Platelet antibody analysis by three different tests.

BACKGROUND: Platelet-reactive antibodies lead to thrombocytopenia and bleeding disorders, and diverse assays are used for their detection. In this retrospective analysis, the applicability of three different test systems was compared and antibody specificities were assessed. METHODS: Sera of 1,234 patients were tested with an enzyme-linked immunosorbent assay (ELISA; Lifecodes PAKPLUS(®) or PAK 12(®), Gen-Probe) and a solid-phase assay (Capture-P Ready Screen(®), Immucor Inc.). In cases of suspected anti-HLA class I antibodies, a specific lymphocytotoxicity test (LCT, Bio-Rad(®)) was performed. RESULTS: Platelet antibodies were detected in 366 of 1,234 samples (29.7%). In 70.3% concordant negative but only in 8.4% concordant positive results were obtained with both the methods; 185 of 1,053 in the solid-phase assay negative samples were positive in the ELISA (15.0%). In samples positive in both methods, most antibodies reacted against HLA class I antigens. Glycoprotein (GP) specific platelet antibodies, mainly against GPIIb/IIIa and GPIa/IIa, were more frequently detectable in the ELISA than in the solid-phase assay, whereas weakly positive results have to be interpreted cautiously. CONCLUSION: ELISA, solid-phase assay, and LCT showed highly divergent results. Due to several limitations, the additional analysis by the "monoclonal antibody-specific immobilization of platelet antigen" (MAIPA)-assay is highly recommended.

Patatin-like phospholipase domain-containing protein 3 rs738409-G in recipients of liver transplants is a risk factor for graft steatosis.

BACKGROUND & AIMS: The G-allele in position rs738409 of patatin-like phospholipase domain-containing protein 3 (PNPLA3) is associated with an increased hepatic concentration of triglyceride and is a risk factor for advanced liver disease. We investigated the association of donor and recipient risk alleles with the development of graft steatosis after liver transplantation. METHODS: PNPLA3 genotypes were determined in 237 transplant recipients and in 255 organ donors. Macrovesicular steatosis was assessed by unenhanced computed tomography 5 years after liver transplantation in 95 patients and correlated with donor and recipient PNPLA3 genotype. RESULTS: The risk allele was significantly more frequent in transplant recipients than in donors (42% vs 28%; P < .001). A prevalence of graft steatosis of 30% or greater significantly increased from 11.6% at 1 year after liver transplantation to 32.6% at 5 years after transplantation. Five years after liver transplantation, steatosis was present in 63.2% of patients homozygous for the rs738409-G allele, in 31.4% of heterozygous recipients, and in 12.0% of rs738409-CC recipients (P = .002). Donor genotypes were not associated with the development of graft steatosis. In multivariate regression analysis, recipients who carried rs738409-GG had a 13.7-fold higher risk of graft steatosis than recipients who carried rs738409-CC (P = .022), independent of recipient age, weight gain after liver transplantation, or the underlying disease. CONCLUSIONS: Liver transplant recipients who carry rs738409-G in PNPLA3 are at increased risk for hepatic triglyceride accumulation, independent of the graft PNPLA3 genotype.

Thermometry of red blood cell concentrate: magnetic resonance decoding warm up process.

