Platelet count | 40 | ||
| Platelet count in health | 40 | ||
| Mean platelet volume | 40 | ||
| Reticulated platelets and immature platelet fraction | 42 | ||
| Reticulocyte count | 42 | ||
| Immature reticulocyte fraction | 42 | ||
| Reticulocyte counts in health | 43 | ||
| Measurement of reticulocyte haemoglobin | 43 | ||
| Point-of-care instruments | 43 | ||
| Calibration of automated blood cell counters | 43 | ||
| Flagging of automated blood counts | 44 | ||
| Microscopy | 45 | ||
| Microscope components | 45 | ||
| Setting up the microscope illumination | 46 | ||
| Examination of slides | 46 | ||
| Low power ( × 10) | 46 | ||
| Medium power ( × 40) | 46 | ||
| High power – oil immersion ( × 100) | 46 | ||
| Routine maintenance of the microscope | 46 | ||
| References | 46 | ||
| Chapter 4 Preparation and Staining Methods for Blood and Bone Marrow Films | 50 | ||
| Preparation of blood films on slides | 50 | ||
| Manual method | 50 | ||
| Automated methods | 51 | ||
| Labelling blood films | 51 | ||
| Fixing blood films | 51 | ||
| Bone marrow films | 52 | ||
| Staining blood and bone marrow films | 52 | ||
| Preparation of solutions of Romanowsky dyes | 52 | ||
| May–Grünwald stain | 52 | ||
| Jenner stain | 52 | ||
| Giemsa stain | 52 | ||
| Azure B–Eosin Y stock solution | 53 | ||
| Leishman stain | 53 | ||
| Buffered water | 53 | ||
| Staining methods | 53 | ||
| May–Grünwald–Giemsa stain | 53 | ||
| Standardised Romanowsky stain | 55 | ||
| Jenner–Giemsa stain | 55 | ||
| Leishman stain | 55 | ||
| Automated staining | 55 | ||
| Rapid staining method | 55 | ||
| Stains | 55 | ||
| Stain A (Polychromed methylene blue) | 55 | ||
| Stain B (Eosin) | 55 | ||
| Method | 56 | ||
| Mounting of coverslip | 56 | ||
| Examination of wet blood film preparations | 56 | ||
| Red cells | 56 | ||
| Cryoglobulinaemia | 56 | ||
| Leucocytes | 57 | ||
| Separation and concentration of blood cells | 57 | ||
| Making a buffy coat preparation | 57 | ||
| Utility of the buffy coat | 57 | ||
| Separation of specific cell populations | 58 | ||
| Bacteria and fungi detectable in blood films | 58 | ||
| Parasites detectable in blood, bone marrow or splenic aspirates | 58 | ||
| Examination of blood films for parasites | 58 | ||
| Making thick films | 58 | ||
| Staining thick films for parasites | 58 | ||
| Field staining | 59 | ||
| Giemsa staining | 59 | ||
| Azure B–Eosin Y staining | 59 | ||
| Staining thin films for parasites | 59 | ||
| Leishman stain | 59 | ||
| Method | 59 | ||
| References | 59 | ||
| Chapter 5 Blood Cell Morphology in Health and Disease | 61 | ||
| Examination of blood films | 62 | ||
| Red cell morphology | 62 | ||
| Abnormal erythropoiesis | 63 | ||
| Anisocytosis ( α ν ι σ ο ζ, unequal) and poikilocytosis ( π ο ι κ ι λ ο ζ, varied) | 63 | ||
| Macrocytes | 65 | ||
| Microcytes | 65 | ||
| Basophilic stippling | 66 | ||
| Inadequate haemoglobin formation | 66 | ||
| Hypochromia (Hypochromasia) ( υ π ο ρ, under) | 66 | ||
| Anisochromasia ( α υ ι σ ο ζ, unequal) and dimorphic red cell population | 68 | ||
| Damage to red cells after formation | 68 | ||
| Hyperchromia (Hyperchromasia) ( υ π ε ρ, over) | 68 | ||
| Spherocytosis ( σ φ α ι ρ α, sphere) | 68 | ||
| Irregularly contracted red cells | 69 | ||
| Elliptocytosis and ovalocytosis | 71 | ||
| Spiculated cells and red cell fragmentation | 72 | ||
| Schistocytosis (Fragmentation) ( σ χ ι σ τ ο ζ, cleft) | 72 | ||
| Keratocytes ( κ ε ρ α ζ, horn) | 73 | ||
| Acanthocytosis ( α κ α ν θ α, spine) | 74 | ||
| Echinocytosis ( ε χ ι ν ο ζ, sea-urchin or hedgehog) | 74 | ||
| Miscellaneous erythrocyte abnormalities | 74 | ||
| Leptocytosis ( λ ε π τ ο ζ, thin) | 74 | ||
| Target cells | 75 | ||
| Stomatocytosis ( σ τ ο μ α, mouth) | 75 | ||
| Sickle cells | 76 | ||
| Haemoglobin C crystals and SC poikilocytes | 77 | ||
| Erythrocyte inclusions | 77 | ||
| Howell–Jolly bodies | 77 | ||
| Pappenheimer bodies | 77 | ||
| Rouleaux and autoagglutination | 78 | ||
| Changes associated with a compensatory increase in erythropoiesis | 78 | ||
| Polychromasia ( π ο λ θ ζ, many) | 78 | ||
| Erythroblastaemia | 79 | ||
| Effects of splenectomy and hyposplenism | 80 | ||
| Scanning electron microscopy | 80 | ||
| Morphology of leucocytes | 82 | ||
| Polymorphonuclear neutrophils | 82 | ||
| Granules | 83 | ||
| Vacuoles | 83 | ||
| Bacteria and fungi | 84 | ||
| Other neutrophil inclusions | 84 | ||
| Döhle bodies | 84 | ||
| Nuclei | 85 | ||
| Hypersegmentation | 85 | ||
| Pelger–Huët cells | 86 | ||
| Detached nuclear fragments | 86 | ||
| Pyknotic neutrophils (Apoptosis) | 86 | ||
| Eosinophils | 86 | ||
| Basophils | 87 | ||
| Monocytes | 87 | ||
| Lymphocytes | 87 | ||
| Platelet morphology | 89 | ||
| References | 91 | ||
| Chapter 6 Supplementary Techniques Including Blood Parasite Diagnosis | 93 | ||
| Tests for the acute-phase response | 93 | ||
| Erythrocyte sedimentation rate | 94 | ||
| Conventional Westergren method | 94 | ||
| Method | 94 | ||
| Range in health | 94 | ||
| Modified methods | 95 | ||
| Length of tube | 95 | ||
| Plastic tubes | 95 | ||
| Disposable glass tubes | 95 | ||
| Capillary method | 95 | ||
| Test duration | 95 | ||
| Sloping tube | 95 | ||
| Anticoagulant | 95 | ||
| Evaluation of a new routine method | 95 | ||
| Quality control | 95 | ||
| Semiquantitative slide method | 95 | ||
| Mechanism of erythrocyte sedimentation | 96 | ||
| Plasma viscosity | 97 | ||
| Reference values | 97 | ||
| Whole blood viscosity | 97 | ||
| Heterophile antibodies in serum: diagnosis of infectious mononucleosis | 97 | ||
| Screening tests for infectious mononucleosis | 97 | ||
| Clinical value | 98 | ||
| Demonstration of lupus erythematosus cells | 98 | ||
| Erythropoietin | 98 | ||
| Reference range | 98 | ||
| Significance | 99 | ||
| Autonomous in vitro erythropoiesis | 99 | ||
| Thrombopoietin | 99 | ||
| Haematological tests in sports medicine | 99 | ||
| Red cell parameters | 100 | ||
| Reticulocytes | 100 | ||
| Haemoglobin concentration and haematocrit | 100 | ||
| Whole blood viscosity and erythrocyte sedimentation rate | 100 | ||
| Erythropoietin | 100 | ||
| Principles of detection of microorganisms | 100 | ||
| Examination of blood films for parasites | 100 | ||
| General principles | 100 | ||
| Staining thin films | 101 | ||
| Microscopic diagnosis of malaria | 101 | ||
| Fluorescence microscopy | 101 | ||
| Quantitative buffy coat method | 101 | ||
| P. knowlesi infection | 102 | ||
| Rapid diagnostic tests for malaria | 102 | ||
| Leishmaniasis | 106 | ||
| Diagnosis of leishmaniasis in the haematology laboratory | 108 | ||
| Trypanosomiasis | 108 | ||
| African trypanosomiasis | 108 | ||
| American trypanosomiasis | 108 | ||
| Diagnosis of trypanosomiasis in the haematology laboratory | 108 | ||
