Allogeneic cord blood red blood cells: assessing cord blood unit fractionation and validation

BackgroundFor neonates and preterm infants, in whom a transfusion dose is low, the use of red blood cells (RBC) from cord blood appears to be feasible. Standardisation of fractionation and identification and assessment of quality control parameters for such RBC are still lacking.Materials and methodsWe describe the process used to obtain RBC from cord blood for transfusion purposes, including quality controls to evaluate fractionation performance and the effects of storage. The cord RBC, to which SAG-M was added, were sampled on the day of fractionation, and 7 and 14 days (end of storage) later in order to measure the complete blood count, biochemical parameters and residual white blood cells. We also assessed microbial contamination.ResultsData relative to 279 cord blood units were evaluated. The median gestational age at collection was 40 weeks (interquartile range [IQR] 39.1–40.7) and the median volume was 90 mL (IQR 81–103). Units were subjected to automated fractionation with Compomat, and packed RBC were suspended in SAG-M solution. The median volume of the SAG-M-suspended units was 31 mL (IQR 24.0–38.1) and the median haematocrit was 54.2% (IQR 49.4–59.5). The median volume after leukoreduction was 22 mL (IQR 17–28), with the volume decrease being similar in units leukoreduced before (n=75) or after (n=204) storage. The haematocrit of leukoreduced units was higher than that of buffy coat-depleted units. Storage at 2–6 °C for 14 days was accompanied by an increase of potassium levels and percentage of haemolysis. Microbial cultures were positive for 2.9% of the collected units.DiscussionFractionation of whole cord blood can provide RBC concentrates with similar baseline characteristics as units from adults. The transfusion dose and quality of the units appear safe and suitable for clinical use in neonates, with a satisfactory haematocrit and residual white blood cell content, despite a very variable collection volume.     

Umbilical cord blood transplants

With the establishment of cord blood banks, the number of related and unrelated umbilical cord blood transplants is increasing worldwide. Close links have been established with the cord blood banks. Available data showed that umbilical cord blood transplants offer overall results comparable to those obtained with related or unrelated bone marrow transplants. Several differences were found: engraftment with cord blood was delayed, resulting in an increased incidence of early transplant complications, and the incidence of acute and chronic graft-versus-host disease was significantly reduced with cord blood grafts, even in HLA-mismatched transplants and in adults. In patients with leukemia, the rate of relapse appeared to be similar to that documented in bone marrow transplant recipients. These data confirm the potential benefit of using umbilical cord blood hematopoietic stem cells for allogeneic transplants.     

Umbilical cord blood transplantation

PURPOSE OF REVIEW: Familiar and unrelated umbilical cord blood is an appealing alternative source of hematopoietic stem cells patients undergoing transplantation for a wide variety of diseases. In the unrelated donor transplant setting, shorter time to transplant, which is particularly relevant to patients requiring urgent transplantation, and tolerance of 1-2 human leukocyte antigen mismatch, which increases the chance of finding a suitable donor, are evident advantages over bone marrow transplantation. The speed of engraftment is slower after cord blood transplantation but it is counterbalanced by a lower incidence of severe graft-versus-host disease. Cell dose and human leukocyte antigen are major factors influencing outcome after umbilical cord blood transplantation. RECENT FINDINGS: Unrelated donor cord blood transplantation is considered an acceptable option to bone marrow for pediatric transplantation, and recent data in adults point the same way. SUMMARY: This review describes the recent clinical results of cord blood transplantation and discusses developing research strategies aimed at optimizing this kind of transplantation.     

