الأربعاء، 21 يوليو 2021
WHO laboratory manual for the Examination and processing of human semen (Fifth Edition)
WHO laboratory manual for the Examination and processing of human semen
FIFTH EDITION
Chapter 1 Background 1
1.1 Introduction 1
1.2 The fi fth edition 1 1.3 Scope of the manual 3PART I. SEMEN ANALYSIS
Chapter 2 Standard procedures 7
2.1 Introduction 7
2.2 Sample collection 10
2.2.1 Preparation 10 2.2.2 Collection of semen for diagnostic or research purposes 11 2.2.3 Sterile collection of semen for assisted reproduction 11 2.2.4 Sterile collection of semen for microbiological analysis 11 2.2.5 Collection of semen at home 12 2.2.6 Collection of semen by condom 12 2.2.7 Safe handling of specimens 132.3 Initial macroscopic examination 13
2.3.1 Liquefaction 13 2.3.2 Semen viscosity 14 2.3.3 Appearance of the ejaculate 15 2.3.4 Semen volume 15 2.3.5 Semen pH 162.4 Initial microscopic investigation 17
2.4.1 Thorough mixing and representative sampling of semen 17 2.4.2 Making a wet preparation 18 2.4.3 Aggregation of spermatozoa 19 2.4.4 Agglutination of spermatozoa 19 2.4.5 Cellular elements other than spermatozoa 212.5 Sperm motility 21
2.5.1 Categories of sperm movement 22 2.5.2 Preparing and assessing a sample for motility 22 2.5.3 Worked examples 25 2.5.4 Lower reference limit 262.6 Sperm vitality 26
2.6.1 Vitality test using eosin–nigrosin 27 2.6.2 Vitality test using eosin alone 29 2.6.3 Vitality test using hypo-osmotic swelling 302.7 Sperm numbers 32
2.7.1 Types of counting chamber 34 2.7.2 The improved Neubauer haemocytometer 34 2.7.3 Using the haemocytometer grid 35 2.7.4 Care of the counting chamber 35 2.7.5 Fixative for diluting semen 36 2.7.6 Importance of counting suffi cient spermatozoa 362.8 Routine counting procedure 37
2.8.1 Determining the required dilution 38 2.8.2 Preparing the dilutions and loading the haemocytometer chambers 39 2.8.3 Assessing sperm numbers in the counting chambers 41 2.8.4 Calculation of the concentration of spermatozoa in semen 43 2.8.5 Worked examples 43 2.8.6 Lower reference limit for sperm concentration 44 2.8.7 Calculation of the total number of spermatozoa in the ejaculate 44 2.8.8 Lower reference limit for total sperm number 442.9 Low sperm numbers: cryptozoospermia and suspected azoospermia 45
2.10 When an accurate assessment of low sperm numbers is not required 45
2.10.1 Taking no further action 45 2.10.2 Examination of centrifuged samples to detect spermatozoa 45 2.10.3 Examination of non-centrifuged samples to detect motile spermatozoa 462.11 When an accurate assessment of low sperm numbers is required 48
2.11.1 Assessing low sperm numbers in the entire improved Neubauer chamber (phase-contrast microscopy) 48 2.11.2 Assessing low sperm numbers in large-volume disposable chambers (fl uorescence microscopy) 522.12 Counting of cells other than spermatozoa 55
2.12.1 Calculation of the concentration of round cells in semen 55 2.12.2 Sensitivity of the method 56 2.12.3 Worked examples 562.13 Sperm morphology 56
2.13.1 The concept of normal spermatozoa 57 2.13.2 Preparation of semen smears 582.14 Staining methods 62
2.14.1 Traditional fixation and sequential staining 62 2.14.2 Papanicolaou staining procedure for sperm morphology 63 2.14.3 Shorr staining procedure for sperm morphology 65 2.14.4 Rapid staining procedure for sperm morphology 662.15 Examining the stained preparation 67
2.15.1 Classification of normal sperm morphology 67 2.15.2 Classifi cation of abnormal sperm morphology 692.16 Morphology plates 70
2.17 Analysing a sperm morphology smear 99
2.17.1 Assessment of normal sperm morphology 99 2.17.2 Worked examples 100 2.17.3 Lower reference limit 100 2.17.4 Assessment of abnormal sperm morphology 101 2.17.5 Worked example 101 2.17.6 Assessment of specifi c sperm defects 1022.18 Assessment of leukocytes in semen 102
2.18.1 Staining cellular peroxidase using ortho-toluidine 1032.19 Assessment of immature germ cells in semen 107
2.20 Testing for antibody coating of spermatozoa 108 2.20.1 The mixed antiglobulin reaction test 109 2.20.2 The direct immunobead test 111 2.20.3 The indirect immunobead test 113Chapter 3 Optional procedures 115
3.1 Indices of multiple sperm defects 115
3.1.1 Calculation of indices of multiple morphological defects 115 3.1.2 Worked example 1163.2 Panleukocyte (CD45) immunocytochemical staining 117
