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Journal of cellular and molecular medicine 26(16), 4463-4478 (2022). doi:10.1111/jcmm.17471, Published by Wiley-Blackwell, Hoboken, NJ

The members of a Caucasian family were genetically analyzed on suspicion of hereditary protein S deficiency. A novel mutation, c.1904TC, associated with severe quantitative protein S deficiency was found. The novel PROS1 mutation was identified by sequencing of the PROS1 gene coding sequence. The identified c.1904TC point mutation results in p.Phe635Ser amino acid exchange, which is located in the Laminin G-like 2 domain of protein S. Computational analysis indicates that this amino acid exchange affects the correct folding of the protein S antigen. Furthermore, this mutation is located in a region of the Laminin G-like 2 domain where changes in the amino acid sequence often result in decreased secretion. We postulate that the novel p.Phe635Ser mutation might lead to an incorrect folding, and thus, to a strongly impaired secretion of this protein S variant. We named this novel variant protein.

Introduction: For the regeneration of large volume tissue defects, the interaction between angiogenesis and osteogenesis is a crucial prerequisite. The surgically induced angiogenesis by means of a...

For the purpose of skeletal muscle tissue engineering, different cell types have been investigated regarding their myogenic differentiation potential, including co-cultured myoblasts and adipogenic mesenchymal stromal cells (Mb/ADSC). As neural cells enhance synaptic junction formation, the aim of this study was to co-culture Schwann cells (SCs) with Mb/ADSC on biocompatible electrospun aligned poly-ε-polycaprolacton (PCL)-collagen I-nanofibers. It was hypothesized that SCs, as part of the peripheral nervous system, promote the myogenic differentiation of Mb/ADSC co-cultures. Mb/ADSC were compared to Mb/ADSC/SC regarding their capacity for myogenic differentiation via immunofluorescent staining and gene expression of myogenic markers. Mb/ADSC/SC showed more myotubes after 28 days of differentiation (p ≤ 0.05). After 28 days of differentiation on electrospun aligned PCL-collagen I-nanofibers, gene expression of myosin heavy chains (MYH2) and myogenin (MYOG) was upregulated in Mb/ADSC/SC compared to Mb/ADSC (p ≤ 0.01 and p ≤ 0.05, respectively). Immunofluorescent staining for MHC showed highly aligned multinucleated cells as possible myotube formation in Mb/ADSC/SC. In conclusion, SCs promote myogenic differentiation of Mb/ADSC. The co-culture of primary Mb/ADSC/SC on PCL-collagen I-nanofibers serves as a physiological model for skeletal muscle tissue engineering, applicable to future clinical applications.

Primary myoblasts (Mb) and adipose derived mesenchymal stromal cells (ADSC) can be co-cultured and myogenically differentiated in the process of skeletal muscle tissue engineering. Electrospun composite nanofiber scaffolds represent suitable matrices for tissue engineering of skeletal muscle, combining biocompatibility and stability. Although growth differentiation factor 11 (GDF11) has been proposed as a rejuvenating circulating factor, restoring skeletal muscle function in aging mice, some studies have also described a harming effect of GDF11.Therefore the aim of the study was to analyze the effect of GDF11 on co-cultures of Mb and ADSC on poly-ε-caprolacton (PCL)-collagen I-polyethylene oxide (PEO)-nanofibers.Human Mb were co-cultured with ADSC two-dimensionally (2D) as monolayers or three-dimensionally (3D) on aligned PCL-collagen I-PEO-nanofibers. Differentiation media were either serum-free with or without GDF11, or serum containing as in a conventional differentiation medium. Cell viability was higher after conventional myogenic differentiation compared to serum-free and serum-free + GDF11 differentiation as was creatine kinase activity. Immunofluorescence staining showed myosin heavy chain expression in all groups after 28 days of differentiation. Gene expression of myosin heavy chain (MYH2) increased after serum-free + GDF11 stimulation compared to serum-free stimulation alone. The results of this study show that PCL-collagen I-PEO-nanofibers represent a suitable matrix for 3D myogenic differentiation of Mb and ADSC. In this context, GDF11 seems to promote myogenic differentiation of Mb and ADSC co-cultures compared to serum-free differentiation without any evidence of a harming effect.

The microvascular endothelial network is essential for bone formation and regeneration. In this context, endothelial cells not only support vascularization but also influence bone physiology via cell contact‐dependent mechanisms. In order to improve vascularization and osteogenesis in tissue engineering applications, several strategies have been developed. One promising approach is the coapplication of endothelial and adipose derived stem cells (ADSCs). In this study, we aimed at investigating the best ratio of human umbilical vein endothelial cells (HUVECs) and osteogenic differentiated ADSCs with regard to proliferation, apoptosis, osteogenesis and angiogenesis. For this purpose, cocultures of ADSCs and HUVECs with ratios of 25%:75%, 50%:50% and 75%:25% were performed. We were able to prove that cocultivation supports proliferation whereas apoptosis was unidirectional decreased in cocultured HUVECs mediated by a p‐BAD‐dependent mechanism. Moreover, coculturing ADSCs and HUVECs stimulated matrix mineralization and the activity of alkaline phosphatase (ALP). Increased gene expression of the proangiogenic markers eNOS, Flt, Ang2 and MMP3 as well as sprouting phenomena in matrigel assays proved the angiogenic potential of the coculture. In summary, coculturing ADSCs and HUVECs stimulates proliferation, cell survival, osteogenesis and angiogenesis particularly in the 50%:50% coculture.

Simple Summary Patients undergoing radiation therapy for the treatment of various types of cancer often experience side effects such as radiation dermatitis. A gold standard treatment is still lacking. The objective of the present study was to find novel therapeutic strategies for the regeneration and repair of damaged skin areas after irradiation. An in vitro 2D and 3D primary keratinocyte model was used to test the effect of insulin-like growth factor I (IGF-I), keratinocyte growth factor (KGF), platelet lysate (PL), hyaluronic acid (HA), and adipose-derived stem cell (ADSC) conditioned medium on the functional abilities (viability, migration) and the gene expression of irradiated keratinocytes. Hyaluronic acid and IGF-I effectively reduced the irradiation damage of primary keratinocytes by stimulating viability and migration and reducing cell apoptosis and necrosis. These findings indicate that the negative effects of irradiation on keratinocytes located in the patient’s skin can be counterbalanced with HA and IGF-I treatment. Abstract Ionizing radiation has become an integral part of modern cancer therapy regimens. Various side effects, such as radiation dermatitis, affect patients in acute and chronic forms and decrease therapy compliance significantly. In this study, primary keratinocytes were irradiated in a 2-dimensional (2D) culture as well as on a 3-dimensional (3D) collagen-elastin matrix with doses of 2 and 5 Gy. The effect of different concentrations of IGF-I, KGF, platelet lysate (PL), high and low molecular weight hyaluronic acid (H-HA, L-HA), and adipose-derived stem cell (ADSC) conditioned medium was analyzed in respect to cell viability (WST-8), wound closure (migration), and the gene expression (quantitative real-time PCR) of 2D cultures. The 3D culture was evaluated by WST-8. A mixture of H-HA and L-HA, as well as IGF-I, could significantly stimulate the keratinocyte viability and migration which were severely reduced by irradiation. The MKI67and IL6 gene expression of irradiated keratinocytes was significantly higher after H-HA/L-HA treatment. The stimulating effects of H-HA/L-HA and IGF-I were able to be confirmed in 3D culture. A positive influence on cell viability, migration, and gene expression was achieved after the treatment with H-L-HA and IGF-I. These results open the possibility of a novel therapeutic method for both the prevention and the treatment of radiation dermatitis.

