Meta-Analysis of the AXP and Sepax Automated Cord Blood Processing Systems

Transcription

1 TM Meta-Analysis of the AXP and Sepax Automated Cord Blood Processing Systems CHRISTY KIM, MINDY WILKE-DOUGLAS, AND MITCHEL SIVILOTTI

2 Introduction Automation is the key to standardizing cord blood processing. The AutoXpress Platform (Thermogenesis ) available from CESCA Therapeutics Inc. and the Sepax (Biosafe SA) system both provide automated cord blood (CB) processing systems in a functionally closed environment to ensure safety and sterility. Both processing systems address manual processing limitations such as technician dependent variability and inefficiencies with respect to processing time and cost. Automation is the key to standardizing CB processing to ensure efficient, reliable, and reproducible high quality cord blood units (CBUs) without compromising the consistency of stem and progenitor cell viability and potency. To further evaluate the performance of the systems and to determine if our previous findings were supported by the available literature, we conducted a comprehensive literature search of all documented studies published by cord blood banks around the world. A meta-analysis was performed from all published data (journals, posters, and abstracts) obtained in order to increase the statistical power and effect size of our former conclusions over individual studies. Thus, the results from this study may be generalized to not just one bank but to all banks using these processing methods. In our recent paper, Automated Cord Blood Processing: A Follow-up to the Pilar Solves Study Comparing the AXP vs Sepax Cord Blood Processing Systems, 1 we evaluated both systems looking at stem cell recoveries and red blood cell (RBC) depletion performance based on published studies. 2-5 We concluded that the AXP system exhibits superior performance for volume reduction of CBUs because it provides consistently high stem cell recoveries while delivering reliably low RBC contamination without the addition of hydroxyethyl starch (HES), thereby minimizing the risks and complications associated with this additive Methods Based on our search, we found 32 references, representing various public and private cord blood banks and institutions ranging from The most published automated systems, the AXP system without HES and the Sepax system with and without HES, were compared to each other and to the manual processing method (if data was provided). Studies were further narrowed to only those providing stem cell recoveries reported as mononuclear cell (MNC) and CD34 + cell recoveries and RBC depletion percentages (Table 1) Giving value to the breadth of the different studies included, MNC and CD34 + percentages and RBC reduction were recorded for each method regardless of CB age (- 48 hrs) and mean processing volume ( ml) with a maximum final volume of 25 ml. The sample size (n) for each study was weighted by multiplying each value with the n; if the n was not given, the value was excluded from the final analysis. The summation of the values for the stem cell recoveries (MNC and CD34 + ) and RBC reduction percentages were then divided by the total N to achieve an average value per N across all studies (Table 1). PROPERTY OF CESCA THERAPEUTICS, INC [A] PAGE 2 OF 8

3 Results and Discussion Table 1. Meta-Analysis of the AXP system without HES and the Sepax system with and without HES compared to manual CB processing. Cord Blood Bank Institute for Transplantation Diagnostics and Cell Therapeutics, Germany CB Age Max (hr) Mean Processing Volume (ml) AXP System Sepax System Manual Study Details n Parameter without Ref. # without with with HES HES HES HES 29 ml CPD, cell count was not MNC sufficient for banking Sepax S MNC 86.9 Jose Carreras Cord Blood Bank Sepax II 100 MNC London Cord Blood Bank, UK Sepax 1156 MNC New York Blood Center, USA MNC CBR, San Bruno, CA USA g 10 MNC Saint Louis Cord Blood Bank 48 >45 35 ml CPD, TNC> 9.0x MNC Dusseldorf Cord Blood Bank TNC mean =1.1x (Sepax); 7415 (manual) MNC Anthony Nolan, UK Sepax1 333 MNC Hellenic Cord Blood Bank, Athens, ( )x2 21ml CPD, 8ml CPD for flushing in satellite bag, 4C 106 MNC MNC 79.9 Greece Seoul National University 20 MNC CBR, San Bruno, CA USA mean age 23.59hr, mean TNC 9.94x10 8, mL 1414 MNC New York Blood Center, USA MNC New York Blood Center, USA MNC University of Texas M.D. Anderson Cancer Center, Houston, TX, USA MNC Japanese Red Cross Tokyo Blood Centre Gasthuisberg University Hospital Leuven, Leuven, Belgium Cord Blood Bank, Transfusion Centre, Valencia, Spain Japanese Red Cross Tokyo Blood Centre 28ml CPD, >10x10 8 TNCs, n= (split from same CB) MNC MNC MNC (manual) ; (Sepax) >60 ml 2-6C, WBC>5x10 8 ; Sepax CS MNC Jose Carreras Cord Blood Bank Sepax II 16 CD London Cord Blood Bank, UK Sepax 1156 CD Newcastle Centre for Cord Blood, 15(Sepax); 98.7 >40mL United Kingdom 19(Manual) CD Etablissement Francais du Sang Aquitaine-Limousin Bordeaux, Sepax CS CD France New York Blood Center, USA CD New York Blood Center, USA CD Geneva University Hospital and Basel University Hospital x10 7 TNC CD Dusseldorf Cord Blood Bank TNC mean =1.1x (Sepax); 7415 (manual) CD Besancon Cord Blood Bank, Besancon, France >2x10 6, 14-20C 213 (Sepax); >80 CD34 447(Manual) CD Sepax CD Cord-Blood Bank Basel, CH Sepax 2; After 2011, minimal CD TNC 150x10 7 Anthony Nolan, UK Sepax1 333 CD Hellenic Cord Blood Bank, Athens, ( )x2 21ml CPD, 8ml CPD for flushing in satellite bag, 4C 106 CD CD Greece New York Blood Center, USA CD C; TNC > x (AXP) ; 670 (Sepax); (Sepax); TNC > x (Sepax) 1000 (AXP) (AXP); CD ml CPD, >10x10 8 TNCs, n= (split from same CB) CD CD (manual) ; (Sepax) >60 ml 2-6C, WBC>5x10 8 ; Sepax CS CD PROPERTY OF CESCA THERAPEUTICS, INC [A] PAGE 3 OF 8

