As the history of the cord blood banking system has lengthened, the number of cord blood units (CBUs) cryopreserved for years has increased. The global expansion of cord blood banking resulted in active international exchange of CBUs. To determine whether long-term cryopreservation and international shipment of CBUs affect the quality of the units and outcome after transplantation, we retrospectively analyzed the quality of 95 CBUs and the hematologic recovery of 127 patients with hematological malignancy following single-unit cord blood transplantation. Of the 127 CBUs used to transplant, 42 units were cryopreserved for long periods (5–11.8 years), and 44 units were shipped from distant countries. We found that length of cryopreservation and origin of CBUs did not affect the ratio of viable total-nucleated cells after thawing. Also, neutrophil engraftment was not affected by long-term cryopreservation (> 5 years) or origin (from distant countries), (hazard ratio, 0.91 and 1.2; P=0.65 and 0.41; respectively). The number of CD34+ cells before freezing (> 1.4 cells/kg recipient) was the only factor that enhanced neutrophil engraftment (hazard ratio, 1.8; P<0.01). This suggests that length of cryopreservation and origin need not be prioritized over the CD34+ cell dose when selecting CBUs.
Recent studies have shown that the number of umbilical cord blood transplantations (CBT) has been steadily increasing, and the outcomes of CBT are getting closer to those obtained from bone marrow transplantation.1, 2, 3 Over the past 20 years, a worldwide large-scale cord blood banking system has enabled immediate access to cryopreserved cord blood units (CBUs) for patients who require an alternative stem cell source for transplantation. As the history of the cord blood banking system becomes longer, the number of cord blood units that are cryopreserved for years has increased.4 Global expansion of this banking has resulted in active international exchange of CBUs; >40% of CBUs are shipped beyond country borders.5
Although long-term preservation of cord blood was shown to not influence hematopoietic reconstitution potential in the mouse model,6, 7 it is unclear whether preservation length has an impact on hematologic recovery following CBT in humans. The fact that not all banks have adopted international guidelines,4 such as NetCord-FACT International Standards,8 raises an additional issue, the potential difference among banks in quality control, which might result in impairment of reconstitution potential during a prolonged period of cryopreservation or international shipment. Moreover, incomplete standardization of the processing method for cord blood9 provokes questions about whether bank-provided information such as number of CD34+ cells reflects clinical outcomes after CBT.
The aim of this study is to evaluate the effect of long-term cryopreservation of CBUs and the region of the banks from which the cord blood originates on the quality of CBUs and hematologic recovery after CBT. We retrospectively analyzed the quality of 95 units obtained from various countries and hematological recovery in 127 CBTs. Also, we investigated whether information about the pre-freezing CBUs that is issued by banks stays reliable and can predict the clinical outcome regardless of the length of cryopreservation or the origin of the units.
Subjects and methods
Cord blood units
CBUs were selected to infuse the most closely matched donor unit/recipient pair: minimum requirements were 4/6 considering difference for HLA-A, B and DRB1. CBUs with >1.0 × 107 cells/kg recipient-body weight of total nucleated cells (TNCs) were selected. Units from the following countries were used in San Martino Hospital (Genoa, Italy): the United States (40), Italy (25), Germany (10), Australia (8), France (5), Belgium (3), Spain (2), Brazil (1) and Taiwan (1). Units of domestic origin were transplanted in University of Tsukuba Hospital (Tsukuba, Japan).
Measurement of TNCs and CD34+ cells
Ninety-five CBUs used in San Martino Hospital were analyzed. Before transplant, each cord blood unit was thawed at 37 °C and cells were washed according to the Rubinstein method.10 Then, cells were resuspended in 20 mL of thawing solution (saline solution+5% dextran+2.5% human albumin). A sample of the final volume was used for quality controls: TNC count and CD34+ cell numbers. Nucleated cells were counted using a Neubauer chamber for the WBC counting; CD34+ cell numbers were evaluated by flow cytometry. Samples were stained with the following antibodies: PE-conjugated anti-CD34 and FITC-conjugated anti-CD45. Nucleic acid dye 7-aminoactinomycin D was used to distinguish dead cells. Flow cytometry was performed using a FACSCalibur instrument (Becton Dickinson, San Jose, CA, USA), and the Cell Quest software (Becton Dickinson) was used for analysis. The CD34+ subpopulation was identified by co-staining of CD45, according to the single platform guidelines of the International Society of Hematotherapy and Graft Engineering (ISHAGE).11 The recovery rates of TNCs and CD34+ cells were determined as the ratios of each post-thawing cell number measured in San Martino Hospital and the pre-freezing cell number provided by each bank.
