Factor XIII deficiency in two Spanish families with a novel variant in gene F13A1 detected by next-generation sequencing; symptoms and clinical management.

Coagulation factor XIII (FXIII) has a major role in coagulation stabilizing the haemostatic clot. FXIII deficiency is associated with an increased risk of bleeding. Severe phenotypes lead to spontaneous, traumatic and surgical bleeding. Umbilical cord bleeding is especially common, and intracranial bleeding may occur in up to one third of patients without prophylaxis. In this work, we used NGS for screening all the coding and intronic boundary regions of F13A1 and F13B genes in two families affected by severe FXIII deficiency. Outcome confirmation analysis and variant studies in related patients was done by Sanger sequencing. Two variants were found: c.34A > G (p.Arg12Gly; NM_00129.3) and c.514C > T (p.Arg172Ter; NM_00129.3), both located in the F13A1 gene. The variant p.Arg172Ter is already described in literature and was found in homozygosis in one family and in compound heterozygosis in the other family. The variant p.Arg12Gly variant has not been described previously. This variant is located in the activation peptide of the FXIII A-subunit which is highly conserved among FXIII homologs. Given the high risk of dangerous bleeding and early manifestation in severe FXIII-deficient patients, a prompt genetic confirmation is imperative. In this sense, NGS technology allows a rapid and simultaneous analysis of all regions of all the genes involved in the pathology.

Transmission of human immunodeficiency virus Type-1 by fresh-frozen plasma treated with methylene blue and light.

BACKGROUND: The risk of transfusion-transmitted infection (TTI) has been minimized by introduction of nucleic acid testing (NAT) and pathogen inactivation (PI). This case report describes transmission of human immunodeficiency virus Type 1 (HIV-1) to two recipients despite these measures. STUDY DESIGN AND METHODS: In March 2009 a possible TTI of HIV-1 was identified in a patient that had received pooled buffy coat platelet concentrate (BC-PLT) in November 2005. The subsequent lookback study found two more patients who had received methylene blue (MB)-treated fresh-frozen plasma (FFP) and red blood cells (RBCs) from the same donation. In November 2005 the donor had tested negative for both HIV antibodies and HIV-1 RNA by 44 minipool (44 MP) NAT. Repository samples of this donation and samples from the recipients were used for viral load (VL) and sequence analysis. RESULTS: HIV-1 RNA was detectable by individual donation (ID)-NAT in the repository sample from the 2005 window period donation and a VL of 135 copies/mL was measured. HIV-1 infection was confirmed in both recipients of both BC-PLT (65 mL of plasma) and MB-FFP (261 mL of plasma), but not in the patient that had received 4-week-old RBCs (20 mL of plasma). The sequence analysis revealed a close phylogenetic relationship between the virus strains isolated from the donor and recipients, compatible with TTI. CONCLUSIONS: Approximately 17,600 and 4400 virions in the MB-FFP and BC-PLT were infectious, but 1350 virions in the RBCs were not. ID-NAT would have prevented this transmission, but the combination of MP-NAT and MB-PI did not.

Qualitative and quantitative cell recovery in umbilical cord blood processed by two automated devices in routine cord blood banking: a comparative study.

BACKGROUND: Volume reduction is a widely used procedure in umbilical cord blood banking. It concentrates progenitor cells by reducing plasma and red blood cells, thereby optimising the use of storage space. Sepax and AXP are automated systems specifically developed for umbilical cord blood processing. These systems basically consist of a bag processing set into which cord blood is transferred and a device that automatically separates the different components during centrifugation. METHODS: The aim of this study was to analyse and compare cell recovery of umbilical cord blood units processed with Sepax and AXP at Valencia Cord Blood Bank. Cell counts were performed before and after volume reduction with AXP and Sepax. RESULTS: When analysing all the data (n =1,000 for AXP and n= 670 for Sepax), the percentages of total nucleated cell recovery and red blood cell depletion were 76.76 ± 7.51% and 88.28 ± 5.62%, respectively, for AXP and 78.81 ± 7.25% and 88.32 ± 7.94%, respectively, for Sepax (P <0.005 for both variables). CD34(+) cell recovery and viability in umbilical cord blood units were similar with both devices. Mononuclear cell recovery was significantly higher when the Sepax system was used. DISCUSSION: Both the Sepax and AXP automated systems achieve acceptable total nucleated cell recovery and good CD34(+) cell recovery after volume reduction of umbilical cord blood units and maintain cell viability. It should be noted that total nucleated cell recovery is significantly better with the Sepax system. Both systems deplete red blood cells efficiently, especially AXP which works without hydroxyethyl starch.

Abacavir and didanosine induce the interaction between human leukocytes and endothelial cells through Mac-1 upregulation.

OBJECTIVE: Abacavir and didanosine are nucleoside reverse transcriptase inhibitors (NRTI) widely used in therapy for HIV-infection but which have been linked to cardiovascular complications. The objective of this study was to analyze the effects of clinically relevant doses of abacavir and didanosine on human leukocyte-endothelium interactions and to compare them with those of other NRTIs. DESIGN AND METHODS: The interactions between human leukocytes - specifically peripheral blood polymorphonuclear (PMN) or mononuclear (PBMC) cells - and human umbilical vein endothelial cells were evaluated in a flow chamber system that reproduces conditions in vivo. The expression of adhesion molecules was analyzed by flow cytometry. RESULTS: Abacavir induced a dose-dependent increase in PMN and PBMC rolling and adhesion. This was reproduced by didanosine but not by lamivudine or zidovudine. Both abacavir and didanosine increased Mac-1 expression in neutrophils and monocytes, but produced no effects on either lymphocytes or the expression of endothelial adhesion molecules. The PMN/PBMC rolling and adhesion induced by abacavir or didanosine did not occur when antibodies against Mac-1 or its ligand ICAM-1 were blocked. CONCLUSION: Abacavir induces significant human leukocyte accumulation through the activation of Mac-1, which in turn interacts with its endothelial ligand ICAM-1. The fact that didanosine exhibits similar effects and that lamivudine and zidovudine do not points to a relationship between the chemical structure of NRTIs and the induction of leukocyte/endothelial cell interactions. This mechanism may be especially relevant to the progression of the vascular damage associated with atherosclerosis and myocardial infarction in abacavir and didanosine-treated patients.

