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Despite major advances in high-throughput and computational modelling techniques, understanding of the mechanisms regulating tissue specification and differentiation in higher eukaryotes, particularly man, remains limited. Microarray technology has been explored exhaustively in recent years and several standard approaches have been established to analyse the resultant datasets on a genome-wide scale. Gene expression time series offer a valuable opportunity to define temporal hierarchies and gain insight into the regulatory relationships of biological processes. However, unless datasets are exactly synchronous, time points cannot be compared directly. Here we present a data-driven analysis of regulatory elements from a microarray time series that tracked the differentiation of non-immortalised normal human urothelial (NHU) cells grown in culture. The datasets were obtained by harvesting differentiating and control cultures from finite bladder- and ureter-derived NHU cell lines at different time points using two previously validated, independent differentiation-inducing protocols. Due to the asynchronous nature of the data, a novel ranking analysis approach was adopted whereby we compared changes in the amplitude of experiment and control time series to identify common regulatory elements. Our approach offers a simple, fast and effective ranking method for genes that can be applied to other time series. The analysis identified ELF3 as a candidate transcriptional regulator involved in human urothelial cytodifferentiation. Differentiation-associated expression of ELF3 was confirmed in cell culture experiments and by immunohistochemical demonstration in situ. The importance of ELF3 in urothelial differentiation was verified by knockdown in NHU cells, which led to reduced expression of FOXA1 and GRHL3 transcription factors in response to PPARγ activation. The consequences of this were seen in the repressed expression of late/terminal differentiation-associated uroplakin 3a gene expression and in the compromised development and regeneration of urothelial barrier function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Network inference deals with the reconstruction of biological networks from experimental data. A variety of different reverse engineering techniques are available; they differ in the underlying assumptions and mathematical models used. One common problem for all approaches stems from the complexity of the task, due to the combinatorial explosion of different network topologies for increasing network size. To handle this problem, constraints are frequently used, for example on the node degree, number of edges, or constraints on regulation functions between network components. We propose to exploit topological considerations in the inference of gene regulatory networks. Such systems are often controlled by a small number of hub genes, while most other genes have only limited influence on the network's dynamic. We model gene regulation using a Bayesian network with discrete, Boolean nodes. A hierarchical prior is employed to identify hub genes. The first layer of the prior is used to regularize weights on edges emanating from one specific node. A second prior on hyperparameters controls the magnitude of the former regularization for different nodes. The net effect is that central nodes tend to form in reconstructed networks. Network reconstruction is then performed by maximization of or sampling from the posterior distribution. We evaluate our approach on simulated and real experimental data, indicating that we can reconstruct main regulatory interactions from the data. We furthermore compare our approach to other state-of-the art methods, showing superior performance in identifying hubs. Using a large publicly available dataset of over 800 cell cycle regulated genes, we are able to identify several main hub genes. Our method may thus provide a valuable tool to identify interesting candidate genes for further study. Furthermore, the approach presented may stimulate further developments in regularization methods for network reconstruction from data.

Although circulating hematopoietic progenitor cells (HPCs) are frequently used in therapeutic approaches, many aspects of their cellular biochemistry are still unclear. In the present study, the effects of cyclic nucleotide-elevating agents on HPC proliferation and differentiation were investigated. HPCs from different sources, including healthy persons, patients with tumors (medulloblastoma, seminoma, or multiple myeloma), and patients with chronic myelocytic leukemia (CML), were compared. HPCs were isolated by standard leukapheresis procedures and analyzed for proliferation and differentiation into the megakaryocytic and granulocytic lineages. HPCs contained high concentrations of cyclic guanosine monophosphate (cGMP)-dependent and cyclic adenosine monophosphate (cAMP)-dependent protein kinases G and A (PKG and PKA, respectively). Whereas PKG was partly down-regulated during culture, the PKA level remained constant. Stimulation of PKG in HPCs isolated from healthy donors or tumor patients resulted in a biphasic reaction: low cGMP concentrations inhibited proliferation and stimulated differentiation into megakaryocytes, whereas high concentrations revealed the opposite effect. In contrast, differentiation into granulocytes was inhibited in a concentration-dependent manner. Stimulation of PKA inhibited HPC differentiation; however, HPC proliferation was inhibited in controls and stimulated in HPCs from tumor patients. HPCs isolated from CML patients showed a nonhomogeneous reaction pattern to both cyclic nucleotides with high variability between the individual donors. We demonstrated the importance of the source of HPCs for the investigation of proliferation and differentiation. Cyclic nucleotide-regulated pathways are clearly involved in HPC proliferation and differentiation. Pharmacological strategies using cyclic nucleotide-elevating substances to influence HPC growth and differentiation in the bone marrow might support current strategies in HPC recovery from the peripheral blood.

