Histotechnology: A Self-Instructional Text [3 ed.] 0891895817, 9780891895817

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Histotechnology A Self-Instructional Text 3rd Edition

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Histotechnology A Self-Instructional Text 3rd Edition

Freida L Carson PhD, HT(ASCP) Department of Pathology (retired) Baylor University Medical Center Dallas, Texas

Christa Hladik AA, HT(ASCP)'m, QIHC Clinical Laboratory Manager, Neuropathology and Immunohistochemistry, UT Southwestern Clinical Laboratories, University of Texas Southwestern Medical Center Dallas, Texas

•

American Society for Clinical Pathology Press

Publishing Team Adam Fanucci (Illustrations) Erik N Tanck & Tae W Moon (Design/Production) Joshua Weikersheimer (Publishing direction)

Notice Trade names for equipment and supplies described are included as suggestions only. In no way does their inclusion constitute an endorsement of preference by the Author or the ASCP. The Author and ASCP urge all readers to read and follow all manufacturers' instructions and package insert warnings concerning the proper and safe use of products. The American Society for Clinical Pathology, having exercised appropriate and reasonable effort to research material current as of publication date, does not assume any liability for any loss or damage caused by errors and omissions in this publication. Readers must assume responsibility for complete and thorough research of any hazardous conditions they encounter, as this publication is not intended to be all-inclusive, and recommendations and regulations change over time.

Cover Images Image (left) : Hematoxylin eosin (H&E) - small intestine Image (middle): Papanicolaou- cervical smear Image (right): Aldan yellow-toluidine blue- gastric biopsy showing H pylori

•

American Society for Clinical Pathology Press Copyright© 2009 by the American Society for Clinical Pathology. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the publisher.

Printed in Hong Kong 13 12 11 10 09

iv

Table of Contents xvi

Preface

............................................ Chapter I ............................................ Fixation

16

POTASSIUM DICHROMATE (K 2Cr20 7 )

16

ZINC SALTS (ZnS0 4)

17

Other Fixative Ingredients

17 17 17 17 19

2

Definition

19

20

2

Functions of Fixatives

20

2

Actions of Fixatives

4

Factors Affecting Fixation

20 20 20

4

TEMPERATURE

4

SIZE

4

VOLUME RATIO

5

TIME

21 21

21 21

7

CHOICE OF FIXATIVE

21

7

PENETRATION

22

7

TISSUE STORAGE

7

pH

7

OSMOLALITY

8

Reactions of the Cell with Fixatives

8

THE NUCLEUS

8

PROTEIN

8

LIPIDS

9

CARBOHYDRATES

9

Simple Aqueous Fixatives or Fixative Ingredients

9

ACETIC ACID

9 10 10

12 12 12 10 10

12 12

FORMALDEHYDE 12% Aqueous Formalin 12% Formalin Saline Calcium Formalin Formalin Ammonium Bromide Acetate Formalin 12% Neutralized Formalin 12% Neutral-Buffered Formalin Modified Millonig Formalin Alcoholic Formalin

13

GLUTARALDEHYDE

13 14

Phosphate-Buffered Glutaraldehyde GLYOXAL (C 2 H 20 2)

14

MERCURIC CHLORIDE (HgCl 2)

15

OSMIUM TETROXIDE (OsO 4 )

15

PICRIC ACID

Compound or Combined Fixatives B-5 FIXATIVE Stock Solution Working Solution Bouin Solution Gendre Solution Hollande Solution ZENKER AND HELLY (ZENKER-FORMOL) SOLUTIONS Zenker and Helly Stock Solution Zenker Working Solution Helly Working Solution Orth Solution Zamboni Solution (Buffered Picric Acid-Formaldehyde, or PAF) ZINC FORMALIN SOLUTIONS Aqueous Zinc Formalin (original formula) Unbuffered Aqueous Zinc Formalin Alcoholic Zinc Chloride Formalin

22

Nonaqueous Fixatives

22

ACETONE

22

ALCOHOL

22

23 23 23 23 23 23 25

Carnoy Solution Clarke Fluid

Transport Solutions Michel Transport Medium PBS Buffer Stock Solution (also used in immunohistochemistry) PBS-10% Sucrose Solution

Removal of Fixation Pigments Lugo) Iodine Solution

24

Troubleshooting Fixation Problems

24

AUTOLYSIS

24

INCOMPLETE FIXATION

27

References

Histotechnology 3rd Edition v

............................................ Chapter 2 ............................................ Processing

Special Techniques in Processing

46 46

DECALCIFICATION

49

TROUBLESHOOTING DECALCIFICATION

49

FROZEN SECTIONS

50

TROUBLESHOOTING PROCESSING TISSUE FOR

33

Dehydration

33

ALCOHOLS

34

ACETONE

34

UNIVERSAL SOLVENTS

35 35

Clearing

35

TOLUENE

35

BENZENE

36

CHLOROFORM

54

Microscopes

36

ACETONE

54

LIGHT MICROSCOPE

36

ESSENTIAL OILS

55

POLARIZING MICROSCOPE

36

LIMONENE REAGENTS (XYLENE SUBSTITUTE)

55

PHASE-CONTRAST MICROSCOPE

36

ALIPHATIC HYDROCARBONS (XYLENE

55

DARKFIELD MICROSCOPE

56

FLUORESCENCE MICROSCOPE

56

ELECTRON MICROSCOPE

XYLENE

SUBSTITUTE)

FROZEN SECTIONS

51

References

............................................ Chapter 3 ................ ................... ......... Instrumentation

