Bio SB TintoStainer Manual Automated System for IHC & ICC Flexible Open Affordable. phone:

Transcription

1 Bio SB TintoStainer Manual Automated System for IHC & ICC Flexible Open Affordable

2 TintoStainer Index Topic Page Overview 3 Terminology and Definitions 6 Operating Procedure 9 Label Printing and Customization 13 Protocol Programming 18 Reagent Programming 22 Additional Resources 27 Supplementary Resources Thermo Scientific Immunohistochemistry Training Guide Version Bio SB TintoStainer Brochure 2

3 TintoStainer Overview 3

4 Overview The Bio SB TintoStainer is an automated 36, 48, or 72 slide system for use in Immunohistochemistry (IHC) and Immunocytochemistry (ICC). The TintoStainer is perfect for laboratories that have a need increase work-flow efficiency through automation, yet want to maintain low operating costs. The Bio SB TintoStainer is a rapid, flexible, open and affordable solution which allows for fast implementation of laboratory protocols while keeping a compact footprint. The TintoStainer features an integrated slide and reagent bar-coding system to ensure that the right reagents and slides are used with a selected protocol, while reducing incidences of user-error. Systems from competing companies are not only expensive to purchase but are closed, limiting the detection systems and antibodies available for use. This leaves laboratories at the mercy of supplier price hikes and a limited catalog of reagents to choose from. The TintoStainer is an open system which allows for the implementation of any ancillary, antibody, detection system, or chromogen from any supplier. To ensure successful integration into your laboratory, the TintoStainer comes bundled with Bio SB ancillaries, Tinto prediluted antibodies, and detection kits. Additionally, the TintoStainer comes with complete setup, support, repair, and training for 1 year so that laboratories feel confident that the TintoStainer is always up and running. TintoStainer Material Check List TintoStainer System Slide Printer Reagent Printer Computer Interface Unit Auto-stainer Report Printer Power Cable (5) Reagent Tray (40) Power Cable (5) Reagent Vials (100) 12-Slide Rack (3) Slide Labels (1 Roll) Reagent Labels (1 Roll) Washer, Waste, and Distilled H20 containers Supplemental IHC Materials Bio SB TintoRetriever or equivalent epitope retrieval system Detection System Antibodies Substrate Chromogen Counterstainer Aqueous or Xyelene Based Mounting Media Coverslips Missing equipment? Call Bio SB at for assistance ordering supplementary materials. 4

5 TintoStainer Material Check List C B D A A. TintoStainer System B. Slide Bar-code Printer C. Computer Unit D. Reagent Bar-code Printer E. 12 Slide Rack F. Reagent Vial (12 ml) E F 5

6 Terminology & Definitions 6

7 Key Concepts and Terminology The following terms are used throughout this training guide and our training process. This section is not meant to be a comprehensive review of all terms but instead to define key concepts used throughout this guide. Reagent Specific reagent as stored in the reagents lists or in a specific protocol. It generally includes a name, bar-code, lot number/expiration date and incubation time. Antibodies are a form of reagent that also include antigen retrieval information. Antibody A single reagent step that also includes antigen retrieval information. An antibody must be inserted into a protocol to be used on a slide. Protocol A protocol is all IHC steps that will take place on a slide to complete a given antibody stain. The details for each unique protocol are stored as a file called an Auto-program (*.aut file format) in the auto-stainer operating directory. The protocol is made up of unique reagents, incubation times and antigen retrieval. Example Shown Below: Protocol Template A protocol template is the number and arrangement of programmable steps that Protocols (antibody protocols/auto-programs) can be created in. Protocol templates are configured in the protocol template screen. Protocols must match the default protocol template in order to be used. 7

8 Default Template The primary protocol template. Protocols must match the default template in order to be used (labels printed and run). Optimization Determining the correct working protocol for a given antibody. This does not included validation. Validation Ensuring an optimized protocol meets desired results via laboratory defined testing. See the Assay Validation Recommendations section for more information. Detection System Protocol This is a protocol that has been saved with the antibody column left blank. Detection System Protocols can be assigned to specific antibody reagents in the antibody edit lists screens to create a full protocol. 8

