THE PATHOLOGY OF DEVIL FACIAL TUMOUR DISEASE IN TASMANIAN DEVILS (SARCOPHILUS HARRISII).

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1 THE PATHOLOGY OF DEVIL FACIAL TUMOUR DISEASE IN TASMANIAN DEVILS (SARCOPHILUS HARRISII). Richmond Loh Cern-Wan BSc BVMS MACVSc Murdoch University Western Australia A dissertation submitted to Murdoch University for the degree of Master of Philosophy 2006

2 DECLARATION OF ORIGINALITY The work described in this thesis is that of the author alone unless otherwise stated in the text. No part of this work has been submitted for any other qualification at this or any other university. Richmond Loh Murdoch University Western Australia 2006 i

3 STATEMENT OF AUTHORITY OF ACCESS This thesis may be made available for loan and limited copying in accordance with the Copyright Act Richmond Loh Murdoch University Western Australia 2006 ii

4 PREFACE Give thanks to the creatures of the world for they bring beauty, joy and peace. We mourn the loss of certain species and pray for the deliverance of endangered ones. Grant them shelter, food, water and fair weather. Matthew 10:29 For only a few cents you can buy two sparrows, yet not one sparrow falls to the ground without your Father s consent. The study of things caused precedes the study of the cause of things. Richmond Loh Murdoch University Western Australia 2006 iii

5 CONTENTS DECLARATION OF ORIGINALITY...I STATEMENT OF AUTHORITY OF ACCESS...II PREFACE...III ABSTRACT... VII ACKNOWLEDGMENTS... VIII PUBLICATIONS ARISING FROM THIS WORK... XI ABBREVIATIONS... XII LIST OF FIGURES... XIII LIST OF TABLES...XVIII CHAPTER GENERAL INTRODUCTION TASMANIAN DEVIL BIOLOGY DISEASES IN TASMANIAN DEVILS DISCOVERY OF DFTD RAMIFICATIONS OF DFTD INVESTIGATION INTO DFTD THE IMPORTANCE OF DISEASE DIAGNOSIS DIFFERENTIAL DIAGNOSES FOR DFTD AIM...15 CHAPTER 2 THE PATHOLOGY OF DEVIL FACIAL TUMOUR DISEASE (DFTD) IN THE TASMANIAN DEVIL (SARCOPHILUS HARRISII) INTRODUCTION MATERIALS & METHODS Animals and Sampling Aging Tasmanian Devils Gross Pathology Cytology Histology Transmission Electron Microscopy (TEM) RESULTS Geographic and Demographic Distribution of DFTD Gross Pathology Findings Cytology Results Histopathology Results Special Histological Stains Results TEM Results Other Neoplasms in Wild Tasmanian Devils DISCUSSION...56 iv

6 CHAPTER 3 THE IMMUNOHISTOCHEMICAL CHARACTERISATION OF THE NEOPLASM IN DEVIL FACIAL TUMOUR DISEASE (DFTD) IN THE TASMANIAN DEVIL (SARCOPHILUS HARRISII) INTRODUCTION MATERIALS AND METHODS Animals and sampling Histology and immunohistochemistry Antigen retrieval Primary antibody incubation Amplification and application of chromogens Quantification of cells showing antigen expression Photography RESULTS DISCUSSION...83 CHAPTER 4 GENERAL DISCUSSION, LIMITATIONS & FUTURE DIRECTIONS...92 REFERENCES...97 APPENDIX 1: FIGURES AND TABLES CASES OF DFTD FOR THE PERIOD INCIDENCE OF METASTASES IN DFTD CASES THE OCCURRENCE OF NEOPLASIA IN OTHER SPECIES CANINE TRANSMISSIBLE VENEREAL TUMOUR COMPARED WITH DEVIL FACIAL TUMOUR DISEASE GROSS SCORING OF DFTD HISTOLOGICAL SCORING OF DFTD APPENDIX 2: COMPOSITION OF FIXATIVES NEUTRAL BUFFERED FORMALIN SOLUTION (NBF) CACODYLATE BUFFER KARNOVSKY S FIXATIVE PARAFORMALDEHYDE/FORMALDEHYDE 20% APPENDIX 3: HISTOLOGICAL STAINS ALCIAN BLUE AT PH 2.5 FOR ACID MUCINS ALDEHYDE FUCHSIN (MODIFIED) FOR NEUROENDOCRINE CELLS CONGO RED (ALKALINE) METHOD (PUCHTLER, SWEAT AND LEVINE 1962) HAEMATOXYLIN & EOSIN GORDON AND SWEET S METHOD GRIMELIUS FOR ARGYROPHILIC ELEMENTS MASSON-FONTANA METHOD FOR MELANIN GRANULES MASSON S TRICHROME STAIN METHYL GREEN PYRONIN FOR DNA/RNA PERIODIC ACID SCHIFF (PAS) REACTION (MCMANUS 1946) SINGH S ARGENTAFFIN TECHNIQUE TOLUIDINE BLUE METHOD VAN GIESON STAIN VERHOEFF S METHOD v