3.2.1 Principle 117 3.2.2 Reagents 118 3.2.3 Procedure 1183.3 Interaction between spermatozoa and cervical mucus 122
3.3.1 In-vivo (postcoital) test 122 3.3.2 In-vitro tests 125 3.3.3 In-vitro simplifi ed slide test 1263.3.4 Capillary tube test 127
3.4 Biochemical assays for accessory sex organ function 130 3.4.1 Measurement of zinc in seminal plasma 130 3.4.2 Measurement of fructose in seminal plasma 132 3.4.3 Measurement of neutral -glucosidase in seminal plasma 1343.5 Computer-aided sperm analysis 136
3.5.1 Introduction 136 3.5.2 Use of CASA to assess sperm motility 137 3.5.3 Use of CASA to estimate sperm concentration 1403.5.4 Computer-aided sperm morphometric assessment 140
Chapter 4 Research procedures 142
4.1 Reactive oxygen species 142
4.1.1 Introduction 142 4.1.2 Measurement of reactive oxygen species generated by sperm suspensions 1434.2 Human sperm–oocyte interaction tests 146
4.3 Human zona pellucida binding tests 146
4.4 Assessment of the acrosome reaction 147
4.4.1 Procedure for the fluorescence assessment of acrosomal status 147 4.4.2 Induced acrosome reaction assay 150 4.5 Zona-free hamster oocyte penetration test 1524.5.1 Protocol 152
4.6 Assessment of sperm chromatin 157PART II. SPERM PREPARATION
Chapter 5 Sperm preparation techniques 161
5.1 Introduction 161
5.1.1 When spermatozoa may need to be separated from seminal plasma 161 5.1.2 Choice of method 161 5.1.3 Effi ciency of sperm separation from seminal plasma and infectious organisms 1625.2 General principles 162
5.3 Simple washing 163
5.3.1 Reagents 163 5.3.2 Procedure 1635.4 Direct swim-up 164
5.4.1 Reagents 164 5.4.2 Procedure 1645.5 Discontinuous density gradients 165
5.5.1 Reagents 165 5.5.2 Procedure 1665.6 Preparing HIV-infected semen samples 166
5.7 Preparing testicular and epididymal spermatozoa 167
5.7.1 Enzymatic method 167 5.7.2 Mechanical method 167 5.7.3 Processing sperm suspensions for intracytoplasmic sperm injection 1675.8 Preparing retrograde ejaculation samples 168
5.9 Preparing assisted ejaculation samples 168
Chapter 6 Cryopreservation of spermatozoa 169
6.1 Introduction 169
6.2 Semen cryopreservation protocols 172
6.2.1 Standard procedure 172 6.2.2 Modifi ed freezing protocols for oligozoospermia and surgically retrieved spermatozoa 175 6.2.3 Labelling of straws and records 176PART III. QUALITY ASSURANCE
Chapter 7 Quality assurance and quality control 179
7.1 Controlling for quality in the andrology laboratory 179
7.2 The nature of errors in semen analysis 179
7.3 Minimizing statistical sampling error 180
7.4 The quality assurance programme 182
7.5 Laboratory procedures manual 182
7.6 Internal quality control 182
7.6.1 Purchased QC samples 183 7.6.2 Laboratory-made QC samples 183 7.6.3 Stored samples (purchased or laboratory-made) 183 7.6.4 Fresh QC samples (laboratory-made) 1847.7 Statistical procedures for analysing and reporting within- and among-technician systematic errors 185
7.7.1 The Xbar chart 185 7.7.2 The S chart 1887.8 QC for percentages 189
7.9 Assessing Xbar and S charts 189
7.9.1 How to recognize out-of-control values 189 7.9.2 Causes of out-of-control values 190 7.9.3 Responses to out-of-control values 1917.10 Statistical procedures for analysing and reporting among-technician variability 191
7.10.1 Comparing results from two or more technicians 191 7.10.2 Monitoring monthly means 1947.11 External quality control and quality assurance 194
7.11.1 Assessment of EQC results 196 7.11.2 Responses to out-of-control results 1977.12 Frequency and priority of quality control 197
7.13 Training 198
7.13.1 Practical hints when experiencing difficulty assessing sperm concentration 198 7.13.2 Practical hints when experiencing diffi culty assessing sperm morphology 200 7.13.3 Practical hints when experiencing diffi culty assessing sperm motility 200 7.13.4 Practical hints when experiencing diffi culty assessing sperm vitality 202REFERENCES 205
APPENDICES
Appendix 1 Reference values and semen nomenclature 223
A1.1 Reference values 223 A1.2 Nomenclature 225Appendix 2 Equipment and safety 227
A2.1 Basic supplies needed in an andrology laboratory 227 A2.2 Potential biohazards in an andrology laboratory 230 A2.3 Safety procedures for laboratory personnel 230 A2.4 Safety procedures for laboratory equipment 232 A2.5 Safety precautions when handling liquid nitrogen 233Appendix 3 Microscopy 234
