42nd Annual Meeting of German Society for Transfusion Medicine and Immunohematology (DGTI)

2009 Transfusion Medicine and Hemotherapy  
Carriers of partial D may produce anti-D following exposure to D positive blood. Current D typing strategies applied in several European countries protect recipients carrying the partial D DVI from immunization by normal D. However, carriers of other partial D can still get immunized, which is particularly relevant in pregnancies. While most of the clinically important partial D were comprehensively characterized, a full record was missing for DIV. We describe the molecular basis and clinical
more » ... portance of DIV variants. RHD nucleotide sequences were determined from genomic DNA. D epitope patterns were established with commercial monoclonal anti-D panels. Cases of D positive patients with anti-D were collated in an internet-based survey. 123 patients with anti-D were reported to our institution between 1998 and 2008, 73 of whom carried partial D. Allo-anti-D was observed in 12 cases with DVI, 12 with DIV, 11 with R0Har, 9 with DNB, 6 with DVII, 5 with DIII, 4 with weak D type 4.2 (DAR) and 2 with DV. Among 12 carriers of DIV with anti-D were 10 females, 7 of whom were likely immunized by pregnancy. DIV and its 2 subgroups DIVa and DIVb differ not only by serology, but also by molecular structures, evolutionary origins and ethnic prevalences. The DIVa phenotype is expressed by the allele DIV type 1.0 which in addition to 350H harbors the 3 dispersed amino acids 62F, 137V and 152T. The DIVb phenotype is expressed by the alleles DIV type 2 to type 5 which represent RHD-CE-D hybrids. 350H is common to all DIV types. We detected new D variants which provided information on the properties of DIV. DWN differs from DIV type 4 by 350D and lacks the epitope pattern characteristic of DIV. DNT carries the primordial substitution N152T of the DIVa cluster which causes a high D antigen density on RBC. D category IV comprises alleles of different evolutionary origin. While DIV type 1 with DIVa phenotype belongs to the oldest extant human RHD alleles, DIV type 2 to type 5 with DIVb phenotype arose from more recent gene conversions. 350H is the only variant amino acid residue shared by all DIV and determines the DIV phenotype. The current serological D typing strategy for recipients, using two monoclonal antibodies not recognizing DVI, could be modified. A set of 2 monoclonal anti-D could be used with one not recognizing DVI, and another not recognizing further clinically important D variants, including DIV. Discrepant results should prompt transfusion of D negative RBC units and anti-D prophylaxis in pregnancies. Panels of monoclonal anti-D and genotyping methods are available to identify the recognized aberrant D. V 1.2 Molecular prediction of antigens may fail in carriers of rare alleles. To identify such alleles, we checked donors predicted to be Co(a+b+) or Lu(a+b+) for their true serologic phenotype. Here we report on the results of the molecular analysis of discrepant samples. Samples predicted by pooled capillary electrophoresis to be Co(a+b+) and Lu(a+b+) but found to be Co(a-b+), Co(a+b-), Lu(a-b+) or Lu(a+b-) were analyzed by sequencing of the exons of the CO and LU genes from genomic DNA, respectively. Adsorption/elution was used to check for faint antigen expression. Of three Lu(a+b-) samples, two showed previously unknown missense mutations, one an in frame two amino acid deletion (table 1). In one of two Co(a-b+) and one of two Co(a+b-) samples, a missense mutation was identified. Of three Lu(b-), one Co(a-) and two Co(b-) tested by adsorption/elution, all but one Lu(b-) sample were positive. The molecular background of Lu(b-), Co(a-), and Co(b-) samples missed by single SNP testing is diverse. Most of these seemingly antigen negative samples are positive on adsorption/elution. Missing such samples does not relevantly reduce the efficacy of molecular donor screening. Sample Phenotype Adsorption/ elution Assumed causative variation Additional variations A Positive 6 bp deletion 98 to 103 (34_35 del RL) Recently, high throughput methods have been developed to predict minor blood group antigens. Information on the impact of systematic donor typing for minor