7 APPENDIX 4: IMMUNOHISTOCHEMICAL SOLUTIONS & PROTOCOLS LIST OF IMMUNOHISTOCHEMICAL STAINS AND THEIR USE SUMMARY TABLE OF IHC PROTOCOLS SOLUTIONS ,3 -Diaminobenzidine (DAB) EDTA ph 8 (for antigen retrieval bath) Hydrogen peroxide working solution (for quenching endogenous peroxidase) M Phosphate buffered saline, ph 7.6 (for washing slides) Sorenson's Phosphate Buffer Sucrose citrate buffer, ph 6.5 (for antigen retrieval bath) Tris Buffered Saline (TBS) ph 7.8 (for washing slides) Tris buffer ph 9 (for antigen retrieval bath) M Tri-sodium-citrate buffer ph IHC PROTOCOLS IHC Protocol SMA (labelled streptavidin biotin2 only) IHC Protocol CD16 (EDTA ph8, Steamer & Envision) IHC PROTOCOL EMA (PROTEINASE K/TRIS ANTIGEN RETRIEVAL) IHC Protocol GFAP (Proteinase K/Tris Antigen Retrieval) IHC Protocol CD57 (citric ph 6.5 retrieval) IHC Protocol Melan A, Desmin and vwf (Sucrose citric ph6.5, microwave & LSAB2) IHC Protocol CD3 (TES ph9, Microwave and Envision) IHC Protocol using TES ph9, Microwave and LSAB PREPARATION OF TISSUE SECTIONS FIXATION AND DEWAXING COUNTER STAINING APPENDIX 5: PRODUCT INFORMATION SHEETS (ENCLOSED CD) CD CD CD CD79a CHROMOGRANIN A CYTOKERATIN DESMIN EPITHELIAL MEMBRANE ANTIGEN GLIAL FIBRILLARY ACID PROTEIN MELAN A NEURON SPECIFIC ENOLASE S SMOOTH MUSCLE ACTIN SYNAPTOPHYSIN VIMENTIN VON WILLEBRAND FACTOR ANTIBODY DILUENT PROTEINASE K PROTEIN BLOCK LSAB 2 SYSTEM HRP ENVISION SYSTEM HRP LABELLED POLYMER APAAP SYSTEM vi

8 ABSTRACT The pathology of a disfiguring and debilitating fatal disease affecting a high proportion of the wild population of Tasmanian Devils (Sarcophilus harrisii) that was discovered is described. The disease, named devil facial tumour disease (DFTD), has been identified in devils found across 60% of the Tasmanian landscape. The prevalence of this disease was extremely variable, possibly reflecting seasonal trapping success. Between 2001 and 2004, 91 DFTD cases were obtained for pathological description. Grossly, the tumours presented as large, solid, soft tissue masses usually with flattened, centrally ulcerated and exudative surfaces. They were typically multi-centric, appearing first in the oral, face or neck regions. Histologically, the tumours were composed of circumscribed to infiltrative nodular aggregates of round to spindle-shaped cells often within a pseudocapsule and divided into lobules by delicate fibrous septae. They were locally aggressive and metastasised in 65% of cases. There was minimal cytological differentiation amongst the tumour cell population under light and electron microscopy. The diagnostic values of a number of immunohistochemical stains were employed to further characterise up to 50 representative cases. They were negative for cytokeratin, epithelial membrane antigen, von Willebrand factor, desmin, glial fibrillary acid protein, CD16, CD57, CD3 and LSP1. DFTD cells were positive for vimentin, S-100, melan A, neuron specific enolase, chromogranin A and synaptophysin. In conclusion, the morphological and immunohistochemical characteristics together with the primary distribution of the neoplasms indicate that DFTD is an undifferentiated neoplasm of neuroendocrine histogenesis. vii

