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Background and Objectives This study aimed to describe motives as well as donation experiences and the intention to return for further donations of German whole blood donors who donated at the beginning of the COVID‐19 pandemic. Materials and Methods To describe motives and donor experiences, a retrospective survey was conducted among whole blood donors that had a donation appointment at the German Red Cross Blood Donation Service in the first 4 weeks of the pandemic. A donor questionnaire including 17 retrospective questions was sent to 7500 donors. Donor motivation and donor experiences were compared for different donor groups using chi‐square statistics. Finally, in an ordinal logistic regression model predictors for the intention to return were identified. Results More than half of the participating donors (56.9%) wanted to contribute to the fight against the pandemic by donating blood. Most of the donors were satisfied with their last donation experience and felt safe during the blood donor appointment. However, some donors would have liked more information on how to deal with the pandemic (20.3%). Intention to return for further donations was strongly associated with overall satisfaction (OR: 1.67, CI: 1.47–1.90) and the feeling of being safe during blood donation (OR: 1.33, CI: 1.05–1.68). Conclusion Donor satisfaction with the last donation was high and the vast majority of donors felt very safe. However, those donors who felt unsafe expressed a low intention to return and blood donation services should therefore carefully monitor donor satisfaction.

We present a novel automated system for morphology analysis of red blood cells (RBC) under flow. RBC concentrates collected by blood banks for transfusions are stored for periods of up to several weeks, during which time a number of changes occur, collectively termed the storage lesion. Typically the extent of hemolysis is the defining criterion to determine the acceptability of the RBCs for transfusions. Morphological changes are related with biochemical alteration during the storage of RBCs. The typical blood smear procedure for determining such changes is a labor-intensive and potentially biased manual process. The advantage of the flow morphometry system presented here is that it provides fully automated morphological classification of RBCs with large sample numbers in a short time. Our system uses a commercially available flow cell and flow conditions that prevent adhesion of RBCs, thus eliminating the need for blocking agents such as albumin that affect the distribution of cell shapes. Our morphometry results are validated by comparison with standard biochemical assays (hemolysis, ATP) for blood from 17 donors stored under blood bank conditions for 13 weeks. We show that the percentage of spherocytes present can be used to estimate the status of RBC concentrates. © 2017 International Society for Advancement of Cytometry.

Major problems with biomedical devices in particular implants located in nonsterile environments concern: (i) excessive immune response to the implant, (ii) development of bacterial biofilms, and (iii) yeast and fungi infections. An original multifunctional coating that addresses all these issues concomitantly is developed. A new exponentially growing polyelectrolyte multilayer film based on polyarginine (PAR) and hyaluronic acid (HA) is designed. The films have a strong inhibitory effect on the production of inflammatory cytokines released by human primary macrophage subpopulations. This could reduce potential chronic inflammatory reaction following implantation. Next, it is shown that PAR, due to its positive charges, has an antimicrobial activity in film format against Staphylococcus aureus for 24 h. In order to have a long-term antimicrobial activity, a precursor nanoscale silver coating is deposited on the surface before adding the PAR/HA films. Moreover, the PAR/HA films can be easily further functionalized by embedding antimicrobial peptides, like catestatin (CAT), a natural host defense peptide. This PAR/HA+CAT film proves to be effective as an antimicrobial coating against yeast and fungi and its cytocompatibility is also assessed. Finally, this all-in-one system constitutes an original strategy to limit inflammation and prevents bacteria, yeast, and fungi infections.

Background Within the coming decades, a steadily growing demand for blood products will face a shrinking blood donor population in many countries. After increasing the donor age of repeat donors for whole blood donation (WB) from 68 to 70 years in 2009 in our Blood Service, we investigated whether this is sufficient as a safe and effective strategy to sustain future blood supply. Materials and Methods Between 1 March 2009 and 28 February 2011, WB donations from donors aged between 69 and 70 and their proportion of total donations in 2010 were determined. We analysed adverse reaction rates in donors with respect to sex and age and calculated mean annual donation frequencies. Results Of all invited donors, 32·5% responded and contributed 0·98% (men) and 0·56% (women) to all WB units collected in 2010. The overall and systemic adverse reaction rate per 1·000 WB donations declined by age [men: 1·10 (95%CI: 0·84–1·35) vs. 0 (0–0·8), P