Introduction: Abnormally shortened activated partial thromboplastin times (aPTT) are associated with significantly increased risk of thrombotic disorders and in-hospital mortality. Shortened aPTTs have been related to increased levels of factor (F) VIII and thrombin-antithrombin complex (TAT). In the current study, four different commercial aPTT reagents were evaluated for their performance to detect shortened aPTTs., Materials and Methods: aPTT of 400 patients was determined using Actin-FS (Dade Behring), APTT-SP (Instrumentation Laboratory), Automated-APTT (bioMerieux) and Platelin-LS (bioMerieux) reagents. FVIII, FIX, FXI and TAT levels were measured in shortened and normal aPTT samples., Results: An association between shortened aPTTs and elevated levels of coagulation factors (FVIII, FIX and FXI) and thrombin generation (TAT) was found with all tested aPTT reagents. Method-comparison studies demonstrated good agreement between Instrumentation Laboratory and bioMerieux reagents. However, 53 to 59% of the patients with a shortened aPTT measured with Actin-FS reagent was determined as a normal aPTT with APTT-SP, Automated-APTT and Platelin-LS reagents. These patients had increased levels of FVIII, FIX and FXI and moderately increased levels of TAT., Conclusion: Overall, an acceptable agreement between the different commercial reagents was found with respect to detection of short aPTTs. However, a disparity between some of reagents existed. Actin-FS reagent appeared to be more sensitive in inducing shortened aPTT reactions than APTT-SP, Automated-APTT and Platelin-LS reagents.

Background: Transfusion-related acute lung injury (TRALI) is an immune-mediated transfusion reaction that can cause severe complications or even death. It is now the leading cause of transfusion-related death in the United States., Methods: The TRALI syndrome is presented in two cases in a surgical intensive care unit and discussed against the background of the present literature. In both cases, concomitant diseases led to an extremely difficult course of TRALI., Conclusions: Knowledge of the TRALI syndrome is necessary to enable early diagnosis and treatment. It should be taken into consideration at any time when cardiopulmonary instability occurs after transfusion of blood products, which is a frequent event on surgical Intensive Care Units. TRALI remains a clinical diagnosis supported by serologic studies if these are available. Against the background of this potentially life-threatening complication, every single indication to transfuse blood products needs to be scrutinized carefully.

Prognosis of disseminated melanoma remains gloomy as neither chemotherapeutic nor unspecific immune modulatory approaches were able to improve the overall survival of these patients. Hence, specific immunotherapy has received increasing attention. Disappointing clinical results, however, indicate that the choice of suitable antigens is of special importance. To this end, the inhibitor of apoptosis (IAP) protein survivin, which is over-expressed in several tumours but is largely undetectable in adult tissues, appears to be a promising target for vaccination purposes, since down-regulation or loss of expression is associated with impaired tumour progression. Consequently, five heavily pretreated stage IV melanoma patients were vaccinated with the HLA-A2 restricted survivin(96-104) epitope presented by autologous dendritic cells (DCs) in a compassionate use setting. Four of these patients mounted strong T cell responses to this epitope as measured by ELISPOT assay. Furthermore, in situ peptide/HLA-A2 multimer staining confirmed that these survivin reactive cells infiltrated both visceral and soft tissue metastases.

Today, platelet concentrates are generally produced from whole blood by differential centrifugation (buffy coat-derived platelet concentrates--PCs) or by plateletpheresis (apheresis-derived platelet concentrates--APCs). As PCs are characterized by a lower number of platelets than APCs, four to six PCs are customarily combined in order to obtain an equivalent dose. In the 1970s and 1980s, the use of PCs exceeded that of APCs by far; in contrast, since the beginning of the 1990s, APCs comprise more than half of all transfused platelets. However, the selection of PCs or APCs for transfusion to thrombocytopenic patients is still a matter of debate. The present paper compares biochemical and functional properties of both platelet preparations in vitro. Besides plasma parameters (e.g. platelet factor 4 (PF4), P-selectin, C3a-desarginin, plasma coagulation factors), platelet function was analysed by aggregometry and the PFA 100 system. APCs are characterized by a better preservation of ADP and collagen-induced platelet aggregation, and shorter closure times of the PFA 100 test system during storage. The improved primary in vitro haemostatic capacity of APCs is presumed to be owing to a lower cellular activation rate in these preparations. This hypothesis is supported by the higher plasma concentrations of PF4, beta-thromboglobulin and P-selectin found in PCs compared with APCs. The concentrations of C3a-desarginin in PCs exceed those in APCs by far. Additionally, thrombin generation is higher in PCs than in APCs. These data suggest that APCs are characterized by a superior haemostatic capacity over PCs in vitro. However, in vivo studies should be performed to confirm these findings in the patients' circulation also.

