Hi, Gail,

to start with, what were the issues of apparatus before realising it was valve, fluidics, etc. problem? What were the indications for particular problem- solution?

We have valve and motherboard problem. There are view examples of what happens in our situation: 1) cytometer takes the sample to fast, 2) before taking the sample it adds sheath, decontamination or cleaning fluids for around 100 uL and then starts to measure the sample, 3) the main filter gets emptied and cytometer starts to take irrational amounts of a sample in order to start measurement, 4) huge and unusual amounts of events even though deionised water is under the SIP.

Was there anything similar in situation of yours?

In addition, we use BD accuri C6 flow cetometer (15-01-2010 make), but bought it on 2014. The problems started around may of 2017, almost 3 years after usage. Filter maintenance and fluids were used as denoted in manuals.

I will appreaciate any additional information because we tried to fix it but with very less of positive results.

With best regards,

Baltramiejus

Baltramiejus Jakstys,

We have fluidics issues (leaking out of the sip before taking up sample was the latest). I've been told that the valves are notoriously bad on these machines and that was our most recent problem. Not only can they go bad, but if they leak then they can ruin the electronic valve board that they are built on. There may be other companies out there that would replace these valves with others, but you'd have to ask around. I know one in near Oregon/Washington now. BD won't sell these parts to you directly, but there are other valves that could work (sorry, I don't know which ones myself). I've also heard that even with the suggested maintenance, buildup can occur and cause issues. The company I've dealt with said that running cleaner through it for an hour or two (or even more) can sometimes clean it up enough to solve some problems. I would not suggest either the C6 or the C6Plus, but that's just my opinion. Good luck!

Thanks for the answer,

actually we have solved, i guess, all of our problems during this period and would know how to solve them in the future. We figured out how the peristaltics work and what cause these problems.

Firstly, if you have problem with leaking some fluid into your sample before taking it you should clean or replace the 3rd valve from left to right side (there are 5 of them).

Secondly, we have purchased additional valves from "The Lee Company" http://www.theleeco.com/. These valves are not the same because they can't sell them because of the agreement with BD Accuri. On the other hand, these valves work just fine because have very similar specifications as original valves.

Usualy, if there is a problem with peristaltics, from the first steps you can remove the valves and clean them, that's the fastiest way. To say the truth, very much of additional events are being recorded in in the begining of measurement, that works as an indicator of clogging.

Importantly, be carefull when cleaning the valves because 1'st and 2'nd holes are connected when innactive, 2'nd and 3'rd holes are connected when under voltage.

Best of luck,

Baltramiejus

I had an question as a future user of flow cytometer. In my Animal research laboratory, I want to procure a flow cytometer. Among CytoFlex and BD Accuri C6 Plus which one is best.

Does anyone have a SOP that mitigates any of these issues with leakage on the BD Accuri C6? (not the plus)? Recommendations for cleaning between runs, etc?

Probalms occur due to clogging of valves. Usually clogging appears on 1'st to 3'rd valves. Fastest way is to take out the valves and then clean them with syringe. Be careful, you will need ~5 V DC energy source which will open different holes in the valves.

Valves tend to clogg due to improper maintenance and poor cleaning of the system. It gets decontaminated by bacterias and debris of the samples.

We started using agressive cleaning every time before measurements and after. Potocol- 5 min with 5,95 potassium hipochloride, 5 min with cleaning solution, 5 min with deionised water, cytometer ready to use. Problam is that after longer period of stagnation (for example 2 weeks) the aparatus gets clogged again. This problam might be solved with extended cleaning solution when leaving cytometer to soak at least for the weekend.

BD is no longer making ANY parts available to customers and/or third parties, so the cost of repair is now thousands upon thousands on a case by case basis. So, if you do not have a service contract it's almost not worth repairing. I wish we had never bought one - seems to be nothing but problems.

Dear, Andor,

we have figured it out of how to solve the problems with BD Accuri C6 flow cytometer.

1) disassemble the machine and check the valves for cloging,

2) usually it is possible to clean the valves and keep them running if you don't use FBS, or other very sticky mediums in the future. Otherwise valves will clog again,

3) in case to have additional valves buy them from Accuri. They have designed new valves according to this problem and sell them ~460 $ each, or around ~2000 $ all the chip with all 5 valves,

4) there is another choice, to buy equivalent valves from The Lee Company and insert them in place of old clogged ones.

