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Introduction: The molecular diagnosis of the A1 blood group is based on the exclusion of ABO gene variants causing blood groups A2, B, or O. A specific genetic marker for the A1 blood group is still missing. Recently, long-read ABO sequencing revealed four sequence variations in intron 1 as promising markers for the ABO*A1 allele. Here, we evaluated the diagnostic values of the 4 variants in blood donors with regular and weak A phenotypes and genotypes. Methods: ABO phenotype data (A, B, AB, or O) were taken from the blood donor files. The ABO genotypes (low resolution) were known from a previous study and included the variants c.261delG, c.802G>A, c.803G>C, and c.1061delC. ABO variant alleles (ABO*AW.06,*AW.08,*AW.09,*AW.13, *AW.30, and *A3.02) were identified in weak A donors by sequencing the ABO exons before. For genotyping of the ABO intron 1 variants rs532436, rs1554760445, rs507666, and rs2519093, we applied TaqMan assays with endpoint fluorescence detection according to a standard protocol. Genotypes of the variants were compared with the ABO phenotype and genotype. Evaluation of diagnostic performance included sensitivity, specificity, positive (PPV), and negative predictive value (NPV). Results: In 1,330 blood donors with regular ABO phenotypes and genotypes, the intron 1 variants were significantly associated with the proposed A1 blood group. In 15 donors, we found discrepancies to the genotype of at least one of the 4 variants. For the diagnosis of the ABO*A1 allele, the variants showed 98.79–99.48% sensitivity, 99.66–99.81% specificity, 98.80–99.31% PPV, and 99.66–99.86% NPV. Regarding the A phenotype, the diagnostic values were 99.02–99.41% sensitivity, 99.63–99.76% specificity, 99.41–99.61% PPV, and 99.39–99.63% NPV. The *A1 marker allele of all intron 1 variants was also associated with the *AW.06, *AW.13, and *AW.30 variants. Samples with *AW.08, *AW.09, and *A3.02 variants lacked this association. Conclusion: The ABO intron 1 variants revealed significant association with the ABO*A1 allele and the A phenotype. However, the intron 1 genotype does not exclude variant alleles causing weak A phenotypes. With the introduction of reliable tag, single nucleotide variants for the A1, A2, B, and O blood groups and the genotyping instead of phenotyping of the ABO blood group are getting more feasible on a routine basis.

BackgroundRecent data on immune evasion of new SARS-CoV-2 variants raise concerns about the efficacy of antibody-based COVID-19 therapies. Therefore, in this study the in-vitro neutralization capacity against SARS-CoV-2 variant B.1 and the Omicron subvariants BA.1, BA.2 and BA.5 of sera from convalescent individuals with and without boost by vaccination was assessed.Methods and findingsThe study included 313 serum samples from 155 individuals with a history of SARS-CoV-2 infection, divided into subgroups without (n=25) and with SARS-CoV-2 vaccination (n=130). We measured anti-SARS-CoV-2 antibody concentrations by serological assays (anti-SARS-CoV-2-QuantiVac-ELISA (IgG) and Elecsys Anti-SARS-CoV-2 S) and neutralizing titers against B.1, BA.1, BA.2 and BA.5 in a pseudovirus neutralization assay. Sera of the majority of unvaccinated convalescents did not effectively neutralize Omicron sublineages BA.1, BA.2 and BA.5 (51.7%, 24.1% and 51.7%, resp.). In contrast, 99.3% of the sera of superimmunized individuals (vaccinated convalescents) neutralized the Omicron subvariants BA.1 and BA.5 and 99.6% neutralized BA.2. Neutralizing titers against B.1, BA.1, BA.2 and BA.5 were significantly higher in vaccinated compared to unvaccinated convalescents (pConclusionsThese findings confirm substantial immune evasion of the Omicron sublineages, which can be overcome by vaccination of convalescents. This informs strategies for choosing of plasma donors in COVID-19 convalescent plasma programs that shall select specifically vaccinated convalescents with very high titers of anti-S antibodies.

Background: Transfusion of red cell concentrates (RCCs) is an integral therapy after severe hemorrhage or trauma. Prehospital transfusion offers an immediate intervention in emergency cases. Air ambulance-based prehospital transfusion, already used in different countries, is currently established in Germany. Limited information is available for regulatory-compliant transport logistics of RCCs and their quality after repeated air rescue missions. Thus, the aim of this study was (i) to validate regulatory-compliant logistics and (ii) to assess product quality, analyzing biochemical parameters and RBC morphology. Study Design and Methods: Due to regulatory requirements, we adapted a rotation system of 1 day transport, 1 day quarantine storage and 1 day storage over the entire RCC shelf life. RCCs transported on air rescue missions (flight group) were compared against a control group, treated identically except for helicopter transport. RCCs were visually inspected, and their temperature was documented throughout the entire rotation cycles. RCCs at the end of shelf life (end point samples) were assessed for levels of hemoglobin, hematocrit, free hemoglobin, hemolysis, mean corpuscular volume, potassium and pH. In addition, morphological changes were assessed using flow morphometry. Results: In total 81 RCCs were assessed in the flight group and 50 in the control group. Within the flight group, 30 RCCs were transfused. RCCs were dispatched on average 11 times (7–13 times). The average flight time was 18.3 h (6.6–28.8 h). The rotation system ensured adherence to regulatory guidelines, especially compliance to storage conditions of +2 to +6°C of intermediate storage. Biochemical and morphological quality parameters did not exhibit any changes upon repeated air rescue missions. A correlation with respect to the flight time was not observed either. Discussion: The quality of RCCs after repeated air rescue missions is noninferior to control samples regarding biochemical and morphological parameters. The product quality is within German regulations for up to 42 days of storage. The logistics and maintenance of the thermal conditions are safe and feasible. Thus, a rotation system of RCCs offers a regulatory-compliant option to supply air rescue missions with RCCs to allow life-saving prehospital transfusions at the incident scene.

Background: The stem cell niche in human bone marrow provides scaffolds, cellular frameworks and essential soluble cues to support the stemness of hematopoietic stem and progenitor cells (HSPCs). To decipher this complex structure and the corresponding cellular interactions, a number of in vitro model systems have been developed. The cellular microenvironment is of key importance, and mesenchymal stromal cells (MSCs) represent one of the major cellular determinants of the niche. Regulation of the self-renewal and differentiation of HSPCs requires not only direct cellular contact and adhesion molecules, but also various cytokines and chemokines. The C-X-C chemokine receptor type 4/stromal cell-derived factor 1 axis plays a pivotal role in stem cell mobilization and homing. As we have learned in recent years, to realistically simulate the physiological in vivo situation, advanced model systems should be based on niche cells arranged in a three-dimensional (3D) structure. By providing a dynamic rather than static setup, microbioreactor systems offer a number of advantages. In addition, the role of low oxygen tension in the niche microenvironment and its impact on hematopoietic stem cells need to be taken into account and are discussed in this review. Summary: This review focuses on the role of MSCs as a part of the bone marrow niche, the interplay between MSCs and HSPCs and the most important regulatory factors that need to be considered when engineering artificial hematopoietic stem cell niche systems. Conclusion: Advanced 3D model systems using MSCs as niche cells and applying microbioreactor-based technology are capable of simulating the natural properties of the bone marrow niche more closely than ever before.