PURPOSE: Temperature is a key measure in human red blood cell concentrate (RBC) quality control. A precise description of transient temperature distributions in RBC units removed from steady storage exposed to ambient temperature is at present unknown. Magnetic resonance thermometry was employed to visualize and analyse RBC warm up processes, to describe time courses of RBC mean, surface and core temperatures by an analytical model, and to determine and investigate corresponding model parameters. METHODS: Warm-up processes of 47 RBC units stored at 1-6°C and exposed to 21.25°C ambient temperature were investigated by proton resonance frequency thermometry. Temperature distributions were visualized and analysed with dedicated software allowing derivation of RBC mean, surface and core temperature-time courses during warm up. Time-dependence of mean temperature was assumed to fulfil a lumped capacitive model of heat transfer. Time courses of relative surface and core temperature changes to ambient temperature were similarly assumed to follow shifted exponential decays characterized by a time constant and a relative time shift, respectively. RESULTS: The lumped capacitive model of heat transfer and shifted exponential decays described time-dependence of mean, surface and core temperatures close to perfect (mean R(2) were 0.999±0.001, 0.996±0.004 and 0.998±0.002, respectively). Mean time constants were τmean = 55.3±3.7 min, τsurface = 41.4±2.9 min and τcore = 76.8±7.1 min, mean relative time shifts were Δsurface = 0.07±0.02 and Δcore = 0.04±0.01. None of the constants correlated significantly with temperature differences between ambient and storage temperature. CONCLUSION: Lumped capacitive model of heat transfer and shifted exponential decays represent simple analytical formulas to describe transient mean, surface and core temperatures of RBC during warm up, which might be a helpful tool in RBC temperature monitoring and quality control. Independence of constants on differences between ambient and storage temperature suggests validity of models for arbitrary storage and ambient temperatures.

Four-dimensional temperature distributions in red blood cells withdrawn from storage and exposed to ambient temperature: a magnetic resonance thermometry study.

BACKGROUND: Recommended by current guidelines, red blood cell (RBC) temperature should not exceed 10°C during transport. Since warming is a generically three-dimensional process that is not homogeneous, it is necessary to clarify the term "temperature during warming." The purpose of this study was therefore to investigate laws and relations between surface, mean, and core temperature and the corresponding times when they exceed 10°C during warm-up. STUDY DESIGN AND METHODS: Time-resolved three-dimensional temperature distributions of 53 resuspended RBC units (mean volume, 253 ± 17 mL) were measured noninvasively by magnetic resonance thermometry. Warm-up temperature maps were visualized and analyzed by dedicated software. RESULTS: Mean times when surface, mean, and core temperature exceeded 10°C were 16 ± 4, 24 ± 5, and 36 ± 7 minutes, respectively. Times strongly correlated with each other (r = 0.78-0.95) and their variances mainly depended on RBC storage temperature and RBC pouch width (R(2) = 0.81-0.89). Measured mean temperature time courses were well described by a lumped capacitive model of heat transfer with a sample width-dependent time constant τ(RBC) = 56.3 ± 3.5 minutes (mean R(2) = 0.996). CONCLUSION: Times when RBC surface, mean, and core temperature exceed 10°C can be estimated from each other. Moreover RBC mean temperature can be calculated for arbitrary storage and ambient temperatures. Findings might serve as a helpful tool in RBC temperature monitoring.

Adverse events and safety issues in blood donation--a comprehensive review.

Although blood donation is generally safe, a variety of risks and complications exist, the most common being iron deficiency, vasovagal reactions and citrate-related events. In the last decades, extensive efforts have significantly improved recipient and product safety, but there is still great potential to optimise donor care. Many therapies in modern medicine depend on the prompt availability of blood products, therefore it is crucial to maintain a motivated and healthy donor pool in view of a limited number of healthy volunteers willing and able to give blood or blood components. We present a comprehensive review on adverse events addressing all types of blood donation including whole blood, plasma, platelet, peripheral blood stem cell, leucocyte and bone marrow donation. In addition, we outline strategies for the prevention and treatment of these events and give a blueprint for future research in this field.

Impact of 13.56-MHz radiofrequency identification systems on the quality of stored red blood cells.

