We do at first Ficoll to obtain the MNCs but without Erythrocte lysis and then go for CD138+ beads for purification from Miltenyi. We do this manually with a magnet from Miltenyi and the respective column which is working as good as automated system, only requires some person to do it. The number of CD138+ cells strongly depends from the patient, often we get either nothing (everything below 50.000 cells is considered in our lab as nothing), or in between 100.000 and 200.000 cells from 20 ml of bone marrow. In rare cases we obtain more than 1 mio cells (up to approx. 10 mio cells was the maximum I got), this is strongly associated with disease progress. Try to get patients history and cases with disease progress for method establishment as this can only be successful when you have a proper amount of cells inside. And also do not hesitate to contact Miltenyi that they should come and help you, my experience in the past was that they did this and also gave free kit samples for testing purpose or borrowed even a magnet for 4 weeks for testing.

You can use the MACS Whole Blood CD138 MicroBeads in combination with Whole Blood Column Kit.

Thank you for the answer. The problem is we are routinely banking and freezing ficoll-isolated cells from bone marrow of myeloma patients and would like to use this material for separation and subsequent experiments as well.

were isolated by use of Isopaque–Ficoll separation medium and PCs were sorted by immunomagnetic separation using Macs CD138 MicroBeads

We do at first Ficoll to obtain the MNCs but without Erythrocte lysis and then go for CD138+ beads for purification from Miltenyi. We do this manually with a magnet from Miltenyi and the respective column which is working as good as automated system, only requires some person to do it. The number of CD138+ cells strongly depends from the patient, often we get either nothing (everything below 50.000 cells is considered in our lab as nothing), or in between 100.000 and 200.000 cells from 20 ml of bone marrow. In rare cases we obtain more than 1 mio cells (up to approx. 10 mio cells was the maximum I got), this is strongly associated with disease progress. Try to get patients history and cases with disease progress for method establishment as this can only be successful when you have a proper amount of cells inside. And also do not hesitate to contact Miltenyi that they should come and help you, my experience in the past was that they did this and also gave free kit samples for testing purpose or borrowed even a magnet for 4 weeks for testing.

Dear Dr Druennert,

Thank you for the answer. I have contacted Miltenyi as well and will try to implement their as well as your recommendations. Could i ask you a few questions? Specifically:

How many cells do you usually enter into the column? In my case it was about 8 million cells.

Which kit and column do you use? Miltenyi recommended using Whole Blood CD138 Microbeads.

The bone marrow samples came from patients with progressive disease and, so I expected a good yield.

Dear Filip Garbicz, we don't use a whole kit, we use the CD138 microbeads order no 130-051-301, buy the colums and needles for the colums separately and also prepare our MACS buffer on our own.

Commonly we get in between 10 mio up to 50 mio PBMCs out of 20 ml bone marrow. This also depends if the bone marrow has a lot of blood clots. Only in rare cases we get yields with more than 100 mio cells, but then we don't use all cells, but maximum 100 mio cells if available.

Dear Filip, in the past I've used anti CD138+ ab and magnetic beads to sort malignant plasma cells from multuple myloma patient. We used Miltenyi MACS sorter which worked quite well. Nevertheless we also observed a drastic reduction of CD138 expression in the positive sorted population which was time and handling dependent. Maybe this is also your case. Apparently the marker is lost pretty fast so you should work with very fresh material and try to reduce at minimum the step before the sorting.

Good luck!

Thank you for the answers, I will try to work according to your tips next time I have bone marrow from MM patients.

Hello,

I would like to sort PC from 3 ml bone marrow multiple myeloma patient. 3 ml it's enough for extract micro RNA with qiagen kit? I would like to have your experience in this field. thank you.

Tough to answer, Mohamed, normally we purify CD138+ cells in our lab from 20 ml bone marrow aspirate. And depending on the patient status, I have seen everything in between no cells and up to 10-12 mio cells.

So a few questions: is it the first aspirate that is taken? if not you may get a lot of blood and very less bone marrow so less primary cells.

Disease status? very critical indeed - under progress, you will get sufficient cells, but when the patient is in remission or in a stable position - you will most probably hardly get anything to work with.

And generally - primary cells can contain very less RNA, this depends a bit from the respective cell size, and treatment condition of the patient, could get in between 300 ng and almost 1 µg total RNA out of 250.000 cells.

And it also depends on what or how much you want to analyze...

Thank you very much Daniela.

clinicians told me that we can not have a very large volume for that I just asked 3 ml. I have to see again with the doctors the possibility of having more volume. Also we will recover the bone marrow cell of newly diagnosed patient and so maybe we will have a large number of cells. For my project I want to see the level of expression of some micro-arn in healthy donors, recently diagnosed patients and relapsed patients. so I'm going to sort the plasma cells and then I'm going to extract the micro-RNA with a Qiagen kit and then do quantitative PCR. if I can have one microgram of RNA will be enough I think.

Update: After a couple of isolations I seem to see what was critical for obtaining myeloma cells.

Most importantly - the bone marrow must be processed immediately (up to 4-6h after biopsy) - after that time MM cells lose CD138 and CD38 antigens

I usually avoid putting the bone marrow into +4C and just let it wait in cell incubator.

I use a syringe before ficoll separation in order to homogenize marrow "clumps" - myeloma cells reside in those clumps and using a syringe with a small needle results in 2x the number of plasmocytes!

If anyone still has problems with primary myeloma isolation, I think I finally got it working very well - ask me.