| Wet preparations | 108 | ||
| Thick blood films or chancre aspirates | 108 | ||
| Concentration techniques | 109 | ||
| Quantitative buffy coat method | 109 | ||
| Capillary tube method | 109 | ||
| Filariasis and loiasis | 109 | ||
| Diagnosis of filariasis in the haematology laboratory | 110 | ||
| Wet preparation | 110 | ||
| Concentration techniques | 110 | ||
| Filtration method | 110 | ||
| Quantitative buffy coat and microhaematocrit methods | 110 | ||
| Lysed capillary blood | 110 | ||
| Babesiosis | 110 | ||
| Ehrlichiosis and anaplasmosis | 110 | ||
| References | 110 | ||
| Chapter 7 Bone Marrow Biopsy | 112 | ||
| Aspiration of the bone marrow | 113 | ||
| Consent and safety | 113 | ||
| Performing a bone marrow aspiration | 113 | ||
| Puncture of the ilium | 114 | ||
| Puncture of the sternum | 114 | ||
| Comparison of different sites for marrow puncture | 114 | ||
| Aspiration of the bone marrow in children | 114 | ||
| Marrow puncture needles | 115 | ||
| Processing of aspirated bone marrow | 116 | ||
| Preparing films from bone marrow aspirates | 116 | ||
| Concentration of bone marrow by centrifugation | 116 | ||
| Preparation of films of postmortem bone marrow | 117 | ||
| Examination of aspirated bone marrow | 117 | ||
| Principles of marrow aspirate examination | 117 | ||
| Quantitative cell counts on aspirated bone marrow | 118 | ||
| Differential cell counts on aspirated bone marrow | 118 | ||
| Sources of error and physiological variations | 118 | ||
| Cellular ratios | 119 | ||
| Reporting bone marrow aspirate films | 119 | ||
| Systematic scheme for examining bone marrow aspirate films | 119 | ||
| Low power ( × 10) | 119 | ||
| Higher power ( × 40, × 100 oil-immersion) | 119 | ||
| Reporting results | 120 | ||
| Preparation of sections of aspirated bone marrow fragments | 120 | ||
| Percutaneous trephine biopsy of the bone marrow | 120 | ||
| Principles behind marrow trephine biopsy examination | 120 | ||
| Imprints from bone marrow trephine biopsy specimens | 122 | ||
| Processing of bone marrow trephine biopsy specimens | 123 | ||
| Staining of sections of bone marrow trephine biopsy specimens | 123 | ||
| References | 125 | ||
| Chapter 8 Molecular and Cytogenetic Analysis | 126 | ||
| Methodologies | 127 | ||
| DNA extraction | 127 | ||
| DNA extraction kits | 127 | ||
| Polymerase chain reaction | 127 | ||
| Principle | 127 | ||
| Methodology | 128 | ||
| Reagents | 128 | ||
| Taq polymerase and oligonucleotide primers | 128 | ||
| PCR buffers | 128 | ||
| Method | 129 | ||
| Modifications and developments | 129 | ||
| Interpretation | 129 | ||
| Problems | 130 | ||
| Presence or absence of a PCR product and sizing PCR products | 130 | ||
| Amplification refractory mutation system (ARMS) | 130 | ||
| Principle | 130 | ||
| Interpretation | 130 | ||
| Gap-PCR | 130 | ||
| Fusion gene analysis | 131 | ||
| Restriction enzyme digestion | 131 | ||
| Principle | 131 | ||
| Methodology | 131 | ||
| Reagents | 131 | ||
| Method | 132 | ||
| Allele-specific oligonucleotide hybridisation | 132 | ||
| Chapter 9 Iron Deficiency Anaemia and Iron Overload | 165 | ||
| Iron metabolism | 166 | ||
| Dietary iron absorption | 166 | ||
| Dietary and luminal factors | 166 | ||
| Iron absorption at the molecular level | 167 | ||
| Regulation of iron absorption | 167 | ||
| Cellular iron uptake and release | 167 | ||
| Iron storage | 168 | ||
| Regulation of iron metabolism | 168 | ||
| Plasma iron transport | 168 | ||
| Iron status | 168 | ||
| Disorders of iron metabolism | 169 | ||
| Methods for assessing iron status | 169 | ||
| Serum ferritin assay | 169 | ||
| Immunoassay for ferritin | 170 | ||
| Selecting an assay method | 170 | ||
| Interpretation | 170 | ||
| Estimation of serum iron concentration | 173 | ||
| Reagents and materials | 173 | ||
| Preparation of glassware | 173 | ||
| Protein precipitant | 173 | ||
| Chromogen solution (ferrozine) | 173 | ||
| Iron-free water | 173 | ||
| Iron standard 80 μ mol/l | 173 | ||
| Method | 173 | ||
| Calculation | 173 | ||
| Alternative procedure: serum iron without protein precipitation | 174 | ||
| Reagents and materials | 174 | ||
| Iron standards 80 μ mol/l and 40 μ mol/l (see p. 173) | 174 | ||
| Phosphate–ascorbate buffer (stock) | 174 | ||
| Chromogen solution | 174 | ||
| Microtitre trays | 174 | ||
| Control serum | 174 | ||
| Method | 174 | ||
| Calculations | 174 | ||
| Automated methods for serum iron | 174 | ||
| Serum iron concentrations in health and disease | 175 | ||
| Estimation of total iron-binding capacity | 175 | ||
| Principle | 175 | ||
| Reagents | 175 | ||
| Method | 175 | ||
| Determination of unsaturated iron-binding capacity | 175 | ||
| Reagents and materials | 175 | ||
| Saturating solution | 175 | ||
| Tris buffer (stock) | 175 | ||
| Tris–ascorbate–iron buffer | 175 | ||
| Chromogen solution | 176 | ||
| Microtitre trays | 176 | ||
| Control serum | 176 | ||
| Method | 176 | ||
| Calculations | 176 | ||
| Fully automated methods | 176 | ||
| Serum transferrin | 176 | ||
| Normal ranges of transferrin and total iron-binding capacity | 176 | ||
| Transferrin saturation | 177 | ||
| Serum transferrin receptor | 177 | ||
| Assays for the serum transferrin receptor | 178 | ||
| Reference ranges | 178 | ||
| Samples | 178 | ||
| Transferrin receptor concentrations in diagnosis | 178 | ||
| Erythropoiesis | 178 | ||
| Iron deficiency | 178 | ||
| Iron overload | 179 | ||
| Erythrocyte protoporphyrin | 179 | ||
| Analysers | 180 | ||
| Diagnostic applications | 180 | ||
| Units | 180 | ||
| Hepcidin | 180 | ||
| Methodological and biological variability of assays | 180 | ||
| Predictive value of blood tests for iron deficiency | 181 | ||
| Iron deficiency anaemia in adults | 181 | ||
| Detection of iron deficiency in acute or chronic disease | 182 | ||
| Functional iron deficiency | 182 | ||
| Iron deficiency in infancy and childhood | 183 | ||
| Pregnancy | 183 | ||
| Evaluation of suspected iron overload | 183 | ||
| Conclusion | 183 | ||
| References | 184 | ||
| Chapter 10 Investigation of Megaloblastic Anaemia: Cobalamin, Folate and Metabolite Status | 187 | ||
| Cobalamin absorption and metabolism | 188 | ||
| Folate absorption and metabolism | 191 | ||
| Haematological features of megaloblastic anaemia | 193 | ||
| Differential diagnosis of macrocytic anaemia | 194 | ||
| Metabolic insufficiency | 194 | ||
| Testing strategy for suspected cobalamin or folate deficiency | 194 | ||
| Utility of serum vitamin B 12 assays | 194 | ||
| Sensitivity and specificity of cobalamin assays | 198 | ||
| Utility of receiver operator characteristic curves | 198 | ||
| Utility of holotranscobalamin II assay | 198 | ||
| Utility of methylmalonic acid and homocysteine assays | 198 | ||
| Clinical and diagnostic pitfalls of folate assays | 199 | ||