transfusion relevant antigens on routine transfusion care is still emerging. Here we report on our experiences with a typed database of more than 25,000 donors. An IT solution was introduced guiding typed units with useful typing results (molecular or serologic) to a specific laboratory inventory and to locate typed units in the blood service. As baseline, typed units delivered in a 6 month period in 2008 by one of our laboratories were compiled from the laboratory documentation. Units guided to the laboratory inventory, blood service stock and laboratory stock were determined. In the baseline period of 6 month, 480 Jk(a-), 472 M-, 447 Fy(a-), 137 S-, 95 Jk(b-), 44 N-, 21 Fy(b-) and 2 s-units were released. Currently, typing information of about 199 donations is available per day. In 2 month, 751 units were guided to this laboratory inventory, including 434 M neg, 399 S neg, 377 Fy(a-) and 348 Jk(a-) units. Although not used as guidance criterium, 110 Jk(b-), 96 N neg, 67 Fy(b-) and 34 s neg units were scheduled, too. At May 3rd, the laboratory inventory consisted of 250 units, of which the following negative phenotypes were known: 44 Fy(a-), 30 Jk(a-), 63 M-, 55 Abstracts 3 RhD phenotyping were caused by weak or partial RHD alleles. Eleven different weak D types and four different partial RHD alleles were found and, in addition, five new RHD alleles were identified. Purpose: Beside anti-D antibodies, anti-C (or anti-G), anti-c and anti-E are frequently involved as a cause of haemolytic disease of the fetus and newborn. Recently, we have published a large scale evaluation of an automated extraction method for fetal DNA from maternal plasma. In order to establish a clinical service for the determination of the fetal RhC-, Rhc and RhE-status the published DNA extraction method was evaluated in combination with two different PCR protocols. Methods: Fetal DNA from maternal plasma was extracted using the Chemagic viral DNA/RNA kit in conjunction with the Magnetic Separation Module I (Chemagen AG, Baesweiler, Germany). Thereafter, real-time PCR was applied for the detection of the C, c, E alleles with two different published PCR protocols. A total of 233 specimen (46 for C, 87 for c, 100 for E) were included from volunteer, not immunized women at weeks 10 to 22 of gestation. The mother was always negative for the respective Rh antigen. Fetal genotyping results from amnion fluid were used as a reference. Results: The sensitivity obtained from the first PCR protocol was 100% for RHC, 38% for RHc, 59% for RHE, respectively. With the second PCR protocol using a smaller size of amplicons sensitivity could be increased: The sensitivity for RHC was 100%, for RHc 100%, and for RHE 100%. The specificity for all assays was found between 99% and 100%. Conclusions: Automated routine analysis of fetal RHCE alleles from maternal plasma with current protocols is feasible. Furthermore, results demonstrate that sensitivity is dependent on the PCR protocol and amplicon size used for amplification and detection. Purpose: Although it is generally accepted that the central immunopathological disturbance in immune thrombocytopenia is the destruction of antibody-coated platelets by phagocytic cells in the reticuloendothelial system, no in vitro mean is available to determine the phagocytosis. The aim of this study is to develop a new phagocytosis assay to investigate phagocytosis induced by platelet-reactive antibodies via Fc receptors on leukocytes and to test new therapeutic approaches. Methods: Human platelets (PLTs) were labeled with the fluorescent dye CMFDA, opsonized with sera from patients with autoimmune thrombocytopenia (ITP) and neonatal alloimmune thrombocytopenia (NAIT). Opsonized PLts were then incubated with monocytes isolated from whole blood of healthy donors using the automated magnetic cell sorter (autoMACS). Intracellular fluorescence of monocytes was subsequently analyzed by flow cytometry. Results: Compared with NAIT-antibodies opsonized platelets, phagocytosis of ITP-antibodies opsonized platelets was significantly slower. Additionally, while all sera from NAIT-patients induced efficiently phagocytosis of opsonized platelets, not all ITP-sera were capable of this, suggesting that another mechanism