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Leica ST5020-CV5030 Routine Staining & Coverslipping

Leica ST5020-CV5030 Stainer Integrated Workstation

ST5020-CV5030

Recommended maintenance of the dispenser nozzle:

Before starting the daily routine replace or refill daily solvent in brush holder housing (volume: approx. 5.5 mL). Remove nozzle cleaner from instrument at the end of the work week or if it is necessary, at an earlier point of time. Place brush in clean solvent and leave until first day of next work week.   Clean all mounting media and solvent from plastic casing for brush and sit out to dry until the next working week begins. When brush appears too worn to clean the nozzle, please replace with a new one.

The coverslip mountant is dispensed uneven (either mainly in the label area or on the front edge of the coverslip)

  • The system settings must be adapted. Please refer to the instruction manual (Configuration menu). When adjusting the settings, please refer to the recommended parameter sets.
  • Please be aware that (environmental) conditions, specimen type, section thickness, fill level of bath, quality of slides and coverslips, all have influence on the chosen settings and may vary individually!
  • Test the new parameter settings first with test slides. Pay attention to the correct dispenser nozzle height, size and the correct loading bath fill level.
  • Use a priming cycle to ensure that the mounting media is applied without any bubbles. Therefore check that no air bubbles are in the hose of the dispense system.

The Leica CV5030 does not detect the specimen slides. The instrument display shows ‘Check Slides’ message.

  • A minimum grade/quality of specimen slides is necessary. For that reason, we recommend using specimen slides in accordance with the ISO 8037/1 standard.
  • Damaged specimen slides and slide racks can also cause this problem.
  • Check whether the slide racks are deformed (please refer to instruction manual)
  • If “check slides” message is repeatedly shown and persists, please call your local Leica Microsystems contact person.

The upper part of the sections on the specimen slides is dried out

  • The stroke of the mounting media may need to be adjusted toward the top of the slide.
  • The volume of mounting media may need to be increased.
  • The solvent level in the xylene bath may not be sufficiently high enough to cover the top of the slides to help with the even dispensing of the mounting media, and is therefore creating bubbles or drying artefact.
  • If the maximum fill level is not sufficient to cover entire specimens on slides, you may need to use the higher load container:
14047839657 Load container for slides racks (Xylene bath), deep
Keep in mind, however, that by default the higher container is compatible only with the stand-alone Leica CV5030. In operation as a workstation (Leica ST5020/CV5030 or ST5010 (Autostainer XL)/CV5030) the higher container is compatible only after corresponding modifications. In this case, please call the Leica Microsystems Service.

Why do air bubbles appear on the specimen slides?

  • The coverslip mountant and solvent used in the loading bath and/or the solvents used at/in the last staining protocol area might be incompatible.
  • The height of the dispense nozzle is not correct.
  • The wrong size dispense nozzle is used.
  • The nozzle is bent.
  • The dispense system including the nozzle is clogged by dried coverslip mountant.
  • The parameter settings used are unsuitable for the used mounting media.
  • The fill level inside the loading bath is too low.

The coverslips are handled incorrectly by the instrument

The coverslip magazine might be inserted incorrectly. Check whether magazine is correctly inserted. Make sure that the coverslips fit snugly against the back of the coverslip magazine. The back is the part of the magazine that is closest to the user. Make sure that the coverslip magazines are free from loose glass shards and other debris. Use only high quality non-stick coverslips. Leica Microsystems offers a selection of high-quality Surgipath coverslips for your country.

We have a problem with broken specimen slides. How should we proceed?

  • Slides normally break when they are incorrectly inserted due to debris (e.g. breakage of glass, dried mounting media).
  • Labelling (sticker on the slides) which doesn’t fit correctly also may cause this problem.
  • Check whether the bath inserts and the slide racks are correctly positioned in the loading bath. Make sure that no defective or deformed slide racks are used within the instrument.
  • Further check the following instrument components for dirt:
Output racks
Slide transport arms (e.g. specimen slide transport to the output racks)
Coverslip catch tray and magazine
  • Check whether the height of the dispense nozzle and the settings are correct. If problem persists, please call Leica Microsystems’ Service.

The slide racks do not fit into the rack holder (bath insert)

Check whether the bath inserts and the slide racks are correctly positioned in the loading bath. Furthermore, look for dirt and broken glass. If you use slide racks from other manufacturers, also check whether compatible bath inserts are used (see Instruction manual). Make sure that no defective or deformed slide racks are used within the instrument.

The stained specimen slides are available in the last station of the Leica ST5020. Why doesn’t the transport arm move the slide rack to the coverslipper, when it is programmed to do so?

The CV5030 is busy or requires operator intervention. For example, a slide rack is still in the bath or the transport station is occupied with three slide racks and was not emptied by the user.

What precautions should be taken when cleaning or filling the coverslip mountant bottle?

