Diagnostic Techniques in Veterinary Dermatology E-Book

Ariane Neuber

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 449

Veröffentlichungsjahr: 2017

Beschreibung

The first book devoted solely to the techniques used to investigate skin problems in animals A practical everyday reference for veterinary practitioners, Diagnostic Techniques in Veterinary Dermatology focuses on contemporary techniques for investigating skin problems in small animals, horses and exotic pets. Written by experienced specialists in veterinary dermatology, this book offers clear, step-by-step guidance on how to perform tests and interpret their results. The first book devoted exclusively to the subject, this hands-on guide demonstrates how to carry out and interpret a huge range of dermatology tests, as well as how to avoid common mistakes and pitfalls. Featuring full colour photographs and illustrations throughout, key topics include: looking for parasites, hair plucks and trichograms, dermoscopy, cytology, fungal and bacterial cultures, histopathology, allergy testing, immune-mediated skin diseases, endocrine and metabolic skin diseases, infectious diseases, diagnostic imaging, otoscopy and examination of the ear, genetic tests, and more. Diagnostic Techniques in Veterinary Dermatology is a valuable working resource for busy practitioners in first opinion practice, as well as veterinary nurses and technicians. It is also an ideal reference for veterinary students and specialists in-training.

Leseprobe

CONTENTS

Cover

Title Page

Copyright

Chapter 1: Introduction to Dermatological Tests

What Equipment Will You Need?

Use and Abuse of the Practice Microscope

Stains

Using Internal and External Laboratories

Sensitivity, Specificity, and Positive and Negative Predictive Value

Chapter 2: Looking for Parasites

Introduction

Visual Inspection

Coat Brushings

Tape-Strips

Hair Plucks

Skin Scrapes

Pustules and Draining Sinus Tracts

Potassium Hydroxide Versus Liquid Paraffin

Vacuuming

Faecal Examination

Parasites in the Environment

Specificity and Sensitivity

Common Mistakes and Pitfalls

Parasite Identification Guide

Chapter 3: Hair Plucks and Trichograms

Introduction

Technique

Hair Pluck Findings and their Significance

Chapter 4: Dermoscopy

Dermoscopes

Uses of Dermoscopy

Chapter 5: Cytology

Introduction

Indications for Cytology

Cytological Techniques

Cytological Findings

Algorithmic Approach

Common Pitfalls and Mistakes

Chapter 6: Fungal and Bacterial Cultures and Identification

Introduction

Commensal and Transient Organisms

When to Perform Cultures

Obtaining Material for Culture

How to Interpret Bacterial Culture and Sensitivity Results

Common Pitfalls for Bacterial Cultures

Wood's Lamp

Dermoscopy

Fungal Culture Media

Identifying Common Fungal Pathogens

Detecting Unusual Fungal and Bacterial Organisms (e.g. Deep Fungal Infections and Mycobacteria)

Antifungal Susceptibility Testing

Chapter 7: Introduction to Histopathology

Indications for Performing a Biopsy

Considerations Before Performing a Biopsy

Biopsy Technique

Submission of the Samples

Does the Histopathology Make Sense?