36

UNIVERSAL SOLVENTS

37

OTHER CLEARING AGENTS

57

Microtomes

37

Infiltration

57

ROTARY MICROTOME

37

PARAFFIN

57

SLIDING MICROTOME

40

WATER-SOLUBLE WAXES

57

CLINICAL FREEZING MICROTOME

40

CELLOIDIN

58

MICROTOME BLADES

41

PLASTICS

58

TROUBLESHOOTING MICROTOMY

41

AGAR AND GELATIN

30

41% SUCROSE

64

Cryostat

41 41

Troubleshooting Processing

65 65

Tissue Processors

66

MICROWAVE PROCESSOR

41

OVERDEHYDRATION

42

POOR PROCESSING

42

SPONGE ARTIFACT

67 67

AUTOMATIC STAINER

42

TISSUE ACCIDENTALLY DESICCATED

67

MICROWAVE STAINING OVEN

68

AUTOMATIC COVERSLIPPER

PRECIPITATE IN THE PROCESSOR CHAMBER AND IN THE TUBING

CONVENTIONAL PROCESSOR

Stainers and Coverslippers

42

Embedding and Specimen Orientation

44

69 69

Miscellaneous Equipment

Troubleshooting Embedding

44

SOFT MUSHY TISSUE

70

CHROMIUM POTASSIUM SULFATE-COATED

45

INCORRECT ORIENTATION

46

TISSUE CARRYOVER

70

POLY-L-LYSINE-COATED SLIDES

46

TISSUE NOT EMBEDDED AT THE SAME LEVEL

70

AMINOALKYLSILANE-TREATED SLIDES

46

PIECES OF TISSUE MISSING FROM THE BLOCK

71

DRYERS AND OVENS

72

CIRCULATING WATER BATH

72

FREEZERS AND REFRIGERATORS

72

pH METERS

74

BALANCES AND SCALES

74

EMBEDDING CENTER

vi

FLOTATION BATHS SLIDES

75

MICROMETER PIPETTES

75

SOLVENT RECYCLER

75 75

Instrument Quality Control

75

QUALITY CONTROL PROGRAM

16

............................................ ~~~~t:r. s. .................................... .

Laboratory Mathematics and Solution Preparation

NEW INSTRUMENT VALIDATION 94

Percentage Solutions

95

Use of the Gravimetric Factor in Solution Preparation

96

Hydrates

96

Normal and Molar Solutions

References

............................................ ~~~J:!t~r. ~ .................................... .

Safety 82

Biological or Infectious Hazards

82

TUBERCULOSIS EXPOSURE

82

CRYOGENIC SPRAYS

83

HIV, HEPATITIS C VIRUS (HCV), AND HBV

83

CREUTZFELDT-JAKOB DISEASE (CJD)

83

HANDLING TISSUE WASTE

83

Mechanical Hazards

84

ERGONOMICS

84

Chemical Hazards

86

PARTICULARLY HAZARDOUS SUBSTANCES (REPRODUCTIVE TOXINS, SELECT CARCINOGENS, AND SUBSTANCES WITH A HIGH DEGREE OF ACUTE TOXICITY)

86

CARCINOGENS

86

CORROSIVE SUBSTANCES

86

FIRE AND EXPLOSION HAZARDS

87

HAZARDOUS CHEMICAL SPILLS AND STORAGE

88

CHEMICAL STORAGE

88

HAZARDOUS CHEMICAL DISPOSAL

89

Hazard Identification General Safety Practices

90 90

EMPLOYEES

90

SUPERVISORS

91

References

97

The Metric System

97

TEMPERATURE CONVERSION

98

Buffers

98

General Guidelines for Solution Preparation, Use, and Storage

99

Stability of Solutions

99

References

101

ANSWERS TO PROBLEMS IN CHAPTER

101

ANSWERS TO PROBLEMS IN LEARNING ACTIVITIES

............................................ ~~~~t:r. ~ .................................... .

Nuclear and Cytoplasmic Staining 104

Ultrastructure of the Cell

104

THE NUCLEUS

105

THE CYTOPLASM

101

Staining Mechanisms

107

NUCLEAR STAINING

107

CYTOPLASMIC STAINING

108

The Dyes

109

FACTORS AFFECTING DYE BINDING

109

DIFFERENTIATION

109

THE NUCLEAR DYES

110

Harris Hematoxylin Delafield Hematoxylin Mayer Hematoxylin Ehrlich Hematoxylin

111 111

111

Histotechnology 3rd Edition vii

112 112 113 113

Gill Hematoxylin Scott Solution Weigert Hematoxylin Celestine Blue

114

PLASMA STAINS Eosin Counterstain Eosin-Phloxine B Counterstain

114

H&E Staining

114

MANUAL PROGRESSIVE STAINING METHOD

115

MANUAL REGRESSIVE STAINING METHOD

116

AUTOMATED STAINING

113 113

130

'troubleshooting Mounted Stained Sections

130

WATER BUBBLES NOTED IN MOUNTED SECTIONS

130

ALL AREAS OF SECTION CANNOT BE BROUGHT INTO FOCUS

130

CORN-FLAKING ARTIFACT SEEN ON MOUNTED

131

MOUNTED STAINED SECTIONS ARE NOT

SECTIONS AS CRISP AS USUAL WHEN VIEWED MICROSCOPICALLY RETRACTED MOUNTING MEDIUM

References

11 7

FROZEN SECTION STAINING

131

118

Troubleshooting the H&E Stain

132

118

INCOMPLETE DEPARAFFINIZATION

118

NUCLEAR STAINING IS NOT CRISP

11 9

PALE NUCLEAR STAINING

119

DARK NUCLEAR STAINING

120

RED OR RED-BROWN NUCLEI

120

PALE CYTOPLASMIC STAINING

136

Carbohydrates

120

DARK CYTOPLASMIC STAINING

136

GROUP 1: NEUTRAL POLYSACCHARIDES

120

EOSIN NOT PROPERLY DIFFERENTIATED

121

BLUE-BLACK PRECIPITATE ON TOP OF SECTIONS

121

HAZY OR MILKY WATER AND SLIDES

122

UNEVEN H&E STAINING

122

DARK BASOPHILIC STAINING OF NUCLEI AND CYTOPLASM, ESPECIALLY AROUND TISSUE

............................................ Chapter 7 ............... .... .. ............ .. ...... ... Carbohydrates and Amyloid (NONIONIC HOMOGLYCANS) 136

GROUP II: ACID MUCOPOLYSACCHARIDES (ANIONIC HETEROGLYCANS)

136

GROUP III: GLYCOPROTEINS (MUCINS, MUCOID, MUCOPROTEIN, MUCOSUBSTANCES)

136

GROUP IV: GLYCOLIPIDS

137

Special Staining Techniques

EDGES 122

POOR CONTRAST BETWEEN NUCLEUS AND CYTOPLASM

137 137

123 123 123 123 123

Nucleic Acid Stains FEULGEN REACTION Hydrochloric Acid, IN Schiff Reagent (De Tomasi Preparation) Sulfurous Acid

125

METHYL GREEN-PYRONIN Y Solution a-0.2M Acetic Acid Methyl Green-Pyronin Y Staining Solution

126

Polychromatic Stains

124 125

138 139 140 140 140 141 141 142 142

127 127 127 127 127 127

MAY-GRUNWALD GIEMSA STAIN Stock Jenner Solution Working Jenner Solution Stock Giemsa Solution Working Giemsa Solution Acetic Water, 1%