9 Operating Procedure 9

10 Bio SB Internal Use Operating Procedure Barmeth = 2 **Note: TintoStainer does not perform epitope retrieval. Ensure that slides undergo epitope retrieval process by undergoing treatment with a citrate or EDTA based system in addition to use of Bio SB TintoRetriever Pressure Cooker or PT Module. Powering On & Logging In Turn on Computer unit, TintoStainer Machine, Reagent Printer, Slide Printer, and HP Printer Ensure that buffer and distilled water containers contain at least 3 Liters of reagent. Also ensure waste containers are less than 50% full. Launch Auto-stain 360.exe Enter Username and Password. By default the username is a and the password is a On Welcome screen, tap Program button. Figure 1 - Welcome Screen Main Programming Grid will appear on screen. You now have to decide whether you will be running a 3 step protocol (ImmunoDetector and HRP Plus) or a 2 Step protocol (PolyDetector). Press Protocol Template button in bottom left hand corner. If building a new protocol, select New template. If you are using a protocol previously programmed into the machine, press Get Template button. Pick protocol from list. **Note: Reference Protocol section, which details steps preprogrammed into TintoStainer. Figure 2 - Programming Grid Programming Slides (Fast Methodology) This methodology is preferable if you do not wish to incorporate data such as the Slide ID, Case, Doctor, Block ID, or Tissue Used into the experiment. It is a faster methodology, aimed at research institutions that use a wide variety of tissues and reference tissue data using a separate in-house documentation procedure, or prefer to skip printing slide labels and manually write slide data. Press Slides in top left of programming grid Enter number of slides for experiment Programming grid will now populate with number of slides to be treated with protocol. Proceed to Operating procedure section 10

11 Programming Slides (Slide, Tissue and Block Info) This methodology is preferable if you need to incorporate data such as the Slide ID, Case, Doctor, Block ID, or Tissue Used into the experiment. This methodology incorporates data onto the slide which can be customized in the Initialize screen at startup. This methodology is ideal if you are using a small number of tissue types or if you are using the TintoStainer in clinical applications and need to incorporate patient data. Press Slide Info in bottom left of programming grid. Enter Slide ID, Case Number, Doctor Name, Block ID, and Tissue #. Figure 3 - Selecting Reagent Enter Number of slides and click Finish Entry Repeat the above steps for every different tissue Type and block. Populating the Programming Grid & Printing Labels Programming grid will now populate with number of slides to be treated with protocol. The elements of the detection system protocol appear at the top of the columns (outlined in green, figure 3). The individual tiles beneath the elements will flash and a box will appear with the specific reagents available for use. Clicking on the reagent from the drop down menu will fill in tile. The next tile will flash and again show the reagents available for use in a drop down menu. Move across the grid filling in the tiles with your selected reagents. If you do not see an antibody, detection system component, or substrate chromogen, see section [Reference Reagent] on Page 12 to program the reagent in properly. Once you are finished programming protocol, press next button on bottom. TintoStainer will prompt user to print labels for slides. Select print all. Affix labels to appropriate slides. Pay attention, as the wrong label affixed to the wrong slide result in incorrect application of reagent to slide. TintoStainer will prompt user to print labels for reagents. If labels are already affixed to reagent, you need not print another. Open up front door of TintoStainer and place reagents and slides wherever you please. Barcode scanner on arm will ensure that the proper reagent is applied to slide. 11

12 Operating Procedure Select Prime pump option if you notice any air bubbles in wash buffer or distilled water tubing. Check the Check Volumes option if you would like the TintoStainer to check reagent volume. Select Start Run Option. TintoStainer will then check reagent volume and prompt user if there is not enough of a reagent. TintoStainer will start protocol. Note start and end time. If a batch protocol was selected, TintoStainer will display when the substrate chromogen will be needed. Figure 4 - Printing Reagent Labels If batch protocol was selected, make sure to allow adequate time for chromogen preparation. When prompted load batch chromogens, TintoStainer will then scan reagents and apply to slide. Once protocol is over, TintoStainer will stop the run. Tissues will be continually hydrated until End Run option is selected. Save/Print run protocol using in-house experiment tracking procedures. Unload slides from auto-stainer Counter-stain slides with Hemtoxylin, Methyl Figure 5 - Reagent Vial Scan Green, Nuclear Fast Red, Etc. Treat and coverslip slides. Observe slides under microscope. Routine Maintenance Ensure Waste Containers are regularly emptied Every 240 Slides, Autostainer should be cleaned using provided DAB-Away. Autostainer will inform user if instrument needs to be cleaned and will provide step-by-step instructions. Ensure that Distilled water and Buffer tanks contain reagent. Figure 6 - Starting Autostainer Run 12