The Histopathology Report

Basic Histopathological Terminology

Pattern Analysis

Hair Follicle Pathology

Chapter 8: Allergy Testing

Food Trials

Food-Allergen Serology and Patch Tests

Allergy Testing for Environmental Allergens

Chapter 9: Immune-Mediated Skin Diseases

Skin Biopsy and Histopathology

Specific Serology Tests

Anti-Nuclear Antibody Test

Coomb's Tests

Anti-Platelet Antibodies

Rheumatoid Factor

Cold Agglutinin Titres

Urinary Protein

Other Tests

Chapter 10: Endocrine and Metabolic Skin Diseases

Clinical Signs Associated with Endocrinopathies

Hair Plucks

Routine Haematology and Biochemistry

Urine Analysis

Analysis of Thyroid Function for Dogs with Suspected Hypothyroidism

Analysis of Thyroid Function for Cats with Suspected Hyperthyroidism

Tests to Confirm Hyperadrenocorticism

Sex Hormone Assays

Miscellaneous Endocrine and Metabolic Tests

Chapter 11: Infectious Diseases

Laboratory Techniques

Diagnosis of Infectious Diseases

Virology Techniques

Diagnosing Viral Diseases

Chapter 12: Diagnostic Imaging

Imaging for Patients with Ear Disease

Imaging for Patients with Endocrine Disease

Imaging for Patients with Paraneoplastic Associated Skin Disease

Other Uses for Imaging

Chapter 13: Otoscopy and Examination of the Ear

Examination of the Pinnae, Ear Canal and Tympanic Membrane

Techniques to Assess the Integrity of the TM

Sample Collection for Cytology

Interpretation of Otic Cytology

Bacterial Culture and Antimicrobial Sensitivity Testing

Myringotomy and Evaluation of the Middle Ear

BAER Testing

Biopsy

Chapter 14: Which Test to Choose When

Introduction

Pruritus

Otitis

Alopecia

Scaling and Crusting Dermatoses

Pigmentary Changes

Macules, Papules and Pustules

Ulcers and Erosions

Importance of Making the Correct Diagnosis and Specialist Referral

Chapter 15: Genetic Tests for Skin Diseases

Patterns of Inheritance

Genetic Tests

Breeding Advice

References

Index

End User License Agreement

List of Tables

Table 1.1

Table 4.1

Table 6.1

Table 6.2

Table 6.3

Table 6.4

Table 7.1

Table 7.2

Table 8.1

Table 8.2

Table 8.3

Table 8.4

Table 8.5

Table 8.6

Table 8.7

Table 9.1

Table 9.2

Table 12.1

Table 12.2

Table 14.1

Table 14.2

Table 14.3

Table 14.4

Table 14.5

Table 14.6

Table 14.7

Table 14.8

Table 15.1

Table 15.2

Table 15.3

Table 15.4

Table 15.5

Table 15.6

Table 15.7

List of Illustrations

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

Figure 1.7

Figure 1.8

Figure 1.9

Figure 1.10

Figure 1.11

Figure 1.12

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Figure 2.9

Figure 2.10

Figure 2.11

Figure 2.12

Figure 2.13

Figure 2.14

Figure 2.15

Figure 2.16

Figure 2.17

Figure 2.18

Figure 2.19

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Figure 3.10

Figure 3.11

Figure 3.12

Figure 3.13

Figure 3.14

Figure 3.15

Figure 4.1

Figure 4.2

Figure 4.3

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

Figure 5.10

Figure 5.11

Figure 5.12

Figure 5.13

Figure 5.14

Figure 5.15

Figure 5.16

Figure 6.1

Figure 6.2

Figure 6.3

Figure 6.4

Figure 6.5

Figure 6.6

Figure 6.7

Figure 6.8

Figure 6.9

Figure 6.10

Figure 6.11

Figure 6.12

Figure 6.13

Figure 6.14

Figure 6.15

Figure 6.16

Figure 6.17

Figure 7.1

Figure 7.2

Figure 7.3

Figure 7.4

Figure 7.5

Figure 7.6

Figure 7.7

Figure 7.8

Figure 7.9

Figure 7.10

Figure 7.11

Figure 7.12

Figure 7.13

Figure 7.14

Figure 8.1

Figure 8.2

Figure 8.3

Figure 8.4

Figure 8.5

Figure 8.6

Figure 8.7

Figure 8.8

Figure 8.9

Figure 8.10

Figure 8.11

Figure 8.12

Figure 9.1

Figure 9.2

Figure 9.3

Figure 9.4

Figure 9.5

Figure 9.6

Figure 9.7

Figure 9.8

Figure 9.9

Figure 9.10

Figure 10.1

Figure 10.2

Figure 10.3

Figure 10.4

Figure 10.5

Figure 10.6

Figure 10.7

Figure 10.8

Figure 10.9

Figure 10.10

Figure 10.11

Figure 10.12

Figure 10.13

Figure 10.14

Figure 10.15

Figure 10.16

Figure 10.17

Figure 10.18

Figure 10.19

Figure 11.1

Figure 11.2

Figure 11.3

Figure 11.4

Figure 11.5

Figure 11.6

Figure 11.7

Figure 11.8

Figure 11.9

Figure 11.10

Figure 11.11

Figure 12.1

Figure 12.2

Figure 12.3

Figure 12.4

Figure 12.5

Figure 12.6

Figure 12.7

Figure 12.8

Figure 12.9

Figure 12.10

Figure 12.11

Figure 12.12

Figure 12.13

Figure 12.14

Figure 13.1

Figure 13.2

Figure 13.3

Figure 13.4

Figure 13.5

Figure 13.6

Figure 13.7

Figure 13.8

Figure 13.9

Figure 13.10

Figure 13.11

Figure 13.12

Figure 13.13

Figure 13.14

Figure 13.15

Figure 13.16

Figure 13.17

Figure 13.18

Figure 13.19

Figure 13.20

Figure 13.21

Figure 13.22

Figure 13.23

Figure 14.1

Figure 14.2

Figure 14.3

Figure 14.4

Figure 14.5

Figure 14.6

Figure 14.7

Figure 14.8

Figure 15.1

Figure 15.2

Figure 15.3

Figure 15.4

Guide

Cover

Table of Contents

Chapter 1

Pages

iii

iv

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

Diagnostic Techniques in Veterinary Dermatology

Ariane Neuber

DrMedVet CertVD DECVD MRCVSDirector, Derm4PetsHead of Dermatology, Chiltern Referral ServicesChalfont St Giles, UK

Tim Nuttall

BSc BVSc CertVD PhD CBiol MRSB MRCVSHead of DermatologyRoyal (Dick) School of Veterinary StudiesUniversity of EdinburghEdinburgh, UK

This edition first published 2017

© 2017 Ariane Neuber and Tim Nuttall.

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, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions.

The right of Ariane Neuber and Tim Nuttall to be identified as the authors of this work has been asserted in accordance with law.

Registered Office(s)

John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA

John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK

Editorial Office

9600 Garsington Road, Oxford, OX4 2DQ, UK

For details of our global editorial offices, customer services, and more information about Wiley products visit us at www.wiley.com.

Wiley also publishes its books in a variety of electronic formats and by print-on-demand. Some content that appears in standard print versions of this book may not be available in other formats.

Limit of Liability/Disclaimer of Warranty

The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting scientific method, diagnosis, or treatment by physicians for any particular patient. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

Library of Congress Cataloging-in-Publication Data

Names: Neuber, Ariane, 1970- author. | Nuttall, Tim, 1967- author.

Title: Diagnostic techniques in veterinary dermatology : a manual of diagnostic techniques / Ariane Neuber, Tim Nuttall.

