When we undertake hematopoietic stem cell transplantation from a previously cryopreserved peripheral blood or a donor lymphochyte infusion we don't usually perform any additional flow cytometry (FC) after thawing these cells and we rely on pre-freezing flow cytometry. There is no doubt that elapsed time and freezing-thawing process can vary the final number of viable cells infused. Considering that cell viability is going to be lower by the time to perform the assay, do you think FC is a reliable indicator of the real number of viable CD34 or CD3 cells has passed into the patient? What cell controls do you routinely perform after thawing? Do these include FC? What time frame do we have to start performing FC? Any example of logistics for this? Thank you!
Of course, You need to calculate viable cells and for this purpose FC is more reliable.
I would recommend some kind of viability assay (FC or any other).
There could be vast difference between frozen batches of cells, and you could get diverse cell doses after thawing.
From an academic and practical point of view, you may want to know cell viability and maybe even the CD34 count of your thawed product. From a regulatory point of view, you may need to validate your freezing and thawing method prior to actually treating patients, to demonstrate, that you generally meet the specific criteria defined for your patient. Euch regulatory authority may have slightly different quality control criteria for this.
We used to take small aliquots (4 x 5mL) of the pre-cryopreserved stem cells and freeze them alonside the main packs and store them in the same tanks. The next day we would thaw an aliquot to perform a post-thaw CD34 count and viability check by FC, to ensure the freezing process was not compromised. The other aliquots were used for future counts and viability (by FC and tissue culture) in cases of delayed engraftment or prolonged storage.
Dear German,
We perform standardly immunephenotyping on the thawed transplant (autologous and allogeneic). We determined CD34+, CD3+ and CD19+ cells on allogeneic transplants and CD34 on autologous transplants using the advanced ISHAGE method. I am convinced that this is necessary to proof the viability of the regarding populations after the programmed freezing proces. Furthermore, viability is also related to the quality and the time of freezing of the transplant before cryopreservation. 7-AAD is a good viability dye but DRAQ7 is better. The FC should be performed immediately after the thawing proces (incubation within 15 min after thawing) otherwise the DMSO will cause additional death and apoptotic cells.
Best regards,
Frank Preijers
Viability of cells must clearly been shown before conditioning for transplantaion has started. The method of the viabiltity test may differ from Center to Center. The test must be certified before. FC measurement is one possibilty but results are mainly due to the thawing procedures lower than in the real product. The old trypan blue method is still an easy and well standardised method with reliable results.
Dear dr Pérez
Yes, after thawing it is absolutely nedessary to perform FC to know exactely the amount of cells we have in the product that will be infused, specially for bone marrow transplantation or cell therapy We quantitate routinely, CD45, and CD34 by Flow and of course determine viability after thawing , quantitate total nucleated cells & the cell fraction needed by you. Best regards Clara Gorodezky
Thank you for this first batch of answers,
As you know quality controls are always a must-have issue for certification agencies but there’s always a wide range of ways to implement them depending on every center's peculiarities.
In fact Jacie’s last manual says literally:
“There shall be the establishment of appropriate and validated assays and test procedures for the evaluation of cellular therapy products:
-For all cellular therapy products, a total nucleated cell count and viability measurement shall be performed.
-For HPC products, a CD34 assay shall be performed.
-For cellular therapy products undergoing manipulation that alters the final cell population, a relevant and validated assay, where available, shall be employed for evaluation of the target cell population before and after the processing procedures”
So, technical aspects are on us, and then heterogenicity could arise...
I forgot (in order to shorten the question) to tell that before infusion we perform viability (acridine orange) and a new cell count from “cryotubes” in a similar way as Mr. Smith describes but without performing FC, so in fact we have certain kind of formal requirement to proceed towards conditioning or whatever, however I think accuracy in cell counts is so important, like in the CD3 DLI setting, and there are so many factors that could vary these figures depending on when and how one perform all these assays and how one read them, that sharing experiences could be helpful.