Background and Objectives Previous studies have shown substantial geographical variation in blood donation within developed countries. To understand this issue better, we identified community characteristics associated with blood donor rates in German municipalities in an ecological analysis. Materials and Methods We calculated an aggregated rate of voluntary blood donors from each of 1533 municipalities in south-west Germany in 2007 from a database of the German Red Cross Blood Service. A multiple linear regression model estimated the association between the municipality-specific donor rate and several community characteristics. Finally, a spatial lag regression model was used to control for spatial autocorrelation that occurs when neighbouring units are related to each other. Results The spatial lag regression model showed that a relatively larger population, a higher percentage of inhabitants older than 30 years, a higher percentage of non-German citizens and a higher percentage of unemployed persons were associated with lower municipality-specific donor rates. Conversely, a higher donor rate was correlated with higher voter turnout, a higher percentage of inhabitants between 18 and 24 years and more frequent mobile donation sites. Conclusions Blood donation appears to be a highly clustered regional phenomenon, suggesting the need for regionally targeted recruiting efforts and careful consideration of the value of mobile donation sites. Our model further suggests that municipalities with a decreasing percentage of 18- to 24-year-olds and an increasing percentage of older inhabitants may experience substantial declines in future blood donations.

Objective Granulocyte-associated antibodies can cause several clinical granulocytopenic disorders. The monoclonal-antibody-specific immobilization of granulocyte antigens (MAIGA) is currently used as the standard assay to specify these antibodies. Here we describe an assay for specific analysis of granulocyte antibodies (SASGA) which is able to simultaneously detect and specify granulocyte IgG- and IgM-antibodies using flow cytometry. Methods Bead populations with distinct fluorescence intensities were used as solid phase for immobilization of mAb. Typed granulocytes were incubated with human sera and a mix of three distinct mouse monoclonal antibodies against specific granulocyte antigens (for example CD16, CD11a, HLA class I). After cell lysis and incubation of lysate with beads, goat antibodies against human IgG and IgM antibodies were added. Seventy-one frozen sera of donors and patients previously implicated in transfusion reactions and various underlying disorders were analysed for specific granulocyte-binding antibodies using MAIGA and SASGA. Results The SASGA assay was able to simultaneously detect granulocyte-specific antibodies for different glycoproteins. Overall, the results of MAIGA and SASGA were concordant in 92·9%. 5 sera containing anti-HNA-1b (n = 2) and -HLA class I (n = 3) were not detected by MAIGA, but were recognized by the SASGA. In serial dilution tests with sera containing anti-HNA-1a, -1b, -2a and HLA class I, the SASGA assay detected the antibodies at higher dilutions than MAIGA. Conclusion The SASGA assay permits reliable detection of specific granulocyte antibodies. Six distinct antibodies can be simultaneously determined. This method will potentially open the way to investigations on additional specific antibodies as it facilitates laboratory diagnosis.

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 and objectives Mirasol pathogen reduction technology (PRT) for platelet concentrates uses riboflavin and ultraviolet light. Previously, we described increased metabolism and activation for PRT platelets stored in 100% plasma. To improve platelet quality, we resuspended platelets in a mixture of plasma and platelet additive solution (PAS). Materials and methods Single-donor platelets were resuspended in plasma and split into an untreated control and a PRT-treated single product. One hundred and fifty millilitre PAS (SSP+) was added to both. Over 7 days, we assayed pH, glucose consumption-, lactate production rate and CD62p with and without TRAP. Results On day 5, PRT units showed a significantly lower pH (7.087 +/- 0.105 vs. 7.288 +/- 0.200) accompanied by a higher lactate production (0.104 +/- 0014 vs. 0.063 +/- 0.017 mmol/10(12)/h) and glucose consumption rate (0.039 +/- 0005 vs. 0.028 +/- 0.009 mmol/10(12) platelets/h). CD62p expression was higher in treated units (44.5 +/- 13.0 vs. 16.5 +/- 7.6%). Conclusion In comparison to PRT platelets resuspended in 100% plasma, a mixture of plasma and PAS improves pH and platelet metabolism but not platelet activation. Prolonged shelf-life for up to 7 days may be possible

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: 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.

