Laboratory Processing of Cord Blood

1. TNC, MNC, and stem cells -- what are they?

2. How many stem cells are in a cord blood collection?

3. How are stem cells separated from cord blood?

4. Manual versus Automated Processing.

5. Pros and Cons of separating stem cells from cord blood.

6. How are stem cells frozen?

There are many types of cells circulating in the blood, and our purpose here is not to give a tutorial on blood.  Stem cells which can form all the components of blood are called "hematopoietic" stem cells, from the Greek terms "haima" for blood, and "poiesis", to make. 

Stem cells happen to be Mono-Nuclear Cells or MNC: when you look at them under a microscope there is only one nucleus.  Unfortunately, one of the most difficult aspects of stem cell medicine is that you can't identify a stem cell just by looking at it.  There are other types of blood cells which are also MNC, such as nucleated red blood cells.  The only proof that a cell is a stem cell comes from how it behaves when it multiplies.  As a short cut, scientists and laboratory technicians have worked for years to develop various chemical stains which have a high affinity for stem cells.  The best known marker for blood-forming stem cells is that they test positive for CD34, a protein found on the surface of stem cells.  But, CD34+ is not an accurate test: test results vary between labs, they can vary within a single lab, and only 1-2% of the MNC that test positive for CD34 are actually stem cells.

When a laboratory processes a cord blood collection for storage, the test which is most often reported is the Total Nucleated Cell count or TNC.  The main advantage of measuring TNC is that the count is highly reproducible within and among labs, so it can be used accurately throughout the blood banking community.  Even better, the TNC count can be automated with the use of a flow cytometer.

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Cord blood collections from full-term, singleton, babies range from 70 to 130 mL volume.  The concentration of stem cells per unit of volume is variable (smaller collections generally have higher concentrations) but the average is 8.6 million TNC per mL.  Hence, a large cord blood collection can have close to a billion TNC, millions of MNC, and thousands of blood-forming stem cells. 

Cord blood is a much more concentrated source of stem cells than almost all other tissues in the adult body.  When you hear the endless media reports about stem cells being found in various parts of the body, bear in mind that the existence of a few cells does not make the tissue a potent or viable source for developing stem cell therapies.

In public cord blood banking, only large collections are processed.  The goal is to only spend financial resources saving units which are big enough to provide a stem cell transplant for a small adult.  The threshold set by HRSA to save cord blood donations for the USA federally-funded public banks is 900 million TNC.

In family banking, parents can choose to save smaller collections with a volume of 30-40 mL, which would only be enough to transplant a young child.  However, these smaller collections may be big enough for use in regenerative medicine applications, and there is the possibility that the cell count could be expanded in the laboratory if necessary.

The topic, "Scientific Factors Necessary to Define a Cord Blood Unit (CBU) as High Quality" is an active area of research.  The USA federal agency HRSA, which funds the national network of public banks, has appointed a Working Group of its Advisory Council to study this question.  The Working Group published a preliminary report dated May 2009 (see pages 6-8 of the pdf file), but research is ongoing as of the close of 2009.  At present, draft FDA guidelines, which are based on the assumption of a patient weighing 20 kg (44 pounds), define a high quality CBU as one which has at least:

• 500 million TNC (HRSA reimbursement requires 900 million TNC)
• 1.5  million viable CD34+ cells
• 85% viability, as measured by Colony Forming Units (CFU) or a correlated assay
  

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The earliest cord blood transplants were performed with whole cord blood.  Thus, it is not necessary to separate the components of the blood in order to save patient lives.  There has never been a prospective randomized trial to compare transplant patient outcomes with processed cord blood versus unmanipulated cord blood.

Over the years, it has become standard practice in the cord blood banking industry, both public and private, to at least reduce the volume of the blood so that the final storage unit is smaller, requiring less cryogenic nitrogen for preservation and enabling more units to be stored in each storage tank.  Volume reduction means that the plasma is removed before storage.

The three main components of cord blood, like any blood collection, can be separated by weight:  the heaviest layer is the red cells, the lightest is the plasma (a clear white liquid), and in the middle is a pinkish layer called the "buffy coat" which contains the white blood cells (WBC), including stem cells.   Picture courtesy of Journal of Visualized Experiments:

Today (Dec 2009) the vast majority of cord blood banks store the buffy coat (see the summary table), regardless of what processing method they used to separate it.  There are several cord blood banks in Europe which store whole blood.  The biggest exception to the rule is StemCyte, a USA-based bank which provides both public and private banking in several countries.  StemCyte stores both the buffy coat and the red cells, in contrast to conventional medical practice that red cells should be removed before transplant.  StemCyte has published a study of how their cord blood collections performed in transplant patients.

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Because the components of cord blood have different density (see image above), the layers can easily be separated either by sedimentation, by spinning in a centrifuge, or a combination of these methods.

Manual processing of cord blood simply means that it is done by hand by trained technicians.  The original processing method employed by public cord blood banks was a manual method developed in the early 1990's under Dr. Pablo Rubinstein in the lab of the NY Blood Center.  It is a combination of sedimentation with a reagent and gentle spin in a centrifuge.  The reagent is Hydroxy Ethyl Starch (known by the brand names "Hespan Starch" or "Hetastarch").  Many banks still follow the Rubinstein protocol, which has been used for thousands of collections that went to transplant.  In parts of the world where skilled labor is less expensive, cord blood banks are more likely to use this standard processing method.

