What are the Pros and Cons of Separation of Mononuclear Cells?

Email This Article to Friends & Family | By Cord Blood Advisor | CordBlood: Stem Cells | 0 Responses to this post

Financial Con: Why bother? Common sense suggests there is no reason to process a sample when you don’t know if it will ever be used. Separation of MNC adds to the laboratory cost of processing, and this cost is passed on to the consumer. Financial Pro: Lower storage costs

The smaller the volume of the final sample, the less cryogenic nitrogen it takes to preserve it.
What is really amusing in this regard, is that those banks which have smaller final volumes do NOT pass that savings on to consumers, they charge whatever storage fee the market is currently bearing!

Medical Pro: Advantages of removing red blood cells

This minimizes any reaction of the patient to incompatibilities with the ABO/Rh blood type of the donor. (Strange but true: two people can have “matching” HLA types but different blood types).
Moreover, the cryo-preservation solvent (see below) that is added before freezing tends to rupture red cells. Thus, when the Stem Cells are thawed for transplant they must be washed to remove the solvent and the hemoglobin released from the ruptured cells (it is toxic to kidneys).
Given the above, some banks will brag about having a very low “final hematocrit”, which refers to the red cell count after processing.

Medical Counter-argument: Red cells don’t have to be removed Cord Blood transplants are less sensitive to blood type incompatibilities than transplants of adult bone marrow. The earliest Cord Blood transplants used whole blood. Nonetheless, at this time it has become standard medical practice in the USA to separate out red cells prior to freezing. References:

Gluckman E et al., 1989, N Engl J Med. 321(17):1174-1178 “Hematopoietic reconstitution in a patient with FanconiÂ´s anemia by means of umbilical-Cord Blood from an HLA-identical sibling” - This was the first cord blood transplant proceded by chemotherapy.
Wagner, JE et al., 1995, Lancet, 346(8969):214-219, “Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease.” - Reports on 44 cord blood transplants; at least 28 of them unmanipulated UCB.
Hahn, t, et al., 2003, Bone Marrow Transplant 32(2):145-50, “Use of nonvolume-reduced (unmanipulated after thawing) umbilical cord blood Stem Cells for allogeneic transplantation results in safe engraftment” - Compares 18 unmanipulated transplnats to 8 which were volume-reduced.

Medical Question: Are Stem Cells are lost during processing? It is inevitable that some Stem Cells will be lost in the process of skimming off the buffy coat or separating the MNC. Just what fraction of the Stem Cells are lost is hotly debated. Everybody likes to claim that their method of processing is better because they lose fewer cells — BUT — studies in the medical literature (see below) do not even find a significant difference between the viability of processed versus unprocessed cord blood. There is a large uncertainty, about 20%, in measuring the recovery of viable stem cells, and this uncertainty exceeds the difference between one processing method versus another. References:

Rubinstein P, et al., 1995, Proc Natl Acad Sci USA 92(22):10119-22, “Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.” - Reports “almost total recovery” of viable hematopoietic progenitor cells after volume reduction, freezing, and thawing.
Sato, J, et al., 1995, Stem Cells 13(5):548-55, “Quantitative and qualitative comparative analysis of gradient-separated hematopoietic cells from cord blood and chemotherapy-mobilized peripheral blood.” - After Ficoll gradient separation of cord blood, the recovery rates of MNC and CD34+ cells were, respectively, 55% and 107% (a recovery over 100% indicates this experiment is not very accurate –editor)
Kletzel, M. et al., 1997, J. Hematotherapy, 6(3):269-272, “Red cell depletion of umbilical cord blood (UCB): comparison between unmanipulated and red cell-depleted UCB by Ficoll-Paque density gradient separation.” - This study found no statistically significant difference between separated UCB versus unmanipulated UCB regarding cell viability or recovery of MNC.
Alonso JM 3rd, et al., 2001, Cytotherapy 3(6):429-33, “A simple and reliable procedure for cord blood banking, processing, and freezing: St Louis and Ohio Cord Blood Bank experiences.” - The methodology is a modification of the hetastarch sedimentation and volume reduction approach of Rubinstein at the New York Placental Blood Program. The average yield of total nucleated cells pre- and post-processing was 90% for the first 4055 units banked, with a processing time of 3 h for a single cord.
Eichler H, et al., 2001, Z Geburtshilfe Neonatol. 205(6):218-23, “Aspects of donation and processing of stem cell transplants from umbilical cord blood” - (Report on the Mannheim public cord blood bank; article in German) Achieved an average MNC recovery of 93.4% by the buffy coat preparation method.
Fietz T, et al., 2002, J Hematother Stem Cell Res 11(2):429-35, “Flow cytometric CD34+ determination in stem cell transplantation: before or after cryopreservation of grafts?” - The average recovery was 93.7% (SD +/- 23.1) of all viable cells and 100% (SD +/- 22.3) of viable CD34+ cells. “Thus, CD34 data from different laboratories, for example, within multicenter trials, should be comparable independent of the different time points of acquisition.”
Timeus F, et al., 2003, Haematologica 88(1):74-9, “Recovery of cord blood hematopoietic progenitors after successive freezing and thawing procedures.” - Most of the world’s cord blood storage is stored in single bags. Is it feasible to thaw the whole bag, divide the blood in two parts, use one and refreeze the other? The authors found cell viability was 90% of baseline values after the first thawing and decreased significantly only after the third thawing.

next step…

What is a cryo-preservation solvent?

When living tissues are frozen, they are immersed in a solvent which protects the cells against the formation of ice crystals, because ice crystals would rupture cell membranes. The solvent most commonly used for cryopreservation of cord blood is “DMSO” (DiMethylSulfOxide, usually in 10% solution). However, there is a HUGE amount of research in the medical literature on alternate chemical combinations. Since no concensus has emerged, these articles are not quoted here.