BACKGROUND: Radiofrequency identification (RFID) technology is emerging as one of the most pervasive computing technologies due to its broad applicability. Storage of red blood cells (RBCs) is a routine procedure worldwide. Depending on the additive solution, RBCs can be stored at 4 ± 2°C up to 49 days. To support the decision of discarding or further using a blood product, temperature measurement of each unit could be provided by RFID application. The safety evaluation of RFID devices was demonstrated in a regulatory agency required study. It has been concluded in limit tests that high frequency-based RFID technology performed safely for blood products; therefore, a longer exposure of radiofrequency (RF) energy on blood units was performed in this study to detect any biologic effects in RBC samples. STUDY DESIGN AND METHODS: Buffy coat-depleted, in line-filtered RBCs were used as standard products in all tests. Various variables like pH, potassium, glucose, lactate, hemoglobin (Hb), hematocrit, free Hb, and hemolysis rate were measured in a test group with RFID tags placed on their surface and continuously radiated with 13.56-MHz RFID reader radiation for 42 days while stored at 4 ± 2°C and compared to a control group by two-sample t test. RESULTS: In both groups glucose and pH levels decreased while lactate, free Hb, and potassium increased within the expected levels. The hemolysis rate showed increase after the 25th day but remained below the maximum acceptable threshold of 0.8%. CONCLUSION: It is feasible to implement RFID-enabled processes, without detecting any known biologic effects of longer exposure of RF energy on the quality of RBCs.

Pro-angiogenic induction of myeloid cells for therapeutic angiogenesis can induce mitogen-activated protein kinase p38-dependent foam cell formation.

BACKGROUND AIMS: Clinical trials for therapeutic angiogenesis use blood- or bone marrow-derived hematopoietic cells, endothelial progenitor cells (EPC) and mesenchymal stromal cells (MSC) for vascular regeneration. Recently concerns have emerged that all three cell types could also contribute to atherosclerosis by foam cell formation. Therefore, we asked whether human myelomonocytic cells, EPC or MSC can accumulate lipid droplets (LD) and develop into foam cells. METHODS: LD accumulation was quantified by flow cytometry, confocal microscopy and cholesterol measurement in each of the cell types. The impact of an initial pro-angiogenic induction on subsequent foam cell formation was studied to mimic relevant settings already used in clinical trials. The phosphorylation state of intracellular signaling molecules in response to the pro-angiogenic stimulation was determined to delineate the operative mechanisms and establish a basis for interventional strategies. RESULTS: Foam cells were formed by monocytes but not by EPC or MSC after pro-angiogenic induction. Mitogen-activated protein kinase (MAPK) p38 phosphorylation was enhanced and kinase inhibition almost abrogated intracellular LD accumulation in monocytes. CONCLUSIONS: These data suggest that hematopoietic cell preparations containing monocytes bear the risk of foam cell formation after pro-angiogenic induction. Instead, EPC and MSC may drive vascular regeneration without atherogenesis aggravation. A thorough understanding of cell biology is necessary to develop new strategies combining pro-angiogenic and anti-atherogenic effects during cell therapy.

Apheresis affects bone and mineral metabolism.

BACKGROUND: Apheresis is a procedure to selectively obtain blood components. For the collection process citrate is routinely used. It inhibits coagulation by binding to ionized calcium and leads to metabolic alkalosis. OBJECTIVE: Whether regular apheresis affects bone and mineral metabolism is unknown. The intention of this study was to investigate 1) the acute effects of apheresis on acid-base balance, bone and mineral metabolism and 2) to compare bone mineral density (BMD) at the lumbar spine and hip of donors to matched control subjects. DESIGN: In this open, observational, single-center, cross-sectional study we enrolled 102 regular plasma and thrombocyte donors to pursue objective 1) and compared those to 102 matched controls (CTR) for objective 2). RESULTS: Platelet donation led to significant decreases in serum ionized calcium (-17%) and phosphate (-18%), to marked increases in base excess (57%) and PTH levels (192%) during apheresis. Baseline biochemical comparisons between donors and CTR revealed significantly lower values for donors for serum calcium, albumin, and 25-hydroxyvitamin D levels. Mean Z-score at the lumbar spine adjusted for BMI, average physical activity and daily calcium intake was lower for donors (-0.28+/-0.11) when compared to CTR subjects (0.06+/-0.11, P<0.05). Total and neck femoral BMD was also lower in the donor group, however, this difference was not significant. CONCLUSIONS: Exposure to citrate during the apheresis procedure acutely affects mineral and bone metabolism. Regular donations of blood components compromised BMD at the lumbar spine. If confirmed, strategies to prevent long-term effects on bone need to be formulated.