Family-directed umbilical cord blood banking

Umbilical cord blood transplantation from HLA-identical siblings provides good results in children. These results support targeted efforts to bank family cord blood units that can be used for a sibling diagnosed with a disease which can be cured by allogeneic hematopoietic stem cell transplantation or for research that investigates the use of allogeneic or autologous cord blood cells. Over 500 patients transplanted with related cord blood units have been reported to the Eurocord registry with a 4-year overall survival of 91% for patients with non-malignant diseases and 56% for patients with malignant diseases. Main hematologic indications in children are leukemia, hemoglobinopathies or inherited hematologic, immunological or metabolic disorders. However, family-directed cord blood banking is not widely promoted; many cord blood units used in sibling transplantation have been obtained from private banks that do not meet the necessary criteria required to store these units. Marketing by private banks who predominantly store autologous cord blood units has created public confusion. There are very few current validated indications for autologous storage but some new indications might appear in the future. Little effort is devoted to provide unbiased information and to educate the public as to the distinction between the different types of banking, economic models and standards involved in such programs. In order to provide a better service for families in need, directed-family cord blood banking activities should be encouraged and closely monitored with common standards, and better information on current and future indications should be made available.     

Neonatal platelets from cord blood and peripheral blood

The haemostatic system in neonates is different from that of adults, possibly contributing to an increased incidence of bleeding disorders, such as intracranial hemorrhage. In this study, we analyzed platelets from cord blood and peripheral blood, collected at three time points after delivery from 20 term and 37 preterm neonates as well as blood from 20 healthy adults. Platelet membrane glycoproteins (GP) were quantified and P-selectin expression and PAC-1 binding ability before and after stimulation with TRAP were analyzed by whole blood flow cytometry. We found no significant differences in neonatal platelets from cord blood and peripheral blood within the first 24?h of life. Platelets from infants less than 30 weeks of gestation expressed lower levels of GP (33271?±?9381 vs. 44085?±?17287 for GPIIIa, P?<?0.05) and were less reactive than platelets from term newborns (4.3?±?3.3 vs. 20.1?±?11.8% PAC-1 positive platelets after stimulation with TRAP, P?<?0.05). A significantly lower level of GPIIb/IIIa expression on platelets from peripheral blood was seen in term newborns as well as preterm infants, compared to adults. There was only a partial enhancement in the degranulation ability (α-granules) (13.4?±?12.3 vs. 50.3?±?16.1% P-selectin positive platelets, P?<?0.05) and no significant increase for PAC-1 binding (13.6?±?10.9 vs. 15.3?±?5.9% PAC-1 positive platelets, P?=?0.8) during the first 12 days of life. In conclusion, we could demonstrate that neonatal platelet reactivity increases with gestational age.     

Neonatal platelets from cord blood and peripheral blood

The haemostatic system in neonates is different from that of adults, possibly contributing to an increased incidence of bleeding disorders, such as intracranial hemorrhage. In this study, we analyzed platelets from cord blood and peripheral blood, collected at three time points after delivery from 20 term and 37 preterm neonates as well as blood from 20 healthy adults. Platelet membrane glycoproteins (GP) were quantified and P-selectin expression and PAC-1 binding ability before and after stimulation with TRAP were analyzed by whole blood flow cytometry. We found no significant differences in neonatal platelets from cord blood and peripheral blood within the first 24h of life. Platelets from infants less than 30 weeks of gestation expressed lower levels of GP (33271+/-9381 vs. 44085+/-17287 for GPIIIa, P<0.05) and were less reactive than platelets from term newborns (4.3+/-3.3 vs. 20.1+/-11.8% PAC-1 positive platelets after stimulation with TRAP, P<0.05). A significantly lower level of GPIIb/IIIa expression on platelets from peripheral blood was seen in term newborns as well as preterm infants, compared to adults. There was only a partial enhancement in the degranulation ability (alpha-granules) (13.4+/-12.3 vs. 50.3+/-16.1% P-selectin positive platelets, P<0.05) and no significant increase for PAC-1 binding (13.6+/-10.9 vs. 15.3+/-5.9% PAC-1 positive platelets, P=0.8) during the first 12 days of life. In conclusion, we could demonstrate that neonatal platelet reactivity increases with gestational age.     