In general, it was terribly exhausting and money wasting years trying to understand and fix the machine but it works.

If there are people in a process of deciding whether to buy Accuri and save, or to buy other, possibly better but more expensive aparatus, don't save.

With respect,

Baltramiejus

Attached are my notes for disassembly, testing and replacing any faulty valves.

Many heartfelt thanks to Baltramiejus and Brad for the tips for repairing the Accuri. Your hard work is appreciated. I have just dismantled ours and I'll be trying to establish which are malfunctioning when I have a bit more time. Your notes and the guide are a great help.

One thing I did was number the valves (1 to 5 - then I saw they were marked 0 to 4 on the circuit board). I then put labels on each of the tubing ends (e.g. 1A, 1B, 1C etc. left to right or top to bottom as per Brad's guide) to ensure I don't reconnect them wrongly.

We used to have one in my previous job and had to send it back to Belgium twice in about 4 months as it was on the "loaner" deal. I understand at the moment the wait time has got longer and longer with more instruments failing.

Hello everyone, again,

months ago i couldn't tell the exact number of valves because we were not sure if those will be working. By telling it I didn't want to direct you wrong way ordering wrong valves. We have ordered:

http://www.theleeco.com/products/electro-fluidic-systems/solenoid-valves/control-valves/lhd-series/3-port/ported/

LHDA0533415H valves and replaced old ones with these new ones. The apparatus seems to work properly without any issues. Hope this is the end of the problems. The LHDA0533415H valves bought from The Lee company are equivalent for original LHDX valves which are not sold by The Lee company by the agreement with BD Accuri., Firstly check the parameters and codes before buying valves because those might be not the one. Price for 5 of the LHDA valves was 600 Euros.

Please consider that all the information i shared is optional and not necessarily suits all the conditions. You take your own risk of trying operations not mentioned in the official BD Accuri protocols.

Best of luck,

B

Again many thanks for the advice. I'm glad to say our Accuri is now back up and running.

It turned out one or more of the valves was stuck although I couldn't determine which one and I didn't observe any debris that came out of them.

Initially, I couldn't think of a way to get a 5V DC source until I remembered that a USB cable from a PC delivers that exact voltage. I temporarily converted the Accuri cable by sticking 2 pieces of stiff wire into the USB type B connector (the 2 pins on the left hand side as you look at it with the longer side to the bottom are the ones that carry the current) and ran it out of the PC.

I didn't have a multimeter handy so I checked it was working by putting the wires on my tongue. Not recommended!

We have routinely replaced our own C6 valves with Lee valves. The valves we buy have straight pins so you have to bend those and trim them as they are too long. The valve that fails the most is the one that has cleaning solution sitting at one of its ports so I think it gets microcorrosion, not debris. We just tape 3 1.5V D batteries together and tape wires to each end. 4.5 V is fine for testing the valves. We remove the valves (with instrument unplugged and one at a time so we don't forget the configuration) and test them at a sink. We use a 3ml syringe with distilled water and small tubing to hook up to the common port and with voltage applied or not applied see how fast the water flows (or not) out the appropriate port. We don't clean the valves, but rather replace them. Excessive flow of sheath fluid out of the sip tube at the start of running a sample is due to a valve failure. My understanding is that the original valves were made for use with gasses and materials corrode or swell in aqueous solution. Sometimes taking them out and letting them dry out will result in them working again but usually for a short time. We have two new problems now, however. The C6 seems to clog easier that it used to and sometimes we have to run 'unclog' and 'backflush' very often while analyzing samples. Secondly, the Forward Scatter is compressed sometimes, sometimes to an extreme degree. Any thoughts on these last two issues would be appreciated.