Meanwhile, in the mid-1990's many of the earliest private cord blood banks used a type of processing which is standard in stem cell research labs, called Ficoll-Hypaque processing.  This procedure relies on two stages of centrifugation.  The first stage is to separate the buffy coat, and the second stage is a harder spin to separate components of the buffy coat.  The final product is a layer rich in stem cells which is only a few mL in volume and can be stored in vials.  At one time this procedure was commonly used in private cord blood banks because the final volume is so small.  Dozens of successful transplants have been performed with cord blood that was processed this way.  However, in 2005 the AABB accreditation requirements were changed to require that testing segments be "integrally attached" to the main collection.  As a result, the industry moved away from vial storage to blood bags, and Ficoll-Hypaque processing was no longer used in cord blood banks which store collections for clinical use.

Automated processing of cord blood is now preferred by large banks because it is a more economical, efficient, and reproducible way to process collections if you have a high volume of inventory going into storage.  The two main technology platforms used for automated processing are the Sepax manufactured by BioSafe in Switzerland, and the AutoXpress Platform, or AXP, manufactured by Thermogenesis in the USA.

Both the Sepax and AXP systems operate on the same scientific principle: that a light beam looking through a thin tube of blood can sense the density gradient between separated layers of the collection.  The two systems differ in how they are configured to utilize that concept.  The Sepax system has been around longer and has been adopted by many large public banks.  The AXP system is newer and claims to be better at processing the smaller samples that are common in family banking.

There are many competing claims as to whether Sepax or AXP is "better" at recovering the TNC and/or MNC in the original cord blood collection.  No head-to-head comparison by an independent  agency has been published.  Plus, depending on what method is used to measure the "viable cells", the uncertainty in the measurement may exceed any differences resulting from the processing method.

Finally, there is one more processing option:  The company BioE in Minnesota has developed a manual cord blood processing system which is completely based on sedimentation with a proprietary reagent.  It is called PrepaCyte-CB.  The USA public bank Cryobanks International has adopted this processing system.

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Pros

• Removing the blood plasma reduces the collection volume so that less freezer space and cryogenic nitrogen are required for preservation.
• Removing the red cells minimizes any reaction of a patient to incompatibilities with the ABO/Rh blood type of the donor.  Two people can have "matching" HLA types for a transplant, but have different blood types.
• Moreover, the cryo-preservation solvent that is added before freezing (see below) tends to rupture the red cells.  When the stem cells are thawed for transplant they are often "washed" to remove both the solvent and any hemoglobin released from the ruptured red cells, because hemoglobin is kidney toxic.  Thus, if the red cells are removed in advance it is one less concern during transplant.

Cons

• In a family bank, the cord blood is usually stored "in case" of need, so it does not make sense to perform extra processing steps that add to the cost.
  
• Cord blood transplants are less sensitive to blood type incompatibilities than transplants of adult bone marrow, so removing red cells isn't as important.

Most blood bankers feel that the pros outweigh the cons, which is why all public banks and most family banks process the original cord blood collection to remove plasma and red cells.

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When living tissues are frozen, they must first be immersed in a cryo-preservation solvent which penetrates the cells and replaces the water in them.  This is because when water freezes it forms ice crystals which rupture cell membranes.  The solvent most commonly used for cryopreservation of cord blood is "DMSO" (DiMethylSulfOxide).  There is a huge medical literature on alternate chemical cocktails, but no consensus has emerged, and DMSO remains the gold standard in the cord blood industry.  When the cells are thawed for transplant, they are usually washed to remove the cryo-preservation solvent and return water to the cells.

There are two stages to the cooling process: The first stage is to place the processed stem cells in a controlled rate freezer which gradually lowers their temperature to about -80 C.  The final step is to place the cells in long-term storage in a cryogenic nitrogen freezer near -196 C.

The reason for gradually lowering the temperature in a controlled rate freezer is to avoid the formation of ice crystals.  Laboratory scientists generally agree that once cells are cooled below the "glass transition" temperature, they become solid, there is no molecular movement, and thus no damage can occur even if the storage temperature fluctuates.  However, studies disagree on exactly where the glass transition occurs for stem cells, since it depends on the choice of cryo-preservation solvent and its concentration.  Temperatures between -105 C and -145 C have appeared in the literature.

The cryogenic storage freezer may contain either liquid or vapor nitrogen.  Liquid nitrogen has the advantage that the temperature in the storage tank is more uniform.  However, there are several documented cases where an infectious disease was transferred from the surface of one storage bag to another in liquid nitrogen.   Hence, biologic samples stored in liquid nitrogen should first be encased in an over-wrap to add a layer of protection against contamination.

Actually, in most public cord blood banks and some private banks, there are two stages of cryogenic storage: the first one is a "quarantine" tank where the collection goes pending test results for infectious diseases.  If the collection passes all the test results, it is moved into the long-term storage freezer.

There is a lot of concern in the cryogenic storage industry about "Transient Warming Events" or TWE.  The concern is that when cells are subjected to brief, or transient, warming events, they warm up above the glass transition temperature, and cell damage occurs.  It is inevitable that all cryogenic cells are subjected to some TWE during routine laboratory handling: when transferring the stem cells from the controlled rate freezer to the quarantine freezer, from the quarantine freezer to the long-term freezer, and from the long-term freezer to the dry shipper for transfer to a transplant center. 

Some cord blood laboratories have invested in robotic freezers which can ensure that the cord blood does not experience TWE during long term storage.  The best known robotic freezer is the BioArchive manufactured by Thermogenesis (Nasdaq: KOOL).  However, many banks continue to rely on storage tanks with a lid, called "Dewars".  Studies have shown that trained laboratory technicians can transfer cell collections between tanks in a matter of seconds, whereas it would take several minutes for a small bag of frozen cells to warm above the glass transition.

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