A Practical Guide to Frozen Section Technique (eBook)

Preface 5
Contents 8
Contributors 9
Understanding and Maintaining the Cryostat 10
1.1 Operation of the Cryostat 14
1.2 Specimen Preparation 14
1.3 Specimen Orientation 18
1.4 Cryostat Disinfection 18
1.5 Cryostat Maintenance 19
1.5.1 Checklist 19
Gross Examination of Tissues in the Frozen Section Room 21
2.1 Gross Examination 22
2.1.1 Verify Specimen Labeling and Patient Identification 22
2.1.2 Review Clinical Information 22
2.1.3 Examine and Palpate All External Surfaces of the Specimen Carefully 23
2.1.4 Understand the Resection Margins 23
2.1.5 Inking Resection Margins 24
2.1.6 Application of Ink 29
2.1.7 Dissecting and Sectioning the Specimen 32
2.1.8 Examining the Cut Specimen 34
2.1.9 Taking the Sections for Frozen Sectioning 36
2.1.10 Cytology Preparations 40
Embedding of Tissue for Frozen Section 44
3.1 Traditional Embedding Techniques 45
3.1.1 Face Up Embedding 45
3.1.2 Pre-freezing Tissue 47
3.1.3 Face Down Embedding 47
3.2 Face Down Cryoembedding in Well Bars: The Precision Cryoembedding System 49
3.3 Apparatus 3.3.1 Embedding Well Bars 50
3.3.2 Chucks 50
3.3.3 Over-chuck Freezing Blocks 50
3.3.4 Dispensing Slides 52
3.3.5 Embedding Shelf 52
3.4 Face Down Cryoembedding in Well Bars: Technique 52
3.5 Face Down Cryoembedding in Well Bars – The Elements in Detail 3.5.1 Use of the Dispensing Slide 55
3.5.2 Application of Embedding Medium and Tissue 56
3.5.3 Super Flat Embedding 56
3.5.4 Looking Through the Dispensing Slide 56
3.5.5 Flimsy Tissues and Friction 57
3.5.6 Dealing with Multiple Samples 57
3.5.7 Use Your Imagination 58
3.5.8 Placement of Tissue in the Well 58
3.5.9 Using Forceps to Embed 59
3.5.10 Standing Tissue on Edge and on Point 59
3.5.11 Use of the Flattening Forceps 60
3.5.12 Filling the Well with Medium 60
3.5.13 Parallel Faces 61
3.5.14 Use of the Over-chuck Freezing Block 62
3.5.15 Releasing the Block 62
3.6 Reducing Freezing Artifact 63
3.6.1 Colder Well Bar Temperature 63
3.6.2 Pre-chilling Tissue 63
3.6.3 Cold Embedding Medium 63
3.6.4 Freezing Semi-liquid Samples 64
3.6.5 Embedding Snap Frozen Tissue Samples 64
3.7 The Cut Off Technique 64
3.8 Plastering Technique 66
3.9 Paper Embedding 68
3.9.1 Paper Embedding Multiple Tissue Sections on Edge: The Book 70
3.9.2 Paper Embedding Membrane Roll 70
3.9.3 Paper Embedding very Thin Tissues on Face 70
3.9.4 Further Details on Paper Embedding 71
3.10 Frozen Block Cryoembedding 73
3.11 Apparatus 3.11.1 Cutting Board/Freezing Griddle 73
3.11.2 Elevated Freezing Block 74
3.12 Frozen Block Cryoembedding Technique 74
3.12.1 Bowel on the Griddle 75
3.13 Frozen Block Cryoembedding Details 3.13.1 Making the Frozen Block 76
3.13.2 How Do I Know When the Block Is Frozen? 77
3.13.3 Removing the Frozen Block 77
3.13.4 Cutting the Frozen Block Fig. 3.22g–i 77
3.13.5 Putting the Tissue Pieces in the Wells Fig. 3.22j 78
3.13.6 Filling the Well 78
3.14 Orienting the Anatomy of Tissue Relative to the Blade 79
References 81
Cutting the Frozen Section 82
4.1 Taking Sections on the Cryostat: Brush or Antiroll Device? 83
4.2 Continuous Motion Frozen Section Brush Technique 4.2.1 Insert the Chuck and Check the Cryostat 84
4.2.2 The Frozen Section Brush 85
4.2.3 The Blade 86
4.2.4 Body Position 87
4.2.5 Holding the Brush 88
4.2.6 Trimming the Block 90
4.2.7 Cutting the Final Sections 95
4.2.8 Retrieving the Section 98
4.2.9 Teaching Continuous Motion 100
