INTERCEPT Pathogen Reduction in Platelet Concentrates, in Contrast to Gamma Irradiation, Induces the Formation of trans-Arachidonic Acids and Affects Eicosanoid Release during Storage.

Pathogen inactivation techniques for blood products have been implemented to optimize clinically safe blood components supply. The INTERCEPT system uses amotosalen together with ultraviolet light wavelength A (UVA) irradiation. Irradiation-induced inactivation of nucleic acids may actually be accompanied by modifications of chemically reactive polyunsaturated fatty acids known to be important mediators of platelet functions. Thus, here, we investigated eicosanoids and the related fatty acids released upon treatment and during storage of platelet concentrates for 7 days, complemented by the analysis of functional and metabolic consequences of these treatments. Metabolic and functional issues like glucose consumption, lactate formation, platelet aggregation, and clot firmness hardly differed between the two treatment groups. In contrast to gamma irradiation, here, we demonstrated that INTERCEPT treatment immediately caused new formation of trans-arachidonic acid isoforms, while 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-HETE were increased and two hydroperoxyoctadecadienoic acid (HpODE) isoforms decreased. During further storage, these alterations remained stable, while the release of 12-lipoxygenase (12-LOX) products such as 12-HETE and 12-hydroxyeicosapentaenoic acid (12-HEPE) was further attenuated. In vitro synthesis of trans-arachidonic acid isoforms suggested that thiol radicals formed by UVA treatment may be responsible for the INTERCEPT-specific effects observed in platelet concentrates. It is reasonable to assume that UVA-induced molecules may have specific biological effects which need to be further investigated.

De novo Vessel Formation Through Cross-Talk of Blood-Derived Cells and Mesenchymal Stromal Cells in the Absence of Pre-existing Vascular Structures.

BACKGROUND: The generation of functional blood vessels remains a key challenge for regenerative medicine. Optimized in vitro culture set-ups mimicking the in vivo perivascular niche environment during tissue repair may provide information about the biological function and contribution of progenitor cells to postnatal vasculogenesis, thereby enhancing their therapeutic potential. AIM: We established a fibrin-based xeno-free human 3D in vitro vascular niche model to study the interaction of mesenchymal stromal cells (MSC) with peripheral blood mononuclear cells (PBMC) including circulating progenitor cells in the absence of endothelial cells (EC), and to investigate the contribution of this cross-talk to neo-vessel formation. MATERIALS AND METHODS: Bone marrow-derived MSC were co-cultured with whole PBMC, enriched monocytes (Mo), enriched T cells, and Mo together with T cells, respectively, obtained from leukocyte reduction chambers generated during the process of single-donor platelet apheresis. Cells were embedded in 3D fibrin matrices, using exclusively human-derived culture components without external growth factors. Cytokine secretion was analyzed in supernatants of 3D cultures by cytokine array, vascular endothelial growth factor (VEGF) secretion was quantified by ELISA. Cellular and structural re-arrangements were characterized by immunofluorescence and confocal laser-scanning microscopy of topographically intact 3D fibrin gels. RESULTS: 3D co-cultures of MSC with PBMC, and enriched Mo together with enriched T cells, respectively, generated, within 2 weeks, complex CD31+/CD34+ vascular structures, surrounded by basement membrane collagen type-IV+ cells and matrix, in association with increased VEGF secretion. PBMC contained CD31+CD34+CD45dimCD14- progenitor-type cells, and EC of neo-vessels were PBMC-derived. Vascular structures showed intraluminal CD45+ cells that underwent apoptosis thereby creating a lumen. Cross-talk of MSC with enriched Mo provided a pro-angiogenic paracrine environment. MSC co-cultured with enriched T cells formed "cell-in-cell" structures generated through internalization of T cells by CD31+CD45 dim⁣/ - cells. No vascular structures were detected in co-cultures of MSC with either Mo or T cells. CONCLUSION: Our xeno-free 3D in vitro vascular niche model demonstrates that a complex synergistic network of cellular, extracellular and paracrine cross-talk can contribute to de novo vascular development through self-organization via co-operation of immune cells with blood-derived progenitor cells and MSC, and thereby may open a new perspective for advanced vascular tissue engineering in regenerative medicine.

The assessment of platelet function by thromboelastometry as a point-of-care test to guide Intercept-treated platelet support in hemato-oncological patients and hematopoietic stem cell transplantation recipients.