143 143 143 145 145 146 146

128

Mounting Stained Sections

128

RESINOUS MEDIA

129

AQUEOUS MOUNTING MEDIA

129

COVER SLIPS

viii

146 146 147 147 147

PAS REACTION Periodic Acid, 0.5% Solution Schiff Reagent PAS REACTION WITH DIASTASE DIGESTION Periodic Acid, 0.5% Solution Potassium Metabisulfite, 0.55% Solution Phosphate Buffer, pH 6 BEST CARMINE Carmine Stock Solution Working Carmine Solution MAYER MUCICARMINE Mucicarmine Stock Solution Mucicarmine Working Solution Weigert Iron Hematoxylin ALCIAN BLU E, PH 2.5 Acetic Acid, 3% Solution ALCIAN BLUE, PH 1.0 O.lN Hydrochloric Acid Solution 1% Alcian Blue Solution, pH 1.0 Nuclear-Fast Red Solution ALCIAN BLUE WITH H YALURONIDASE O.lM Potassium Phosphate, Monobasic Nuclear-Fast Red Solution

148 148 149 149 149 150 150 150

ALCIAN BLUE-PAS-HEMATOXYLIN

152 152 153 154 154 155 155

Amyloid

157

Acetic Acid, 3% Solution Aldan Blue, pH 2.5 Schiff Reagent MULLER-MOWRY COLLOIDAL IRON Ferric Chloride, 29% Solution Working Colloidal Iron Solution Nuclear-Fast Red Solution

172 173 173 173 174 174 175 175 176 177

ALKALINE CONGO RED METHOD Stock 80% Alcohol Saturated with Sodium Chloride CRYSTAL VIOLET Stock Saturated Crystal Violet Solution

Iodine-Iodide Solution Crocein Scarlet-Acid Fuchsin Solution Phosphotungstic Acid, 5% Solution Alcoholic Safran Solution SILVER TECHNIQUES FOR RETICULAR FIBERS GOMORI STAIN FOR RETICULAR FIBERS Silver Nitrate, 10% Solution Potassium Permanganate, 0.5% Solution Sodium Thiosulfate, 2% Solution GORDON AND SWEETS STAIN FOR RETICULAR FIBERS

178 178 178

Silver Nitrate, 10% Solution Potassium Permanganate, 1% Solution Ferric Ammonium Sulfate, 2.5% Solution

179 179

MALLORY PTAH TECHNIQUE FOR

THIOFLAVINE T FLUORESCENT METHOD Thioflavine T, 1% Solution

References

............................................ Chapter 8 ............................................ Connective and Muscle Tissue

Staining Techniques for Muscle CROSS-STRIATIONS AND FIBRIN

180 180 180 180 181 181

PTAH Solution Gram Iodine Sodium Thiosulfate, 5% Solution Potassium Permanganate, 0.25% Solution PTAH WITHOUT MERCURIC SOLUTIONS Acidic Dichromate Solution

160

Connective 'tissue

182

Staining Technique for Basement Membranes

161

Basement Membrane

182

PERIODIC ACID-METHENAMINE SILVER MICROWAVE PROCEDURE FOR BASEMENT

161

Muscle

162

~taining

162 163 164 165 165 166 166 167 168 168 168 168 170 170 171 171 171

MASSON TRICHROME STAIN

172 172 172

Techniques for Connective Tissue Fibers

Bouin Solution Light Green Counterstain GOMORI 1-STEP TRICHROME STAIN Bouin Solution Acetic Acid, 0.5% Solution VAN GIESON PICRIC ACID-ACID FUCHSIN STAIN Acid Fuchsin, 1% Solution

MEMBRANES

182 182 183 183 184 184 185 185 186 186

VERHOEFF ELASTIC STAIN

187

Lugo) Iodine Ferric Chloride, 10% Solution Sodium Thiosulfate, 5% Solution

187

ALDEHYDE FUCHSIN ELASTIC STAIN Aldehyde Fuchsin Solution Alcoholic Basic Fuchsin, 0.5% Solution Aldehyde Fuchsin Solution NOTES ON OTHER ELASTIC STAINS RUSSELL MODIFICATION OF THE MOVAT PENTACHROME STAIN Aldan Blue, 1% Solution Alkaline Alcohol Solution

Stock Methenamine Silver Gold Chloride, 0.02% Solution Methenamine Silver Solution Gold Chloride, 0.2% solution

Staining Techniques for Lipid OIL RED 0 METHOD FOR NEUTRAL FATS Oil Red 0 Stock Solution Oil Red 0 Working Solution SUDAN BLACK B IN PROPYLENE GLYCOL Calcium-Formalin Solution OSMIUM TETROXIDE PARAFFIN PROCEDURE FOR FAT Osmium Tetroxide, 1% Solution

188

Staining Techniques for Connective 'tissue Cells

188 188 188

TOLUIDINE BLUE FOR MAST CELLS

190

References

Toluidine Blue Solution METHYL GREEN-PYRONIN Y

Histotechnology 3rd Edition ix

Chapter 9 ............................................ Nerve

202

NERVE FIBERS, NEUROFIBRILLARY TANGLES, AND SENILE PLAQUES: MICROWAVE MODIFICATION OF BIELSCHOWSKY METHOD

203 203

194

The Nervous System

194

Neurons

194

NISSL SUBSTANCE

204

194

NERVE CELL PROCESSES

205

194

Neuroglia

204

Silver Nitrate, 1% Solution Sodium Thiosulfate, 2% Solution NERVE FIBERS, NEUROFIBRILLARY TANGLES, AND SENILE PLAQUES: THE SEVIER-MUNGER MODIFICATION OF BIELSCHOWSKY METHOD

205

194

OLIGODENDROGLIA

194

ASTROCYTES

207

195

MICROGLIA EPENDYMAL CELLS

Myelin

195

Special Staining Techniques

195

NISSL SUBSTANCE: CRESYL ECHT VIOLET METHOD I

195 195 196

Cresyl Echt Violet Solution Balsam-Xylene Mixture NISSL SUBSTANCE: CRESYL ECHT VIOLET METHOD II

196 196 197

Stock Cresyl Echt Violet Solution Working Cresyl Echt Violet Solution, pH 2.5

197 197 198 199

Protargol, 1% Solution Oxalic Acid, 2% Solution Aniline Blue Solution NERVE FIBERS AND NEUROFIBRILS: HOLMES SILVER NITRATE METHOD

