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After recovery, mild and severe COVID-19 diseases are associated with long-term effects on the host immune system, such as prolonged T-cell activation or accumulation of autoantibodies. In this study, we show that mild SARS-CoV-2 infections, but not SARS-CoV-2 spike mRNA vaccinations, cause durable atopic risk factors such as a systemic Th2- and Th17-type environment as well as activation of B cells responsive of IgE against aeroallergens from house dust mite and mold. At an average of 100 days post mild SARS-CoV-2 infections, anti-mold responses were associated with low IL-13 levels and increased pro-inflammatory IL-6 titers. Acutely severely ill COVID-19 patients instead showed no evidence of atopic reactions. Considering convalescents of mild COVID-19 courses and mRNA-vaccinated individuals together, IL-13 was the predominant significantly upregulated factor, likely shaping SARS-CoV-2 immunity. Application of multiple regression analysis revealed that the IL-13 levels of both groups were determined by the Th17-type cytokines IL-17A and IL-22. Taken together, these results implicate a critical role for IL-13 in the aftermath of SARS-CoV-2 mild infections and mRNA vaccinations, conferring protection against airway directed, atopic side reactions that occur in mildly experienced COVID-19.

ObjectiveGenetic determinants of fibrin clot formation and fibrinolysis have an impact on local and systemic inflammatory response. The aim of the present study was to assess whether coagulation-related genotypes affect the predictive value of C-reactive protein (CRP) in regards of radiographic spinal progression in axial spondyloarthritis (axSpA).MethodsTwo hundred and eight patients with axSpA from the German Spondyloarthritis Inception Cohort were characterised for genotypes of α-fibrinogen, β-fibrinogen (FGB) and γ-fibrinogen, factor XIII A-subunit (F13A) and α2-antiplasmin (A2AP). The relation between CRP levels and radiographic spinal progression defined as worsening of the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) by ≥2 points over 2 years was assessed in dependence on the respective genetic background in logistic regression analyses.ResultsOverall, CRP was associated with mSASSS progression ≥2 points: time-averaged CRP ≥10 mg/L, OR: 3.32, 95% CI 1.35 to 8.13. After stratification for coagulation-related genotypes, CRP was strongly associated with mSASSS progression in individuals predisposed to form loose, fibrinolysis-susceptible fibrin clots (FGB rs1800790GG, OR: 6.86, 95% CI 2.08 to 22.6; A2AP 6Trp, OR: 5.86, 95% CI 1.63 to 21.0; F13A 34Leu, OR: 8.72, 95% CI 1.69 to 45.1), while in genotypes predisposing to stable fibrin clots, the association was absent or weak (FGB rs1800790A, OR: 0.83, 95% CI 0.14 to 4.84; A2AP 6Arg/Arg, OR: 1.47, 95% CI 0.35 to 6.19; F13A 34Val/Val, OR: 1.72, 95% CI 0.52 to 5.71).ConclusionsElevated CRP levels seem to be clearly associated with radiographic spinal progression only if patients are predisposed for loose fibrin clots with high susceptibility to fibrinolysis.

The variability in resolution of SARS-CoV-2-infections between individuals neither is comprehended, nor are the long-term immunological consequences. To assess the long-term impact of a SARS-CoV-2-infection on the immune system, we conducted a prospective study of 80 acute and former SARS-CoV-2 infected individuals and 39 unexposed donors to evaluate autoantibody responses and immune composition. Autoantibody levels against cyclic citrullinated peptide (CCP), a specific predictor for rheumatoid arthritis (RA), were significantly (p = 0.035) elevated in convalescents only, whereas both acute COVID-19 patients and long-term convalescents showed critically increased levels of anti-tissue transglutaminase (TG), a specific predictor of celiac disease (CD) (p = 0.002). Both, anti-CCP and anti-TG antibody levels were still detectable after 4–8 months post infection. Anti-TG antibodies occurred predominantly in aged patients in a context of a post-SARS-CoV-2-specific immune composition (R2 = 0.31; p = 0.044). This study shows that increased anti-CCP and anti-TG autoantibody levels can remain long-term after recovering even from mildly experienced COVID-19. The inter-relationship of the lung as viral entry side and RA- and CD-associated autoimmunity indicates that a SARS-CoV-2-infection could be a relevant environmental factor in their pathogenesis.

Adenovirus (HAdV) infections confer a high risk of morbidity and mortality for immunocompromised patients after stem cell transplantation (SCT). Treatment with standard antiviral drugs is of limited efficacy and associated with a high rate of adverse effects. HAdV-specific T cells are crucial for sustained viral elimination and the efficacy of adoptive T-cell therapy with donor-derived HAdV-specific T cells has been reported by several investigators. Here, we report our experience with the transfer of HAdV-specific T cells specific for penton, which was recently identified as an immunodominant target of T cells, and hexon in a 14-year-old boy after T-cell-depleted haploidentical SCT for myelodysplastic syndrome (MDS). He developed severe HAdV-associated enteritis complicated by acute graft-versus-host disease (GvHD). The patient received ten infusions of allogeneic HAdV-specific T cells manufactured from the haploidentical stem cell donor using the CliniMacs Interferon-γ (IFN-γ) cytokine capture and immunomagnetic selection. Initially, T cells were generated against the immunodominant target hexon and in subsequent transfers dual antigen-specific T cells against hexon and penton were applied. T-cell transfers were scheduled individually tailored to current immunosuppressive treatment. Each transfer was followed by reduction of HAdV load in peripheral blood and clinical improvement. Importantly, T-cell responses to both penton and hexon pools emerged in patient blood after repetitive transfers. Unfortunately, the patient experienced bacterial sepsis, and in this context, severe GvHD requiring intensive immunosuppression followed by secondary progression of HAdV infection. The patient succumbed to multiorgan failure 283 days after SCT. This case demonstrates the feasibility of HAdV-specific T-cell transfer even in the presence of immunosuppressive treatment. Targeting of multiple immunodominant viral proteins may prove valuable in patients with complicated HAdV infections.

Background Adoptive transfer of virus-specific T cells (VSTs) represents a prophylactic and curative approach for opportunistic viral infections and reactivations after transplantation. However, inadequate frequencies of circulating memory VSTs in the T-cell donor’s peripheral blood often result in insufficient enrichment efficiency and purity of the final T-cell product, limiting the effectiveness of this approach. Methods This pilot study was designed as a cross-over trial and compared the effect of a single bout (30 min) of high-intensity interval training (HIT) with that of 30 min of continuous exercise (CONT) on the frequency and function of circulating donor VSTs. To this end, we used established immunoassays to examine the donors’ cellular immune status, in particular, with respect to the frequency and specific characteristics of VSTs restricted against Cytomegalovirus (CMV)-, Epstein–Barr-Virus (EBV)- and Adenovirus (AdV)-derived antigens. T-cell function, phenotype, activation and proliferation were examined at different time points before and after exercise to identify the most suitable time for T-cell donation. The clinical applicability was determined by small-scale T-cell enrichment using interferon- (IFN-) γ cytokine secretion assay and virus-derived overlapping peptide pools. Results HIT proved to be the most effective exercise program with up to fivefold higher VST response. In general, donors with a moderate fitness level had higher starting and post-exercise frequencies of VSTs than highly fit donors, who showed significantly lower post-exercise increases in VST frequencies. Both exercise programs boosted the number of VSTs against less immunodominant antigens, specifically CMV (IE-1), EBV (EBNA-1) and AdV (Hexon, Penton), compared to VSTs against immunodominant antigens with higher memory T-cell frequencies. Conclusion This study demonstrates that exercise before T-cell donation has a beneficial effect on the donor’s cellular immunity with respect to the proportion of circulating functionally active VSTs. We conclude that a single bout of HIT exercise 24 h before T-cell donation can significantly improve manufacturing of clinically applicable VSTs. This simple and economical adjuvant treatment proved to be especially efficient in enhancing virus-specific memory T cells with low precursor frequencies.

Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD) with central nervous system (CNS) involvement is a severe complication after solid organ transplantation. Standard treatment with reduction of immunosuppression and anti-CD20 antibody application often fails leading to poor outcome. Here, we report the case of an 11-year-old boy with multilocular EBV-positive CNS PTLD 10 years after liver transplantation. Complete remission was achieved by repeated intravenous and intrathecal anti-CD20 antibody rituximab administration combined with intrathecal chemotherapy (methotrexate, cytarabine, prednisone) over a time period of 3 months. Due to the poor prognosis of CNS PTLD and lack of EBV-specific T-cells (EBV-CTLs) in patient's blood, we decided to perform EBV-directed T-cell immunotherapy as a consolidating treatment. The patient received five infusions of allogeneic EBV-CTLs from a 5/10 HLA-matched unrelated third-party donor. No relevant acute toxicity was observed. EBV-CTLs became detectable after first injection and increased during the treatment course. Next-generation sequencing (NGS) TCR-profiling verified the persistence and expansion of donor-derived EBV-specific clones. After two transfers, epitope spreading to unrelated EBV antigens occurred suggesting onset of endogenous T-cell production, which was supported by detection of recipient-derived clones in NGS TCR-profiling. Continuous complete remission was confirmed 27 months after initial diagnosis.

Background Donors with short interdonation intervals (e.g., apheresis donors) have an increased risk of window period donations. The frequency of cytomegalovirus (CMV) window period donations is important information to decide whether selection of seronegative donors might be advantageous for patients at risk for transfusion-transmitted CMV infections (TT-CMV). Study Design and Methods CMV seroconversion in 93 donors with positive results in routine CMV antibody testing within at most 35 days after the last seronegative sample was evaluated by Western blot and/or a second antibody test. In donors with unconfirmed seroconversion, an additional later sample was tested. Concentration of CMV DNA was determined in pre- and postseroconversion samples. Results CMV seroconversion was confirmed in 12 donors (13%). Among these, the last seronegative sample was CMV DNA positive in three donors (25%, below 30 IU/mL). The first seropositive sample was CMV DNA positive in 10 donors (83%, maximum 1600 IU/mL). Both prevalence and median concentration of CMV DNA were higher in the first seropositive sample (p = 0.004 and p = 0.02), with maximum concentrations being reached about 2 weeks after seroconversion. No CMV DNA was detected in samples from donors with unconfirmed seroconversion. Conclusion At least in donors with short interdonation intervals, most suspected CMV seroconversions are due to false-positive results of the screening test. As window period donations are rare and contain less CMV DNA than the first seropositive donation, avoidance of blood products from primarily seropositive donors is especially helpful to avoid TT-CMV if donors with short interdonation intervals are concerned.

Background and aims. The infusion of enriched CMV-specific donor T-cells appears to be a suitable alternative for the treatment of drug resistant CMV reactivation or de novo infection after both solid organ and hematopoietic stem cell transplantation. Antiviral lymphocytes can be selected from apheresis products using the CliniMACS Cytokine-Capture-System® either with the well-established CliniMACS® Plus (Plus) device or with its more versatile successor CliniMACS Prodigy® (Prodigy). Methods. Manufacturing of CMV-specific T-cells was carried out with the Prodigy and Plus in parallel starting with 0.8-1*109 leukocytes collected by lymphapheresis (n=3) and using the MACS GMP PepTivator® HCMVpp65 for antigenic re-stimulation. Target and non-target cells were quantified by a newly developed single-platform assessment and gating strategy using positive (CD3/CD4/CD8/CD45/IFN-gamma), negative (CD14/CD19/CD56), and dead cell (7-AAD) discriminators. Results. Both devices produced largely similar results for target cell viabilities: 37.2%-52.2% (Prodigy) vs. 51.1%-62.1% (Plus) CD45+/7-AAD- cells. Absolute numbers of isolated target cells were 0.1-3.8*106 viable IFN-gamma+ CD3+ cells. The corresponding proportions of IFN-gamma+ CD3+ cells ranged between 19.2% and 95.1% among total CD3+ cells and represented recoveries of 41.9%-87.6%. Within two parallel processes predominantly IFN-gamma+ CD3+CD8+ cytotoxic T-cells were enriched compared to one process that yielded a higher amount of IFN-gamma+ CD3+CD4+ helper T lymphocytes. T-cell purity was higher for the Prodigies products that displayed a lower content of contaminating IFN-gamma- T-cells (3.6%-20.8%) compared to the Plus products (19.9%-80.0%). Conclusions. The manufacturing process on the Prodigy saved both process and hands-on time due to its higher process integration and ability for unattended operation. Although the usage of both instruments yielded comparable results, the lower content of residual IFN-gamma- T-cells in the target fractions produced with the Prodigy may allow for a higher dosage of CMV-specific donor T-cells without increasing the risk for graft versus host disease.

BACKGROUND: The objective was to investigate potential risks for apheresis donors associated with a triple-plateletpheresis (TP) program. STUDY DESIGN AND METHODS: Eleven hemapheresis centers randomly assigned 411 repeat donors (ratio, 1:1.2) to either double plateletpheresis (DP; 185 donors) or TP (226 donors) with a platelet (PLT) target content of at least 5.0 × 1011 PLTs/DP and at least 7.5 × 1011 PLTs/TP. The primary endpoint was procedure-related postapheresis PLT count of at least 150 × 109/L (probability, ≥98%). Secondary endpoints were apheresis characteristics and donor adverse reactions. RESULTS: In 6 of 1133 DPs (0.5%) in 4 of 185 donors (2.2%) and in 20 of 1020 TPs (2.0%) in 14 of 226 donors (6.2%), postapheresis PLT counts were below 150 × 109/L. There were marginal but significant differences in collection efficiency (DP, 69.2 ± 9.1%; TP, 70.9 ± 9.0%; p ≤ 0.0001) and collection rate (DP, 10.4 × 109 ± 2.3 × 109 PLTs/min; TP, 10.8 × 109 ± 2.3 × 109 PLTs/min; p ≤ 0.005). The PLT yields were 5.9 × 1011 ± 0.8 × 1011 PLTs for DP and 8.3 × 1011 ± 0.9 × 1011 PLT for TP (p ≤ 0.0001) at processing times of 59 ± 13 minutes (DP) versus 80 ± 16 minutes (TP; p ≤ 0.0001). Significant PLT recruitment (1.10 ± 0.14 vs. 1.20 ± 0.23; p

The reactivation of the human cytomegalovirus (CMV) can be prevented or controlled by the adoptive transfer of ex vivo expanded donor-derived CMV-specific T lymphocytes. Several methods for expansion and adoptive transfer of CMV-specific T cells have been developed using either defined CMV peptides or peptide pools for antigen-specific T-cell stimulation. The majority of studies have focused on the lower matrix protein (pp65) and the immediate-early protein-1 (IE-1) of CMV as immunodominant targets. We investigated the behavior of secretory CMVpp65 (sCMVpp65) with respect to its capacity to stimulate pp65-specific T cells independently of human lymphocyte antigen (HLA) type and even in donors unresponsive to the immunodominant HLA-A*0201-restricted CMVpp65495-503 peptide. To facilitate the eukaryotic expression and isolation procedures, we constructed an HLA-A*0201/CMVpp65 fusion protein that is secreted into the supernatant of human embryonic kidney 293 (HEK293) cells. CMV-specific CD4 and CD8 T cells generated by culturing unfractionated peripheral blood mononuclear cells in the presence of recombinant sCMVpp65 did not differ in function with regard to cytotoxicity and interferon-gamma (IFN-gamma) production compared with cytotoxic T cells induced using the well-studied HLA-A*0201-restricted CMVpp65495-503 peptide. We demonstrated that polyclonal CMV-specific T cells could be generated from CMV-seropositive individuals expressing HLA alleles for which no immunogenic epitopes have been identified so far. The production of recombinant sCMVpp65 can easily be adapted to good manufacturing practice conditions and can be used to generate large numbers of immunogenic pathogen-derived proteins for therapeutic applications.