13 Label Printing and Customization 13

14 Slide and Reagent Label Printing The TintoStainer allows for automated printing of barcodes for both reagents and individual slides. This process not only reduces user error but allows the TintoStainer to ensure that a proper reagent or slide is loaded after a protocol is programmed. Some users may choose to omit this step if an automated or manual label printing process is incorporated in another laboratory process (e.g. Histology). Reagent labels may hold 3 lines of data, while slides may hold 4 lines of data. Labels or HIER and Xyelene resistant, and may be affixed before epitope retrieval. When handling reagents and slides, ensure that the user does not touch the printing area excessively, as this may cause data to rub off the surface. Customization of Data Presented on Slide and Reagent Labels To customize the data printed on slides and reagents... Login to TintoStainer At startup menu, click Initialize Click Slide Label or Reagent Label option (see Figure 7) The Design Slide or Design Reagent Label box will appear with drop down menus, along with the Length, or number of characters you want displayed on the label (see figure 8). Figure 7 - Label Customization Items that may be printed on label include Case Number User Slide ID Short Name Todays Date Lot Number Pretreatment Program File Doctor Tissue Incubation Time Figure 8 - Slide Label Drop Down Menu 14

15 Customization of Slide and Reagent Labels (continued.) Slides Label Printing 1. Data Customized in Design Slide Label in initialize screen... Reagent Label Printing 1. Data Customized in Design Reagent Label in initialize screen Is retrieved from data input by user on Slide Information menu on programming grid 2. Is retrieved from Edit Reagent list menu on programming grid 3. Which is incorporated onto slide printout. 3. Which is incorporated onto reagent printout. 15

16 Label Adjustment If you encounter mis-alignment of labels when printing, you may adjust where the label maker prints for both slides and reagents. Reagent & Slide Label Printing Adjustment If you encounter mis-alignment of labels when printing reagent labels, you may adjust where the label maker prints for both slides and reagents. Load Autostainer Software. Login. Click Initialize Screen. Click on Reagent Labels or Slide Labels on Right Side of Screen. Click Adjust Label option (see Figure 8B) Using bottom and side cursors, adjust printing area (See Figure 8C). Select Test Print option to ensure label printing properly (See Figure 8C) Adjust Y axis Figure 8B Figure 8C Adjust X axis 16

17 Reagent Drop Zone Make sure that the proper Drop Zone is applied to the tissue, depending on the part of the slide your tissue is located. Default drop zone and volume can be changed in itiliaze screen. To pick a reagent drop zone, simply click on the Dispense column in the protocol template. The area highlighted in yellow is where reagent will be dispersed. The ul symbol corresponds to the top of the slide. More than one drop zone can be picked, although more reagent will be used. Figure 10 - Reagent Drop Zone Customization If highlighted, reagent applied to bottom of slide. If highlighted, reagent applied to top of slide. If highlighted, reagent applied to middle of slide. 17

18 Protocol Programming 18

19 Programmed Bio SB Protocols To load a protocol template... Press Protocol Template button in bottom left hand corner. Press Get Template button. Pick protocol from list. All protocols below have Immunowasher Rinses between each step unless otherwise indicated. Protocol 1A - Bio SB ImmunoDetector AP/HRP 5 minute blocking solution 30 minute Antibody Application 10 minute Link 10 minute Label 5 minute chromogen Protocol 1B - Bio SB ImmunoDetector AP/HRP Batch (Same as 1A, For use with unstable chromogens) 5 minute blocking solution 30 minute Antibody Application 10 minute Link 10 minute Label Autostainer prompts user to prepare chromogen 5 minute chromogen Protocol 1C - Bio SB ImmunoDetector AP/HRP + Background Blocker (For use primarily with Antibodies that have large amounts of background or Infectious Diseases) 5 minute blocking solution 10 minute background blocker application (no rinse after) 30 minute Antibody Application 10 minute Link 10 minute Label 5 minute chromogen 19