Glycoprotein (GP)-specific platelet antibodies can cause allo-immune and auto-immune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Here, we describe an improved method based on simultaneous detection of various platelet-specific immunoglobulin G (IgG) and IgM antibodies. Bead populations with distinct fluorescence intensities, coated with monoclonal antibodies specific for mouse heavy chain isotypes, were used for the simultaneous immobilization of platelet-GP [IIb/IIIa, Ib/IX, human leucocyte antigen (HLA) class I, or Ia/IIa, CD32, GPIV or CD109, Ib/IX, HLA class I]. In order to detect human IgG and IgM antibodies simultaneously, phycoerythrin- and fluorescein isotiocyanate-conjugated goat anti-human IgG and IgM were added. On this basis, the abundance of six distinct antibodies (three anti-GP, each with subclasses IgG and IgM) were simultaneously analysed without cross-reaction by flow cytometry. For evaluation, sera and platelets from 169 patients with platelet-binding and/or platelet-associated antibodies were investigated. The monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay was performed in parallel as reference test. The simultaneous analysis of platelet-specific antibodies (SASPA) assay was able to detect all platelet-specific IgG and IgM that were also recognized by MAIPA with a comparable sensitivity. SASPA proved to be a rapid and reliable assay that required less platelets than other methods. This method has the potential to pave the way for new investigations of platelet-specific antibodies.

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.

Background and objectives: The integrity of granulocytic cells and platelets is compromised within cryopreserved stem cell transplants, and consequent DNA release during the thawing procedure can therefore lead to clotting phenomena or microaggregate formation and that in turn may cause loss of progenitor cells. To circumvent this problem a new processing protocol was introduced using recombinant human deoxyribonuclease (rhDNase) to prevent cell aggregate formation. In addition, the impact of this new processing protocol on CD34+ umbilical cord blood (UCB) cells was assessed. Materials and methods: Fifty samples derived from 7 buffy coat (BC) volume reduced UCB units were cryopreserved, thawed, and processed with washing solutions that were supplemented with rhDNase in various concentrations. Thereafter, clotting and microaggregate formation was scored microscopically. In addition, expression of the adhesion molecules leukocyte function-associated antigen 1 (LFA-1, n = 6), intercellular adhesion molecule-1 (ICAM-1, n = 11), and L-selectin (n = 11) on CD34+ UCB cells was analyzed by flow cytometry after incubating the samples with either dimethyl sulfoxide (DMSO) 5.5%, rhDNase 10 or 50 U/mL, or a combination of DMSO 5.5% and rhDNase 50 U/mL. Results: At a minimal concentration of 10 U rhDNase/mL, clotting or microaggregate formation could be prevented for all tested samples, whereas cell clots could be observed for concentrations up to 8 U/mL. The expression of adhesion molecules on untreated CD34+ UCB cells (L-selectin: 64.6 ± 18.8%; LFA-1: 62.6 ± 7.5%; ICAM-1: 14.8 ± 4.1%) did not show any significant difference compared with cells that were incubated with up to 50 U/mL rhDNase (L-selectin: 62.2 ± 19.3%; LFA-1: 63.1 ± 5.9%; ICAM-1: 17.5 ± 6.7%). However, after a combined treatment with DMSO 5.5% and rhDNase 50 U/mL, a slight but significant decrease in L-selectin expression could be observed (P

BACKGROUND AND OBJECTIVES Inflammatory cytokines in platelet concentrates (PC) may cause side-effects such as febrile non-haemolytic transfusion reactions. The maximum white blood cell (WBC) content tolerable to avoid the accumulation of cytokines, and whether these cytokines originate from degranulating leucocytes or de novo synthesis during storage, had not been investigated prior to this study. MATERIAL AND METHODS We investigated the secretion of interleukin (IL)-1beta, IL-2, IL-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) and quantified the appropriate expression of corresponding mRNA in PC with regard to different levels of WBC contamination and storage times. In addition we tested the viability of WBCs during PC storage (by staining with 7-aminoactinomycin D) and their ability to perform de novo cytokine synthesis (by using superantigen stimulation). RESULTS We detected a statistically significant increase of IL-1beta, IL-6, IL-8 and TNF-alpha in PC with > or = 108 WBCs. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed increasing mRNA expression of the respective cytokines depending on the number of WBC present. On day 5 of storage, WBC viability was > 80% and the leucocytes were still able to produce cytokines de novo. CONCLUSIONS These data show clear evidence for de novo synthesis of cytokines in PC. The cytokine pattern supports the hypothesis that activated monocytes are responsible for this cytokine synthesis. PC with a WBC contamination of > or = 108 contain inflammatory mediators in clinically relevant concentrations.

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: 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.