Background: We present a multivariate test system using flow cytometry after in-vitro lymphocyte stimulation using 5 mitogens and 7 antigens to describe in-vitro immunofunction. Methods: The present work is a crucial step towards establishing a simple, CFSE-based, multivariate test system that can describe the dynamics of stimulus-induced lymphocyte proliferation with considerably more precision than is possible with the radionucleotide method using H-3-thymidine. Using multicolour flow cytometry, our method allows additional phenotyping of the proliferating cells and quantifies the proliferation behaviour by precisely resolving daughter generations and determining the precursor frequency. Results and Conclusions: Taking the calculated apoptosis parameters into account not only provides additional information about the stimulus-specific response behaviour but also improves the validity of the commonly used proliferation indices. Not only can we confirm previous findings that healthy people have marked differences in a multivariate test system in terms of the individual in-vitro reactivity to various stimuli but also substantiate that the response pattern of an individual is remarkably constant. In follow-up studies we can show for the first time that the results of immunofunctional testing do not change over a period of at least 6 months and appear to be an inherent characteristic of the individual and thus possibly have a genetic basis.

Background Quantification of CD34+ cells in peripheral blood stem cell apheresis is normally performed by single platform flow cytometric measurements according to the ISHAGE protocol. Peripheral blood stem cell concentrates (PBSC) produced by apheresis normally contain many T cells. Those T cells can be used for production of donor lymphocyte infusion doses, if abundant amounts of CD34+ cells have been collected. Therefore, it is of interest to know both the CD3+ and the CD34+ cell count of allogeneic PBSC. This is the first study comparing the performance of a modified ISHAGE protocol allowing additional quantification of CD3+ cells on two different flow cytometers, the FACSCalibur and the FACSVerse, respectively. Methods CD45+ and CD34+ cell concentrations were measured using a standard and a modified ISHAGE protocol including CD3+ cell quantification on both machines. All cell concentrations were measured using a Trucount bead based stem cell enumeration kit. The FACSVerse machine can additionally be equipped with a sample volume sensor allowing cell quantification without using beads. The samples analysed were taken from granulocyte-colony-stimulating factor mobilized peripheral blood stem cell apheresis procedures (pre- and post-apheresis, and apheresis concentrate). Results There were no significant differences in cell concentrations measured by the standard and modified ISHAGE protocol, regardless of which machine had been used when using bead quantification. No significant differences between the results of the two flow cytometers using the modified ISHAGE protocol were observed. Pearson´s correlation was always > 0.96, and regression coefficients were higher than 0.93. The only significant differences were observed between bead quantification and volume sensor quantification on the FACSVerse machine. Conclusions The modified ISHAGE protocol can effectively be used on both flow cytometers tested, especially if bead quantification is used.

In most cases, the amount of hematopoietic stem and progenitor cells (HSPCs) in a single cord blood (CB) unit is not sufficient for allogenic transplantation of adults. Therefore, two CB units are usually required. The ex vivo expansion of HSPCs from CB in coculture with mesenchymal stroma cells (MSCs) might be an alternative. It was investigated, whether bone marrow-derived MSCs, which have to be obtained in an invasive procedure, introduce a further donor and increases the risk of transmissible infectious diseases for the patient can be replaced by MSCs from amnion, chorion, Wharton's jelly, amniotic fluid, and CB, which can be isolated from placental tissue which is readily available when CB is sampled. In a two-step ex vivo coculture mononuclear cells from cryopreserved CB were cultured with different MSC-feederlayers in a medium supplemented with cytokines (stem cell factor, thrombopoietin [TPO], and granulocyte colony-stimulating factor). Expansion rates were analyzed as well, by long-term culture-initiating cell (LTC-IC) and colony-forming unit (CFU) assays, as by measuring CD34(+)- and CD45(+)-cells. Due to the comparably low number of 5×10(2) to 1×10(4) CD34(+)-cells per cm(2) MSC-monolayer, we observed comparably high expansion rates from 80 to 391,000 for CFU, 70 to 313,000 for CD34(+)-, and 200 to 352,000 for CD45(+)-cells. Expansion of LTC-IC was partly observed. Compared to the literature, we found a better expansion rate of CD34(+)-cells with MSCs from all different sources. This is probably due to the comparably low number of 5×10(2) to 1×10 CD34(+)-cells per cm(2) MSC-monolayer we used. Comparably, high expansion rates were observed from 80 to 391,000 for CFUs, 70 to 313,000 for CD34(+)-, and 200 to 352,000 for CD45(+)-cells. However, the expansion of CD34(+)-cells was significantly more effective with MSCs from bone marrow compared to MSCs from amnion, chorion, and Wharton's jelly. The comparison of MSCs from bone marrow with MSCs from CB and amniotic fluid showed no significant difference. We conclude that MSCs from placental tissues might be useful in the expansion of HSPCs, at least if low numbers of CD34(+)-cells per cm(2) MSC-monolayer and a high TPO concentration are implemented in the expansion culture.

Background There is a lack of data on the usage of plasma derived/recombinant coagulation factor concentrates (PD/RCFC) regarding diagnostic categories. An audit of PD/RCFC and blood component usage at a tertiary care teaching hospital in northern Bavaria was conducted. Study design and methods All blood components and PD/RCFC transfused at a university hospital (Erlangen, Germany) during the year 2006 were analysed. Transfused blood products were listed by major diagnostic categories (MDC) formed from principal diagnoses of recipients according to the International Classification of Diseases, tenth revision and German modification. Results Blood component usage has markedly increased since last surveyed in 1994 through 1996. The diagnostic categories responsible for most transfusions have not changed since. Antithrombin is the PD/RCFC used most, whereas most money for PD/RCFC was spent on FVIII concentrates. Polytrauma patients need most fibrinogen, whereas most of FXIII is needed in patients with malignancies. Patients with prolonged artificial ventilation receive PCC most often. Altogether, three MDCs (Pre, 17, 05) accounted for 80·5% of costs created by PD/RCFC transfusion. Conclusion This study provides for the first time combined data on blood component and PD/RCFC usage in a German university hospital. It shows that the MDCs responsible for most of the costs in transfusion therapy with blood components and with PD/RCFC are few, and are the same. At the same time, blood bank information software should be further improved in order to be able to identify new trends in hemotherapy in more detail.