A3.1 Loading the sample 234 A3.2 Adjusting the oculars 236 A3.3 Focusing the image 236 A3.4 Focusing the oculars 236 A3.5 Focusing the light condenser 236 A3.6 Centring the condenser 237 A3.7 Adjusting the phase rings 237 A3.8 Fluorescence microscopy 237Appendix 4 Stock solutions 238
A4.1 Biggers, Whitten and Whittingham 238 A4.2 Dulbecco’s phosphate-buffered saline 238 A4.3 Earle’s medium 239 A4.4 Ham’s F-10 medium 239 A4.5 Hanks’ balanced salt solution 240 A4.6 Human tubal fl uid 240 A4.7 Krebs–Ringer medium 240 A4.8 Tris-buffered saline 241 A4.9 Tyrode’s solution 241 A4.10 Papanicolaou stain 241Appendix 5 Cervical mucus 245
A5.1 Introduction 245 A5.2 Collection and preservation of cervical mucus 246 A5.3 Evaluation of cervical mucus 247Appendix 6 Record forms for semen and cervical mucus analyses 251
A6.1 Template for a semen analysis recording form 251 A6.2 Template for a cervical mucus recording form 253Appendix 7 Sampling errors and quality control 254
A7.1 Errors in measurement of sperm concentration 254 A7.2 The importance of understanding sampling errors 256 A7.3 Errors in measurement of percentages 257 A7.4 Production of semen samples for quality control 260 A7.5 Preparation of a video-recording for internal quality control of analysis of sperm motility 261 A7.6 Preparation of diluted semen for internal quality control of determination of sperm concentration 265 A7.7 Preparation of slides for internal quality control of assessment of sperm morphology 268 A7.8 Calibration of equipment 269Appendix 8 National external quality control programmes for semen analysis 271
A Concise Manual of Pathogenic Microbiology
A Concise Manual of Pathogenic Microbiology
Contents
1 Introduction
Koch’s Postulate 2
Terminology 3
Major Categories of Pathogenic Microorganisms 4
Transmission of Infectious Disease (Mode of Dissemination) 5
Universal Precautions 6
2 Host-Microbe Interactions
Resident Microbiota 9
Host Defenses 11
3 Antibiotics and Other Chemotherapeutic Agents 17
Classification of Antibiotics 17
Summary of the Mechanisms of Action 24
4 Antiseptics and Disinfectants
Physical Control of Microorganisms 25
Chemical Control of Microorganisms 27
5 Gram-Positive Cocci
Bacterial Taxonomy (An Overview) 31
Clinically Important Gram-Positive Cocci 32
Gram-Positive Cocci Related to Streptococcus Species 38
6 Gram-Positive Bacilli
Clostridium Species 41
Lactobacillus Species 46
Bacillus Species 46
Listeria Species 49
7 Gram-Positive Bacteria with Rudimentary Filaments
Corynebacterium diphtheriae 53
Mycobacterium Species 54
8 Gram-Negative Cocci
Neisseria Species 61
Moraxella catarrhalis 64
Haemophilus influenzae 65
An Overview of Gram-Negative Bacteria 67
9 Gram-Negative Bacilli
Specimen Collection 69
Media and Laboratory Diagnosis 69
Enterobacteriaceae 71
Glucose Nonfermenters 78
Uncommon Nonfermentative Taxa 81
10 Miscellaneous Gram-Negative Bacteria
Brucella melitensis 83
Bordetella pertussis 85
Francisella tularensis 86
Pasteurella Species 87
Vibrio cholerae 88
Aeromonas Species 90
Campylobacter Species 90
Legionella Species 92
Gardnerella vaginalis 93
Chlamydia Species 94
Rickettsia rickettsii 95
Bacteroides Species 96
Calymmatobacterium granulomatis 96
Cardiobacterium hominis 96
Streptobacillus moniliformis 96
Spirillum minus 97
11 Spirochetes and Bacteria without a Cell Wall
Spirochetes 99
Bacteria without a Cell Wall 103
12 Actinomycetes
Anaerobic Actinomycetes 108
Aerobic Actinomycetes 108
Thermophilic Actinomycetes 112
13 Introduction to Pathogenic Fungi and Superficial Mycoses
Yeast-Like Fungi 113
Molds or Filamentous Fungi 114
Dimorphic Fungi 114
Superficial Mycoses 115
Mucocutaneous Mycoses 121
14 Subcutaneous and Systemic Mycoses
Subcutaneous Mycoses 125
Systemic Mycoses 127
Diseases Caused by Dimorphic Fungi 127
Diseases Caused by Yeast-Like Fungi 135
Diseases Caused by Filamentous Fungi 138
Diseases Caused by Miscellaneous Filamentous Fungi 143
15 Unicellular Parasites
Laboratory Methods in Parasitology 145
Diseases Caused by Lumen-Dwelling Protozoa 146
Blood- and Tissue-Dwelling Protozoa 149
16 Multicellular Parasites
Lumen-Dwelling Helminths 155
Blood- and Tissue-Dwelling Helminths 161
17 Viruses and Prions
Laboratory Diagnosis 166
Double-Stranded DNA Viruses 166
Single-Stranded DNA Viruses 171
Double-Stranded RNA Viruses 171
Single-Stranded RNA Viruses 171
Prions 179
Bibliography and Suggested Reading 181
الأحد، 18 يوليو 2021
الاثنين، 12 يوليو 2021
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