Identification of 14 new alleles at the fucosyltransferase 1, 2, and 3 loci in Styrian blood donors, Austria.

BACKGROUND: Genes for fucosyltransferases 1 (FUT1:H), 2 (FUT2:Secretor), and 3 (FUT3:Lewis) encode enzymes crucial for ABH and Lewis blood group antigen synthesis. They are highly polymorphic and ethnically and geographically specific. STUDY DESIGN AND METHODS: Genetic variations and allele frequencies of FUT1, FUT2, and FUT3 encoding regions and flanking sequences were analyzed in 100 Styrian blood donors by systematic sequencing. Haplotypes were verified with sequence-specific primers. To identify discrepancies, serologically determined ABO and Lewis blood groups were correlated to respective genotypes. RESULTS: Two novel FUT1 alleles were defined by 9C>T (silent) and 991C>A (P331T) mutations, the latter located in the catalytic domain of the enzyme. Five new alleles of FUT2 were found: three were characterized by new variants and two resulted from new combinations of known polymorphisms. The new 412G>A (G138S) mutation also is located in the catalytic domain. A new nonsecretor allele, based on the presence of 428G>A (nonsense), was found. Another FUT2 allele may have resulted from an intragenic crossover event. FUT3 analysis revealed seven novel alleles, partly based on the new mutations 41G>A (R14H), 1060C>G (R354G), 735G>C (silent), and 882C>T (silent). While 41G>A is placed in the cytoplasmic domain and functional, 1060C>G is placed in the catalytic domain. CONCLUSION: Multiple common and sporadic sequence variations including 14 new alleles at FUT1, FUT2, and FUT3 loci were identified. Four novel mutations result in amino acid substitution in the protein. Three of them are predicted to have adverse effects on the enzyme activity. A novel nonsecretor allele was found.

Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.

Endothelial progenitor cells are critically involved in essential biologic processes, such as vascular homeostasis, regeneration, and tumor angiogenesis. Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells with robust proliferative potential. Their profound vessel-forming capacity makes them a promising tool for innovative experimental, diagnostic, and therapeutic strategies. Efficient and safe methods for their isolation and expansion are presently lacking. Based on the previously established efficacy of animal serum-free large-scale clinical-grade propagation of mesenchymal stromal cells, we hypothesized that endothelial lineage cells may also be propagated efficiently following a comparable strategy. Here we demonstrate that human ECFCs can be recovered directly from unmanipulated whole blood. A novel large-scale animal protein-free humanized expansion strategy preserves the progenitor hierarchy with sustained proliferation potential of more than 30 population doublings. By applying large-scale propagated ECFCs in various test systems, we observed vascular networks in vitro and perfused vessels in vivo. After large-scale expansion and cryopreservation phenotype, function, proliferation, and genomic stability were maintained. For the first time, proliferative, functional, and storable ECFCs propagated under humanized conditions can be explored in terms of their therapeutic applicability and risk profile.

Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum.

Adult mesenchymal stem cells (MSCs) are considered as valuable mediators for tissue regeneration and cellular therapy. This study was performed to develop conditions for regularly propagating a clinical quantity of > 2 x 10(8) MSCs without animal serum from small bone marrow (BM) aspiration volumes within short time. We established optimized culture conditions with pooled human platelet lysate (pHPL) replacing fetal bovine serum (FBS) for MSC propagation. MSC quality, identity, purity, and function were assessed accordingly. Biologic safety was determined by bacterial/fungal/mycoplasma/endotoxin testing and genomic stability by array comparative genomic hybridization (CGH). We demonstrate that unmanipulated BM can be used to efficiently initiate MSC cultures without the need for cell separation. Just diluting 1.5-5 mL heparinized BM per 500 mL minimum essential medium supplemented with L-glutamine, heparin, and 10% pHPL sufficiently supported the safe propagation of 7.8 +/- 1.5 x 10(8) MSCs within a single 11- to 16-day primary culture under defined conditions. This procedure also resulted in sustained MSC colony recovery. MSC purity, immune phenotype, and in vitro differentiation potential fully matched current criteria. Despite high proliferation rate, MSCs showed genomic stability in array CGH. This easy single-phase culture procedure can build the basis for standardized manufacturing of MSC-based therapeutics under animal serum-free conditions for dose-escalated cellular therapy and tissue engineering.