Background Convalescent plasma is one of the treatment options for COVID-19 which is currently being investigated in many clinical trials. Understanding of donor and product characteristics is important for optimization of convalescent plasma. Methods Patients who had recovered from CO­VID-19 were recruited as donors for COVID-19 convalescent plasma (CCP) for a randomized clinical trial of CCP for treatment of severe COVID-19 (CAPSID Trial). Titers of neutralizing antibodies were measured by a plaque-reduction neutralization test (PRNT). Correlation of antibody titers with host factors and evolution of neutralizing antibody titers over time in repeat donors were analysed. Results A series of 144 donors (41% females, 59% males; median age 40 years) underwent 319 plasmapheresis procedures providing a median collection volume of 850 mL and a mean number of 2.7 therapeutic units per plasmapheresis. The majority of donors had a mild or moderate course of COVID-19. The titers of neutralizing antibodies varied greatly between CCP donors (from 1:640). Donor factors (gender, age, ABO type, body weight) did not correlate significantly with the titer of neutralizing antibodies. We observed a significant positive correlation of neutralization titers with the number of reported COVID-19 symptoms and with the time from SARS-CoV-2 diagnosis to plasmapheresis. Neutralizing antibody levels were stable or increased over time in 58% of repeat CCP donors. Mean titers of neutralizing antibodies of first donation and last donation of repeat CCP donors did not differ significantly (1:86 at first compared to 1:87 at the last donation). There was a significant correlation of neutralizing antibodies measured by PRNT and anti-SARS-CoV-2 IgG and IgA antibodies which were measured by ELISA. CCP donations with an anti-SARS-CoV-2 IgG antibody content above the 25th percentile were substantially enriched for CCP donations with higher neutralizing antibody levels. Conclusion We demonstrate the feasibility of collection of a large number of CCP products under a harmonized protocol for a randomized clinical trial. Titers of neutralizing antibodies were stable or increased over time in a subgroup of repeat donors. A history of higher number of COVID-19 symptoms and higher levels of anti-SARS-CoV-2 IgG and IgA antibodies in immunoassays can preselect donations with higher neutralizing capacity.

Macrophages are key innate immune cells that mediate implant acceptance or rejection. Titanium implants degrade over time inside the body, which results in the release of implant wear-off particles. Titanium nanoparticles (TiNPs) favor pro-inflammatory macrophage polarization (M1) and lower tolerogenic activation (M2). GDF-15 regulates immune tolerance and fibrosis and is endocytosed by stabilin-1. How TiNPs affect the healing activities of macrophages and their release of circulating cytokines is an open question in regenerative medicine. In this study for the first time, we identified the transcriptional program induced and suppressed by TiNPs in human pro-inflammatory and healing macrophages. Microarray analysis revealed that TiNPs altered the expression of 5098 genes in M1 (IFN-γ-stimulated) and 4380 genes in M2 (IL-4–stimulated) macrophages. 1980 genes were differentially regulated in both M1 and M2. Affymetrix analysis, confirmed by RT-PCR, demonstrated that TiNPs upregulate expression of GDF-15 and suppress stabilin-1, scavenger receptor of GDF-15. TiNPs also significantly stimulated GDF-15 protein secretion in inflammatory and healing macrophages. Flow cytometry demonstrated, that scavenging activity of stabilin-1 was significantly suppressed by TiNPs. Confocal microscopy analysis showed that TiNPs impair internalization of stabilin-1 ligand acLDL and its transport to the endocytic pathway. Our data demonstrate that TiNPs have a dual effect on the GDF-15/stabilin-1 interaction in macrophage system, by increasing the production of GDF-15 and suppressing stabilin-1-mediated clearance function. In summary, this process can result in a significant increase of GDF-15 in the extracellular space and in circulation leading to unbalanced pro-fibrotic reactions and implant complications.

Introduction: Recently, we identified a huge discrepancy between the collection practice and the actual utilization of cryopreserved peripheral blood stem cells (PBSCs) for high-dose chemotherapy (HDCT) and autologous blood stem cell transplantation (ABSCT). Specifically, patients with Burkitt lymphoma, acute leukemia, and myeloproliferative neoplasms (MPN) were frequently not referred for ABSCT after successful PBSC collection. Objective: The aim of this study was to identify variables that are associated with the non-utilization of PBSC grafts. Methods: We retrospectively analyzed the collection, storage, and disposal of PBSC grafts in Burkitt lymphoma (n = 18), acute lymphoblastic leukemia (ALL, n = 22), MPN (n = 18), and acute myeloid leukemia (AML, n = 71) patients. Patients who underwent autologous PBSC collection at 2 collection and transplantation centers between 2001 and 2012 were included and followed up until 2016. Results: None of the Burkitt lymphoma patients were referred for ABSCT. Only in 1 (6%) patient, the graft was discarded after the patient’s death. In all other patients (n = 17, 94%), the grafts were stored independently of the patient’s status (death, n = 4, 22%; no follow-up, n = 6, 33%; no indication for ABSCT given, n = 7, 39%). In ALL patients, 4 (18%) patients underwent ABSCT after a median follow-up of 74 (1–182) months. In the remaining patients, PBSC grafts were either discarded (8 patients, 36%) or stored until the reference date (10 patients, 45%). Seven of 18 MPN patients (39%) underwent ABSCT. ABSCT was performed in 24 (34%) AML patients. In 20 (28%) patients who were not referred to ABSCT, an allogeneic transplantation (TPL) was performed. Fifteen (21%) patients received palliative care or deceased, and their grafts were discarded in all but 1 patient. Additional grafts were discarded in 21 (31%) patients and stored in 9 (13%) patients who underwent ABSCT or allogeneic TPL (n = 44). Conclusions: As the role and efficacy of autologous HDCT/ABSCT are not established in the analyzed entities, the indication for PBSC collection should be reanalyzed in regular intervals. Moreover, PBSC grafts from patients who have deceased, have insufficient grafts, or have already undergone an allogeneic TPL should be considered for disposal or (if applicable) for research use, to economize storage costs on a rational basis.

Background: The provision of compatible blood products to patients is the most essential task of transfusion medicine. Besides ABO and Rh, a number of additional blood group antigens often have to be considered for the blood supply of immunized or chronically transfused patients. It also applies for platelet antigens (HPA) and neutrophil antigens (HNA) for patients receiving platelet or granulocyte concentrates. Here, we describe the molecular screening for a number of blood group, HPA, and HNA alleles. Based on the screening results we are building up a regional blood donor registry to provide extended matched blood products on demand. Methods: We developed and validated TaqMan™ PCR and PCR-SSP methods for genetic markers defining 37 clinically relevant blood group antigens (beyond ABO and Rh), 10 HPA, and 11 HNA. Furthermore, we describe a feasible method for fast molecular screening of the HNA-2null phenotype. All data were statistically evaluated regarding genotype distribution. Allele frequencies were compared to ExAC data from non-Finnish Europeans. Results: Up to now more than 2,000 non-selected regular blood donors in south-west Germany have been screened for blood group, HPA, and HNA alleles. The screening results were confirmed by serology and PCR-SSP methods for selected numbers of samples. The allele frequencies were similar to non-finnish Europeans in the ExAC database except for the alleles encoding the S, HPA-3b and HNA-4b antigens, with significantly lower prevalence in our cohort, as well as the LU14 and the HNA-3b antigens, with a higher frequency compared to the ExAC data. We identified 71 donors with rare blood groups such as Lu(a+b-), Kp(a+b-), Fy(a-b-) and Vel-, and 169 donors with less prevalent HPA or HNA types. Conclusion: Molecular screening for blood group alleles by using TaqMan™ PCR is an effective and reliable high-throughput method for establishing a rare donor registry.