199 200 200

Aqueous Silver Nitrate, 20% Solution Reducing Solution NERVE FIBERS, NEUROFIBRILLARY TANGLES, AND SENILE PLAQUES: BIELSCHOWSKY-PAS

GLIAL FIBERS: MALLORY PHOSPHOTUNGSTIC ACID HEMATOXYLIN (PTAH) STAIN

208 208

GLIAL FIBERS: HOLZER METHOD

208

209 210 210

Aqueous Phosphomolybdic Acid, 0.5% Solution ASTROCYTES: CAJAL STAIN

Formalin Ammonium Bromide

211

MYELIN SHEATH: WEIL METHOD

211

Ferric Ammonium Sulfate, 4% Solution

212

213 214

MYELIN SHEATH: LUXOL FAST BLUE METHOD

Luxol Fast Blue, 0.1% Solution MYELIN SHEATH AND NISSL SUBSTANCE COMBINED: LUXOL FAST BLUE-CRESYL ECHT

NERVE FIBERS, NERVE ENDINGS, NEUROFIBRILS: BODIAN METHOD

Potassium Permanganate, 0.25% Solution

PTAH Solution Lugol Iodine Potassium Permanganate, 1% Solution Oxalic Acid, 5% Solution

207 2 07

195

NEUROFIBRILLARY TANGLES AND SENILE PLAQUES: THIOFLAVIN S (MODIFIED)

2 06

195

Silver Nitrate, 20% Solution Sodium Thiosulfate, 5% Solution

214 215

VIOLET STAIN Acetic Acid, 10% Solution MYELIN SHEATHS AND NERVE FIBERS COMBINED: LUXOL FAST BLUE-HOLMES SILVER NITRATE METHOD

216 216 216 218 218 218

Aqueous Silver Nitrate, 20% Solution Impregnating Solution Lithium Carbonate, 0.05% Solution LUXOL FAST BLUE-PAS-HEMATOXYLIN

Luxol fast blue, 0.1% Solution Periodic Acid, 0.5% Solution

STAIN 201 201 201 2 01 201 201 20 I

x

Aqueous Silver Nitrate, 20% Solution Ammoniacal Silver Solution Developer Gold Chloride, 0.5% Solution Sodium Thiosulfate, 5% Solution Periodic Acid, 1% Solution Schiff Reagent

219

References

Chapter 10 ............................................ Microorganisms

222

Bacteria

222

Fungi

223

Viruses

239

GROCOTT METHENAMINE-SILVER NITRATE FUNGUS STAIN

240 240 240 242

Chromic Acid, 5% Solution Silver Nitrate, 5% Solution Sodium Thiosulfate, 2% Solution MICROWAVE METHENAMINE-SILVER NITRATE PROCEDURE FOR FUNGI

242 244

Chromic Acid, 10% Solution MAYER MUCICARMINE AND ALCIAN BLUE TECHNIQUES E FOR CRYPTOCOCCUS NEOFORMANS

224

Protozoans

244

224 224 224 226

Special Staining Techniques

244 245 245

WARTHIN-STARRY TECHNIQUE FOR SPIROCHETES

KINYOUN ACID-FAST STAIN Kinyoun Carbol-Fuchsin Solution

226 227

Ziehl-Neelsen Carbol-Fuchsin Solution MICROWAVE ZIEHL-NEELSEN METHOD FOR ACID-FAST BACTERIA

227 228

ORGANISMS

228 229 230

246 246 246 247

Xylene-Peanut Oil Ziehl-Neelsen Carbol-Fuchsin Solution

247 248 248 249

Auramine 0-Rhodamine B Solution Acid Alcohol, 0.5% Solution BROWN-HOPPS MODIFICATION OF THE GRAM STAIN

231 231 233

GIEMSA METHODS

233

MODIFIED DIFF-QUIK GIEMSA STAIN FOR

Crystal Violet, 1% Solution Gram Iodine

HELICOBACTER PYLORI

233 233 233 234

Diff-Quik Solution I Diff-Quik Solution II Acetic Acid Water ALCIAN YELLOW-TOLUIDINE BLUE METHOD FOR H PYLORI

234 235 235 236 236 237 237 238 238 238

Periodic acid, 1% Solution HOTCHKISS-MCMANUS PAS REACTION FOR FUNGI Periodic Acid, 1% Solution IN Hydrochloric Acid CHROMIC ACID-SCHIFF STAIN FOR FUNGI (CAS) Chromic acid, 5% Solution Fast Green, 1:5000 Solution

DIETERLE METHOD FOR SPIROCHETES AND Alcoholic Uranyl Nitrate, 5% Solution Alcoholic Gum Mastic, 10% Solution Formic Acid, 10% Solution MICROWAVE STEINER AND STEINER PROCEDURE FOR SPIROCHETES,

MICROWAVE AURAMINE-RHODAMINE

HELICOBACTER, AND LEGIONELLA ORGANISMS

FLUORESCENCE TECHNIQUE

230 230 231

Glycine-Acetic Acid Stock Solution Silver Nitrate, 2% Solution Hydroquinone, 0.1% Solution LEGIONELLA ORGANISMS

Carbol-Fuchsin Solution FITE ACID-FAST STAIN FOR LEPROSY

MICROWAVE MODIFICATION OF THE WARTHIN-STARRY METHOD FOR BACTERIA

ZIEHL-NEELSEN METHOD FOR ACID-FAST BACTERIA

Citric Acid, 1% Solution Gelatin, 5% Solution

249 251

Uranyl Nitrate, 1% Solution

References

............................................ Chapter 11 ............................................ Pigments, Minerals, and Cytoplasmic Granules 254 254

Pigments

254

EXOGENOUS PIGMENTS

254

ENDOGENOUS HEMATOGENOUS PIGMENTS

255

ENDOGENOUS NONHEMATOGENOUS PIGMENT

255

Endogenous Deposits

256

Minerals

256

Cytoplasmic Granules

256 256 257 257

Special Staining Techniques

ARTIFACT PIGMENT S

GRIDLEY FUNGUS STAIN Chromic Acid, 4% Solution Aldehyde Fuchsin Solution

PRUSSIAN BLUE STAIN FOR FERRIC IRON Potassium Ferrocyanide, 2% Solution Nuclear-Fast Red (Kernechtrot) Solution Histotechnology 3rd Edition xi

