Processing Cord Blood for Storage

Video Tours:
Cord Blood Registry:  private bank in Tucson, Arizona USA
VITA34: private cord blood bank in Leipzig, Germany

A few private cord blood banks freeze the blood whole, while most process it. There are two stages of processing: Volume Reduction and Separation.

Volume Reduction can be accomplished either by sedimentation or spinning in a centrifuge. (Note: sedimentation uses a chemical called "Hespan Starch" or "Hetastarch" which is made by Dupont Pharmaceuticals.) Afterwards, the blood components are separated so that: red cells are on the bottom, plasma (a clear white liquid) is on top, and in the middle is a pinkish layer called the "buffy coat" which contains the white blood cells (WBC), including stem cells. At this point, a gentle spin in a centrifuge is sufficient to isolate the white blood cells (WBC). Most public banks stop processing at this point and store the WBC in bags.

Separation of mononuclear cells is accomplished by further centrifuge spinning of the white cells. One protocol for doing this which is routinely used by stem cell researchers is called "Ficoll-Hypaque" density gradient centrifuge. The separation step reduces the volume of the collection to only a few milliLiters, which makes storage in vials feasible.

MonoNuclear Cells: Hematopoietic stem cells are identified with a characteristic surface molecule called "CD34+", but only 1-2% of the MNC are actually stem cells. When parents bank cord blood blood, and they receive a follow-up lab report saying that so many billions of cells were stored, the lab is actually counting MNC, not stem cells. This is the standard medical procedure.

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 Fanconi´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.

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.

To further protect cord blood cells against the formation of ice crystals, it is important that the cells freeze slowly. This is done in a special controlled-rate freezer, and then the blood is transfered to long-term storage. The final storage temperature should remain stable at -196 C. Freezers and handling procedures are designed to avoid "Transient Warming Events (TWEs)". In particular, the cells must stay below -130 C while they are in storage. If the temperature fluctuates above -130 C and then below again, the blood will suffer irreparable cell damage.

Freezer Types
A few public cord blood banks with big budgets use "BioArchive" freezers from Thermogenesis (Nasdaq: KOOL). These freezers were originally developed at the New York Blood Center. This liquid nitrogen freezer uses a proprietary, computer-driven robotic system to cryopreserve and archive up to 3,626 bag units. The location and retrieval of each sample is computerized. The goal of using a robotic arm to circulate and retrieve samples is to avoid "Transient Warming Events (TWEs)" when samples are added or removed from storage.

Most of the public cord blood banks in the USA are using "dewars", which are basically insulated tanks with lids. The disadvantage of these units is the need to open the lid when adding or removing samples. However, temperatures are monitored constantly and they have been proven effective for long-term storage.

All private cord blood banks in the USA are also using "dewars", like most of the public banks.

Dewar Photos

Freezer Contents
Blood is stored in either bags or vials. When the racks in the freezer are loaded with blood containers, they should be spaced evenly to ensure temperature stability. A given freezer will have racks designed to hold either bags or vials, you cannot mix containers in the same freezer. Hence, most banks build their business around one storage container or the other.

The cryogenic temperatures are maintained with cold nitrogen. Some banks use the liquid phase of nitrogen and others use cold nitrogen vapor (gas). The pros and cons of the nitrogen options are given in the table below.   
 

Liquid Nitrogen	Vapor Nitrogen
Uses more nitrogen	Cheaper (only fill the bottom of the tank)
Constant temperature	Possible Transient Warming Events
Documented chance of infectious disease leaking from one bag onto the outside of another and infecting the recipient	Safer for Quarantine Tank
long-term viability confirmed Broxmeyer, H. E., et al. 2003 pnas.0237086100	long-term viability not studied