20 Protocols (Continued) Protocol 2A - Bio SB PolyDetector AP/HRP 5 minute blocking solution 30 minute Antibody Application 30 minute polymer application 5 minute chromogen Protocol 2B - Bio SB PolyDetector Batch AP/HRP 5 minute blocking solution 30 minute Antibody Application 30 minute polymer application Autostainer prompts user to prepare chromogen 5 minute chromogen Protocol 3A - Bio SB PolyDetector PLUS HRP 5 minute blocking solution 30 minute Antibody Application 10 minute Polymer A Incubation 10 minute Polymer B Incubation 5 minute chromogen Protocol 3B - Bio SB PolyDetector PLUS HRP + Batch 5 minute blocking solution 30 minute Antibody Application 10 minute Polymer A Incubation 10 minute Polymer B Incubation Autostainer prompts user to prepare chromogen 5 minute chromogen 20

21 Programming a Custom Protocol The TintoStainer comes programmed with protocols for the Bio SB ImmunoDetector HRP/AP, Bio SB PolyDetector HRP/AP, and Bio SB PolyDetector PLUS HRP Detection Systems. However, some laboratories may need to to build a protocol template customized to their needs. The TintoStainer has a easy to use methodology for this. On the programming grid screen, press Protocol Template button in bottom left hand corner. Left side of screen will list steps that you can add to your protocol. Select the steps you wish to incorporate, including any necessary washes. Before the chromogen option is incorporated into your template, ensure that you select the *Switch step. This step ensures that the waste generated from chromogen goes into the waste container. When done building protocol, select New Template button. Select save to disk now option. Select Yes. Type in protocol name. All protocols below have Immunowasher Rinses between each step unless otherwise indicated. Figure 9 - Protocol Outline 21

22 Reagent Programming 22

23 Reagent Programming Although the Bio SB TintoStainer comes with reagents already pre-programmed, the Bio SB TintoStainer is an open platform, and reagents can be programmed in from any supplier. Coupled with the ability to program in custom protocols, the TintoStainer allows the end user ultimate flexibility. Additionally, laboratories can use the TintoStainer in either clinical or research settings, and it is important for technicians and laboratories to set internal protocols as to how to program in reference reagents, reagents to be used in quality control procedures, and other reagents in procedures the TintoStainer will be used. Implementing a standardized methodology will ensure faster work-flow and turnaround time while redeucing user-error. The following pages are used as an example of how Bio SB has incorporated the TintoStainer into certain laboratory protocols. They are included to stimulate discussion within the laboratory as to which protocols may work best with your existing work-flow. The following pages display how reference, QC, and R&D components may be programmed into the TintoStainer. Reference Reagent Programming QC Reagent Programming Antibody Optimization Programming 23

24 Reference Reagent Programming For use with all reagents and antibodies that are verified control reagents and have passed all QC procedures. All Reference reagents should have a Green sticker. 1. Load desired protocol using previous Operating Procedure in Section 1 2. Select Edit Lists option from top 3. Select the category of reagent you would like to add (Antibody, reagent, etc). See figure Follow the following naming protocol... Reagent Name Supplier + Reagent Name/Antibody Name + Antibody Clone (If applicable) Figure 11 - Edit Lists Menu Short Name (10 character Max) Reagent: Reagent Name (If not Bio SB, use a 3 letter code to identify manufacturer) Antibody: Antibody + Clone Bar-code (10 character max) 3 letter code for Manufacturer + Catalog Number Figure 12 - Entering in Reagent info *For Bio SB Reagents, use 15 ml catalog # *For Predilutes, use 15mL catalog # *See Side Panel for Manufacturer codes Compatibility - Don t fill in Lot # - Lot Number Expiration - Manufacturers expiration date 24