BACKGROUND: Pathogen inactivation (PI) techniques for platelet concentrates (PCs) are one of the latest innovations to improve blood safety and reduce the risk of transfusion-transmitted infections (TTIs). An impaired function and in vivo recovery of platelets as well as an increased PC demand are concerns regarding these techniques. The intent of this study was to evaluate the hemostatic effect of PCs treated with the Intercept™ System by thromboelastometry (TEM) and to assess the clinical validity of its results in comparison to post-transfusion increase (PTI) and corrected count increment (CCI). STUDY-DESIGN AND METHODS: This prospective-observational study included 47 patients (m:f = 25:22; median age: 54 years [21-70]) of our Bone Marrow Transplantation unit with hemato-oncological malignancies transfused with Intercept™-treated PCs. Serial TEM measurements were performed just before and 1 hour after PC transfusion and were analyzed for their correlation with PTI and CCI as well as for clinical variables. RESULTS: The majority of our patients had received a hematopoietic stem cell transplantation (HSCT) (n = 41; 87%). In median 9 (1-50) PCs were transfused. Serial TEM, PTI, and CCI measurements were available for 150 transfusion episodes. The median platelet dose transfused was 2.65 × 1011 /unit (1.8-6). The median CCI was 9.250 (0-28.000). We observed a significant improvement in TEM parameters (p < 0.05) after transfusion of PI PCs, which did not mandatory correlate with the 1-hour PTI and CCI. CONCLUSION: Serial TEM measurements indicate the hemostatic effect of Intercept™-treated PCs. The 1-hour PTI and CCI may not appropriately reflect the in vivo function of platelets after PI PC transfusion.

Transfusion of standard-issue packed red blood cells induces pulmonary vasoconstriction in critically ill patients after cardiac surgery-A randomized, double-blinded, clinical trial.

BACKGROUND: Experimental and volunteer studies have reported pulmonary vasoconstriction during transfusion of packed red blood cells (PRBCs) stored for prolonged periods. The primary aim of this study was to evaluate whether transfusion of PRBCs stored over 21 days (standard-issue, siPRBCs) increases pulmonary artery pressure (PAP) to a greater extent than transfusion of PRBCs stored for less then 14 days (fresh, fPRBCs) in critically ill patients following cardiac surgery. The key secondary aim was to assess whether the pulmonary vascular resistance index (PVRI) increases after transfusion of siPRBCs to a greater extent than after transfusion of fPRBCs. METHODS: The study was performed as a single-center, double-blinded, parallel-group, randomized clinical trial. Leukoreduced PRBCs were transfused while continuously measuring hemodynamic parameters. Systemic concentrations of syndecan-1 were measured to assess glycocalyx injury. After randomizing 19 patients between January 2014 and June 2016, the study was stopped due to protracted patient recruitment. RESULTS: Of 19 randomized patients, 11 patients were transfused and included in statistical analyses. Eight patients were excluded prior to transfusion, 6 patients received fPRBCs (10±3 storage days), whereas 5 patients received siPRBCs (33±4 storage days). The increase in PAP (7±3 vs. 2±2 mmHg, P = 0.012) was greater during transfusion of siPRBCs than during transfusion of fPRBCs. In addition, the change in PVRI (150±89 vs. -4±37 dyn·s·cm-5·m2, P = 0.018) was greater after transfusion of siPRBCs than after transfusion of fPRBCs. The increase in PAP correlated with the change of systemic syndecan-1 concentrations at the end of transfusion (R = 0.64,P = 0.034). CONCLUSION: Although this study is underpowered and results require verification in larger clinical trials, our findings suggest that transfusion of siPRBCs increases PAP and PVRI to a greater extent than transfusion of fPRBCs in critically ill patients following cardiac surgery. Glycocalyx injury might contribute to pulmonary vasoconstriction associated with transfusion of stored blood.

Plerixafor as preemptive strategy results in high success rates in autologous stem cell mobilization failure.

Plerixafor in combination with granulocyte-colony stimulating factor (G-CSF) is approved for autologous stem cell mobilization in poor mobilizing patients with multiple myeloma or malignant lymphoma. The purpose of this study was to evaluate efficacy and safety of plerixafor in an immediate rescue approach, administrated subsequently to G-CSF alone or chemotherapy and G-CSF in patients at risk for mobilization failure. Eighty-five patients mobilized with G-CSF alone or chemotherapy were included. Primary endpoint was the efficacy of the immediate rescue approach of plerixafor to achieve ≥2.0 × 106 CD34+ cells/kg for a single or ≥5 × 106 CD34+ cells/kg for a double transplantation and potential differences between G-CSF and chemotherapy-based mobilization. Secondary objectives included comparison of stem cell graft composition including CD34+ cell and lymphocyte subsets with regard to the mobilization regimen applied. No significant adverse events were recorded. A median 3.9-fold increase in CD34+ cells following plerixafor was observed, resulting in 97% patients achieving at least ≥2 × 106 CD34+ cells/kg. Significantly more differentiated granulocyte and monocyte forming myeloid progenitors were collected after chemomobilization whereas more CD19+ and natural killer cells were collected after G-CSF. Fifty-two patients underwent transplantation showing rapid and durable engraftment, irrespectively of the stem cell mobilization regimen used. The addition of plerixafor in an immediate rescue model is efficient and safe after both, G-CSF and chemomobilization and results in extremely high success rates. Whether the differences in graft composition have a clinical impact on engraftment kinetics, immunologic recovery, and graft durability have to be analysed in larger prospective studies.