| Standards, accuracy and precision of cobalamin and folate assays | 200 | ||
| Genetic factors | 200 | ||
| Pre-analytical sample preparation | 201 | ||
| Analytical factors | 201 | ||
| Limitations and interference | 201 | ||
| Post-analytical factors | 201 | ||
| Methods for cobalamin and folate analysis | 201 | ||
| General principles of competitive protein-binding assays | 201 | ||
| Serum B 12 assays | 201 | ||
| Release from endogenous binders and conversion of analyte to appropriate form | 201 | ||
| Binding of B 12 to kit binder | 202 | ||
| Separation of bound and unbound B 12 | 202 | ||
| Signal generation | 202 | ||
| Electrochemiluminescence immunoassay | 202 | ||
| Enzyme-linked fluorescence generation | 202 | ||
| Holotranscobalamin assays | 202 | ||
| Principle | 202 | ||
| Holotranscobalamin ‘active B 12' immunoassay | 203 | ||
| Holotranscobalamin radioimmunoassay | 203 | ||
| Quantification of transcobalamin saturation | 203 | ||
| Serum folate methods | 203 | ||
| Release from endogenous binders | 203 | ||
| Binding of folate to folate-binding protein | 203 | ||
| Separation of bound and unbound folate | 204 | ||
| Signal generation | 204 | ||
| Red cell folate methods | 204 | ||
| Manual haemolysate preparation | 204 | ||
| Calculation of red blood cell folate from haemolysate folate result | 204 | ||
| Serum vitamin B 12 and folate and red cell folate assay calibration | 205 | ||
| Whole blood folate standards | 205 | ||
| Primary instrument calibration | 205 | ||
| Internal adjustment calibration | 205 | ||
| Internal quality control | 205 | ||
| Direct measurement of 5-methyltetrahydrofolate in plasma, red cells and cerebrospinal fluid by high performance liquid chro ... | 205 | ||
| Analytical procedure | 206 | ||
| Preparation | 206 | ||
| Extraction | 206 | ||
| High performance liquid chromatography | 206 | ||
| Methylmalonic acid measurement | 206 | ||
| Principle | 206 | ||
| Methods | 206 | ||
| Automated liquid chromatography tandem mass spectrometry with electrospray ionisation-based method | 206 | ||
| Gas chromatography–mass spectrometry-based method | 207 | ||
| High performance liquid chromatography-based method | 207 | ||
| Homocysteine measurement | 207 | ||
| Principle | 207 | ||
| Immunoassay for homocysteine measurement | 207 | ||
| Other enzyme assays for homocysteine | 208 | ||
| Standardisation of homocysteine methods | 208 | ||
| Reference methods for homocysteine | 208 | ||
| Pre-analytical variables in homocysteine testing | 208 | ||
| Dynamic testing of cobalamin folate metabolism | 208 | ||
| Investigation of the cause of cobalamin deficiency | 208 | ||
| Intrinsic factor antibody measurement | 208 | ||
| Principle | 208 | ||
| Intrinsic factor antibody kits | 209 | ||
| Principle of binding assay for type I intrinsic factor antibodies | 209 | ||
| Enzyme-linked immunosorbent assay methods for type I and type II intrinsic factor antibodies | 209 | ||
| Interpretation | 209 | ||
| Investigation of absorption of B 12 | 209 | ||
| Development of non-isotopic B 12 absorption tests using holotranscobalamin levels and recombinant intrinsic factor | 209 | ||
| Non-isotopic B 12 absorption test utilising recombinant intrinsic factor in combination with holotranscobalamin levels | 210 | ||
| Urinary excretion of radiolabelled B 12 with and without intrinsic factor (Schilling test) | 210 | ||
| B 12 Binding capacity of serum or plasma: transcobalamin measurement | 210 | ||
| Principle | 210 | ||
| Unsaturated B 12 binding capacity and transcobalamin identification and quantification | 210 | ||
| Reference ranges for transcobalamins | 210 | ||
| Acknowledgements | 210 | ||
| References | 210 | ||
| Chapter 11 Laboratory Methods Used in the Investigation of the Haemolytic Anaemias | 214 | ||
| Investigation of haemolytic anaemia | 215 | ||
| Is there evidence of increased haemolysis? | 215 | ||
| What is the haemolytic mechanism? | 216 | ||
| What is the precise diagnosis? | 216 | ||
| Plasma haemoglobin | 216 | ||
| Sample collection | 216 | ||
| Peroxidase method 3 | 216 | ||
| Spectrophotometric method | 216 | ||
| Normal range | 217 | ||
| Significance of increased plasma haemoglobin | 217 | ||
| Serum haptoglobin | 217 | ||
| Electrophoretic method 6, 7 | 217 | ||
| Principle | 217 | ||
| Reagents | 217 | ||
| Buffer (pH 7.0, ionic strength 0.05) | 217 | ||
| Haemolysates. | 217 | ||
| Stain. | 218 | ||
| Clearing solution. | 218 | ||
| Acetic acid rinse. | 218 | ||
| Method | 218 | ||
| Interpretation. | 218 | ||
| Radial immunodiffusion method | 218 | ||
| Principle | 218 | ||
| Reagents | 218 | ||
| Single diffusion plates * . | 219 | ||
| Reference sera. | 219 | ||
| Test serum. | 219 | ||
| Method | 219 | ||
| Normal ranges | 219 | ||
| Significance | 219 | ||
| Serum haemopexin | 220 | ||
| Examination of plasma (or serum) for methaemalbumin | 220 | ||
| Schumm test | 220 | ||
| Method | 220 | ||
| Significance of methaemalbuminaemia | 220 | ||
| Quantitative estimation by spectrometry | 220 | ||
| Calibration graph | 220 | ||
| Demonstration of haemosiderin in urine | 221 | ||
| Method | 221 | ||
| Significance of haemosiderinuria | 221 | ||
| Chemical tests of haemoglobin catabolism | 221 | ||
| Serum bilirubin | 221 | ||
| Urobilin and urobilinogen | 222 | ||
| Qualitative test for urobilinogen and urobilin in urine | 222 | ||
| Schlesinger zinc test | 222 | ||
| Porphyrins | 222 | ||
| Demonstration of porphobilinogen in urine | 223 | ||
| Chapter 12 Investigation of the Hereditary Haemolytic Anaemias: Membrane and Enzyme Abnormalities | 228 | ||
| Investigation of membrane defects | 229 | ||
| Osmotic fragility as measured by lysis in hypotonic saline | 229 | ||
| Principle | 229 | ||
| Reagents | 229 | ||
| Method | 229 | ||
| Notes | 229 | ||
| Osmotic fragility after incubating the blood at 37 °C for 24 H | 230 | ||
| Method | 230 | ||
| Factors affecting osmotic fragility tests | 231 | ||
| Recording the results of osmotic fragility tests | 231 | ||
| Alternative methods of recording osmotic fragility. | 231 | ||
| Interpretation of results | 232 | ||
| Flow cytometric (dye-binding) test | 233 | ||
| Principle | 233 | ||
| Reagents | 233 | ||
| Eosin-5-maleimide (EMA) | 233 | ||
| Bovine serum albumin (30%) solution (BSA) | 233 | ||
| Phosphate buffered saline | 233 | ||
| Method | 233 | ||
| Interpretation of results | 234 | ||
| Glycerol lysis-time tests | 234 | ||
| Acidified glycerol lysis-time test | 234 | ||
| Principle | 234 | ||
| Reagents | 234 | ||
| Phosphate buffered saline | 234 | ||
| Glycerol reagent | 234 | ||
| Method | 234 | ||
| Results | 234 | ||
| Significance of the acidified glycerol lysis-time test | 234 | ||
| Cryohaemolysis test | 235 | ||
| Chapter 13 Acquired Haemolytic Anaemias | 254 | ||
