Recently, the flow cytometer (BD FACSort) that I am using (with other labmates) is having some problems.
The Drain, Fill and Backflush functions are working fine. However, the Run function is not working properly.
When the vent valve of the sheath fluid is closed, liquid is not taken up by the machine, and even there is some leakage from the machine into the sample tube. However, if the vent valve is opened, liquid can be taken up.
We tried using a wire to clean up any blockage in the acquisition tube. It did solve the problem for a while but the problem came back during the washes with bleach solution right after that.
Can anyone suggest any solution to solve the problem?
Thank you.
-MC
http://www.cytekdev.com/kb/questions/28/Flow+Cytometer+Fluidics+troubleshooting+flow+Chart
Try the above troubleshooting procedure.
Thank you for your reply. I also found this guide yesterday but went wrong into flushing the droplet containment module (DCM) tubing.
At the end of the day, it turns out to be insufficient pressurization of the sample. When the vent valve of the sheath tank is opened (no sheath pressure), the sample pressurization is fine and the sample voltage is a bit lower than it should be under normal condition. However, when the valve is closed, the pressurization becomes a bit insufficient so that the sheath fluid is even forced out from the sample injection port (SIP). Is there any explanation and solution to this? Moreover, the pressurization of the sample was becoming more and more inefficient (takes longer time to pressurize) in the past runs. Does this help to give any conclusion?
Thanks a lot.
-MC
I will make some assumptions: The instrument status box within Cell Quest says standby while in Standby and will go to Ready when you rotate fluidic control to Run. The FACSort does not have front panel light like the predecessor FACScan did for this.
When you have the instrument in Run with no sample on the SIP (Sample Injection Port) and the outer droplet containment tube is removed, you see back dripping. This is normal because the sheath fluid is flowing back down the Sample Injection tube. This is good, no total blockage present from debris.
From your description, sounds like when a sample tube is installed on the SIP, instrument in RUN, there is not enough Sample pressure differential between the Sheath pressure/ Sample pressure to aspirate the sample up into the flow cell (for hydrodynamic focusing, and result failure back fills the sample tube with sheath fluid).
Check for small sample tube air supply leaks. This can be a simple ball seal replacement. The Ball seal is the small black Teflon coated spring ring around the top of the SIP accessed by removal of the Droplet containment Tube (the outer tube you had to remove to run a wire up the SIP). Also, for the age of the instrument, FACSort, check to see if the sample barbed port, the one the green sample air tubling goes to on the flow cell, is not cracked. Just tub a little on the tubing to this threaded barbed port to see if it fall apart. Also, there are two ways the instrument vents the sample tube to atmosphere when in standby, through a pinch value in the fluidic nest (front knob mech) and the clicking pinch valve behind the DCM (Droplet containment pump) when you move the Sample tube swing arm to one side. Check to see if these are partially opening while in Run mode.
You should be able to TEE a pressure gauge (analog is fine) into the sample air line at the flow cell to monitor what the Sample pressure is doing when going into Run. The pressure should boost momentarily then settle to a steady pressure, slightly higher than the Sheath tank pressure, 4.5 to 5 PSI about, depends on what your sheath regulator is locked down to. If the pressure creeps up and settles low than I might think you have a pressure leak.
One last item. the LO/MED/HIGH switch used on the front panel actually incudes the feedback loop gain from the Sample control circuit board. I have had a bad contamination of Sheath fluid form carless removal of fluidic nest tubing troubleshooting (salt water) compromise this electrical connection and affect sample rate. Make sure the integrity of this switch assemble is intact through inspection with a strong focusable flashlight.
Ed
Many many thanks for your really detailed and helpful reply.
1. Check for small sample tube air supply leaks. This can be a simple ball seal replacement.
-The technician and I also thought of this possibility. However, we don't have any spare for this and not sure if there is any available for sale. I will check. Is there alternative method to have a simple and temporary check for this?
2. the one the green sample air tubing goes to on the flow cell, is not cracked.
-The troubleshoot guide also mentioned checking the green tube but we didn't know what to do earlier today. Will check tomorrow. (The machine is only 1 year younger than me, hope it will continue working fine...)
3. Also, there are two ways the instrument vents the sample tube to atmosphere when in standby
-Not so sure where they are right now, will have a look tomorrow.
4.You should be able to TEE a pressure gauge (analog is fine) into the sample air line at the flow cell to monitor what the Sample pressure is doing when going into Run
-Not so sure if we have the tool for this. Will ask tomorrow. For the momentary pressure boost, is it observable from the sample voltage displayed in the software? From the sample voltage, I can see it drops slowly from 10.23V (corresponds to the sheath pressure) to around 6.4V after a minute (which is still higher than 6.33V corresponds to the expected voltage of LO speed). Also, I can see back-filling of sheath fluid into the sample tube right after putting back the support arm when the machine is in RUN. Does this imply the boost is problematic? Is the boost immediate after putting back the arm or does it take time to rise gradually to a high pressure?
