Umbilical Cord Blood Cytomegalovirus Serostatus Does Not Have an Impact on Outcomes of Umbilical Cord Blood Transplantation for Acute Leukemia.

Several studies have reported an impact of adult hematopoietic stem cell donor cytomegalovirus (CMV) serostatus on allogeneic hematopoietic cell transplantation outcomes. Limited data, however, are available on the impact of cord blood unit (CBU) CMV serostatus on allogeneic umbilical cord blood transplantation (UCBT) outcomes. We analyzed, retrospectively, the impact of CBU CMV serostatus on relapse incidence (RI) and 2-year nonrelapse mortality (NRM) of single-unit CBU transplantation for acute leukemia. Data from 1177 de novo acute leukemia pediatric and adult patients transplanted within European Group for Blood and Marrow Transplantation centers between 2000 and 2012 were analyzed. CBUs were provided by the European Cord Blood Banks. The median follow-up time for live patients was 59.9 months. The recipients of CMV-seropositive and -seronegative CBUs showed a comparable RI (33% versus 35%, respectively, P = .6) and 2-year cumulative incidence of NRM (31% versus 32%, respectively, P = .5). We conclude that CBU CMV serostatus did not influence RI and NRM in de novo acute leukemia patients after allo-UCBT and should not be included as a criteria for cord blood choice.

Impact of cord blood banking technologies on clinical outcome: a Eurocord/Cord Blood Committee (CTIWP), European Society for Blood and Marrow Transplantation and NetCord retrospective analysis.

BACKGROUND: Techniques for banking cord blood units (CBUs) as source for hematopoietic stem cell transplantation have been developed over the past 20 years, aimed to improve laboratory efficiency without altering the biologic properties of the graft. A large-scale, registry-based assessment of the impact of the banking variables on the clinical outcome is currently missing. STUDY DESIGN AND METHODS: A total of 677 single cord blood transplants (CBTs) carried out for acute leukemia in complete remission in centers affiliated with the European Society for Blood and Marrow Transplantation were selected. An extensive set of data concerning CBU banking were collected and correlations with clinical outcome were assessed. Clinical endpoints were transplant-related mortality, engraftment, and graft-versus-host disease (GVHD). RESULTS: The median time between collection and CBT was 4.1 years (range, 0.2-16.3 years). Volume reduction (VR) of CBUs before freezing was performed in 59.2% of available reports; in half of these the frozen volume was less than 30 mL. Cumulative incidences of neutrophil engraftment on Day 60, 100-day acute GVHD (II-IV), and 4-year chronic GVHD were 87, 29, and 21 ± 2%. The cumulative incidence of nonrelapse mortality (NRM) at 100 days and 4-year NRM were, respectively, 16 ± 2 and 30 ± 2%. Neither the variables related to banking procedures nor the interval between collection and CBT influenced the clinical outcome. CONCLUSION: These findings indicate a satisfactory validation of the techniques associated with CBU VR across the banks. Cell viability assessment varied among the banks, suggesting that efforts to improve the standardization of CBU quality controls are needed.

An update on methods for cryopreservation and thawing of hemopoietic stem cells.

The aim of this article is to review a number of variables that may affect the cryopreservation of minimally manipulated products containing allogeneic or autologous hemopoietic progenitor cells (HPC) used for transplantation, with particular reference to processing, type and addition of cryoprotectant, cell concentration, volume, freezing procedure, cooling rate, storage, thawing, and quality management. After defining final product's requirements in compliance with norms, laws and regulations, it is crucial to define the critical control points of the process. New approaches of processing were developed in the last few years such as automatic devices for volume reduction and high cell concentration in the frozen product. DMSO at 10% final concentration is still the most used cryoprotectant for HPC cryopreservation. Although controlled rate freezing is the recommended method for HPC cryopreservation, alternative methods may be used. Last generation vapor storage vessels ensure temperature stability better than older tanks. Their use may reduce risks of cross-contamination. Finally we review advantages and disadvantages of thawing procedures that may be carried out in the laboratory or at the patient's bedside.

Engraftment kinetics and graft failure after single umbilical cord blood transplantation using a myeloablative conditioning regimen.