I will try to look up the valves we use from Lee and mention it here. They may be the LDA0533415H ones mentioned by Baltramiejus above. We tried different ones that are made of different materials and the one we use now was the best. They last longer than the original C6 valves and cost MUCH less than buying a valve from BD (not sure you can now). However, I didn't want to forget to mention that when you take valves out to test, ALWAYS cut a few mm off of the tubing that were attached to them before putting the valve (or a new valve) back in. The tubing that was attached to them gets stretched and is much more likely to leak if you just put the same ends back onto the valves. By cutting a few mm off of each tubing line you make it so some unstretched tubing goes over each port, providing a tight seal. Eventually the tubings get too short to use so you may need to order some replacement tubing, but just cutting a few mm each time allows the tubing to last a while before needing replacement. Oh, also make sure you push the tubing all the way onto each port or cutting the mm off won't help. Finally (for this installment), it sounds like Baltramiejus solved his problems but I'm not sure the motherboard itself is the sole cause of any problems so far (maybe I misunderstood one). The valves seem to be the culprit in most cases. Too much fluid coming out of the SIP at the start of a run is a valve problem (sorry I don't have the valve numbers in front of me), but a little (a drop) is not a big deal since you can just let it push out, quickly wipe it off when it stops pushing out further, and then quickly put your sample tube on. Sucking too much sample is a valve problem. Usually one of the valves the sheath fluid has to go through is the culprit, because if the machine can't suck sheath fluid well enough, it will pull more of your sample instead. Filter emptying (in line filter) can be due to valve problems as well. Tubing problems at the pumps can enhance imbalance, but they are more rare. One pump pushes and the other pulls. If there is a valve problem in the supply then you can get negative pressure that the balancers can't handle and suck tiny bubbles in the lines, giving you very high background events. Anyway, this is not guaranteed to all be 100% correct, but I think it is pretty close. Check valves first and go from there. ;-)

Hi,

I checked and we tried around four different valves from Lee Co. The one that worked the longest has been LHDA0533315H. I don't know if I have this valve in all positions or whether there is a different one that work better at another position but I've replaced a few so far and used them longer than the original valves were lasting. They were around $75 last time I bought them with a 10+ discount. You need to carefully bend (don't crack where the prongs come out of the valve) the two prongs to match the original valve prongs (distance from the valve body to the bend) and then use wire cutters to clip some of the two prongs off so they are similar to the original valves. There is some wiggle room, so it does not have to be perfect. Just a note on leaking into your sample from the SIP. This is a problem for live cells due to the leaking fluid being water, which can lyse your cells. However, i really consider a single drop 'normal' and live with that, wiping it off quickly on the side of my tube when it stops coming out and quickly sliding the tube up. More than that can be solved with changing the right valve. All the best and make sure you unplug your machines before you work on them AND remove the tube of water under the SIP tube or you may accidentally dump it out on your circuit board!

Very informative input, David. You are very much right. Most of the problems occur due to valve failure. Then failure of the valves is quite unexpected and can happen due to various reasons: 1) to much of FBS in your sample, 2) poor cleaning and maintenance of the apparatus, 3) valves got overheated (our case), 4) improper function of the valve due to its wear. High background count number means your valves contain a lot of debris which continues to grow. We changed all of 5 valves at once because it was really big trouble disassembling and reassembling the BD Accuri every two weeks. After changing all valves it works for three-four months without a problem even after long standing unused periods. No leaking into sample, low background, nice and accurate sample collection. We tried to save up by changing 3 valves which seemed to cause the problems, but still new symptoms used to appear and used to change one after another: 1) taking to much sample, 2) pouring some liquid before sample got started to be measured, 3) high background, 4) emptying sheath filter.

By the way, David is correct, if BD adds some liquid into sample before measurement cells get lysed rapidly (especially it depends on the quantity of added liquid).

In our case the BD Accuri got a short circuit somewhere in the motherboard, smokes appeared but machine kept working like nothing changed. We fixed the board because but left the lever (there is a lever on the lower side of main switch near the motherboard) on purge or waste (it has to be in the middle) and our valves got overheated. That was the reason of our future problems (another problem was we tried to fix it by saving money- bought 3 valves). After we realized problems continue we changed all the valves and problems disappeared straight after. Remember, Purge and Waste is for specific cleaning and maintenance only when voltage introduced to valves is doubled (7 V), while normal voltage on valves is 3,5 V. Do not do this mistake and do not overheat the valves. If Purge or Waste will be ON you will notice the red light near the switch.

In addition, I have no idea of how efficient is changing one by one valve, but it didn't work for us so we changed all of them. I believe it is possible to get them cheaper but that was amount The Lee Company charged us for valves and delivery.