Patients and transplant procedures
One hundred and twenty-seven consecutive CBTs performed on adult patients with hematologic malignancies from April 2007 to September 2014 were retrospectively analyzed. Eighty-three were transplanted in San Martino Hospital and 44 were transplanted in University of Tsukuba Hospital. Patients were prepared for transplant with myeloablative conditioning for younger patients or reduced-intensity conditioning for older patients or those with comorbidities. Cord blood was transplanted into the bone marrow in 92 cases, or intravenously in 35 cases. G-CSF was given after transplant until neutrophil recovery. The time of neutrophil engraftment was defined as the first day of three consecutive days after transplantation when the ANC was maintained at 0.5 × 109/L or higher. The time of platelet engraftment was defined as the first day of seven consecutive days when the platelet count was maintained at 20 × 109/L or higher without transfusion support. Graft failure was defined as no sign of hematological recovery by post-transplant day 100.
Recovery rates of TNCs and CD34+ cells were evaluated with the Student's t-test for length of cryopreservation and bank of origin. Cumulative incidence of neutrophil and platelet recovery was assessed with the Gray test, with deaths from other causes as competing risk factors.12 Multivariate analyses were performed with the Fine and Gray proportional hazards regression model. All P-values were two-sided with type I error fixed at 0.05. Statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan),13 a graphical user interface for R (R Foundation for Statistical Computing, Vienna, Austria. Version 3.0.2).
The median cryopreservation period of 127 CBUs was 3.2 (range, 0.1–11.8) years. The median number of TNCs and CD34+ cells before cryopreservation was 2.0 × 109 (range, 0.9 to 4.5) cells and 7.5 × 106 (range, 2.1–27), respectively.
To analyze the influence of the length of cryopreservation, we divided the evaluable 95 cord blood units into 50 ‘younger’ units, which were cryopreserved for <5 years, and 45 ‘older’ units, which were cryopreserved for >5 years. The TNC recovery rate of younger and older units was 74.2±18.9 and 76.1±15.6%, respectively (P=0.61, Figure 1a). The mean difference was 1.9 (95% CI, −5.5 to 9.2). Also, the CD34+-cell recovery rate of older units (74.3±28.1%) was not different from that of younger units (76.2±34.6%) (P=0.79, Figure 2a). The mean difference was −1.9 (95% CI, −15.9 to 12.1). Next, we analyzed the influence of region of bank on the quality of CBUs. TNC recovery rates of units from European countries and other distant countries were 74.8±17.4 and 75.3±17.5%, respectively. The mean difference was 0.5 (95% CI, −6.9 to 7.9). CD34+-cell recovery was 76.7±30.7 and 73.9±32.7%, respectively. The mean difference was −2.8 (95% CI, −16.7 to 11.1). Hence, distance between the transplant facility and banks did not have statistically significant impact on recovery of TNCs and CD34+ cells (P=0.89 and 0.69, respectively). The correlation coefficients between pre-freezing and post-thawing numbers of TNCs and CD34+ cells were not different regardless of the length of cryopreservation and the origin (data now shown).
The overall cumulative incidence of neutrophil engraftment was 86% (95% CI, 78–91) and median neutrophil engraftment day was day 23 in 127 evaluable patients following CBT performed in Genoa and Tsukuba. Of the 127 CBUs used to transplant, 42 units were cryopreserved over 5 years (5–11.8 years), and 44 units were shipped from distant countries. When we compared the cumulative incidence of neutrophil engraftment after CBT, length of cryopreservation did not have a significant impact (84 vs 91%; P=0.95, Figure 2a). Moreover, neutrophil recovery after CBT with units from distant countries was not different from that with domestic and neighboring country-origin units (European-origin units used in Genoa and Japanese-origin units used in Tsukuba) (84 vs 87%; P=0.66, Figure 2b). In our series of transplants, the pre-freezing number of TNCs per recipient body weight did not influence neutrophil recovery, namely, cumulative engraftment of patients receiving units of larger and smaller than 2.5 × 107/kg TNCs were 86 and 85%, respectively (P=0.36; Figure 2c). In contrast, a pre-freezing CD34+ cell dose >1.4 × 105/kg significantly promoted neutrophil recovery (P=0.002, Figure 2d), namely, cumulative incidence and median day of engraftment were 92% (95% CI, 79–97) and day 21 in the larger CD34+ cell dose group, and 81% (95% CI, 70–88) and day 25 in the smaller CD34+ cell dose group, respectively.