BACKGROUND: Delayed donor red blood cell chimerism (DRCC), pure red blood cell aplasia (PRCA), and autoimmune hemolytic anemia (AIHA) are poorly documented complications after hematopoietic cell transplantation (HCT). The clinical variable “prolonged isolated red blood cell transfusion requirement” (PRTR) was evaluated as a trigger for an extended diagnostic workup. STUDY DESIGN AND METHODS: PRTR was defined as the need for red blood cell (RBC) transfusions beyond Day 60 after HCT. We analyzed 487 patients transplanted between 2000 and 2006. Median age was 37 years (range, 0-70 years). Peripheral blood stem cells (n = 344), marrow (n = 138), and cord blood (n = 5) from 278 unrelated and 209 family donors were used. RESULTS: Univariate analysis identified age (incidence of 18.3% among elderly patients, 10.5% in adults, and 2.0% among children [p = 0.002]), ABO incompatibility (16.4% after major incompatible, 2.9% after minor incompatible, and 9.4% after ABO-compatible transplantations [p = 0.003]), conditioning (15.2% after reduced-intensity regimens vs. 7.3% after myeloablative conditioning; p = 0.006), donor type (13.2% after HLA-matched unrelated, 13.6% after mismatched unrelated, 5.7% after matched related, and 0.0% after mismatched related grafts; p = 0.026), and acute graft-versus-host disease (aGVHD; 7.1% with aGVHD vs. 12.5% without aGVHD; p = 0.046) as predisposing factors. In multivariate analysis minor ABO incompatibility (odds ratio [OR] = 0.2, p = 0.01), younger age (OR = 0.1, p = 0.02), and matched related HCT (OR = 0.4, p = 0.02) remained independent protective factors. CONCLUSIONS: PRTR could serve as a trigger for a standardized screening for DRCC, PRCA, and AIHA after HCT.

We compare the actual with the potential donor exposure and possible infection rates in the Hanover Medical School (MHH) platelet (PLT) transfusion recipients if the current MHH standard of apheresis PLT concentrate (A-PC) supply would be replaced by a pooled PLT concentrate (P-PC) transfusion regimen.The electronic records of the MHH Institute of Transfusion Medicine and the MHH Department of Medical Controlling were evaluated to assess the development of PLT needs and supply at MHH from 2003-2006. For 2006, we evaluated all PLT transfusion recipients with respect to their overall transfusion needs, classified them for low and high PLT transfusion needs, and related them to the diagnostic groups that underlie their PLT demands. We assumed a P-PC preparation procedure using 4 whole blood-derived buffy coats for all calculations for potential donor exposure. To predict the possible infection rates of an unrecognized viral infection with low prevalence in the general population to A-PC or to P-PC recipients and the influence of neutralizing agent specific antibodies (NAB), we established a mathematical contamination/infection model based on the current PLT transfusion mode and data about GBV-C virus infection among Hanover blood donors.From 2003 to 2006, the 1,300-1,400 persons comprising MHH apheresis donor pool covered a 36% increase in PC transfusions. The exclusive use of P-PCs instead of A-PC would require a total of 36,240-49,276 whole blood donations to meet MHH demands, corresponding to a more than 1 log step increase in donor exposure. For individual hematological patients, the change to P-PCs would imply an 80-125%, for individual surgical patients a 40-50% higher donor exposure. Our infection model revealed an approximately 4 times higher infection.A change to P-PC would imply a more than one log step higher donor exposure, and an unrecognized infection with a prevalence around 1% leads to an up to 4 times higher infection rate. A general change in the PC transfusion policy that favors P-PCs is dangerous and must be avoided.Ziel der Studie war die Erfassung der tatsächlichen im Vergleich zur potentiellen Spenderexposition sowie der möglichen Infektionsraten bei Empfängern von Thrombozytenkonzentraten (TK), falls der gegenwärtige Transfusionsstandard an der Medizinischen Hochschule Hannover (MHH) – die Patientenversorgung mit Apheresekonzentraten (A-TK) – von einer Transfusionsstrategie mit gepoolten Thrombozytenkonzentraten (P-TK) abgelöst würde.Wir haben die elektronischen Datenbanken des MHH-Instituts für Transfusionsmedizin sowie der MHH-Abteilung für Medizinkontrolling bezüglich der Entwicklung des Thrombozytenbedarfs und der Thrombozytenversorgung für die Jahre 2003–2006 ausgewertet. Bezogen auf das Jahr 2006 wurden alle MHH-Thrombozytenempfänger hinsichtlich des Gesamttransfusionsbedarfs, niedrigen bzw. hohen Thrombozytenbedarfs bzw. auf die dem Thrombozytenbedarf zugrunde liegenden Erkrankungen untersucht. Auf der Basis von 4 Buffy Coats aus Vollblutkonserven als Ausgangsmaterial für die Herstellung eines P-TK berechneten wir die potentielle Spenderexposition für die MHH-TK-Empfänger. Wir entwickelten ein mathematisches Modell, um mögliche Infektionsraten bei einer unerkannten viralen Infektion mit niedriger Prävalenz in der Allgemeinbevölkerung bei A-TK-oder bei P-TK-Empfängern unter Einschluss neutralisierender virusspezifischer Antikörper vorherzusagen. Dieses Modell basiert auf der gegenwärtigen Transfusionspraxis für TK in Hannover und bekannten Daten zu GBV-C-Virusinfektionen bei Hannoveraner Blutspendern.Der 1,300–1,400 Personen umfassende MHH-Apheresespenderpool stellte für die Jahre 2003 bis 2006 sicher, dass der 36%ige Anstieg der Thrombozytentransfusionen bewältigt werden konnte. Die ausschlieβliche Anwendung von P-TK anstelle von A-TK hätte 36.240–49.276 Vollblutspender erfordert, was einem Anstieg der Spenderexposition um mehr als eine Logstufe entsprochen hätte. Individuelle Thrombozytenempfänger mit niedrigem, moderatem, hohem und sehr hohem A-TK-Bedarf hätten eine um 42, 67, 84 und 109% höhere Spenderexposition gehabt. Für individuelle hämatologische Patienten hätte der Wechsel des Transfusionsregimes zu P-TK eine um 80–125%, für individuelle chirurgische Patienten eine um 40–50% höhere Spenderexposition zur Folge gehabt. Unser Rechenmodell zur Abschätzung von Infektionsfolgen zeigt eine zirka viermal höhere Infektionsrate.Ein Wechsel der Transfusionsstrategie auf Pool-TK würde mit einer um mehr als eine Log-Stufe erhöhten Spenderexpositition einhergehen, und eine unerkannte Infektion mit einer Prävalenz von zirka 1% führt zu einer um den Faktor 4 erhöhten Infektionsrate bei Pool-TK-Empfängern. Ein genereller Wechsel in der Transfusionsstrategie für Thrombozytenkonzentrate ist daher als gefährlich anzusehen und muss unbedingt vermieden werden.

Intracoronary application of BM-derived cells for the treatment of acute myocardial infarction (AMI) is currently being studied intensively. Simultaneously, strict legal requirements surround the production of cells for clinical studies. Thus good manufacturing practice (GMP)-compliant collection and preparation of BM for patients with AMI was established by the Cytonet group.As well as fulfillment of standard GMP requirements, including a manufacturing license, validation of the preparation process and the final product was performed. Whole blood (n=6) and BM (n=3) validation samples were processed under GMP conditions by gelafundin or hydroxyethylstarch sedimentation in order to reduce erythrocytes/platelets and volume and to achieve specifications defined in advance. Special attention was paid to the free potassium (6 mmol/L), some rheologically relevant cellular characteristics (hematocrit0.45, platelets450 x 10(6)/mL) and the sterility of the final product.The data were reviewed and GMP compliance was confirmed by the German authorities (Paul-Ehrlich Institute). Forty-five BM cell preparations for clinical use were carried out following the validated methodology and standards. Additionally three selections of CD34+ BM cells for infusion were performed. All specification limits were met. Discussion In conclusion, preparation of BM cells for intracoronary application is feasible under GMP conditions. As the results of sterility testing may not be available at the time of intracoronary application, the highest possible standards to avoid bacterial and other contaminations have to be applied. The increased expense of the GMP-compliant process can be justified by higher safety for patients and better control of the final product.