Very helpful, Beltramiejus! I'm glad I didn't have the motheroboard problem on top of the repeated valve problems. One thing that should be noted is that I have one of the older C6 units, bought from Accuri. There could be differences with the newer units in terms of valves used (and therefore whether the valves/advice above apply to newer units). I'll have to look for the purge/waste light. I'm not sure my unit has one or I have not noticed it. I don't think I have a purge or waste option or I've never used them. My unit has unclog and backflush. Perhaps they are related. All the best. It was a great concept to have a flow cytometer for which the voltage does not need to be adjusted. The design flaws, especially the valves, detracted from the benefit...

Thank you very much for your tips @Bradley Elmore. We were having problem with our Accuri C6 and we could not send to repair because the BD is no longer providing assistance for our machine series. We detected that one of our valves was clogged and we disassembled the machine to get the valve, and according to your instructions we boiled for 15 min and sonicated for 20min (we did twice this two process) and the machine started to work very well again. Thank you very much. In attachment is the video after the cleaning process. The blue pipelines were not having fluids to supply the filter with the sheath fluid before the cleaning.

I read through all of the discussion above, thank you! Very helpful!

If anyone finally retires their older Accuri C6, please consider donating it or a part to us as our photomultiplier tube for forward scatter has died. It may just be the small circuit board on that part since it appears it got wet from a leak at some point and corroded. BD will not sell us the part and their minimum repair charge is huge and not affordable, especially with my lab having a lapse in major grants. You can reply here or my email is [email protected]. Thanks for the consideration.

A company called "Certified Genetool" claims they can repair Accuri cytometers, faster and cheaper than BD. They also say they'll quote prices in advance of shipping out the machine! I found then on the used equipment site LabX.

I don't know how good they are, but might be worth checking.

Good luck!

I have a first gen Accuri, I have had many problems with my Accuri until I realized it's the valves. I buy the valves from the Lee Company and when I do I change all 5 + all of the tubing. So the cost of fixing it doesn't exceed $500 which is a far cry from what BD would charge IF they still fixed them.

Now I have a problem with what I believe the circuit board that is attached to the FL2. If anyone knows who made those circuit board or if anyone can figure out the schematic for that circuit board, please help me out.

Hi Joy,

Thank you for the suggestion. Certified Genetool is trying to help but still are expensive and don't have a supply of circuit boards. I'll be interested if anyone has an answer to Saada Eid's question above about who made the circuit boards. Still interested if anyone is considering dumping their old first gen C6... Finally regarding Saada Eid's comments above, when I change valves, I only cut about 2-3mm off of each tubing so I have a tight seal. No need to change the entire tubing. Eventually the tubing gets too short and you do have to change it.

[email protected]

Today we opened our ACCURI C6 for valve changing, I found that the connection of the tubing is different from the one it showed on the protocol which Mr. Bradley Elmore showed in his file. Our machine is the second generation, maybe the tubing is different from that in the first generation (?). Also, an important lesson to learn is: take pictures at each step before you dissemble the valve, otherwise it is easy to mis-reconnection.

David C Pallas if you are having problems with your motherboards or other circuit boards, consider asking someone from your electrical engineering department for their expertise. I did and thankfully they were able to help.

Thank you. That is a good idea. I took the PMT board to a board level repair person outside our university but they said they need a schematic. Does anyone have schematics for the Accuri C6?

Hello Everyone,

Thank you for all your helpful answers! We are attempting to revive an Accuri C6 that appears to be suffering from a myriad of valve issues (wildly variable flow rates, back flow into the sample tube, high background counts, etc). Upon opening the unit, we found some corrosion around the electrical pins connecting the first two valves to the valve board. In your opinion, is this unit fixable with gentle cleaning and replacement valves? Can the corroded electrical contacts on the board itself be replaced?

David C Pallas Saada Eid

Thanks!

Best,

Dustin

Hi, Dustin,

yes, it can be fixed. The problem is that durability of changed valves will depend on many conditions. You can order new valves from The Lee company, 5 valves = 600 Eur, other than that you can buy a new valve board that will cost -+6000 Eur at least. As i got to know from other scientists, the new valve board does the job much better than those new replaced valves. There is something wrong with the valves them self. They have a higher chance to clog probably due to being to small and debris forms on them very fast. You can clean the contacts with the contact cleaner because it has a very low conductivity, which means you can spray on the contacts with no fear of a short cut. You don't need to change the board.

