Within 24 hours of collection, cord blood units are transported to the New York Blood Center's National Cord Blood Program Processing Laboratory where technicians "log in" new units into the NCBP database, obtain aliquots (small samples) for testing and storage, reduce the volume by removing excess red blood cells and plasma and freeze and store each unit. Processing is completed within 36 hours of collection.

Processing:

Processing involves a series of steps to remove excess red blood cells (RBCs) and plasma and bring each collection down to a uniform single unit volume of 20 ml. (large collections may be frozen in two separate 20 ml. containers). A uniform volume assures that freezing conditions are uniform for all units.

1. Upon arrival in the Processing Laboratory, cord blood units are "logged-in" to the NCBP database and weighed to estimate the collected blood volume.
2. Before processing, small samples are removed to count the total nucleated cells (TNC) and the number of nucleated cells that are positive for CD34, a marker of very early cells that include the hematopoietic stem cells.
3. Sterile hetastarch (Hespan) is added (according to the unit volume) to facilitate RBC sedimentation.
4. The unit is centrifuged gently to sediment RBCs.
5. The remaining plasma and nucleated cells (along with some red cells) are transferred into a sterile blood processing bag.
6. The processing bag is then centrifuged to separate the nucleated blood cells from the plasma.
7. Excess plasma is removed and a 20 ml. final volume (containing more than 90% of the original nucleated cells and stem cells, on average) is transferred to a freezer bag.
8. Small samples are taken to test for the number of cells left after the processing steps and for bacterial culture.
9. Cold sterile Dimethylsulfoxide (DMSO, 5 ml of a 50% solution) is added slowly. DMSO is a kind of "anti-freeze" or cryoprotectant that prevents ice crystals from forming and destroying the cells during freezing and when the unit is thawed.
   
   NOTE: To prevent mix-ups and labeling errors, samples are taken from only one unit at a time. Similarly, when the unit is transferred into the freezer bag, only the one unit is handled at a time ("one-at-a-time" rule). To prevent transcription and labeling errors, no labels are handwritten. New labels for sample tubes and for processing and freezer bags are printed "on demand" by scanning the original bar code label into a computer that automatically prints new "copy-cat" labels.

Quarantine:

Before freezing, each cord blood unit is sealed in a teflon overwrap that acts to quarantine each unit individually within the freezer, preventing possible contamination.

Freezing:

Each cord blood unit is frozen (one at a time) in an individual controlled-rate freezer that is part of the BioArchive™. The freezing rate has been documented to maintain cell viability. A record of the freezing curve is maintained on each unit.

Storage of cord blood units:

Since 1999, all newly acquired units have been stored in BioArchive™ freezers under liquid nitrogen (at -196° Centigrade). Units collected earlier are stored in standard freezer tanks (also under liquid nitrogen) in racks of 3-5 units. Storage in the BioArchive™ minimizes the number of exposures to room temperature and, thus, to transient warming events (TWEs) that can progressively damage cell viability. This is possible because each unit is placed in the freezer one by one, in its own individual location rather than in a rack with other units. Thus, a unit can be placed in the freezer or taken out when it is requested for a transplant without disturbing any other unit.

Routine tests of cord blood:

1. Typing for HLA-A, -B and -DRB1 is completed on both the cord blood and mother's blood. Typing of the mother is critical to ensure that the "mother's" sample was actually taken from the baby's mother and not from someone else by mistake. The cord blood must have at least one HLA-A, -B and -DRB1 antigen in common with the mother's HLA-type. HLA typing is done by the New York Blood Center's Fred H. Allen Laboratory of Immunogenetics (ASHI Accredited). Typing is done both at low resolution (serological) and at higher resolution (molecular) levels, with final testing at the time of confirmatory typing by DNA sequencing (highest resolution possible).
   
2. Total nucleated cell enumeration.
   Since mid-2000, cell counts have been done with an automated hematology analyzer (Sysmex XE-2100) that includes a count of nucleated red blood cells.
   
3. CD34+ cell number has been assessed on all units since February 2000. Before that time most, but not all, units had counts of colony forming cells (CFU).
   
4. Bacterial culture (of the unit after processing) for both aerobic and anaerobic organisms uses the ESP Culture System (TREK). The identification of any bacteria isolated and the antibiotic sensitivity of aerobic organisms is established through a Microbiology Reference Laboratory.
   
5. Infectious disease testing of both the mother's blood and cord blood is performed for antibody to HIV-1 and 2, HTLV-1 and 2, HCV, HBV (anti-HBc) and syphilis and for the hepatitis B virus surface antigen (HBsAg) by Blood Systems Laboratory (BSL), Tempe, AZ.
   
6. Nucleic Acid Testing (NAT) for HIV, HCV and West Nile Virus is done on cord blood and maternal blood samples of units selected for transplantation (by BSL or by the NYBC's Special Diagnostics Laboratory).
   
7. Cytomegalovirus (CMV) culture of the infant's saliva is done at the University of Alabama Viral Diagnostic Laboratory, Birmingham AL.
   
8. ABO blood group and Rh type of the cord blood is determined by Blood Systems Laboratory (BSL), Tempe, AZ.
   
9. Hemoglobinopathies (sickle cell hemoglobin and hemoglobins C and E) are detected by high performance liquid chromatography (HPLC).
   (BioRad Inc.)
   
10. Stored Samples: Aliquoted samples of plasma, viable cells and DNA from the cord blood unit and from the mother's blood are available for special testing as needed.