258

TURNBULL BLUE STAIN FOR FERROUS IRON

258

Hydrochloric Acid, 0.06N Solution SCHMORL TECHNIQUE FOR REDUCING

259

SUBSTANCES 259 259 260

Ferric Chloride, 1% Stock Solution Metanil Yellow, 0.25% Solution FONTANA-MASSON STAIN FOR MELANIN AND ARGENTAFFIN GRANULES

261 261 261 262 262 262 263 264 264 265

Silver Nitrate, 10% Solution Gold Chloride, 0.2% Solution MICROWAVE FONTANA-MASSON STAIN

Fontana Silver Nitrate Solution Gold Chloride, 0.2% Solution Sodium Thiosulfate, 2% Solution GRIMELIUS ARGYROPHIL STAIN

Silver Nitrate, 1% Solution Nuclear-Fast Red Solution CHURUKIAN-SCHENK METHOD FOR ARGYROPHIL GRANULES

265

Citric Acid, 0.3% Solution

266

MICROWAVE CHURUKIAN-SCHENK METHOD

266

Citric Acid-Glycine Stock Solution GOMORI METHENAMINE-SILVER METHOD FOR

Chapter 12 ............................................ Immunohistochemistry 278

Introduction

278

General Immunology

278

ANTIBODY

278

ANTIGEN

278

POLYCLONAL ANTISERA

278

MONOCLONAL ANTIBODIES

279

RABBIT MONOCLONAL ANTIBODIES

219

Tissue Handling

279

FROZEN TISSUE FIXATION AND PROCESSING

280

FIXATIVES FOR PARAFFIN-PROCESSED TISSUE

280

PROCESSING

280

MICROTOMY

281

EPITOPE ENHANCEMENT OR RETRIEVAL

283

Methods of Visualization

283

IMMUNOFLUORESCENCE

283

ENZYME IMMUNOHISTOCHEMISTRY

FOR ARGYROPHIL GRANULES 267

URATES 267 268 268 269

269 270 270 271 271 272 272 273 273

214

Silver Nitrate, 5% Solution Stock Methenamine-Silver Nitrate Solution

284

BILE STAIN

Ferric Chloride, 10% Solution VON KOSSA CALCIUM STAIN

Silver Nitrate, 5% Solution ALIZARIN RED S CALCIUM STAIN

284

DIRECT METHOD

284

INDIRECT METHOD

284

UNLABELED, OR SOLUBLE ENZYME IMMUNE COMPLEX, METHOD

Alizarin Red S Stain, 2% Solution RHODANINE METHOD FOR COPPER

Saturated Rhodanine Solution (Stock)

Immunohistochemical Staining Methods

284

AVIDIN-BIOTIN METHODS

285

POLYMERIC DETECTION

MICROWAVE RHODANINE COPPER METHOD

Saturated Rhodanine Solution (Stock) Sodium Borate (Borax), 0.5%

285

Controls

285

POSITIVE CONTROLS

285

NEGATIVE CONTROLS

285

Antibody Evaluation and Validation

285

ANTIBODY SPECIFICATION SHEET

285

PREDILUTED AND CONCENTRATED

References

ANTIBODIES 286

ANTIBODY VALIDATION

287

STORAGE OF ANTIBODIES

287

BLOCKING REACTIONS

289

MULTILINK BIOTINYLATED SECONDARY ANTISERA

xii

289

DAB REACTION PRODUCT INTENSIFICATION

289

BUFFER SOLUTIONS

289

Commonly Used Antibodies and Their Applications

289

NEOPLASTIC TERMINOLOGY

289

Quality Control

290

RECOMMENDED QC FOR AN ANTIBODY

291

POSITIVE AND NEGATIVE TISSUE CONTROLS

292

RECOMMENDED QC FOR A TISSUE BLOCK

292

DAILY QC OF IMMUNOHISTOCHEMISTRY

292

STORAGE OF CONTROL SLIDES

292

Standardization

296

'troubleshooting Immunoperoxidase Techniques

291 297 297 298 298 299 300 300 300

308

Muscle Histology

308

Pathologic Changes in Muscle

309

Enzyme Histochemistry

310

Oxidation and Reduction

310

Properties of Enzymes

310

Preservation of Enzymes

Staining Techniques

310

Classification of Enzymes

BASIC PAP IMMUNOPEROXIDASE PROCEDURE

311

HYDRO LASES

312

OXIDOREDUCTASES

312

TRANSFERASES

312

Freezing Muscle Biopsy Specimens

314

a-NAPHTHYL ACETATE ESTERASE STAIN FOR

Modified PBS Buffer (Stock Solution) Primary Antibodies Swine Antirabbit Linking Serum ABC-IMMUNOPEROXIDASE PROCEDURE

Modified PBS Buffer (Stock Solution) AEC Acetate Buffer (O.OSM, pH 5.2)

301

HRP ENZYME-LABELED POLYMER PROCEDURE

301

Tris-Buffered Saline Solution (with Tween-TBST), pH 7.6 Ready to Use Primary Antibodies Chromogen Solution Retrieval Solution (pH 6.0)

302 302 302 303

Chapter 13 ............................................ Enzyme Histochemistry

References

315 315 315 315 315 315 315 316

MUSCLE BIOPSIES 0.2N Phosphate Buffer, pH 7.2 Pararosaniline Stock Solution Sodium Nitrite, 4% Solution a-Naphthyl Acetate in Acetone, 1% Solution lNHCl IN Sodium Hydroxide Incubation Solution (prepare just before use) NAPHTHOL AS-D CHLOROACETATE ESTERASE TECHNIQUE

316 317 317 317 317 317

Esterase Solution A Esterase Solution B Esterase Solution C O.lN HCl O.IM Sodium Barbital (Sodium Diethylbarbiturate) Working Esterase Solution

317

MAYER HEMATOXYLIN

318

ATPASE STAIN

318 318 318 318 319 320 321 321 322 322 323

Barbital Acetate Buffer Stock Solution A Barbital Acetate Buffer Stock Solution B (O.lN HCl) Barbital Acetate Buffer Working Solution Sodium Barbital Solution (use to make 10.4, 9.4, and incubation solutions) Incubating Solution ACID PHOSPHATASE IN MUSCLE BIOPSIES

2N HCl Incubating Medium ALKALINE PHOSPHATASE STAIN FOR MUSCLE BIOPSIES

0.2M Tris Incubating Medium

Histot.echnology 3rd Edition xiii

323 323 324 325 325 326 326 326

NADH DIAPHORASE Saline Solution Phosphate Buffer, pH 7.4 SUCCINIC DEHYDROGENASE (SDH) Phosphate Buffer, 0.2M, pH 7.6 Physiological Saline PHOSPHORYLASE STAIN FOR MUSCLE