In the course of reconstruction of peripheral arterial occlusion processes, two gelatin-coated Dacron grafts and one collagen-coated Dacron patch were implanted in a 52-year-old male patient. Eight days following low-dose heparinization (5 days prior to surgery, 3 days postoperatively) with unfractionated heparin, with no clinical symptoms present, a dramatic isolated thrombocyte depression occurred, from 212 Gpt/l prior to surgery to 14 Gpt/l on postoperative day 3. Laboratory tests verified an HIT type II [heparin-induced platelet aggregation assay (HIPAA) and ELISA]. Despite immediate discontinuation of heparin and commencement of an anticoagulant therapy with Revasc and Refludan, an 8-week thrombocyte depression occurred which was eliminated only temporarily by administration of gammaglobulin. The specific antibody tests turned out positive for more than 5 months. Having ruled out other causes of thrombocytopenia, we assume that the case presented was either due to an interaction not elucidated to date or triggered by the grafts (gelatin/collagen/Dacron). The manufacturers of the grafts have disputed a heparinoid action.

Aim: The incidence of heparin-induced thrombocytopenia (HIT type II) as a consequence of postoperative thrombosis prophylaxis after hip or knee prosthesis was investigated in this study. Furthermore the platelets count was postoperatively analysed in patients without HIT II., Patients and Methods: Patients with knee and hip prosthesis were included in a prospective study during an 8 months period. All patients received 3 x 5,000 Liquemin (Hoffmann-LaRoche) from the day of the operation until discharge. In cases with a platelet count drop of more than 40% and in patients with clinically manifest thrombosis or embolism a HIT type II test was initiated., Results: 5 of 252 patients included in this study developed a HIT type II. The platelet count drop was on average 65.7% (40.9-81.6). One patient died of a lung embolism (lethality 20%). Four patients were treated with Hirudin and 1 patient with Danaproid-Natrium. There was no drop of the platelet count between the 5th and 7th postoperative day of more than 15% in the other patients without HIT type II., Conclusion: In operative departments not enough attention is paid to HIT type II. Knowing the risks with an appropriate monitoring HIT type II can be early detected. Under these conditions the advantages of a heparin prophylaxis outweigh the risk of developing a HIT type II with it's life threatening sequelae.

History and Admission Findings: At the age of 55 years a now 70-year-old man had his aortic valve replaced by a prosthetic (Björk-Shiley) valve, and 11 years later a VDD pacemaker had been implanted. 18 months before the latest admission he had been hospitalized for treatment of staphylococcal endocarditis involving the aortic prothesis. At that time thrombocytopenia developed during heparin administration, diagnosed clinically and with the heparin-induced platelet activity (HIPA) test as type II heparin induced thrombocytopenia. His latest admission was for the diagnosis and treatment of peripheral arterial disease of the right leg (Fontaine stage IIb)., Investigations: Right popliteal and pedal pulses were not palpable. He was able to walk pain-free for only 70 m. Doppler sonography demonstrated an arm-leg index on the right of 0.7. Angiography revealed marked stenosis in the right superficial femoral artery and a filiform stenosis in the right popliteal artery., Treatment and Course: Both stenoses were relieved by percutaneous transluminal balloon angioplasty, in the course of which 5000 IU heparin were administered as a bolus intraarterially. Postoperative anticoagulation was maintained for 2 days with recombinant hirudin. There was no evidence of platelet reduction or heparin-induced antibodies despite the renewed infusion of heparin., Conclusion: Single re-administration of heparin in a patient who had developed a type II heparin-induced thrombocytopenia several years before does not necessarily lead to a booster of antibodies and thus to a reduction of platelets in the peripheral blood. It is a moot point whether the course in this case was an exception or the rule.

The PFA-100(R) (PFA) diagnostic system for the detection of platelet dysfunction was evaluated to determine reference ranges in a normal population. The PFA determines the primary haemostasis capacity (PHC) of anticoagulated whole blood, expressed by the system's closure time (CT). In this study the CT reference ranges were determined for blood samples collected in 105 mmol/l (3.2%) buffered citrate and the effect of gender, smoking, and use of oral contraceptives on reference ranges was assessed. Each of the 309 healthy blood donors from five blood centres was confirmed to have normal platelet function before inclusion in the study. Blood samples were tested in duplicate with both the collagen/epinephrine (Col/Epi) and collagen/ADP (Col/ADP) test cartridges. PFA reference ranges (90% central intervals of measured closure times) for both cartridge types were similar for all groups. Subgroup analysis showed that neither gender nor oral contraceptive usage had any effect on PHC. The 95% cut-off value for the Col/Epi CT was slightly higher for smokers than for non-smokers, an effect more pronounced in female than in male donors. However, the small difference did not justify establishment of specific reference ranges for smokers. Data from all included subjects were pooled to calculate the CT reference ranges for blood samples collected in 105 mmol/l buffered citrate (Col/Epi 82-150 s; Col/ADP 62-100 s). Normal levels of fibrinogen, as well as normal platelet counts and normal haematocrit levels, appeared not to influence the PHC. Because slight but significant differences of the reference ranges were observed between some of the participating sites, in-house confirmation of these reference range guidelines is recommended.