Description: Hoboken, NJ, USA : Wiley-Blackwell, 2017. | Includes bibliographical references and index. |

Identifiers: LCCN 2017005977 (print) | LCCN 2017015845 (ebook) | ISBN 9781119233046 (Adobe PDF) | ISBN 9781119233060 (ePub) | ISBN 9781405139489 (paperback)

Subjects: LCSH: Veterinary dermatology. | MESH: Skin Diseases–veterinary | Dog Diseases–diagnosis | Cat Diseases–diagnosis

Classification: LCC SF901 (ebook) | LCC SF901 .N48 2017 (print) | NLM SF 992.S55 | DDC 636.089/65–dc23

LC record available at https://lccn.loc.gov/2017005977

Cover images: courtesy of the authors

1Introduction to Dermatological Tests

Recent studies show that skin and ear diseases comprise 25% of all veterinary consultations. They are often complex and ongoing conditions that are a challenge to manage. Very few can be diagnosed on history and appearance alone. The modern approach to dermatology emphasises using the history and clinical signs to construct a logical differential diagnosis list. The diagnosis is then achieved by utilising appropriate tests to eliminate and/or confirm conditions in the differential diagnosis. On the other hand it is all too easy to become over-reliant on tests; it is most important that the clinical pathology is made to fit the history and clinical signs, not vice versa.

There is a very wide range of tests that can be used to investigate skin problems, but selecting, performing and interpreting the most appropriate tests in each case requires some experience. Many textbooks and journals, however, concentrate on individual skin conditions and assume that the reader is experienced enough to undertake the relevant diagnostic procedures. In practice, some of the most common reasons for poor management of skin conditions involve the inappropriate use of diagnostic tests, suboptimal execution of test procedures, inadequate sample choice and misinterpretation of results. The aim of this book, therefore, is to provide an illustrated, step-by-step guide to help you select, perform and interpret clinical tests and procedures for a range of dermatological presentations.

What Equipment Will You Need?

A wide range of equipment is necessary for thorough examination of the skin, which may seem daunting. The vast majority, however, are inexpensive, non-specialist items that are common to virtually all veterinary practices and do not need special skills to operate. The few items that are expensive and/or need specific training to use are all optional; they are undoubtedly useful, especially to dermatology specialists, but are not necessary to successfully practise veterinary dermatology.

Essential Equipment

Good lighting is essential for proper examination of the skin, lesions and collected material. Good fluorescent room lighting is a minimal requirement and a high-intensity spotlight is necessary for any serious examination.

Flea comb for coat combings (

Figure 1.1

).

Hand lens or magnifying glass for close examination of the skin, coat and collected material; the large illuminated lenses sold for reading are most useful (

Figure 1.2

).

A good-quality binocular microscope for examining hair plucks, skin scrapes and cytology (

Figure 1.3

).

Glass slides for mounting material; frosted slides are easier to label (

Figure 1.1

and

Figure 1.4

).

Cover-slips are essential for any microscopic examination (

Figure 1.4

).

Immersion oil for using the ×100 oil immersion microscope lens; different types of oil with different viscosities are available. Type A is the least viscous and is often preferred as it is the least messy and the cheapest. Type NVH is the most viscous. For in-house use, type B would also be suitable also but it is messier than type A (

Figure 1.4

).

Lens tissue or cloth and cleaning fluid or alcohol for cleaning microscope lenses without damage.

Otoscope for examining the ears (

Figure 1.5

).

Wood's lamp for screening for fluorescent dermatophytes (

Figure 1.2

).

Electric clippers for removing hair, allowing access to the skin.

Curved scissors for more precise and less traumatic hair removal.

Fine-tipped curved artery (mosquito) forceps for hair plucks (

Figure 1.1

).

Liquid paraffin for skin scrapes (

Figure 1.1

).

20% potassium hydroxide as a clearing agent when looking for parasites or dermatophytes (optional).

No. 10 and No. 15 scalpel blades for skin scrapes and biopsies (

Figure 1.1

).

Cotton buds for collecting material from the ears (

Figure 1.6

).

Adhesive tape (Sellotape®, Scotch Tape® etc.) for skin surface parasites and cytology (

Figure 1.1

).

Sterile bacteriology swabs with and without transport media; fine-tipped ENT swabs are useful for taking samples from narrow sites or when using otoscopes etc. (

Figure 1.6

).

Sterile universal (30 mL) and bijoux (5 mL) containers for storing and transporting tissue samples (

Figure 1.7

).

Diff-Quik® type stain for routine cytology (

Figure 1.4

).

Toothbrushes to collect material for dermatophyte culture (

Figure 1.8

).

Syringes and needles of various sizes for taking blood samples and aspirates (

Figure 1.9

).

Blood collection tubes: plain, EDTA, heparin and gel-clotting tubes.

Indelible marker pen to mark biopsy and skin test sites.

4-, 6- and 8-mm skin biopsy punches (

Figure 1.7

).

Basic surgical kit and suture material for performing skin biopsies and closing skin wounds.

10% neutral buffered formalin for fixing biopsy specimens (

Figure 1.7

).

Figure 1.1 Some equipment needed for taking samples for skin parasitology: a flea comb, clear sticky tape, No. 10 scapel blades, cotton buds, liquid paraffin, artery forceps for taking hair pluckings and microscopic slides.

Figure 1.2 A magnifying lens with illumination. In this case the lamp doubles up as a Wood's lamp for dermatophyte detection.

Figure 1.3 A good-quality microscope is essential to be able to correctly identify parasites, cells and microorganisms in the samples examined. The image shows a trinocular microscope with the option to attach a camera to document the findings.

Figure 1.4 Essential microscope equipment: glass slides with a frosted edge for easy labelling, glass cover-slips to improve the optic performance under the microscope, immersion oil and a modified Romanowsky-type rapid staining solution kit.

Figure 1.5 Equipment used for otoscopy: a handheld otoscope, various sizes of cones to be attached to the otoscope (all packaged individually after autoclaving to sterilise the cones after each use) and a cone cleaner to remove otic debris after use prior to sterilising.