| Assessing the likelihood of acquired haemolytic anaemia | 254 | ||
| Assessment of the blood film and count in suspected acquired haemolytic anaemia | 254 | ||
| Immune haemolytic anaemias | 255 | ||
| Types of autoantibody | 256 | ||
| Warm autoantibodies | 256 | ||
| Cold autoantibodies | 257 | ||
| Combined warm and cold autoantibodies | 257 | ||
| Methods of investigation | 258 | ||
| Collection of samples of blood and serum | 258 | ||
| Storage of samples | 258 | ||
| Scheme for serological investigation of haemolytic anaemia suspected to be of immunological origin | 258 | ||
| Detection of incomplete antibodies by means of the direct antiglobulin (coombs) test | 260 | ||
| Principle. | 260 | ||
| Precautions. | 260 | ||
| Method. | 260 | ||
| Direct antiglobulin test using column agglutination technology | 260 | ||
| Significance of positive direct antiglobulin test | 260 | ||
| Positive direct antiglobulin tests in normal subjects | 261 | ||
| Positive direct antiglobulin tests in hospital patients | 261 | ||
| False-negative antiglobulin test results | 261 | ||
| Direct antiglobulin test-negative autoimmune haemolytic anaemia | 261 | ||
| Manual direct polybrene test | 262 | ||
| Reagents | 262 | ||
| Method. | 262 | ||
| Positive control. | 262 | ||
| Negative control. | 262 | ||
| Determination of the blood group of a patient with autoimmune haemolytic anaemia | 262 | ||
| ABO grouping | 262 | ||
| RhD grouping | 263 | ||
| Demonstration of free antibodies in serum | 263 | ||
| Identification by adsorption techniques of coexisting alloantibodies in the presence of warm autoantibodies | 263 | ||
| Use of ZZAP reagent in autoadsorption techniques | 263 | ||
| Reagents | 263 | ||
| Method | 263 | ||
| Notes. | 263 | ||
| Alloadsorption using papainised R 1 R 1, R 2 R 2 and rr cells | 263 | ||
| Method for testing alloadsorbed sera | 264 | ||
| Example of alloantibody detection using the alloadsorption technique in a recently transfused patient with AIHA. | 264 | ||
| Explanation of the results of testing alloadsorbed sera, A, B and C | 264 | ||
| Additional notes on adsorption techniques | 264 | ||
| Preparing and testing a concentrated eluate | 264 | ||
| Elution of antibodies from red cells | 265 | ||
| Notes | 265 | ||
| Heat elution | 265 | ||
| Freeze and thaw elution (Lui) | 265 | ||
| Screening eluates | 265 | ||
| Determination of the specificity of warm autoantibodies in eluates and sera | 265 | ||
| Titration of warm antibodies in eluates or sera | 265 | ||
| Determination of the specificity of cold autoantibodies | 265 | ||
| Titration of cold antibodies | 266 | ||
| Normal range. | 266 | ||
| Cold agglutinin titration patterns | 266 | ||
| Determination of the thermal range of cold agglutinins | 266 | ||
| Detection and titration of the Donath–Landsteiner antibody | 266 | ||
| Direct Donath–Landsteiner test | 267 | ||
| Indirect Donath–Landsteiner test | 267 | ||
| Two-stage indirect Donath–Landsteiner test | 267 | ||
| Titration of a Donath–Landsteiner antibody | 267 | ||
| Detection of a Donath–Landsteiner antibody by the indirect antiglobulin test | 267 | ||
| Method. | 267 | ||
| Thermal range of Donath–Landsteiner antibody. | 268 | ||
| Specificity of the Donath–Landsteiner antibody. | 268 | ||
| Treatment of serum with 2-mercaptoethanol or dithiothreitol | 268 | ||
| Method | 268 | ||
| 2-mercaptoethanol. | 268 | ||
| Dithiothreitol. | 268 | ||
| Drug-induced haemolytic anaemias of immunological origin | 268 | ||
| Drug-induced autoimmune haemolytic anaemias | 269 | ||
| Detection of antipenicillin antibodies | 269 | ||
| Reagents | 269 | ||
| Penicillin-coated normal red cells | 269 | ||
| Control normal red cells. | 269 | ||
| Method. | 270 | ||
| Note. | 270 | ||
| Detection of antibodies against drugs other than penicillin | 270 | ||
| Interpretation | 270 | ||
| Oxidant-induced haemolytic anaemia | 270 | ||
| Microangiopathic and mechanical haemolytic anaemias | 271 | ||
| Paroxysmal nocturnal haemoglobinuria | 271 | ||
| Acidified-serum lysis test (Ham test) | 272 | ||
| Principle | 272 | ||
| Chapter 14 Investigation of Variant Haemoglobins and Thalassaemias | 282 | ||
| The haemoglobin molecule | 283 | ||
| Structural variants of haemoglobin | 284 | ||
| Haemoglobins with reduced solubility | 284 | ||
| Haemoglobin S | 284 | ||
| Sickle cell disease | 284 | ||
| Haemoglobin C | 285 | ||
| Other sickling haemoglobins | 285 | ||
| Unstable haemoglobins | 285 | ||
| Haemoglobins with altered oxygen affinity | 285 | ||
| Haemoglobin M | 285 | ||
| Thalassaemia syndromes | 286 | ||
| β thalassaemia syndromes | 286 | ||
| α thalassaemia syndromes | 287 | ||
| Thalassaemic structural variants | 288 | ||
| Increased haemoglobin F in adult life | 288 | ||
| Inherited abnormalities that increase haemoglobin F concentration | 288 | ||
| Investigation of patients with a suspected haemoglobinopathy | 289 | ||
| Laboratory detection of haemoglobin variants | 289 | ||
| Blood count and film | 290 | ||
| Collection of blood and preparation of haemolysates | 291 | ||
| Preparation of haemolysate for qualitative haemoglobin electrophoresis | 291 | ||
| Preparation of haemolysate for the quantification of haemoglobins and stability tests | 291 | ||
| Control samples | 291 | ||
| Quality assurance | 291 | ||
| Cellulose acetate electrophoresis at alkaline pH | 292 | ||
| Principle | 292 | ||
| Equipment | 292 | ||
| Reagents | 292 | ||
| Method | 292 | ||
| Interpretation and comments | 292 | ||
| Agarose gel electrophoresis | 294 | ||
| Reagents and method | 294 | ||
| Interpretation | 294 | ||
| Automated high performance liquid chromatography | 294 | ||
| Principle | 294 | ||
| Method | 294 | ||
| Interpretation and comments | 295 | ||
| Capillary electrophoresis | 296 | ||
| Principle | 296 | ||
| Chapter 15 Erythrocyte and Leucocyte Cytochemistry | 312 | ||
| Erythrocyte cytochemistry | 312 | ||
| Siderocytes and sideroblasts | 312 | ||
| Method for staining siderotic granules | 313 | ||
| Significance of siderocytes | 313 | ||
| Haemoglobin derivatives | 315 | ||
| Heinz bodies in red cells | 315 | ||
| Demonstration of Heinz bodies | 316 | ||
| Unstained preparations | 316 | ||
| Stained preparations | 316 | ||
| Carboxyhaemoglobin and methaemoglobin | 316 | ||
| Fetal haemoglobin | 316 | ||
| Reagents | 316 | ||
| Method | 316 | ||
| Haemoglobin S and other haemoglobin variants | 317 | ||
| Leucocyte cytochemistry | 317 | ||
| Myeloperoxidase | 318 | ||
| Method with 3,3 ′ -diaminobenzidine | 318 | ||
| Reagents | 318 | ||
| Method | 318 | ||
| Technical considerations. | 318 | ||
| Results and interpretation. | 318 | ||
| Pathological variations. | 319 | ||
| Sudan Black B | 319 | ||
| Reagents | 319 | ||
| Method | 319 | ||
| Technical considerations | 319 | ||