Trends in cord blood banking

Background

Umbilical cord blood (UCB) is a source of hematopoietic precursor cells for transplantation. The creation of UCB banks in 1992 led to the possibility of storing units of UCB for unrelated transplants. The distribution of cell contents in historical inventories is not homogenous and many units are not, therefore, suitable for adults. The aim of this study was to analyse our UCB bank inventory, evaluate the units released for transplantation and calculate the cost of the current process per unit of UCB stored.

Methods

Three study periods were defined. In the first period, from January 1996 to January 2006, the total nucleated cell (TNC) count acceptable for processing was 4–6×10⁸ and a manual processing system was used. In the second period, from October 2006 to July 2010, processing was automated and the acceptable TNC count varied from 8–10×10⁸. In the third period, from January 2009 to June 2010, an automated Sepax-BioArchive procedure was used and the accepted initial TNC count was >10×10⁸. Within each period the units were categorised according to various ranges of cryopreserved TNC counts in the units: A, >16.2×10⁸; B1, from 12.5–16.1×10⁸; B2, from 5.2–12.4×10⁸; and C, <5.1×10⁸.

Results

The third period is best representative of current practices, with homogenous TNC acceptance criteria and automated processing. In this period 15.7% of the units were category A and 25.5% were category B. Overall, the mean TNC count of units released for transplantation was 14×10⁸ (range, 4.6×10⁸ to 36.5×10⁸). The cost of the processed UCB in 2009 was 720.41 euros per unit.

Conclusion

An UCB bank should store units of high-quality, in terms of the TNC count of units issued for transplantation, have a training programme to optimise the selection of donors prior to delivery, use similar volume reduction systems and homogenous recovery indices, express its indicators in the same units, use validated analytical techniques, and bear in mind ethnic minorities.     

CFU-F circulating in cord blood

Summary CFU-F (colony forming units-fibroblast) were studied from cord blood and, as controls, from normal bone marrow of older children and adults. Numbers of CFU-F in cord blood buffy coat cells are lower by a factor of 10 in comparison to bone marrow CFU-F. Cytomorphology and staining with monoclonal antibody identify the progeny cells of CFU-F as fibroblasts. Cord blood CFU-F derived fibroblasts have properties supporting hematopoiesis: They produce CSF (colony stimulating factor) to which fresh cord blood CFU-GM (colony forming units-granulocytic, monocytic) react by colony formation in a dose-response manner. In addition, fibroblast colonies discharge clonogenic round cells into the medium forming CFU-GM and CFU-F colonies in secondary methyl cellulose cultures. We conclude that fetal blood contains clonogenic stromal cells (CFU-F) that give rise to fibroblasts with properties of hematopoietic support.This work was supported by the Deutsche Forschungsgemeinschaft (Pr 75/12-1)     

Cord blood transplantation and cord blood bank in Japan

The first cord blood transplantation (CBT) was performed in a 5-year-old boy with acute myelogenous leukemia from his HLA-identical sibling donor in 1994. Since then there have been 17 related and 131 unrelated CBTs in Japan. Overall survival and disease-free survival (DFS) were over 70% in sibling donor CBT. DFS of unrelated CBT in leukemia and other hematological malignancies was 43%, and OS of UCBT in non-malignant diseases was 63%. HLA disparity between the donor and the recipient did not affect the incidence and severity of acute graft-versus-host disease (GVHD) or survival. Cell dose was the most important factor for engraftment and survival both in malignant and in non-malignant diseases. The Japanese government has recently established the nationwide cord blood bank network, and eight local cord blood banks are financially supported by the government. 20,000 units of CB are planned to be collected and stored in the next 5 years by this network.     

Haematopoietic progenitors from umbilical cord blood

BACKGROUND/AIM: The aim of this study is to improve the obstetrician-based cord blood collection system and an efficient recovery of CD34+ haematopoietic progenitor stem cells. METHODS: CD34+ cells were purified from total blood using a positive selection enrichment method, called Mini-Macs. RESULTS: The final yield of CD34+ cells we obtained was 10(4) cells/ml, with a CD34+ purity of 99%. CONCLUSION: Our results confirm that, by using this method, it is possible to get a significant stem cell number, thus improving transplanting both peripheral stem cells and umbilical cord ones.     