BACKGROUND: The standard mononuclear cell (MNC) program of the COM.TEC device (Fresenius HemoCare GmbH) showed excellent collection efficiency of CD14+ monocytes. A major disadvantage was high content of residual cells in MNC harvests, which could influence dendritic cell (DC) culture. STUDY DESIGN AND METHODS: The autoMNC program (COM.TEC) was compared with the standard MNC program (n = 12). Additionally, two cycle volumes (300 mL vs. 450 mL, n = 19) were compared (standard MNC program). Samples were assayed for white blood cells (WBCs), red blood cells (RBCs), granulocytes (PMNs), hematocrit, and platelets (PLTs) on an automated blood cell counter (Sysmex K 4500, TAO Medical). CD14+ cells were analyzed by flow cytometry (FACSCalibur, BD). RESULTS: The autoMNC program produced 1.33 × 109 ± 0.36 × 109 CD14+ cells, 5.60 × 1011 ± 0.97 × 1011 PLTs, and 1.43 × 1011 ± 0.37 × 1011 RBCs. Compared to the standard MNC program, significantly higher PLT yields but lower RBC yields and product volume were harvested. Increasing the CV from 300 to 450 mL dropped the product volume, residual PLTs, and RBCs significantly, whereas WBC and monocyte yields did not change. The WBC predonation counts of donors correlated significantly with monocyte yields. CONCLUSIONS: The autoMNC program reduced the buffy coat (BC) volume and RBC yields in products compared to the standard MNC program. Increasing the CV (standard MNC program) reduced residual PLTs, RBCs, and the BC volume of MNC harvests. The donor WBC predonation count was a good predictor for the monocyte yield of products.

Background The aim of this study was to investigate the effect of gamma irradiation with 30 Gy on the coagulation system in leukoreduced fresh-frozen plasma (FFP). Study design and methods In 74 FFP units that had been stored for 352 +/- 103 days below -30 degrees C, the following variables were determined in parallel in an irradiated and not irradiated half: prothrombin time (PT); activated partial thromboplastin time (APTT); thrombin time; antithrombin III; protein C; protein S; von Willebrand factor antigen; ristocetin cofactor; plasminogen-alpha(2)-antiplasmin; the coagulation factors fibrinogen, factor (F)II, FV, FVII, VIII, F IX, FX, FXI, FXII, FXIII, and activated factor XII (FXIIa); D-dimer; fibrin monomer; thrombin-antithrombin complex; prothrombin fragment 1 + 2 (F1+2); plasmin-alpha(2)-antiplasmin complexes (PAPs); and platelet factor 4. The FVII activity ratio was assayed to quantify activation of FVII. Results Irradiation with 30 Gy resulted in a reduction of APTT (35.0 +/- 4.1 sec vs. 34.4 +/- 4.1 sec; p = 0.00000006) and PT (89.8 +/- 8.2% vs. 90.7 +/- 8.0%; p = 0.002) and a significant increase of the activities of the coagulation factors FII, FV, FVII, F IX, FX, and FXII. FVIII activity decreased from 118 +/- 31 to 116 +/- 32 percent (p = 0.02). Activation of the coagulation system was shown by an increase in the FVII activity ratio (1.19 +/- 0.29 vs. 1.31 +/- 0.34; p = 0.0000001), FXIIa (0.81 +/- 0.50 ng/mL vs. 0.90 +/- 0.51 ng/mL; p = 0.006), and F1+2 (1.19 +/- 0.20 nmol/L vs. 1.24 +/- 0.20 nmol/L; p = 0.000005) after irradiation with 30 Gy, whereas an increase of PAP (16.2 +/- 11.5 ng/mL vs. 20.2 +/- 12.0 ng/mL; p = 0.0004) demonstrated activation of the fibrinolytic system. No negative influence of irradiation with 30 Gy on inhibitors of coagulation was observed. Conclusion Gamma irradiation of leukoreduced FFPs with 30 Gy results in a significant but very weak activation of the coagulation and fibrinolytic system in FFPs.

BACKGROUND: The Fresenius COM.TEC cell separator is a new device for producing white cell concentrates (WBCs) and leukoreduced single-donor platelet concentrates (SDPs) and performing therapeutic cytapheresis and plasmapheresis that might replace the Fresenius systems AS104 and AS.TEC 204. This novel system's performance was evaluated for producing leukoreduced SDPs. STUDY DESIGN AND METHODS: In an investigational phase, each of 200 donors underwent plateletpheresis with the AS.TEC 204 and the COM.TEC systems. The collection efficiency (CE) and WBC contamination of the different techniques were compared. After some hard- and software modifications, the system was evaluated in an additional 800 procedures in the confirmatory phase. RESULTS: In the investigational phase, the CE of the COM.TEC device was increased significantly in comparison to the AS.TEC 204 device's CE (by 45 ± 32% when collecting 1 unit of platelets [PLTs] and 1 unit of fresh-frozen plasma and by 43 ± 42% when collecting only 1 unit of PLTs). Although all AS.TEC products proved to be leukoreduced, 2 percent of the COM.TEC procedures led to PLT concentrates containing more than 1 × 106 WBCs. In the confirmatory phase, all 1300 products from 800 COM.TEC procedures proved to be leukoreduced. Furthermore, the CE increased significantly from 53.5 ± 4.6 percent in the investigational phase to 55.5 ± 4.9 percent (p

Background: Sensitive and accurate methods to detect hematopoietic chimerism after hematopoietic stem cell transplantation (HSCT) are essential to evaluate engraftment and to monitor response to therapeutic procedures such as donor lymphocyte infusion. Continuous long-term follow up, however, requires large amounts of pre-HSCT samples limiting the application of many widely used techniques for sensitive chimerism monitoring. Methods: DNAs from 42 normal healthy donors and 16 HSCT donor/recipient pairs were employed to validate the use of allele-specific insertion/deletion (indel) quantitative real-time polymerase chain reaction (qPCR) to quantify chimerism in samples with low amounts of DNA. Consequently, indel-qPCR analyses of samples from 16 HSCT patients were compared to short-tandem repeat (STR) specific PCR analyses. Results: Typing with reduced amounts of input DNA (15 vs. 60 ng) allowed for the reliable distinction of positive (mean threshold cycle (ct) 28.05) and negative (ct >36) signals. The high informativity of primer/probe sets, with 12 out of 19 markers exceeding 20% informativity, was confirmed in our cohort (n = 74). Importantly, a fourfold reduction of input DNA compared to published protocols did not alter PCR efficiencies and allowed for a more sensitive detection of chimerism in 7 of 16 HSCT patients compared to results obtained by STR-PCR. Conclusions: Our data suggest that indel-qPCR is a more sensitive technique for the detection of hematopoietic chimerism compared to STR-PCR and works efficiently for samples with low amounts of DNA.

Objectives The hepatitis B vaccination policy in Germany was intensified by the implementation of hepatitis B vaccination for adolescents in the vaccination calendar in 1995. To investigate the effect of this measure on the hepatitis B vaccination coverage of healthy adults, we analysed the hepatitis B vaccination status of blood donors. Furthermore, the reasons for vaccination and the relationship between vaccination status and age, sex, and current profession were studied. Method Using a standardised questionnaire, randomly incoming whole blood donors were asked for hepatitis B vaccination status, the reason for vaccination, gender, age and current profession. Multiple logistic regression analysis with vaccination status as dependent variable and age, gender and current profession as explanatory variables was performed. Results Overall, 1519 (22.3%) of the 6812 interviewed whole blood donors were vaccinated against hepatitis B. Younger age was significantly associated with higher acceptance of hepatitis B vaccination with already 44.1% of whole blood donors aged 18–29 years being hepatitis B immunised. Beside health care workers, teaching professions and students showed the highest hepatitis B vaccination rate. Foreign travel was nearly an equivalently important reason for hepatitis B vaccination as occupational risks. Conclusion The high hepatitis B vaccination coverage among young and healthy adults indicates the success of the intensified hepatitis B vaccination policy since 1995. However, concentrating education measures on individuals with lower educational level and intensifying hepatitis B vaccination in the context of foreign travel could further increase the acceptance of hepatitis B vaccination.