That being said my original question can be broken down a little more:
1. Are thawed cryotubes reliable to use as a surrogate for the measure of the number of CD34 or CD3 infused (percentage method or truecount)?
2. Does FC make any help if time from thawing to start working with the samples is 15-20 minutes, taking into account what Mr. Preijers stated about DMSO…? (Our cytometer is located far from the transplantation unit, just in the opposite side of the hospital)
3. Should numbers be recalculated taking into account viability results? And if so, how to do this? Do you assume that viability is affected on every subpopulation with the same intensity?
Thank you for all your comments.
Yes you need to do FC after freezing. The viability drop after freezing specially if you don't use suitable freeing method for your cell types. You can look for viability by 7-AAD staining. In addition, you need to look for additional markers like CD34 to make sure that the property of your cells didn't change specially if you passage your cells for long or multiple times, because it is known that if you passage or grow stem cells for certain time they will differentiate and lose their properties.
Post thaw flow cytometric assessments of cellular products is difficult to interpret and may not bear any relationship to the pre cryopreserved results. This is especially true for CD 34 enumeration. It is important to ascertain the viability of a thawed cellular product for quality control reasons. This assures you that your cryopreservation protocol is appropriate for the production of clinical grade products. Until now, the pre cryopreservation data of cell count, viability and CFU determination seem to be of more value in predicting successful outcomes.
Viability of Thawed Hematopoietic precursor is a complex question. In our lab we perform viability test by FC using 7-AAD, Tru- count tubes and ISHAGE protocols, so we check also the number of CD34 and we re-calculate the target cells /CD34+/kg) on the base of the viability results. The viability is performed using satellite samples but frequently we checked the viability on the bag left over following the infusion and we found camparable results neverthless the interval of time between thawing and test performing. In my experience the viability can vary if you are testing CD34 from an autologous harvest which means from a patient previuosly treated or if you check a donor harvest eventually freezed. Another question is do you wash the cells to remove DMSO? This is another point which can influence your results, generally washed cells show a better viability. If your lab is far from the freezing unit you need to transport your samples freezed and thawing them just before to perform the viability test. Last thing we performed a study by adding Annexin V to 7-AAD and CD34 mo-Ab to evaluate apoptosis...it seems that apopototic cells do not move to death by the time!!
Hoping to give you any useful suggestion.
Hi German,
With regards to your questions [and bearing in mind I left Stem Cells and Immunotherapies, NHS Blood and Transplant, England, three years ago so my recollections would need verification].
1. We did some work comparing packs with cryovials from patients no longer needing their donations and who had consented to them being used for research. If I remember there was quite a variation, but I left the laboratory and, unfortunately, it was not published. You could ask NHSBT in England if they did anything with the work.
2. I have an old procedure that says we would store thawed samples up to one hour at 4C, but my recollection is we used a shorter limit [more in line with what has been reported]
3. Work done within NHSBT showed CFU count related to viable CD34 count by FC, and increasing dose of viable cells by either method correlated with speed of engraftment.
Hope this helps!
Viability of the cells have to be performed before infusion. (Please bear in mind that if you are a patient would you take that chance?). You have to strictly adhere to the quality control. Viability can be achieved by simple Trypan blue exclusion test.. If there are more than 2% we usually flow sort the cells. Hope you know that DMSO used in the cell freezing technique is toxic to the cell when kept not frozen. Usually from mere freeze thawing once you can considerably loose 5-10% of cells. To avoid this you have to reconstitute it quickly to efficiently recover cells and FC is done to check various markers. Please be aware that a prolonged freezing and repeated freeze thawing has a tremendously impact on the cell receptors as well. When I was in the transplantation unit, I had an opportunity to study the HLA antigens and the expression weakening. You have to understand that you are awakening the cells from dormancy. So please do every precaution to maintain the quality control and flow sort them if you observe more than 2% cell death.
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