Background: In the recent past, the discrepancy between blood supply and future demand may have been overestimated. As medical progress develops rapidly, it will be essential to monitor ongoing demographic changes in the donor population regularly and to re-evaluate retention and recruiting strategies. The aim of the current study was to compare first-time donor (FTD) characteristics and their return rates. We therefore compared whole blood (WB) donations in total and the annual donation frequencies in 2010 and in 2015/2016. Furthermore, we evaluated whether over the same observation period, medical reasons for deferral underwent a change (2010 vs. 2015). Methods: The return probability of FTD within 12 months was analysed in 2010 and 2015 with respect to successful donation versus deferral and with regard to age. The total number of WB donations was investigated, and age distribution was compared between 2010, 2013 and 2016. WB donation frequencies were calculated with respect to age and gender in 2010 and 2016. In a second analysis, medical reasons for deferral were differentiated into 14 categories and a possible impact of time (2010 vs. 2015) on the respective percentage was studied. Results: We observed a significant decline of the FTD return rate from 42.5% to 38.8% in donors that successfully donated WB while the rate remained unchanged in deferred FTD. At the same time the mean FTD age decreased from 29.1 ± 11.6 to 28.5 ± 11.7 years in 2016. Analysis of total WB donations revealed an increase of all donations from donors ≥60 years, a constant percentage from donors Conclusion: There is ongoing demographic change in our WB donor population. Our data prove a need for a re-evaluation of retention and recruitment strategies since previous marketing campaigns seem to have neglected the age group 30-59 years. This must be addressed in further studies as this age group will be highly relevant for assuring future blood supplies since donor recruitment from adolescents will be limited due to declining birth rates. Furthermore, deferral due to travel history is increasing significantly. Thus we will require further studies on the possible impact on donor retention.

Background: Noninvasive prenatal testing (NIPT) for fetal antigens is a common standard for targeted immune prophylaxis in RhD-mediated hemolytic disease of the fetus and newborn, and is most frequently done by quantitative PCR (qPCR). A similar approach is considered for other blood group and human platelet alloantigens (HPA). Because of a higher sensitivity compared to qPCR for rare molecule detection, we established and validated digital PCR (dPCR) assays for the detection of RHD exons 3, 5 and 7, KEL1, HPA-1a, and HPA-5b from cell-free DNA (cfDNA) in plasma. The dPCR assays for the Y-chromosomal marker amelogenin and autosomal SNPs were implemented as controls for the proof of fetal DNA. Methods: Validation was performed on dilution series of mixed plasma samples from volunteer donors with known genotypes. After preamplification of the target loci, two-color (FAM and VIC) TaqManTM probe chemistry and chip-based dPCR were applied. The assays for RHD included GAPDH as an internal control. For the diallelic markers KEL1/2, HPA-1a/b, HPA-5a/b, and AMEL-X/Y and 3 autosomal SNPs, the probes enabled allelic discrimination in the two fluorescence channels. The dPCR protocol for NIPT was applied to plasma samples from pregnant women. Results: The RHD exon 5 assay allowed the detection of a 0.05% RHD target in an RhD-negative background, whereas the exon 7 assay required at least a 0.25% target. The exon 3 assay showed the highest background and required at least a 2.5% RHD target for reliable detection. The dPCR assays for the diallelic markers revealed similar sensitivity and enabled the detection of at least a 0.5% target allele. The HPA-1a assay was the most sensitive and allowed target detection in plasma mixtures containing only 0.05% HPA-1a. The plasma samples from 13 pregnant women at different gestational ages showed unambiguous positive and negative results for the analyzed targets. Conclusion: Analysis of cfDNA from maternal plasma using dPCR is suitable for the detection of fetal alleles. Because of the high sensitivity of the assays, the NIPT protocol for RhD, KEL1, and HPA can also be applied to earlier stages of pregnancy.

Background: There is an increasing demand for products containing mononuclear cells (MNCs) for cellular immune therapy. Hence, leukapheresis is increasingly performed in healthy volunteer donors. Methods: We evaluated 147 low-volume leukapheresis procedures from 77 healthy non-cytokine-stimulated donors. Complete blood counts (CBCs) of the donors were measured before and directly after the procedures as well as from the MNC products. Follow-up CBCs were collected from donors within 21 days. Results: The product hematocrit within a range from 1.2 to 6.0% did not correlate with the collection efficiency of any cell population or the granulocyte and platelet yield. There was a strong correlation between the CBC values before leukapheresis and the cell yield of lymphocytes and monocytes as well as a perfect negative correlation between cell recruitment and cell loss in all cell populations. Furthermore, we observed a significant decrease in the CBC values in all cell populations directly after leukapheresis, which recovered within a mean of 16.1 days (SD ± 2.1 days) and even showed a significant increase in granulocytes and platelets. Conclusion: Low-volume leukapheresis is feasible for the collection of MNCs in which the product hematocrit is negligible for the collection efficiency, cell yield, or contamination of residual cells under operational settings recommended by the manufacturer. Our data suggests that cell recruitment is regulated by the number of cells removed, which may also be the stimulus to induce granulo- and thrombopoiesis within the first days after leukapheresis.

In breast cancer, the tumor microenvironment plays a critical role in the tumor progression and responses to therapy. Tumor-associated macrophages (TAMs) are major innate immune cells in tumor microenvironment that regulate intratumoral immunity and angiogenesis by secretion of cytokines, growth factors as well as chitinase-like proteins (CLPs), that combine properties of cytokines and growth factors. YKL-39 is a chitinase-like protein found in human and absent in rodents, and its expression in TAMs and role in breast cancer progression was not studied to date. Here for the first time we demonstrate that YKL-39 is expressed on TAMs, predominantly positive for stabilin-1, but not by malignant cells or other stromal cells in human breast cancer. TGF-beta in combination with IL-4, but not IL-4 alone was responsible of the stimulation of the production of YKL-39 in human primary macrophages. Mechanistically, stabilin-1 directly interacted with YKL-39 and acted as sorting receptor for targeting YKL-39 into the secretory pathway. Functionally, purified YKL-39 acted as a strong chemotactic factor for primary human monocytes, and induced angiogenesis in vitro. Elevated levels of YKL-39 expression in tumors after neoadjuvant chemotherapy (NAC) were predictive for increased risk of distant metastasis and for poor response to NAC in patients with nonspecific invasive breast carcinoma. Our findings suggest YKL-39 as a novel therapeutic target, and blocking of its activity can be combined with NAC in order to reduce the risk of metastasis in breast cancer patients.

Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytoldne profile that can support of diabetes-associated inflammation and development of vascular complications. (C) 2016 Elsevier GmbH. All rights reserved.

Background: The aim of this study was to describe donor satisfaction regarding different aspects of the new German blood donor questionnaire (BDQ) and to assess whether donor satisfaction is associated with the intention to return for further donations. Methods: A random number of 6,600 blood donors, donating at the German Red Cross Blood Service Baden-Wuerttemberg - Hessen, were asked to rate their satisfaction with four different aspects of the BDQ. Chi-square statistics was used to test for associations between satisfaction and the intention to return. Results: Most of the donors were satisfied with format and layout (72.7%) and the clarity of the questions (72.5%). However, only 39.5% of the donors were satisfied with the scope of the BDQ and 44.3% with the questions about sexual risk behavior. The lowest satisfaction seemed to be among experienced donors and among donors from small municipalities. Among experienced and very experienced donors, a significant association between the satisfaction with the different aspects and the intention to return became apparent. Conclusion: When considering the implementation of the BDQ, Blood Donor Services have to weigh up the advantages of increased deferral rates among donors with high-risk behavior against the potential drop-out of dissatisfied blood donors.

Background aims Mesenchymal stromal cells (MSC) are promising candidates for innovative cell therapeutic applications. For clinical-scale manufacturing, different supplements have been evaluated as alternatives for the commonly used fetal bovine serum (FBS). We have reported previously that pooled human AB serum (HS) accelerates the proliferation of adipose tissue-derived MSC (ASC) while maintaining key functions of MSC biology such as differentiation, immune suppression and growth factor secretion. ASC expanded in FBS-supplemented culture media undergo replicative aging that is associated with a progressive loss of differentiation capacity but without indications of cellular transformation. The effects of HS media on ASC long-term culture, however, remain poorly characterized. Methods Long-term cultures of ASC in FBS and HS media were analyzed with respect to proliferation, marker expression, differentiation and immune suppression. Results Despite signs of an accelerated proliferation, extended life span and clonogenic capacity of ASC cultivated in HS-supplemented media, HS and FBS cultures revealed no significant differences with respect to differentiation potential and expression of senescence markers. Anchorage-independent growth, which is indicative of tumorigenic properties, was not observed in either culture conditions. Similarly, immune suppressive activities were maintained. Donor variation regarding differentiation potential and marker expression became apparent in this study independent of the culture supplement or culture duration. Conclusions We have demonstrated that the use of pooled allogeneic HS maintains the characteristics of ASC even after long-term expansion, further demonstrating that the use of HS is an alternative to FBS.

BACKGROUND: The ABO blood groups result from DNA sequence variations, predominantly single nucleotide and insertion/deletion polymorphisms (SNPs and indels), in the ABO gene encoding a glycosyltransferase. The ABO blood groups A(1), A(2), B and O predominantly result from the wild type allele A1 and the major gene variants that are characterized by four diallelic markers (261Gdel, 802GA, 803GC, 1061Cdel). Here, we were interested to evaluate the impact of ABO genotyping compared to ABO phenotyping in paternity testing. METHODS: The major ABO alleles were determined by PCR amplification with sequence-specific primers (PCR-SSP) in a representative sample of 1,335 blood donors. The genotypes were compared to the ABO blood groups registered in the blood donor files. Then, the ABO phenotypes and genotypes were determined in 95 paternity trio cases that have been investigated by 12 short tandem repeat (STR) markers before. We compared statistical parameters (PL, paternity likelihood; PE, power of exclusion) of both blood grouping approaches. RESULTS: The prevalence of the major ABO alleles and genotypes corresponded to the expected occurrence of ABO blood groups in a Caucasian population. The low resolution genotyping of 4 diallelic markers revealed a correct genotype-phenotype correlation in 1,331 of 1,335 samples (99.7%). In 60 paternity trios with confirmed paternity of the alleged father based on STR analysis both PL and PE of the ABO genotype was significantly higher than of the ABO phenotype. In 12 of 35 exclusion cases (34.3%) the ABO genotype also excluded the alleged father, whereas the ABO phenotype excluded the alleged father only in 7 cases (20%). CONCLUSION: In paternity testing ABO genotyping is superior to ABO phenotyping with regard to PL and PE, however, ABO genotyping is not sufficient for valid paternity testing. Due to the much lower mutation rate compared to STR markers, blood group SNPs in addition to anonymous SNPs could be considered for future kinship analysis and genetic identity testing.

BACKGROUND: Glycoprotein (GP)-specific platelet (PLT) antibodies can cause allo- or autoimmune thrombocytopenia. Their detection is of high diagnostic value. The simultaneous analysis of specific PLT antibodies (SASPA) assay is based on simultaneous detection of various PLT-specific antibodies by flow cytometry and has entered routine use in our Mannheim institution. In this study, we performed an interlaboratory comparison investigation of PLT-specific antibodies using SASPA versus the “gold standard,” the monoclonal antibody–specific immobilization of PLT antigen (MAIPA) assay. STUDY DESIGN AND METHODS: Sera from 194 patients with suspected PLT allo- or autoantibodies were tested against GPIIb/IIIa, IX, Ia/IIa, IV, and HLA Class I by SASPA (in Mannheim) and MAIPA (in Vienna). All data were reported blinded to those from the respective other method. Sensitivity studies included dilution studies with known antibodies against HPA-1a, -1b, -3b, -5b, and -15b and HLA Class I. RESULTS: Overall, results were concordant in 78.9%. The specificity and sensitivity of SASPA, based on the MAIPA results, were 97.3 and 86.3%, respectively, for the detection of alloantibodies. The respective results for the detection of autoantibodies were 95.3 and 44.9%. Serial dilution experiments with sera containing anti-HPA1a, -1b, -3b, -5b, and -15b and anti-HLA Class I revealed a higher sensitivity of the SASPA assay with all alloantibodies. CONCLUSION: In this first blind interlaboratory comparison, SASPA yielded similar results to those of MAIPA. The SASPA assay may be superior to the MAIPA assay for the detection of weak alloantibodies while simultaneous detection of a variety of antibody specificities or immunoglobulin classes and the need of fewer PLTs are obvious advantages.

BACKGROUND: White blood cell (WBC)-associated antibodies can lead to severe pulmonary transfusion reactions (transfusion-related acute lung injury [TRALI]). Investigation of a large number of blood donor samples using the standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) proved to be difficult to perform due to the time-consuming process and the large quantity of test cells required. This study describes the novel flow cytometric GIFT (Flow-GIFT) method for a rapid detection of granulocyte antibodies by flow cytometric analysis. STUDY DESIGN AND METHODS: A total of 141 sera were analyzed for the presence of granulocyte antibodies that were previously associated with suspected TRALI. As test cells whole blood samples from human neutrophil antigen (HNA)-typed donors were isolated using cell sedimentation in a ficoll density gradient. WBCs were incubated with the respective serum and binding of antibodies to the test cells was detected using fluorescein isothiocyanate―conjugated anti-human antibody. Standard GIFT and GAT were performed as reference methods. RESULTS: Seven sera containing anti-HNA-3a, CD16, and HLA Class I were negative in the standard GIFT and eight sera containing anti-HNA-2a, anti-CD16, and anti-HLA Class I were not detected in the GAT. The novel Flow-GIFT was able to detect all granulocyte antibodies, which were only detectable in a combination of standard GIFT and GAT. In serial dilution tests, the Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. CONCLUSION: The Flow-GIFT method permits rapid detection of granulocyte antibodies requiring fewer donor test cells. This method is ideal for automation and will potentially open the way for screening of granulocyte antibodies in a large donor population.