9 ACKNOWLEDGMENTS This project was funded by the Department of Primary Industries & Water and also by the Commonwealth Research Training Scheme, and has been supported by the Australian Wildlife Health Network. I am deeply indebted to Margaret Williams, my manager at DPIW, for giving me this opportunity to do the work and study. I wish to thank my academic supervisors, Shane Raidal and Amanda O Hara for their constant support, enthusiasm and encouragement during the course of this project and for many valuable insights into the work described in this thesis. They deserve my immense gratitude. It has been a collaborative effort and many bodies and minds have contributed to my effort. Stephen Pyecroft, my work-place supervisor, has generously shared his time and knowledge to produce publishable manuscripts and has been a great mentor in issues surrounding work, social and life in general. I would like to thank Dane Hayes especially for all his untiring work in churning out countless histological slides and preparing the samples for electron microscopy. He is a machine! Jemma Bergfeld and Robyn Sharpe helped collect samples from the field trips to add statistical weight to the pathology investigation (and doing some sight-seeing at the same time). I would like to thank Ashkan Mahjoor who assisted with the tabulation of histological data and for helping with the selection of relevant immunostains. viii

10 I am greatly indebted to Michael Slaven and Gerard Spoelstra for their help and advice on the immunostaining. It was an information mine-field and they helped me develop IHC protocols for the various antibodies and with trouble-shooting. I also owe my gratitude to the many private veterinary clinicians at clinics in Launceston, Montrose, Kingston, Smithton, Penguin, Devonport and Longford as well as the wildlife parks that have provided samples and cases for examination. Testing was performed in the Animal Health Laboratories of DPIW Tasmania; Murdoch University, WA; University of Sydney, NSW; AAHL, Victoria and at Royal Hobart Hospital, Tasmania. Many thanks also go to Catherine Marshall, Alex Hyatt, Jamie Chapman, Andrew Parker, and Peter Fallon for their assistance with histological and electron microscopic examination and interpretations. Thanks are due to Judy Rainbird, Kathryn Medlock, David Pemberton at Queen Victoria Museum and the Tasmanian Museum and Art Gallery for allowing me to examine the archived materials. I would like to thank Phillip Clark for his advice on the cytology section. My heartfelt thanks to Phil Ladds, Brad Chadwick, Roy Mason, Majid Ghoddusi, Karrie Rose, Bruce Rideout, Ray Lowenthal, Tony Ross, Paul Canfield, Jane Sammons, Bruce Jackson, David Obendorf, Paul Tucker, Susan Hemsley, Mark Krockenberger, Rupert Woods, Vanessa Di Giglio, Tim McManus, Philip Nicholls, Sandy Mclachlan, Jo Meers, Rachael Tarlinton, Jon Hanger, Jeff McKee, John Rasko, Chuck Bailey, Tim Holton, Kelly O Sullivan and David Middleton for providing data and feedback on the results. ix

11 The project has also been strongly supported by my colleagues. I am particularly grateful to Nick Mooney, Clare Hawkins, Billie Lazenby, Menna Jones, Heather Hesterman and Jason Wiersma at the Resource Management Branch of DPIW for the collection of samples. All this work could not have been accomplished without great technical support from Nolan Fox, Kate Swift, Leah Parker, Erin Noonan, Lisa Edwards, Denise Wells, Robyn Aylmer, Megan Barney, Kate Young, Bronwyn Gardner, Jim Talbot, Chris Emms, Anne-Lucaudo Wells, Margaret Quill, Anne-Marie Pearse, Peter Verwey, Karen Nutter, Bonnie Bealle, Jim Lentern, Pat Statham, Bruce Cullen, Glenn Maclaren, Margaret Sharp and Sofia Obradovich. My parents, John and Agatha, have helped to do the proof reading and my brothers Des and Ray have provided me with a lot of sound advice and encouragement throughout my work. I would also like to thank Amy for her patience with all my work. I thank God for making it possible for me to complete this thesis and allowing me to meet so many wonderful people in the process. x