Background: We prospectively investigated lepirudin for further parenteral anticoagulation in patients with heparin-induced thrombocytopenia (HIT)., Methods and Results: Patients with confirmed HIT (n=112) received lepirudin according to need for 2 to 10 days (longer if necessary): A1, treatment: 0.4 mg/kg IV bolus, followed by 0.15 mg. kg(-1). h(-1) intravenous infusion, n=65; A2, treatment in conjunction with thrombolysis: 0.2 mg/kg, followed by 0.10 mg. kg(-1). h(-1), n=4; and B, prophylaxis: 0.10 mg. kg(-1). h(-1), n=43. Outcomes from 95 eligible lepirudin-treated patients were compared with those of historical control patients (n=120). Complete laboratory response (activated partial thromboplastin time ratio >1.5 with /=1 outcome (cumulative incidence 30.9% versus 52.1%; relative risk [RR] 0.71; P=0.12, log-rank test). Bleeding events were more frequent in the lepirudin group than the historical control group (cumulative incidence at 35 days, 44.6% versus 27.2%; RR 2.57; P=0.0001, log-rank test). No difference was observed in bleeding events requiring transfusion (cumulative incidence at 35 days, 12.9% versus 9.1%; RR 1.66; P=0.23, log-rank test); no intracranial bleeding was observed in the lepirudin group., Conclusions: Lepirudin effectively prevents death, limb amputations, and new thromboembolic complications and has an acceptable safety profile in HIT patients. Treatment should be initiated as soon as possible if HIT is suspected.

Background: During the past decade much work has been carried out towards establishing the optimum method for cryopreservation of platelets. Among the various cryoprotectants dimethyl sulfoxide (DMSO) has been shown to be the most effective. This report describes ultrastructural and functional changes of platelets during the deep-freezing process with DMSO., Materials and Methods: Single-donor platelet concentrates were cryopreserved in liquid nitrogen by use of DMSO. Before, during and after the freezing process samples were taken for analysis of ultrastructure and platelet function., Results: While after isolation and addition of DMSO a normal ultrastructure of platelets could be observed, clear signs of beginning cell necrosis were detected after thawing and resuspension in autologous plasma. Fibrinogen-binding capacity and platelet aggregation were significantly diminished., Conclusions: Although cryopreserved platelets are characterized by hemostatic effects in vivo, it seems conceivable that these effects could be improved by further development of platelet-freezing techniques.

It has been well established that cryopreservation of red cells with glycerol is a suitable method for long-time storage. Therefore, many data for quality control have been published. Most measurements, however, are restricted to the final product. Less information is available about the particular steps of cryopreservation. The present paper describes in detail the results of quality control measurements during the procedure. Although the final product meets the demand of the WHO for cryopreserved red cells, it could be demonstrated that erythrocytes are remarkably damaged by the deep-freezing process. Further experiments seem to be necessary in order to improve the details of the deep-freezing procedure.

Objective: Aim of the present analysis was the evaluation of clinical conditions and product-specific parameters influencing posttransfusion platelet increment., Design: 400 single-donor platelet transfusions were analyzed for patient- and concentrate-specific factors influencing posttransfusion platelet increment. Statistical analysis was performed by the General Mixed Model Analysis of Variance., Setting: Department of hematology and oncology at a university hospital., Patients: 46 patients (24 male, 22 female; age 17-80 years)., Interventions: Single-donor platelet transfusions., Results: As demonstrated earlier, splenomegaly, body temperature, and bone marrow transplantation could be proven as factors reducing posttransfusion platelet increment. In addition, hepatomegaly and application of antibiotics had negative effects on platelet increment. Among the product-specific parameters leukocyte contamination and pretransfusion storage time reduced transfusion success significantly., Conclusions: Clinical factors influencing posttransfusion platelet increment can hardly be controlled. In contrast, concentrate-specific parameters can be influenced by preparation technique and storage procedure. Therefore, high value should be set on low leukocyte contamination and short pretransfusion storage time of platelet concentrates.