BACKGROUND: The aim of this study was to investigate the occurrence of hypotension in the 24-hour period after preoperative autologous blood donation (PABD) in patients with and without hypertension. STUDY DESIGN AND METHODS: In 20 patients, 24-hour ambulatory blood pressure monitoring (ABPM) was performed before PABD was started and on every donation day in two repeated phlebotomies. RESULTS: Seven patients had no hypertension and 11 patients had hypertension. In 2 additional patients, hypertension was diagnosed during the study. Overall, the mean systolic BP (SBP) decreased from 131 ± 15 mmHg before donation to 128 ± 13 and 127 ± 10 mmHg after Donations 1 and 2; the corresponding values for the diastolic BP (DBP) were 77 ± 9, 75 ± 9, and 73 ± 7 mmHg, both without significant differences between the groups with and without hypertension. In single patients, substantial decreases of BP occurred, especially during the night. Two patients with and 2 without hypertension showed a nightly decrease in SBP and DBP of more than 10 percent (in 1 of these patients, more than 20%). Concerning diurnal BP variability, 1 patient with and 1 without hypertension, the latter showing a nightly decrease of SBP and DBP of more than 10 percent, also changed to the pattern of a nightly “extreme dipper” after PABD. CONCLUSION: In 25 percent of the patients, changes of BP were observed during the 24-hour period after PABD, especially during the night, which are known to be associated with an increased risk of cerebral or myocardial ischemia. Whether those changes of BP lead to major morbidity or mortality requires further investigation.

BACKGROUND: Only a few systematic studies have examined the effect of variable produced small platelet (PLT) aliquots on PLT function before transfusion to neonates. Although neonatal transfusion could be critical, no standardization of production or systematic quality controls have been introduced so far. STUDY DESIGN AND METHODS: PLT split products were prepared in three different ways (30- or 60-mL bag, syringe aliquot) at different times from the parent unit (Days 1-4) and stored for 2 or 4 hours. The measures of PLT function include pH, lactate, P-selectin expression, and cytokines (β-thromboglobulin [β-TG], PLT-derived growth factor AB [PDGF-AB]). Additionally, syringe passage (0.5 mL/min) was assessed. RESULTS: High product variability of PLT content was found (40% deviation of PLT content from programmed target, 13%-19% PLT loss by product distribution), which resulted in PLT concentrations of split units between 0.94 × 109 and 1.66 × 109 PLTs per mL. Different gas transfer rates (pCO2) of PLT containers caused different pH values of the product (Trima 7.47 ± 0.09 vs. COM.TEC 7.33 ± 0.08; p

BACKGROUND: Rare clinical conditions cause the need for washed platelet (PLT) concentrates (PCs). Saline-washed PCs can only be stored shortly, however, owing to lack of substrates for PLT metabolism. New PLT additive solutions (PASs) contain such substrates and might be used alternatively. The in vitro quality of apheresis PCs washed with Composol-PS or modified PAS-III (PAS-IIIM) stored up to 48 hours after wash was compared. STUDY DESIGN and METHODS: Twelve blood donors underwent two apheresis procedures (A and B) collecting 6.0 × 1011 PLTs in 500 mL of plasma with a least 2 weeks in between. The PCs collected by Apheresis A were stored for 3 days and then split in two equal units before washing with Composol-PS or PAS-IIIM. The PCs collected by Apheresis B were split after collection. One unit was released for transfusion and 1 unit was stored unwashed up to Day 6 and used as reference unit. In vitro testing was performed before and after washing as well as 24 and 48 hours after wash. RESULTS: After 48 hours of postwash storage, the units washed with either PAS showed acceptable results for hypotonic shock response (HSR), P-selectin expression, and pH, whereas PLT aggregability was significantly impaired. Throughout the storage, unwashed units showed better in vitro quality. HSR and P-selectin expression were similar before and immediately after the washing procedure. CONCLUSION: Based on these in vitro results, 48-hour postwash storage of washed PCs with the two PASs seems to be feasible. In vivo recovery studies, however, must confirm this finding in the future.

In this study we investigated human metabolism and excretion of DEHP after intravenous exposure. For this purpose we determined the five major DEHP metabolites in urine samples of a volunteer before and after a platelet donation (dual-needle technique). Plateletpheresis procedures are known to cause a significant DEHP exposure. We observed a sharp increase in urinary DEHP metabolite concentrations after the procedure. Maximum concentrations of 5OH-MEHP, 5oxo-MEHP, 5cx-MEPP and MEHP observed 4 h after the procedure were 822, 729, 577 and 388 microg/l respectively. 2cx-MMHP was excreted at highest concentrations after 8 h (201 microg/l). Due to longer elimination half-times, 5cx-MEPP and 2cx-MMHP were the major metabolites excreted in urine 24 h after the exposure. The 24-h-cumulative excretion of 363 microg 5cx-MEPP, 353 microg 5OH-MEHP, 309 microg 5oxo-MEHP, 178 microg MEHP and 133 microg 2cx-MMHP indicates an absolute exposure of our volunteer of about 2.6 mg DEHP. Related to the body weight this equals a dose of 31.6 microg/kg body weight/day. This indicates that current risk or preventive limit values for DEHP such as the RfD of the US EPA (20 microg/kg/day) and the TDI of the European Union (20-48 microg/kg/day) can be exceeded on the day of the plateletpheresis. The amount of the dose excreted in urine, distribution of the metabolites in urine and all other elimination characteristics after intravenous DEHP exposure are comparable to oral exposure. There are no indications that toxicokinetic behaviour and the toxicity of DEHP are fundamentally different after the two routes of exposure. Therefore, toxicological endpoints observed for DEHP after oral application should also be considered relevant for medical procedures causing intravenous DEHP exposure, like apheresis procedures. Especially women in their reproductive age need to be protected from DEHP exposures exceeding the above mentioned preventive limit values.

BACKGROUND: Standard plateletpheresis techniques and effects on platelet (PLT) donors were investigated to provide an informative basis for advancement of apheresis software. STUDY DESIGN AND METHODS: Three paired groups with 33 male and 22 female blood donors were prospectively investigated by analyzing blood counts of donors and products. Four apheresis platforms, the COBE Spectra LRS and the Trima v4 (Gambro BCT) and the AS.TEC204 and the COM.TEC (Fresenius Hemocare), were compared. Deviations of the collected from programmed PLT targets and donor PLT recruitment were calculated for single-unit PLT concentrates (SU-PCs; 3 × 1011 PLTs) and double-unit PLT concentrates (DU-PCs; 6 × 1011 PLTs). RESULTS: Regarding SU-PCs, the productivity of the COM.TEC machine was superior to the AS.TEC204 machine, because of shorter processing time (54 min vs. 67 min) and higher yields (2.90 × 1011 PLTs vs. 2.75 × 1011 PLTs). Compared to the Spectra machine, the Trima v4 machine showed higher collection efficiencies (CEs) and shorter processing time and complied better with the programmed target (SU-PCs, 3.24 × 1011 PLTs vs. 3.70 × 1011 PLTs; DU-PCs, 6.87 × 1011 PLTs vs. 7.56 × 1011 PLTs). Harvests of the Spectra machine (DU-PCs) exceeded the target by 40 percent, which resulted in high PLT loss for donors. A longer processing time resulted in some higher CEs (SU-PCs, 53%; DU-PCs, 58%), which could contribute to this result. PLT recruitment compensated PLT loss to some extent. CONCLUSION: The major finding was that the newer devices (COM.TEC and Trima) gave more predictable yields than the older devices (AS.TEC204 and Spectra) and resulted in lower PLT deficit. PLT software should be improved to minimize relevant variations of collected yields regarding the programmed target.