12 PUBLICATIONS ARISING FROM THIS WORK Loh R, Bergfeld J, Hayes D, O Hara A, Pyecroft S, Raidal S and Sharpe R. The pathology of Devil Facial Tumor Disease (DFTD) in Tasmanian Devils (Sarcophilus harrisii). Veterinary Pathology 2006, 43 (September). Loh R, Hayes D, Mahjoor A, O Hara A, Pyecroft A and Raidal S. The immunohistochemical characterization of Devil Facial Tumor Disease (DFTD) in the Tasmanian Devil (Sarcophilus harrisii). Veterinary Pathology 2006, 43 (September). Loh R, The pathology of Devil Facial Tumour Disease in Tasmanian Devils (Sarcophilus harrisii). Australian Veterinary Association Annual Conference Proceedings, May (CD) Loh R, O'Hara A, Raidal S: The Pathology & Characterisation of Devil Facial Tumour Disease (DFTD) in Tasmanian Devils (Sarcophilus harrisii). Murdoch University, Perth, November (poster) Loh R, Hayes D, Mahjoor A, O'Hara A, Pyecroft S & Raidal S: The histological, ultrastructural and immunohistochemical characterisation of the neoplasm in Devil Facial Tumour Disease (DFTD) in Tasmanian Devils (Sarcophilus harrisii). Wildlife Disease Association Conference, Cairns, June (poster) Loh R, Raidal S, O'Hara A, Pyecroft S & Sharpe R: Devil Facial Tumour Disease. In: What is happening to our devils? Murdoch University, Perth, November (poster) Loh R. Devil Facial Tumour Disease: Devastating the Tasmanian Icon. Proceedings of the Annual Conference of the Australian Association of Veterinary Conservation Biologists May 2004; xi

13 ABBREVIATIONS ARWH CD CgA CK DAB DFTD DFTDH DFTHG DIW DPIW DPX EDTA EMA ES FFPE GFAP hpf IHC LSP1 Mel A MGP MVA NBF NET NSE PBS RMC SMA TAHL TBS TEM TVT vwf Australian Registry of Wildlife Health cluster differentiation chromogranin A cytokeratin 3,3 -diaminobenzidine Devil Facial Tumour Disease DFTD Histology score DFTD Gross score deionised water Department of Primary Industries & Water di-butyl-polystyrene-xylene (hydroxymethyl) aminomethane ethylenediaminotetraacetic acid epithelial membrane antigen Ewing s sarcoma formalin-fixed paraffin-embedded glial fibrillary acidic protein high power field (equivalent to 400x magnification) immunohistochemistry leucocyte specific antigen melan A methyl green pyronin motor vehicle accident 10% neutral buffered formalin neuroendocrine tumour neuron specific enolase phosphate buffered saline Resource Management Branch, DPIW smooth muscle actin Tasmanian Animal Health Laboratory, DPIW tris buffered saline transmission electron microscopy canine transmissible venereal tumour von Willebrand factor xii

14 LIST OF FIGURES Fig. 1.1 Fig. 2.1 Fig. 2.2 Fig. 2.3 Fig. 2.4a Fig. 2.4b Fig. 2.5 Fig. 2.6 Fig. 2.7 Fig. 2.8 Healthy Tasmanian devils fighting over a carcass, Road-kill and injured animals make up a large proportion of their diet. Picture courtesy of Christo Baars, Netherlands. Polyurethane pipes with side slots for ventilation and a sliding trap door are favoured over the traditional metal traps which can cause traumatic injuries to the Tasmanian devils when they try to escape. General anaesthesia of Tasmanian devils allows quicker and safer sample collection and causes less stress to the animals so they can be released soon after the procedure. Upper canines and the ventral molars are reliable tools for aging Tasmanian devils. This cast was taken from a 2 year old Tasmanian devil. Location and number of Tasmanian devils that have been sampled for histological examination for the period January 1991 to December 2004 (n = 459). For the period pre-2001, n = 11; in 2001, n = 4; in 2002, n = 5; in 2003, n = 64 and in 2004, n = 375. Map showing the 41 locations where DFTD was confirmed by laboratory diagnosis in this study (n = 91), reflecting the extent of Devil Facial Tumour Disease (DFTD) for the period spanning 2001 and Bar = 50km. An early case of DFTD in an animal. A small nodule (arrow) located at the rostral part of the chin at the central midline. Multicentric tumours. DFTD lesions occurred subcutaneously and form circumscribed masses with a flat ulcerative surface. Extensive DFTD lesion affecting the lower jaw in this Tasmanian devil. The cut surface of the tumour showing a multifocal coalescing solid mass of glistening pale tissue, often with central necrosis. Bar = 4 cm xiii

