CFU assays on proficiency surveys

[kate murphy]

We seem to be doing abyssmal with our CFU assays on proficiency surveys. Our assays are okay with 'live' products and correlate well with engraftment. But we are not comparing well with our peers on surveys - we've actually tried 2 different vendors for these.

Can anyone share what they do for CFU assays for surveys - do you wash the cells pre-plating? What concentration are you using? Or can someone share their procedure for CFUs? Many thanks!

[applejw]

It warms my heart to know that other real people have problems with these tests, too! I cringe every time we send results in - we've done both CAP and StemCell technologies and of the two, StemCell Technologies seemed to be better since we followed their instructions. The difficulty for us is that the results are reported as colonies per 100,000 cells - we plate at numbers much lower - 20,000 cells - because it makes the plates easier to evaluate and enumerate. For the PT samples, we try not to plate lower that 50,000 cells per ml. We cannot plate at 100,000 cells/ml or the plates would be un-readable. There's a reason that it takes 6 months for CAP to post the evaluations- there seems to be little concensus for CFU. This probably didn't help you with your problem but it sure did make me feel a little better.

[Sarah Dickerson]

CFUs are usually report per 100,000 cells plated. You can make a dilution of 20,000 cells/ml and multiple the answer by five. Many people do that to make the plates readalbe.

Sarah

[applejw]

CFUs are usually report per 100,000 cells plated. You can make a dilution of 20,000 cells/ml and multiple the answer by five. Many people do that to make the plates readalbe.

Sarah

We've done it that way and it seems that the calculation multiplies 5 fold any inherent "errors" in counting - so it's easy to go out of the 2 or 3 SDI that they use for "passing". That's why we suffer with the 50,000 cells/ml which is only multiplied by a factor of 2 even though it is more difficult to read. We have a better track record doing it that way.

[kate murphy]

Many thanks for the replies! You're right, applejw, it's so nice not to feel so alone!

We too have done CAP and Stem Cell Technologies. I will try to play with our plating conentration and see how that works.

Thanks!!

[dnielsen]

The purpose of the PT test is to compare data from different labs. In order for this to work it requires that everyone follows the same procedure, plating the same number of cells. The instructions that come with the kit need to be followed in order to be 'successful'.

I run the PT program at STEMCELL Technologies. Problems with making the dilution and plating the appropriate number of cells are commonly the source when a participant is consistently out of sync with the rest of the group. I realize that the procedure we give is likely very different from your standard procedure. The problem is that there is not just one way to set up a CFC assay. We have chosen a method that has been used by some labs for many years. We are always open to feedback, so please feel free to let me know how we can make this program better.

I would be happy to talk with any of you about your particular issues. We can go through the calculations together and troubleshoot where things might be going wrong for you. We do have a video on our website and a technical support team that is also ready to help at any time.