Pharmacological- and non-pharmacological therapeutic approaches in inflammatory bowel disease in adults.

Inflammatory bowel diseases (IBDs) are a group of chronic inflammatory conditions mainly of the colon and small intestine. Crohn's disease (CD) and ulcerative colitis (UC) are the most frequent types of IBD. IBD is a complex disease which arises as a result of the interaction of environmental, genetic and immunological factors. It is increasingly thought that alterations of immunological reactions of the patients to their own enterable bacteria (microfilm) may contribute to inflammation. It is characterized by mucosal and sub mucosal inflammation, perpetuated by infiltration of activated leukocytes. CD may affect the whole gastrointestinal tract while UC only attacks the large intestine. The therapeutic goal is to achieve a steroid-free long lasting remission in both entities. UC has the possibility to be cured by a total colectomy, while CD never can be cured by any operation. A lifelong intake of drugs is mostly necessary and essential. Medical treatment of IBD has to be individualized to each patient and usually starts with anti-inflammatory drugs. The choice what kind of drugs and what route administered (oral, rectal, intravenous) depends on factors including the type, the localization, and severity of the patient's disease. IBD may require immune-suppression to control symptoms such as prednisolone, thiopurines, calcineurin or sometimes folic acid inhibitors or biologics like TNF-α inhibitors or anti-integrin antibodies. For both types of disease (CD, UC) the same drugs are available but they differ in their preference in efficacy between CD and UC as 5-aminosalicylic acid for UC or budesonide for ileocecal CD. As therapeutic alternative the main mediators of the disease, namely the activated pro-inflammatory cytokine producing leukocytes can be selectively removed via two apheresis systems (Adacolumn and Cellsorba) in steroid-refractory or dependent cases. Extracorporeal photopheresis results in an increase of regulatory B cells, regulatory CD8(+) T cells and T-regs Type 1. Both types of apheresis were able to induce clinical remission and mucosal healing accompanied by tapering of steroids.

Granulocyte collection using a novel apheresis system eases the procedure and provides concentrates of high quality.

BACKGROUND: The latest technical innovation for granulocyte (PMN) collections is the fully automated Spectra Optia (Optia) device (TerumoBCT). In a retrospective investigation we evaluated the impact of the technical automation on the product quality in a routine working field. STUDY DESIGN AND METHODS: A total of 71 granulocyte collections (GCs) were collected from either routine random blood donors, mobilized with prednisolone (P; two females/23 males; median age, 42 years; range, 25-63 years), or family donors (three females/12 males; median age, 29 years; range, 21-59 years) who were mobilized with recombinant human granulocyte-colony-stimulating factor (rHuG-CSF) at a dose of 5 µg/kg body weight. All collections were performed with the Optia device. Fifty-nine concentrates (GTX) produced with the Cobe Spectra (Cobe; TerumoBCT) served as a historical control. RESULTS: In total a mean of 452 ± 60 mL with a mean purity of 83 ± 9.6% PMNs was collected with the Optia. Compared with the Cobe collections (298 ± 52 mL), the product volumes in general as well as the absolute PMN yield in P-mobilized products were significantly higher with the Optia: PMN count, 1.9 × 10(10) ± 0.49 × 10(10) versus 1.5 × 10(10) ± 0.85 × 10(10) , respectively (p < 0.05), due to higher white blood cell (WBC) yields. rHuG-CSF-mobilized products showed no significant differences in the absolute WBC (7.2 ± 3.0/Optia vs. 7.0 ± 2.1/Cobe) and PMN (5.9 ± 2.6/Optia vs. 5.7 ± 1.9/Cobe) yield. The PMN purity was equal in both devices (mean, 83%) although it was slightly lower in the rHuG-CSF group (82 ± 9.2%) than in the P group (84 ± 6.2%). In none of the procedures were side effects recorded. CONCLUSION: GC with the fully automated Optia device is safe for donors, is not inferior to its forerunner Cobe Spectra, produces GTX of a high quality, and requires less manpower than the Cobe Spectra.