A new automatic device for routine cord blood banking: critical analysis of different volume reduction methodologies.

BACKGROUND AIMS: Volume reduction is the usual process in cord blood banking that has some advantages regarding reducing the storage space and dimethyl sulfoxide (DMSO) quantity in the final product. The volume reduction methodology must guarantee high cell recovery and red blood cell (RBC) depletion by reducing all the umbilical cord blood (UCB) units to a standard volume. METHODS: We analyzed and compared critically three different volume reduction methods [hydroxyethylstarch (HES), top and bottom with Optipress II and Compomat G4, and AXP] used at the Valencia Cord Blood Bank over 10 years. RESULTS: The highest significant RBC depletion was achieved with the AXP system (P<0.001), while the top and bottom system with Compomat G4 and an adjusted buffy coat (BC) volume to 41 mL enabled the best total nucleated cell (TNC) recovery (P<0.001). TNC recovery and RBC depletion were similar for AXP and HES with an adjusted volume to 21 mL. In the multivariate analysis, when analyzing all cases, the BC volume set significantly influenced TNC, CD34+ and lymphocyte recoveries and RBC depletion (P<0.001). RBC depletion was significantly influenced by the initial volume and initial RBC content of UCB units (P<0.001). CONCLUSIONS: AXP is a highly efficient method for RBC depletion, providing the same TNC recovery as HES method with a final volume of 41 mL. AXP has the advantages of being an automatic and functionally closed system that shortens and better standardizes the proceedings. Top and bottom is a closed system that allows better TNC recoveries when the BC volume set is 41 mL.

Characteristics of umbilical cord blood units collected from preterm deliveries.

BACKGROUND: Umbilical cord blood (UCB) banking is a well-established activity supporting the increasing number of UCB transplantations in haematological diseases. Our aim was to analyse the UCB characteristics of UCB units from preterm deliveries and compare them to full-term deliveries. MATERIAL AND METHODS: A prospective study in 194 preterm deliveries occurring at the La Fe University Hospital in Valencia was performed. Patients between 25 and 37 weeks of gestation were included. Those cases were compared to a full-term deliveries control group. RESULTS: The cases were grouped according to the gestational age: between 25 and 33 weeks (group 1), between 34 and 37 weeks (group 2) and between 38 and 42 weeks (group 3). Among obstetric variables, only arterial pH and maternal age variables were similar for all the groups. Higher CD34(+) cell counts were observed in the group 2, while the clonogenic efficiency was higher for the most preterm deliveries. DISCUSSION: UCB from deliveries of at least 34 weeks of gestation contain sufficient hematopoietic stem cell content for unrelated banking and transplantation, even containing higher CD34(+) cell content than UCB units from full-term deliveries. However, UCB from deliveries of less than 33 weeks' gestation contain only sufficient progenitors for children under 20 kg.

Human platelet lysate enhances the proliferative activity of cultured human fibroblast-like cells from different tissues.

Several studies have shown the presence of fibroblast-like cells in the stromal fraction of different tissues with a high proliferative and differentiation potential. Platelet alpha granules contain growth factors released into the environment during activation. The effects of different supplements for culture medium (human serum, bovine serum and platelet lysate) on cultured human fibroblast-like cells from bone marrow, adipose tissue, trabecular bone and dental pulp have been compared. Expression of typical stromal and hematopoietic markers was analyzed and proliferative rates were determined. Flow cytofluorometry showed a homogenous pattern in serial-passaged cells, with a high level of stromal cell-associated markers (CD13, CD90, CD105). The presence of platelet lysate in culture media increased the number of cell generations obtained regardless of cell source. This effect was serum-dependent. Cell-based therapies can benefit by the use of products from human origin for "ex vivo" expansion of multipotent cells.

Optimizing donor selection in a cord blood bank.

OBJECTIVES: The main limitation factor for the wide use of umbilical cord blood (UCB) as a source of hematopoietic progenitor for transplantation is cell dose. One of the specific areas identified by some studies for improvement of UCB collection is donor selection. METHODS: Over a 3-mth period, 391 consecutive maternal-neonatal pairs were evaluated during the pre-partum period in the maternity ward at La Fe University Hospital (Valencia) by the Cord Blood Bank staff. Reasons for discarding umbilical cord blood donors and collected UCB units at the Cord Blood Bank in Valencia have been analysed. Obstetric factors influencing TNC content of 1300 collected UCB units have been determined, in order to establish obstetric criteria for cord blood donors selection in our geographic area. RESULTS: Only 32.5% of potential cord blood donors were refused. Among 1300 UCB collected, 506 (38.9%) were discarded before cryopreservation, mainly due to low cell counts. Multivariate analyses showed that the main significant factors influencing nucleated cell count were the weight of the placenta, sex of newborn and mode of collection. CONCLUSIONS: Our study shows that maternal medical histories must be completely reviewed by medical staff before collection of the UCB. Obstetrical factors influence cell content of UCB and could be added to standard cord blood donor criteria in order to improve the bank efficiency.