Umbilical cord blood transplant recipients are exposed to an increased risk of graft failure, a complication leading to a higher rate of transplant-related mortality. The decision and timing to offer a second transplant after graft failure is challenging. With the aim of addressing this issue, we analyzed engraftment kinetics and outcomes of 1268 patients (73% children) with acute leukemia (64% acute lymphoblastic leukemia, 36% acute myeloid leukemia) in remission who underwent single-unit umbilical cord blood transplantation after a myeloablative conditioning regimen. The median follow-up was 31 months. The overall survival rate at 3 years was 47%; the 100-day cumulative incidence of transplant-related mortality was 16%. Longer time to engraftment was associated with increased transplant-related mortality and shorter overall survival. The cumulative incidence of neutrophil engraftment at day 60 was 86%, while the median time to achieve engraftment was 24 days. Probability density analysis showed that the likelihood of engraftment after umbilical cord blood transplantation increased after day 10, peaked on day 21 and slowly decreased to 21% by day 31. Beyond day 31, the probability of engraftment dropped rapidly, and the residual probability of engrafting after day 42 was 5%. Graft failure was reported in 166 patients, and 66 of them received a second graft (allogeneic, n=45). Rescue actions, such as the search for another graft, should be considered starting after day 21. A diagnosis of graft failure can be established in patients who have not achieved neutrophil recovery by day 42. Moreover, subsequent transplants should not be postponed after day 42.

Effect of HLA-matching recipients to donor noninherited maternal antigens on outcomes after mismatched umbilical cord blood transplantation for hematologic malignancy.

Transplantation-related mortality (TRM) is high after HLA-mismatched umbilical cord blood (UCB) transplantation (UCBT). In utero, exposure to noninherited maternal antigen (NIMA) is recognized by the fetus, which induces T regulator cells to that haplotype. It is plausible that UCBTs in which recipients are matched to donor NIMAs may alleviate some of the excess mortality associated with this treatment. To explore this concept, we used marginal matched-pair Cox regression analysis to compare outcomes in 48 NIMA-matched UCBTs (ie, the NIMA of the donor UCB unit matched to the patient) and in 116 non-NIMA-matched UCBTs. All patients had a hematologic malignancy and received a single UCB unit. Cases and controls were matched on age, disease, disease status, transplantation-conditioning regimen, HLA match, and infused cell dose. TRM was lower after NIMA-matched UCBTs compared with NIMA-mismatched UCBTs (relative risk, 0.48; P = .05; 18% versus 32% at 5 years posttransplantation). Consequently, overall survival was higher after NIMA-matched UCBT. The 5-year probability of overall survival was 55% after NIMA-matched UCBTs versus 38% after NIMA-mismatched UCBTs (P = .04). When faced with the choice of multiple HLA-mismatched UCB units containing adequate cell doses, selecting an NIMA-matched UCB unit may improve survival after mismatched UCBT.

Storage characteristics of cord blood progenitor cells: report of a multicenter study by the cellular therapies team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.

BACKGROUND: Most hematopoietic progenitor cell (HPC) products are infused or processed shortly after collection, but in some cases this may be delayed for up to 48 hours. A number of variables such as temperature and cell concentration are of critical importance for the integrity of HPCs during this time. STUDY DESIGN AND METHODS: We evaluated critical variables using cord blood HPC units that were divided equally and stored at 4 °C versus room temperature (RT) for up to 96 hours. Total nucleated cell (TNC) and mononuclear cell (MNC) counts, viable CD34+ cell counts, and CD45+ cell viability as well as colony-forming unit-granulocyte-macrophage (CFU-GM) present over time at each temperature were determined. RESULTS: Overall, the data indicate that with the exception of viable CD34+ cells, there was a significant decrease in each variable measured for 72 to 96 hours and, with the exception of viable CD34+ cells and CFU-GM, the reductions were significantly greater in RT units than 4 °C units. There was an increase in viable CD34+ count for units where TNC count was greater than 8.5 × 10(9) /L, compared with units where TNC count was less than 8.5 × 10(9) /L, that was different for each storage temperature. CONCLUSIONS: Cord blood HPC collections maintained at 4 °C retained higher TNC counts, MNC counts, and CD45+ cell viability over a 72- to 96-hour storage period.