of antibody-mediated platelet clearance is implicated in ITP than phagocytosis. Conclusion: The results of our study suggest that the kinetics how monocytes engulf opsonized platelets and induce phagocytosis differ between NAIT-and ITP-sera. Further mechanisms than phagocytosis could be involved in autoantibody-mediated platelet clearance in ITP-Patients. The use of an in vitro phagocytosis assay will potentially enhance the understanding of the mechanism of in vivo destruction of platelets and help in developing and testing new therapeutic approaches. Purpose: Heparin-induced thrombocytopenia (HIT) is the most frequent immune mediated adverse drug reaction with a high risk for new thrombotic complications. HIT is caused by antibodies against complexes of platelet factor 4 (PF4) and the polyanion heparin. Even patients without any previous heparin exposure develop anti-PF4/heparin IgG from on day 4 of heparin treatment. This early onset of IgG after first antigen contact is atypical for a classical primary immune response and suggests preimmunization with an antigen that mimics the PF4/heparin complex. The bacterial cell wall consists of polyanions. Therefore we analysed whether PF4 interacts with bacteria thereby exposing the epitope(s) recognized by anti-PF4/heparin antibodies. Methods: First, binding of increasing concentrations of biotinylated PF4 to Gram-positive (S. aureus, S. pneumoniae, L. monocytogenes) and Gramnegative bacteria (E. coli, N. meningitidis) was quantified in the presence of heparin, dextran sulphate, or buffer by using streptavidin-PerCP-Cy5.5 and flow cytometry. Second, HIT sera were adsorbed by a Protein A deficient S. aureus strain (SA113spa) and a non-pathogenic E. coli strain (JM109), preincubated with PF4 or buffer; thereafter, bound antibodies were eluted (pH3), neutralised, and tested by PF4/heparin enzyme immunoassay (EIA). Third, C57BL/6 mice underwent colon ascendens stent peritonitis (CASP) surgery (model for polymicrobial abdominal sepsis) or sham surgery (control for surgical intervention), or were left untreated. At day 1, 3, 7, 14, and 28 after surgery serum was analysed by PF4/heparin EIA. Results: PF4 bound specifically to both Gram-positive and Gram-negative bacteria. PF4 binding was inhibited by heparin and dextran sulphate suggesting a charge dependent mechanism. PF4-coated bacteria adsorbed anti-PF4/heparin IgG of HIT-patient sera and the respective eluates of these bacteria reacted in the PF4/heparin EIA. C57BL/6 mice (not previously exposed to heparin) developed anti-PF4/heparin IgM already at day 3 after induction of bacterial infection and low IgG levels from on day 14. Conclusions: PF4/polyanion complexes on the bacterial surface prime the immune response. This might be an explanation for the rapid anti-PF4/heparin IgG production after heparin medication in HIT. HIT seems to be a misguided secondary immune response, during which heparin coated platelets mimic previously encountered bacteria. V2 Hemostasis I V 2.1 Background: Acquired hemophilia A (AHA) is a rare but significant hemostatic disorder caused by autoantibody inhibitors against factor VIII coagulant protein (FVIII:C). The annual incidence of AHA is low with 1.3 to 1.5 cases per one million individuals; however, the lethality due to severe hemorrhages (and comorbidity) was high, reaching 22% in some series related to age, inhibitor levels, and response to treatment. Patients in whom the inhibitor cannot be eliminated may have a mortality rate as high as 42%. Design, Patient Characteristics and Clinical Endpoints: We have performed a monocenter study on 24 consecutive patients with AHA who were referred to the Düsseldorf Hemophilia Center between March 2001 and April 2009. The cohort included 15 males (age ranging from 59 to 87 yrs.) and 9 females (age 28 to 76 yrs.). For laboratory evaluation, a standardized staged protocol of APTT, FVIII:C activity and concentration, mixing studies with patient and normal plasma, characterization and quantitation of inhibitor titers (Bethesda assay) was used. Diagnostic work-up included elaborate examinations for any underlying disorder. Clinical endpoints were control of bleeding, eradication of the inhibitor, remission, relapse or death. Results: Only in 5 of the 24 patients (21%), an underlying disorder (breast cancer; lymphoma; multiple sclerosis; rheumatoid arthritis; postpartum state) was identified, while in 79% AHA was the 24 patients (46%) presented with life-threatening hemorrhages. Control of bleeding was achieved in 10 of these 11 patients by high doses of rFVIIa (NovoSeven), one patient required combined FVIII bypassing agents (rFVIIa plus FEIBA). In the other 13 patients, bleeding also subsided in response to rFVIIa. Concurrent immunosuppression consisted of prednisone (2 mg/kg/day), cyclophosphamide (2 mg/kg/day), and 4 weekly doses of rituximab (375 mg/m 2 ). Fourteen of 24 patients (58%) had high inhibitor titers above 30 Bethesda units (ranging up to 168 BU). Of these 14 patients, 10 individuals required daily large-volume immunoadsorption (Ig-Therasorb) for up to 4 weeks to accelerate inhibitor elimination. Induction of immunotolerance was achieved without administration of exogenous rFVIII:C. In total, 21 patients experienced complete remission, one had a relapse, 2 patients died (one of acute myocardial infarction, one of sepsis). Conclusion: These monocenter data demonstrate that control of lifethreatening bleeding by rFVIIa, eradication of the inhibitor by combined immunosuppression and immunoadsorption and induction of tolerance to endogenous factor VIII have significantly improved the clinical outcome of acquired hemophilia. Purpose: Persisting thrombocytopenia is a diagnostic challenge. In a subgroup of these patients, hereditary thrombocytopenia is the underlying cause. Congenital macrothrombocytopenias are a heterogeneous group consisting of MYH9-related diseases, glycoprotein Ib/IX receptor defects, lack ofgranules in the gray platelet syndrome, and abnormalities in the transcription factors like the Paris-Trousseau thrombocytopenia, or the X-linked macrothrombocytopenia. Patients are often misdiagnosed as having ITP. We developed a method to diagnose the most frequent causes of hereditary macrothrombocytopenia using blood smears. Methods: Air dried blood smears from peripheral blood were fixed, permeabilized, stained by monoclonal antibodies, and visualized by immunofluorescence. Mutation analysis in the MYH-9 gene was performed by standard methods using PCR-RFLP/SSP and sequencing. Results: Blood smears are stable at room temperature for at least one week and can be shipped by regular mail. Expression of glycoprotein Ib/IX and IIb/IIIa as well as the presence of alpha granule proteins, and dense granule expression can be visualized by immunofluorescence semiquantitatively. Background and Objectives: Normal platelet aggregation requires (i) agonist-induced activation of IIb 3, (ii) binding of fibrinogen (Fg), and (iii) postoccupancy events following ligand binding. To assess aggregation defects in these terms, we used flow cytometry and specific antibodies that can distinguish between resting, activated, and ligand-occupied forms of IIb 3. Design and Methods: We studied 30 MPD patients. PAC1 (directed to activated IIb 3) and anti-LIBS-1 (recognizing a ligand-induced binding site on 3) were used. LIBS is either expressed upon receptor occupancy by Fg, a process which requires activation, or by Fg-mimetic RGD peptides, which bind to IIb 3 by an activation-independent manner. This approach offers a strategy to distinguish defects in activation, ligand binding, or postoccupancy events. Results: Among the patients, 25 had aggregation defects in response to epinephrine (EPI) and/or ADP despite normal expression of IIb 3. Of these 25 patients, 15 failed to bind PAC1 upon stimulation with EPI or ADP, but 11 of them bound PAC1 in response to PMA, a signal mimetic which circumvents agonist-induced platelet activation by directly activating protein kinase C. In 11 of the 15 patients, binding of anti-LIBS-1 was concomitantly absent in response to EPI or ADP, but intact upon incubation with RGDS. In the other 4 patients, RGDS failed to induce binding of anti-LIBS1. The remaining 10 patients showed intact binding of PAC1 and anti-LIBS-1 in response to EPI, ADP, or PMA, indicative of postoccupancy dysfunction. Conclusion: Platelet aggregation abnormalities can be classified with regard to defects of activation, ligand