Figure 1.6 Equipment for obtaining samples from the ear canal: cotton buds, bacteriology swabs with transport medium, liquid paraffin and glass slides.

Figure 1.7 Some equipment for obtaining and transporting tissue samples (skin biopsy specimens): single-use biopsy punches in varying sizes and sterile containers with formalin saline for histopathology or empty for tissue culture.

Figure 1.8 Materials to obtain samples for dermatophyte culture/perform in house cultures: clear sticky tape (for direct microscopy), liquid paraffin (for direct microscopy), sterile toothbrushes, artery forceps for hair plucks, glass slides for direct microscopy and a combined dermatophyte test medium (DTM) and Sabouraud agar plate for culture.

Figure 1.9 Materials for fine-needle aspirates: different gauge sterile single-use needles, 5-ml syringes and glass slides.

Optional Equipment

Dermatophyte test medium for in-house dermatophyte culture.

Lactophenol cotton blue is useful for staining dermatophyte colonies for identification if in-house cultures are performed.

Special laboratory stains for cytology: Grams, Leishman, Giemsa, Ziehl–Nielsen etc.

Intradermal allergen test kit for more experienced clinicians with an interest in dermatology to perform allergy testing in atopic dermatitis (

Figure 1.10

).

Video-otoscope (

Figure 1.11

).

Figure 1.10 Allergens for intradermal allergy testing. The relevant allergens will vary regionally.

Figure 1.11 A video-otoscope is very helpful for performing deep ear flushes.

Use and Abuse of the Practice Microscope

No piece of equipment is so vital or subject to so much abuse as a microscope. Robust and inexpensive models with binocular lenses and integral light sources are easily mastered and give good results, but need looking after and must be used correctly for the best results.

Which Microscope Should I Buy?

Monocular microscopes or, even worse, those with mirrors for external lights, belong in a museum. The ideal microscope should have binocular eyepieces (with one capable of independent focusing), an integral light source, a focusing condenser, a mechanical stage, coarse and fine focus, and four lenses – ×4, ×10, ×40 and ×100 oil immersion (eyepieces are usually ×10 giving a final magnification of ×40 to ×1000). Dry (non-oil) ×60 lenses with a final magnification of ×600 are sometimes used instead of the oil immersion lens. Wide-field, stand-off eyepieces with rubber cups are best, as they can also be used when wearing glasses by folding the rubber cups down. The light source should provide white light (e.g. by using an LED) or have a daylight filter to convert the yellow–orange tungsten light to daylight (i.e blue–white). More expensive microscopes have a filter mount above the light source so that a variety of other filters can be used, although this is rarely necessary in general practice.

You essentially get what you pay for with microscopes. Despite this, budget models with the above features are perfectly adequate for most routine practice use. Cheaper lenses, however, can result in a curved edge to the field of view, which can be disorientating and/or trigger motion sickness. More expensive, flat-field (or planform) lenses achieve an even depth of field across the whole image and avoid edge effects. More regular or intensive users may appreciate the increased robustness, finer movement and improved image that come with more expensive, better-quality models. It is often possible to try various models on loan first to select one that best fits your budget and requirements.

Trinocular mounts or multiple-headed microscopes are more expensive and are generally used where hands-on teaching or archiving of images is important. In general practice, however, cameras can also be useful for real-time video to show other staff and/or clients, and taking still images for clinical records or to send for a second opinion. The best results are from using trinocular mounts connected to digital cameras and monitors. It is also possible attach inexpensive CCD cameras to eyepieces and feed images directly to a computer, although the quality is not usually as good as using a dedicated camera and mount. Some camera-equipped mobile phones and tablets placed against an eyepiece can also take fairly decent images.

Microscope Set-Up: Where Should It Be?

It is important that the microscope is situated where it is comfortable to use. Discomfort will lead to you skimming slides in order to finish them as quickly as possible or, in the worst case, not using the microscope at all. There should be a dedicated site for microscopy with enough working surface and shelving for the microscope, clinical notes, clinical slides and ancillary equipment, such as clean slides, cover slips, immersion oil and cleaning materials. The working surface should be firm and stable. An adjustable seat (ideally with lower back support) will allow different users to maintain a comfortable, upright position. Some users may like to use elbow or forearm pads; placing split foam tubing insulation over the edge of the bench can also help. It is important to examine and adjust the arrangements if using the microscope causes strain.

Microscope Set-Up: Eye-Pieces and Illumination

It is also important to set the microscope up correctly. Incorrect adjustment can result in poor-quality images, eye strain, headaches and motion sickness. If these problems cannot be solved by the fairly simple steps outlined below or cleaning, get the microscope professionally serviced.

Alter the separation between the eyepieces until you see a single, circular image.

Focus to get a sharp image with relaxed eyes; don't strain to get the image in focus.

Adjust the focus between the eyepieces for your eyesight. First focus normally on a slide with one eye looking through the fixed (non-adjustable) eyepiece. Next, close your first eye and then correct the focus for your other eye, if necessary, by adjusting the other eyepiece (using its independent focus). You will now have the two binocular images in perfect focus for each eye. Some microscopes enable you to note down the adjustment, which is useful for multiple users with different eyesight.

Koehler illumination (

Figure 1.12

) focuses the light source on the slide, giving the optical illusion that the light comes from the sample. This avoids transillumination and gives you the best light balance and image quality. It is very easy to set up:

– close the light source lens diaphragm so that you can see the edges in the image field using the ×4 or ×10 objective;

– focus the condenser until edges of the diaphragm are sharp;

– adjust the condenser centring screws until the light source is centred in the field of view;

– open the diaphragm so that it is no longer visible and there is even illumination of the field of view;

– some older microscopes do not have a light source lens diaphragm – if this is the case, hold a thin piece of card, paper, a paper-clip, or the tip of a pen or pencil against the light source and focus the condenser until you see a sharp image outline in the image field.