Thrombin generation before and after multicomponent blood collection.

BACKGROUND: Apheresis technology has made tremendous progress up to the development of automated blood component collection, which offers increased efficiency in donor blood use, but the concern about the contact of donor blood with artificial surfaces remains. Activation of the hemostatic system is a major issue in this context and is controversial. The aim of this study was to estimate the effect of apheresis on continuous thrombin generation (TG), representing a new tool to examine the overall function of the plasmatic clotting system. STUDY DESIGN AND METHODS: Twenty-six voluntary blood donors, fulfilling the law requirement for apheresis donation, participated in the study. Two units of platelets (6 x 10(11)) and 1 unit of red cells (250 mL; hematocrit level, 80%) were collected using two types of cell separators (Amicus, Fenwal, Inc.; and Trima Accel, Gambro BCT). Each donor underwent collection on both apheresis systems with at least 8 weeks in between. Samples of blood were collected before, immediately after, and 48 hours after apheresis. TG was measured using a slow fluorogenic substrate by means of calibrated automated thrombography (CAT). RESULTS: CAT data changed only slightly, and no significant changes were seen before, immediately after, and 48 hours after apheresis (p > 0.05). The variables did not differ significantly between the two different apheresis systems (p > 0.05). CONCLUSION: Using a CAT-based technique, no change in variables of continuous TG were observed, suggesting that multicomponent blood collection did not lead to severe alterations in the hemostatic system of the donors.

Blood donor screening for parvovirus B19 in Germany and Austria.

BACKGROUND: Although the main transmission pathway of parvovirus B19 (B19) is typically via the respiratory route, several transfusion-transmitted infections have been reported. To increase blood safety, all blood donations to our blood donor service have been screened by a B19 minipool real-time nucleic acid testing (NAT) since April 2000. Additional customers have been screened since the summer of 2003. STUDY DESIGN AND METHODS: In total, 2.8 million donations from Germany and Austria were screened for B19 by real-time minipool NAT. A subgroup of 50 B19 DNA-positive donors was screened for B19 immunoglobulin G (IgG) and IgM antibodies and B19 DNA over a 6-month period. Results were compared to those of 100 B19 DNA-negative donors. RESULTS: Data accumulated over the past 6 years indicate a high incidence period from May 2004 to January 2006. In total, the incidence was 12.7 and 261.5 per 100,000 donations with high virus loads equal to or above 10(5) and below 10(5) IU per mL, respectively. Median virus concentration in the case group was 4.85 x 10(7) IU per mL at Time Point T0 and was reduced to 4 x 10(2) IU per mL at the time of the next donation (3 months later). Neutralizing antibodies (VP2) were detected in all donations if virus load was reduced to less than 10(5) IU per mL. CONCLUSION: The release of B19 DNA-positive blood products with a concentration of less than 105 IU per mL is thought to be safe due to the high level of neutralizing VP2 antibodies and is currently examined in a donor recipient infectivity study. In contrast, blood products with a high B19 DNA concentration (> or =10(5) IU/mL), some of which did not contain neutralizing antibodies, were discarded to protect at risk individuals.

Two steps to functional mesenchymal stromal cells for clinical application.