binding, or postoccupancy dysfunction of IIb 3. V3 Immunogenetics, Immunohematology V 3.1 Purpose: Semaphorins are a wide family of phylogenetically conserved signalling molecules. Semaphorin 7A (Sema7A) is expressed on a variety of myeloid and lymphoid cells as well as on red blood cells. It has shown the ability to promote axon outgrowth and modulate T cell-mediated immune responses through integrins. In this study, we investigated the influence of Sema7A on NK cell phenotype and function. Methods: Recombinant soluble Sema7A was expressed in HEK cells and used to stimulate primary NK cells. The NK cells were measured for activation of the mitogen-activated protein kinase (MAPK) pathway and changes in the surface expression of KIR2DL1, KIR2DL2, NKp30, NKp44, NKp46, NKG2A and NKG2D receptors. NK cell proliferation assays were performed in the presence or absence of Sema7A. Cytotoxic assays using K562 cells were performed with non-stimulated or Sema7A pre-stimulated NK cells. To study whether the effect of Sema7A on NK cell function was mediated by integrins, a mutated Sema7A protein with an altered integrin-binding motif was used as negativecontrol. Results: Soluble Sema7A showed the ability to bind NK cells and to induce phosphorylation of the non-receptor protein kinase focal adhesion kinase (FAK) and extracellular regulated kinases (ERK) 1 and 2. Sema7A caused a downregulation of NKG2D, NKp30 and NKp46 expression by up to 30%, 47% and 43%, respectively. The proliferation rate of NK cells decreased by 62% in presence of Sema7A, in comparison with non-stimulated NK cells. Pre-incubation of NK cells with Sema7A reduced the cytotoxic activity of NK cells against K562 cells by up to 60%. The mutated Sema7A protein did not show any effect on NK cell proliferation or cytotoxicity suggesting that integrin receptors are involved in Sema7A-mediated NK cell activation. Transfus Med Hemother 2009;36(suppl 1):1-62 Abstracts 7 Conclusion: This study demonstrates for the first time that Sema7A is a potent inhibitor of NK cell function. This observation further highlights Sema7A as an important effector molecule in cell-based immunity. Purpose: T cell activation is an important process of the adaptive immune system, which requires recognition of MHC-associated antigens by antigen presenting cells (APCs) via the T cell receptor (TCR). To induce a productive T cell response the interaction of T cells with APCs needs to be stabilized by adhesion molecules. Junction adhesion molecules (JAMs) are a recently discovered group of immunoglobulin (Ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. The third member of the JAM protein family, JAM-C, is highly expressed in platelets and endothelial cells, whereas expression in T cells is largely unknown. Results: To investigate the regulation of JAM-C in T lymphocytes, we determined JAM-C gene expression in quiescent and activated human T cells. Treatment with the polyclonal T cell activator phytohemagglutinin (PHA) increased surface and total JAM-C expression in T cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. By contrast, no up-regulation of JAM-A in activated T cells was detectable. The highest level of JAM-C up-regulation by PHA was observed in CD3+FoxP3+ and CD4+CD25high T cells. Moreover, T cell receptor activation with combined anti-CD3 and anti-CD28 stimulation induced JAM-C expression in T cells. JAM-C induction occurred at the mRNA level suggesting a transcriptional regulatory mechanism of JAM-C expression. Accordingly, we studied the regulation of the human JAM-C gene promoter in transiently transfected T cells. Luciferase activity of a JAM-C promoter gene construct with three potential consensus sites for the transcription factor NFAT was markedly induced in activated T cells. Finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin A and FK-506, but not with MAPK inhibitors, blocked JAM-C induction in activated T cells. Conclusion: In summary, the present data indicate that JAM-C is induced in activated human T lymphocytes via a transcriptional mechanism and suggests a major regulatory function of JAM-C for the T cell response. Purpose: Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. It has been shown in HO-1 knock out mice and in human