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Processing Methods

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Processing: How is cord blood processed before storage?
The three main components of cord blood, like any blood collection, can be separated by weight:  the heaviest layer is the red blood cells (RBC), 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.  When banks process the cord blood, the final separated component that goes into storage is the buffy coat, even though only about 1% of the cells are actually stem cells.  There is no procedure to separate out the stem cells alone.

The vast majority of blood processing methods rely on the different density of the three main blood components.  They can be separated by sedimentation, or by centrifuge, or by a combination of the two techniques.  The procedure can be performed manually by trained technicians or by automated machine.
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Processing: Must cord blood be processed before storage?
The earliest cord blood transplants were performed with whole cord blood.  Thus, it is not absolutely necessary to process cord blood in order to save patient lives.  There has never been a prospective randomized trial to compare transplant patient outcomes with cord blood that had been stored whole versus processed.

Most cord blood banks, both public and private, now process cord blood to remove both the plasma and the red cells, and cryo-preserve the remaining buffy coat holding stem cells.  Some banks also save the removed red cells and plasma in companion storage.  Some banks save a sample of maternal blood.

The removal of plasma is also called volume reduction.  The volume reduction enables more collection units to fit in a freezer and requires less cryogenic nitrogen per unit.

Also, the majority of banks remove red blood cells prior to freezing, primarily because these cells often burst during freezing and release iron from hemoglobin that can be toxic.  The alternate to removing the red cells before freezing is to wash any broken cells out of the collection upon thaw.  Removing the red cells also removes the donor's blood type (the ABO and Rh types).  When cord blood goes from a donor to a patient for a transplant, the donor and patient can be compatible on all the HLA types used for transplant matching and still have incompatible red blood types.

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What are TNC, MNC, CD34+, and CFU, and why should I care?
These are all ways of counting cell types, and they tell you whether or not your cord blood collection has lots of stem cells and if they are healthy.

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 biology 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. 

Scientists 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+ counts are not an accurate measure of stem cells: CD34+ results vary between labs, they can vary within a single lab, and only 1-2% of the MNC that have CD34+ are actually stem cells.

The Total Nucleated Cell count or TNC is the test most often reported as a measure of the cell count after cord blood processing.  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 device called a flow cytometer.

At present Colony Forming Units or CFU are considered to be the best measure of whether stem cells are "viable", or quite frankly alive.  The TNC count includes both living and dead cells.  In the CFU test a small portion is watched under controlled conditions to see if stem cells divide and form colonies.  This used to be a subjective measure, but recently it has been standardized with technology to image the cells and count colonies in the image.  The only remaining problem with the test is that it takes days for colonies to grow.