The impact of immunogenetics and clinical factors on the outcome of unrelated cord blood transplantation: the Milano Cord Blood Bank data.

In this article we examined the role of HLA incompatibility, of KIR C1 and C2 ligands and of other clinical factors on 99 cord blood transplants performed using single units from Milano Cord Blood Bank (MICB). We analyzed the occurrence of rejection, overall patient survival (OS) and occurrence of acute GvHD >or= 2 grade (severe aGvHD). No correlation was found between the end points and the number of HLA-A,-B, -DRB1 and -DQB1 mismatches. Only HLA-C disparities are associated with the occurrence of rejection (P=0.03). Our results showed that the presence of the C1 ligand in the donor decreased the occurrence of aGvHD (grade >or= 2) in the recipient while recipients of donors expressing the C2 KIR ligand experienced more frequently aGvHD (P=0.03). The HLA-C1 ligand, therefore, proved to have a protective effect towards severe aGvHD. The probability of rejection increased in KIR epitope-mismatched recipient/donor pairs (P=0.01). Finally the stage of disease at transplantation and cell dose were important for patient survival (P=0.003, P=0.048 respectively).

Ten-year quality control of a semiautomated procedure of cord blood unit volume reduction.

BACKGROUND: Volume reduction of cord blood units decreases the cost of cryogenic storage. This study reports the analysis of a 10-year quality control program of a semiautomated cord blood volume reduction procedure. STUDY DESIGN AND METHODS: Cord blood was collected in a plastic bag containing 29 mL citrate-phosphate-dextrose, centrifuged at 2124 x g for 12 minutes, and processed with a semiautomated device. The procedure was aimed at removing most red blood cells and plasma and concentrating hematopoietic progenitors in the buffy coat (BC), thus reducing the unit volume and saving cryogenic space. Finally, the BC was cryopreserved with an equal volume of 20 percent dimethyl sulfoxide. Total nucleated cells (TNCs) were counted before and after processing in the 4311 units banked from 1998 through 2007, whereas CD34+ cells and colony-forming units-granulocyte-macrophage (CFU-GM) were counted in 420 random units from 2001 through 2007. RESULTS: Mean postvolume reduction annual recoveries of TNCs, CD34+ cells, and CFU-GM ranged from 82.8 +/- 12.3 (standard deviation) to 91.4 +/- 6.4 percent, from 87.8 +/- 14.1 to 95.2 +/- 23.8 percent, and from 101.5 +/- 51.4 to 117.8 +/- 59.5 percent, respectively. Very strong correlations were found (r > 0.87) between postprocessing versus preprocessing TNCs, CD34+ cells, and CFU-GM; a moderate correlation between initial TNC count and unit's volume (r = 0.51); and no correlation between TNC percentage of recovery in the BC and initial unit's volume. The latter data indicate that most TNCs concentrate in the BC. CONCLUSIONS: The semiautomated procedure of cord blood unit volume reduction used in this study provides high and stable cellular recoveries during several years of routine cord blood banking.

Development of a mock hemopoietic stem cell component suitable for the validation of cryopreservation procedures.

We developed a laboratory model of a unit of hemopoietic progenitor cells (mock-HPC unit) suitable for the validation of HPC cryopreservation procedures. The project was prompted by the practical and ethical difficulty of using real HPC units collected from healthy donors and patients for validation purposes. Mock-HPC units of different volumes ranging from about 120 to about 540mL were prepared by pooling a routinely discarded by product of our procedure to prepare platelet concentrates from buffy-coats, a standard procedure in most blood centers in Europe. Five ABO/Rh identical buffy coats each of 50mL volume, obtained from 450mL whole blood units by hard spin, were pooled with 300mL of a commercial platelet additive solution. After soft spin, the supernatant platelet concentrate pool was removed. The bottom fraction of this procedure, which contains RBC, WBC, HPC and platelets, constitutes a mock-HPC unit of about 120mL volume. Several bottom fractions may be pooled to obtain a mock-HPC unit of the desired volume. We used 20 mock-HPC units to validate an automatic procedure of HPC cryopreservation with a controlled rate freezer. In particular, we documented the standardization of critical points of the cooling profile, such as the correspondence of the crystallization phase with the theoretical freezing temperature of the product, the temperature peak rise above the theoretical freezing temperature of -5.7 degrees C and the automatic achievement in the chamber of a constant minimum temperature during supercooling (about -37 degrees C) with mean chamber loading volumes ranging from 231.8 to 1027.2mL.