genetic HO-1 deficiency that this enzyme has potent anti-inflammatory functions and may be a major target in transplantation medicine. Although HO-1 has previously been shown to be induced by various stimuli via activation of the p38 MAPK signaling pathway, the role of this protein kinase for HO-1 gene regulation is largely unknown. Results: In the present study it is demonstrated that pharmacological inhibitors of p38 and siRNA-dependent knock down of p38 induced HO-1 expression in monocytic cells. Moreover, basal HO-1 gene expression levels were markedly higher in untreated murine embryonic fibroblasts (MEF) from p38 -/-mice as compared to that from wild type mice. Transfection studies with luciferase reporter gene constructs indicate that increased HO-1 gene expression via inhibition of p38 was mediated by the transcription factor NF-E2-related factor-2 (Nrf2), which is a central regulator of the cellular oxidative stress response. Accordingly, inhibitors of p38 induced binding of nuclear proteins to a Nrf2 target sequence of the HO-1 promoter, but did not affect HO-1 protein expression and promoter activity in Nrf2-/-MEF. Genetic deficiency of p38 led to enhanced phosphorylation of ERK and increased cellular accumulation of reactive oxygen species (ROS). In addition, pharmacological blockage of ERK and scavenging of ROS with Nacetylcysteine reduced HO-1 gene expression in p38-/-MEF, respectively. Taken together, it is demonstrated that pharmacological inhibition and genetic deficiency of p38 induce HO-1 gene expression via a Nrf2-dependent mechanism in monocytic cells and MEF. Conclusion: These findings not only give new insights into the complex signaling events of HO-1 regulation during the inflammatory response, but may also help to develop novel therapeutic approaches for inflammatory diseases and in transplantation medicine. Blood Center Linz, A Purpose: We describe the development of a novel, human leukocyte antigen (HLA) sequence-based typing (SBT) method by exploring the nextgeneration sequencing technology as provided by the Genome Sequencer FLX (GS-FLX) system (Roche/454 Life Sciences, Branford, CT, USA). Methods: The established system allows for mostly ambiguity-free, highthroughput, high-resolution HLA typing with the potential for high-grade automation. The on average 259 bp long sequence reads generated by the Genome Sequencer FLX system in this study, span most of the HLA exons of interest enabling an amplicon sequencing strategy. We developed a list of 11 HLA-A and -B locus-specific intronic primers for exon 1, 2, 3 and 4. Results: In 2005 95,477 RBCs (45.8% to females) were transfused in MV. Median patient age was 68.9 years with a peak at 65-79 years (47% of all transfusions). 37.4% (35,737 RBCs) of all RBCs were transfused to medical patients; 24.6% (23,446 RBCs) to patients in the emergency room and in intensive care units (combined medical and surgical patients); 35.1% (33,530 RBCs) to surgical patients; 0.7% (695 RBCs) to pediatric patients (2.2% (2,069 RBCs) not classified). Projections of the overall blood demand and blood donations predicted a shortfall of 56,083 RBCs for in-hospital patients in 2020 with a demand of ~46,000 RBCs for medical patients; 28,000 RBCs for patients in the emergency room / intensive care units; 42,000 RBCs for surgical patients; 600 RBCs to pediatric patients. Discussion: In 2005 already more than half of the RBCs were transfused to medical patients and to intensive care/emergency patients. These patients will be treated with higher priority than elective surgery patients for medical and ethical reasons. Hence, the shortfall in RBCs will likely lead to a substantial reduction of elective interventions, which could severely affect hospital budget planning and challenge the provision of medical care. Purpose: Information on socioeconomic status of blood donors are scarce. We report results of a systematic assessment of blood donors, the Greifswald DONOR-SHIP Study. Methods: In the DONOR-SHIP study over a 1 year period every 6th person enrolling for blood donation was asked to fill in a structured questionnaire on characteristics of the socio-economic situation, the subjective health status, and the health related behaviour. All data were adjusted for oversampling due to multiple donations. Results: 2503 blood donors participated; females 54.6%; mean age 29.7 years, 23.5% were married; most blood donors had a higher grade of school education: 30% primary (Hauptschule), 30% intermediate (mittlere Reife), 40% secondary education ([Fach-]Abitur). Only 33% were full time working; 32% were in training (total students 24%), unemployed were 16%. 