| Results and interpretation | 319 | ||
| Neutrophil alkaline phosphatase | 319 | ||
| Reagents | 320 | ||
| Method | 320 | ||
| Technical considerations | 320 | ||
| Results and interpretation | 320 | ||
| Acid phosphatase reaction, including tartrate-resistant acid phosphatase reaction | 321 | ||
| Reagents | 321 | ||
| Method | 321 | ||
| Results and interpretation | 321 | ||
| Periodic acid–Schiff reaction | 321 | ||
| Reagents | 322 | ||
| Method | 322 | ||
| Technical considerations | 322 | ||
| Results and interpretation | 322 | ||
| Esterases | 323 | ||
| Naphthol AS-D chloroacetate esterase | 323 | ||
| Reagents | 323 | ||
| Method | 324 | ||
| Technical considerations | 324 | ||
| Results and interpretation | 324 | ||
| α -naphthyl butyrate esterase | 325 | ||
| Chapter 16 Immunophenotyping by Flow Cytometry | 330 | ||
| Principles of flow cytometric immunophenotyping | 330 | ||
| Multicolour flow cytometric immunophenotyping | 331 | ||
| Methods | 332 | ||
| Sample preparation | 332 | ||
| Detection of membrane antigens | 333 | ||
| Detection of surface immunoglobulin | 334 | ||
| Method 1: wash–stain–lyse–wash | 334 | ||
| Method 2: lyse–stain–wash | 334 | ||
| Detection of intracellular antigens | 335 | ||
| Method | 336 | ||
| Simultaneous detection of intracellular and membrane antigens | 336 | ||
| Acquisition and data analysis | 336 | ||
| Immunological markers in acute leukaemia | 336 | ||
| Multiparameter flow cytometry in acute lymphoblastic leukaemia | 337 | ||
| Multiparameter flow cytometry in acute myeloid leukaemias | 338 | ||
| Multiparameter flow cytometry in mixed phenotype acute leukaemia | 341 | ||
| Immunological markers in chronic lymphoproliferative disorders | 342 | ||
| Panels of monoclonal antibodies for screening, classification and diagnosis | 342 | ||
| Secondary B-cell panel and phenotypic profiles | 342 | ||
| Secondary T-cell and NK-cell panel and phenotypic profiles | 344 | ||
| Secondary plasma cell panel | 344 | ||
| Minimal residual disease detection by flow cytometry | 345 | ||
| HIV monitoring | 346 | ||
| Antibody panels | 346 | ||
| Method | 346 | ||
| Data analysis | 347 | ||
| Common pitfalls | 348 | ||
| Acknowledgement | 348 | ||
| References | 348 | ||
| Chapter 17 Diagnostic Radioisotopes in Haematology | 350 | ||
| Sources of radioisotopes | 352 | ||
| Radiation protection | 352 | ||
| Apparatus for measuring radioactivity in vitro | 353 | ||
| Apparatus for measuring radioactivity in vivo | 353 | ||
| Surface counting | 353 | ||
| Imaging | 353 | ||
| Measurement of radioactivity with a scintillation counter | 353 | ||
| Standardisation of working conditions | 353 | ||
| Counting technique | 353 | ||
| Measurement of radioactivity. | 353 | ||
| Correction for physical decay | 353 | ||
| Double radioisotope measurements | 353 | ||
| Differential decay. | 353 | ||
| Physical separation. | 354 | ||
| Blood volume | 354 | ||
| Measurement of blood volume | 355 | ||
| Principle | 355 | ||
| Red cell volume | 355 | ||
| Radioactive chromium method | 355 | ||
| Technetium method | 356 | ||
| Chapter 18 Investigation of Haemostasis | 366 | ||
| Components of normal haemostasis | 367 | ||
| The blood vessel | 367 | ||
| General structure of the blood vessel | 367 | ||
| Endothelial cell function | 367 | ||
| Vasoconstriction | 368 | ||
| Platelets | 368 | ||
| Platelet function in the haemostatic process | 369 | ||
| Platelet aggregation | 369 | ||
| Blood coagulation | 369 | ||
| The contact activation system | 371 | ||
| Tissue factor | 371 | ||
| Cofactors | 371 | ||
| Fibrinogen | 371 | ||
| Factor XIII | 372 | ||
| Inhibitors of coagulation | 372 | ||
| The fibrinolytic system | 372 | ||
| General approach to investigation of haemostasis | 372 | ||
| Clinical approach | 373 | ||
| Principles of laboratory analysis | 373 | ||
| Immunological | 373 | ||
| Assays using chromogenic peptide substrates (amidolytic assays) | 373 | ||
| Coagulation assays | 374 | ||
| Fluorescence resonance energy transfer | 374 | ||
| Other assays | 374 | ||
| Notes on equipment | 374 | ||
| Water baths | 374 | ||
| Refrigerators and freezers | 374 | ||
| Centrifuges | 374 | ||
| Reagents and buffers | 374 | ||
| Plastic and glass tubes | 374 | ||
| Pipettes | 374 | ||
| Stopwatches and clocks | 374 | ||
| Automated coagulation analysers | 375 | ||
| Pre-analytical variables including sample collection | 375 | ||
| Collection of venous blood | 375 | ||
| Blood sample anticoagulation | 375 | ||
| Time of sample collection | 375 | ||
| Transportation to the laboratory | 376 | ||
| Centrifugation: preparation of platelet-poor plasma | 376 | ||
| Storage of plasma and sample thawing | 376 | ||
| Some common ‘technical’ errors | 376 | ||
| Calibration and quality control | 376 | ||
| Reference standard (calibrator) | 376 | ||
| Calibration of standard pools and suggested calibration procedure | 376 | ||
| Control plasma | 377 | ||
| Variability of coagulation assays | 377 | ||
| Performance of coagulation tests | 377 | ||
| Handling of samples and reagents | 377 | ||
| Eliminating a time trend | 377 | ||
| Assay monitoring and end-point detection | 377 | ||
| Manual methods | 377 | ||
| Electromechanical | 378 | ||
| Impedance, steel ball, rotating cuvette | 378 | ||
| Impedance, steel ball, rotating steel ball | 378 | ||
| Photo-optical analysis | 378 | ||
| Scattered light detection for clotting assays | 378 | ||
| Transmitted light detection for chromogenic assays (405 nm, 575 nm, 800 nm) | 378 | ||
| Transmitted light detection for immunoassays (405 nm, 575 nm, 800 nm) | 378 | ||
| Nephelometry (IL ACL analysers) | 378 | ||
| Photo-optical end point determination and analyses | 378 | ||
| Percentage detection method | 378 | ||
| Rate method | 378 | ||
| VLin integral method | 379 | ||
| Analysis time over | 379 | ||
| Turbidity level over | 379 | ||
| Clot signatures: normal and abnormal activated partial thromboplastin time clot waveforms | 380 | ||
| Commonly used reagents | 380 | ||
| CaCl 2 | 380 | ||
| Barbitone buffer | 380 | ||
| Barbitone buffered saline, pH 7.4 | 380 | ||
| Glyoxaline buffer. | 380 | ||
| Factor-deficient plasmas | 380 | ||
| The ‘clotting screen’ | 380 | ||
| Prothrombin time | 380 | ||
| Principle | 380 | ||
| Chapter 19 Investigation of a Thrombotic Tendency | 410 | ||
| Introduction to thrombophilia | 410 | ||
| Pre-analytical factors | 411 | ||
| Tests for the presence of a lupus anticoagulant | 411 | ||
| Sample preparation | 411 | ||
| Dilute Russell’s viper venom time | 412 | ||
| Principle | 412 | ||
| Reagents | 412 | ||
| Reagent preparation | 412 | ||
| Method | 412 | ||
| Interpretation | 412 | ||
| Platelet neutralisation test | 413 | ||
| Principle | 413 | ||
| Reagents for preparation of platelet neutralisation reagent | 413 | ||