You can now adjust the iris diaphragm to give the clearest image for each lens:

– for parasites and dermatophytes it is useful to close the iris diaphragm – the image is poorer but the increased contrast makes the parasites or fungi stand out better on unstained samples;

– for cytology of stained preparations under high power, open the diaphragm to reduce contrast and improve the detail of the nucleus, cytoplasm, granules and microorganisms.

Figure 1.12 Koehler (Köhler) Illumination provides optimum contrast and image quality by focusing and spreading the light source evenly over the field of view. In these figures the light source diaphragm has been closed to set up Koehler illumination (in the absence of a light source diaphragm this can be approximated by holding a paperclip, piece of card or pencil point against the light source). a) The edge of the diaphragm is not in focus with the sample, which will result in poorer-quality images. b) Adjusting the condenser focus wheel (which moves the condenser lens independently of the slide stage) will bring the edge into focus and optimise the image.

Dark Field Microscopy

Dark field microscopy is rarely used in clinical practice and few practice microscopes have dark field capability. However, it can be useful in liquid phase non-stained preparations (e.g. urine sediments, Leptospira, Treponema paraluiscuniculi, some endoparasites, insects in bedding, forage mites in foods etc.). It is also fairly easily to quickly modify most microscopes to do this.

The principle is that the centre of the light source is occluded, leaving the specimen illuminated only by scattered light from the margin. This gives a bright view of the specimen surrounded by a dark background. Dark field microscopes have occluding discs built in to the condenser. However, a similar effect can be achieved using discs of black card or insulation tape fixed to glass slides or discs and held against the light source, fitted to the light source filter holder or held against the base of the condenser. Ideally the edge of the disc should be just wider than the field of view for each lens. The condenser diaphragm and light intensity should be adjusted to give the best contrast for each view.

Scanning the Field

You should always use a cover-slip (or cover glass). The only exceptions are tape-strip preparations where the tape is, in effect, its own cover-slip. Microscope lenses are designed to ‘look’ through a cover-slip and fluid layer, which provides a flat optical surface, puts material in a similar focal field, avoids air–fluid interfaces and reduces contrast. Curved surfaces (including oil on top of the cover-slip) act as mini-lenses and cause serious distortion. Cover-slips can also protect the lenses from scratches and the mounting fluid, and provide a defined search area for skin scrapes etc. Mounting fluids include immersion oil and liquid paraffin or potassium hydroxide, if these were used for skin scrapes or for clearing hairs prior to examination for dermatophytes etc. Cover-slips can be permanently sealed to slides using mounting solutions such as DPX (or, if necessary, clear nail varnish), but this is only really needed if the slides are to be archived. It is important to use enough mounting fluid to seal the cover-slip to the slide without gaps or air bubbles, but not so much that it gets onto the microscope lenses or stage. Be very careful not to get any DPX or similar mountant onto the microscope lenses or stage.

Initially, visually examine the slide to orientate it, and appreciate the depth of material, degree of staining and possible areas of interest. It is useful to check the quality of staining visually or under low power before placing a cover-slip, as the slide can be re-stained at this stage if necessary. You should then study the slide at progressively higher magnification. Low-power lenses (×4–×10) are useful to scan large areas; with scrapes, plucks etc., start at one corner of the cover slip, proceeding down to the opposite edge, across one visual field and back up again to methodically search all the collected material. Use the high-power lens (×40) to close in on areas of interest. Use the oil-immersion lens (×100) last of all to avoid getting oil on the other lenses.

It is important that microscopic preparations are thin enough to allow proper examination without having to continually change focus. This is time consuming, tiring and diagnostic findings are easily missed. It is also time consuming to examine preparations that are too thin and spread out. It further follows that any material outside the area of the cover-slip will not be examined at all.

Cleaning

Cleaning is really, really important. In the first instance, make every effort to avoid contaminating the rest of the microscope with oil and debris. Many non-oil immersion lenses are not oil-proof, and will need replacing if oil gets in. Dried oil and skin debris can also clog up and jam stages and focus wheels. Clean the lenses and stage with lens cleaner and lint-free, fine tissue or lens cloths immediately after use. Commercial lens cleaner is best, but methylated spirits or similar alcohols are also effective. Do not use anything that will leave a residue, or coarse paper or cloths that could damage the lenses or leave fine particles of lint etc. Dried on immersion oil and debris can be removed by carefully rubbing the lens on polystyrene soaked in lens cleaner.

Stains

Diff-Quik® Type Stains

There are several Diff-Quik® type stains available. These are modified Romanowsky or Wright–Giemsa stains used to identify cells and microorganisms. They are the most commonly used stains in practice by virtue of their ease of use and interpretation. There are three pots: fixer (usually methanol or methylated alcohol, which is pale blue to green), stain 1 (eosinophilic – red) and stain 2 (basophilic – deep blue to purple). Staining efficiency declines over time and the pots can also accumulate skin debris, Malassezia and bacteria that can contaminate slides. In a busy laboratory, dispose of old solutions, rinse the pots and replenish from the stock solutions every 1–2 weeks.

There is a variety of ways to dry, fix and stain cytology samples. These have their advantages and disadvantages, and to some extent the method depends on personal preference. However, it is helpful to think about the sample and the method that might be best in each situation.

Two-Stain Method

Dip air-dried smears or tape-strips in each pot five to ten times for 1 second each time. The staining time will need to be longer for thicker, oily or waxy preparations. Rinse under a tap or with distilled water (directing the flow against the back of the slide stops you flushing your preparation down the sink) and gently blot dry using textured paper towel without damaging the stained preparation. Wait for the rest of the water to evaporate or use a hand- or hair-dryer to completely dry the slide before applying a mounting agent and cover slip.

