Processing and cryopreservation of stem cells

Scope of laboratory services

Services provided by Stem Cells and Immunotherapy (SCI) laboratories vary depending on the requirements of different transplantation programmes. Services available include:

• Co-ordination with apheresis units to ensure adequate CD34+ cell doses from haematopoietic progenitor cell (HPC) collections
• The processing of bone marrow, cord blood and apheresis collections by validated methodology to:
  
  - Reduce product volume
  - Deplete red blood cells
  - Concentrate mononuclear cells
  - Purge lymphocytes
  - Concentrate and purify HPC
  - Select other cell types as required e.g. CD34+ or TCR α/β CD19 depletions
  
  
• The preparation of T Cell Therapeutic (TC-T) aliquots (also called donor lymphocyte infusions or DLIs) in measured CD3 doses
• The cryopreservation and long-term storage of HPC and TC-T
• The thawing and washing of cryopreserved products to allow further processing and remove DMSO if necessary
• The use of tissue culture assays to demonstrate progenitor viability and process suitability
• The use of flow cytometric assays to assess the viability and purity of target cell populations and the efficiency of processing operations
• The use of microbiological tests to assure the suitability of donors and the safety of products
• The organisation of safe and prompt transit of HPC products between hospitals and processing facilities in line with national and international guidelines
• The provision of full and timely reports on processes performed

The return of HPC products for transplantation

Reinfusions of cryopreserved products

Reinfusion dates and times for cryopreserved products must be discussed with the SCI laboratory head or nominated deputy and confirmed in writing using the Request for issue of cryopreserved products form (2F)

Reinfusions are performed under the supervision of an appropriately qualified nurse or doctor employed by the Transplant Centre who is responsible for the well-being of the patient being transplanted. It is the responsibility of the supervising nurse or doctor to ensure that:

• The products being reinfused are those requested and are correctly labelled for the patient
• The patient has received suitable pre-transplant cover
• Regular observations are performed during and following the transplant
• The correct HPC dose has been identified

The Transplant Centre provides a suitable waterbath filled with sterile 0.9% sodium chloride solution to thaw the stem cell products. It is the responsibility of the Transplant Centre to ensure the waterbath is disinfected between procedures. Cryopreserved products are transported to the Transplant Centre in a dry shipper at very low temperature, with an in-built temperature logger. Staff are required to sign the transit form to acknowledge delivery of the products.

The supervising nurse or doctor will be informed of the volume of individual bag in the accompanying infusion report so that optimal infusion rates can be calculated. Thawed bags must be infused within 20 minutes to minimise damage to cells by DMSO. The supervising nurse will be required to complete the infusion paperwork to confirm that requisite checks on the product identity have been made. Thaw time and infusion time should also be recorded in the appropriate section of the form.

The empty dryshipper should be returned to the stem cell lab by the locally arranged route.

INF284 - Cryopreserved Haematopoietic Stem Cells - Information and Handling Instructions

Reinfusions of non-cryopreserved products

Non-cryopreserved products will be delivered to a nominated representative of the Transplant Centre by a member of the SCI laboratory or courier as soon as processing and quality checks have been completed unless temperature controlled overnight storage is required. Transit and transit boxes will comply with all relevant national and international guidelines. 

On arrival at the Transplant Centre, a member of the nursing or medical staff will be required to sign the transit form to accept delivery. Any discrepancies between products delivered and those expected or in patient details on pack labels or accompanying paperwork should be noted at this time.

The supervising nurse or doctor will be informed of the volume of individual bag in the accompanying infusion report, so that optimal infusion rates can be calculated. The supervising nurse will be required to complete the infusion paperwork to confirm that requisite checks on the product identity have been made along with the infusion times.

The empty transit box should be returned to the Stem Cell lab by the locally arranged route.

INF288 - Fresh Haematopoietic Progenitor Cells (HPC) or TCells, Apheresis (TCells(A) for infusion

Quality Monitoring

Testing

Routine testing methods include automated full blood count and flow cytometry to determine cell viability and to enumerate the number of cells positive for CD34 and CD3. CD34 estimations are performed after all processes using a single platform methodology recommended by UK NEQAS. The protocol is a modification of the widely recommended ISHAGE method with additional gating to improve distinction between small mononuclear cells and debris present in some bone marrow products. NHSBT SCI laboratories are registered in the UK NEQAS Immunophenotyping Scheme. Performance results are available on request.

Total cell numbers for relevant populations are calculated before and after any process to identify excessive losses within that process. CD3 counts are assessed pre- and post-depletion to demonstrate purging efficiency. All end products are monitored for sterility by broth culture. CFU-GM/BFU-E tissue culture assays are performed routinely on a proportion of all processes as part on ongoing quality assurance. These assays are also used to validate improvements to processing protocols.

Reporting

An interim written report of cell counts, volumes, viability, CD34+ numbers and T-lymphocyte depletion efficiency will be prepared and supplied on the day of process.

Data associated with the process are reviewed by the laboratory head who will then issue a final report within 21 days. These reports will also detail the outcome of bacteriology testing.

Clinical outcome data

The Stem Cell Laboratory is legally obliged to maintain a record of clinical outcome data for all transplants involving products processed in the laboratory. This is an essential aspect of assuring the quality of processing, and a HTA licensing requirement.

A clinical outcome form will be sent to the referring consultant or an agreed representative:

• After a HPC transplant: form 2G engraftment report – 30 days post stem cell transplantation, 
• After a TC-T (DLI) transplant: form 2H clinical outcome report form – 100 days post donor lymphocyte infusion

Both forms are in the drop down for the issue of cryopreserved products.

The completed form must be returned to the stem cell laboratory head:

• approximately 30 days after a HPC transplant
• 100 days after a TC-T (DLI) transplant

Any adverse reactions occurring after this time should also be reported to the Consultant Medical Adviser responsible for the stem cell service.

At 100 days and 1 year after transplantation, a letter will be sent to the referring consultant requesting a copies of EBMT "Minimum Essential Data A" forms.

Storage and disposal

A regular audit of cryopreserved material will be circulated to all Consultants annually. Clinical grade storage will be maintained until a written request for discard (FRM 1960) is received by the SCI laboratory. Consultants will be asked to provide consent to dispose of products from deceased patients (by incineration) unless prior consent has been obtained for the products to be used for non-commercial research purposes.

Unwanted products from living patients can only be discarded with the written consent of both patient and Consultant. Continued clinical grade storage will generate additional charges specified in the annual contract negotiations.

Other services

Tissue culture assays

Suitable samples can be tested for CFU-GM, BFU-E, LTCIC, CFU-F or other systems in discussion with the laboratory. No samples should be sent without prior approval from the laboratory.

Cryopreservation of cells for laboratory use

The SCI laboratory can provide information on processing of cells for laboratory use.

In addition to the routine assays described above, protocols can be developed for research applications on discussion with the SCI laboratory. No samples should be sent without prior discussion with the SCI laboratory.

Clinical data management

Records of transplant processes, cell counts and clinical outcomes are maintained. Data may be made available for the preparation of reports, presentations, publications and trend analyses.