4 Results and Discussion (continued) Table 1. (continued) Cord Blood Bank CB Age Max (hr) Mean Processing Volume (ml) Jose Carreras Cord Blood Bank Sepax II 100 London Cord Blood Bank, UK Sepax 1156 Etablissement Francais du Sang Aquitaine-Limousin Bordeaux, France AXP System Sepax System Manual Study Details n Parameter without Ref. # without with with HES HES HES HES Sepax CS New York Blood Center, USA New York Blood Center, USA Geneva University Hospital and Basel University Hospital x10 7 TNC Anthony Nolan, UK Sepax1 333 Barcelona Cord Blood Bank 82 Cord Blood Bank, Transfusion Centre, Valencia, Spain Cord Blood Bank, Transfusion Centre, Valencia, Spain Japanese Red Cross Tokyo Blood Centre Gasthuisberg University Hospital Leuven, Leuven, Belgium (AXP) ; (Sepax) (manual) ; (Sepax) >60 ml 4C;CD34+ > 30x105 (AXP) ; TNC > x107 (Manual) 4C; TNC > x10 7 (Sepax); TNC > x10 7 (AXP); 28ml CPD, >10x10 8 TNCs, n= (split from same CB) 2-6C, WBC>5x10 8 ; Sepax CS (Manual); 492 (AXP) 670 (Sepax); 1000 (AXP) Data from the meta-analysis shows that the AXP system, when used without HES, outperforms the Sepax system (with or without HES) for cord blood processing as follows: average MNC/N: 94.5% vs 86.7% and average CD34 + /N: 97.3% vs 92.3% as summarized in Figure 1 (A and B). These results are in agreement with our previous comparison between Takanashi et al. and Zingsem et al. in which the AXP without HES delivers higher recoveries of MNCs and CD34 + cells than the Sepax. 1 However, the results here extend our previous finding by revealing that the AXP outperforms the Sepax system irrespective of HES. Since total nucleated cell counts (TNCs) underestimate the ability and capacity of stem and progenitor cells by including RBCs, granulocytes, and other cell impurities, we selectively analyzed MNCs and CD34 + stem cell recoveries because these are more reliable parameters for potency. 28,33 MNCs include hematopoietic stem and progenitor cells at different stages of maturation, as well as lymphocytes, monocytes, and macrophages that better reflect the quality PROPERTY OF CESCA THERAPEUTICS, INC [A] and potency of the CBU than TNCs. This is further evident in that the AXP system can be used with HES to achieve 20% higher TNC recoveries while maintaining consistently high CD34 + recoveries regardless if HES is omitted or added. 25 Although the use of the colony-forming unit (CFU) assay is the gold standard for testing potency, the CD34 + cell count is known to correlate with time to engraftment and transplant related mortality (TRM) and does not require 2 weeks of culture. 28,34,35 Not only are stem and progenitor cell recoveries critical in generating a potent CBU, but equally important and often overlooked is the opposing effect of RBC contamination. The results from this meta-analysis show that the AXP system when used without HES, is comparable to the Sepax system with HES, as noted in previously. 1 In addition, the AXP system without HES provides for superior RBC reduction when compared to the Sepax system when used with or without HES (average RBC reduction %/N=87.5% vs 81.6%, respectively). While both automated systems PAGE 4 OF 8