Background aims The non-exudative form of age-related macular degeneration (ARMD) is characterized by a progressive decay of retinal pigment epithelium cells at the posterior pole of the eye. As mesenchymal stromal cells (MSC) have been shown to differentiate into various cell types from the mesodermal and ectodermal lineages, we investigated whether we can induce a phenotype displaying retinal pigment epithelium (RPE) characteristics. Methods The differentiation of human lipo-aspirate-derived MSC toward the RPE lineage was triggered by exposure to conditioned medium from either human or porcine RPE cells. In a second approach we tested whether adding vasoactive intestinal peptide (VIP) is capable of further modifying differentiation processes. Resulting cell populations were assessed for expression of RPE-specific markers by immunofluorescence, quantitative real time (RT)-polymerase chain reaction (PCR) and Western blotting. The potential for pigment synthesis was assessed by the response to melanocyte-stimulating hormone (MSH). Results Following culture of undifferentiated MSC with RPE-conditioned medium and/or VIP, expression of typical RPE markers bestrophin, cytokeratins 8 and 18 and RPE 65 was induced. MSH induced the formation of pigmented granula in differentiated MSC. Conclusions MSC are shown to express RPE markers upon induction with either RPE-conditioned medium and/or VIP. The gain of basic functional features of RPE cells was indicated by melanin synthesis. This alludes to a differentiation potential of MSC into the neuroectodermal lineage, yielding cells with phenotypic characteristics of RPE cells.

SUMMARY: BACKGROUND: The Mirasol® pathogen reduction technology (PRT) for platelet concentrates (PC) uses riboflavin and UV light (270-360 nm). We evaluated the impact of PRT on platelets in comparison to standard single-donor PC. MATERIAL AND METHODS: Platelets were resuspended in autologous plasma. After 2 h rest without agitation, PC were split into an untreated control unit (C-PC) and an immediately treated unit (T-PC) (series I). In series IV, split PC were stored under agitation over night before PRT was carried out. Platelet quality was assessed by pH, glucose consumption, lactate production rate, LDH, soluble sCD62p and CD62p expression with and without TRAP (thrombin receptor-activating peptide) over 7 days. RESULTS: SERIES I: On day 5, pH values were lower for T-PC (6.8 ± 0.2 vs. 7.4 ± 0.1, C-PC), accompanied by a higher glucose consumption rate of 0.069 ± 0.016 vs. 0.035 ± 0.006 mmol/10(12) platelets/h and lactate production rate of 0.126 ± 0.031 vs. 0.063 ± 0.011 mmol/10(12) platelets/h. CD62p using TRAP was lower for T-PC (50 ± 11 vs. 62 ± 14%). Baseline activation was higher in T-PC (35 ± 12 vs. 28 ± 15%). Longer initial rest time had no impact on these results (series II/III/IV). CONCLUSION: PRT leads to an increase of platelet metabolism and activation independent of the length of the initial rest times. PC resuspended in autologous plasma should be stored at maximum up to day 5.

Over the past few years, the new technologies of transcriptomics and proteomics have been applied to platelet research. Whole-genome profiling of the transcriptome using microarray hybridization can be performed with less time and effort compared to proteome investigation. On the other hand, the significance of RNA data may be limited with regard to actual protein expression. This review compares published platelet transcriptome and proteome data in order to identify the number of discrepancies between the two molecular profiles. With a focus on membrane proteins, we wanted to know whether transcriptomic and proteomic studies may contribute to identifying novel platelet proteins.

Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept.The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values.The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale.The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.

Glycoprotein-specific platelet antibodies can cause alloor autoimmune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Recently, we int

a Institut fur Immunologie und Transfusionsmedizin, Ernst-Moritz-Arndt-Universitat Greifswald, bAbteilung Transfusionsmedizin, Universitat Rostock, c DRK Blutspendedienst Baden-Wurttemberg – Hessen, Institut fur Transfusionsmedizin und Immunologie Mannheim, dDRK Blutspendedienst NSTOB, Institut Dessau, e Institut fur Experimentelle Hamatologie und Transfusionsmedizin, Rheinische Friedrich-Wilhelms-Universitat Bonn, fKlinik fur Hamatologie und Onkologie, Charite Campus Virchow, Berlin, Deutschland

BACKGROUND: The JAHK antigen was first described in 1995 as a low-frequency red blood cell antigen. Family studies confirmed the association of the antigen with the rare rG phenotype of the Rh blood group system, which is associated with weak expression of C and e, but normal G expression. JAHK was allocated the Rh number RH53. The serologic findings indicated the location of the antigen on the RhCE protein, although the molecular basis for JAHK has not been known. STUDY DESIGN AND METHODS: The RHCE gene of eight persons from three unrelated families was analyzed by exon amplification and direct sequencing. Four of the samples were JAHK+ the remaining four were JAHK–. In one JAHK+ sample, the entire RHCE gene was sequenced. The remaining samples were sequenced for exons 1 to 3. A polymerase chain reaction procedure with sequence specific primers was developed for the specific detection of the JAHK allele. RESULTS: Analysis of the entire RHCE gene of one JAHK+ sample showed the expected CcEe-specific nucleotide sequences and revealed an additional nucleotide change (365C>T) in exon 3. This change represented a missense mutation, which led to an amino acid substitution from serine to leucine at position 122 of the RhCE protein. Three JAHK+ samples from two other unrelated families showed the 365C>T mutation and confirmed the association of the Ser122Leu substitution with the JAHK+ phenotype. CONCLUSION: The molecular basis of the JAHK antigen (RH53) is defined by a 365C>T mutation in exon 3 of the RHCE gene leading to the amino acid substitution Ser122Leu. Because the position 122 is predicted to be located in the transmembrane region adjacent to the second loop, the substitution of the neutral serine by the hydrophobic leucine seems to be the cause of the JAHK antigen by a conformational change of the RhCE protein. This structural change may also cause the weakened expression of the C and e antigens observed in JAHK+ individuals. Based on our results it is concluded that the JAHK-specific mutation is associated with a dCe haplotype.