BACKGROUND: Ex vivo expansion of multipotent mesenchymal stromal cells (MSCs) is a prerequisite for evaluating their therapeutic potential in ongoing clinical trials. Even large volumes of starting material and extended culture periods, however, do not necessarily produce 2 x 10(6) MSCs per kg per adult patient. A new two-step procedure has been devised to propagate more than 1 x 10(8) MSCs from small marrow volumes within fewer than 4 weeks. STUDY DESIGN AND METHODS: The influence of log fold decreased MSC seeding (2500, 250, 25, 2.5/cm(2)) on clinical-scale expansion, MSC phenotype, and immunomodulatory function combined with multiplex cytokine display was analyzed. Maintenance of MSC characteristics was tested in fibroblast colony-forming unit and differentiation assays. RESULTS: Reduced seeding density boosted MSC propagation. Low-density expanded MSCs were CD29+, CD73+, CD90+, CD105+, CD14-, CD34-, CD45-, HLA-DR-; retained their differentiation potential; and inhibited lymphocyte proliferation. This was accompanied by deregulated cytokine production. Seeding 0.7 x 10(6) to 1 x 10(6) MSCs derived from a 10- to 13-day primary culture at a low density of 28 to 40 per cm(2) permitted propagation of 1.5 x 10(8) to 3.7 x 10(8) functional MSCs within a 13- to 15-day secondary expansion step. CONCLUSION: Primary seeding of only 10-mL marrow aspirates on approximately 0.2-m(2) culture area (Step 1) followed by expansion on 2.5 m(2) (Step 2) is sufficient to consistently generate at least 1.5 x 10(8) MSCs in fetal bovine serum-supplemented medium within less than 4 weeks. The efficiency of this two-step procedure for clinical-scale MSC propagation may facilitate rational clinical testing of MSC-based therapies.

Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells.

BACKGROUND: Human multipotent mesenchymal stromal cells (MSCs) are promising candidates for a growing spectrum of regenerative and immunomodulatory cellular therapies. Translation of auspicious experimental results into clinical applications has been limited by the dependence of MSC propagation from fetal bovine serum (FBS). STUDY DESIGN AND METHODS: The capacity of human platelet lysate (HPL) to replace FBS for clinical-scale MSC propagation was analyzed. RESULTS: HPL could be efficiently produced from buffy coats. Multiplex analyses allowed a distinct HPL growth factor profile to be delineated. With a previously established two-step clinical-scale procedure, HPL was reproducibly more efficient than FBS in supporting MSC outgrowth. With only 3 x 10(5) primary culture-derived MSCs, a mean of 4.36 x 10(8) HPL-MSCs (range, 3.01 x 10(8)-5.40 x 10(8)) was obtained within a single secondary 11- to 13-day culture step. Although morphologically distinct, HPL-MSCs and FBS-MSCs did not differ significantly in terms of immunophenotype, differentiation potential in vitro, and lack of tumorigenicity in nude mice in vivo. CONCLUSIONS: Replacing FBS with HPL prevents bovine prion, viral, and zoonose contamination of the stem cell product. This new efficient FBS-free two-step procedure for clinical-scale MSC propagation may represent a major step toward challenging new stem cell therapies.

Humanized system to propagate cord blood-derived multipotent mesenchymal stromal cells for clinical application.