One-Stain Method

With this method simply put a drop of the basophilic stain directly onto the cytology preparation, place a cover-slip and examine. Alternatively, the stain can be rinsed off and the preparation dried as above. With tape-strips, place one drop of the stain on the slide, stick the tape down over the stain, blot away the excess and examine. This approach is very quick and easy, and particularly suitable for tape-strips or waxy preparations that are hard to dry and stain. This method, however, leads to a more monochromatic stain. This is perfectly adequate for identifying microorganisms, such as bacteria and Malassezia, but distinguishing different cell types is harder.

Checking Staining

Once the slide is ready, check the staining under low power; if it is adequate add a drop of oil and a cover slip; if not, re-stain. If necessary oil can be removed from slides for re-staining: place a piece of lens tissue on the slide, add one to two drops of xylene, acetone or methanol, allow the oil to pass into the lens tissue, and then carefully move it across the slide to remove the oil without damaging the smear. De-staining may be necessary if the slide is over-stained (the basophilic stain in particular can colour over cellular detail) or if using an alternative stain (e.g. Gram or Ziehl–Nielsen) is appropriate. Slides can be immersed in acetone, ethanol or methanol (you can also use the Diff-Quik® fixer in pot one) until sufficiently decolourised.

Heat Fixing

Waxy or oily preparations can be difficult to air dry and, as they are soluble in alcohol, may be lost during fixing and using the two-stain method described earlier. Heat fixing, by passing the slide through a Bunsen or spirit burner or hand- or hair-dryer until it is just hand hot, and then staining with the eosinophilic and basophilic stains, can avoid this. Heat fixing can easily damage cytology preparations if it overdone, however, and remember to let the slides cool before staining to avoid cracking them. Careful and thorough air drying using a hand- or hair-dryer and then staining in the usual fashion gives good results with most oily or waxy samples.

Staining Tape-Strips

Tape-strips can be stained by directly immersing the tape in the solutions. This is most easily done by attaching the two ends of the tape to a microscope slide to form a loop on the surface or over the end of the slide. After rinsing, detach one end of the loop and stick the tape flat against the slide, blot out the excess water and examine. The tape traps a thin layer of water next to the slide, so it is not usually necessary to use a cover-slip. Most fixatives dissolve adhesive tape and are best avoided – if using the two-stain method (see earlier) just dip the tape in the eosinophilic and then the basophilic stain. The stains will also turn some tapes opaque, and it may be necessary to experiment to find a locally available, compatible combination.

Other Romanowsky-Type Stains

Several other similar but distinct Romanowsky-type stains exist, but their use is mainly restricted to specialist laboratories as they are less straightforward and take longer than Diff-Quik® stains. Wright's (also known as Wright's–Giemsa) and May–Grünwald stains are particularly good at distinguishing fine cellular detail and are most frequently used for examining peripheral blood smears and bone marrow preparations. Leishman's (which is very similar to Wright's stain) and Giemsa are highly effective differential stains used to identify malaria, trypanosomes, other parasites and bacteria in peripheral blood smears, bone marrow specimens and other tissues. The stains are difficult and tedious to make up, and are best obtained from commercial sources.

Gram Staining

Gram staining is a relatively simple technique, but with the advent of Diff-Quik® type stains its use in practice has become less common. The technique is widely used in bacteriology to differentiate Gram-negative (which stain pale red–pink) and Gram-positive (which stain purple–blue) bacteria. Cell wall damage, aging or death may cause Gram-positive bacteria to appear Gram-negative. Gram stains can be performed on smears prepared from tissue samples or cultured colonies. There are various methods, which vary in slight details. One technique is as follows.

Air dry and gently heat fix the smear by passing the slide briefly through a Bunsen flame until just hot to the touch.

Cover the slide with crystal violet solution for 1 minute. Briefly wash in tap water and drain.

Cover with Gram's iodine solution for 1 minute. Wash off with tap water, drain and then blot up remaining water.

To decolourise the preparation, cover with 95% alcohol for 10 seconds, wash off with tap water and drain. Thick smears may need longer, but be careful not to overdo it.

Cover the slide with safranin solution (the counterstain) for 30 seconds, wash off with tap water, and then drain and blot dry prior to microscope examination.

Lactophenol Cotton Blue

Lactophenol cotton blue is widely used to stain fungal hyphae, microconidia and macroconidia for identification. It is commercially available and easy to use with preparations taken from fungal cultures in practice.

Place one to two drops of lactophenol cotton blue onto a microscope slide.

Tease a small amount of hyphae from the surface of a fungal culture into the stain.

Carefully place a cover-slip on the preparation for microscopic examination.

Clear adhesive tape is particularly good for collecting material for transfer to microscope slides and preserving the natural orientation of fungal elements.

Place one to two drops of lactophenol cotton blue onto a microscope slide.

Cut off a small piece of tape (approximately 0.5 cm

2

), taking care not touch the adhesive surface.

Using forceps, gently touch the adhesive side to a fungal colony.

Lower the tape fungal (i.e. adhesive) side down onto the drops of stain and place a cover-slip for microscopic examination.

Alternatively, place the tape fungal side up, add one to two drops of stain onto the tape and then place the cover-slip.