  • To clean the dispenser tubing, either turn the coverslipper off or press”vent mountant” to release the pressure. Carefully remove the hose from the mountant bottle and place in the second bottle with 150 - 200 mL of xylene. Once the bottle has been recapped, continue to press the prime button until a sufficient amount of solvent has passed through the lines to insure that all mounting media has been removed.
  • Once the cleaning step is complete, replace the solvent filled bottle with the bottle of mounting media, press ‘prime’ until air bubbles can no longer be seen in the hose and the mounting media is coming out of the dispensing nozzle bubble-free.
  • To routinely add mounting media, turn instrument off or press “vent mountant”, lift the mounting media hose slightly out of the bottle. Be careful not to turn the assembly upside down as this may cause mounting media to enter the air system. While holding the mounting media hose out of the way, pour a sufficient amount of mounting media to the 150 - 200 mL (maximum) fill line, and then recap.

A new mountant type is used requiring new settings.

The dispenser system should be rinsed beforehand with approx. 150 - 200 mL of a compatible solvent (e.g. xylene). For suggestions on suitable parameter sets for the system setting, refer to the instruction manual.

Why is the dispenser nozzle clogged with dried coverslip mountant?

  • During longer breaks or overnight, the dispenser nozzle should sit in the park position glass bottle. The dispenser nozzle should be totally immersed in solvent.  Fill the provided glass vial ¾ full with a compatible solvent to ensure that the nozzle is sufficiently immersed.
  • Before you start your coverslipper, a priming cycle should be carried out – even if the instrument does not prompt you to do so. Please be aware not to prime into the glass solvent bottle.
  • If using the glass solvent bottle in the park position for priming the mounting media, make sure to change and clean this bottle at least once a week and fill with clean solvent. Otherwise, you may cause additional clogging problems with your dispenser nozzle.

Can the Autostainer and Multistainer Workstations also be operated with the deep load container in the Coverslipper?

Yes, since August 2006, both workstations can be operated with the deep load container. Thus, the covering on the slides will be improved and problems during coverslipping, like the forming of bubbles, will be eliminated. In order to ensure the proper function of the deep load container in the CV/ST combinations, the workstations have to be modified before using the container. A catch plate is already installed and the spacer has to be modified to ensure correct function. Please contact your local Leica service representative for further details. Without prior retrofitting of the instruments, the use of the deep load container will lead to substantial functional problems.

Is there any limitation on programming exact steps?

With the exception of the Exit station, each step can be programmed as an exact step. The last station always needs an interaction, either from the user or the workstation (only for workstation mode).

Why does the dispenser nozzle loosen?

If the dispenser nozzle does not fit securely, make sure that one corner of the edge of the nozzle is rotated so that the square edge of the nozzle rests on the silver retaining plate on the nozzle holder.

Reasons why a rack might not start when loaded into the Leica ST5020

  • The program is not complete; either a station has no defined time or the reagent name in the program does not match the name assigned to the vessel on the instrument status.
  • When starting a program that is displayed as ‘ready to start’, you get the error ‘slide rack cannot be optimally integrated into the staining schedule’.  It is necessary to use the optimization tool to optimize the staining steps in order to integrate this program. If the resources are unavailable to the user, the local Leica Microsystems representative should be contacted.
  • In case the serial cable linking the stainer to the coverslipper is not securely fastened, the stainer is not aware of the coverslipper and every program ending in the coverslipper will not start.
  • The stainer is in Supervisor mode.
  • An oven is the first step in the protocol and for some reason the instrument does not recognize that the oven is installed.
  • The programs are assigned to end in the CV5030, but the coverslipper is switched off (only workstation mode).

Why is coverslip mounting media not evenly dispensed?

The dispenser nozzle is clogged with dried mounting media, or dried mounting media has accumulated around it. There are several reasons why this might happen:
  • The solvent level is too low in the reservoir at the overnight park position so the dispenser nozzle is not fully submerged.
  • Failure to keep nozzle cleaner full of solvent during the working day.
  • Solvent compatibility problems (e.g. when changing mountants, or skipping rinse steps between different mountants)
  • Pressure setting is too low to allow for a proper stream of mountant to be dispensed.
  • Clogging in the dispensing group. We recommend to rinse the dispenser system with 150-200 mL of a compatible solvent.
  • Should your nozzle become clogged with mounting media, we recommend cleaning the dispenser nozzle by removing it and soaking in a compatible solvent overnight. If you are unable to clean sufficiently so that there is a smooth flow of mounting media through the nozzle, replace with a new nozzle. Ensure that a nozzle with the same nozzle size replaces it, otherwise quality problems may be caused as settings are not optimized correctly.
The sizes of dispenser nozzles with the related ordering numbers are listed below:

Order Number

Description

1404740157

Dispenser nozzle 21G, extra-small, pack of 1

1404740158

Dispenser nozzle 20G, small, pack of 1

1404740159

Dispenser nozzle 18G, large, pack of 1

1404740160

Dispenser nozzle 21G, extra-large, pack of 1

Why does the dispenser nozzle touch the specimen slide or hit the glass?

