TissueWave 2. Microwave Protocols. Shandon. B Issue 3. Fixation Processing Take-Backs HIER Staining Decalcification

Transcription

1 7 09:41 Shandon TissueWave 2 Microwave Protocols B Issue 3 Fixation Processing Take-Backs HIER Staining Decalcification WIP-TW2-Protocol Booklet-B /04/ :57:42

2 2007 Thermo Fisher Scientific. All rights reserved. Thermo Shandon Limited is an ISO 9001 and TickIT Accredited Company Thermo Fisher Scientific is the trading name of Thermo Shandon Limited All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries Thermo Fisher Scientific makes every endeavour to ensure that the information contained in its support documentation is correct and clearly stated but does not accept responsibility for any errors or omissions. The development of Thermo Fisher products and services is continuous. Make sure that any published information that you use for reference is up to date and relates to the status of the product. If necessary, check with Thermo Fisher or your local Thermo Fisher representative. This manual may not, in whole or in part, be copied, photocopied, reproduced, translated, or converted to any electronic or machine readable form without prior written consent of Thermo Fisher. All information contained in this manual is proprietary and confi dential, and the exclusive property of Thermo Fisher Scientifi c. This manual is protected by copyright and any reproduction is prohibited. This manual is for use only by the individuals to whom it has been made available by Thermo Fisher Scientific. Contact address Anatomical Pathology 4481 Campus Drive Kalamazoo MI 49008, USA References American Journal of Pathology, 12, 549, Warthin Starry Techniques for Spirochetes Brinn NT: Rapid metallic histologic staining using the microwave oven. J Histotechnol 1983 Gomori G: A rapid one-step trichrome stain. Am J Clin Pathol 20:661, 1950 Grocott, R.G.: American Journal of Clinical Pathology, 25: pp , Histotechnology: A Self Instructional Text. Carson, ASCP Press, Chicago, 1990 Kinyoun, J.J.: Am. J. Pub. Health, 5, 867, Kok and Boon: Microwave Cookbook for Microscopists. Couloomb Press, Leyden 1992 Lev, R. and Spicer, S.S. J. Histochem. Cytochem. 12: 309, Williams & Wilkins Co. Login, G.R., and Dvorak, A. M. (1994). The Microwave Tool Book. A Practical Guide for Microscopists. Boston: Beth Israel Hospital. P.J.: Jour. Technical methods, 12: 75-90, 1929 (AFIP Modification) Valle S: Special stains in the microwave oven. J Histotechnol 9:237, 1986 Tel: Fax: The Shandon TissueWave 2 meets the following CE Mark requirements: In Vitro Diagnostic Directive 98/79/EC Low Voltage Directive 2006/95/EC C US WARNING While the listed protocols have been independently tested and verifi ed, it is the responsibility of the user to follow all printed instructions in both this Protocol Booklet and the TissueWave 2 User Guide. Failure to follow protocols correctly may result in damage to the instrument or specimens, and in extreme cases could cause personal injury. B Iss 3 TissueWave 2 Protocol Booklet 16 WIP-TW2-Protocol Booklet-B /04/ :57:43

3 007 09:41 Solutions for Warthin-Starry: Acidulated Water 1 litre of Distilled Water 0.1 g Citric Acid ph of required Notes: ph 4.0 is ideal for staining spirochetes ph 3.6 is ideal for demonstrating Donovan Bodies of granuloma inguinale 1% Silver Nitrate (Impregnation) 0.5 g Silver Nitrate C.P. crystals 50.0 ml Acidulated Water 2% Silver Nitrate (Developer) 0.5 g Silver Nitrate C.P. crystals 25.0 ml Acidulated Water Contents Tissue Processing Protocols 1 Alternate Tissue Processing Protocols 3 Time-Saving Formalin Alternatives; No HIER Necessary 6 Decalcification 7 Heat Induced Epitope Retrieval (HIER) 8 Specimen Fixation Technique 9 Formalin-Free Microwave Assisted Fixation 9 Take-Back Procedure 9 Microwave Special Stains 10 Solutions for Warthin-Starry: 15 References % Hydroquinone 0.35 g Hydroquinone crystals, photographic quality 25.0 ml Acidulated Water 5% Gelatin 1.5 g Gelatin 25.0 ml Acidulated Water 15 TissueWave 2 Protocol Booklet B Iss 3 WIP-TW2-Protocol Booklet-B /04/ :57:43