Although the risk of transfusion-transmitted hepatitis B virus (TT-HBV) infection is very low, it still exists. Therefore, introduction of further precautions to reduce this risk is discussed at present. However, so far no data are available about the HBV vaccination status among blood donors (BDs). We compared HBV vaccination status of apheresis donors (ADs) of a university based and whole BDs (WBDs) of a Red Cross blood donation service using a standardized questionnaire. On the basis of these data, the estimated costs over 10 years for HBV vaccination were calculated for two different strategies and compared with the costs for HBV nucleic acid amplification technology (NAT) testing. 22.3% of the WBDs and 41.2% of the ADs indicated having received at least one HBV vaccine dose. This difference was related to the different demographic structures of the two BD populations (BDPs). With regard to the primary costs for the blood donation service, HBV vaccination of BDs could be an alternative to HBV NAT testing, especially for BDPs with an already high HBV vaccination rate and a high donation frequency.

Background and objectives No data are currently available on the quality of washed prestorage leucocyte-depleted red blood cell concentrates (RCCs). Materials and methods Five groups of RCCs stored in additive solution (SAG-M) were washed. The groups differed in the age of RCCs (2-5 days or 11-15 days), the temperature during the washing procedure and a 6-h storage period (4 degrees C or room temperature) and the washing solution (saline, SAG-M or 5% albumin). We measured ATP, 2,3-diphosphoglycerate (2,3-DPG), haemolysis, blood cell count, Na(+), K(+), pH, pO(2), pCO(2) and lactate, before and after the washing procedure and hourly during the 6-h postwash storage period. Results The erythrocyte ATP content increased by 2-13%, relative to the baseline value, during the washing procedure. The 2,3-DPG level decreased by 15-35% in 2-6-day-old RCCs and by 30-40% in 11-15-day-old RCCs (relative to baseline values) during the washing procedure. In RCCs that were washed and stored at room temperature, and in 2-week-old RCCs, a further decrease in 2,3-DPG of up to 40%, relative to the baseline value, was observed during the 6-h postwash time-period. Conclusions Washing of RCCs stored in SAG-M results in a considerable, significant loss of erythrocyte 2,3-DPG, especially in older RCCs. This loss increases in during a 6-h storage period postwash, even at 4 degrees C. This loss of erythrocyte quality might well outweigh the benefits of washed SAG-M RCCs during massive transfusion in neonates.

BACKGROUND: In cancer and transplantation therapy apheresis devices and software of optimum standards are required for the collection of high cell yields with high purity of the desired cell fraction. STUDY DESIGN AND METHODS: In a paired study, 15 healthy blood donors underwent four 10-L leukapheresis procedures (197 ± 33 min) with an inlet blood flow rate of 60 mL per minute by use of two different MNC program settings of the COBE Spectra (Gambro BCT) and the AS.TEC 204 (Fresenius Hemocare) cell separators. RESULTS: Use of the standard MNC program of both apheresis devices resulted in significantly higher (p < 0.01) collection efficiencies of CD14+ monocytes, CD3+ cells, CD4+ cells, CD8+ T cells, CD16+ CD56+ natural killer (NK) cells, and residual PLTs (p < 0.001), owing to higher centrifuge speed. The mean MNC purity of all components was more than 90 percent. By use of stan-dard programs of either device, significant correlations (p < 0.01) between donor monocytes and preleukapheresis NK cell counts and the corresponding component cell yields were found. CONCLUSION: Compared to the program modifications with lower centrifuge velocities the standard MNC programs were significantly more efficient regarding CD14+, CD3+, and CD16+ CD56+ cells. Enhanced centrifuge speed and inlet blood flow rate in MNC programs resulted in higher, similar composed MNC concentrations of the products.

It has been the aim of the present prospective clinical study to assess the morbidity following the harvest of bone from the anterior and posterior ilium in elective preprosthetic augmentations. Fifty consecutive healthy patients (30 female, 20 male, mean age 52.5+/-9.3 years, range 31 years to 65 years) underwent augmentations of implant sites by iliac crest bone grafts. The bone harvest was carried out in 25 cases from the anterior and in 25 cases from the posterior ilium. The superficial sensory function of the skin was determined quantitatively preoperatively, 7 and 30 days after surgery with the 'Pain and Thermal Sensitivity' Test (PATH Test). On the same occasions subjective pain on a visual analogue scale (VAS) and gait disturbances were documented. In the PATH Test, for the innervation areas of the lateral femoral cutaneous nerve (LFCN) and the superior and middle cluneal nerves (SMCN) a significant impairment of the superficial sensory function could be found after 1 week and a significant tendency towards recovery after 1 month (warm stimulus(FCNpreop) 37.9+/-3.0 degrees C, warm stimulus(LFCNday7): 38.6+/-3.2 degrees C, warm stimulus(LFCNday30): 37.9+/-2.9 degrees C, P(LFCNwarmpreop/day7)=0.023,P(LFCNwarmpreop/day30) =0.886, warm stimulus(SMCNpreop): 36.1+/-2.4 degrees C, warm stimulus(SMCNday7): 36.6+/-2.3 degrees C, warm stimulus(SMCNday30): 36.3+/-4.0 degrees C,P(SMCNwarmpreop/day7)0.0005,P(SMCNwarmpreop/day30) =0.086). Gait disturbances were seen in seven patients after anterior and in three patients after posterior bone harvest 7 days after surgery (P(gaitdisturbanceanterior/posterior)=0.123). After 1 month none of the patients of both groups showed gait disturbances any longer. The maximum subjective pain level was found on the second postoperative day in both groups. It was significantly higher for the anterior approach (VAS(anteriorday2) 7.0+/-1.5, VAS(posteriorday2) 5.5+/-1.8,P(VASanterior/posteriorday2) =0.004). At day 7 and at day 30, the pain levels did no longer differ significantly (VAS(anteriorday7) 3.7+/-1.4, VAS(posteriorday7) 3.2+/-1.6,P(VASanterior/posteriorday7) =0.165, VAS(anteriorday30) 1.4+/-0.7, VAS(posteriorday30) 1.4+/-0.8,P(VASanterior/posteriorday30) =0.724). Because of the lower morbidity of bone harvest from the posterior ilium in the early postoperative phase compared to the anterior approach it seems that it should be preferred in elective augmentation procedures.