Compensation for acute blood loss requires cardiovascular integrity. In older people, asymptomatic cardiovascular impairment is common. In these subjects, even moderate blood loss is often treated by external volume replacement although its benefits are not clear. We investigated the effect of 450 ml of blood loss on the microcirculation and compensatory mechanisms in healthy older blood donors. Red blood cell count, plasma viscosity and protein concentration were measured. Plasma volume replacement was calculated using haematocrit. We measured microcirculation tissue perfusion by laser Doppler fluxmetry prior to, during and after blood donation. Blood loss was immediately accompanied by a median rapid water shift of 208 ml (interquartile range 134-298 ml). Haemodilution led to a decrease in haematocrit, protein and plasma viscosity. We observed no changes in cutaneous microcirculation. Moderate blood loss is tolerated in older cardiovascularly asymptomatic patients without having an impact on microcirculation. This may reduce the need for external volume replacement.

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.

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.

We describe a flow cytometric method using a newly designed product, fluorochrome-containing microspheres (Flow Count fluorospheres), which facilitates the precise quantification of cells in whole blood samples or heterogeneous cell suspensions on a single-cell level. These microparticles are easily distinguishable from other events of interest and can be detected by their light-scattering and fluorescence properties. In contrast to the traditional manual or automated cell-counting techniques, this method offers the opportunity to quantify cells simultaneously with flow cytometric immunophenotyping without additional cell loss or other cell preparation steps. We evaluated the accuracy and reproducibility of this flow cytometric method on the determination of CD45+ leukocyte counts and compared the results with those obtained by conventional techniques. Particular interest was focused on the behavior of cells and fluorospheres regarding their sedimentation rate over the period of analysis. The data from 48 blood samples with low, normal, and high leukocyte counts confirmed the reliability and comparability of the flow cytometric method, permitting the determination of white blood cell concentration at least to a limit of 100 cells/microL. A broad field of applications will benefit from this flow cytometric supplement because it is easy to perform and highly accurate. The results appear to be transferable to clinical decision-making monitoring of CD4+ lymphocytes in patients infected with human immunodeficiency virus or of CD34+ hematopoietic cells, optimizing the harvest for peripheral blood stem cell transplantation.

International regulations for blood donation recommend a maximum donor age of 65 years. As the average population age is steadily rising in western societies, a considerable group of volunteers is lost to the donor base.In a prospective study we investigated the effect of a 450-ml whole blood donation on the physical fitness and hemorheology of regular elderly allogeneic blood donors (n = 24, aged 63-69 years, mean = 65). Results were compared with a younger group of regular donors (n = 23, aged 55-62 years, mean = 58) and a group of elderly subjects (n = 7, aged 63-66 years, mean = 65), who did not donate blood for this study. Assessing the physical fitness, we determined the submaximal physical working capacity at a heart rate of 130 min-1 (PWC 130) and the maximal working capacity (MWC) by treadmill exercise testing the day before (day -1) and after donation (d +1). The impact of the blood loss on hemorheology was examined by analyzing the plasma viscosity before, during and after donation.We found an increase of mean values of PWC 130 and MWC on day +1 in all study groups, but increases were only significant in the younger group (PWC 130 p = 0.03; MWC p = 0.04). Values did not differ significantly between the three groups. Plasma viscosity decreased significantly directly after donation in both groups of donors.A single blood donation did not alter the physical fitness of otherwise healthy elderly people. The older blood donors and the younger controls showed a similar compensation mechanism to blood loss. We found no general reason for disqualifying blood donors aged 65 years from donating.

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.

We evaluated a new glucose-free citrate-acetate-NaCl platelet additive solution (PAS 2). In series I, platelet concentrates (PC) were prepared by apheresis and subsequently stored either in plasma (n = 16) or in PAS 2 (n = 15). In series II, PCs were prepared from pools of four buffy coats (BC) and stored in plasma (n = 12) or in PAS 2 (n = 11). By means of ultrastructural morphometry, the volume fractions of alpha-granules, the open canalicular system (OCS) and the fraction of storage granules secreted into the OCS were analyzed during storage for up to 8 days. Additionally, we determined pH, glucose, lactate, pCO2, HCO3-, lactate dehydrogenase and platelet factor 4. Apheresis platelets stored in plasma showed no changes in their contents of alpha-granules and in the fractions of the OCS. In contrast, apheresis platelets stored in PAS 2 displayed a decrease of their relative volume fraction of alpha-granules from 9.1 +/- 1% on day 1 to 3.7 +/- 0.9% on day 5. The fraction of the OCS increased from 7.4 +/- 0.8% on day 1 to 17.1 +/- 1.4% on day 3. On day 8, 93 +/- 9% of all platelets were lysed. Levels of glucose were significantly lower in these preparations and after day 3 glucose consumption decreased to zero. Among PC derived from pooled BC, differences between storage in PAS 2 or plasma were less striking. Only the fraction of alpha-granules secreted into the OCS was significantly greater in BC derived PC stored in PAS 2 on all days. These PCs stored in PAS 2 had a higher plasma carryover (30%) in comparison to apheresis PC stored in PAS 2 (10%). We conclude that plasma is superior to PAS 2 for storage of both apheresis and buffy coat platelets. For preservation of the structural integrity of platelets, the use of PAS 2 requires a minimum of 30% plasma carryover.