| Method | 413 | ||
| Interpretation | 413 | ||
| Interpretation of tests for lupus anticoagulant | 413 | ||
| Kaolin clotting time | 414 | ||
| Chapter 20 Laboratory Control of Anticoagulant, Thrombolytic and Antiplatelet Therapy | 425 | ||
| Oral anticoagulant treatment using vitamin K antagonists | 426 | ||
| Selection of patients | 426 | ||
| Methods used for the laboratory control of oral anticoagulant treatment | 426 | ||
| Standardisation of oral anticoagulant treatment | 426 | ||
| Calibration of thromboplastins | 426 | ||
| Principle | 426 | ||
| Reagents | 427 | ||
| Method | 427 | ||
| Calibration | 427 | ||
| Calculation of international sensitivity index | 427 | ||
| Local calibration of thromboplastins and direct INR determination | 428 | ||
| Geometric mean normal prothrombin time | 428 | ||
| Calibration audits | 428 | ||
| Determination of the international normalised ratio | 428 | ||
| Capillary reagents | 428 | ||
| Point-of-care testing | 429 | ||
| Therapeutic range and choice of thromboplastin | 429 | ||
| Management of overanticoagulation | 429 | ||
| Heparin treatment | 429 | ||
| Selection of patients | 430 | ||
| Laboratory control of heparin treatment 13 | 430 | ||
| Activated partial thromboplastin time for heparin monitoring | 431 | ||
| Principle | 431 | ||
| Reagents and method | 431 | ||
| Therapeutic range | 431 | ||
| Near-patient heparin monitoring | 432 | ||
| Chapter 21 Blood Cell Antigens and Antibodies: Erythrocytes, Platelets and Neutrophils | 439 | ||
| Erythrocytes | 439 | ||
| Red cell antigens | 439 | ||
| ABO system | 441 | ||
| ABO antigens and encoding genes. | 441 | ||
| Secretors and non-secretors. | 443 | ||
| ABO antigens and disease. | 443 | ||
| ABO antibodies | 443 | ||
| Anti-A and Anti-B. | 443 | ||
| Anti-A 1 and anti-H. | 443 | ||
| Lewis system | 444 | ||
| Lewis antigens and encoding genes. | 444 | ||
| Lewis antibodies. | 444 | ||
| The P system and globoside collection | 444 | ||
| Antigens. | 444 | ||
| Antibodies. | 444 | ||
| Rh system | 444 | ||
| Rh antigens and encoding genes. | 444 | ||
| Antibodies. | 445 | ||
| Kell and Kx systems | 446 | ||
| Antigens and encoding genes. | 446 | ||
| Kell antibodies. | 446 | ||
| Duffy system | 446 | ||
| Duffy antigens and encoding genes. | 446 | ||
| Duffy antibodies | 446 | ||
| Kidd (JK) system | 446 | ||
| Kidd antigens and encoding genes. | 446 | ||
| Kidd antibodies. | 446 | ||
| MNSs system | 446 | ||
| MNSs antigens and encoding genes. | 446 | ||
| MNSs antibodies. | 447 | ||
| Other blood group systems | 447 | ||
| Lutheran system. | 447 | ||
| Yt (Cartwright) system. | 447 | ||
| Colton system. | 447 | ||
| Dombrock system. | 447 | ||
| Clinical significance of red cell alloantibodies | 447 | ||
| Mechanisms of immune destruction of red cells | 448 | ||
| Antigen–antibody reactions | 450 | ||
| General points of serological technique | 450 | ||
| Serum versus plasma | 450 | ||
| Red cell suspensions | 450 | ||
| Normal ionic strength saline. | 450 | ||
| Low ionic strength saline. | 450 | ||
| Reagent red cells | 451 | ||
| Use of enzyme-treated cells | 451 | ||
| Agglutination of red cells by antibody: a basic method | 451 | ||
| Tube tests. | 451 | ||
| Tubes. | 451 | ||
| Temperature and time of exposure of red cells to antibody | 451 | ||
| Slide tests | 451 | ||
| Reading results of tube tests | 451 | ||
| Microscopic reading. | 451 | ||
| Macroscopic reading | 452 | ||
| Demonstration of lysis | 452 | ||
| Controls. | 452 | ||
| Antiglobulin test | 453 | ||
| Antiglobulin reagents | 453 | ||
| Polyspecific (broad-spectrum) reagents. | 453 | ||
| Monospecific reagents. | 453 | ||
| Quality control of antiglobulin reagents | 453 | ||
| Recommended antiglobulin test procedure | 454 | ||
| Alternative technology for antibody detection by the antiglobulin test | 455 | ||
| Assessment of individual worker performance | 455 | ||
| Titration of antibodies | 456 | ||
| Preparation of serial dilutions of patient’s or other sera | 456 | ||
| Addition of red cell suspensions to dilutions of serum. | 456 | ||
| ABO titration for renal transplant recipients. | 456 | ||
| Test for ABH substance secretion | 457 | ||
| Red cell genotyping | 457 | ||
| Platelet and neutrophils | 457 | ||
| Platelet and neutrophil alloantigen systems | 457 | ||
| Clinical significance of platelet and neutrophil antibodies | 459 | ||
| Alloantibodies | 459 | ||
| Isoantibodies | 459 | ||
| Autoantibodies | 460 | ||
| Drug-induced antibodies | 460 | ||
| Demonstration of platelet and neutrophil antibodies | 460 | ||
| Alloantibodies | 460 | ||
| Autoantibodies | 461 | ||
| Drug-induced antibodies | 461 | ||
| Methods of demonstrating antibodies | 462 | ||
| The immunofluorescent antiglobulin methods | 462 | ||
| Patient’s and screening panel cells | 462 | ||
| Patient’s serum | 462 | ||
| Control sera | 462 | ||
| Eluate from patient’s sensitised cells | 462 | ||
| Heat eluate | 462 | ||
| Platelet preparation | 462 | ||
| Granulocyte preparation | 463 | ||
| Platelet and granulocyte immunofluorescence tests | 463 | ||
| Indirect test | 463 | ||
| Scoring results | 464 | ||
| Use of flow cytometry | 464 | ||
| Chloroquine treatment of platelets and granulocytes | 465 | ||
| Interpretation of results with chloroquine-treated cells | 465 | ||
| MAIPA assay | 465 | ||
| Other methods | 466 | ||
| Molecular genotyping of platelet alloantigens | 466 | ||
| References | 467 | ||
| Chapter 22 Laboratory Aspects of Blood Transfusion | 470 | ||
| Technology and automation in blood transfusion laboratories | 472 | ||
| Pretransfusion compatibility systems | 473 | ||
| Documentation of the transfusion process | 474 | ||
| Blood samples and their storage requirements | 474 | ||
| quality assurance in the transfusion laboratory | 475 | ||
| ABO and RhD grouping | 475 | ||
| ABO grouping | 475 | ||
| Reagents for ABO grouping | 476 | ||
| D grouping | 476 | ||
| Reagents for D grouping | 476 | ||
| Methods | 476 | ||
| Tube and slide tests | 476 | ||
| Slide method | 476 | ||
| Liquid-phase microplate methods | 476 | ||
| Column agglutination techniques | 477 | ||
| Solid-phase techniques | 477 | ||
| Controls for ABO and D grouping | 477 | ||
| Causes of discrepancies in ABO/D grouping | 477 | ||
| False-positive reactions | 477 | ||
| Rouleaux | 477 | ||
| Cold autoagglutination and cold reacting alloantibodies | 477 | ||
| T-activation/polyagglutination | 477 | ||
| Acquired B | 477 | ||
| Potentiators | 477 | ||
| In vitro bacterial contamination | 478 | ||
| False-negative reactions | 478 | ||
| Failure to add reagents | 478 | ||
| Loss of potency | 478 | ||
| Failure to identify lysis | 478 | ||
| Mixed-field appearance | 478 | ||
| D variant phenotypes | 478 | ||
| Antibody screening | 479 | ||