4 Tissue Processing Protocols All the following protocols assume that the tissue is fixed in 10% neutral buffered formalin (NBF). WARNING - Warm formalin may produce vapours in excess of OSHA IDLH levels, and it is recommended that any work with formalin, at any temperature, be carried out in a fume cupboard or hood. Do not breathe vapours from trays containing formalin! Settings used for the following protocols are: Notes: Mode - Temperature (unless otherwise stated) Power - 80% Agitation - On (Unless Vacuum is used) If vacumm is On, use 16 inhg maximum unless otherwise stated. Dehydrants or reagents are interchangeable with Ethyl Alcohol. Biopsies 0.8 mm 1 hr. 10 min. Small Biopsies WARNING - This protocol uses formalin (see warning above). 1 Formalin Off 2 90% Dehydrant Off 3 100% Dehydrant Off Propanol Off Propanol Off 6 Wax Optional 7 Wax Optional Warthin-Starry Requirements: 4 µm thick, formalin fixed, wax sections Use tissue containing spirochetes as a control Procedure: De-wax and hydrate sections using an automatic stainer Rinse in distilled water twice 1 1% Silver Nitrate (impregnation) Preheat: Developer, Hydroquinone and Gelatin 45 sec. 3 Mix 1.5 ml Developer, 3.75 ml Gelatin and 2.0 ml Hydroquinone* 4 Remove slides from 1% Silver Nitrate - Do not rinse 5 Place slides flat, cover with Developer sec. * Mix in order given in a warm, empty flask. When the protocol is complete: Rinse slides in 50 ml tap water at 56 C Rinse in distilled water Dehydrate Clear Mount with xylene-soluble media Results: Spirochetes - Black Background - Pale Yellow to Light Brown 1 TissueWave 2 Protocol Booklet B Iss 3 B Iss 3 TissueWave 2 Protocol Booklet 14 WIP-TW2-Protocol Booklet-B /04/ :57:43

5 Microwave Rapid Mucin Requirements: 4 µm thick, formalin fixed, wax sections Use mucin-containing epithelial or connective tissue as a control Procedure: De-wax and hydrate sections using an automatic stainer 1 Wiegert Iron Hematoxylin 1 2 Rinse in running water 1 3 Fast Green FCF or Metanil Yellow Acidified Solution sec. 4 Rinse in Glacial Acetic Acid 1-2 sec % Basic Fuchsin sec. When the protocol is complete: Dehydrate Clear Mount with xylene-soluble media Results: Mucin, Cartilage and Mast Cells - Pink Cytoplasm - Yellow to Green Nuclei - Blue to Black Thick and Fatty 5.0 mm 2 hrs. 58 min. Breast Brain Large excisional biopsies Adipose tissue Note: Take usual precautions such as a cooler water bath to prevent fragmentation. Use with muscular tissue may result in undesirably hard or crunchy sections and is not recommended % Dehydrant Off 2 100% Dehydrant Off 3 Presolve Clearant inhg 4 Wax On 5 Wax On PolarHeat Wax Melt Protocol This protocol requires a Pyrex dish containing a properly positioned PolarHeat sheet and Shandon Excelsior baskets, encased in solidified wax to a depth just covering the basket centre hubs. Notes: Do not place the temperature probe or air agitator in the wax. If any wax remains solid after the protocol has finished, let the dish stand until all the wax has melted. Time may be increased if necessary and temperature is unimportant. WARNING - Never increase power above 50% Step Detail Power Time, min. Vacuum 1 No agitation 50% 22 Off 13 TissueWave 2 Protocol Booklet B Iss 3 B Iss 3 TissueWave 2 Protocol Booklet 2 WIP-TW2-Protocol Booklet-B /04/ :57:43

