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Production

 

Cord Blood Production At Our Facility

In this context, ‘production’ includes all aspects of manipulation, cryopreservation, packaging, and labelling of our cellular therapy products. It includes microbial testing, preparation for storage and removal from storage. On receipt, we process and categorise cords. This is according to how many hours the material was in storage before receipt, and the initial total nucleic cell (TNC) counts obtained as follows:

The Anthony Nolan Cord Blood Bank (ANCBB) uses methods to ensure the cord products safety and efficacy in order to optimise clinical transplant outcomes. We also want to minimise the risk of transmission of disease. We use bags designed for storage of blood stem cells and are able to maintain integrity at temperatures of -196°C.

Samples are stored at this temperature with a plastic over-wrap prior to the initial long-term storage of the unit. Units are also cryopreserved within 48 hours of collection using controlled rate freezers validated to maintain cell viability. Temperatures within the tanks are monitored continuously and are equipped with an automated recording system. Maximum temperature registered in the warmest part of the tank never exceeds -150°C and after retrieval and transfer not higher than -135°C. The centre documents duration and cord blood unit (CBU) temperature of any transient warming event using nonconformity notes. Cord blood unit testing includes at a minimum: blood grouping, tissue typing, Infectious Disease (IDM) Marker testing, haemoglobinopathy testing, cell counts (total nucleated cells and viable CD34+ cells), CFU, and sterility.
 
Donor mother sample testing list includes at a minimum, the following IDM assays: HIV-1 & 2, HTLV-I & II, HBV (antigen & core), HCV, Syphilis, and CMV. These are tested for by serological and molecular means.
 
After initial testing, sample aliquots are stored for future testing for both CBU and Donor Mother Sample. The ANCTC has established and maintains procedures for the control of CBU storage areas which helps prevent cross contamination and improper release of CBUs. The ANCTC has also established and maintains procedures for storing, selecting, retrieving and transporting CBU to transplant centres. Roles and responsibilities of each entity and chain of custody of the CBU are defined.
In order to obtain the highest quality product the ANCBB operates the following CBU criteria: CBU must meet the following minimum requirement to be validated and transferred to the Register:

  1. Total nucleated cells count prior to processing > 120x10^7.

  2. Total viable CD34+ cells count prior to cryopreservation > 1.5x10^6.

  3. Total Colony Forming Units prior to cryopreservation must show growth higher than 10% of the CD34+ cell counts.

  4. Viability of CD45+ cells must be higher than 85% prior to cryopreservation.

  5. Sterile for bacteria and fungi.

  6. Tissue typing for intermediate or high resolution for A, B, C, and DQB1, and high resolution for DRB1.

  7. Haemoglobinopathy testing: confirmatory test results not showing clinically significant abnormal haemoglobin.

  8. IDM: negative for HIV, HCV, HBV (HBsAg and HBC), Lues (antigen) and HTLV I & 2.

  9. CBU must have at least 2 (preferably 3), attached segments. Each segment usually contains between 70-100μL to provide adequate amounts to perform HLA typing and future testing which may include viability, cell counts, CFU, or IDMs.

The key technology used in our facility is the SepaxNet® system. The Sepax cell separation device is a turn-key system that allows safe and efficient processing of blood and its components. The combination of the compact Sepax main processing unit and an application-specific single-use kit provides a fully automated, GMP compliant and closed environment for use in a stem cell laboratory. No additional equipment or operator intervention is required during the separation procedure. The protocol used with the Sepax system is the ‘UCB protocol’. It is designed for routine processing of umbilical cord blood (UCB) to concentrate the buffy-coat fraction that is rich in hematopoietic stem cells. Highly efficient separation and Total Nucleic Cell recovery is achieved without addition of any sedimentation agent. The UCB protocol allows a volume reduction of UCB in approximately 35 minutes to a predetermined fixed volume ranging from 10 to 50 ml.

Research samples are also processed by a range of other methods. These are:

  • Manual HES Hydroxyethyl starch (HES) is used to isolate the nucleated white blood cell layer which contains stem cells from cord blood by sedimentation of red blood cells.

  • Manual Ficoll® Ficoll-PaqueTM solution is a synthetic polysaccharide used to separate granulocytes and erythrocytes from mononuclear cells. It does this by exploiting the differences in the cells physical properties such as size and density. The higher density cells such as erythrocytes and granulocytes sediment through the Ficoll® solution layer during centrifugation. This leaves the mononuclear cells at the plasma-Ficoll® interface. They can then be extracted, washed, re-suspended in plasma and frozen.

  • We are also currently developing and validating other processing methods.

 

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