Best regards,

Baltramiejus

Baltramiejus Jakštys

Thank you! We will proceed with cleaning the contacts and ordering replacement valves.

in continuation to the above discussion , can you kindly mention of the sites or places from where we could buy the 1 year maintenance kit parts cheaper, the tubings, the filter etc.. most of the prices of BD & the its distributor is quite high...

Hello,

I wanted to buy valves from Lee Company, those dedicated to Accuri BD C6, but it turned out that I needed an authorization letter. Is there a chance that you can buy valves with similar parameters, but matching this equipment? Unless you received authorization letters and only then you made the purchase.

Thank You in advance for Your help!

Best regards,

Marta

Dear Marta,

That's a good point you mentioned. I didn't think of an authorisation letter. I have discussed with some people.

That's what I know:

1) the valves with the code ( LHDA0533415H ) are the best fit. We have bought 15 of them and will buy 10 of them again. 5 of them cost ~600 $.

2) the LHDA0533415H valves are primary valves while manufacturing the original C6 valves which are protected by an agreement of The Lee company and BD. So their durability is much lower. I can tell that from our experience. Since starting buying these valves there is no end. Every few months, some valves still fail.

3) the good news is that cytometer runs, despite much higher expenses than expected.

4) I know from experience of other people who were lucky about getting a new fully preassembled valve board of C6+ and installed it on C6, they have no problem ever since. It's more than one year from this moment.

5) the problem of getting the new C6+ valve board is now much complicated since it costs around 6000-7000 $. In this situation, you have to calculate, whether you want to spend 1200-2400 $ each year on unoriginal C6 valves or take a risk and buy the valve board of C6+.

6) as far as I know, BD now offers of purchasing the new BD C6+ cytometer with a discount of ~11.000 Eur if you give back the old C6. Marta, please, get in touch with me in case you would work it out with the authorisation letter. We would join the purchase. Some additional information. We had a situation when our detection chamber poorly clogged. Don't be afraid of taking off the hoses and cleaning the chamber with pressure and hot water/ethanol.  Don't forget taking off the SIP needle firstly and cleaning it with a syringe under pressure. GOOD LUCK and please share if any news in the field of C6 cytometer.

Dear Baltramiejus,

Thank You for the answer and for Your advices. We have decided to buy replacement valves for now. If something changes and the authorization letter will be needed, I'll let You know.

When it comes to cleaning the cytometer, we have already done it several times in the way You described. Unfortunately, after some time it ceased to be sufficient and the cytometer stopped working.

Best regards,

Marta

For what it is worth, I have had bad experiences in general with BD as a company and BD Accuri flow cytometers. Long story short, during my postdoc we had constant problems with our BD Accuri C6 flow cytometer (pump issues, leaky valves, etc.) and the BD technicians were never able to fix it properly because they were not familiar with the machine (BD acquired Accuri after our model was made). Now I am a PI and my lab is using a BD Accuri flow cytometer and the service (at least here in Canada) from BD is horrible! For instance, I have been waiting several months for a 1-maintenance kit that I ordered. My next flow cytometer will definitely not be a BD Accuri.

Following

We have also replaced the valves in our Accuri C6.

We are now looking for the tubing replacement for the entire instrument. Does anyone know the specs of the blue red yellow and green tubing for the instrument?

Thank you in advance,

Megan H

Dear Megan L Heinrich ,

I have bought some tubing just on the internet because the specs are unclear. We didn't change the tubing though.

However, my colleague did a risky move and with a pressure built up by syringe cleaned all inlets of the detection chamber. Then cleaned the SIP. They really get clogged there.

After a few rough clean ups as mentioned above, the cytometer works just fine and only ads a drop of liquid before starting to take a sample.

Best regards,

Baltramiejus

We have had similar problems with our Accuri C6 plus since we bought it in 2016. It's been sent for repair 6 times within 4 years we've owned it, depiste regular and meticulous maintenance. It's a waste of money, time, energy and your precious samples and reagents. I'd be happy to spend more on a more robust flow cytometer. If you buy Accuri C6 make sure you also buy extended warranty otherwise you will end up spending a lot of money on repair and shipping costs every few months.

