We are using the 5 x 60 mL reagent trough carrier and the 60 mL reservoirs for one of our protocols. We use liquid level detection and liquid following when aspirating from these reservoirs since the max volume we use fills to the top of the reservoir (73 mL).
The aspirating step runs smoothly until the span-8 needs to aspirate from the reservoir for the third time. On the third and following times the span-8 aspirates, it will detect the liquid and submerge the tips at the defined height but will not liquid follow it, leading to an improper aspiration error due to not being able to aspirate all of the requested volumes at the submerge height.
The issue still occurs regardless if I use capacitive or pressure LLD and high or low sensitivity. It also occurs when the tip is approaching the carrier (indicated by the red arrow when the liquid level is at that height), which I suspect is the cause of the issue since the aspiration steps work fine if the starting volume is below the height of the carrier. I do not know how to bypass this and was wondering if anyone has any ideas?
Setting a fixed submerge height without liquid following is not feasible since the transfer volume varies for each run, and we currently do not have the budget to get a reagent carrier for bigger reservoirs.
Any help is greatly appreciated,
Could you please upload the method pkg and example trace files? They can be uploaded to the Shared Temp Folder here. Password is: Hamilton23
I uploaded the package folder as “playground”, which should override the previous file that was already there. The corresponding trace file is “playground_1c9”. Let me know if you need anything else.
I suspect there may be a couple of things going on.
First, I would recommend using a different labware definition which is slightly higher in the z:
50/60mL Reagent Carrier definition
Second, the error that you’re getting is error 80 or improper aspiration error which is a MAD (Monitored Air Displacement) error. So, it’s detecting the liquid using LLD just fine, but generates an error during the aspirate. As you are re-using tips, it is generally recommended to disable MAD as any residual liquid left in the tip could add noise to the pressure signal and inadvertently trigger MAD errors. You can disable MAD locally within the method using the HSLML_STARLib Aspiration Monitoring Off function:
Finally, you will need to have some dead volume in the trough to ensure sufficient liquid is present. At least 1mL or so depending on the liquid for the 50/60mL trough.
I hope this helps!
Thank you for the help, especially providing the correct file for the carrier I was using! Unfortunately, even locally disabling the MAD (regardless if I’m disabling the “Aspiration_monitoring” of PLLD or CLLD or both), the problem of the liquid following still persists during run. I have tried doing the aliquot steps as single steps instead of a smart step to see if the problem is with the smart step itself.
The liquid following problem did not arise when I transitioned to single steps to do the aliquot. This confirms my suspicion that the smart step could not recognize the local disabling of aspiration monitoring. However, I was using “surface part volume,” which is not the current dispense mode since I am dispensing the reagent into an empty plate. Plus, the dispense speed was much slower than what was defined in the liquid class. It also did not matter if the “Aspiration_monitoring” disabled or not.
I switched the dispense mode to “jet part volume” but it gave me a hardware error when it was going back to aspirate from the reservoir for the second time. The error it generated was that the max volume was reached and it didn’t matter if the “Aspiration_monitoring” disabled or not.
I currently do not know how to efficiently program an aliquot step as single steps. Hence, is why I opted for the smart step. If you have any other suggestions on how to make it work with the smart step, then feel free. I am open to using single steps but I’m also running into issues as described above
It sounds like there are several issues so it is challenging to disentangle them all without more information on each (method pkg and trace file).
While the library function gives you local control on turning MAD on or off, it is actually toggling the setting globally and would disable it for single steps or smart steps. So if you are still encountering the same error 80 then something else is going on and I would need to see the trace files to confirm.
To program in single steps, you would need one aspirate step picking up the total volume and then multiple dispense steps (or a dispense step in a loop corresponding to # of aliquots).
Thank you for in-depth insights on how the library works regarding to MAD. I provided the new trace file named as “playground_7a4”. I also reuploaded the package file (in case), albeit it’s not much difference other than the addition of the “aspiration_monitoring” step. In the meantime, I will work on doing the method as single steps and troubleshooting in case using the smart step is no longer an option.
I greatly appreciate your help!
From the trace, it looks like you used the ClotDetectionMonitoring_1000ulChannel_Off function (which uses the cLLD sensor) and not the AspirationMonitoring_1000ulChannel_Off function which would turn MAD off.
The STAR platform has several similar process controls that use either the capacitive or the pressure sensor.
I would also recommend placing the command after an Initialize step. The smart step includes an initialization so you would have to add an Initialize single step at the beginning of the method.
Apologies for the confusion. I enabled and disabled the wrong functions when running the protocol again to obtain TRC file. I did run the protocol with the AspirationMonitoring_1000ulChannel_Off function alongside with the other suggestion you provided, but the liquid following issue still persists. The corresponding TRC file is “playground_f0c” and below is a screenshot of the method.
Could it be that, because the smart step already have an initialization step, it would restart the MAD back to on? If that is the case then I will resort to single pipetting steps.
The function is a global toggle so I would not expect such behavior. I will follow up with you separately to troubleshoot some more and then post an update to the thread.
So upon further investigation, this was confirmed to be a reported bug that was resolved in VENUS 5 onwards. See link to the version history and below for the excerpt.
Using gold or single steps should work without issue.
My apologies for the back and forth!
Here is the logic for how to perform your process using the Gold steps.
- Uncheck Auto increment in aspirate
- Add tip sequence to the outside loop over sequence (set it to never for reset)
- Changed inside loop to loop over the destination sequence
- Added a break when inside loop reaches 4
- Removed the reset source sequence from outside loop
For those who need to use this method, I want to add (with the help from Eric) that you need to add an additional dispensing step that will dispense the remaining volume to reset the plunger, especially when reusing the same tips. There’s a couple of ways to do it but, in my case, I add an additional step so that span-8 moves to the aspiration source and dispenses the “remaining” volume (air at this point) at a fixed height above the container before it loops back to the next aspiration step.