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 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 must be completed and cord blood units frozen within 36 hours of collection.
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 and greatly facilitates storage and automation.
- Upon arrival in the Processing Laboratory, cord blood units are "logged-in" to the NCBP database and weighed to estimate the collected blood volume.
- The cord blood is transferred using a sterile-docking device, from the collection bag into a sterile, disposable processing bag set in preparation for its automated processing using the AXP (AutoXpress) platform (510 k-approved by FDA). AXP use was implemented by NCBP in 2006.
- Before and after 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 a very early class of cells that include the hematopoietic stem cells. These samples are removed within plastic bubbles (pre-set in the tubing), sealed off from the processing bag set without exposing the products to possible infectious agents in the air of the laboratory.
- The bag set containing the cord blood unit is placed into the computerized AXP device, which is automatically centrifuged twice. The first centrifugation stratifies the cellular components [RBCs, white blood cells (WBC) and platelets] separated from the plasma. The second spin harvests the blood components into the three different bags of the AXP sterile bag set: RBC bag, freezing bag [containing the WBCs, predominantly the mononuclear cells (MNCs)] and plasma bag.
- The freezing bag contains the 20 ml final product (on average, more than 90% of the original MNCs and stem cells in some of the plasma, along with some red cells).
- Cold, sterile, Dimethylsulfoxide (DMSO, 5 ml of a 50% solution) is then added slowly. DMSO is a kind of "anti-freeze" or cryoprotectant that prevents ice crystals from forming inside of and destroying cells during freezing. It is also protective when the unit is eventually thawed.
NOTE: To prevent mix-ups and labeling errors, samples are taken and labeled from only one unit at a time. To prevent transcription and labeling errors, no labels are handwritten. New labels (for example for sample tubes and for the bag set components) are printed "on demand" by scanning the original bar code label into a computer or stand alone printer that automatically prints new, or "copy-cat", labels, respectively.
Before freezing, each cryoprotected cord blood unit is sealed in a Teflon overwrap bag that insulates and quarantines each unit individually within the freezer, preventing possible contamination from infected units, plastic surfaces and from organisms “swimming” in the liquid nitrogen.
Each cord blood unit is frozen (one at a time) in an individual “controlled-rate freezer” module within the BioArchive™ system. We use a freezing rate documented to maintain cell viability. A record of the freezing curve is printed and maintained in the BioArchive™ computer memory.
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 were and remain 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 or being disturbed by any other unit.
Testing of cord blood units:
- Typing for HLA-A, -B and
-DRB1. Typing for human leukocyte antigens (HLA) is performed on all cord blood units. The testing is done at the New York Blood Center’s Fred H. Allen Laboratory of Immunogenetics, which is ASHI/CLIA accredited and licensed under the New York State Department of Health. Typing is performed at low intermediate resolution for HLA-A and -B and at high resolution (sequence based typing) for HLA-DRB1. In addition, before releasing a cord blood unit for transplantation, confirmatory HLA typing is performed on cells from a continuous segment for HLA-A, -B, -C at low intermediate resolution and HLA-DRB1 at high resolution. The mother’s HLA typing is also determined prior to releasing a cord blood unit, if not done previously. Typing of the mother ensures that the “mother’s” sample was actually taken from the baby’s mother and not from someone else by mistake; verifying the correct sample is important because all testing for infectious diseases is performed on the mother also. The cord blood must have at least one of each antigen (HLA-A, -B, -C, and -DRB1) in common with the mother’s HLA-type.
- Total nucleated cell enumeration is performed on all cord blood units before and after processing. 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. The total nucleated cell count (TNC) of a unit includes all nucleated cells: white blood cells and nucleated red cells. The results are reviewed to make certain that all values are within normal range. Out-of-range values (e.g. low MCV, high nucleated red blood cell count) are reported with the other information on the cord blood unit.
- CD34+ cell numberhas been assessed on all units since February 2000. Currently, CD34+ cell count and viability are assessed in all NCBP cord blood units by flow cytometry before and after cord blood processing.
- Colony forming unit (CFU) assay. In 2008, we developed and introduced a new, fully standardized procedure for hematopoietic colony forming cell counting based on digital imaging. As a result, this assay is very reproducible and does not depend on the operator (laboratory technician). In addition, images of CFU from each cord blood unit are stored electronically and can be reviewed at later times.
- Bacterial culture (of the unit after processing) for both aerobic and anaerobic organisms. We are using the ESP Culture System (Versa TREK). The system screens a sample of the cord blood unit for bacterial infection, and if a positive signal is detected, the sample is sent to a Microbiology Reference Laboratory for bacterial identification and antibiotic sensitivity. Units with bacterial contamination are not included in the NCBP searchable inventory.
- Infectious disease testing of both the mother's blood and cord blood is performed by Blood Systems Laboratories (BSL), Tempe, AZ, according to the FDA requirements for donors. Testing includes antibodies to HIV-1 and 2 (with a test that detects antibodies to HIV-O also), HTLV-1 and 2, HCV, HBV (anti-HBc), syphilis and hepatitis B virus surface antigen (HBsAg). Since 2008, mother’s blood is also tested for antibodies to Chagas’ disease.
- Nucleic Acid Testing (NAT) for HIV, HCV and West Nile Virus are done on the maternal blood sample only (the test is not FDA approved for the cord blood sample). In the past we were testing only units that would be released for transplantation. Since 2007, all units are being tested routinely.
- Cytomegalovirus (CMV) testing. Mother’s and cord blood samples are tested for total antibody to CMV, indicating past or recent exposure to the virus. In addition, we used to perform CMV culture of the baby’s saliva. We now test the cord blood sample by PCR amplification of the viral DNA, in all cases that the mother’s antibody testing is positive for CMV.
- ABO blood group and Rh type of the cord blood is determined by Blood Systems Laboratories (BSL), Tempe, AZ.
- Hemoglobinopathies screening. Sickle cell hemoglobin and hemoglobins C and E are detected by high performance liquid chromatography (HPLC) (BioRad Inc.)
- Stored Samples. Aliquoted samples of plasma, viable white cells and DNA from the cord blood unit and from the mother's blood are kept frozen and are available for special testing in the future, as needed.