Nonenzymatic Procedures for Muscle Disorders

328 328

MODIFIED GOMORI TRICHROME Gomori Trichrome Solution

329

Acknowledgment

330

References

............................................ Chapter 14 ............................................ Electron Microscopy 334 334

Fixation

335

FACTORS INFLUENCING FIXATION

335 335 335

FIXATIVE SOLUTIONS

336 336 336 336

Paraformaldehyde with Cacodylate Buffer Paraformaldehyde or Glutaraldehyde with Phosphate Buffer Formaldehyde with Phosphate Buffer (Modified Millonig Fixative) Formaldehyde-Glutaraldehyde (4CF-1G) Buffered PAF (Zamboni Fixative) Osmium Tetroxide with Cacodylate Buffer Osmium Tetroxide with Phosphate Buffer

Processing

337

TRANSITIONAL SOLVENTS

337

EMBEDDING MEDIA

337

PROCEDURE FOR ROUTINE PROCESSING AND

DEHYDRATION

SPURR EMBEDDING PROCEDURE FOR ROUTINE PROCESSING AND EPON EMBEDDING

339

PROCEDURE FOR LR WHITE PROCESSING FOR ELECTRON MICROSCOPY IMMUNOLABELING

340 341

Sectioning

341

KNIVES

342

CORRECTING PROBLEMS ENCOUNTERED IN SECTIONING

xiv

343 343 343 344 344

Staining 0.5-µm Sections

345 345

Staining Thin Sections

346 346

Special Techniques

347

CELL SUSPENSIONS (FLUIDS, CULTURES,

TOLUIDINE BLUE-BASIC FUCHSIN PROCEDURE Staining Solution TOLUIDINE BLUE STAINING Toluidine Blue, 2%

Lead Citrate Solution

BLOOD CELL PREPARATION PARASITES, ETC)

347

Processing Tissues Previously Embedded in Paraffin

347

Processing Tissue from an H&E-Stained Paraffin Section

348

Acknowledgment

348

References

FIXATIVES

337 337

339

Staining

Acetate Buffer, pH 5.9

328

335

343

SECTION THICKNESS

............................................ !=~~J!t~r. 1.s • ••••••••••••••••••••••••••••••••••••

Cytopreparatory Techniques 352

Cytopreparation

352 352

Collection GYNECOLOGIC CYTOLOGY

352

NONGYNECOLOGIC CYTOLOGY

353 354 354

Fixation

354 354

Smear Preparation

355

FLUIDS

366

Glossary

372

Index

PRE-FIXATIVES Saccomanno Fluid

DIRECT SMEARS

355

MUCOID SPECIMENS

356

SPARSELY CELLULAR SPECIMENS

357

FINE NEEDLE ASPIRATIONS

357

SPECIAL PROBLEMS

358

CHOOSING THE BEST METHOD

358

Liquid-Based Cytology

359 360

Cell Blocks

361 361

Cytology Staining

METHODS

HEMATOXYLIN

362

OG-6

362

EA

362 362 363 363 364 364

PAPANICOLAOU STAIN

364

SPECIAL STAINS

364

References

Orange G, 10% Stock Solution Orange G, Working Solution TOLUIDINE BLUE WET FILM Toluidine Blue CROSS CONTAMINATION

Histotechnology 3rd Edition xv

-

--~

Preface The reception of the first two editions of this text has far exceeded my expectations, and I am very grateful that it has found such a welcome place in the field of histotechnology. The field has changed, especially in the areas of immunohistochemistry and instrumentation, since the publication of the second edition, and there was a need to update the text; therefore I have asked Christa Hladik, AA, HT(ASCP)cm, QIHC, clinical laboratory manager, Neuropathology and Immunohistochemistry, UT Southwestern Clinical Laboratories, University of Texas Southwestern Medical Center at Dallas, TX, to join me as an author of the third edition. My experience in these areas has been limited due to my retirement several years ago. All chapters have been carefully reviewed and most have been updated or expanded. We have attempted to increase the emphasis on troubleshooting in many areas and have added numerous illustrations. We are also pleased to add a chapter on cytopreparatory techniques by Beth Cox, who is certified by ASCP as both a histotechnician and a specialist in cytology. It is our hope that this updated edition will continue to serve as a basic guide for all students of histotechnology, or for practicing technicians, technologists, residents, and pathologists seeking to gain a better understanding of the technology utilized in the histopathology laboratory.

We are especially grateful to Agatha Villegas and Nied Duckworth for assisting with the preliminary typing of many chapters; to Charles White III, MD, Director of the Division of

xvi

Neuropathology and Immunohistochemistry and Histology Laboratories, UT Southwestern Medical Center at Dallas, TX, for assistance with photographs, chapter review, and mentoring for the immunohistochemistry and instrumentation chapters; to Dennis Burns, MD, Division of Neuropathology, UT Southwestern Medical Center at Dallas, TX, for photomicrographs; to all the staff at UT Southwestern Medical Center at Dallas, TX, who work in the Neuropathology, Immunohistochemistry, and Histology Laboratories and in the gross room at St Paul University Hospital for their assistance with tissue preparation and staining. Major contributions were made by the following: Amy Davis, HTL(ASCP), Debra Maddox, HT(ASCP)QIHC, Ping Shang, HT(ASCP)QIHC, Pattie Seward, HT(ASCP), Dawn Bogard, HT(ASCP), Courtney Andrews, HTL(ASCP), Gwen Beasley, HT(ASCP), Eva Osborn, PA(ASCP), and Chan Foong, PA(ASCP), and Steve Lee, BS, HT(ASCP). Our thanks also go to Maureen Doran, HTL(ASCP), Chair of the Health and Safety Committee of the National Society for Histotechnology, for reviewing the Safety chapter and offering many helpful suggestions, and to Robert Lott, HTL(ASCP), who was able to provide help with images when needed. Again, to all of you who are students ofhistotechnology, who continue to search for answers in this field of part art and part science, and who care first and foremost about the quality of your work on the specimens entrusted to you, we dedicate this third edition.

.

I ..CHAPTER .....- . ............................. ..................................................... . ~

-·

Fixation

. . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . .. . .

•••••••••••••••••••••••••••••••••

~ -- !l

••••••••••••••••••••

~. ~. ~. ~..~. ! ..•. ~ .~ .~.................................................................... . On completing this chapter, the student should be able to do the following: 1.