Ziehl–Nielsen Stain

Ziehl–Nielsen (ZN) staining is used to identify mycobacteria that stain poorly or not at all with Gram or Romanowsky stains because of the mycolic acid waxes in their cell wall. Heating allows the carbol fuchsin stain to penetrate the cell wall, where it forms a complex with the mycolic acids. This fails to decolourise with mild acid treatment – hence the term acid fast. ZN stains can be used on cytology smears, histopathology sections and culture material, but are rarely performed outside of histopathology or cytology laboratories.

Cover the sample preparation with carbol fuchsin stain.

Heat the stain so that it gently steams for at least 5 minutes. Add more stain if necessary to prevent the preparation drying out and do not let the sample boil.

Remove from the flame and allow to cool slightly to prevent the glass slide cracking in the next step.

Cover with acid–alcohol for 20 seconds then rinse in tap water to stop the decolourisation.

Counterstain with methylene blue for 60 seconds, then rinse in tap water and blot dry.

Other Stains

There are various other stains that, again for reasons of time, expense and ease of use, are rarely performed outside of histopathology or cytology laboratories. Some stains that may be used include:

Haematoxylin and eosin (H&E)

is the most common tissue stain used in histopathology. Haematoxylin stains cell nuclei blue, while eosin stains cytoplasm, connective tissue and erythrocytes pink or red;

Periodic acid-Schiff

(

PAS

) stains carbohydrates such as glycogen, glycoprotein and proteoglycans. It is used to distinguish glycogen storage diseases and identify fungi in tissue sections;

Sudan stains

(such as Sudan III, Sudan IV, Oil Red O, and Sudan Black B) are used to detect lipids;

Methylene blue

is occasionally used to highlight nuclei in cytology specimens. It can also be used to stain hyphae, microconidia and macroconidia from cultured fungi.

Different staining methods are suitable for different types of samples and to examine the specimen for different types of cells and microorganisms. Table 1.1 summarises the uses of the various types of stain.

Table 1.1 Indications for the most common stains used on cytology samples and histopathology sections.

Stains

Uses

Modified Romanowsky or Wright-Giemsa (e.g. Diff-Quik®, Rapi-Diff®)

Routine rapid in-clinic staining of cytology samples and blood smearsDifferentiates cells, nucleus, cytoplasm, cytoplasmic granules, bacteria and fungiAll organisms that stain with these stains will appear dark blue and the stains do not differentiate Gram-negative and Gram-positive bacteria

Haematoxylin and eosin (H&E)

Routine first-line histopathology stainMay be combined with other stains to highlight specific organisms and substances

Gram

To identify bacteria in cytology samples and histopathology sectionsDifferentiates Gram-negative and Gram-positive bacteria

Ziehl–Nielsen

To identify acid-fast bacteria, mainly mycobacteria but also

Nocardia

and

Rhodococcus

, and some protozoa (e.g.

Cryptosporidium

)

Grocott–Gomori's methenamine silver

Stains both living and dead fungal organisms

Periodic acid–Schiff (PAS)

Detects polysaccharides (e.g. glycogen), glycoproteins, glycolipids and mucins in histopathology sectionsWill stain living but not dead fungi

Giemsa

Accurate identification and differentiation of cells in cytology samples, bone marrow aspirates and blood smearsTo identify certain microorganisms (e.g. trypanosomes, spirochaetes,

Histoplasma

,

Chlamydia

and

Chlamydophila

, and protozoa)

Wright

Accurate identification and differentiation of cells in blood smearsCombination Wright–Giemsa staining is widely used

Leishman

To identify leucocytes, blood-borne parasites and trypanosomes in blood smears

Sudan (Sudan II, Sudan III, Sudan IV, Oil Red O, and Sudan Black B)

To identify fat in cytology samples, histopathology sections and faeces

Congo red

To detect amyloid in histopathology sections

Toluidine blue

Detects mast cell granules in cytology samples and histopathology sections

Calcofluor-white

Fluorescent stain that identifies fungi, algae and related organisms on hairs, cytology samples and histopathology sectionsMost commonly used with fluorescent microscopes but stained hair samples on a standard microscope can be illuminated with a Wood's lamp to detect dermatophytes

Using Internal and External Laboratories

Using your in-house laboratory is quicker, but may not be any cheaper once the full running costs are taken into account, and may not be any easier or more accurate depending on your experience and the equipment available. It is also important that all equipment is maintained properly and that quality control is conducted where appropriate (e.g. haematology and biochemistry analysers).

There are some tests – including serology, endocrine assays, microbial culture and sensitivity, and histopathology – that can only be performed in specialised laboratories. Most reputable laboratories perform these tests to a high standard and are open about their quality assurance measures. It is useful to develop a relationship with your laboratory; you will come to be familiar with their format and results and they will know what to expect from you. It also makes it easier to call in for advice – many laboratories now also have a network of clinical specialists that can give you advice on particular cases and results. It is also perfectly appropriate to use different laboratories for different tests, depending on their field of expertise, e.g. haematology and biochemistry, serological assays, endocrine assays, histopathology etc.

Dermatohistopathology, in particular, is becoming an increasingly specialised field and it is crucial that biopsy specimens go to a pathologist with a particular interest in skin diseases and who is clinically up to date. Many skin conditions can only be confirmed on histopathology and a number of new conditions are described each year. Some of these may be quite rare, leaving the pathologist in the ideal position to collate and disseminate information from their clinical colleagues.

How to Get the Best From an External Laboratory

Take the appropriate samples. The bulk of this book is concerned with this, but check with the laboratory first if you are unsure. Some samples, for instance, may need special treatment or storage before despatch.

Fill the submission forms in correctly, concisely and accurately. The amount of information will vary depending on the sample, but identification, age, sex and breed should be a minimum. Objective tests (e.g. blood tests) will require little else, but more clinical information can help the laboratory staff interpret the results.