Multiple-laboratory comparison of in vitro assays utilized to characterize hematopoietic cells in cord blood.

BACKGROUND: Understanding the variability in results obtained by multiple laboratories is important because cord blood units are distributed worldwide for transplantation. STUDY DESIGN AND METHODS: Four exercises were conducted by multiple laboratories to assess assay variability on nucleated cell (NC), mononuclear cell (MNC) by hematology analyzers [HAs], and CD34+ cell (flow cytometry) measurements. Exercise 1 was an intralaboratory exercise in which the reproducibility of cell measurements was determined. Exercises 2 and 3 involved the shipment of identical processed cord blood samples. In Exercise 2, laboratory-specific methods were utilized. In Exercise 3, two commercial CD34+ cell methods (Stem-Kit and TruCOUNT) were used. In Exercise 4, CD34+ cell levels were determined on repetitive regating of identical list-mode files. RESULTS: Intralaboratory reproducibility was highest for NC measurements and lowest for CD34+ cell measurements. In Exercise 2, all laboratories except one utilized HA with an impedance technology and determined comparable results for NC and MNC levels, whereas the other laboratory utilized a HA with an optical counting method. Substantial variation was observed on measuring CD34+ cells with ranges of 32 to 141, 32 to 66, and 25 to 116 CD34+ cells per microL for the three identical samples. In Exercise 3, on the use of one specific commercial assay, the ranges of CD34+ levels were 214 to 411 and 62 to 178 cells per microL for the two identical samples. Nearly all participating laboratories determined comparable CD34+ levels on the use of identical list-mode files. CONCLUSION: These studies indicate that substantial variability in CD34+ cell levels were determined with flow cytometry. The variability in NC and MNC levels was minimal with HA methodology.

Recipient cytokine genotypes for TNF-alpha and IL-10 and the minor histocompatibility antigens HY and CD31 codon 125 are not associated with occurrence or severity of acute GVHD in unrelated cord blood transplantation: a retrospective analysis.

BACKGROUND: In HLA-identical sibling bone marrow transplantation, certain recipient cytokine gene polymorphism genotypes and minor histocompatibility differences influence the occurrence and severity of acute graft-versus-host disease (aGvHD). The present study investigated the role of cytokine tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 gene polymorphisms HY, HA-1, and CD31 minor histocompatibility antigen (mHag) mismatch in the development of aGvHD after unrelated cord blood (CB) transplant (CBT). METHODS: DNA samples of 115 CB recipients and their unrelated CB grafts were analyzed for genotype associated with TNF-alpha (TNFd3/d3) and IL-10 (IL-10(-1064), 11-16) and for disparities in major and three minor histocompatibility antigens, HY, HA-1, and CD31 codon 125. Results were correlated with the incidence of aGvHD grades II to IV. RESULTS: Neither the donor nor the recipient GvHD risk alleles TNFd3/d3 and IL-10(-1064) (11-16) were associated with the development of aGvHD grades II to IV and I to IV. Because of the heterogeneity of CBTs, the data were reanalyzed separately for patients with malignancies (n=83) or with inborn errors (n=24). No significant association was observed between the severity of aGvHD and the possession of either TNFd3/d3 or IL-10 (11-16) genotypes. Mismatches for the mHags HY, HA-1, and CD31 exon 125 between donor and recipient did not associate with aGvHD grades II to IV. CONCLUSIONS: In contrast to HLA-identical sibling bone marrow transplantation, in mismatched unrelated CBT, neither the cytokine genotypes TNFd3/d3 alone or in combination with IL-10(-1064) alleles nor the minor histocompatibility antigens HY, HA-1, and CD31 exon 125 were associated with aGvHD grades II to IV. Further determination of the cytokine gene polymorphism genotypes in CBTs compared with bone marrow transplants may identify those polymorphisms that could be potential predictive markers for the occurrence of aGvHD.