Clinically the diagnosis of an autoimmune hemolytic anemia (AIHA) often starts with a patient with an unclear anemia, while in the laboratory the first hint is a blood sample with a positive direct antiglobulin test (DAT). Both anemia and positive DAT are non-specific findings that raise many questions. These include a wide range of laboratory characteristics such as immunoglobulin (sub-)class of the red blood cell (RBC) autoantibodies (Au-ab), presence of warm or cold Au-ab, thermal range of cold Au-ab, capability of Au-ab to activate complement, specificity of warm or cold Au-ab, and masking of additional RBC alloantibodies by Au-ab. Moreover, the clinical findings have to be scrutinized critically: Was there a sudden onset or a gradual beginning of hemolytic anemia? Is the hemolysis life-threatening? Does the patient suffer from a persisting systemic autoimmune or tumor disease, in particular a hematological malignancy? Has the patient been transfused within a certain time span before the first occurrence of the anemia symptoms? Was there a recent infection? Does the patient take drugs, newly or regularly? If a clinician is confronted with a newly diagnosed AIHA, each of these questions has to be answered to distinguish ‘idiopathic AIHA’ as a disorder of its own from ‘secondary AIHA’ as a symptom of a detectable underlying disease (e.g. chronic lymphocytic leukemia, systemic lupus erythematosus, Mycoplasma pneumoniae infection). This classification is important for choosing the appropriate medical treatment of AIHA. In idiopathic AIHA that is present in about 45% of the AIHA cases [1] immunosuppressive drugs (corticosteroids and others) are most frequently applied to cope with severe hemolysis [2]. In secondary AIHA the ‘drug of choice’ is governed by the underlying disease and may range from ‘wait and see’ in self-limiting cases of infection-related AIHA, over immunosuppressive drugs in cases with an overt systemic autoimmune disorder to chemotherapy in patients with lymphoproliferative malignancies [2,3]. Regardless of the clinical background, many AIHA patients have to be transfused, at least temporarily, as none of the available drugs are able to stop severe hemolysis immediately [4]. In view of these facts, each newly diagnosed ‘AIHA’ may become a challenging experience for the doctor who is responsible for the patient for several reasons: i) There is no close relationship between the laboratory findings and the clinical symptoms of the patient. ii) Hypoxic anemia may require urgent transfusion. iii) A deliberate search for an underlying disease often leads to the somewhat dissatisfactory ‘idiopathic’ AIHA. Moreover, none of the administered drugs - neither corticosteroids that are in routine use for decades nor newly introduced monoclonal antibodies such as rituximab - are always effective, and their beneficial immediate or long-term effects are not safely predictable [5,6]. The present issue of Transfusion Medicine And Hemotherapy is an attempt to summarize and review theories, ideas, and medical expert knowledge on AIHA. John Freedman [7] and Wilma Barcellini [8] present the history of AIHA and discuss the current concepts that contribute to RBC destruction. Barcellini reports on the roles of cytokines and cytotoxicity effector cells (CD8+ T cells, natural killer cells, and activated macrophages) in the AIHA disease process, thereby explaining the great clinical heterogeneity of AIHA ranging from a fully compensated (‘uneventless') clinical course to rapidly evolving severe life-threatening hemolysis. The contributions of Sigbjorn Berentsen [9] and Thilo Bartolmas et al. [10] deal with the role of the complement system in the development of AIHA and describe the main mediators of complement-driven hemolysis, in particular in cold Au-ab-associated AIHA. Charles H. Packman [11] gives an overview on the clinical manifestations of AIHA while Beate Mayer et al. [12] present more than 70 cases with drug-induced hemolysis, mainly as a result of diclofenac, piperacillin and cephalosporin ingestion. Abdulgabar Salama [13] reviews pitfalls that may hamper the diagnose ‘AIHA’ and summarizes his professional experience with the medical treatment of primary (‘idiopathic') AIHA over a period of 30 years. The latter contribution might provoke intense discussions among transfusion medicine specialists as the author attenuates the therapeutic enthusiasm with regard to newer drugs such as rituximab as the new ‘gold standard’ for medical AIHA treatment. When looking at the contents of this issue, you might wonder why one name seems to be nearly omnipresent in this special topic. It is not the task of this editorial to fully appreciate the tremendous scientific work of Abdulgabar Salama in the field of immunohematology over the last 30 years. However, as obvious for every specialist in transfusion medicine, Abdulgabar Salama has not only significantly influenced the field but has clearly dominated (together with his Californian colleague George Garratty) the diagnosis and the pathogenic background of drug-induced AIHA. His most important contribution is the discovery that many drugs supposed to be involved in severe AIHA are detectable in laboratory investigations only when their degradation products, either in serum/plasma or urine, are available (e.g. nomifensine [7], cephalosporin antibiotics such as ceftriaxone or cefotaxime [8,9], or the non-steroid antirheumatic diclofenac [10], just to mention some frequently used medications). Another similarly important contribution to the understanding of the pathogenicity of AIHA is his observation that transfusion itself is able to induce Au-ab formation, often in coexistence with transfusion-associated alloantibody formation, thereby complicating a delayed serologic/hemolytic transfusion reaction [11]. These are only a few reasons why we are very glad to have allured such an exposed representative of AIHA research to significantly contribute to this special topic.

BACKGROUND Inherited and acquired marrow failure syndromes most commonly lead to defect in myeloid and/or neutrophil differentiation and/or function. Besides this, neutropenia induced by cancer-adjusted chemotherapy is a frequent clinical problem. In both cases, cell replacement therapy is a well-established, but due to necessity of donors limited and perilous procedure. Therefore, autologous cell replacement from patients' own marrow-derived cells lowers risk and bares new possibilities for therapy. Since the immune system of the marmoset monkey is known to show high similarity to humans, preclinical studies with these animals bare high hopes for immunologic research and cell replacement therapy. STUDY DESIGN AND METHODS Marmoset-induced pluripotent stem (iPS) cells (cj-iPSC) were first cultivated on mouse embryonic feeder cells in medium containing recombinant human vascular endothelial growth factor. After 13 days, CD34+/vascular endothelial growth factor receptor-2 (VEGFR2)– cells were sorted, treated with interleukin (IL-3), thrombopoietin, and stem cell factor for 20 days and further cultivated with granulocyte–colony-stimulating factor (G-CSF) and IL-3 for 10 days. RESULTS CD34+/VEGFR2– cells could be generated in high amounts (39.65 ± 6.01%; 2.31 × 105 cells). Afterward, these hematopoietic progenitors could be successfully differentiated into mature cj-iPSC–derived neutrophils showing similar morphology, specific surface antigens, and neutrophil-specific gene products and in vitro phagocytic activity. CONCLUSION cj-iPSC–derived neutrophils bare high hopes in hematologic cell replacement therapy. They exhibit high morphologic similarity to native neutrophils and present neutrophil-specific surface antigens, antimicrobial proteins, and gene products yielding an auspicious approach for continuative experiments including tests in living animals.

Background: Antigen-specific immunoadsorption combined with rituximab offers the possibility for ABO-incompatible kidney transplantation without splenectomy. Patient and method: An 8-year-old mentally retarded girl with steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis due to mitochondriopathy poorly tolerated hemodialysis. Paternal blood group A1B was incompatible with blood group B of the child. Therefore, we decided to perform the first ABO-incompatible renal transplantation in a child in Germany using antigen-specific immunoadsorption. Rituximab (1 × 375 mg/m2) was administered 2 weeks before the first immunoadsorption (Glycosorb® ABO A-column). Triple-drug immunosuppression (tacrolimus, mycophenolate mofetil and prednisolone) was simultaneously started with immunoadsorption. Initial tacrolimus levels were targeted between 15 and 20 ng/ml. Before transplantation, six immunoadsorptions were applied on days −9, −7, −4, −3, −2 and −1. Intravenous immunoglobulin (0.5 g/kg) was administered preoperatively. After transplantation, three immunoadsorptions were performed on days +4, +6 and +8. Results: Before transplantation, antibody (Ab) titers against paternal erythrocytes (20°C) were reduced from 1 : 64 to 1 : 4 by six antigen-specific immunoadsorptions. After transplantation, we performed three more immunoadsorptions and the Ab titers were stable between 1 : 1 and 1 : 8. One, 2 and 8 months later we observed increases in the Ab titer up to 1 : 32 requiring no change in immunosuppressive therapy. No side effects of immunoadsorption were observed. The girl had excellent initial graft function with a serum creatinine of 55 to 70 μmol/l. Two weeks after transplantation, graft biopsy showed no signs of rejection; there was focal positivity for C4d only. Twelve months after transplantation, renal function was stable, with a serum creatinine of 117 μmol/l. Episodes of rejection or severe infections were absent. Conclusion: ABO-incompatible transplantation using antigen-specific immunoadsorption and rituximab may serve as a suitable alternative for children urgently needing renal transplantation and missing a blood group-compatible donor.