Background: The shelf life of liquid-stored platelet concentrates is limited to 5 days. Therefore, much work has been carried out in an attempt to establish the optimum method for cryopreservation. Among the various cryoprotectants, dimethyl sulfoxide (DMSO) has been shown to be the most effective. However, DMSO-frozen platelets are characterized by a number of cell lesions. This report describes metabolic and functional changes that should give rise to some concern about the functional integrity of these cells., Study Design and Methods: Single-donor platelet concentrates were frozen in liquid nitrogen by use of DMSO (5%). After thawing, the cells were washed and resuspended in autologous plasma. Before, during, and after the freezing process, samples for analysis of metabolic measures (e.g., pH; calcium, potassium, and lactate dehydrogenase concentrations; plasma complement factors) and functional measures (e.g., aggregometry, in vitro bleeding time, alpha-granule membrane protein-140 expression) were taken., Results: Mean platelet volume increases during the deep-freezing process. Potassium, calcium, and lactate dehydrogenase are released from the intracellular space to the extracellular space. A strong activation of complement, which is mainly due to the addition of DMSO, is observed. Platelets become activated as indicated by the expression of alpha-granule membrane protein-140. Accordingly, decreased platelet function can be observed., Conclusion: DMSO-frozen platelets are characterized by several metabolic and functional changes. Although these cells have been shown to exert hemostatic effects in vivo, it is conceivable that those effects could be improved by further development of platelet-freezing techniques.

Leukocytes contaminating donated blood are considered to be responsible for many of the side effects associated with blood transfusions. These include HLA sensitization and its sequelae, as also graft versus host reaction, transmission of CMV. The present article summarizes the indications for leukocyte depletion and its technical execution.

Quality control of platelet concentrates (PC) is an important prerequisite for good transfusion praxis. However, direct measurement of platelet function is complex, since available methods (e.g. aggregometry, serotonin release) are time consuming and require special equipment. Therefore a test system is needed, which is easy to handle, fast, and achieves reliable results. The present paper compares the results of conventional platelet function tests with those of a modified in-vitro bleeding test (IVBT) (Thrombostat 4000) in liquid-stored and cryopreserved PCs. A high correlation between aggregometry, serotonin release, GMP 140 expression upon stimulation, and IVBT was demonstrated. Therefore IVBT seems to be a good alternative to the conventional platelet function tests for quality control of PCs. In addition, a good correlation between the results of IVBT of patients' blood after PC transfusion and IVBT of patients blood before transfusion supplemented with platelets of the respective PC could be found. Therefore IVBT seems to be able to predict PC transfusion success. However, since these data were obtained in a small sample undergoing bone marrow transplantation, further studies are needed to verify this hypothesis.

Storage of single-donor platelets is currently limited to 5 days. During this period, however, numerous morphologic and biochemical changes have been observed. The present study describes increased binding of anti-GMP 140 to stored single-donor platelet concentrates; this reveals a progressive activation process. In contrast, when stored platelets are stimulated with ADP, GMP 140, expression is reduced indicating a diminished release reaction. Additionally, the GP IIb/IIIa receptor complex and its fibrinogen-induced binding site (LIBS1) are reduced by storage time, demonstrating a diminished fibrinogen binding. Further experiments have to clarify whether these changes observed in vitro translate into a reduced hemostatic capacity after transfusion in vivo.

Single-donor platelets are stored up to 5 days prior to transfusion. Since contact of plasma to plastic surfaces may lead to complement activation, we investigated whether there is any increase in the complement factors C3a, C4a and C5a in routinely stored single-donor platelet concentrates. C3a levels increased about 40-fold during a 7-day storage. C4a levels also increased with storage time but to a lesser extent. By contrast, C5a levels remained stable throughout this period. ADP- and collagen-induced aggregation was impaired after storage of platelets, indicating severe functional injury. In platelet-poor plasma stored under identical conditions a comparable increase in C3a and C4a concentrations was observed. The loss of platelet function during storage might at least in part be due to the excessive anaphylatoxin concentrations observed.

Stored single-donor platelets are characterized by a progressive cell activation. The data presented indicate that activation of plasma coagulation and complement system could contribute to this storage lesion process.

The results of platelet transfusions depend upon a variety of different conditions; besides alloimmunization, a lot of clinical factors are responsible for transfusion success. The present paper tries to work out clinical findings, which are related to successful or unsuccessful platelet transfusions. The following criteria could be identified to influence posttransfusion platelet increment: hepatomegaly, splenomegaly, diagnosis, antibiotics, number and time of previous platelet transfusions. AB0 compatibility, pretransfusion storage time and leucocyte contamination. Therefore, in platelet transfusion therapy a high value should be set on AB0 compatibility, brief storage time and low leucocyte contamination, since these parameters--in contrast to the other influencing factors--can easily be controlled.

The transfusion of platelet concentrates now represents one of the most common therapeutic measures employed in transfusion medicine. In particular the development of new, aggressive forms of chemotherapy has rapidly increased the need for platelet concentrates over the last few years. The aim of the present study is to describe the major indications and discuss briefly the side effects and problems associated with the transfusion of platelet concentrates.