The properties of red blood cell (RBC) concentrates stored in different additive solutions have been previously examined under laboratory conditions at the end of shelf-life. However, whether these data are representative for RBC units used in clinical practice has not been shown. Therefore, we examined 164 RBC units from six manufacturers outdated after clinical usage in a hospital-based transfusion service for cellular content, hemolysis, adenosin triphosphate, 2,3-DPG, pH, oxygen saturation and levels of beta-thromboglobulin and proinflammatory cytokines interleukin (IL) 1beta (IL-1), IL-6, IL-8 and tumor necrosis factor alpha (TNFalpha). Results were correlated with the number of interruptions of recommended storage conditions and with different manufacturers. TNFalpha and IL-8 levels in the supernatant of RBC concentrates showed a weak correlation with the number of interruptions of recommended storage conditions (TNFalpha: r = 0.25, P < 0.01; IL-8: r = 0.20, P < 0.01) for the whole series. We detected no significant correlation between hemolysis and interruptions of recommended storage conditions or any of the remaining studied parameters. However, we found significant differences between RBC concentrates supplied by different manufacturers with respect to cellular content and most of the studied parameters. RBC concentrates containing SAG-M from one single manufacturer had higher in vitro hemolysis at the end of shelf-life compared to all other manufacturers (P < 0.05). We conclude from our data that interruptions of optimal conditions for storage of red cell components during cross-match testing and transport in our setting play a minor role for in vitro properties of RBC units at the end of shelf-life. The influence of processes of production, storage and/or transport until entry of RBC units into our blood component depot seems to be much more important for final product quality at the end of shelf-life than subsequent events.

BACKGROUND: Monocytes collected by leukapheresis are increasingly used for dendritic cell (DC) culture in cell factories suitable for DC vaccination in cancer. STUDY DESIGN AND METHODS: Using modified MNC programs on two apheresis systems (Cobe Spectra and Fresenius AS.TEC204), leukapheresis components collected from 84 patients with metastatic malignant melanoma and from 31 healthy male donors were investigated. MNCs, monocytes, RBCs, and platelets (PLTs) in donors and components were analyzed by cell counters, WBC differential counts, and flow cytometry. RESULTS: In 5-L collections, Astec showed better results regarding monocyte collection rates (11.0 vs. 7.4 × 106/min, p = 0.04) and efficiencies (collection efficiency, 51.9 vs. 31.9%; p < 0.001). Both devices resulted in monocyte yields at an average of 1 × 109 (donors) and 2.5 × 109 (patients), whereas Astec components contained high residual RBCs. Compared to components with low residual PLTs, high PLT concentration resulted in higher monocyte loss (48 vs. 20%, p < 0.0001) before DC culture. CONCLUSION: The Astec is more efficient in 5-L MNC collections compared to the Spectra. Components with high residual PLTs result in high MNC loss by purification procedures. Thus, optimizing MNC programs is essential to obtain components with high MNC yields and low residual cells as prerequisite for high DC yields.

BACKGROUND: A cell separator (Spectra, Gambro BCT) with an integrated leukoreduction system (LRS) for producing WBC-reduced single-donor platelet concentrates has been shown to result in a slightly reduced collection efficiency as compared to the former Spectra system without LRS. A novel modified system for improved collection efficiencies (LRS Turbo, Gambro BCT) was evaluated. STUDY DESIGN AND METHODS: Each of 37 donors underwent plateletpheresis using the LRS Turbo (LRS-T) and the standard LRS (LRS) of the Spectra cell separator. The collection efficiency and WBC contamination of the different techniques were compared. Platelets were counted automatically and WBCs were counted by using one or two full grids of a Nageotte chamber. RESULTS: The preseparation and postseparation numbers of RBCs, WBCs, and platelets, as well as the number of collected platelets, did not differ for the two techniques. In the LRS-T separations, the collection efficiency was 112 percent of that in the LRS procedures. Median residual WBCs in the platelet components were 0.0256 × 106 per LRS-T procedure and 0.0253 × 106 per LRS procedure. The purity of the LRS-T components was not less than that of the standard LRS components, whereas the collection efficiency of the LRS-T was significantly greater, 44.9 percent versus 40.7 percent. CONCLUSIONS: The LRS-T procedures produced platelet concentrates with WBC-reduction capacity that is comparable to that obtained with the standard LRS procedures, which have previously been described as satisfying the most stringent criteria for WBC-reduced platelets. The new technique significantly improved the collection efficiency of the plateletpheresis procedure.

BACKGROUND: Compensatory RBC production during repeated preoperative autologous blood donation (PABD) shows marked interindividual variability. This study was performed to reveal variables that might be useful to predict the amount of the erythropoietic response to PABD in an individual patient who was not iron deficient. STUDY DESIGN AND METHODS: In a retrospective study, 104 adult patients, 48 women and 56 men (mean age, 59.9 years; range, 18-82 years) who donated 3 units (450 mL) of autologous blood at weekly intervals for major surgery were investigated. Blood counts, ferritin, and net preoperative RBC production (net RBC production) were determined in all patients, and soluble transferrin receptor and endogenous levels of EPO, SCF, and IL-1β were measured in 63 patients. Multiple linear regression analysis was used to determine whether the variance of net RBC production was attributable to baseline values of these variables. RESULTS: Net RBC production was not different in patients who received oral iron and patients who did not (384 ± 222 mL vs. 356 ± 158 mL). In both groups, the same two variables consistently showed a significant relationship to net RBC production: the length of the period between the third donation and the last visit was positively related (p = 0.00001 vs. p = 0.0002) and the Hct at baseline was negatively related (p = 0.0002 vs. p = 0.02) with net RBC production. The proportion of variance in net RBC production that was attributable to these two variables was 48.1 percent (r2 = 0.481) and 34.9 percent (r2 = 0.349), respectively. CONCLUSION: RBC production after PABD increases with increasing interval from last donation to surgery. This suggests that the interval from last donation to surgery should be maximized. This can be achieved by organizational measures in combination with the preparation of RBC concentrates in additive solution with a maximum shelf life.

Background This study was performed to evaluate the capacity of oral and intravenous (i.v.) iron administration during autologous blood donation (ABD) to improve the efficacy of ABD and to prevent the need for allogeneic blood transfusion in patients without iron deficiency who are undergoing major elective surgery for which a minimum of 3 autologous units have been ordered. Study design and methods One hundred twenty-three patients were enrolled in an open-labeled, randomized, controlled trial and assigned to three treatment groups: patients in Group 1 received 3 x 100 mg of Fe2+ per day given orally for 5 weeks before operation; patients in Group 2 received 200 mg of Fe3+ given intravenously after each donation combined with initial i.v. iron supplementation in patients with hemoglobin under 15 g per dL; and patients in Group 3 were in the control group that received no iron medication. A modest ABD program involving weekly phlebotomy and threshold hemoglobin values for donation of 11.5 g per dL in women and 12.0 g per dL in men was performed. Results Ninety patients, 15 women and 15 men in each of the three groups, completed the study. The mean net red cell production during ABD was no higher (p>0.2) in the iron-treated groups (Group 1: 473 +/- 178 mL; Group 2: 436 +/- 170 mL; Group 3 (controls): 397 +/- 174 mL). The mean number of autologous units donated per patient did not differ (p>0.7) among the groups (Group 1: 3.1 +/- 0.6; Group 2: 2.9 +/- 0.7; Group 3: 3.0 +/- 0.7). The proportion of patients who needed allogeneic blood transfusion showed no significant (p>0.4) advantage for iron treatment, (Group 1: 7%; Group 2: 20%; Group 3: 10%). Conclusion In non-iron-deficient patients undergoing modest ABD without erythropoietin therapy, neither oral nor i.v. application of iron during the preoperative period enhances the success of preoperative ABD.