| Red cell reagents | 480 | ||
| Methods | 480 | ||
| Indirect antiglobulin techniques | 480 | ||
| Column agglutination | 480 | ||
| Solid-phase systems | 481 | ||
| Liquid-phase techniques – tubes and microplates | 481 | ||
| Controls for antibody screening | 481 | ||
| Antibody identification | 481 | ||
| Principles | 481 | ||
| Phenotyping | 482 | ||
| Additional panels/techniques | 482 | ||
| Reagents | 482 | ||
| Antibody cards | 482 | ||
| Selection and transfusion of red cells | 482 | ||
| Crossmatching | 483 | ||
| Indirect antiglobulin crossmatch | 483 | ||
| Saline spin crossmatch | 484 | ||
| False-negative results in the saline spin crossmatch | 484 | ||
| False-positive results in the saline spin crossmatch | 484 | ||
| Electronic issue | 484 | ||
| Emergency blood issue | 485 | ||
| Rapid ABO and D typing | 485 | ||
| Confirmation | 485 | ||
| Selection of units | 485 | ||
| Compatibility testing | 485 | ||
| Antibody screening | 485 | ||
| Major or massive haemorrhage | 485 | ||
| Blood components and tranexamic acid in major haemorrhage | 486 | ||
| Compatibility testing in special transfusion situations | 486 | ||
| Neonates and infants in first 4 months of life 52 | 486 | ||
| Investigations on the maternal sample | 486 | ||
| Investigations on the infant sample | 486 | ||
| Direct antiglobulin test | 486 | ||
| Selection of blood and other components | 486 | ||
| Intrauterine (fetal) transfusion | 487 | ||
| Patients receiving transfusions at short intervals | 487 | ||
| Chronic transfusion programmes | 487 | ||
| Allogeneic haemopoietic stem cell transplantation | 488 | ||
| Investigation of a transfusion reaction | 488 | ||
| Acute transfusion reactions | 488 | ||
| Acute intravascular haemolysis | 489 | ||
| Documentation check | 489 | ||
| Serological investigations | 490 | ||
| Tests for haemolysis | 490 | ||
| Microbiological tests | 490 | ||
| Delayed haemolytic transfusion reaction | 490 | ||
| Haematological investigation | 490 | ||
| Serological investigation | 490 | ||
| Antenatal serology and haemolytic disease of the FETUS AND newborn | 490 | ||
| Haemolytic disease of the fetus and newborn | 491 | ||
| Antenatal serology | 491 | ||
| ABO and D grouping and antibody screening | 491 | ||
| Antibody titration and quantitation | 491 | ||
| Antibody quantitation | 491 | ||
| Follow-up antibody screening | 491 | ||
| Prediction of fetal blood group | 491 | ||
| Partner testing | 491 | ||
| Testing fetal DNA in the maternal circulation | 492 | ||
| Fetal blood sampling | 492 | ||
| Antenatal assessment of the severity of haemolytic disease of the fetus and newborn | 492 | ||
| Assessment of fetal anaemia | 492 | ||
| Tests on maternal and cord blood at delivery | 492 | ||
| Anti-D immunoglobulin prophylaxis | 492 | ||
| Anti-D prophylaxis | 492 | ||
| Measurement of fetomaternal haemorrhage | 493 | ||
| Recommended action at delivery (or potentially sensitising event) | 493 | ||
| ABO haemolytic disease of the newborn | 494 | ||
| ABO titrations | 494 | ||
| References | 494 | ||
| Chapter 23 Approach to the Diagnosis and Classification of Blood Cell Disorders | 497 | ||
| Common presentations of haematological diseases | 497 | ||
| Initial screening tests | 497 | ||
| Interpretation of screening tests | 497 | ||
| Quantitative abnormalities of blood cells | 498 | ||
| Increased numbers of cells | 498 | ||
| Increases affecting more than one cell line | 498 | ||
| Erythrocytosis | 498 | ||
| Leucocytosis | 498 | ||
| Neutrophilia | 498 | ||
| Lymphocytosis | 498 | ||
| Monocytosis | 499 | ||
| Eosinophilia | 499 | ||
| Basophilia | 499 | ||
| Thrombocytosis | 499 | ||
| Reduced numbers of cells | 499 | ||
| Reductions in more than one cell line | 499 | ||
| Anaemia | 499 | ||
| Microcytic anaemia | 500 | ||
| Macrocytic anaemia | 501 | ||
| Normocytic anaemia | 502 | ||
| Leucopenia | 502 | ||
| Neutropenia | 502 | ||
| Reduced numbers of lymphocytes, monocytes, eosinophils and basophils | 502 | ||
| Thrombocytopenia | 502 | ||
| Pancytopenia | 502 | ||
| Qualitative abnormalities of blood cells | 502 | ||
| Abnormalities of all cell lines | 502 | ||
| Abnormalities of individual cell lines | 503 | ||
| Red cells | 503 | ||
| White cells | 503 | ||
| Platelets | 503 | ||
| Specific tests for common haematological disorders | 503 | ||
| Red cell disorders | 503 | ||
| Microcytic hypochromic anaemias | 503 | ||
| Macrocytic anaemias | 503 | ||
| Aplastic anaemia 10 | 504 | ||
| Haemolytic anaemias | 504 | ||
| White cell disorders | 504 | ||
| Acute leukaemia | 504 | ||
| Neutropenia | 504 | ||
| Chronic myelogenous leukaemia | 504 | ||
| Chronic lymphoproliferative disorders and/or lymphadenopathy | 504 | ||
| Myelomatosis (plasma cell myeloma) 12 | 504 | ||
| Other disorders | 505 | ||
| Myeloproliferative neoplasms | 505 | ||
| Myelodysplastic syndromes | 505 | ||
| Pancytopenia with splenomegaly | 505 | ||
| Classification of haematological neoplasms | 505 | ||
| Classification of acute myeloid leukaemia and related neoplasms | 505 | ||
| Classification of the myelodysplastic syndromes | 506 | ||
| Classification of acute lymphoblastic leukaemia | 507 | ||
| Classification of myeloproliferative neoplasms and related conditions | 508 | ||
| References | 509 | ||
| Chapter 24 Laboratory Organisation, Management and Safety | 511 | ||
| Management structure and function | 512 | ||
| Staff appraisal | 512 | ||
| Continuing professional development | 513 | ||
| Strategic and business planning | 513 | ||
| Workload assessment and costing of tests | 513 | ||
| Financial control | 514 | ||
| Calculation of test costs | 514 | ||
| Test reliability | 515 | ||
| Test selection | 516 | ||
| Likelihood ratio | 516 | ||
| Receiver–operator characteristic analysis | 516 | ||
| Test utility | 516 | ||
| Instrumentation | 517 | ||
| Equipment evaluation | 517 | ||
| Principles of evaluation | 517 | ||
| Precision | 517 | ||
| Linearity | 518 | ||
| Carryover | 518 | ||
| Accuracy and comparability | 518 | ||
| Maintenance logs | 519 | ||
| Data processing | 519 | ||
| Laboratory computers | 519 | ||
| Pre-analytical and postanalytical stages of testing | 519 | ||
| Test requesting | 520 | ||
| Specimen collection and delivery | 520 | ||
| Pre-analytical phase | 521 | ||
| Postanalytical phase | 521 | ||
| Test turnaround time | 522 | ||
| Point-of-care testing | 522 | ||
| Point-of-care testing beyond the laboratory | 523 | ||
| Patient self-testing | 523 | ||
| Laboratory services for general practitioners | 523 | ||
| Pre-analytical service | 523 | ||
| Postanalytical service | 523 | ||
| Standard operating procedures | 523 | ||
| Laboratory audit and accreditation | 523 | ||
| Audit | 523 | ||
| Accreditation | 525 | ||
| International standards of practice | 525 | ||
| Benchmarking | 526 | ||