Platelets contain several mediators, belonging to a family of proinflammatory cytokines named chemokines, that are stored in the organelles. Release and accumulation of these chemokines during storage of platelet concentrates (PCs) might be responsible for nonhemolytic transfusion reactions.Analysis was done of pH and the levels of platelet factor 4, beta-thromboglobulin, interleukin 8, RANTES, macrophage-inflammatory protein-1 alpha, lactate dehydrogenase, and serotonin in the supernatant of stored PCs on Days 1, 3, 5, and 8. PCs were prepared by apheresis or from pools of four buffy coats. Buffy coat PCs were filtered before storage.Nonfiltered apheresis PCs, which had a higher white cell contamination (p0.01), contained significantly more platelets than did buffy coat PCs (p = 0.02). The pH decreased significantly in apheresis PCs (p = 0.01), whereas there was a significant increase in lactate dehydrogenase (p0.001). In buffy coat PCs, pH remained stable and lactate dehydrogenase increased moderately. Concentrations of platelet factor 4 and beta-thromboglobulin increased steadily in both preparations over the storage period (p0.001). Macrophage-inflammatory protein-1 alpha was hardly detectable in the supernatant of both PCs, while RANTES levels increased significantly with storage time (p0.001). Interleukin 8 was not found in the supernatant of any PCs, with the exception of one apheresis PC with high white cell contamination (10(9)/ L). Serotonin levels were higher in apheresis PCs (p = 0.01), but the levels did not correlate with storage time.Platelet factor 4, beta-thromboglobulin, and RANTES were released from platelets during storage and accumulated over time in the PCs. These chemokines might play a causative role in nonhemolytic transfusion reactions because of their inflammatory potential, but the clinical effects of the transfusion of PCs with high chemokine contents remain to be investigated.

There has been some discussion whether the atopic disposition of a blood donor is associated with a potentially higher incidence of hypersensitivity nonhemolytic transfusion reactions (NHTRs). Serum samples from patients who had suffered from NHTRs and samples from the platelet concentrates (PCs) responsible for the reactions were examined for total and specific IgE as diagnostic markers for allergic events. In addition, the allergy prevalence among 1,088 blood donors was determined to analyze the allergy prevalence among our blood donors. Our results indicate that in 90% of cases, allergic NHTRs were associated with specific IgE antibodies in the recipient's serum, indicating the allergic disposition of the patient. In contrast, specific IgE antibodies were detected in only 22% in the transfused PCs. However, among all investigated NHTRs, there was not a single case in which specific IgE antibodies were detected exclusively in the PC. The allergy prevalence among our blood donors was about 26%. In our opinion, the few cases in which the allergic disposition of blood donors in combination with the allergic disposition of the recipients was associated with NHTRs reflects the allergy prevalence among our blood donors in general (26%). On the basis of these findings, we conclude that allergy diagnosis for blood donors is only of minor value in the prevention and prediction of NHTRs, whereas allergy diagnosis for patients who require multiple PC transfusion might be helpful.

We describe the identification and molecular characterization of a novel variant O 1 allele of the ABO blood group locus. The allele was found in a young child and by analyzing the maternal DNA we were able to show that ameiotic recombination event between the maternal O 1 v - 3 and B 1 - 1 alleles recreated a O 1 /B hybrid allele. Further characterization of intron 6 sequences delineated the putative recombination breakpoint between nucleotide position 42 and 163 of the intron. We propose that the novel O 1 v a r i a n t allele should be named O 1 v - 7 and is a combination of exon 6 from a O 1 v - 3 allele and exon 7 from a B 1 - 1 allele.