15 Fig. 2.9 Fig Fig Fig Fig Fig Fig Fig Fig Cytological preparation of a fine needle aspirate of DFTD. All cells pictured are DFTD cells. They are large round cells and tended to clump. Diff quick. Bar = 25µm. Cytological preparation of a fine needle aspirate of DFTD showing anisocytosis. Diff quick. Bar = 25µm. DFTD in facial skin. The neoplasm occurs in the dermis and present as well circumscribed masses, compressing the surrounding connective tissue. E = epithelium, SC = stratum compactum, H = hair follicle, N = DFTD neoplasm. H&E, Mag x4. Bar = 300µm. Architectural variations of DFTD with most cells forming bundles (a) and some presenting as palisades (b), clumps (c), nests (d), or sheets (e). H&E, Mag x10. Bar = 100µm. Neoplastic cells in DFTD are essentially round to pleomorphic cells (left) with fibrillar cytoplasmic material and indistinct cytoplasmic borders (right). H&E, Mag x40 & x100 and Bar = 40µm & 15µm respectively. Necrosis usually appear to occur centrally at first (left) and then progresses peripherally (right). H&E, Mag x4. Metastatic locations of DFTD (asterisk): submandibular lymph node (a), lung (b), spleen (c), heart (d), ovary (e), rib serosa (f), kidney (g), mammary (h), adrenal (i) and pituitary gland (j). H&E Mag. x4. Positive reaction on a section of Tasmanian devil intestinal mucosa (inset) and negative reaction on a section of DFTD neoplasm using the Alcian blue technique for acid mucins. Mag. x40. Positive reaction on a section of Tasmanian devil pancreas (inset) and negative reaction on a section of DFTD neoplasm using the aldehyde fuchsin technique for insulin. Mag. x xiv

16 Fig Fig Fig Fig Fig Fig Fig Fig Positive reaction on a section of bovine metastatic melanoma to the liver (inset) and negative reaction on a section of DFTD neoplasm using the Masson s Fontana technique. Mag. x40. Positive reaction on a section of Tasmanian devil skeletal muscle (inset) and negative reaction on a section of DFTD neoplasm using the Gordon and Sweet technique for reticulin. Mag. x40. Positive reaction on a section of Tasmanian devil intestine (inset) and negative reaction on a section of DFTD neoplasm using the PAS technique for carbohydrates. Mag. x40. Positive reaction on a section of Tasmanian devil skin (inset) and negative reaction on a section of DFTD neoplasm using toluidine blue technique for mast cell granules. Mag. x40. Positive reaction on a section of Tasmanian devil artery (inset) and negative reaction on a section of DFTD neoplasm using Verhoeff technique for elastin. Mag. x40. Tasmanian devil spleen was used for the positive (top, inset) and negative (bottom, inset) control tissue using the methyl green pyronin technique for RNA and DNA. DFTD stained positive (top) which was confirmed with the contrast in RNAse stained DFTD cells (bottom). Mag. x40. Positive reaction on a section of Tasmanian devil intestinal mucosa (inset) and negative reaction on a section of DFTD neoplasm using the Mason s trichrome technique for connective tissue. Mag. x40. Positive reaction on a section of Tasmanian devil muscle (inset) and negative reaction on a section of DFTD neoplasm using the Van Gieson technique for collagen. Mag. x xv

17 Fig Transmission electron microscopic view of DFTD showing the 53 close apposition of DFTD cells and relative sparseness of ultrastructural features. Bar = 10 µm. DFTD are characterised by vacuolated mitochondria (a, bar = 1µm), occasional ribosome-lamellar complexes (b = 3µm), myelin bodies (c, bar = 1µm) and low numbers of desmosome-like junctions (d, bar = 500nm). Fig Periosteal osteoblastoma. H&E Mag. x Fig Cutaneous lymphosarcoma. H&E Mag. x4. 55 Fig. 3.1 Positive reaction on a section of duck liver (inset) but negative 75 on a section of DFTD neoplasm using the Congo red technique for amyloid. Mag x40. Fig. 3.2 Positive argentaffin reaction on a section of Tasmanian devil 76 adrenal medulla (inset) and negative reaction on a section of DFTD neoplasm using the Singh s silver technique. Mag x40. Fig. 3.3 Positive argyrophilic reaction on a section of Tasmanian devil 76 adrenal medulla (inset) but negative on a section of DFTD neoplasm using the Grimelius technique. Mag x40. Fig. 3.4 Vimentin stain showing homogenous moderate to high 80 intensity cytoplasmic staining in all DFTD cells (inset, positive control tissue: Tasmanian devil intestinal blood vessel endothelium). Mag x40. Fig. 3.5 S-100 showing patchy homogenous moderate cytoplasmic 80 staining of DFTD cells (inset, positive control tissue: Tasmanian devil lymph node). Mag x40. Fig. 3.6 Melan A was positive in 28% of cases with an average of 51% 81 of cells in each case being positive (inset, positive control tissue: Tasmanian devil hair follicle). Mag x40. Fig. 3.7 Neuron specific enolase showed diffuse homogenous expression in DFTD cells (inset, positive control tissue: Tasmanian devil pancreatic islets). Mag x xvi