In the multivariate models, >1.4 × 105/kg recipient body weight of pre-freezing CD34+ cell dose was the unique variable affecting neutrophil recovery (hazard ratio 1.8; 95% CI, 1.2–2.8, P=0.005, Table 1) and platelet recovery (hazard ratio, 2.0; 95% CI, 1.3–3.0, P=0.002, data not shown). HLA compatibility or intensity of conditioning regimens did not have any impact on neutrophil and platelet recovery in both univariate and multivariate analysis (data not shown).
As the history of CB banking has become longer, the number of cord blood units stored for more than a decade has increased,14 while whether or not there is an expiry date for cord blood units has been unclear. In addition, the system of cord blood banking has spread worldwide, and over 40% of cord blood units is currently exported to another country.5 Not all banks are yet accredited by global standards such as NetCord-FACT International Standards,8 and, moreover, long-distant transportation from banks could affect cord blood quality.15 Marked differences in the CD34+ cell viability of units obtained from different individual CB banks were demonstrated,16 and more than 10% of units was reported to have quality problems that might be a risk for patients undergoing CBT.15 Thus, the influence of long-term cryopreservation and bank origin need to be investigated to know whether the units remain useful for clinical use.
The numbers of TNCs and CD34+ cells are good indicators of cord blood quality, because they have been reported to be associated with engraftment.17, 18 However, is the information about the pre-freezing number of cells reliable? The information provided by banks might not reflect actually infused viable cells, because various factors could influence the cell viability, such as long-term cryopreservation, impaired quality control during cryopreservation and long-distance shipment of cord blood units. Previous studies showed that long-term cryopreservation did not compromise the number of hematopoietic progenitor cells for up to 12 years,19 or the recovery of TNCs and CD34+ cells from CBUs.20 But in those studies conditions of cryopreservation were homogeneous, which may not mimic the actual banking system in which preservation conditions may differ from bank to bank. We measured post-thawing TNC and CD34+-cell doses in each unit by a standardized method. Deterioration of viability and dispersion of the bank-provided cell dose can result in alteration in the recovery rate of TNCs and CD34+ cells, which is the ratio between the pre-freezing and post-thawing values. That is why we chose the recovery rate as a quality indicator of cord blood units. Consequently, recovery rates of TNCs and CD34+ cells were not statistically different regardless of length of cryopreservation or distance between the transplant facility and banks.
With regard to the function of long-term cryopreserved units, the hematopoietic reconstitution potential of CB cells stored for 15 years,6 and for up to 23.5 years,7 has been proved by in vitro assay and transplantation in immunodeficient mice. It has been reported that long-term cryopreservation did not influence hematological recovery after CBT using units of at most 5 years old21 and by analysis of child recipients,22 although these data are based on a limited number of cases. Our retrospective study provides confirmative information that length of cryopreservation for up to 11.8 years, and bank of origin had little impact on engraftment ability in adult recipients. In addition, these results implied that the quality control of banks is working well.
Since cord blood was transplanted directly into the bone marrow in majority of our cases,23 the homing capacity of hematopoietic stem cells could not fully be evaluated by this study. Although equivalence of TNC recovery rate could be shown, our sample size might not be large enough to strictly prove equivalence in CD34+ cell recovery in Figure 1, judged from the relative wideness of 95% CIs of the mean differences. Moreover, we could not exclude potential selection bias because our study was a retrospective analysis. Further large-scale prospective multicenter analysis is needed.
In conclusion, the factor that had impact on hematological recovery after CBT in adults was neither length of cryopreservation nor bank of origin, but instead the number of pre-freezing CD34+ cells provided by the cord blood bank. Given that the recovery rate of TNCs and CD34+ cells after thawing and engraftment serve as indicators of cord blood quality, quality control is working well regardless of length of cryopreservation or bank of origin. These results provide the useful information that the number of pre-freezing CD34+ cells provided by banks is reliable and can serve as a basis for selection of suitable cord blood units.
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This study was supported by a grant from the European Hematology Association—Japanese Society of Hematology Fellowship Exchange Award in 2011. We thank Dr M Gosho (CREIL Center, University of Tsukuba) for statistical advice, and Brian K. Purdue (Medical English Communications Center, University of Tsukuba) for grammatical review and advice. This study was supported by a grant from the European Hematology Association—Japanese Society of Hematology Fellowship Exchange Award in 2011.
The authors declare no conflict of interest.
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Kurita, N., Frassoni, F., Chiba, S. et al. Impact of length of cryopreservation and origin of cord blood units on hematologic recovery following cord blood transplantation. Bone Marrow Transplant 50, 818–821 (2015) doi:10.1038/bmt.2015.56
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