Toxoplasmosis in cord blood transplantation recipients

Toxoplasmosis is a devastating opportunistic infection that can affect immunocompromised patients such as cord blood transplantation (CBT) recipients. The clinical characteristics of 4 toxoplasmosis CBT patients treated at our institution are reviewed, together with 5 cases collected from the literature. The rate of toxoplasmosis in our hospital was 6% in CBT recipients and 0.2% in other types of allogeneic hematopoietic stem cell transplantation (P < 0.001). Five patients (56%) presented disseminated toxoplasmosis and 4 patients (44%) had localized infection in the central nervous system. In 5 of the 9 patients considered (56%), cytomegalovirus viral replication had been detected before the clinical onset of toxoplasmosis. Seven patients (78%) had previously developed graft‐versus‐host disease. All patients who exhibited disseminated disease died due to Toxoplasma infection. Pre‐transplant serology was positive in 1 patient, negative in 3 patients, and not performed in another. Only 1 of these 5 patients with disseminated disease had received Toxoplasma prophylaxis with cotrimoxazole. It could be concluded that mortality in CBT patients with disseminated toxoplasmosis is unacceptably high. The negative results of serology in the majority of these cases, and its unspecific clinical presentation, makes diagnosis exceedingly difficult. Better diagnostic tests and prophylaxis strategy are needed in CBT recipients.     

Indirect evidence that maternal microchimerism in cord blood mediates a graft-versus-leukemia effect in cord blood transplantation

During pregnancy women can develop B- and T-cell immunity against the inherited paternal antigens (IPAs) of the fetus, such as HLA, peptides of minor histocompatibilty antigens, and possibly onco-fetal antigens. The biological and pathological role of these pregnancy-induced immunological events is only understood in part. However, anti-IPA immunity in the mother persists for many decades after delivery and may reduce relapse in offspring with leukemia after HLA-haploidentical transplantation of maternal hematopoietic stem cells (HSC). We hypothesized that maternal anti-IPA immune elements cross the placenta and might confer a potent graft-versus-leukemia effect when cord blood (CB) is used in unrelated HSC transplantation. In a retrospective study of single-unit CB recipients with all grafts provided by the New York Blood Center, we show that patients with acute myeloid or lymphoblastic leukemia (n = 845) who shared one or more HLA-A, -B, or -DRB1 antigens with their CB donor's IPAs had a significant decrease in leukemic relapse posttransplantation [hazard ratio (HR) = 0.38, P < 0.001] compared with those that did not. Remarkably, relapse reduction in patients receiving CB with one HLA mismatch (HR = 0.15, P < 0.001) was not associated with an increased risk of severe acute graft-versus-host disease (HR = 1.43, P = 0.730). Our findings may explain the unexpected low relapse rate after CB transplantation, open new avenues in the study of leukemic relapse after HSC transplantation (possibly of malignancies in general), and have practical implications for CB unit selection.     

Umbilical cord blood transplant in human

Human cord and placental blood provides a rich source of hematopoietic stem cells. On the basis of the finding, umbilical cord blood stem cells have been used to reconstitute hematopoiesis in children with malignant and non malignant diseases after treatment with myeloablative doses of chemoradiotherapy. Early results show, that a single cord blood provides enough hematopoietic stem cells to provide short and long term engraftment and, that the incidence and severity of graft versus host disease has been low even in HLA mismatched transplants. These results are encouraging enough to embark on large scale banking of cord blood for purposes of future allogeneic and autologous stem cell transplantation, to promote studies on the unique properties of fetal and neonatal hematopoiesis, to study the immunological properties of cord blood cells and, to initiate investigations on gene transfer into human cord blood cells for future gene therapy trials. This review will briefly summarize the current knowledge on cord blood transplantation as well as the future development of research on this unique source of hematopoietic stem cells.This work was supported by Eurocord Transplant Biomed II Program