During the last decade, the trend toward intensifying chemotherapeutic regimens in patients with hematologic malignancies rapidly increased the demand for single-donor platelet concentrates (PCs). The logistics of such supply, however, necessitated the storage of these blood components prior to transfusion. Today, most blood centers use di(2-ethylhexyl)phthalate-free blood bags, which are assumed to allow a storage period of up to 5 days. This report describes biochemical and functional changes of stored single-donor PCs, which may influence the expected quality of PCs. The acid-base status is characterized by an initial respiratory alkalosis compensated by a metabolic acidosis. Changes in extracellular electrolyte, lactate dehydrogenase, glucose, lactate, elastase, and complement levels, as well as in the release of alpha granule content and the initial activation of plasma coagulation, are demonstrated. These changes result in a functional impairment of stored PCs as reflected by thromboxane and serotonin release reaction and by aggregation and in vitro bleeding time studies. In contrast, in vivo recovery and survival rates have been reported to be unaffected. Whether the good recovery and survival rates are caused by a rejuvenescence of stored PCs in vivo or are due to injured circulating platelets has not yet been proven.

Background: The increasing demand for single-donor platelet concentrates necessitates the storage of these blood products prior to transfusion. Quality control of these platelets, however, is still a problem. Most of the available techniques are time-consuming and require sophisticated equipment and specifically trained personnel. The present paper describes a new method for quality control of stored platelet concentrates., Materials and Methods: Single-donor platelet concentrates were stored for 7 days; daily aliquots were taken and the in vitro bleeding time (Thrombostat 4000) and platelet aggregation (aggregometer) were determined., Results: The in vitro bleeding test can be handled simply and fast. The results are comparable with those of platelet aggregation tests., Conclusion: The in vitro bleeding test provides a good alternative to the conventional methods commonly used for quality control of platelet concentrates.

The routine use of platelet concentrates has greatly increased during the last years. Most of the concentrates are transfused to hematologic patients, who frequently receive additional red blood cells. These polytransfusion regimens often result in the formation of HLA- or platelet-specific antibodies, which lead to refractoriness to further platelet support. In order to avoid this problem, the number of HLA antigens transmitted should be reduced. Therefore, a consequent leukocyte depletion of all blood products administered to multitransfused patients seems to be necessary. If antibodies are already preformed in the patient's serum, compatible platelets have to be selected for transfusion.

Equine blood may contain salicylic acid (SA) taken up as free acid or represents the metabolite of acetylsalicylic acid (ASA). To obtain information of SA in race horses we screened blood samples of trotting-horses routinely drawn to be analyzed for doping substances. The individual values determined followed a Gaussian distribution displaying a geometric mean of 19 ng SA per ml serum. A probit analysis revealed linear relationship (r = 0.995). Additional studies examined the antithrombotic efficacy of ASA in the horse. An oral dose of 300 mg ASA considerably elevated the bleeding time for more than 2 hours with concomitant SA serum levels between 800 and 1000 ng/ml. It is concluded that salicylate levels even below 1 microgram/ml serum bring about considerable pharmacologic effects such as prolongation of bleeding time, decrease in blood viscosity and possibly dilatation of blood vessels. These effects may improve the tissue supply with blood including oxygen.

Leukocyte-depleted red cell concentrate (RCC) and plasma were separated by a hollow fiber filter system combined with a leukocyte filter without any additional devices. The RCC with 100 ml additive solution had a weight of 329 g; hematocrit was 0.55, free hemoglobin 16 mg/dl; leukocytes were (0.6 +/- 0.6) x 10(9)/l. The plasma (268 g) contained 5.4 g/dl of total protein, and only a few blood cells; clotting factor VIII activity 75%, all satisfying the guidelines.

A variety of antibody screening tests are available to detect immunization in patients' sera. However, antibodies can be observed only if corresponding antigens are present on test cells used by an antibody screen. By comparing available test cell kits of different manufacturers we revealed various specificities of antigens present or absent on these cells. Using by an antibody screen Wr(a+), Co(b+) and Kp(a+) test cells additionally to available test cells, we detected in 1000 sera of patients 13 anti-Wr(a) and 1 anti-Co(b) besides 17 antibodies with different specificities.

Transfusion-associated graft-versus-host disease (TA-GVHD) resulting from the engraftment of competent lymphocytes contained in blood products has been well described in immunocompromised patients and more recently in immunocompetent patients. Prophylactic irradiation of blood products prior to transfusion is the most efficient way to prevent TA-GVHD. Standard blood bank measures to reduce mononuclear cell contamination in red blood cell units, such as freezing, washing and filtration, may reduce the number of viable lymphocytes to prevent immunizations. However, it is unknown whether the depletion of leukocytes with these techniques would decrease the risk of TA-GVHD. In this report we describe the first case of TA-GVHD following transfusion of filtrated red blood cells given to a patient receiving cytotoxic therapy for Hodgkin's disease.