BACKGROUND: Quality assurance of blood transfusion covers institutions, personnel, and procedures involved in preparing, issuing, and using blood components. The accuracy of data related to blood component transfusions is a tool for quality control in the transfusion service. STUDY DESIGN AND METHODS: A study of the accuracy of data records of the transfusion service at the University Hospital of Erlangen, Germany, between June 1994 and May 1996 was carried out. All returned blood component transfusion report forms were examined for discrepancies between primary data records and clinical transfusion reports. RESULSTS: Blood components (n = 49,224) from allogeneic and autologous donations, packed red cells, fresh-frozen plasma, and platelet components that had been issued for transfusion were included in this evaluation. For 27.3 percent of all components issued, no transfusion report was returned to the blood bank. For the remaining 35,786 units, errors were found in 3.8 percent of the records. For 1.24 percent of all components, discrepant information related to the recipient's identity or the component's status was found; this affected the feasibility of lookback or traceback searches. CONCLUSION: A remarkably high frequency of discrepancies exists between computerized blood bank records and the information recorded on returned blood transfusion forms. The processes of data acquisition and entry must be included in quality assurance efforts in transfusion medicine.

Background: The consequences of the new edition of the ‘German Guidelines for Blood Grouping and Blood Transfusion (Haemotherapy)’ for aptitude tests of haemapheresis donors were studied. Material and Methods:During a 19-month period the reasons for exclusion and deferral of first-time donors were retrospectively evaluated, and the resulting exclusion and deferral rates in accordance with the old and the new edition of the German guidelines were calculated. Results:From January 1995 to July 1996 979 first-time donors for haemapheresis donations were tested. Based on the old edition of the guidelines, 4.6% of the first-time donors were excluded and 16.8% were temporarily deferred after taking the health history. From 770 persons blood and urine samples and ECGs were taken. Due to pathological results 5% of the first-time donors were excluded and 13.5% were temporarily deferred. Using the new edititon of the guidelines, 12.5% of the first-time donors would have been deferred after taking the health history and 7.9% because of pathological laboratory tests. The new edition of the guidelines did not change the exclusion rate of first-time donors. Conclusion: The new edition of the ‘German Guidelines for Blood Grouping and Blood Transfusion (Haemotherapy)’ reduces the deferral rate of voluntary first-time donors. Nevertheless, the deferral rate of first-time donors in Germany exceeds the deferral rates reported from other countries.

The Marburg I polymorphism of factor VII–activating protease and the risk of venous thromboembolism

BACKGROUND AND OBJECTIVES Because of widespread use of leukocyte reduction in platelet concentrates (PCs) and the need to store such concentrates, we investigated the effects of leukocyte depletion on the quality of stored PCs. MATERIALS AND METHODS Ten double-sized PCs were divided into 2 equal units which were tested simultaneously. One half was stored for 5 days after filtration through a polyester filter, the other one was stored unfiltered. RESULTS The volume of the 10 "oversized' PCs was 483 +/- 40 ml (mean +/- standard deviation) and they contained 5.9 +/- 1.5 x 10(11) platelets and 80 +/- 23 x 10(6) leukocytes. Filtration significantly reduced the leukocyte concentration (168 +/- 56/microliter before, 6 +/- 4 /microliter after filtration) and leukocyte count (39.9 +/- 11.3 x 10(6) vs. 1.3 +/- 0.9 x 10(6); p < 0.0005). Filtration caused a platelet loss of 16%, the platelet count decreasing not significantly from 2.91 +/- 0.75 x 10(11) to 2.40 +/- 0.94 x 10(11) (p = 0.26). After 5 days of storage all parameters of platelet function (platelet aggregation to several stimuli, hypotonic shock reaction [HSR] and platelet retraction), mean platelet volume, and pH and pCO2 showed no advantage for PCs filtered prior to storage compared to PCs stored unfiltered. Moreover, platelet aggregation on day 5 using 4 agonists at 10 concentrations showed worse results in 4 assays in prestorage filtered PCs (collagen [4 micrograms/ml: p < 0.05, ADP [0.2 mM]: p < 0.05, ADP [0.3 mM]: p < 0.05, thrombin [0.6 E/ml]: p < 0.05). But there is no convincing trend in all aggregation tests, and HSR, presumably the most useful parameter, was not different or day 5. CONCLUSIONS There is no advantage in terms of improved quality for prestorage leuko-depletion of PCs. Taking into account the obvious disadvantages of filtration, such as platelet loss and increasing costs per transfusion, we conclude that pre- or post-storage filtration of single-donor PCs should be done only for patients who have a clear indication for the transfusion of leukocyte-poor blood products.

Rationale:Oral anticoagulants and painkillers, some with an additional effect on the coagulation system, are widely used and are therefore prone to abuse and (intentional) overdose. We report the case of a patient with a massive mixed anticoagulant intoxication.Patient concerns:The patient h

Posttranslational modifications (PTMs) have been reported in hemoglobin (Hb) treated with ROS/RNS in cell-free experiments. However, little is known about oxidative PTMs of Hb occurring within the erythrocytes. The aim of this study is to characterize the patterns of Hb PTMs in erythrocytes under oxidative stress. Using mass spectrometry, we investigated specifically methionine/tryptophan oxidation, tyrosine nitration, and the modification via 4-hydroxynonenal (HNE), a product of lipid-peroxidation, on Hb. We demonstrated that the treatment with H(2)O(2)/nitrite induced higher levels of Hb oxidation/nitration in purified Hb preparations than in unpurified hemolysates and erythrocytes, indicating that ROS/RNS are primarily removed by antioxidative mechanisms. We further studied Hb from erythrocytes exposed to γ-irradiation. An irradiation of 30-100 Gy triggered a remarkable increase of intracellular ROS. However, 30 Gy did not induce apparent changes in Hb oxidation/nitration and hemolysis, while Hb oxidation/nitration and hemolysis were significantly enhanced by 100 Gy, suggesting that Hb oxidation/nitration are the consequence of overwhelmed antioxidative mechanisms after oxidative attack and reflect the severity of the oxidative damage of erythrocytes. Although irradiation was known to induce lipid-peroxidation, we could not detect HNE-Hb adducts in irradiated erythrocytes. Analyzing PTM patterns suggests Hb nitration as a more suitable indicator of the oxidative damage of erythrocytes.