6 Alternate Tissue Processing Protocols These protocols are intended to allow tissue processing without the use of formalin, or in cases where there is no requirement to process 5 mm thick, fatty samples. Settings used for the following protocols are: Notes: Mode - Temperature (unless otherwise stated) Power - 80% Agitation - On (Unless Vacuum is used) If vacumm is On, use 16 inhg maximum unless otherwise stated. Dehydrants or reagents are interchangeable with Ethyl Alcohol. Fatty Tissue 2 hrs 45 min. Fatty tissue 4 mm thick Tissue pre-fixed with 10% NBF 1 90% Dehydrant Off 2 100% Dehydrant Off Propanol Off 4 Wax On 5 Wax On When the protocol is complete: Dehydrate Clear Mount with xylene-soluble media Results: Muscle fibres, Cytoplasm - Red Collagen - Green Nuclei - Blue to black Alcian Blue This protocol stains acid mucopolysaccharides. Requirements: 4 µm thick, formalin fixed, wax sections or 8 µm eye sections Use appendix as a control Procedure: De-wax and hydrate sections using an automatic stainer 1 Alcian Blue (2.5 ph)* sec. 2 Rinse in distilled water 3 Place in 2nd Eosin in automatic stainer * The ph of the acetic acid buffer can drift due to temperature; it is advisable to set the ph at 60 C. When the protocol is complete: Dehydrate Clear Mount with xylene-soluble media Results: Acid mucopolysaccharides - Blue Nuclei - Pink 3 TissueWave 2 Protocol Booklet B Iss 3 B Iss 3 TissueWave 2 Protocol Booklet 12 WIP-TW2-Protocol Booklet-B /04/ :57:43

7 When the protocol is complete: Rinse slides in running tap water Rinse slides in distilled water Dehydrate Clear Mount with xylene-soluble media Results: Glycogen, Mucopolysaccharides and Fungi - Rose to purplish-red Nuclei, Background - Blue Microwave Gomori Trichrome A time-saving procedure with colour differentiation similar to Masson s. WARNING - Bouin s contains Formaldehyde and Picric Acid and it is recommended that any work with Bouin s, at any temperature, be carried out in a fume cupboard or hood. Do not breathe vapours from trays containing Bouin s! Requirements: 4 µm thick, formalin fixed, wax sections Use striated muscle as a control Procedure: De-wax and hydrate sections using an automatic stainer 1 Bouin s Rinse in running water until yellow has gone ~5 3 Gill #3 Hematoxylin (Sigma) Rinse in running water 1 5 Gomori Trichrome* 15 sec. 6 Incubate in Gomori Solution 1 7 Rinse in 1.0% Acetic Acid sec. * Microwave set to 800 Watts Non-Fatty Tissue 2 hrs Non-fatty tissue 4 mm such as: colon uterus gallbladder uterine content cervical cones prostate 1 100% Dehydrant Off Propanol Off 3 Wax Optional 4 Wax Optional Skin, Endocervical Tissue 33 min. Small skin biopsies Endocervical material 1 100% Dehydrant Off Propanol 74 8 Off 3 Wax 74 5 Optional 4 Wax Optional 11 TissueWave 2 Protocol Booklet B Iss 3 B Iss 3 TissueWave 2 Protocol Booklet 4 WIP-TW2-Protocol Booklet-B /04/ :57:43