Variables Affecting the Cutting Properties of Tissues and the Resulting Artifacts 103
5.1 Temperature of the Block 104
5.1.1 Adjusting the Temperature of the Block 106
5.1.2 Starting at the Crumple Stage 108
5.1.3 Cutting Ribbons 108
5.2 Cutting Behavior of Specific Tissues 108
5.2.1 Softer Non-fatty Tissues 108
5.2.2 Watery Tissues 108
5.2.3 Tough Collagenous Tissues 109
5.2.4 Bony Hard Tissues 109
5.2.5 Necrotic and Liquifactive Tissues 109
5.2.6 Fatty Tissues 110
5.3 Curling Away 114
5.4 How Much Tissue Can be Put in a Single Block? 116
5.4.1 The Ability of the Cryostat to Cut Through Large Tough Portions of Tissue 116
5.4.2 The Toughness or Hardness of the Tissue 117
5.5 Thickness of the Section 117
5.5.1 Thick and Thin Sections 118
5.6 Stripes and Chatter 119
Reference 121
6.1 Fixation of Frozen Section Slides 122
Fixation, Staining and Coverslipping of Frozen Section Slides 122
6.2 Air Dried Preparations 126
6.3 Staining of Frozen Section Slides 126
6.4 H& E Staining Procedure
6.5 Toluidine Blue 128
6.6 Special Stains for Intraoperative Consultation 128
6.7 What Holds Our Tissue to the Slide and Why Does It Fall Off? 129
6.8 Coverslipping 131
6.9 Wiping the Slide 133
References 134
7.1 Microscopic Interpretation and Intra-operative Consultation 135
Microscopic Interpretation in the Frozen Section Setting 135
7.1.1 The Zen of Reading Slides 136
7.2 Observations on Observation 137
7.2.1 You Only See What You Are Looking For 137
7.2.2 You Can Only Look Carefully for One Thing at a Time 137
7.2.3 You Can Only Make a Diagnosis if You Think of It 138
7.2.4 A Simple Plan 139
7.2.5 Reading with Scanning Objectives 140
7.3 Struggling with the Tough Ones 140
7.3.1 Don’t Forget to Think About 141
7.3.2 Common Sense 141
7.3.3 Study the Obvious Examples to Learn the Subtleties: Stop and Smell the Roses 142
7.4 Cytologic Preparations 142
7.5 The Kinetics of the Smear 143
7.6 The Cytology of the Nucleus 144
7.7 The Many Faces of Necrosis: A Potential Pitfall 148
7.8 Freezing Artifacts 148
7.8.1 Ice Crystals “Bubbles” in Edematous Stroma 151
7.8.2 Compression Artifacts 151
7.8.3 Nuclear Chromatin Changes in Frozen Control 151
7.8.4 Nuclear Ice Crystals 152
7.9 Reporting the Diagnosis 152
8.1 Mohs Surgery Procedure 154
Frozen Section Techniques Used in Mohs Micrographic Surgery 154
8.2 Relaxing the Specimen 155
8.3 Notating the Map 158
8.4 Embedding the Specimen 160
8.5 Slide Technique 161
8.6 Miami Special 162
8.7 Cryo-embedder 163
8.8 Heat Extractor/Cryostat Stage Method 165
8.9 The Precision Cryoembedding System 166
8.10 Embedding in Plastic Molds 168
8.11 Embedding Samples from Secondary and Latter Stages of Mohs Surgery 168
8.12 Sectioning the Block 169
8.13 Staining the Sections 170
8.14 Suggestions for Sectioning Specific Difficult to Handle Tissues 8.14.1 Fatty Specimens 171
8.14.2 Cartilage 171
8.14.3 Mucosa and Soft Tissues 172
Reference 172
Frozen Section Technique in the Animal Research Setting 173
9.1 Introduction 174
9.2 Sacrif ice Perfusion 174
9.3 Avoiding Shrinkage 177
9.4 Tonicity of Perfusion Fluids 179
9.5 Tissue Orientation 181
9.6 Tape Transfer Technique 184
9.6.1 Choice of Sectioning Method 184
9.7 Blade Angle 186
9.8 Vibrating Microtome 187
9.9 Freezing of Biological Tissue 188
References 190
Index 192
We must know what excellence looks like and sounds like in order to begin to approximate it. We can only achieve excellence if we are aware of all the ways to make mistakes and are aware when we are making them. Stephen Peters MD 195