BACKGROUND: Umbilical cord blood (UCB) is an easily accessible alternative source for multipotent mesenchymal stromal cells (MSCs) and is generally believed to provide MSCs with a higher proliferative potential compared with adult bone marrow. Limitations in cell number and strict dependence of expansion procedures from selected lots of fetal bovine serum have hampered the progress of clinical applications with UCB-derived MSCs. METHODS: We analyzed the isolation and proliferative potential of human UCB MSCs compared with bone marrow MSCs under optimized ex vivo culture conditions. We further investigated human platelet lysate as an alternative to replace fetal bovine serum for clinical-scale MSC expansion. Clonogenicity was determined in colony-forming units-fibroblast assays. MSC functions were tested in hematopoiesis support, vascular-like network formation and immune modulation potency assays. RESULTS: MSCs could be propagated from UCB with and without fetal bovine serum. MSC propagation was effective in 46% of UCB samples. Once established, the proliferation kinetics of UCB MSCs did not differ significantly from that of bone marrow MSCs under optimized culture conditions, resulting in more than 50 population doublings after 15 weeks. A clinical quantity of 100 million MSCs with retained differentiation potential could be obtained from UCB MSCs within approximately 7 weeks. Ex vivo expansion of hematopoietic UCB-derived CD34+ cells as well as immune inhibition and vascular-like network formation could be shown for UCB MSCs propagated under both culture conditions. CONCLUSION: We demonstrate for the first time that human MSCs can be obtained and propagated to a clinical quantity from UCB in a completely bovine serum-free system. Surprisingly, our data argue against a generally superior proliferative potential of UCB MSCs. Functional data indicate the applicability of clinical-grade UCB MSCs propagated with human platelet lysate-conditioned medium for hematopoiesis support, immune regulation and vascular regeneration.

Immune cells mimic the morphology of endothelial progenitor colonies in vitro.

Endothelial progenitor cells (EPC) are considered powerful biologic markers for vascular function and cardiovascular risk, predicting events and death from cardiovascular causes. Colony-forming units of endothelial progenitor cells (CFU-EC) are used to quantify EPC circulating in human peripheral blood. The mechanisms underlying colony formation and the nature of the contributing cells are not clear. We performed subtractive CFU-EC analyses to determine the impact of various blood cell types and kinetics of protein and gene expression during colony formation. We found that CFU-EC mainly comprise T cells and monocytes admixed with B cells and natural killer cells. The combination of purified T cells and monocytes formed CFU-EC structures. The lack of colonies after depletion or functional ablation of T cells or monocytes was contrasted with effective CFU-EC formation in the absence of CD34+ cells. Microarray analyses revealed activation of immune function-related biological processes without changes in angiogenesis-related processes during colony formation. In concordance with a regenerative function, soluble factors derived from CFU-EC cultures supported vascular network formation in vitro. Recognizing CFU-EC formation as the result of a functional cross between T cells and monocytes shifts expectations of vascular regenerative medicine. Our data support the move from a view of circulating EPC toward models that include a role for immune cells in vascular regeneration. Disclosure of potential conflicts of interest is found at the end of this article.

Immune reconstitution after purified autologous and allogeneic blood stem cell transplantation compared with unmanipulated bone marrow transplantation in children.

Immune reconstitution is critical for the long-term success of haematopoietic stem cell transplantation (HSCT). We prospectively analysed immune reconstitution parameters after transplantation of autologous (group 1; n = 10) and allogeneic (group 2; n = 12) highly purified CD34+ peripheral blood stem cells (PBSC) and unmanipulated allogeneic bone marrow (BM) (group 3; n = 9) in children. Median follow-up after HSCT was 56 (group 1), 61 (group 2), and 40.5 months (group 3). Median CD34-cell dose transplanted in the three groups was 9.4 x 10(6)/kg, 20.3 x 10(6)/kg, and 4.25 x 10(6)/kg recipient's body weight (BW) respectively. Complete haematopoietic engraftment was seen in all patients without any significant differences between the three groups. T-cell reconstitution at 6 months was significantly delayed in autologous peripheral blood stem cell transplantation (PBSCT) compared with allogeneic BM transplantation (P < 0.028) and allogeneic PBSCT (P < 0.034). At 3 months after transplantation numbers of CD56+/3- natural killer cells were higher in the allogeneic PBSC group (P < 0.01) compared with the BM group. The numbers of proven bacterial and viral infections were equally distributed between the three groups. In conclusion, recipients of allogeneic highly purified CD34+ PBSC or unmanipulated BM have higher lymphocyte subset counts at 6 months after transplantation than recipients of autologous CD34-selected PBSC. Infection rates and outcome, however, were not significantly different.