18 Fig. 3.8 Fig. 3.9 Chromogranin A was expressed as a low intensity scattered positivity in the cytoplasm of DFTD cells (inset, positive control tissue: Tasmanian devil adrenal medulla). Mag x40. Synaptophysin showing high intensity granular staining of DFTD cell cytoplasm for of DFTD was strong and homogenous (inset, positive control tissue: Tasmanian devil pancreatic islets). Mag x xvii

19 LIST OF TABLES Table 1.1 Demographics of neoplasia in captive Tasmanian devils at San Diego Zoo. 4 Table 2.1 The frequency of diagnosing DFTD. 27 Table 2.2 Demographics of DFTD in wild Tasmanian devil populations. 30 Table 2.3 Colorimetric characteristics of DFTD cells using a suite of special histochemical stains. 45 Table 3.1 Tabulated results for primary antibody reactivities to DFTD. 77 Table 3.2 Differential diagnoses for DFTD based on IHC findings and published literature. 86 xviii

20 / Ready-To-Use DAKO Antibody Diluent CODE NO.: S 0809 PRESENTATION: INTENDED USE: REACTIVITY: APPLICATIONS: LIMITATIONS: STORAGE: Tris-HCl buffer containing carrier protein and 0.015M sodium azide. This product is intended for the preparation of primary and secondary antibody dilutions and negative control reagents for use in immunohistochemical (IHC) staining procedures. FOR LABORATORY USE. In the demonstration of tissue antigens by IHC, optimal staining performance usually requires the use of diluted antibodies. The interaction between antibodies and epitopes consists mainly of relatively weak forces such as van der Waals forces, electrostatic forces, and hydrophobic forces, which are readily disturbed by extreme ph, and ionic strength. Consequently to assure optimal performance of the antibody, the use of a proper diluent is imperative. This product provides an appropriately buffered medium for the dilution of both polyclonal and monoclonal antibodies and for the preparation of negative control reagents in IHC. It may be especially useful when staining connective tissue, epithelial tissue, adipocytes, or other types or tissues which are susceptible to high background. DAKO Antibody Diluent can also stabilize diluted antibodies when stored at 2-8 C. Tissue staining is dependent on the handling and processing of the tissue prior to staining. Aldehyde-containing fixatives, such as formalin and glutaraldehyde frequently result in increased background staining. Improper fixation, freezing, thawing, washing, drying, heating, or sectioning may produce artifacts, antibody trapping, or false negative results. False positive staining may also be caused by cross-reactivity of other IHC staining reagents to e.g. endogenous alkaline phosphatase, endogenous peroxidase, pseudoperoxidase, endogenous avidin-binding activity or by nonspecific reaction with necrotic or degenerated cells. Store at 2-8 C. Do not freeze. PL CF:tt