Administration of recombinant human granulocyte-colony-stimulating factor does not induce long-lasting detectable epigenetic alterations in healthy donors.

BACKGROUND: The short-term safety profile of recombinant human granulocyte-colony-stimulating factor (rHuG-CSF) in the allogeneic stem cell setting seems acceptable; only few data on long-term safety are available. To further study possible epigenetic alterations, we investigated prospectively the influence of rHuG-CSF on DNA methyltransferase (DNMT) activity and on changes in DNA methylation of candidate genes in peripheral blood cells of healthy unrelated stem cell donors within an observation period of 1 year. STUDY DESIGN AND METHODS: In this study, 20 stem cell donors (14 male/six female; median age, 40 years; range, 22-54 years) and 20 sex- and age-matched blood component donors (controls) were included. Sampling was performed before rHuG-CSF administration; at the time of donation; and on Days (+1), 7, 30, 100, 180, and 360 in both groups. Analysis of DNMT activity in nuclear extracts was performed using a modified radionuclide assay. We performed methylation-specific polymerase chain reaction to detect the methylation status of promoter CpG islands of the genes of the retinoic acid receptor beta (RAR-B) and the Ras association domain family 1A (RASSF1A). RESULTS: DNMT activity increased significantly on the day of donation and 1 day after (p < 0.05). By Day +7 baseline values were reached. No further significant alterations of DNMT activity in the treated group compared to the controls were observed. We could not detect any differences in the gene methylation of RAR-B and RASSF1A between both groups. CONCLUSION: In our prospective study no evidence of long-lasting increased DNMT activity or enhanced DNA methylation in a limited panel of target genes after recombinant human G-CSF administration was observed in healthy stem cell donors.

Automated red blood cell exchange as an adjunctive treatment for severe Plasmodium falciparum malaria at the Vienna General Hospital in Austria: a retrospective cohort study.

BACKGROUND: Severe falciparum malaria is associated with considerable rates of mortality, despite the administration of appropriate anti-malarial treatment. Since overall survival is associated with total parasite biomass, blood exchange transfusion has been proposed as a potential method to rapidly reduce peripheral parasitaemia. However, current evidence suggests that this treatment modality may not improve outcome. Automated red blood cell exchange (also referred to as "erythrocytapheresis") has been advocated as an alternative method to rapidly remove parasites from circulating blood without affecting patients' volume and electrolyte status. However, only limited evidence from case reports and case series is available for this adjunctive treatment. This retrospective cohort study describes the use of automated red blood cell exchange for the treatment of severe malaria at the Medical University of Vienna. METHODS: Epidemiologic data for imported malaria cases in Austria are reported and data of patients treated for malaria at the General Hospital/Medical University of Vienna were extracted from electronic hospital records. RESULTS: Between 2000 and 2010, 146 patients were hospitalized at the Medical University of Vienna due to malaria and 16 of those were classified as severe malaria cases. Eleven patients of this cohort were potentially eligible for an adjunctive treatment with automated red blood cell exchange. Five patients eventually underwent this procedure within a period of seven hours (range: 3-19 hours) after hospital admission. Six patients did not undergo this adjunctive treatment following the decision of the treating physician. The procedure was well tolerated in all cases and rapid reduction in parasite counts was achieved without occurrence of haemodynamic complications. One patient died within seven days, whereas four patients survived without any sequelae. DISCUSSION AND CONCLUSION: Automated red blood cell exchange was a safe and efficient procedure to rapidly clear peripheral parasitaemia. Whether the fast reduction in parasite biomass may ultimately improve patient survival remains however unclear. Randomized controlled trials are needed to conclusively appreciate the value of this adjunctive treatment.

Effects of endothelin-1 and phenylephrine on plasma levels of von Willebrand factor and protein S.