I have also encountered problems with Accuri C6 since I purchase it in 2016 related to the valve. Currently, BD is charging me an exorbitant price for the valve (approximately 10,000 euros). Do you have a solution to change it with a better price? Thank you

I believe the BD Accuri Flow cytometers become obsolete April 15th of this year and BD will not offer service after that. If anyone is interested in selling theirs for parts or donating it (I'll pay shipping), I'd love to keep ours going as we are on a low budget. You can contact me at [email protected]. For those looking for valves but not being able to get them from Lee Company because they are proprietary, we use the equivalent Lee Company valves but cut off the two prongs some and bend them to make them fit the Accuri setup. They work fine but will get stuck eventually, especially the one that has decontamination (bleach) fluid coming into it first. We use the LHDA0533315H version, as we found it lasted longer than the original perfect match valve. That is our experience but they all have to be changed more than one would like. Secondly, has anyone had a problem with the FSC-A collapsing towards the x-axis? We can't afford to send the machine to BD as their minimum repair is so costly now so was wondering if anyone had this problem and solved it and knew what it was due to. Thanks much,

David

Megan L Heinrich - I believe it is generic 1/16 ID/ 3/16 OD polyurethane tubing. You can order the tubing, tubing harnesses and various connectors from CPC worldwide. https://www.cpcworldwide.com/

David C Pallas That is super helpful info. I too got the 'it's proprietary' answer from Lee, and they were unwilling to point me to the equivalent. I second your offer to buy parts though :)! I ended up jut being extra thorough cleaning the valves out, and that seems to have worked so far.

I recently detailed (vented?) in another thread, but I'm in the middle of my third full teardown/cleanout (after a $23k rebuild by BD that took 3 months). We do a lot of cell cycle with PI stained, ethanol fixed nuclei so we get a lot of clogs, primarily on the valves in the waste line. My standard "fluidics stability error' protocol for clearing blockages now is to hang them them in a jewler's sonicator with hot water for an hour, then hook up to a 12V DC power supply and switch the valves while pushing DI through the ports with a syringe. This has which has generally been able to clear blockages in the valves. I've also had clogs in the waste out line at the tubing harness and at the quick disconnect on the waste bottle, as well as the pulse dampener (aluminum-acrylic blocks) on the waste line. Similar approach to the valves, sonicate and purge with hot DI, but to be honest I'm not sure they were ever the issue that was ever at issue. I'm not sure exactly what you mean by the FSC-A falling toward the X-axis (decreasing cell size? size), but that sounds optical or electrical to me, unless you mean your number is decreasing, that might be the either dampener on the sheath side. Some have also suggested running filtered DI as sheath fluid, and running water through all lines at shutdown if not used daily.

Thanks. I meant condensing towards the y-axis (FSC-A on the x-axis) What I mean by FSC-A going towards the y-axis is that it condenses for some samples (not as spread out) but then will go back to normal and the same sample that was 'condensed' rerun 10minutes later will be spread out. It is clearly a machine error, perhaps photomultiplier tube or ? No problem with differences in cell number. When the FSC-A profile is condensed, the cell populations are harder to gate and cells are harder to gate from debris.

David C Pallas try unclogging the detection chamber by unscrewing the SIP and building up the pressure by a syringe filled with DI water. One should see porridge dripping out of the hole for the SIP. The unusual distribution of cell population (spreadness) happen due to the changes in the core size and sample speed through the detection chamber due to the changes of cells mechanical properties. The build-up in the detection chamber has caused these problems for us.

We now run our C6 for maybe half a year without problems other than pouring some water into the sample before suction. However, that's not a problem at all, you just clean the droplet and put the sample tube under the SIP before the machine starts sucking the sample.

Asma Gati all the information you will find above in the discussion. Check for my answers.

Thanks. I've done that but this seems to be electronic. I will change the flow rate and see if I can spread it out some when it happens.

I think I have a clog in the clear tube port of the flow cell on my BD C6 SORP. The clear line going into the flow cell is empty. During startup/shutdown/decon cycles some fluid is pushed in this line but it doesn't make it into the flow cell, it just flows back through the clear line after I hear a valve switch. This is not normal operation right?

BD replaced the valve board on this machine a couple years ago now with the new larger valve version after I inherited this instrument, but I don't think it has ever been quite right.