Preparation of cord blood for infusion: bedside thaw, dilute and wash, or somewhere in between
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1 Preparation of cord blood for infusion: bedside thaw, dilute and wash, or somewhere in between Donna Wall, MD Director, Manitoba Blood and Marrow Transplant Program ISCT 2012
2 Disclosures: none
3 The problem: Cord blood grafts contain 1/10 th the number of hematopoietic progenitors (TNC, CD34, CFU) compared to other HSPC sources and are all previously frozen. Progenitor cell loss at time of thaw must be minimized.
4 Impact on graft characteristics on engraftment Duke retrospective review of 435 primarily pediatric (median age: 5.3 years) patients receiving a single-unit unrelated UCBT after myeloablative conditioning from 2000 to 2008 Cumulative incidences of neutrophil and platelet engraftment were 76.9% and 55% respectively Page BBMT 2011
5 Precryopreservation graft characteristics on the probability of platelet engraftment. Page BBMT 2011
6 Post-thaw graft characteristics and the probability of platelet engraftment Page et al. BBMT 2011
7 Higher CFU dosing was the only precryopreservation graft characteristic predictive of neutrophil (P =.0024) and platelet engraftment (P =.0063). In the post-thaw model, CFU dose best predicted neutrophil and platelet engraftment (both P <.0001). Page et al. BBMT 2011 Yoo et al. BMT 2007
8 Objectives: To review the 3 main approaches to CBU thaw and compare transplant outcomes To highlight special issues in the red cell replete units and pediatrics
9 Historical perspective: In the early days products were frozen without red cell depletion in bags that had a high propensity to break That led to thawing in the laboratory with a wash stem to remove red cell lysate and DMSO. Product volumes were ml
10 The Rubinstein method Rationale was that during the freeze process cells became dehydrated from the hyperosmolar DMSO solution. Thus by thawing with a hyperosmolar combination of albumin and dextran one allowed cells to equilibrate more gently improving survival. The osmotic damage inflicted by the severe solute concentration gradient, however, can be averted by a simple 2-fold dilution after thawing, providing almost total recovery of viable hematopoietic progenitor cells. Rubinstein PNAS 1995
11 Products evolved Red cell and plasma depletion product volume down to 25 ml with 5 ml of DMSO Improved cryopreservation bag integrity Smaller products, often with 2 compartments
12 Bedside Thaw Adult transplant centers looked at small product (small red cell lysate and DMSO content) and concern of cell loss with wash Greatest risk was poor or non- engraftment This looks like our autologous PBSC products Hahn et al. BMT, 2003 Chow et al. BBMT 2007 Stiff et al. Blood 2005
13 Alternate approach: Dilute only Simple dilution with albumin/dextran would support laboratory thaw and potential benefit of stabilization with hyperosmolar solution 2-5 fold dilution in laboratory with 25% albumin/40% dextran solution Stability of product for at least 8 hours facilitating remote lab thaw Barker et al, BBMT 2007
14 Thaw approach Pro Con Bedside thaw Dilute only Dilute and wash No lab issues Similar to practice for other frozen products Controlled thaw Theoretical advantage of hyperosmolar reequilibration Removal of 80% of DMSO and red cell lysate Manipulation of small product at bedside Immediate infusion necessary High load of DMSO and red cell/white cell lysate Larger volume of infusion Same load of red cells and DMSO Risk of cell loss with centrifugation (bag breaking, Cell aggregation with centrifugation
15 Comparison of the 3 thaw methods St. Louis Cord Blood Bank thawed 10 products in a 37 degree water bath and immediately split product into 3 groups: Thaw only 2 fold dilution with dextran-albumin 2 fold dilution with dextran-albumin followed by centrifugation at 400g for 20 min.
16 Viable CD34 recovery between thaw methods (n = 10 per group) Dilution Wash Thaw p< 0.01 Regan et al, Transfusion 2010
17 CFC mean percent recovery (n = 10 per group), Dilute/Wash Thaw P< 0.01 Regan et al, Transfusion 2010
18 Transplant outcomes of 401 cord blood units exported from St. Louis Cord Blood Bank between All units were red cell and plasma depleted All used in single cord transplants
19 Patient characteristics Thaw N = 36 Dilute N = 163 Wash N = 202 P value Age (median) <0.01 Weight (kg) <0.01 TNC recovery 99% 78% 79% <0.01 TNC /kg <0.01 Post thaw viability <0.1 Regan et al, Transfusion 2010
20 No difference in time to neutrophil recovery Regan et al, Transfusion 2010
21 Infusional toxicity reported to the SLCBB ( ) No serious adverse events reported
22 Cell processing laboratory The cell product is much smaller than the lab is used to working with. Important to practice thaws Minimize cell loss Contingency plan to handle broken bags Careful with testing sample volumes Validated thaw procedure for the variety of products being used. Requires early communication between transplant team and laboratory
23 Pediatric considerations: DMSO load: <1 gram DMSO/kg Red cell lysate: <1 ml of red cell lysate/kg Volume: although young children can handle volume better than older adults, in general want to keep volume <20 ml/kg.
24 The red cell replete unit The products are plasma depleted resulting in hematocrit in 80% range. At thaw there is a lot of red cell and neutrophil lysate Reports of cardiac and renal toxicity when high cell numbers infused (double cord, small recipient) 120 pediatric ( y) transplants with 40% washed and 60% thaw only OS was 79% and DFS was 70 % at 3 years Petz et al. Transfusion on line 2011
25 Conclusions: For cord blood units that have been banked after red cell and volume reduction there are similar transplant outcomes with the 3 thaw approaches Special consideration is needed for pediatric recipients and red cell replete units Retrospective review of CIBMTR experience is ongoing (Ballen et al)
26 Acknowledgements: Donna Regan and Jonathan Woffard St. Louis Cord Blood Bank
27
28 Improved hematopoietic progenitor cell reservoir following cord blood vs. bone marrow transplant 2 groups of children: 12 CB recipients and 12 adult BM recipients Recipients of BM transplants received 10-fold more cells and had significantly faster neutrophil and platelet recovery At 1 year after transplantation, the frequency of colonyforming cells (CFCs) and long-term culture initiating cells (LTC-ICs) were compared Frassoni et al, Blood 102:1138
29 Comparison of marrow hematopoietic progenitors following CB vs BM transplant Cord Blood Recipients (n=12) Bone Marrow Recipients (n=12) CFC/2 x 10 4 BM mononuclear cells P =.007 LTC-IC/10 6 BM mononuclear cells P =.001
30 Public cord blood banking Despite heavy support by national bodies the cost of units is high $25-40,000/unit Chance of finding a donor increases if you collect from families of similar ethnic background Hema-Quebec is the largest public bank in Canada Smaller banks in Toronto and Edmonton
31
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