Endothelial dysfunction is considered a major factor in the pathogenesis of a wide array of diseases and often leads to increased production of, or increased responsiveness to endogenous vasoconstrictive mediators, such as endothelin-1 (ET-1) or adrenergic agonists. Based on previous studies in animals and in-vitro, we hypothesized that ET-1 and alpha adrenergic stimulation by phenylephrine (PE) may alter plasma levels of von Willebrand factor-Ag (vWF) and protein S in humans. Ten healthy men were studied in a prospective randomized double blind trial: we investigated the effects of infusions of ET-1 and PE on plasma levels of vWF-Ag and protein S. Aditionally, we examined the effects of these vasoconstrictors on thrombomodulin, tissue plasminogen activator and on protein C. ET-1 increased plasma levels of vWF-Ag by 19% (CI: 9-36%; p=0.008) and increased plasma levels of protein-C by 7% (CI: 0.2 -12 %; p=0.028), even at doses which produced no increase in blood pressure. Plasma levels of protein S and TPA were not significantly affected by ET-1. After PE-infusion we observed a relative increase in plasma levels of protein S by 20% (CI: 5-28%; p=0.036) and of TPA by 14 % (CI: 2-39%; p=0.036). Also, platelet counts increased by 36% (CI: 12-53%; p=0.028) which correlated (r(2)=0.86; p=0.014) with an increase in leukocytes by 49 % (CI: 18-84%; p=0.028). Plasma levels of vWF and protein C were not affected by PE and neither drug had a significant effect on plasma levels of thrombomodulin. In conclusion, our results support the study hypothesis, that ET-1 and PE increase the plasma levels of vWF and protein S, respectively. ET-1 may induce a procoagulant status in various disease states by increasing vWF-Ag levels.

Surface disinfection of packed red blood cells with 70% ethanol.

No data or recommendations are available on feasibility of surface disinfection of blood bags, but some circumstances can make such procedures inevitable. Impact of immersion of blood bags in 70% ethanol for 30min was investigated with respect to alcohol penetration and changes of hemolysis parameters in the product, and bag material changes influencing material stability and composition. After immersion ethanol concentration in blood bags was below detection limit. Hemolysis parameters did not differ between blood products that had been exposed to ethanol and a control group. Inner surface of the bag material was unchanged according to our infrared spectrometry results. Also endurance testing showed no altered results. We conclude that immersion of blood bags in 70% ethanol for surface disinfection is a safe procedure for the quality of the blood product and the bag material.

Collection and storage of leukocyte depleted whole blood in autologous blood predeposit in elective surgery programs.

INTRODUCTION: The aim of this investigation was to provide evidence that leukocyte depleted whole blood meets the requirements for transfusion of the European Council and thus may be an alternative to leukocyte and plasma depleted packed red blood cells in autologous blood predeposit for patients undergoing elective surgery programs. MATERIAL AND METHODS: Standard units of 450mL blood were collected from 25 healthy male volunteers. Leukocyte depletion was done via inline filtration 4h after collection. Storage lesion was assessed by measuring the release of K(+), LDH, free hemoglobin, and lactate into the storage medium, as well as by the increase of hemolysis, the decrease of pH and consumption of glucose over a storage period of 35 days. As surrogate marker for red cell quality the intracellular concentrations of adenine nucleotides [ATP, ADP, AMP] were determined. RESULTS: The extent of storage lesion remained within the ranges of standard liquid storage conditions. Hemolysis was far below the threshold of 0.8% in all WB units at the end of their shelf life. Only minor changes of intracellular adenine nucleotide levels were measured indicating a preserved function of red blood cells in leukocyte depleted whole blood. At the end of shelf life 70%+/-18% of initial ATP levels were detected. CONCLUSION: Based on our data we propose that leukocyte depleted whole blood, stored for 35 days can be an option in the autologous blood supply as it meets the requirements for transfusion of the European Council.

The influence of human platelet antigen match on the success of allogeneic peripheral blood progenitor cell transplantation following a reduced-intensity conditioning regimen.

BACKGROUND: Allogeneic transplantation in elderly patients requires a dose-reduced conditioning regimen. Owing to reduced-intensity conditioning, host- and donor-type immune responses may affect the early posttransplant period, whereas only later on donor-derived reactions may ensue. Mismatches in the HLA system are known to be detrimental for the outcome of transplantation. Mismatches between donor and recipient for human platelet antigens (HPAs) may also affect the success of transplantation owing to serving as minor histocompatibility antigens and therefore rendering recipients at risk for graft-versus-host disease (GVHD) or graft rejection and inhibition of thrombopoiesis attributed to platelet (PLT) antibodies. PATIENTS AND METHODS: Therefore, the occurrence of GVHD, incidence of relapse, need of PLT support, and outcome by analysis of 45 donor-recipient pairs for HPA-1, -2, -3, and -5 allotypes and screening for PLT antibodies were evaluated before transplantation and again 1 year thereafter. RESULTS: Mismatches within the HPA system were not associated with an increased occurrence of transplant-related mortality or GVHD, the onset of thrombopoiesis, the frequency of PLT transfusions, or the incidence of relapse. Neither were settings of homozygous donors versus heterozygous recipients (graft-vs.-host direction) nor homozygous recipients versus heterozygous donors (host-vs.-graft direction) associated with any adverse effects on the outcome of the transplantation. CONCLUSION: Thus, the HPA match does not affect the outcome of transplantation after reduced-intensity conditioning.