Define the purposes of fixation

2.

Define: a. b. c. d. e.

autolysis fixation artifact pigment nonaqueous fixative f. coagulating fixative g. additive fixative - co1v>h 1 ~ •n h. hypertonic i. isotonic

3.

4.

Identify the factors that affect the quality of fixation and describe the effect of each factor on tissue (eg, temperature, size of tissue, time of fixation, or osmolality of fixative) Identify the properties, functions, and actions, and determine whether each action is an advantage or disadvantage of each of the following fixative reagents or solutions: a. b. c. d. e. f. g.

h. i.

j. k.

1. m. n. o. p. q. r. s. t.

5.

acetic acid acetone alcohols B-5 fixative Bouin solution Carnoy and methacarn solutions formalin (aqueous, buffered, neutralized, acetate formalin, formalin alcohol, calcium formalin, and formalin ammonium bromide) Gendre solution glutaraldehyde glyoxal Helly solution Hollande solution mercuric chloride Orth solution osmium tetroxide paraformaldehyde potassium dichromate Zamboni solution Zenker solution zinc formalin

d. e. f. g. h. i.

Gendre solution Helly solution Hollande solution Orth solution Zamboni solution Zenker solution

6.

Identify any special indication for use of each of the fixatives listed in objectives 4 and 5

7.

Identify which fixatives require postfixation washing, and identify the preferred washing agent

8.

Identify the fixation pigments and the conditions under which the pigment may be formed

9.

Identify which of the fixation pigments can be prevented and which of the fixation pigments can be removed

10. For fixation pigments that can be removed, state the method(s) of removal; for fixation pigments that can be prevented, state the method(s) of prevention 11. Explain the difference between buffered and neutralized formalin 12. State how paraformaldehyde differs from formaldehyde 13. Describe the difference between the terms formalin and formaldehyde 14. Identify the percentage and volume of formaldehyde in 1,000 mL of a 10% formalin solution

Identify the chemicals in: a. B-5 fixative b. Bouin solution c. Carnoy and methacarn solutions

15. Compare and contrast Zenker and Helly fixatives

16. List 2 methods of fixation other than using chemical reagents 17. Identify the preferred method of fixation (or lack of fixation) for a. enzyme histochemistry b. immunofluorescence c. skeletal muscle cross-striations (nonimmunohistochemical staining) d. pheochromocytomas e. electron microscopy f. urates l· mmunohistochemical methods tissue for trichrome stains

f'.

18. Identify which fixative reagents are protein coagulants and which are noncoagulants 19. Identify which fixative reagents are additive fixatives and which are nonadditive 20. If the reagent is an additive compound, identify the site or group with which the reagent reacts (if known) 21. Describe the effect of acetic acid on erythrocytes and collagen 22. Identify any reagents that have associated safety hazards and state the hazard and any special precautions required 23. Describe the action of zinc in fixation 24. Give the 2 major problems associated with fixation, and identify at least 3 corrective actions for each

•••• •

Histotechnology 3rd Edition

I

Definition A fixative alters tissue by stabilizing the protein so that it is resistant to further changes. Baker [1958] uses the following example to explain fixation: When a door is opened, its position can be changed easily, but if the door is fixed open, it is altered in such a way that it is stabilized and is resistant to change. A fixative must change the soluble contents of the cell into insoluble substances so that those substances are not lost during the subsequent processing steps. This change occurs by either chemical (fixative solutions) or physical (heat, desiccation) means in a process called denaturation. Denaturation causes the protein molecule to unfold and the internal bonds to become disrupted. In the process known as additive fixation, this disruption enables the proteir!.JQ__cQmbine chemically with a fixative molecule. and the protein then hPmmes insoluble [Feldman 1980] . With nonadditive fixatives (eg, alcohol, acetone), denaturation causes the protein to become less cap::ihle of maintaining an intimate rel0.5 ppm (action level), monitoring must be repeated every 6 months. If monitoring reveals employees at or above the STEL, monitoring must be repeated at least once a year under "worst case conditions." If the TWA is ::>0.5 ppm and also within the STEL limits, then monitoring does not have to be repeated, and a medical surveillance program does not have to be established unless there is a change in procedures or conditions that might change the exposure, or unless an employee exhibits signs or symptoms of exposure. Employers may discontinue exposure monitoring if results from 2 consecutive samplings, -;z.7 days apart, indicate exposure below the STEL and action levels. Monitoring results must be provided to the employees within 15 days, either by individual distribution or by posting. A medical surveillance program is required for all employees exposed to formaldehyde at or exceeding the action level or STEL, those workers exhibiting signs or symptoms of exposure, and those employees exposed during an emergency situation. The employer shall establish regulated areas in which the concentration of airborne formaldehyde exceeds either the TWA or STEL and post all entrances and access ways with signs bearing the following information shown in [f4.l].

DANGER: FORMALDEHYDE Irritant and Potential Cancer Hazard Authorized Personnel Only

[f4.l] Example of warning sign to be posted at all access points to areas where concentration of airborne formaldehyde exceeds the TWA or STEL. Histotechnology 3rd Edition

85

Employers shall institute engineering and work practice controls to reduce and maintain employee exposure to formaldehyde at or below the TWA and the STEL. Whenever engineering controls and work practice controls cannot reduce employee exposure to the PEL or below it, then approved respirators can be used to satisfy the exposure requirements of this standard. A written hazard communication program must be developed and available to the employees. Employers are required to provide training to all employees assigned to workplaces where there is exposure to formaldehyde at or above 0.1 ppm. For a more comprehensive discussion of the Formaldehyde Standard, either the text by Montgomery [1995] or by Dapson and Dapson [1995] should be consulted. The text by Dapson and Dapson [1995] and the NIOSH Pocket Guide to Chemical Hazards [1990] are handy references for the laboratory; together they provide synonyms, exposure limits, health hazards, physical hazards, handling precautions, incompatibilities, monitoring methods, skin protection, first aid, spill procedures, recommended respirators, disposal, target organ effects, and the EPA number of chemicals commonly used in histology.