Fungal infections represent a life-threatening complication for patients receiving chemotherapy or undergoing hematopoietic stem cell transplantation. Historically, antifungal monotherapy is associated with a poor outcome. We treated three children with hematological malignancies and proven fungal infections (one cerebral mold infection, one disseminated Candida infection, one naso-pharyngeal mucor infection) with combination antifungal therapy plus granulocyte-colony-stimulation-factor-mobilized granulocyte transfusions as secondary prophylaxis during subsequent neutropenic episodes. With this approach, the fungal infection was effectively treated, and the anticancer therapy was completed without major delay. All children survived the fungal infection and the underlying malignancy. These experiences illustrate the feasibility of this approach using more than one antifungal agent together with immune-therapy in high-risk patients.

Background and Objectives The aim of this study was to analyse the platelet function, over a 5-day time-period, of apheresis-derived platelet concentrates obtained from smokers and non-smokers. Materials and Methods Smoker and non-smoker plateletpheresis products were investigated on days 1, 3 and 5 of storage. Receptor expression (as evaluated by flow cytometry) and the platelet aggregation response were measured. Results There was only a slight loss of platelet function in apheresis products from smokers compared to non-smokers. Conclusions Smoking does not significantly change the quality of platelet preparations. The current practice, of not to exclude smokers from platelet donation, can be continued.

BACKGROUND The objective of this study was to establish a mobilization and apheresis regimen for collection of two consecutive polymorphonuclear neutrophil (PMN) concentrates from the same donor. STUDY DESIGN AND METHODS In this prospective study, 111 healthy unrelated volunteers underwent either one (Group 1, n = 57) or two consecutive granulocyte apheresis procedure (Group 2, n = 54) using the a cell separator (Spectra). Both Group 1 and 2 donors were initially mobilized with glycosylated G-CSF 6.0 micro g per kg (range, 5.2-7.0 micro g/kg) subcutaneously plus oral dexa-methasone (DXM, 8 mg) and underwent granulocyte apheresis (GA-1) 16 hours (range, 13-18 hr) after initial G-CSF+DXM. Group 2 donors were remobilized with a second DXM dose of 8 mg (n = 13), 4 mg (n = 15), 1.5 mg (n = 13), or none (n = 13), and a second apheresis (GA-2) was run 40 hours (range, 37-42 hr) after G-CSF+DXM administration and 12 hours after remobilization with DXM alone. RESULTS Based on equivalent median preapheresis WBC and PMN counts of around 35 x 10(9) WBCs per L and 33 x 10(9) PMNs per L after initial mobilization the GA-1 yields were 85 x 10(9) PMNs per U (range, 34-150) in Group 1 and 75 x 10(9) PMNs per U (range, 35-135) in Group 2 (p = 0.14, NS). In Group 2, median preapheresis values of 19.6 x 10(9) WBCs per L (range, 9.5-37.0) and 16.6 x 10(9) PMNs per L (range, 8.8-34.8) were measured after remobilization and GA-2 yields of 49 x 10(9) WBCs per U (range, 26-113) and 42 x 10(9) PMNs per U (range, 21-95) were obtained. Borderline statistical differences in the GA-2 yields were observed from the remobilized donors: 8 mg: 36 x 10(9) PMNs per U (range, 23-60); 4 mg: 47 x 10(9) PMNs per U (range, 21-56) (p

BACKGROUND: A new cell separator (COM.TEC, Fresenius) was recently developed aimed at efficient collection of WBC-reduced single-donor PLT concentrates (SDPs). STUDY DESIGN AND METHODS: Five German centers collected 554 WBC-reduced SDPs with help of the COM.TEC cell separator. Two multicenter cell counting studies were performed at the beginning and at the end of the study to document uniform counting results among the participating centers. RESULTS: A total of 441 (79.6%) PLT collections were included in the study according to the protocol. A total of 342 single-dose and 99 double-dose SDPs were collected. For single-dose SDPs, an average blood volume of 2826 ± 409 mL was processed in a donation time of 55 ± 11 minutes. Mean PLT yield of these products was 3.11 × 1011± 0.40 × 1011 and the WBC contamination was 0.11 × 106± 0.20 × 106. For double-dose SDPs (PLT count, 5.29 ± 0.93 × 1011), 3943 ± 639 mL was processed. The average difference between the target and the collected PLT concentration was −2.8 ± 12.0 percent for single-dose SDPs and −1.8 ± 9.5 for double-dose SDPs, respectively. The collection efficiency was 53.7 ± 5.8 percent for single-dose SDPs and 58.2 ± 6.2 percent for double-dose SDPs. If all results of each sample from the counting study were set to unity (to the mean over all centers), most PLT determinations were very similar to the mean, for example, near or 1 if set to unity. CONCLUSION: The COM.TEC machine makes it possible to obtain WBC-reduced SDPs that comply with current standards.

Background The adoptive transfer of allogeneic antiviral T lymphocytes derived from seropositive donors can safely and effectively reduce or prevent the clinical manifestation of viral infections or reactivations in immunocompromised recipients after hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). Allogeneic third party T-cell donors offer an alternative option for patients receiving an allogeneic cord blood transplant or a transplant from a virus-seronegative donor and since donor blood is generally not available for solid organ recipients. Therefore we established a registry of potential third-party T-cell donors (allogeneic cell registry, alloCELL) providing detailed data on the assessment of a specific individual memory T-cell repertoire in response to antigens of cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (ADV), and human herpesvirus (HHV) 6. Methods To obtain a manufacturing license according to the German Medicinal Products Act, the enrichment of clinical-grade CMV-specific T cells from three healthy CMV-seropositive donors was performed aseptically under GMP conditions using the CliniMACS cytokine capture system (CCS) after restimulation with an overlapping peptide pool of the immunodominant CMVpp65 antigen. Potential T-cell donors were selected from alloCELL and defined as eligible for clinical-grade antiviral T-cell generation if the peripheral fraction of IFN-γ+ T cells exceeded 0.03% of CD3+ lymphocytes as determined by IFN-γ cytokine secretion assay. Results Starting with low concentration of IFN-γ+ T cells (0.07-1.11%) we achieved 81.2%, 19.2%, and 63.1% IFN-γ+CD3+ T cells (1.42 × 106, 0.05 × 106, and 1.15 × 106) after enrichment. Using the CMVpp65 peptide pool for restimulation resulted in the activation of more CMV-specific CD8+ than CD4+ memory T cells, both of which were effectively enriched to a total of 81.0% CD8+IFN-γ+ and 38.4% CD4+IFN-γ+ T cells. In addition to T cells and NKT cells, all preparations contained acceptably low percentages of contaminating B cells, granulocytes, monocytes, and NK cells. The enriched T-cell products were stable over 72 h with respect to viability and ratio of T lymphocytes. Conclusions The generation of antiviral CD4+ and CD8+ T cells by CliniMACS CCS can be extended to a broad spectrum of common pathogen-derived peptide pools in single or multiple applications to facilitate and enhance the efficacy of adoptive T-cell immunotherapy. Electronic supplementary material The online version of this article (doi:10.1186/s12967-014-0336-5) contains supplementary material, which is available to authorized users.

A novel DNA virus (TTV) was identified recently in Japanese patients with posttransfusion hepatitis non-A-E and has been implicated as a cause of acute and chronic liver diseases of unknown etiology in some patients. The frequency of TTV infections was investigated in 284 blood donors, 105 patients with different liver disorders before and after liver transplantation (OLT), as well as in 64 patients with chronic hepatitis C who received antiviral therapy. TTV infections were found more frequently by nested-PCR in patients with liver disorders (15%) as compared to blood donors (7%). TTV occurred independently of the aetiology of the liver disease (e.g., cryptogenic cirrhosis [12.5%], alcoholic cirrhosis [16%], fulminant hepatic failure non-A-E [35%], and chronic hepatitis C [12.5%]; p=n.s.). After OLT, a high rate of TTV de novo infections (44%) was observed. However, TTV viremia after OLT (in 56 out of the 105 patients) was not associated with graft hepatitis. Analysis of patients with chronic hepatitis C coinfected with TTV who have been treated with interferon alpha alone or in combination with ribavirin revealed that TTV is an interferon-sensitive virus. Phylogenetic analysis of TTV sequences suggest that at least four different genotypes and several subtypes exist in Germany. In conclusion, the high prevalence of TTV infections observed in patients with parenteral risk factors is an argument in favour of transmission of the virus via blood and blood products. A relevant hepatitis-inducing capacity of TTV, however, seems unlikely, considering the observation that in the majority of patients, TTV infection after OLT was not accompanied by graft hepatitis.