68% came to blood donation from home, 19% from work. Median single way to the donation center were 4km (quar ation about blood donation were obtained in 90% of donors by family members, 30% by newspaper, and 20% in school, while radio broadcast and internet had a minor role (5% and 7%). Motivation for blood donation were (multiple answers possible): I want to help 98%; wanted my blood assessed 80%; I feel more satisfied 67%; renumeration (Aufwandsentschädigung) 64%; feel physically better 43%; to know my blood group 40%; to receive a donor passport 40%; wanted to be seen by physician 38%; other blood donors took me with them 31%; family member required blood transfusion 11%; wanted HIV test 6.6%. Conclusion: Most blood donors are well educated. To help others but also personal benefits (blood assessment and renumeration) are strong motivators for blood donation. Full time employees are underrepresented. These data may be used to design blood donor campaigns. 1 German red cross blood transfusion service, Bremen, D, 2 German red cross blood transfusion service, Springe, D, 3 German red cross blood transfusion service, Oldenburg, D Background: The transportation from blood banks to the recipients is difficult to standardize and potentially affects the quality of PC. We developed a model to examine the effects of trans reduction of the surface of the storage bag available for gas exchange combined with agitation or non-agitation. The quality of the PC was evaluated by measurement of pH and the levels of glucose, lactate, beta-thromboglobulin and solube p-selectin. Purpose: Nitric oxide (NO) is a diffusible, short-lived, diatomic free radical ubiquitously produced by mammalian cells. The NO signaling pathway involves mainly the activation of soluble guanylyl cyclase to produce cyclic GMP (cGMP) as a second messenger and downstream mediator. Nitric oxide (NO) is known to inhibit platelet function by this induction of cGMP production. However, there exists only very few data on the discriminative effect of nitric oxide on the in-vitro aggregation response of human platelets to different agonists as measurable by the classical Born aggregometry. Methods: We studied the aggregation response to arachidonic acid, collagen, ristocetin, and thrombin receptor-activating peptide (TRAP) using Born Aggregometry and a PAP-4 aggregometer. The effect of nitic oxide (NO) on the different aggregation responses was measured using diethylamine NONOate sodium salt hydrate (DEA-NONOate) as a NO donor. Results: DEA-NONate inhibits completely the aggregation response to arachidonic acid. The response to collagen is decelerated and decreased in the presence of NO. Using TRAP as agonist, platelet aggregates can be induced even in the presence of NO but these aggregates disaggregate. Only the aggregation response to ristocetin is not inhibited by NO aside from very high NO cencentrations. Conclusions: This study is the first presenting data on the discriminative influence of NO on the aggregation response to different platelet agonists. Nitric oxide does not inhibit all aggregation responses at the same NO concentration. The aggregation response to ristocetin is much less sensitive to the inhibition by NO than the aggregation responses to arachidonic acid, collagen, and TRAP. P 6.04 Purpose: The most common mode of transmission of hepatitis A virus (HAV) is person-to-person, resulting from fecal contamination and oral ingestion. However, a few HAV transfusion-transmissions have been reported. This case report describes the first possible transmission of HAV via the transfusion of a thrombocyte concentrate in our blood donation service. Methods: All donations were routinely screened by in-house RT-PCR for HAV in pools of 96 with a sensitivity (95% cut off) of 155IU/ml in the
doi:10.1159/000242471 fatcat:zrs5oxt7nzbxxcntpk54engkjm