8 Small Biopsies - STAT 16 min. G.I. Prostate Cervical Material Note: This protocol is only suitable for small biopsies % Dehydrant 67 5 Off Propanol 74 3 Off 3 Wax 74 3 Optional 4 Wax 82 5 Optional Microwave Special Stains The following protocols have been tested using the TissueWave 2 and a plastic Coplin jar. Settings used for the following protocol are: Periodic Acid Schiff (PAS) Mode - Temperature Power - 80% Agitation - On Requirements: 4 µm thick, formalin fixed, wax sections Tissue should contain PAS positive material as a control Example: If staining for Glycogen, use liver containing glycogen as a control. Procedure: De-wax and hydrate sections using an automatic stainer Rinse in distilled water For PAS reaction with digestion: Place sections in a Coplin jar in 0.5% diatase Microwave for 45 seconds For PAS reaction with non-digestion: Place sections in a Coplin jar in distilled water Microwave for 45 seconds 1 0.5% Periodic Acid Schiff s Reagent Rinse in 55% K 2 S 2 O 5 30 sec. 4 Rinse in 55% K 2 S 2 O 5 30 sec. 5 Rinse in running water 10 6 Harris Hematoxylin 30 sec. 5 TissueWave 2 Protocol Booklet B Iss 3 B Iss 3 TissueWave 2 Protocol Booklet 10 WIP-TW2-Protocol Booklet-B /04/ :57:43

9 Specimen Fixation Technique Cut the tissue section thickness to 3-4 mm or bread-load larger specimens. 1 GlyoFixx RT 30 2 GlyoFixx Formalin-Free Microwave Assisted Fixation Tissue is placed in 10% NBF for collection and transport. Gross to be no thicker than 3 mm M PBS, ph &0% Dehydrant or Ethanol RT - Continue with either conventional or microwave histoprocessing. Time-Saving Formalin Alternatives; No HIER Necessary Microwave processing of tissue using GlyoFixx can result in patient-toembedded sample times of less than 2 hours. Fresh tissue or tissue fixed with most common fixatives can be used. Specimens received unfixed or in zinc formalin, Z-Fix or Prefer usually do not need epitope retrieval. Initial fixation in GlyoFixx - 30 min. at room temperature 1 mm Biopsies 19 min. 1 GlyoFixx 55 4 Off 2 100% Dehydrant 67 4 Off Propanol 74 4 Off 4 Wax 82 7 Optional 3 mm Samples 1 hr. 16 min. Take-Back Procedure This protocol is used to recover or take-back specimens. Settings used for the following protocol are: Mode - Temperature Power - 80% Agitation - On WARNING - Do not microwave Xylene. traditionally processed 1 GlyoFixx Off 2 100% Dehydrant 67 8 Off 3 100% Dehydrant 67 8 Off Propanol 74 8 Off Propanol 74 8 Off 6 Wax On 1 Xylene RT Propanol Propanol Wax Wax TissueWave 2 Protocol Booklet B Iss 3 B Iss 3 TissueWave 2 Protocol Booklet 6 WIP-TW2-Protocol Booklet-B /04/ :57:43

10 Decalcification Bone Marrow Decalcification < 3 mm Preset 1 Zinc Formalin Wash in water 10 3 Decal (TBD2) Bone Marrow Decalcification 3-5 mm Preset 1 Zinc Formalin Wash in water 10 3 Decal (TBD2) 40 30* 12 * Decalcification time may vary depending on sample thickness or bone density; up to 2 hrs. Heat Induced Epitope Retrieval (HIER) Initial: De-wax and re-hydrate to distilled water. This protocol requires: Vented Coplin jar or slide staining container. Note: A pressure cooker is NOT used in this protocol. Settings used for the following protocol are: Note: Mode - Temperature Power - 80% Agitation - On 10mM Sodium Citrate or Citrate buffer; ph 6.0 Sodium Citrate buffer often gives a better stain with less background. IMPORTANT - Allow samples to cool to room temperature before IHC to avoid inconsistent results. 1 Pre-heat Sodium Citrate or Citrate buffer sec. 2 Place slides in buffer Incubate (allow to cool) RT TissueWave 2 Protocol Booklet B Iss 3 B Iss 3 TissueWave 2 Protocol Booklet 8 WIP-TW2-Protocol Booklet-B /04/ :57:43