| Laboratory safety | 527 | ||
| Principles of safety policy | 527 | ||
| Design of laboratory | 528 | ||
| Electrical and radiation safety | 528 | ||
| Fire hazard | 528 | ||
| Chemical safety | 528 | ||
| Eyewash facilities | 528 | ||
| Biohazardous specimens | 528 | ||
| Universal precautions | 529 | ||
| Disinfectants | 529 | ||
| Sodium hypochlorite (chlorine) | 529 | ||
| Alcohols | 529 | ||
| Applications of disinfectants | 530 | ||
| Automated equipment | 530 | ||
| Centrifuges | 530 | ||
| Syringes and needles | 530 | ||
| Gloves | 530 | ||
| Laundry | 530 | ||
| Waste disposal | 531 | ||
| Specimen shipping | 531 | ||
| Acknowledgement | 531 | ||
| References | 532 | ||
| Chapter 25 Quality Assurance | 533 | ||
| Standardisation | 533 | ||
| Reference preparations and control materials | 535 | ||
| Haemoglobin reference preparations | 535 | ||
| Quality control preparations | 536 | ||
| Quality assurance procedures | 536 | ||
| Internal quality control procedures | 537 | ||
| Control charts | 537 | ||
| Duplicate tests on patients’ specimens | 538 | ||
| Use of patient data for internal quality control | 538 | ||
| Correlation check | 539 | ||
| External quality assessment procedures | 539 | ||
| Analysis of external quality assessment data | 540 | ||
| Target values | 540 | ||
| Quantitative tests | 541 | ||
| Bias | 541 | ||
| Deviation index | 541 | ||
| Consecutive performance assessment | 541 | ||
| Out of consensus method | 542 | ||
| Youden ( xy) plot | 542 | ||
| Methodology check | 542 | ||
| Clinical significance in performance assessment | 542 | ||
| Semiquantitative tests | 542 | ||
| Qualitative tests | 543 | ||
| Interpretation of results | 543 | ||
| Preparation of extended-life material for use in quality assessment | 543 | ||
| Preparation of preserved whole blood | 543 | ||
| Method 2 | 543 | ||
| Preparation of haemolysate | 543 | ||
| Method | 543 | ||
| Preparation of stabilised whole blood control material | 544 | ||
| Method 16 | 544 | ||
| Simple method for producing quality control preparations for individual types of blood cell material | 544 | ||
| Chapter 26 Haematology in Under-Resourced Laboratories | 546 | ||
| Introduction: types of laboratories | 547 | ||
| Organisation of clinical laboratory services | 547 | ||
| Level 1: primary and subdistrict laboratories | 548 | ||
| Level 2: intermediate (district) level laboratories | 548 | ||
| Level 3: regional and provincial hospitals | 548 | ||
| Level 4: national referral and teaching hospitals | 548 | ||
| Availability of HAEMATOLOGY tests at each level | 548 | ||
| Level 1 | 548 | ||
| Level 2 | 548 | ||
| Levels 3 and 4 | 549 | ||
| Microscopes | 549 | ||
| Care of the microscope | 549 | ||
| Essential haematology tests | 549 | ||
| Cost per test | 550 | ||
| Diagnostic reliability | 550 | ||
| Clinical usefulness | 550 | ||
| Maintaining quality and reliability of tests | 550 | ||
| Quality control of a test method (technical quality) | 550 | ||
| Internal quality control | 550 | ||
| External quality assessment | 551 | ||
| Basic haematology tests | 551 | ||
| Haemoglobinometry | 551 | ||
| Direct reading haemoglobinometers | 551 | ||
| HemoCue blood haemoglobin system | 551 | ||
| Haemoglobin colour scale * | 551 | ||
| Packed cell volume | 552 | ||
| Manual cell counts using counting chambers | 552 | ||
| Counting chambers | 552 | ||
| Total white blood cell count | 552 | ||
| Method | 553 | ||
| Calculation | 553 | ||
| Range of white blood cell count in health | 553 | ||
| Platelet count | 554 | ||
| Method | 554 | ||
| Calculation | 555 | ||
| Range of platelet counts in health | 555 | ||
| Errors in manual cell counts | 555 | ||
| Standardised counting chambers | 555 | ||
| Accurate dilutions | 555 | ||
| Microscopy artefacts | 555 | ||
| Peripheral blood morphology | 555 | ||
| Modified (one-tube) osmotic fragility test | 556 | ||
| Haemoglobin E screening test | 556 | ||
| Sickle cell screening test | 556 | ||
| Tests for paroxysmal nocturnal haemoglobinuria | 556 | ||
| Reagents for prothrombin time and activated partial thromboplastin time | 556 | ||
| Laboratory support for management of HIV/AIDS: CD4-positive T-cell counts | 557 | ||
| Laboratory management | 557 | ||
| Interlaboratory communication | 557 | ||
| Specimen transport | 557 | ||
| Point-of-care tests | 557 | ||
| Staff training | 557 | ||
| Clinical staff interaction | 558 | ||
| Facility management teams | 558 | ||
| Health and safety | 558 | ||
| References | 559 | ||
| Laboratory organisation and management | 559 | ||
| Practical methods | 560 | ||
| Quality assurance | 560 | ||
| Appendix | 561 | ||
| Preparation of commonly used reagents | 561 | ||
| Water | 561 | ||
| Anticoagulants and preservative solutions | 561 | ||
| Acid–citrate–dextrose (ACD) solution – NIH-a | 561 | ||
| Acid–citrate–dextrose (Alsever) solution | 561 | ||
| Citrate–phosphate–dextrose (CPD) solution, pH 6.9 | 561 | ||
| Citrate–phosphate–dextrose (CPD) solution, pH 5.6–5.8 | 562 | ||
| Citrate–phosphate–dextrose–adenine (CPD-a) solution, pH 5.6–5.8 | 562 | ||
| Low ionic strength saline (LISS) 1 | 562 | ||
| K 2 EDTA | 562 | ||
| Neutral EDTA, pH 7.0, 110 mmol/l | 562 | ||
| Neutral buffered Na 2 EDTA, pH 7.0 | 562 | ||
| Saline (normal ionic strength) | 562 | ||
| Trisodium citrate (Na 3 C 6 H 5 O 7 .2H 2 O), 109 mmol/l | 562 | ||
| Heparin | 562 | ||
| Buffers | 562 | ||
| Barbitone buffer, pH 7.4 | 562 | ||
| Barbitone buffered saline, pH 7.4 | 562 | ||
| Barbitone buffered saline, pH 9.5 | 563 | ||
| Barbitone–bovine serum albumin buffer, pH 9.8 | 563 | ||
| Citrate–saline buffer | 563 | ||
| Glycine buffer, pH 3.0 | 563 | ||
| HEPES buffer, pH 6.6 | 563 | ||
| HEPES–saline buffer, pH 7.6 | 563 | ||
| Imidazole buffered saline, pH 7.4 | 563 | ||
| Phosphate buffer, iso-osmotic | 563 | ||
| Phosphate buffered saline | 563 | ||
| Phosphate buffer, Sörensen | 563 | ||
| Tris–HCl buffer (200 mmol/l) | 564 | ||
| Tris–HCl bovine serum albumin (BSA) buffer, pH 7.6, 20 mmol/l | 564 | ||
| Buffered formal acetone | 564 | ||
| Preparation of glassware | 564 | ||
| Siliconised glassware | 564 | ||
| Cleaning slides | 564 | ||
| New slides | 564 | ||
| Dirty slides | 564 | ||
| Cleaning glassware | 564 | ||
| Iron-free glassware | 564 | ||
| Sizes of tubes | 565 | ||
| Speed of centrifugation | 565 | ||
| Statistical procedures | 565 | ||
| Calculations | 566 | ||
| Variance (s 2 or SD 2) | 566 | ||
| Standard deviation (SD or s) | 566 | ||
| Coefficient of variation (CV) as percentage | 566 | ||
| Standard error mean (SEM) | 566 | ||
| Standard deviation of paired results | 566 | ||
| Standard deviation of median | 566 | ||
| Confidence interval | 566 | ||
| Analysis of differences by t -test | 566 | ||
| Index | 569 | ||
| Inside Back Cover | ES3 |