BACKGROUND The OrbiSac device, which was developed to automate the manufacture of buffy-coat PLT concentrates (BC-PCs), was evaluated. STUDY DESIGN AND METHODS In-vitro characteristics of BC-PC preparations using the OrbiSac device were compared with manually prepared BC-PCs. For standard processing (Std-PC, n = 20), four BC-PCs were pooled using 300 mL of PLT AS (PAS) followed by soft-spin centrifugation and WBC filtration. The OrbiSac preparation (OS-PC, n = 20) was performed by automated pooling of four BC-PCs with 300 mL PAS followed by centrifugation and inline WBC filtration. All PCs were stored at 22 degrees C. Samples were withdrawn on Day 1, 5, and 7 evaluating PTL count, blood gas analysis, glucose, lactate, LDH, beta-thromboglobulin, hypotonic shock response, and CD62p expression. RESULTS A PLT content of 3.1 +/- 0.4 x 10(11) (OS-PCs) versus 2.7 +/- 0.5 x 10(11) (Std-PCs, p < 0.05) was found. A CV of 19 percent (Std-PC) versus 14 percent (OS-PC) suggests more standardization in the OS group. At Day 7, the Std-PCs versus OS-PCs showed a glucose consumption of 1.03 +/- 0.32 micro mol per 10(9) PLT versus 0.75 +/- 0.25 micro mol per 10(9) PLT (p < 0.001), and a lactate production of 1.50 +/- 0.86 micro mol per 10(9) versus 1.11 +/- 0.61 micro mol per 10(9) (p < 0.001). The pH (7.00 +/- 0.19 vs. 7.23 +/- 0.06; p < 0.001), pO(2) (45.3 +/- 18 vs. 31.3 +/- 10.4 mmHg; p < 0.01), and HCO(3) levels (4.91 +/- 1.49 vs. 7.14 +/- 0.95 mmol/L; p < 0.001) suggest a slightly better aerobic metabolism within the OS group. Only small differences in CD62p expression was observed (37.3 +/- 12.9% Std-PC vs. 44.8 +/- 6.6% OS-PC; p < 0.05). CONCLUSION The OrbiSac device allows an improved PLT yield without affecting PLT in-vitro characteristics and may enable an improved consistency in product volume and yield.

Ausblick Diese berechtigte Hoffnung sowie die wissenschaftliche Eigenstandigkeit, nicht zuletzt aber auch die wissenschaftliche Kooperationsbereitschaft, ohne die eine gedeihliche Weiterentwicklung der universitaren Transfusionsmedizin und klinischen Hamotherapie nicht denkbar sind, rechtfertigen die universitare Transfusionsmedizin in Forschung und Lehre. Die Transfusionsmedizin ist mit ihren verschiedenen, zukunftsweisenden Aufgabengebieten [s. Seifried E, Muller MM: Transfusionsmedizin – von der Blutspende zur Biotechnologie; in diesem Heft] so umfassend geworden, dass zur Wahrnehmung von Forschungs- und Entwicklungsaufgaben im Gebiet der klinischen Hamotherapie ihre Eigenstandigkeit in jeder Beziehung gerechtfertigt erscheint. Mogen auch Interessenskonflikte zwischen verschiedenen Disziplinen auf lokaler Ebene einer selbstandigen wissenschaftlichen Profilbildung der Transfusionsmedizin vor Ort entgegenwirken, zumal wenn wirtschaftliche Nutzung und seltener wissenschaftliche Leistungen im Vordergrund stehen, so kann doch zur Weiterentwicklung moderner Therapiestrategien in Ganze nur der wissenschaftliche Charakter der universitaren Transfusionsmedizin beitragen. Nur beide Aspekte – Wissenschaftlichkeit und Wirtschaftlichkeit mit hoher transfusionsmedizinischer Effizienz – konnen die aktuellen und zukunftigen Herausforderungen der klinischen Hamotherapie bewaltigen und das Fortbestehen dieses wichtigen Bausteins der modernen Hochleistungsmedizin in Diagnostik und Therapie, den Ausbau interdisziplinarer Forschung und Entwicklung sowie die wissenschaftliche Reputation eines Universitatsklinikums sichern.

Background: Hereditary haemochromatosis (HH) is the most common genetic disease with a prevalence of 1:200–1:400 in Northern Europe. In most cases HH is caused by two mutations of the HFE gene (H63D and C282Y). As haemochromatosis leads to elevated iron uptake it is conceivable that blood donors with HFE mutation might be refused from giving a donation less often than donors without the mutation. Therefore, HFE mutations might be overrepresented among frequent blood donors. This study aimed to determine the prevalence of HFE mutations in a blood donor population from Southwestern Germany. Material and Methods: The HFE mutations H63D and C282Y were analysed in 3,015 samples by PCR-SSP typing. Age and sex of the donors were considered as well as the number of blood donations. Results: No differences in frequency of both mutations could be observed between first-time and frequent donors. The prevalence of the H63D mutations was higher among female than male donors: 28.6 vs. 24.3%; p = 0.007, whereas the genotype frequencies containing the C282Y mutation were similar between male and female donors. Compared to populations from Australia or South Wales the prevalence of the C282Y mutation was lower in our blood donor population (14.8–15.8 vs. 9.0%; p Conclusions: The proposed overrepresentation of HFE mutations among frequent donors could not be confirmed. However, we found a higher prevalence of the H63D mutation in female donors than in male donors. One might therefore presume that blood donation in addition to the loss of blood due to menstruation is compensated more adequately in female H63D mutation carriers than in females without H63D mutation. This mutation seems to elevate the iron uptake so that female mutation carriers may have a higher chance to be admitted for blood donation. Individuals with diagnosed haemochromatosis-linked mutations should be informed about the potential health benefit from periodical blood donation and the admittance of such donors should be considered in Germany.

BACKGROUND: Dendritic cells (DCs) for immunotherapy of malignant melanoma can be generated from partially enriched monocytes prepared from PBMNCs. The feasibility of a single steady-state leukapheresis procedure to enrich monocytes for a complete vaccination series with up to 10 vaccinations was investigated. STUDY DESIGN AND METHODS: Thirty-eight patients (27 males and 11 females) with metastatic melanoma were enrolled in the study. All leukapheresis procedures were performed by a continuous flow method (Spectra, Cobe BCT) with a standard MNC program. RESULTS: An average of 11.7 L (range, 8-14 L) of whole blood was processed within 197.3 ± 23.7 minutes, and a mean of 13.5 ± 5.7 × 109 WBCs in a final volume of 191.0 ± 24.2 mL was collected. The MNC purity in the apheresis component was 81.5 ± 15.1 percent, from which 29.8 ± 14.7 percent were monocytes. Thus, 11.0 ± 5.0 × 109 MNCs and 3.2 ± 2.0 × 109 monocytes were collected per procedure. Linear regression analysis revealed a high correlation between the absolute number of monocytes in peripheral blood before the apheresis procedure and the number of monocytes in the collected component (r = 0.74, p < 0.0001). For the generation of DCs, 1.6 ± 0.8 × 109 MNCs were plated into culture dishes; 3.2 ± 1.8 percent of the cultured cells matured to DCs, which resulted in 56.5 ± 49.4 × 106 DCs (range, 6.3-178) per patient for the complete vaccination series. CONCLUSION: A target dose of monocytes for the complete vaccination series could be obtained by a single convenient, safe, steady-state leukapheresis procedure in each patient without the need for G–CSF mobilization. The absolute number of monocytes in peripheral blood before the apheresis procedure is the best predictive variable for the yield of monocytes in the apheresis component.