21 APAAP, Mouse Monoclonal Code No./ Code/ Code-Nr. D 0651 Edition/ Edition/ Ausgabe ENGLISH Intended use Introduction Reagent provided Immunogen Precautions Storage Staining procedure FRANÇAIS Intérêt Introduction For in vitro diagnostic use. APAAP, Mouse, Monoclonal, is a visualization reagent intended for use in immunocytochemistry (1, 2), immunoblotting and ELISA. APAAP, Mouse, Monoclonal, consists of soluble immunocomplexes of calf intestinal alkaline phosphatase and monoclonal mouse anti-alkaline phosphatase. In immunocytochemistry, the use of APAAP, Mouse, Monoclonal - in conjunction with primary mouse antibodies - is particularly advantageous for staining of preparations rich in endogenous peroxidase, for example, blood and bone marrow smears, and cell smears prepared from serous effusions. APAAP, Mouse, Monoclonal, may also be used successfully together with peroxidase-labelled reagents for immunocytochemical double staining. Endogenous alkaline phosphatases in tissues other than the small intestine and the placenta are easily inhibited by adding levamisole to the enzyme substrate. Alkaline phosphatase in APAAP, Mouse, Monoclonal, is not affected by levamisole. D 0651 is provided in liquid form as cell culture supernatant dialysed against 0.05 mol/l Tris/HCl, 0.1 mol/l NaCl, and containing 1 mmol/l MgCl 2, 0.1 mmol/l ZnCl 2, and 15 mmol/l NaN 3, ph 7.2. Clone: AP7/6/7. Isotype: IgG1, kappa. Mouse IgG concentration g/l: See label on vial. Purified calf intestinal alkaline phosphatase. 1. For professional users. 2. This product contains sodium azide (NaN 3), a chemical highly toxic in pure form. At product concentrations, though not classified as hazardous, sodium azide may react with lead and copper plumbing to form highly explosive build-ups of metal azides. Upon disposal, flush with large volumes of water to prevent azide build-up in plumbing. 3. As with any product derived from biological sources, proper handling procedures should be used. Store at 2-8 C. Do not use after expiration date stamped on vial. If reagents are stored under any conditions other than those specified, the user must verify the conditions. If unexpected results are observed which cannot be explained by variations in laboratory procedures and a problem with the reagent is suspected, contact our Technical Services. For immunocytochemistry the following procedure may serve as a guideline: 1. Mouse antibody, optimally diluted. Incubate for 30 minutes at room temperature. 2. DakoCytomation Polyclonal Rabbit Anti-Mouse Immunoglobulins, code No. Z 0259, diluted 1:25. Incubate for 30 minutes at room temperature. 3. APAAP, Mouse, Monoclonal, code No. D 0651, diluted 1:25 to 1:50. Incubate for 30 minutes at room temperature. 4. Chromogenic substrate. Incubate for minutes at room temperature. The staining intensity can be enhanced by repeating steps 2 and 3 before adding the chromogenic substrate. Tris-buffered saline, ph 7.6, is a suitable diluent for steps 1, 2 and 3. Phosphate buffers must be avoided as phosphate is a substrate for alkaline phosphatase and, therefore, competes with the chromogenic substrate. Additionally, phosphate in the dilution buffer for APAAP, Mouse, Monoclonal, will form an insoluble precipitate with magnesium. Unless the stability in the actual test system has been established, it is recommended to dilute the product immediately before use. Pour diagnostic in vitro. APAAP, Mouse, Monoclonal, est un réactif de révélation destiné à être utilisé en immunocytochimie (1, 2), en immunobuvardage et ELISA. APAAP, Mouse, Monoclonal, est composé d'immunocomplexes solubles de phosphatase alcaline intestinale de veau et de phosphatase anti-alcaline de souris monoclonale. En immunocytochimie, l'utilisation d'apaap, Mouse, Monoclonal, associé à des anticorps primaires de souris, est particulièrement intéressante pour la coloration de préparations riches en peroxydase endogène, par exemple des frottis de sang et de moelle osseuse et des frottis cellulaires préparés à partir d'épanchements séreux. APAAP, Mouse, Monoclonal, peut également être utilisé avec des réactifs marqués à la peroxydase pour double coloration immunocytochimique. Dans les tissus autres que le petit intestin et le placenta, les phosphatases alcalines endogènes sont facilement inhibées par l'ajout de lévamisole au substrat enzymatique. Le lévamisole n'a pas d'effet sur la phosphatase alcaline contenue dans APAAP, Mouse, Monoclonal. Réactif fourni Immunogène Précautions d'emploi Conservation D 0651 est fourni sous forme liquide, comme surnageant de culture cellulaire dialysé contre 0,05 mol/l de Tris/HCl, 0,1 mol/l de NaCl et contenant 1 mmol/l de MgCl 2 0,1 mmol/l de ZnCl 2 et 15 mmol/l de NaN 3, ph 7,2. Clone: AP7/6/7. Isotype : IgG1, kappa. Concentration en IgG de souris, en g/l: Voir l'étiquette sur le flacon. Phosphatase alcaline intestinale de veau purifiée. 