Skin biopsy specimens should be sent with a complete history, description of the clinical signs and differential diagnosis list. It can also be very helpful to attach photographs (it is very easy now to submit digital images by email) of the lesions. Each specimen should be submitted in a separate pot, clearly labelled with the biopsy site, to help match the histopathology to the clinical lesions.

Package and label the samples correctly. You must ensure that legal requirements for posting pathological specimens are adhered to. Most laboratories now provide appropriate packaging and mailing containers, often with prepaid postage or a courier service. Guidelines for regulations in the UK can be found on:

http://www.royalmail.com/sites/default/files/Guidance-Document-Infectious-Substances-171012.pdf

.

Always consider test results in light of the history and clinical signs. If they do not fit, then discuss them with the laboratory. It may be necessary to re-evaluate the clinical data, test results or re-submit samples, but an incorrect diagnosis can lead to inappropriate and potentially life-threatening treatment. Very few diagnoses come out of the blue – virtually all rely on clinicopathological correlation of all the available data. Remember to use your clinical skills, and make the diagnosis fit the data – do not twist the findings to justify a diagnosis.

Sensitivity, Specificity, and Positive and Negative Predictive Value

Many journal articles and textbooks will refer to these statistical terms when referring to diagnostic tests. They can be qualified in absolute terms if figures are known (e.g. a test may have a sensitivity of 83%) or estimated in more relative terms (e.g. a test is highly sensitive but poorly specific). They describe the reliability with which tests can be used to confirm or rule out particular conditions. It is therefore important that clinicians have some understanding of these concepts when considering test results or trying to interpret published papers etc.

Sensitivity refers to the probability that a test will be positive if an animal has the condition tested for – in other words, how likely are false-negative results? Specificity refers to the probability that if animal does not have the disease, the test will be negative – in other words, how likely are false-positive results? An ideal test would have 100% sensitivity and specificity, but in practice virtually all tests will have some false-negative and false-positive results. An example of this would be using skin scrapes to detect Sarcoptes mites: if you find mites, then you can be nearly 100% sure that the animal has scabies, i.e. skin scrapes are highly specific for Sarcoptes infestation – there are no false-positives. Negative skin scrapes on the other hand do not rule out the possibility of scabies as you will not find mites in a large proportion of affected animals; i.e. skin scrapes are poorly sensitive for Sarcoptes infestations – there are many false-negatives.

The predictive values refer to the proportion of individuals for whom the test result is true. The positive predictive value (PPV) is the proportion of animals with a positive test that actually have the condition – i.e. that are correctly diagnosed. Conversely, the negative predictive value (NPV) is the proportion of animals with a negative test that do not have the condition.

The difference between these two sets of figures is that the sensitivity and specificity refer to inherent properties or accuracy of the test, whereas the PPV and the NPV give you some idea of the clinical usefulness of the test in a defined population that is tested. This is only useful, however, if the prevalence of the condition in the population is known as this has an effect on predictive values. For this reason, the sensitivity and specificity are more commonly quoted.

Presence of the condition

Positive

Negative

Test result

Positive

True positive

False positive

PPV

Negative

False negative

True negative

NPV

Sensitivity

Specificity

Sensitivity = true positive/(true positive + false negative)Specificity = true negative/(true negative + false positive)PPV = true positive/(true positive + false positive)NPV = true negative/(true negative + false negative)

Worked Example

Let us say that 100 dogs with pruritus have a serology test for Sarcoptes performed. Of these, 10 dogs have Sarcoptes: nine are positive on an enzyme-linked immunosorbent assay (ELISA) and one is negative. Of the remaining 90 dogs with another diagnosis, five are also positive on the ELISA.

Presence of the condition

Positive

Negative

Test result

Positive

9

5

PPV

Negative

1

85

NPV

Sensitivity

Specificity

Sensitivity = 9/(9 + 1) = 90%Specificity = 85/(85 + 5) = 94%PPV = 9/(9 + 5) = 64%NPV = 85/(85 + 1) = 99%

The test has a high sensitivity detecting 90% of Sarcoptes cases, and is highly specific, as 94% of animals with other dermatoses had a negative test. In this population of 100 tested animals the PPV is 64%, meaning that approximately two thirds of animals with a positive test have Sarcoptes, and the NPV is 99%, virtually excluding Sarcoptes in animals with a negative ELISA. In this example, the prevalence of Sarcoptes in the population is 10%. See how changing the prevalence (but keeping the sensitivity and specificity) affects the PPV and NPV. The PPV, in particular, can be poor for conditions with low prevalences, and, where possible, positive results for rare diseases should be confirmed with an alternative test.

When considering sensitivity and specificity it is also important to understand why the test has been used, the clinical presentation and what the expected outcome is. For example, urinary cortisol:creatinine ratios (UCCR) are quick, cheap and easy tests that can be used to diagnose canine hyperadrenocorticism (HAC). They are highly sensitive, as virtually all dogs with HAC have an elevated UCCR, but poorly specific, as UCCRs are also elevated in many other conditions. The diagnostic value in sick, polydipsic/polyuric animals can therefore be low; the UCCR is often elevated, but this can be of little clinical significance. It can, however, be a useful test in an otherwise healthy animal with alopecia, as a negative test will effectively eliminate HAC.

Before considering which test to perform, it is, therefore, important to think carefully about which differential diseases need to be investigated and which questions the clinician would like to have answered. Without careful consideration of these factors, the tests cannot give useful answers and this can effectively cost a lot of the client's money without reaching a diagnosis, which is most frustrating both for the attending vet and client. Although packages offered by commercial laboratories can be useful in some cases, using them without reflection is probably not the most effective use of the resources available to manage any given case.