Background: By inhibiting nucleic acids, photochemical treatment (PCT) using amotosalen hydrochloride (S-59) in combination with UVA light provides a new method for the inactivation of pathogens and leukocytes, potential contaminants of platelet concentrates (PCs). Material and Methods: We evaluated the effect of PCT on function and viability of PCs derived from apheresis over a storage period of 5 days. Double-dose PCs containing 6.5–7.0 × 1011 platelets (n = 8) were collected in 600 ml of approximately 35% autologous plasma and 65% platelet additive solution. Each collection was split into two equal-sized portions for paired analysis. One day after collection, test PCs were mixed with 15 ml of 3 mmol/l S-59 and illuminated with UVA light for 3 min, followed by a 6- to 8-hour incubation in a compound adsorption device (CAD) for the reduction of residual S-59 and unbound photoproducts. In vitro platelet function measurements included platelet count, pH, lactate dehydrogenase (LDH), β-thromboglobulin (β-TG), hypotonic shock response (HSR), extent of shape change (ESC) and the expression of p-selectin. Platelet morphology and ultrastructure were analyzed by light and electron microscopy (EM). Results: We found no statistically significant differences on day 5 of the storage between the control and test platelets for pH, platelet count, p-selectin, ESC, HSR, morphology, and the percentage of lysed platelets analyzed by EM. The level of β-TG was reduced in the test units by the CAD step. Significant differences (p Conclusion: These in vitro results indicate that PCT with amotosalen and UVA light is applicable for split double-dose platelets derived from apheresis as it leads only to a transient activation of platelets immediately after the procedure, without affecting the function and structure of PCs over the 5-day storage period.

Background/Aims: The aim of this study was to compare donor return patterns of non-compensated and compensated German first-time donors to assess the effect of monetary reward on donor return. Methods: We conducted a retrospective analysis of a donor survey of 3,077 non-compensated and 738 compensated German first-time donors. Survey data were pooled and linked with blood donor return rates within the 1st, 2nd, and 3rd year. Logistic regression models were used to estimate differences in the probability of donor return between non-compensated and compensated donors. Results: In the first 2 years following the initial donation, compensated donors were more likely to return with the odds of giving at least one further donation 1.86 (1st year) and 1.32 (2nd year) times higher for compensated donors than for non-compensated donors. In the 3rd year, there were no significant differences in donor return. Conclusion: This report, which was based on two non-randomized donor samples, suggests that monetary compensation may increase the likelihood of donors returning in the first months after the initial donation. Monetary reward may therefore be used as a short-term strategy to recruit new donors. The long-term commitment, however, seems not to be affected by monetary reward, and complementary donor retention strategies are needed.

BACKGROUND: The objective of this work was to develop a novel and highly sensitive RT-PCR method that is suitable for HCV RNA screening of blood donations according to the criteria released by the Paul Ehrlich Institute, the federal licensing agency of Germany, for routine HCV NAT. STUDY DESIGN AND METHODS: RNA was prepared from plasma pools of up to 20 single blood donations using an automated nucleic acid isolation system (NucliSens Extractor, Organon Teknika). For reverse transcription, amplification, and simultaneous detection of PCR products, a novel approach based on the TaqMan technology was developed. Glyceraldehyde-3-phosphate dehydrogenase messenger RNA, which is detectable in human plasma, was coamplified in each reaction as an internal positive control. RESULTS: The HCV genotypes and subtypes 1a, 1b, 2a, 2b, 2c, 2i, 3a, 4, and 5a were detected in parallel with comparable amplification efficiency. The 95-percent detection limit related to the WHO HCV RNA standard preparation was calculated to be 389 IU per mL of plasma of the single blood donation. Total CVs (%) were

DNA samples isolated from corresponding uncultured amniotic fluid, cord blood and maternal blood (n=5) were subjected to low resolution typing of the HLA-A, -B and -DRB loci by the polymerase chain reaction using sequence-specific primers (PCR-SSP). Furthermore, the effect of ethylene diamine tetraacetate disodium salt (EDTA) on the quality of genomic DNA isolated from amniotic fluid samples after long-term storage was evaluated. Unambiguous results of HLA typing could be achieved from all amniotic fluid samples stabilized with EDTA. PCR-SSP typing failed in DNA samples from amniotic fluid without the addition of EDTA. In all cases the fetal HLA type could be confirmed by the result from the corresponding cord blood typing. Contamination with maternal DNA led to additional weak PCR-SSP bands in one case, but data interpretation was still unambiguous. Reliable fetal HLA typing can be achieved directly from amniotic fluid and culturing of amniocytes is not required.

The interest in the quantitative analysis of cytokine mRNA profiles has increased substantially in recent years. This is based on the potential use of basal cytokine mRNA expression as sensitive markers for in vivo lymphocyte activation in a variety of clinical settings. However, it is less well known to what extent differences in blood collection and preparation techniques may cause ex vivo alteration of quantitative cytokine mRNA levels. We therefore evaluated the effect of blood sampling and the impact of cell separation on interleukin (IL)-2, IL-4, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha mRNA expression in an intraindividual study design (n=8). Two different blood sampling procedures were applied. A whole blood sample 1 was collected by constant moderate blood flow into a blood collection tube containing lithium-heparin. Moreover, a second sample from the same donor was collected by a 5-fold acceleration of blood flow. Furthermore, peripheral blood mononuclear cell (PBMC) were isolated from the first whole blood sample by density separation over Ficoll-Hypaque. The quantification of cytokine mRNA expression was performed by real-time PCR in native whole blood/PBMC samples or unstimulated cultures. We found a significant increase of IL-2, IL-4 and TNF-alpha mRNA expression (P=0.018, P=0.028, P=0.018) in whole blood samples collected by rapid sampling. The isolation of PBMC by density gradient separation prompted on upregulation of the mRNA levels of IL-2, IL-4 and TNF-alpha 5-9-fold (P=0.018, P=0.018, P=0.018). In contrast, IFN-gamma mRNA expression was not significantly influenced by differences in blood sample preparation. Our data clearly demonstrate that differences in the blood sampling technique or cell separation should be considered as important factors for non-physiological ex vivo induction of cytokine mRNA expression. The current data emphasize the need for data on the impact of ex vivo variation in order to extract reliable and consistent information, particularly when cytokine mRNA expression data from healthy blood donors are included in clinical studies.

1 Current Knowledge about the Pathogen 1.1 Characteristics of Hepatitis A Virus The clinical features of hepatitis A have been known for a long time. However, the pathogen (hepatitis A virus (HAV)) was firstly identified in 1973 with the aid of immune electron microscopy [1]. HAV is classified in the family of Picornaviridae , genus Hepatovirus . It is a small, spherical, non-enveloped virus with a diameter of approximately 30 nm. The capsid of the viral particle is made up of 60 copies of the three viral structural proteins VP1–VP3. The genome consists of a single- stranded (+)sense RNA molecule of 7,500 bases in length. The 5'' end of the genome is covalently bound to a small protein (VPg). The genome serves as messenger RNA and can be divided into three regions: a long open-reading frame (ORF) is flanked by the non-coding 5'' region and the non-coding 3'' region. The ORF encodes a polyprotein which is cleaved by proteolysis into the mature viral proteins. Two thirds of the genome code for non-structural proteins with enzymatic and regulatory functions. Translation is controlled by an ‘internal ribosomal entry site’ (IRES) located within the non-coding 5'' region [2]. Only one serotype of HAV has been identified so far. Analysis of HAV genome sequences has revealed 6 genotypes to date [3], with genotype I predominating worldwide. Genotypes IV–VI have only been observed in Old World monkeys. ...