From November 1994 to November 1997 we investigated 133 patients with alloantibodies directed against high-frequency red cell antigens (Ab-HFA). More than 90% of these antibodies were found in females

Platelet dysfunction can cause clinically relevant bleeding. Treatment with DDAVP is advocated for this condition. DDAVP increases von Willebrand factor (VWF) on endothelial cells (ECs) and in plasma. VWF could facilitate platelet deposition on subendothelial collagen. VWF also facilitates platelet/EC interactions. Therefore DDAVP could precipitate thromboembolic events. We used a flow chamber model to study in vitro and ex vivo if DDAVP alters recruitment of platelets to EC and collagen. Resting or TRAP-activated platelets and EC were treated individually or simultaneously with 0.4 ng/ml DDAVP. Fluorophor-labeled platelets (10(6)/ml) were resuspended in reconstituted blood and superfused across EC and collagen in an in vitro flow chamber model at arterial shear (320 s(-1)). Adhesion of platelets to the respective surface was recorded fluorescence microscopically and platelet covered area was assessed. TRAP significantly induced adhesiveness of platelets for collagen and EC. DDAVP pretreatment of platelets did not affect adhesiveness of resting or TRAP-activated platelets for collagen or EC. Adhesiveness of resting but not TRAP-activated platelets was induced on DDAVP-treated EC. DDAVP-conditioned EC supernatant contained vWF and significantly increased platelet deposition on collagen. Platelets from patients with clinically suspected platelet dysfunction undergoing aortic valve replacement exhibited decreased platelet deposition on collagen surfaces. In summary, our data confirm that DDAVP can induce release of platelet adhesion promoting factors from EC, which is most likely vWF. DDAVP has no direct effect on platelets. Blood samples from DDAVP-treated patients do not exhibit significantly augmented platelet deposition on collagen ex vivo. This influence of released promoting factors might cause an increase of undesirable interactions of platelets with EC.

Adoptive immunotherapy with virus-specific T lymphocytes can efficiently reconstitute antiviral immunity against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (ADV) without causing acute toxicity or increasing the risk of graft-versus-host disease. To gain insight into antiviral T cell repertoires and to identify the most efficient antigens for immunotherapy, the frequencies of CMV-, EBV- and ADV-specific T cells in 204 HLA-typed healthy donors were assessed using viral peptides and peptide pools. Confirmatory testing for CMV serology by Western blot technique revealed 19 of 143 (13%) false-positive results. We observed highly significant individual and overall differences in T cell frequencies against CMV, EBV, and ADV antigens, whereas antigen-specific T cells were detected in 100% of CMV- seropositive donors, 73% of EBV- seropositive donors, and 73% of ADV-seropositive donors. At least 124 (61%) potential T cell donors were identified for each virus. Among the tested antigens, frequencies for CMVpp65 and EBVBZLF1 peptide pools were highest. Short-term in vitro peptide stimulation revealed that a donor response to a certain ADV- and EBV-derived peptide may not be determined without prior stimulation. A modified granzyme B ELISpot was used to detect T cell specificity and alloreactivity. Treatment with allogeneic virus-specific cytotoxic T lymphocytes from seropositive third-party donors may be a feasible therapeutic option for infections following cord-blood stem cell transplantation or hematopoietic stem cell transplantation from virus-seronegative donors.

BACKGROUND: Photodynamic virus inactivation of fresh-frozen plasma (FFP) may result in its impaired coagulation capability. STUDY DESIGN AND METHODS: Double-volume plasmapheresis samples from 11 donors were divided in pairs of 250 mL. One group underwent methylene blue (MB) phototreatment (MB-FFP). The other group was treated according to the standards of the American Association of Blood Banks for preparation and storage of FFP. Parameters of hemostasis and clinically important plasma proteins were tested in native plasma, thawed MB-FFP, thawed FFP, and twice-frozen and thawed FFP (FFP-II). RESULTS: Mean activities of factor V (73.4 vs. 94.5%; p < 0.01), factor VIII (58.1 vs. 86.7%; p < 0.001), and fibrinogen (1.8 vs. 2.8 g/L; p < 0.001) were reduced in MB-FFP as compared to those in FFP. The comparison of MB-FFP to FFP-II revealed reduced activities of factor VIII (58.1 vs. 85.2%; p < 0.001) and fibrinogen (1.8 vs. 2.8 g/L; p < 0.001) but no changes in factor V. Activated partial thromboplastin time in MB-FFP was prolonged beyond the upper normal range (+5.3 sec; p < 0.001) and prothrombin time increased in MB-FFP versus FFP (+0.96 sec; p < 0.001). CONCLUSION: MB phototreatment reduces the in vitro coagulation capacity of FFP, most likely as a result of the effects of an additional freezing and thawing procedure and photooxidation-induced protein damage.

Objectives: To determine the optimal time schedule for neutrophil collection after single mobilization with glycosylated recombinant granulocyte colony-stimulating factor (G-CSF, lenograstim) with or without dexamethasone (DXM). Donors and Methods: In this prospective randomized trial, 26 healthy volunteers were randomly assigned to a single subcutaneous dose of lenograstim 6 μg/kg plus 8-mg DXM (G-CSF/DXM, n = 13) or placebo (G-CSF/placebo, n = 13). Hematological and biochemical parameters were analyzed before and 12, 15, 18, 21, 24, 27, 29, 36, 48, 60, 72, and 84 h and 7 and 30 days after mobilization. Six G-CSF/DXM subjects underwent standard neutrophil apheresis (NA) 12 and 36 h after mobilization. Results: Polymorphonuclear neutrophil (PMN) counts 12 and 21 h after mobilization were 22.7 (16.6−32.8) × 109/L and 22.4 (18.6−30.6) × 109/L for G-CSF/placebo versus 33.1 (24.2–44.9) × 109/L and 32.5 (17.4–39.6) × 109/L for G-CSF/DXM. This mobilization plateau was followed by slow normalization at 72–84 h. The six NA subjects had median PMN yields of 62 (47–101) × 109 and 39 (23–42) × 109 per therapeutic unit. After the first apheresis, PMN counts sharply decreased to 21.1 (14.8–26.3) × 109/L and then temporarily recovered to 25.9 (18.9–36.5) × 109/L (P ≤ 0.001) over the next 8 h. Conclusions: Single doses of lenograstim with or without DXM induced a PMN plateau that lasted 9 h (12–21 h after mobilization), with PMN counts suitable for neutrophil collection. Lenograstim plus DXM made it possible to perform NA twice, 12 and 36 h after mobilization. © 2011 Wiley Periodicals, Inc.

The transfusion of rhesus positive (D+) red blood cells to a rhesus negative (D-) person usually induces the development of an irregular anti-D antibody in the recipient. This can lead to a hemolytic reaction in subsequent transfusions, and, in women of childbearing age, can lead to fetal erythroblastosis in any future pregnancy. The recommended interventions to avoid the immunization of the recipient include the administration of intravenous rhesus immune globulin within 72 h after the transfusion. We report the case of a D- woman who received one unit of D+ red blood cells and a total of 40 units of D- red blood cells after severe trauma. In spite of treatment with rhesus immune globulin, the patient developed anti-D antibodies.