1. Pour utilisateurs professionnels. 2. Ce produit contient de l'azide de sodium (NaN3), un agent chimique extrèmement toxique à l'état pur.bien qu il ne soit pas classé comme dangereux aux concentrations présentes dans le produit, l'azide de sodium est susceptible de réagir avec les parties en plomb et en cuivre des tuyauteries pour former des azides métalliques extrêmement explosifs. Lors de l'élimination des réactifs, rincer avec de grandes quantités d eau pour éviter toute accumulation d'azides métallisés dans la tuyauterie. 3. Comme pour tout dérivé d origine biologique, sa manipulation doit être précise. Conserver entre 2 et 8 C. Ne pas utiliser au-delà de la date de péremption mentionnée sur le flacon. Si les réactifs sont conservés dans d'autres conditions que celles préconisées, les conditions doivent être vérifiées par l'utilisateur. En cas de résultats imprévus qui ne peuvent pas être expliqués par des changements de procédures de laboratoire et si un problème avec le produit est suspecté, contacter nos Services Techniques. Procédure La procédure suivante peut servir de guide pour l'immunocytochimie : d immunomarquage 1. Anticorps de souris, à dilution optimale. Incuber à température ambiante pendant 30 minutes. 2. DakoCytomation Polyclonal Rabbit Anti-Mouse Immunoglobulins, code Z 0259, à dilution 1:25. Incuber pendant 30 minutes à température ambiante. 3. APAAP, Mouse Monoclonal, code D 0651, à dilution 1:25 à 1:50. Incuber pendant 30 minutes à température ambiante. 4. Substrat chromogène. Incuber à température ambiante pendant minutes. Il est possible d'augmenter l'intensité de la coloration en répétant les étapes 2 et 3 avant d'ajouter le substrat chromogène. La solution TBS, à ph 7,6, est un diluant adapté aux étapes 1, 2 et 3. Il faut éviter d'utiliser des tampons phosphate car le phosphate est un substrat de la phosphatase alcaline et peut donc entrer en compétition avec le substrat chromogène. De plus, le phosphate dans le tampon de dilution pour APAAP, Mouse, Monoclonal, formera un précipité insoluble avec le magnésium. A moins que la stabilité du système d'analyse n'ait été établie, il est recommandé de diluer le produit juste avant l'utilisation. DEUTSCH Zweckbestimmung Zur Verwendung für In-vitro-Untersuchungen. Einleitung Geliefertes Reagenz Immunogen Hinweise und Vorsichtsmaßnahmen Lagerung APAAP, Mouse, Monoclonal ist ein Visualisierungsreagenz, das in der Immunzytochemie (1,2), beim Immunblotting und in ELISA-Verfahren verwendet wird. APAAP, Mouse, Monoclonal, besteht aus löslichen Immunkomplexen aus intestinaler alkalischer Phosphatase vom Kalb und monoklonaler Maus-Anti-Alkalischer Phosphatase. In der Immunozytochemie erweist sich der Gebrauch von APAAP, Mouse, Monoclonal -in Verbindung mit primären Mausantikörpern- als besonders vorteilhaft bei der Anfärbung von Präparaten mit einem hohen Anteil an endogener Peroxidase, wie z.b. Blut- und Knochenmarkausstriche sowie aus serösen Ergüssen gewonnene Zellausstriche. APAAP, Mouse, Monoclonal kann ebenfalls zusammen mit peroxidasemarkierten Reagenzien erfolgreich zur immunzytochemischen Doppelfärbung verwendet werden. Endogene alkalische Phosphatasen werden in anderen Geweben als dem Dünndarm und der Plazenta leicht durch Zugabe von Levamisol zum Enzymsubstrat inhibiert. In APAAP, Mouse, Monoclonal wird die alkalische Phosphatase von Levamisol nicht beeinflusst. D 0651 liegt als Zellkulturüberstand, dialysiert gegen 0,05 mol/l Tris/HCl und 0,1 mol/l NaCl, in Lösung vor und enthält 1 mmol/l MgCl 2, 0,1 mmol/ll ZnCl 2, und 15 mmol/l NaN 3, ph 7,2. Klon: AP7/6/7. Isotyp: IgG1, Kappa. Maus IgG-Konzentration g/l: Siehe Produktetikett. Gereinigte intestinale alkalische Phosphatase vom Kalb. 1. Für geschultes Fachpersonal. 2. Dieses Produkt enthält Natrium-Azid (NaN 3), eine in reiner Form hoch toxische chemische Verbindung. Bei den in diesem Produkt verwendeten Konzentrationen kann Natrium-Azid, obwohl nicht als gefährlich klassifiziert, mit in Abflussrohren enthaltenen vorhandenem Blei oder Kupfer reagieren und zur Bildung von hochexplosiven Metall-Azid- Anreicherungen führen. Nach der Entsorgung muss mit reichlich Wasser nachgespült werden, um Metall-Azid- Anreicherung in den Abflussrohren zu vermeiden. 3. Wie bei allen aus biologischen Materialien gewonnenen Produkten müssen die ordnungsgemäßen Handhabungsverfahren eingehalten werden. Bei 2 8 C lagern. Nicht nach dem auf dem Fläschchen angegebenen Verfallsdatum verwenden. Sollten die Reagenzien unter anderen Bedingungen als den beschriebenen aufbewahrt worden sein, so müssen diese vom Anwender verifiziert werden. Wenn unerwartete Resultate beobachtet werden, welche durch Änderungen in den ( ) D 0651/EFG/KB/ p. 1/3 ( ) D 0651/EFG/KB/ p. 2/3