Optimal storage of platelet concentrates is still an unsolved problem. The present paper demonstrates changes in morphology and function of stored platelets. Transmission microscopy reveals a loss of organelles as well as a progredient destruction of cell membranes during storage. At the same time in vitro aggregability is clearly diminished. Therefore, further investigations seem to be necessary to improve storage conditions of platelets.

A newly developed filter-system (PL 100) for the removal of leukocytes from platelet concentrates was tested. It removes contaminating leukocytes by 99.7 +/- 0.6%. The platelet loss was calculated as 25.7 +/- 9.5%. The filtration process does not influence the filtered platelets; plasma-electrolytes, LDH-concentrations, platelet aggregation curves and serotonin-release remain constant. In addition, platelet ultrastructure is not influenced by the filtration. In spite of the high platelet loss, the tested filter-system seems to be a suitable alternative to the conventional methods for the removal of contaminating leukocytes from platelet concentrates.

Clinical and experimental studies indicate, that blood transfusion can modify the recipient's immune system. While beneficial to renal allograft survival, these immunomodulating effects may, however, prove detrimental to cancer patients. Recently, an adverse relationship between blood transfusion and cancer recurrence was reported in colon, lung, breast, kidney and gastric cancer. Moreover, a higher postoperative infection rate was observed when blood units were administered intraoperatively. We retrospectively reviewed the records of 174 patients with squamous cell carcinoma of the larynx undergoing curative resection between 1979 and 1985. One hundred and forty-one patients received blood transfusions, 33 did not. Recurrence rate (16.7%) was significantly related to clinical stage (p = 0.008) and lymph node status (p = 0.000); cumulative survival time depended significantly on clinical stage (p = 0.003), lymph node status (p = 0.004) and tumor size (p = 0.017). In contrast, when corrected for these baseline prognostic factors, no significant correlation could be detected between blood transfusion and cancer relapse. Also, survival time did not depend on blood transfusions. No correlation could be found between postoperative infection rate and intraoperative application of blood transfusion (p = 0.694). The present study does not support the hypothesis that blood transfusion adversely affects the prognosis of patients with laryngeal cancer. It indicates, that risk factors other than blood transfusion have a greater influence on recurrence and survival time.

A newly developed filter-system (Sepacell PL) for the removal of leukocytes from single donor platelet concentrates was tested. It removes contaminating leukocytes by 99.9%, the platelet loss was calculated as less than 1%. Platelet ultrastructure and function do not seem to be altered by the filtration. Therefore, the tested filter system seems to be a suitable device for leukocyte depletion of single donor platelet concentrates. Further clinical studies, however, are necessary to prove the efficiency of the filter system in preventing alloimmunization in vivo.

A commercially available solid phase red cell adherence test (Capture P) for platelet crossmatching was tested. The specificity of the test is 94.8%, the sensitivity 47.9%. Whereas the predictive value for negative results is low (52.9%), the predictive value for positive results was calculated as 93.8%. Therefore, the Capture P test seems to be useful for the exclusion of incompatible single donor platelet concentrates from transfusion. When negative results are obtained, however, no precise prediction of the transfusion success can be made.

Binding experiments were performed with [3H]ouabain on plasma membranes derived from several types of isolated and cultivated endothelial cells. Identical saturation curves for [3H]ouabain binding to endothelial cells from pig aorta, caval vein, and pulmonary artery were obtained with a dissociation constant (KD) of 3.29 +/- 0.31 nmol/l and a binding capacity (Bmax) of 5.22 +/- 0.12 pmol/mg protein. On guinea-pig coronary endothelial cells, saturation of [3H]ouabain revealed much lower affinity (KD 95 +/- 15 nmol/l, Bmax 2.08 +/- 0.09 pmol/mg protein). All Scatchard plots were linear, indicating a homogeneous class of binding sites. In competition experiments, cardiac glycosides and their aglycons displaced the radioligand with a structure-activity relationship typical for interaction with Na+/K+-ATPase (proscillaridin A greater than ouabain greater than digoxin greater than g-strophanthidin greater than digoxigenin greater than dihydrodigoxin); in particular, removal of the sugar moiety results in considerable reduction of affinity. Furthermore, K+ displayed a steep inhibition curve with a half-maximal inhibitory constant of 2 mmol/l. All these findings suggest the presence of endothelial ouabain receptors linked to Na+/K+-ATPase. However, direct measurement of this enzyme was not possible due to an extremely high Mg2+-ATPase activity.

