Estimating potency of cord blood transplants: Current standards and challenges

Transcription

1 Cord Blood Infusions: Current Gold Standards in Preparing and Testing of Cord Blood Products for Transplantation Estimating potency of cord blood transplants: Current standards and challenges Teja Falk Radke José Carreras Cord Blood Bank University Medical Center Düsseldorf, Germany

2 Primary task: The cord blood bank`s challenge Banking cord blood transplants with the best quality achievable according to current standards. but also: 1.) Providing quality data on these units! 2.) Providing clear instructions for infusion! Transplant centers/physicians should be able to estimate potency of the CBU and must be aware of potential risks. Data/Instructions provided must be reliable, reproducible, and unmistakable.

3 Data provided Final report : (Exemplary single volume-reduced CB unit for pediatric patient) HLA-data Volume & additives Cell counts Disease markers etc.

4 Data provided Repository labeling : (Exemplary for double-bag volume-reduced CB unit) Data on content, including active components (TNC, erythrocytes, platelets etc.) and reagents (e.g. DMSO, Hespan etc.)

5 4. The transplant center must agree that the CB unit will be thawed and processed prior to the transplantation using the thawing procedure from Düsseldorf, which is identical to the Rubinstein protocol (it is possible to receive a dummy CB unit` for practice use or training). T.F.Radke Estimating potency Instructions provided Instruction Insert and Product Information 10 pages with comprehensive instructions Identification, Indication, Information on use, Precautions, Side effects, Pharmacological and toxicological properties Instructions for washing (manually as well as with automated Sepax100). Composition Formal Request for Procurement

6 Current methods

7 Current methods for estimating potency Cell count Total cell count of nucleated cells (TNC) with and without erythroblasts using haematology analyzers Content CD34 + -cells : Amount of haematopoietic stem and progenitor cells (CD34+) by flow cytometry according to ISHAGE protocol. Additionally, viability is assessed by 7-AAD. Colony-forming cells: Ability to form colonies in an in vitro colony-forming unit (CFU)-assay

8 Cell count Current methods for estimating potency... and their pitfalls WBC : White blood cell count, either based on WOC or WIC/NOC WOC : White blood cells Optical Count all cells Can be falsely increased by resistant red blood cells (ResRBC) or decreased by fragile WBC (e.g. after thawing) WIC/NOC : White blood cells Impedance Count / Nucleated cells Optical Count all nucleated cells Can be falsely increased by nucleated red blood cells (Cord blood contains high amounts of NRBC!)

9 Current methods for estimating potency... and their pitfalls BUT: Together with WBC and CFU dose, NRBC content is a significant predictor of time to myeloid engraftment. TNC value might be more important than a perfect WBC value! WIC values are better reflecting CB potency

10 Current methods for estimating potency... and their pitfalls Content CD34 + -cells: Results can be influenced by A) antibody used and B) gating strategy A) Clone 8G12: 860 events... more sensitive? Clone 581: 755 events... more specific? B) Generous: 732 events (CB unit aliquot, post-thawing) Strict: 617 events

11 Current methods for estimating potency... and their pitfalls Colony-forming cells: Ability to form colonies in an in vitro colony-forming unit (CFC)-assay CFU/CD34 + ratio (after volume reduction) CFU/CD34 + ratio (thawed) Though, correlation CFC/HSC varies with seeding-density! CFU per seeded CD CB1 CB2 CB3 CB4 CB5 CB6 CB7 CB8 CB9 CB10 CFU per seeded CD CB1 CB2 CB4 CB6 CB7 CB8 CB9 CB10 2 ideal 2 ideal seeded CD34 + cells/per ml seeded CD34 + cells/per ml

12 Potential improvements

13 Single-platform enumeration of cells Bead-based systems for flow cytometry: Commercially available inert beads are added to sample (defined number/defined volume) Number of beads recorded allows calculation of tested volume.

14 Assessement of apoptotic cells Annexin V (AnnV) : Stains phosphatidylserine (PS). PS is present on the cytosolic side of the cell membrane, but gets exposed on the outside in case of apoptosis. 7-AAD only vs. 7-AAD + AnnV True viability might be much lower!

15 Factors affecting potency

16 Recovery [%] Recovery [%] T.F.Radke Estimating potency Factor affecting potency: Cell loss A certain loss of total cells is connected to processing, resulting in a recovery of app. 85% after volume reduction and app. 75% after thawing and washing. Recovery total nucleated cells (CellDyn) * * Whole blood Whole blood + HES Post reduction Pre freeze + DMSO Thawed Post wash Recovery CD34 + -cells (CellDyn + FCM) * * This loss also affects the population of CD34-positive cells on a comparable level. 0 Whole blood Whole blood + HES Post reduction Pre freeze + DMSO Thawed Post wash

17 Viability [%] Viability [%] T.F.Radke Estimating potency Factor affecting potency: Loss in viability Using Annexin V, it clearly shows that viability of HSC is not as high as estimated with 7-AAD alone. Viability CD34 (7-AAD) Viability CD34 (AnnV + 7-AAD) Whole blood Whole blood + HES Post reduction Pre freeze + DMSO Bag thawed undiluted Bag thawed post wash 0 Whole Blood Whole blood + HES Post reduction Pre freeze + DMSO Bag thawed undiluted Bag thawed post wash

18 Factor affecting potency: Temperature/Time There are no definite requirements in regard to ambient temperature prior to processing. Though, storage at 4 C resulted in less non-viable leukocytes as compared to room temperature (20-24 C). Analyzing apoptosis in CD34-positive cells over time prior to processing, it could be confirmed that storage at 4 C or room temperature showed no significant difference. Storage exceeding 48h, however, resulted in increased apoptosis.

19 Segments and aliquots

20 Cell count recovery [%] Viability [%] T.F.Radke Estimating potency Problem: How to predict potency ahead of thawing Can segments/aliquots reproducibly give information on quality? Cell count recovery segments vs. aliquots vs. bag Live CD34 (AnnV/7-AAD) in bag (thawed/washed), aliquot and segments Segments Aliquots Thawed bag Washed bag 0 Segments Aliquots Thawed bag Washed bag

21 Acknowledgements Gesine Kögler Riccardo Saccardi Sergio Querol Richard Duggleby Svenja Peters David Barbosa