5. One last item. the LO/MED/HIGH switch used on the front panel actually incudes the feedback loop gain from the Sample control circuit board.
-I guess this would not be the problem as we have never done any manipulation on this...
Sorry for raising too many questions and again thanks a lot for your reply. I really appreciate it.
-MC
Usually, if you are using Becton Dickinson Falcon tubes and the tube seems to fit snug on the SIP, then the Ball Seal might be ok. Doesn't mean it is not compromised. They are available as a consumable replacement part from Becton Dickinson for about $20 US. There's always Teflon tape but you can damage the Ball Seal if compressed to much.
If the sample tube is back filling filling with fluid (its going to be sheath fluid) when in Run then you have a sample pressurization problem.
When was the last time this instrument had its pressure transducer replaced? Its black mounted on the back of the fluidic nest. It monitors and reports via voltage the pressure difference between the sheath pressure and sample pressure. The membrane diaphragms rot out over the years inside of the unit.
You need to monitor the Sample air, green line to the flow cell to see what is going on. Get a 0-10 or 0-15PSI analog pressure gauge from your Bio-Med bone yard or try your local swimming pool supply store.
Ed
1. They are available as a consumable replacement part from Becton Dickinson for about $20 US.
-Thanks for the information!
2. If the sample tube is back filling filling with fluid (its going to be sheath fluid) when in Run then you have a sample pressurization problem.
-This problem was seen before when some bad tubes were used (we are re-using the tubes) but now seems happening even with good tubes. That's why I am not sure if this is due to the ring or the pressurizing parts.
3. When was the last time this instrument had its pressure transducer replaced?
-I guess we have never replaced this before as the technician following the machine seems have no experience on this. Also, how to make sure the problem is in the pressure transducer but not in the differential pressure regulator? I had a check online and there should be pressure transducer available for ordering but the other one seems not available...
4.You need to monitor the Sample air, green line to the flow cell to see what is going on. Get a 0-10 or 0-15PSI analog pressure gauge from your Bio-Med bone yard or try your local swimming pool supply store.
-Will ask the technician tomorrow.
Thank you so much!!
-MC
We tried troubleshooting the machine again today.
In the morning, the machine suddenly became perfectly fine without any manipulation. (Last night was not working).
Throughout the hours, we kept the machine at RUN and didn't switch to other functions (trying to reduce changes made to the machine, though the pinch valves in the fluidics box seems to be alright), but the sample voltage gradually increased with time.
The green gas tubing is in good condition (no detachment, no hardening or cracking).
For measuring the sample pressure, unfortunately we don't have the apparatus for measuring that small pressure. We only got those for higher pressure according to what the technician told me.
The machine seems to be able to recover after a long break but goes wrong again within ~30min of running. If thing are going wrong in the simple hardware, the machine should be not working all the time, right? It's a bit hard to find out a suitable explanation for this......
-MC
Also, check the sheath tank for cracks, especially where it would flex when the black top plate is locked into place on top (cracks on top), also that the probe orange gasket (inside threaded top of probe) integrity is ok. You can substitute your own sheath tank by making one from a 1 liter polycarbonate vessel if you are handy. Remember, the sheath tank is pressurized with air and the fluid is drawn out through a tube. Just duplicate with the 1 liter bottle, your own tubing, probe sensor needs to be fooled by placing 1K ohm resistor across sensor socket, and some fittings with sealant. If you had a pressure gauge you can monitor the current sheath tank pressure for any change, should be constant.
It turns out to be the problem of the joint between the gas tube and the flow cell.
The problem is solved now!
Thanks a lot for your patient and helpful explanations!
-MC
Dear Yan,
First, different models have different design in the inside; second, there are quite a lot joints inside the machine connecting different tubes or different parts together. You will need to do a more detailed troubleshooting to find out the problem.
To determine if there is any leakage, barometer may not be that necessary. You can look for the sample voltage shown in the status inside the software (again, different softwares may have the voltage shown at different places, you may need to find it out by yourself.) check the detailed user manual for the proper value of the sample voltage at different running speed and see the actual value shown by the software is correct. If the value is very different from the proper value, then basically you can confirm the gas leakage, but still need to find out the exact place that leaks. You can have both the FACS machine and the software on, at the same try to look for the leakage by different means :
1. Look for any obvious breakage in the tubings or the joints;
2. Try to listen to any little sound for leakage;
3. More accurately, clamp different parts of different tubes (one at a time) and look at the sample voltage.
If you can find out the leaking part, replace it and you should be able to fix the problem. However, you may need to first confirm if the problem is really due to a leakage. There could be many different causes for a machine not to take up your sample. More detailed description would help a lot in troubleshooting.
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