5 Results and Discussion (continued) surpass manual processing (77.1%) (Figure 1C), whether differences in CB product regarding RBC content result in a therapeutic difference remains to be determined. In particular, CB RBCs are less osmotically tolerant than adult RBCs and are therefore even more sensitive to post-thaw swelling after cryopreservation; 36 this poses the risk of hemolysis-induced Average cellular RBC damage Reduction to the %/N carefully processed 100 and stored CBUs. High concentrations of RBCs have 95 been shown to negatively affect stem cell viability and functionality 90 in both CB 27,37,38 and bone marrow (BM) 39 via secreted inhibitory factors Figure 1. (A,B) Average stem cell recovery % per N (MNC and CD34 + ) and (C) 65 average RBC reduction % per N for AXP without HES and Sepax with HES compared to manual CB processing across all studies with recorded n. *Statistical significance via a two-tailed Student s t-test was further confirmed with all the data sets reporting standard deviations 55 from Table 1 for all three parameters (MNC, CD34 +, and RBC reduction) comparing the AXP to the SEPAX (p<0.0001) and to manual processing (p<0.0001). 50 SEPAX with HES MANUAL N=1539 N=3110 N=905 % Recoveries A % Recoveries Average MNC Recovery %/N 94.5 AXP without HES N= SEPAX with HES N= MANUAL N=8263 B % Recoveries Average CD34+ Cell Recovery %/N 97.3 AXP without HES N= SEPAX with HES N= MANUAL N=7945 C % Recoveries Average %/N 87.5 AXP without HES N= SEPAX with HES N= MANUAL N=905 PROPERTY OF CESCA THERAPEUTICS, INC [A] PAGE 5 OF 8

6 Summary The results of our comprehensive meta-analysis demonstrate that the AXP system is a superior automated CB processing system that outperforms not only manual CB processing but also other automated technologies such as the Sepax system, including the Sepax 2 which uses the same core technology with modified external design, user interface, and software. 40 Further studies using Sepax 2 confirm that there is no statistical difference between Sepax 2 and Sepax 1 for stem cell recoveries (MNC and CD34 + ) because they use the same technology. 17,20,22 Although there have been noted volume discrepancies in the Sepax 2 vs the Sepax 1 due to enhanced sensitivity of available Sepax 2 protocols, the resulting product is at the expense of an unacceptable level ( %) of hematocrit in a 20 ml volume sample. 22 Therefore, the conclusions based on this meta-analysis are relevant to the AXP system and all current Sepax systems. When we consider the advancements in cord blood processing, we have crossed milestones in time, cost, efficiency, and quality. The AXP system is a FDA-cleared, functionally closed automated system with technologies that allow for greater throughput by reducing processing time. It can be easily and cost effectively scaled up to run 6 devices at a time in one centrifuge and does not require HES for efficient stem cell recoveries and RBC depletion, eliminating the additional risks associated with HES The disparity between current global CBU inventories and physician selection criteria of CBUs for hematopoietic stem cell transplantation remains a major concern impacting CB banking sustainability. 46,47 The ever increasing demands for higher TNC requirements call into question the value of the current CBUs banked, particularly in comparison to increasing competition from other cell sources in the wake of improving patient conditioning regimens and immune suppression (i.e. haploidentical). It is well known that TNC homing and viability are negatively affected by RBC contamination and yet, it is not a banking requirement to report RBC levels. 27,37-39 The further reduction of RBCs is possible using current technology at the risk of also reducing TNC numbers, however, we are left to ponder whether or not ultra-low RBC CBUs would perform better than their high RBC (and higher TNC) counterparts. The answer to this question may never be answered due to the maturity and inertia surrounding the drive for high TNCs. Additionally, we should also call into question the current standards for determining cell viability. When reported/ registered pre-cryopreservation, methods such as PI and 7AAD do not reliably quantify functionally viable and actively metabolizing cells; dye penetration methodologies do not adequately assess more subtle damage leading to irreversible apoptosis. 33,47 Although the selection of CBUs for transplantation is by convention linked with high TNCs, more relevant parameters like MNCs and CD34 + cells better predict time to engraftment and TRM than TNCs. 28,33-35 Why, then, does the CB banking industry continue to place such high value on TNC numbers when assessing CBUs, inextricably dismissing the significance of the true predictors of transplant outcomes and encouraging the faulty selection of CBUs for transplantation? The education of prospective parents and transplant centers to select based on MNCs and CD34 + cells may be the only way to encourage a change in the banking industry to adjust their registering parameters to those that will better reflect the true potential of the CBU. To advance the field of CB transplantation, quality standards relative to viable and functional stem and progenitor cells and not just empty counts must be continually pursued and improved in order to guarantee the safety, purity, potency and consistency of each CBU. PROPERTY OF CESCA THERAPEUTICS, INC [A] PAGE 6 OF 8

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