25 QC Reagent Programming This programming method is useful for people wishing to qualify reagents. Reagents to undergo quality control procedures should have a yellow sticker. All reagents undergoing QC proecures should have a yellow sticker. 1. Load desired protocol using previous Operating Procedure in Section For Reference (control) reagents, select from drop down menu. If Reference (control) reagent is not input, program reference reagent in. 3. For reagent to undergo QC procedure, select QC Reagent from the drop down menu for secondary, tertiary, or chromogen component. 4. List QC Reagent Name, Lot number and expiration on any appropriate forms. 5. Once QC procedure is comeplete, select Save Run option, and type experiment number. Example. If you are QCing a lot Biotin Link and M/R HRP Label vs. a Control, the Biotin Link lot would be QC Secondary Reagent 1 and M/R HRP Label would be QC Tertiary Reagent 1. Note: By Default, Secondary Reagents and Tertiary Reagents are programmed for 10 minute incubation times. Chromogens are by default 5 minute incubations. Polymers are by default 30 minute incubations. 25

26 Antibody Optimization Programming For Use with Antibodies to be optimized in TintoStainer. 1. Load desired protocol using previous Operating Procedure in Section 1 2. For antibody to be optimized, select Antibody Optimization followed by an integer 3. If testing more than 1 antibody, it is recommended that you pick the lowest integer for the highest dilution (i.e 1:50 Dilution = Antibody Opt 1, 1:100 = Antibody Opt 2, etc.) 4. Ensure that all relevant data is input on internal document forms. 5. Once Antibody optimization is finished, select Save Run option, and type experiment number. Example. If you are running an experiment to optimize ER at 1:50, 1:100, and 1:200 from manufacturer X, you would select... Control Lot of ER (Reference component with Green Label) for first set of slides. Antibody Optimization 1 (ER 1:50) -> Input Lot, Expiration, Dilution and Clone on form. Antibody Optimization 2 (ER 1:100) -> Input Lot, Expiration, Dilution and Clone on form. Antibody Optimization 3 (ER 1:200) -> Input Lot, Expiration, Dilution and Clone on form. Note: Default incubation time for Antibody Optimization is 30 minutes. If an antibody has passed QC, feel free to program the reagent as a reference reagent and affix a green sticker. 26

27 Additional Resources 27

28 Additional Resources The Bio SB TintoStainer Manual should be sufficient to help get most laboratories up and running. For additional support, we have also included a copy of the Thermo Scientific Immunohistochemistry training Guide version This guide includes routine operation procedures, recommendations for validation, Advanced operation, and trouble shooting information. If you still need assistance, feel free to contact Bio SB Support using the information listed below. Bio SB is open Monday through Friday from 9am - 5pm PST. Bio SB Support info@biosb.com Phone (International): Phone (United States): Bio SB online trouble-shooting form: Reordering: info@biosb.com 28

29 TintoStainer Accessories All the components below compliment the TintoStainer. TintoRetriever Pressure Cooker BSB 7008 The TintoRetriever Pressure Cooker with built in temperature gauge allows for quick and efficient epitope and nucleic acid retrieval for all IHC, ICC, IF, CISH and FISH protocols. Bio SB IHC Detection Systems Bio SB TintoAntibodies All Bio SB IHC ancillaries, chromogens, counter-stains, biotin & polymer detection systems are compatible with the TintoDetector. Additionally all Bio SB TintoAntibodies are compatible and ready-to-use with the TintoDetector. CISH/FISH Probes and Kits All CISH and FISH probes, ancillaries and reagents are compatible with the TintoDetector. If you would like to see a list of CISH and FISH Probes distributed by Bio SB visit our ISH site at TintoDetector BSB 7034 A capillary gap based system, the TintoDetector allows a technician to perform IHC, ISH, and ICC. The built in incubator and reagent container allow for a wide variety of protocols to be implemented in the laboratory. 29

30 Additional Information If you would like additional resources on how your TintoStainer system can be used, feel free to visit the following sites: You Tube Our You Tube channel contains up to date information about Bio SB products. Channel URL: Web Our website strives to have the most up to date information for your needs. Additionally our website lists IHC, ICC, IF, FISH and CISH reagents that can be used with your TintoStainer system. Channel URL: Support Phone (International): Phone (United States): Bio SB online trouble-shooting form: Reordering: International Distribution Our most up to date list of distributors is available at 30