BACKGROUND The objective was to evaluate the dose-response relationship of lenograstim plus dexamethasone for neutrophil mobilization and collection. STUDY DESIGN AND METHODS In a prospective study, 260 healthy volunteers received oral dexamethasone (8 mg) plus a single subcutaneous injection of glycosylated granulocyte-colony-stimulating factor (G-CSF; lenograstim) at medians of 1.5 (1.0-2.3) microg per kg (n = 43), 3 (2.4-4.1) microg per kg (n = 73), 6 (4.3-7.9) microg per kg (n = 123), and 12 (8.2-17.2) microg per kg (n = 21) and underwent neutrophil collections with a polymorphonucleated neutrophil (PMN) program. White blood cell (WBC) counts and PMN mobilization and collection results were compared, and the severity and clinical significance of donor adverse reactions were evaluated. Fifty-two neutropenic patients (29 children, 23 adults) underwent 271 neutrophil transfusions (GTXs) every other day to maintain WBC levels continuously above 0.5 x 10(9) per L. RESULTS Within the dose range 1.5, 3, and 6 microg per kg, each doubling step was associated with a 10 to 15 percent PMN increase in peripheral blood up to 32.8 (19.1-49.2) x 10(9) per L (6 microg/kg; p

Both granulocyte colony-stimulating factor (G-CSF) and dexamethasone (DXM) are used for neutrophil (PMN) mobilization and collection. This prospective study was aimed to evaluate and compare the rate, severity and clinical significance of adverse reactions of these drugs alone and in combination in healthy donors. PMN mobilization was carried out using dexamethasone alone (8 mg orally; n=25) or glycosylated G-CSF alone (Lenograstim, 5 microg/kg subcutaneously, n=24) or in combination (n=23) prior to a standard granulocyte apheresis on the Spectra cell separator. The number of PMNs counted in the mobilized peripheral blood of the donors was 7.0 (3.6-20.4) x10(9)/L (DXM), 25.2 (15.5-49.7) x10(9)/L (G-CSF), and 31.6 (20.0-43.0) x10(9)/L (G-CSF+DXM), corresponding to PMN apheresis yields of 13 (8-43) x10(9)/U, 56 (34-118) x10(9)/U, and 83 (33-117) x10(9)/U, respectively. The three groups had comparable percentages of donors with at least one adverse effect (ranging from 75 to 80%), but the G-CSF-containing regimens were generally more toxic, as was reflected by higher percentages of donors with moderate to severe adverse reactions and higher overall severity scores of 2.28 (G-CSF) and 2.08 (G-CSF+DXM) compared with 1.33 in the DXM group ( por=0.001). With G-CSF alone, pain symptom complexes were more frequent, more severe, and more often triggered requests for analgesics (9/47 donors; 19%) and unwillingness to give further neutrophil donations (2/47 donors; 4%). The addition of DXM to G-CSF diminished some symptoms, particularly bone pain, headache and the frequency of requests for analgesics. The predominant symptoms in the DXM alone group were mild gastrointestinal complaints. We conclude that G-CSF stimulation improved neutrophil mobilization and apheresis yields at the expense of donor tolerability. Compared with G-CSF alone, the combination G-CSF and DXM did not increase the quantity or the severity of donor symptoms.

BACKGROUND The aim of this study was to find a regimen for mobilization and collection of granulocytes that combines low-dose G-CSF administration with satisfactory PMN mobilization and apheresis at a low rate of donor adverse reactions. STUDY DESIGN AND METHODS In a prospective study, 52 healthy unrelated volunteers received a single subcutaneous injection of glycosylated G-CSF (Lenograstim Chugai-Pharma, Frankfurt, Germany) at medians of 3.1 (range, 2.4-3.6) microg per kg plus dexamethasone (8 mg orally; n = 29) or at 11.8 (7.1-18.5) microg of lenograstim per kg (p < or = 0.0001) without dexamethasone (n = 23) and underwent standard apheresis using the PMN program of a cell separator (Spectra, COBE [now Gambro] BCT). WBC and PMN mobilization results and apheresis yields were compared and the severity and clinical significance of donor adverse reactions was evaluated. RESULTS For the low-dose G-CSF plus dexamethasone versus the high-dose G-CSF alone group, similar mobilization results were observed for WBCs with 31.3 (19.1-44.9) x 10(9) per L versus 27.5 (19.2-44.0) x 10(9) per L (p = 0.21, NS) and PMNs with 29.0 (17.6-42.2) x 10(9) per L versus 25.2 (16.2-39.0) x 10(9) per L (p = 0.08, NS). The PMN apheresis yields were equal with 70 (39-139) x 10(9) per unit with low-dose G-CSF versus 68 (33-120) x 10(9) per unit in the high-dose G-CSF group (p = 0.83, NS). Regarding donor adverse reactions, 7 out of 29 (24%) and 8 out of 23 donors (35%) reported moderate or severe symptoms. The character of these reactions was different; symptoms of greater clinical significance and a higher need for analgesics were observed in the high-dose G-CSF group. CONCLUSIONS A Lenograstim dose of 3 microg per kg plus DXM assures effective PMN mobilization and acceptable apheresis components. The combination of glycosylated G-CSF with DXM allows a significant dose reduction in G-CSF for PMN mobilization and collection as compared with higher G-CSF doses alone. In the high-dose G-CSF mobilization group, adverse reactions were more severe and required more analgesics.

Mesenchymal Stromal Cells (MSCs) have achieved a great potential in Regenerative Medicine, although limited information exists about bioprocess and production on Good Manufacture Practices (GMP)-grade, standardization and quality controls. The present work describes our experience on the validation and GMP production of 25 consecutive bone marrow (BM)-derived MSCs batches for clinical use. BM was obtained from posterior iliac crests of patients. Manufacturing included isolation by density-gradient centrifugation and plating, trypsinization and secondary expansion cultures and, harvesting and formulation of a suspension containing 40 10E6 viable MSCs. Quality controls included cell counting and viability assessment; immunophenotyping; Sterility Test, Mycoplasma detection and Endotoxin Tests conducted according to European Pharmacopeia (Eur. Ph.) and, finally, Gram Staining. All products were shipped according to current EU Regulations. After validation, 25 MSCs batches for clinical application were manufactured. BM harvested volumes ranged from 98 to 163 mL (median 132,1mL) containing 8,38E8 to 4,29E9 viable Nucleated Cells (NCs) (median 2,53E9). Cell concentrations ranged from 5.74E6 to 3.56E7 NCs/mL (median 2.34E7). Final product volume was 10,0 mL (4-10,5 mL) containing a median of 4.02E7 (3.21E7-4.87E7) viable MSCs. Antigen expression was consistent with MSCs phenotype as described by ISCT. In a minority of the batches produced, we observed an increase of HLA-DR expression compatible with the activation on the MSCs. Sterility, Mycoplasma, Gram Staining and Endotoxin tests of the finished products were always negative. We have established a GMP protocol, using human sera as supplement, confirming its safety and feasibility. It is consistent and reproducible and the bioprocess developed demonstrated to be robust, safe and reliable. This work was cofounded by MEDCEL and FACTOCEL projects from the Spanish government (Ministerio de Ciencia e Innovacion).

BACKGROUND: The purpose of this study was to analyze the CD34 cell collection efficiency (CE) of automated leukapheresis protocols of two blood cell separators (Spectra, COBE [AutoPBSC protocol] and AS104, Fresenius [PBSC-Lym, protocol]) for peripheral blood progenitor cell (PBPC) harvest in patients with malignant diseases. STUDY DESIGN AND METHODS: PBPCs were collected by the Spectra AutoPBSC protocol in 95 patients (123 collections) and the AS104 PBSC-Lym protocol in 87 patients (115 harvests). Patients underwent a median of one (range, 1-4) conventional-volume apheresis procedure of 10.8 L (9.0-13.9) to obtain a target cell dose of ≥2.5 × 106 CD34+ cells per kg. RESULTS: The median overall CD34 CE was significantly better on the AS104 than on the Spectra: 55.8 percent versus 42.4 percent (p = 0.000). This was also true below (59.2% vs. 50.1%; p = 0.022) and above (51.2% vs. 41.3%; p = 0.001) the preleukapheresis threshold of 40 CD34+ cells per μL needed to collect a single-apheresis autograft. However, at ≥40 circulating CD34+ cells per μL, both cell separators achieved the target of ≥2.5 × 106 CD34+ cells per kg. The CD34 CE dropped significantly, from 59.2 percent at