Hintergrund: Seit Oktober 1991 steht für den Zellseparator Fresenius AS 104 ein Single-needle-(SN-)Programm zur Verfügung. Wir berichten über unsere Erfahrungen während der ersten 18 Monate im Routinebetrieb mit dem SN-Verfahren. Material und Methodik: Im Zeitraum von 18 Monaten wurden bei 225 Blutspendern 395 Thrombozytapheresen im SN-Verfahren durchgeführt. Der Einfluß unterschiedlicher Flußgeschwindigkeiten, Zyklusvolumina und Trenngrenzenpositionen auf die Effizienz der Thrombozytapherese und auf die leukozytäre Kontamination der Konzentrate wurde untersucht. Ergebnisse: Bei 395 Thrombozytapheresen konnten im Mittel 3,00 ± 0,69 ×1011 Thrombozyten bei einem durchschnittlichen Konzentratvolumen von 283 ± 39 ml und einer Leukozytenkontamination von 1,66 ± 3,1 ×108 Leukozyten pro Konzentrat gewonnen werden. Flußgeschwindigkeiten von 50 ml/min (versus 60 ml/min) sowie eine Trenngrenzenposition von 6:2 (versus 7:1) erbrachten verminderte leukozytäre Kontaminationen der Konzentrate, ohne jedoch statistische Signifikanz zu erreichen. Schlußfolgerung: Das SN-Programm des Zellseparators AS 104 zeichnet sich durch eine hohe Thrombozytenausbeute aus und zeigt im Vergleich zu anderen SN-Systemen geringere Leukozytenwerte im Konzentrat. Eine weitere Verminderung der Leukozyten im Konzentrat ist jedoch anzustreben, da 77% der Konzentrate den empfohlenen Grenzwert von 0,5×108 Leukozyten pro Konzentrat überschreiten.

BACKGROUND: There exists only very few data on in vitro and in vivo effects of gamma irradiation of red blood cells (RBCs) that have been leukoreduced by filtration before a subsequent irradiation. Reported studies reflect neither the current Food and Drug Administration (FDA) nor the European recommendations on timing of irradiation and subsequent storage. STUDY DESIGN AND METHODS: We studied 40 RBC units that were prepared from inline filtered whole blood and 40 RBC units that were filtered after component separation. All RBCs were stored in the additive solution saline-adenine-glucose-mannitol and leukoreduced on the collection day. In both groups, 20 components were irradiated on Day +14 with 30 Gy, and 20 served as nonirradiated controls. In vitro evaluation of both irradiated and nonirradiated RBC units was performed before and after irradiation on Days +1, +7, +14, +21, +28, +35, and +42 from the collection day. RESULTS: Gamma irradiation induced enhanced leakage of potassium ions and lactate dehydrogenase and an enhanced in vitro hemolysis rate in the irradiated components. However, in vitro hemolysis rate of both nonirradiated and irradiated components was remarkably lower than 0.8 percent, and the preservation of adenosine triphosphate over 42 days was satisfying. CONCLUSIONS: This study reflects the current FDA and European recommendations on timing of irradiation and subsequent storage. Our findings together with recent results of other investigations on the effect of gamma irradiation on leukoreduced RBCs allow the proposal that a storage time up to 28 days after irradiation is allowable.

The aim of this study was to investigate the effect of gamma irradiation with 30 Gy on the coagulation system in leukoreduced fresh-frozen plasma (FFP).In 74 FFP units that had been stored for 352 +/- 103 days below -30 degrees C, the following variables were determined in parallel in an irradiated and not irradiated half: prothrombin time (PT); activated partial thromboplastin time (APTT); thrombin time; antithrombin III; protein C; protein S; von Willebrand factor antigen; ristocetin cofactor; plasminogen-alpha(2)-antiplasmin; the coagulation factors fibrinogen, factor (F)II, FV, FVII, VIII, F IX, FX, FXI, FXII, FXIII, and activated factor XII (FXIIa); D-dimer; fibrin monomer; thrombin-antithrombin complex; prothrombin fragment 1 + 2 (F1+2); plasmin-alpha(2)-antiplasmin complexes (PAPs); and platelet factor 4. The FVII activity ratio was assayed to quantify activation of FVII.Irradiation with 30 Gy resulted in a reduction of APTT (35.0 +/- 4.1 sec vs. 34.4 +/- 4.1 sec; p = 0.00000006) and PT (89.8 +/- 8.2% vs. 90.7 +/- 8.0%; p = 0.002) and a significant increase of the activities of the coagulation factors FII, FV, FVII, F IX, FX, and FXII. FVIII activity decreased from 118 +/- 31 to 116 +/- 32 percent (p = 0.02). Activation of the coagulation system was shown by an increase in the FVII activity ratio (1.19 +/- 0.29 vs. 1.31 +/- 0.34; p = 0.0000001), FXIIa (0.81 +/- 0.50 ng/mL vs. 0.90 +/- 0.51 ng/mL; p = 0.006), and F1+2 (1.19 +/- 0.20 nmol/L vs. 1.24 +/- 0.20 nmol/L; p = 0.000005) after irradiation with 30 Gy, whereas an increase of PAP (16.2 +/- 11.5 ng/mL vs. 20.2 +/- 12.0 ng/mL; p = 0.0004) demonstrated activation of the fibrinolytic system. No negative influence of irradiation with 30 Gy on inhibitors of coagulation was observed.Gamma irradiation of leukoreduced FFPs with 30 Gy results in a significant but very weak activation of the coagulation and fibrinolytic system in FFPs.

BACKGROUND: Semaphorin 7A (Sema7A), the protein that carries the JMH blood group antigen, is involved in immune responses and plays an important role in axon growth and guidance. Because previous serologic studies on red blood cells (RBCs) suggested a considerable diversity of Sema7A, the present study was designed to elucidate the complex picture of the molecular diversity of this protein. STUDY DESIGN AND METHODS: The JMH antigen status was determined by serology, flow cytometry, and Western blot. Genomic and transcript analysis of SEMA7A was performed by nucleotide sequencing. Recombinant Sema7A proteins were used for genotype-phenotype correlation. A three-dimensional model of Sema7A was generated for topologic analyses. RESULTS: Our studies on 44 individuals with unusual JMH phenotypes and their family members revealed that aberrant Sema7A expression can be an inherited or an acquired phenomenon and is based on reduced surface expression or qualitative changes in Sema7A. These different phenotypes are caused by variations of the SEMA7A gene or seem to be generated by autoimmune-related or RBC lineage-specific mechanisms. The variant JMH phenotypes were related to the presence of missense mutations in SEMA7A, predicting amino acid changes in the semaphorin domain of Sema7A. Sequence analysis of the variant SEMA7A alleles revealed mutations affecting codons 207 and 460/461. Topologic analyses showed that Sema7A polymorphisms were prominently located on the top and bottom of the semaphorin domain, suggesting a functional relevance of these sites. CONCLUSION: These findings provide a basis with which to delineate the various ligand-binding surfaces of Sema7A.

During recent years eye drops from autologous serum have become increasingly popular for treating ocular surface disorders such as persistent corneal epithelial defects and severe forms of dry eye.1–4 Although very successful, one potential disadvantage of this approach—especially because the drops are used in a domestic setting—is the possibility of transmission of viral infections by the erroneous application of the eye drops to the wrong recipient. In a diagnostic laboratory, one single droplet of serum has been reported to have transmitted HIV to a laboratory technician.5 However, serological testing of patients who donate autologous serum is not generally established and there has been no report published so far analysing the rate of unknown viral infections in potential donors of autologous serum eye drops.1–4 We report the …