Background: The combination of IL-1, IL-6, IL-11, stem cell factor (SCF), and thrombopoietin (TPO) is capable of stimulating megakaryocytopoiesis using a bone marrow CD34+ cell po

Background Nonhemolytic transfusion reactions (NHTRs) frequently occur after platelet transfusions. White cell (WBC)-derived inflammatory cytokines can cause these reactions, but they are rarely found in WBC-poor platelet preparations. Transfusion reactions were investigated with regard to the residual WBC content in the stored platelet concentrate in two consecutive study periods. Study design and methods In the first study period, platelet concentrates were WBC-reduced by bedside filtration. In the second period, all platelet concentrates were filtered before storage. Recipients who experienced transfusion reactions were examined with regard to their main clinical symptoms during and after transfusion. In the supernatant of the involved platelet concentrates, concentrations of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor (TNF)alpha, macrophage inflammatory protein 1alpha, and RANTES were analyzed. Results The incidence of transfusion reactions remained steady when the transfusion regimen was changed from bedside filtration to prestorage WBC filtration (1.63% and 1.56%; p = 0.84). In both periods, NHTRs were predominantly of allergic origin. Inflammatory mediators IL-1beta, IL-6, IL-8, and TNFalpha were detectable in only a minority of platelet components involved in NHTRs. Platelet concentrates involved in allergic reactions contained high concentrations of RANTES (668 +/- 223 ng/mL). Conclusions Prestorage WBC filtration did not reduce the incidence of these reactions, and inflammatory cytokines were of minor relevance. The proinflammatory platelet-derived chemokine RANTES, which accumulates even in WBC-reduced platelet concentrates, was associated with allergic transfusion reactions. Platelet-derived mediators may be a key to understanding NHTRs.

An involvement of immunological events in the process of neurodegeneration has frequently been reported. We investigated the cytokine producing capacity for interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in whole blood cultures of de-novo patients with idiopathic Parkinson's disease (PD) at the time of first diagnosis and after oral amantadine treatment. Before treatment, productions of IL-2 and IFN-gamma were markedly decreased in PD patients compared to patients with major depressive disorder and healthy controls. After amantadine treatment, the in vitro IL-2 secretion defect was corrected to normal levels in half of the patients, and the increase in IL-2 production was correlated with an increase in IFN-gamma secretion. Our findings suggest that immunological abnormalities occur in the course of PD and that a formerly unappreciated therapeutic potential of amantadine may arise from its immunomodulatory effects on altered T cell function in patients with PD.

BACKGROUND: Platelet concentrates were recently used for ophthalmologic treatment of macular holes. This strategy was investigated to define standardized blood bank components. STUDY DESIGN AND METHODS: Two different, highly concentrated autologous platelet components, one from whole-blood preparation and the other from plateletpheresis, were evaluated. In the first procedure, platelet-rich plasma was obtained from 250 mL of whole blood. After storage for 20 hours, platelet-rich plasma was concentrated In a second centrifugation step and adjusted to 10 × 109 platelets per mL. In the second procedure, platelets were collected by apheresis, stored overnight, centrifuged, and adjusted to 20 × 109 platelets per mL. The respective component was instilled during vitrectomy and gas tamponade in patients with stage II to IV macular holes. Patients were followed for 9 months. RESULTS: With regard to the various preparation procedures and final concentrations of platelets in the components, no differences in wound healing were observed. An anatomic closure of the macular hole was achieved in 18 of 19 treated patients. Visual acuity improved in 14 patients. CONCLUSION: Both types of highly concentrated platelet components were effective in achieving high closure rates of macular holes. These autologous platelet components possess the quality standard of blood bank components and could be of great benefit for initiating wound healing in other clinical settings.

Background: In recent years, peripheral blood stem cell transplantation has replaced autologous bone marrow transplantation to a large extent in patients with hem

Background White cell (WBC) reduction prior to storage of red cell (RBC) concentrates may reduce the incidence of HLA alloimmunization and may improve the quality of stored RBCs. Study design and methods An integrated WBC-reduction filter system was tested after various RBC preparation procedures (from whole blood), and the influence of filtration on RBCs during storage for 42 days was investigated. Four additive system RBC preparation protocols were used. Units prepared from conventional triple blood bags were held for 4 to 6 hours at 22 degrees C, and then the RBCs were separated via a hard spin and filtration, performed immediately (Group 1) or after 18 hours' storage at 4 degrees C (Group 2). Units prepared from a top-and-bottom collection system were held at 22 degrees C for 4 to 6 or 22 to 24 hours; the centrifuged RBCs were filtered immediately after preparation (Groups 3 and 4, respectively, by holding time). WBC reduction and filtration time were analyzed. The impact of WBC filtration on pH, hemolysis rate, hemoglobin content, ATP, potassium glucose, and lactate was investigated weekly during storage for 42 days. Results Filtration reduced the mean WBC count by 3 to 4 log10, to 0.19 +/- 0.25 x 10(6), regardless of the RBC preparation method. Mean filtration times differed significantly between the groups and were longest for Group 1. Besides hemolysis and pH values, which were greater in all filtered units, no major differences were found in filtered and unfiltered RBCs during the storage interval. Conclusion The efficiency of prestorage WBC filtration of RBCs was unaffected by the preparation procedure. However, the filtration time for RBCs freshly prepared in the conventional triple blood bag system without buffy-coat depletion was unacceptable. No major metabolic differences between filtered and unfiltered RBCs during 42 days of storage were found.

Background Whole blood-derived buffy coat (BC) has become an alternative source from which to prepare random-donor platelet concentrates. The influence of prolonged storage of BC prior to platelet concentrate preparation is a matter of controversy. The impact of BC storage on cytokine release was evaluated and the platelet activation quantified. Study design and methods BCs were prepared from whole-blood donations after hard-spin centrifugation. After 1, 3, 6, 12, and 24 hours of storage at 22 degrees C without agitation, samples were withdrawn for cell count and blood gas analysis and measurement of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor alpha and platelet factor 4. Platelet surface markers CD41a, CD42b, CD62P, and CD63 were analyzed by flow cytometry, and the antibody-binding sites were quantified by using microbeads. Results Inflammatory cytokines IL-1beta, IL-6, and tumor necrosis factor alpha were hardly detectable in stored BCs but levels of IL-8 increased in 25 percent of BCs after 24 hours. A constant increase in platelet factor 4 was observed, which accelerated after 12 hours of storage. Analysis of platelet surface markers showed an initial decrease of platelet activation, followed by an increase after storage for 12 to 24 hours. Conclusion Storage of BCs for up to 12 hours without agitation showed a good preservation of platelets but storage of BCs for 24 hours resulted in increased platelet activation and significantly higher release of platelet factor 4 and IL-8. Stored BCs might well be suitable for platelet preparation, but their storage time should not greatly exceed 12 hours.