The height of the dispenser nozzle must be adjusted. The recommended distance between the end of the dispenser nozzle and the highest point of the specimen slide out feed is 0.5 - 0.8 mm. The exact procedure for making this adjustment is outlined in the chapter "Aligning the dispense nozzle to the specimen slide" in the instruction manual. The dispenser module sits on a large screw, which can be used to change the nozzle height. If the problem cannot be resolved by changing the nozzle height, you may need to have the system checked by a Leica Microsystems Service technician.

The coverslips are not properly positioned on the specimen.

  • Coverslips may be aligned using the CSP (coverslip position) setting on the control panel.  With every step of +10 that you adjust the coverslip, you are moving the coverslip up towards the labelling field of the slide. If you make a correction backwards (steps of -10), the coverslip will move towards the bottom of the slide.
  • If coverslips are positioned off to one side of the slide, first check to make sure that you have the appropriate size magazine as listed below. Two different coverslip magazine sizes are contained in the Leica CV5030 standard delivery:

Order Number

Description

14047839749

Multisize™ magazine for coverslips with Size 40 – 60 x 24mm

14047839748

Multisize™ magazine for coverslips with Size 40 – 60 x 23mm

  • If the magazine is correct, the instrument may need a left to right adjustment and a service engineer should be called in for this adjustment.

The loading bath does not move.

The transport mechanism of the loading bath consists of a chain module on the floor of the loading area. Check whether the chains move when the loading bath is pulled out. If not, the chains may be blocked, e.g. by breakage of glass and/or dried mounting media. Then check whether pieces of broken glass or specimen slides are clinging to the bottom of the loading bath. Clean the transport area with a suitable solvent, e.g. xylene. If the chain mechanism is still blocked, please call the Leica Microsystems Service.

Undamaged coverslips are being discarded into the coverslip catch tray

It is possible that parts of the coverslip transfer module (pick&place) are dirty, e.g. the suction cups, the coverslip sensor, or the skids of the coverslip transfer module. Please check for adhesive residue or broken glass is in the suction cups, in pick&place or on the glass sensor. Clean all parts carefully with a cloth moistened with a compatible solvent e.g. xylene. Daily cleaning is recommended. If the suction cups are warped, they should be replaced: order number 14047839701 Suction cup, pack of 2.

What are the possible reasons for a program not starting?

One step in the program has no defined station-time. The reagent in the program-step does not correspond with the station or otherwise.

Why does the coverslip transfer module collide with the coverslip magazine (and there is a scraping noise when removing the coverslip)?

Most likely the coverslip magazine is inserted incorrectly. Check for debris, such as broken glass, on the floor and/or under the coverslip magazine. Another reason might be that the quality of coverslips is not consistent throughout the batch being used.

What is the meaning of the red bar in the station button on the display?

The Reagent Management System (RMS) is activated. The longer the red bar the closer the moment the reagent has to be changed.

Why is the rack not picked-up from the load station?

  • SUPERVISOR mode is still activated.
  • The first/next station is already occupied.
  • There is no clip on the rack.
  • The rack is inserted with the clip to the rear side.
  • Programs are not compatible.

Why does the robotic transport arm drop racks?

The handles of the stations are located on the wrong (left) side or they are deformed in shape.

Recommended maintenance of the dispenser nozzle:

  • Before starting the daily routine replace or refill daily solvent in brush holder housing (volume: approx. 5.5 mL).
  • Remove nozzle cleaner from instrument at the end of the work week or if necessary, at an earlier point of time. Place brush in clean solvent and leave until first day of next work week.  
  • Clean all mounting media and solvent from plastic casing for brush and sit out to dry until the next work week begins.
  • When brush appears too worn to clean the nozzle, replace with a new one.

How often does the activated carbon filter need to be replaced?

Every 3 months.

What is the recommended slide drying time?

After a warm up period of approximately 5 minutes it takes 15 minutes to dry slides (bake) at 70 °C.

When does the filling process of the water stations start and how long does it take until the stations are filled?

The filling process starts 40 seconds before the rack is picked up by the transfer arm and it takes approximately 20 seconds to fill the water stations.

What can be done to avoid carryover?

Lower the lift speed (1 = less carryover; 9 = more carryover). Place the staining solutions next to water stations. Avoid long distances between stations.

How many racks can be stained at the same time?

Depending on the staining protocol up to 12 racks can be stained simultaneously - with Leica Microsystems' proposed stain protocols commonly between 4 to 9 racks.