PARTICULARLY HAZARDOUS SUBSTA N CES (REPRODUCTIVE TOX IN S, SELECT CARCINOGENS, AND SUBSTANCES WITH A H IGH DEGREE OF ACUTE TOXICITY)

Reproductive toxins comprise chemicals that affect the reproductive capabilities, including chromosomal damage (mutations) and effects on fetuses (teratogenesis). According to CAP, every chemical that is used in the laboratory must be evaluated for carcinogenic potential, reproductive toxicity, and acute toxicity. Results of those evaluations must be documented, and the policy and procedure manual must define specific handling requirements for these chemicals according to OSHA directives. In defining the toxic dose of a substance, different terms may be encountered in the literature. The toxic dose low is defined as the lowest dose of a substance that will produce any toxic effect in humans when introduced by any route other than inhalation; substances that are toxic by inhalation are reported as toxic concentration low. The lethal dose low is usually reported for a substance and is defined as the lowest dose reported to have caused death in humans, or as the lowest single killing dose reported for animals. The LD511 is the calculated dose of a chemical substance expected to cause the death of 50% of an experimental animal population, as determined by exposure to the substance by any route other than inhalation. Examples of toxic chemicals are the hydrocarbons (eg, xylene and toluene), formaldehyde, and some metallic compounds (eg, mercury, chromium, and silver). Designated area indicates an area that may be used for work with select carcinogens, reproductive toxins, or substances with a high degree of acute toxicity. A designated area may be the entire laboratory, an area of a laboratory, or a device such as a laboratory hood.

CARCINOGENS

Carcinogens are substances that cause or greatly increase the risk of malignant disease. OSHA defines select carcinogens under the 86 Safety I Ch 4

Laboratory Standard as any substance that meets 1 of the following criteria [OSHA 1987]: 1. It is regulated by OSHA as a carcinogen.

2. It is listed as "known to be carcinogen" by the National Toxicology Program (NPT). 3. It is listed by the International Agency for Research on Cancer (IARC) under group 1, 2A or 2B. Bis-chloromethyl ether, formed by the reaction between hydrochloric acid and formaldehyde, induces lung cancer; chloroform, chromic acid, pararosaniline, and benzidine-based dyes are among other probable carcinogens identified in histopathology. Formaldehyde has been identified as a potential human carcinogen and is regulated under the formaldehyde standard.

CORROSIVE SUBSTANCES

For disposal purposes, corrosive hazards are defined officially as those substances that, by direct contact, will corrode SAE 1020 steel at a rate >6.25 mm/year at 55°C, or more simply, as substances that are capable of destroying mild steel under certain defined conditions. The acids are considered corrosive substances by this definition. Contact with most metallic surfaces should be avoided with all substances that pose a physical corrosive threat. As health hazards, corrosive substances are defined as those substances that will cause injury to the skin and eyes by direct contact or severe damage to the tissues of the respiratory and alimentary tracts when inhaled or ingested. The effects of corrosive chemicals lead to disruption of cell membranes, coagulation of proteins, and death of essential cellular components.

FIRE AND EXPLOSION HAZARDS

Although liquid organic compounds are most commonly thought of as fire hazards, certain chemicals such as dry picric acid, benzoyl peroxide, and ammoniacal silver solutions can be explosion hazards. Fire is defined as the rapid oxidation of a fuel in the presence of an ignition source, with liberation of heat and light [Moya 1980]. This sequence of events is called the fire triangle. Air, or oxygen, and fuels are mixed closely everywhere in the environment, but fires will not occur unless an ignition source is present; the 3 elements necessary for a fire are present in the laboratory. Fires are classified into 4 groups (classes A, B, C, and D) [t4.l]. Fire extinguishers are classified and rated in 4 groups corresponding to the class of fire that they are able to extinguish. Class A includes water-based, foam or loaded-stream, and multipurpose dry extinguishers. Class B includes carbon dioxide, dry chemical, foam and loaded-stream, bromotrifluoromethane (Halon 1301), and bromochlorodifluoromethane (Halon 1211) extinguishers. Class C includes the Halon, carbon dioxide, and dry chemical extinguishers. Class D extinguishers contain dry powder media that will not react or combine adversely with the burning materials. Class D fires are unlikely in the

histopathology laboratory. ABC extinguishers (also called allpurpose or tri-class) are preferred, and an extinguisher must be no more than 50 feet away from flammable or combustible liquids [Dapson 2007]. Documentation must verify that all personnel have been instructed in the correct use of extinguishers. Health hazards may be created by the use of automatic fire extinguishing systems because of the concentration of the extinguishing agent or the toxic products of decomposition that may result from exposure of the extinguishing agent to a fire or hot surfaces. OSHA limits the Halon and carbon dioxide concentrations released by fixed extinguishing systems when employees are exposed for only a short period. In areas in which employees would normally remain after the automatic discharge of the extinguishing agent, OSHA prohibits the use of Halon 1211 and carbon dioxide. The OSHA [OSHA 1987] and National Fire Protection Association (NFPA) ratings [NFPA 1980] for flammable and combustible materials are as shown in [t4.2]. The flash point of a liquid is the lowest temperature at which sufficient vapors are produced to form an ignitable mixture with air near the surface of the liquid or within the container used. It is the vapors, rather than the liquid itself, that contribute to fire or explosion. Each flammable liquid has a vapor concentration range in air below which the vapor-air mixture is too lean to burn or explode and above which the mixture is too rich to burn. Personnel who work with flammable solvents should be aware of the important flammability characteristics of each solvent in use, and should be familiar with, and follow, the manufacturer's precautions provided on the labels. Many of the flammable liquids also are toxic, so personnel must be aware of this additional hazard. The hydrocarbons and alcohols used in histopathology are all fire hazards. The flash points of some common laboratory solvents are shown in [t4.3]. Most fires can be avoided if all laboratory personnel follow good safety practices. NFPA and OSHA require that solvents be stored in fire safety cabinets and safety cans. Each laboratory should have an emergency plan that clearly defines actions that both the employer and the employee must take in a life- or injurythreatening emergency. An employee education program is necessary and should designate specific personnel to be responsible for each part of any emergency plan.

[t4. l] Classification of fires Class

Description

Class A

Fires involving ordinary combustible materials such as wood, plastics, paper, and textiles. Class A fires can be extinguished with water or water-based solutions.

Class B

Fires involving flammable liquids and gases, requiring that oxygen be blocked from the fuel in order to be extinguished.

Class C

Electrical fires that must be extinguished with nonconductive media.

Class D

Fires of combustible and reactive elements, such as metallic sodium, potassium, magnesium, and lithium. Class D fires are difficult to control and extinguish because spreading and explosion can occur easily.

[t4.2] Classification of flammable and combustible materials Class

Description

Class I

Flammable liquids (flash point