For doing HLA Cross match T cell and B cell, I need to separate WBC from whole blood using a cell separation media ( HISTOPREP, separation medium for cells). In some case i need to face a problem, where Blood RBC ring formed instead of the WBC ring formation.
Its not a regular phenomenon, but for some samples I am having such problems frequently. I have done re-sampling for such issues, but the problem persisted.
So, What can be the cause for such problems ?
Does it indicate any blood related problem of sample donor ?
Yes, I have encountered some times this problem. I think that this is due to the fact that the red cells in these cases are not so heavy as the normal RBC, perhaps because they have less hemoglobin (in case of hypochromic anemia for example).
However, I think that for cross match purposes it is better to separate T and B cells via magnetic particle separation (Stem Cell company produces such magnetic beads) since so you have much more purified T and B populations.
In my experience the yield of B cells is very poor with the density gradient separation.
Dear Sir, Genc Sulcebe,
Sir, in my lab we are also thinking to change our process to beads system. Sir, it will be convenient for me if you give a best source for such magnetic beads.Sir, link will be helpful.
There could be several reasons for the red cell contamination where the WBC (Buffy coat) is. Besides the intrinsic problem of a donor’s cells health.
The preparation method is also very important. If you leave your cells in the tube for a long time without vigorously re-suspending prior to the undelay method that often results in a poor separation and a RBC contamination. (I assume you are doing underlay and not overlay from your results?)
Things to keep in mind are, your Histoprep should always be in room temperature, 2200rpm for 30 min NO BRAKE.
I also agree with Gene Suicebe that after Histoprep separation a B or a T cell separation with Magnetic beads will be beneficial. We have used Miltenyi Biotech for years both for human and mouse work (The beads are FDA approved for clinical use ).
The company provides a protocol when you buy the beads, but if you need further help, I will be happy to help.
Hope this help you somewhat. Good luck with your research.
I am completely agree with Jeanette Baker. Histoprep should always be in room temperature, 2200rpm for 30 min NO BRAKE and keeping the sample for long time before processing, and moreover donor health specific. I saw the rinf in the patients with immunosuppressive drugs
Dear Jeanette Baker & Sharad K Mittal ,
In my protocol, I normally dilute the whole blood with Normal saline (in 1:1 ratio). Like, 2.5ml Whole blood and 2.5 ml Normal Saline (0.9% NaCl) .
Then Histoprep (3ml) taken in 14 ml polypropylene tube and the mixture (Blood & Normal saline mix) is slowly pour upon the Histoprep solution. Then centrifuge is done at 4000 RPM for 15 minutes (keeping the centrifuge temperature below room temperature) .
But i keep the Histoprep in 4 degree temperature (Because level in the Histoprep bottle written so) . I have added the level of the Histoprep bottle.
Dear Sir, Genc Sulcebe ,
Thank you for the book (Stem cell technologies HLA product). A huge collection of different analysis and procedure related illustration. Really a knowledgeable book.:)
Yes, certainly you should keep the Histoprep in the fridge (4 degrees) but take it out of the fridge before the procedure and take the volume you are going to use so it will reach room temp (about 30 min before, keep it in the Hood).
Centrifugation should also be in room temp.
I would suggest (just for convenience , not for scientific reasons) to try and do underlay.
I use a 10 ml syringe with a Bone Marrow needle aspirate attached (about 12 cm long).
I believe everything else you are doing is fine.
Do try the Miltenyi beads I think you will be happy with the results.
Dear Sir, Jeanette Baker,
Yes,Sir we are trying to change our procedure to beads system to yield fresh culture as much as possible.Hope the new system will work better for every sample with good quality separation and reproducibility.
The immature red cells or red cells with low MCHC do not go down as normal RBC, because they have lower density or do not form rouleaux well. There is a quick and easy way to remove the red cells in the upper layer.
A product called Red-Out (Robbins Scientific, Sunnyvale, CA) works very well. You can simply add 100ul Red-Out to 10ml blood, mix by inverting the tubes several times and incubate at room temperature for 5 minutes. Then perform the isolation as usual. You will get a clean cell prep. It works every time!
Red-Out is a murine mAB against a carbohydrate moiety on red cell membrane. The antibody binds to both mature and immature red cells producing small cell aggregates. Red cell aggregates sediment through the isolation media.
Although Red-Out may no longer available through Robbins Scientific, it is still possible to get through some labs. A little bit goes a long way.
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