Dear Editor, Recently, Schrezenmeier and Seifried [1] presented a review on the question whether pooled buffy-coat-derived platelet concentrates (PPCs) or apheresis PCs (APCs) should be preferred. They claimed that PPCs and APCs have comparable content of platelets (PLTs), purity, safety, and efficacy. They argued that almost all APCs could be replaced by PPCs and that PLT apheresis donations should be restricted to PLT recipients with anti-HLA and ⁄ or anti-HPA antibodies from the donor¢s perspective to drop the apheresis procedure-related donor risks to a ‘zero’ value and to optimize the use of the whole blood (WB) donation. Some weeks ago, Vamvakas [2] gave a systematic review of disparate risks of PPCs vs. APCs. He calculated an annual increase of 1AE2 (best case) to 3AE5 (worst case) additional cases for HIV, 1AE3 to 3AE9 for HCV, and 9AE0 to 27AE1 for HBV transmission, if PPCs would completely replace APCs. For a novel ‘HIV like’ infectious agent with a prevalence of 1:10 000 donations (for comparison, the incidence of HIV in the last year before testing was ten times higher), he predicted 252AE6 to 757AE7 additional transmissions. From our group, a recent risk calculation was performed by Heuft et al. [3], Hannover Medical School. They found that an unrecognized infection with a prevalence of 1% in the donor population would result in three to four times higher infection rates for PPC recipients compared to pure APC supply. Both Vamvakas and Heuft considered shorter donation intervals, the influence of splitting apheresis donations and low numbers WB donations per PPC for their calculations. Schrezenmeier and Seifried [1] cited both studies but voted to ignore their results because ‘so far, no epidemiological study or clinical trial have demonstrated different risk of viral infections transmitted by pooled PCs compared to apheresis PCs.’ This argumentation is on absolutely no account acceptable. Of course, the effects of a novel HIV-like virus cannot be proven epidemiologically or clinically before it has emerged. However, over the past decades, many previously unknown infectious agents have emerged. Recent work on the genetic distance of the two oldest currently known specimens of HIV-1 demonstrated that the first infections of humans by HIV-1 have occurred more than 100 years ago, at least two decades earlier than previously believed [4]. This clearly demonstrates that a dangerous infectious agent, which can be transmitted via transfusion and which kills recipients some years or even decades after transmission, may spread over many decades until becoming uncovered. The only way to meet this typical risk of blood transfusion is to strictly limit the donor exposure for the transfusion recipient and to replace a pooled component by a nonpooled alternative whenever possible. On the strength of past experience with the transmission of HIV and hepatitis viruses by blood and blood products, the consideration of very small and even of fictitious and yet unproven risks is common practice as well in transfusion medicine as in the legislation and the jurisdiction in all developed countries. In addition, we want to point out another relevant, but currently neglected issue: the production of PPCs has negative effects on the overall quality of the red cell supply. WB for PPC production is often held overnight at room temperature. However, at this temperature, red cells lose 2,3diphosphoglycerate (2,3-DPG) extremely rapidly [5]. Red cell concentrates (RCCs) from WB leukoreduced by a PLTsaving filter and stored overnight at room temperature contain remarkable levels of PLT-derived cytokines [6]. Finally, if PCs are produced by the BC method, a relevant proportion of red cells, at least ten per cent, is removed from the WB. This may result in a remarkable proportion of underfilled RCCs [7]. In summary, the production of PPCs impairs the quality of the red cell supply by red cell loss, by the rapid loss of 2,3-DPG, and by the accumulation of PLT-derived cytokines. Therefore, we strongly argue for an integral view on the blood supply that notes and considers the negative effects of PPC production from WB on the red cell supply. In conclusion, we fully agree with Vamvakas and disagree with Schrezenmeier and Seifried. Not APCs should be replaced by PPCs, but PPCs should be replaced by APCs. The most important factor determining the PLT product choice must be the difference of the risk of transfusiontransmitted infections, particularly of the transmission of a new HIV-like agent. In addition, there is sufficient evidence Vox Sanguinis (2010)

Natural killer-like T lymphocytes termed cytokine-induced killer (CIK) cells have been shown to eradicate established tumours in a severe combined immune deficient (SCID) mouse/human lymphoma model. Recently, we demonstrated that CIK cells transfected with cytokine genes possess an improved proliferation rate and a significantly higher cytotoxic activity as compared to non-transfected cells. Here, in a phase I clinical protocol, autologous CIK cells were generated from peripheral blood obtained by leukapheresis in patients with metastatic renal cell carcinoma, colorectal carcinoma and lymphoma. CIK cells were transfected with a plasmid containing the interleukin-2 (IL-2) gene via electroporation. Transfected cells generated IL-2 in the range of 330–1800 pg 10−6 cells 24 h−1 with a mean of 836 pg 10−6 cells 24 h−1. Ten patients received 1–5 intravenous infusions of IL-2-transfected CIK cells; five infusions with transfected CIK cells were given. In addition, the same patients received five infusions with untransfected CIK cells for control reasons. In three patients, WHO grade 2 fever was observed. Based on polymerase chain reaction of peripheral blood transfected cells could be detected for up to 2 weeks after infusion. There was a significant increase in serum levels of interferon gamma (IFN-γ), granulocyte–macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-β) during treatment. Interestingly, there was also an increase in CD3+ lymphocytes in the blood of patients during therapy. In accordance, a partial increase in cytotoxic activity in peripheral blood lymphocytes (PBLs) was documented when patient samples before and after therapy were compared. Concerning clinical outcome, six patients remained in progressive disease, three patients showed no change by treatment, and one patient with lymphoma developed a complete response. In conclusion, we were able to demonstrate that CIK cells transfected with the IL-2 gene can be administered without major side-effects and are promising for future therapeutic trials. © 1999 Cancer Research Campaign

Background and Objectives. Infections remain a major obstacle during chemotherapy induced neutropenic episodes. Despite improvements achieved in antibiotic and antimycotic treatment, mortality is still high in patients with severe infections. In this retrospective analysis we studied the effect of granulocyte-transfusions (GTX) in critically ill pediatric patients with life-threatening infections. Design and Methods. We performed a retrospective review of clinical data, laboratory and microbiological results during chemotherapy induced neutropenia between 2000 and 2003. GTX was considered during neutropenic sepsis not responding to broad-spectrum antibiotic and antimycotic therapy and increasing markers of infection. Granulocytes were collected from unrelated, blood-group matched, healthy and cytomegaly antibody negative donors. All donors received glycosylated G-CSF (Lenograstim™, 1.5 to 6μg/kg BW) plus oral dexamethasone (8mg) and underwent a neutrophil apheresis using the Spectra™ PMN program. Results. In total, 153 (range 1–19) GTX were transfused in 32 children aged 0.25 to 16 years. The underlying diseases included haematological malignancies (n=28), inborn immunological diseases (n=3) and a solid tumor (n=1). In 15/32 patients an allogenic stem cell transplantation (HCT) was performed. Infections consisted of fever of unknown origin (FUO, n=13), bacterial sepsis (n=8), invasive fungal infections (n=6), and viral infections (n=4). In one patient a double infection with virus and fungi was seen. The mean duration of neutropenia prior to the first GTX lasted 22.2 (range 3–85) days; the mean total duration of neutropenia was 36 (range 5–99) days. In three children, no regeneration of the leukocytes was observed. 18/32 children survived the neutropenic sepsis. With a thrice per week delivery of GTX (mean leukocyte count per transfusion 6.3 (range 1.9–13.9) x 1010) the mean increase of the leukocyte count 1 hour after GTX was 3480/μl (1580 to 18300). 16 hours after GTX a mean increase of the leukocyte number to 2560/μl (300–13200) was noted. One child developed a respiratory distress syndrome shortly after her 2nd GTX. Apart from this, no serious adverse events were seen. In 18/32 children the fever / infection was cleared during GTX. Out of 8 children with bacterial infection, 7 children survived. In total, 9 children died in the context of the underlying infection (6 viral, 2 fungal, 1 bacterial, 1 viral+fungal). 8/15 children died after SCT, in 3/8 treatment related toxicity (TRT) was responsible. In the surviving patients we observed a significant decrease in the C-reactive protein (CRP) (mean CRP prior to 1st GTX 183 mg/l, mean CRP 48 hours after the last GTX 116 mg/l) levels during and after GTX, whereas the CRP levels increased in those children who died (mean CRP prior to 1st GTX 186 mg/l, mean CRP 48 hours after the last GTX 231 mg/l). HCT or duration of neutropenia were no significant risk factors in this analysis. Conclusions. GTX were a safe therapeutic measure for critically ill neutropenic patients with progression of sepsis. After initiation of GTX a sustained increase of the leukocyte count could be achieved with beneficial effects on bacterial infections. No effects on viral infections were observed. The CRP-level might serve as a prognostic factor to identify good responders to GTX during sepsis.

In a 73-year-old female with chronic lymphocytic leukemia we observed an acute illness with shivering, high fever, and hemolytic anemia following chlorambucil administration. Reexposure in a controlled clinical situation suggested an allergic drug reaction. In an in vitro assay, we were able to demonstrate chlorambucil as the causative agent of immune hemolysis.