The adenosine hypothesis in its original form neglects other myocardial tissues besides the predominant cardiomyocyte compartment. We could, however, demonstrate that the coronary endothelium comprises a metabolically very active adenosine and adenine nucleotide compartment of the heart, and functions as an impermeable metabolic barrier for interstitially or intravascularly accumulating adenosine if the vasoactive nucleoside is present at concentrations less than 10(-6) M. As a consequence, the vasodilatory action of intracoronarily applied adenosine cannot result from a direct action on the smooth muscle cells of the arterioles, but must be mediated by the endothelium. Since high molecular weight derivatives of adenosine, which are clearly confined to the coronary system, can also induce a very prompt coronary flow increase when applied intravascularly, smooth muscle relaxation must be triggered by an extracellular adenosine receptor at the luminal surface of the endothelium. According to preliminary pharmacological studies, this receptor belongs to the A2-type and thus stimulates the endothelial adenylate cyclase system. On the basis of our findings it is evident that with respect to the vasodilating effect of adenosine one has to distinguish between its action from the interstitial space directly via the putative receptor at the surface of the arteriolar smooth muscle cells, and its action from the intravascular space via the newly detected endothelial A2-receptor. It is a matter of further experimentation to determine to what extent both receptor populations actually participate in the metabolic regulation of coronary flow under physiological and pathophysiological conditions.

A prospective randomized study was carried out on 29 patients undergoing transurethral prostatectomy. In addition to the regional anaesthetic given for the operation, the patients received either: 1 mg morphine intrathecally (spinal group), 0.05 mg/kg body weight of morphine i.v. (i.v. group), or 0.05 mg/kg body weight epidurally (PDA group). Two of the intrathecal group patients had to be given an antagonist because of clinically relevant respiratory depression. In one of these cases, this depression could be documented by a continuous fall in respiratory minute volume, and an increase in PCO2. In the other, bradypnoea and vomiting developed within a few minutes of injection. The presence of a central action of intrathecal and epidural opiates was indicated by the significant increase in reaction time found. In the two instances of respiratory depression, the CSF morphine concentration 24 hrs after injection was markedly lower (0 and 18 ng/ml respectively) than in unaffected patients. It must therefore be assumed that the respiratory depression was caused by a more rapid cephelad transport than that occurring in normal cases.

We compared the manual Polybrene technique to three standard methods (albumin/Coombs, LISS/Coombs, and enzyme/papain) on 113 red cell antibodies. Polybrene identified 8 antibodies of the Rhesus system missed by standard methods, whereas 3 antibodies could only be detected by the standard techniques. Four antibodies were identified only in saline solution at room temperature; 6 were found by use of Polybrene exclusively in the additional Coombs phase. In addition, 61 antibodies were tested by 4 different LISS. No considerable differences in the quality of the various LISS were seen. The manual Polybrene test appears to be suitable for crossmatching and rapid antibody identification in emergency situations.

Cultured coronary endothelial cells and the coronary endothelium of isolated perfused guinea-pig hearts are characterized by a very active adenosine and adenine nucleotide metabolism. Adenosine applied to the endothelium at low concentrations is avidly metabolized and preferentially incorporated into different nucleotide pools--only a minor amount is degraded to uric acid. Physiologically, the coronary endothelium therefore functions as an impermeable metabolic barrier for interstitially or intravascularly accumulating adenosine. Only at concentrations greater than or equal to 10(-6) M adenosine can pass the endothelial barrier. As a consequence, the vasodilatory action of adenosine formed in or administered into the coronary system cannot be induced by a direct association of the nucleoside with the putative adenosine receptor of the arteriolar smooth muscle cells, but must be mediated by the endothelium. High molecular weight derivatives of adenosine, clearly confined to the coronary system, can also induce a coronary dilation. The endothelium-mediated smooth muscle relaxation is therefore obviously due to triggering of an extracellular adenosine receptor at the luminal surface of the endothelium. Since this process is accompanied by a rapid and pronounced activation of the adenylate cyclase system, the endothelial receptor conforms to an A2-type. According to our results it is necessary to reconsider qualitative and quantitative facets of the adenosine hypothesis of metabolic regulation of coronary blood flow, which--in its original formulation--exclusively centers on the cardiomyocyte metabolism. With respect to the vasoactivity of adenosine one obviously has to distinguish between its action from the interstitial space directly via the myocyte receptors of the vessel wall, and/or its action from the intracoronary space via the newly detected endothelial A2-receptor. More information is needed to determine the extent to which both receptor populations actually participate in the metabolic regulation of coronary flow under physiological and pathophysiological conditions.

In mammalian heart tissue beta 2-adrenoceptors are known to coexist with beta 1-adrenoceptors. In the present study, evidence that beta 2-adrenoceptors in guinea-pig and rat ventricles are primarily localized on the coronary endothelium is provided by competition binding studies with the subtype-selective beta-adrenoceptor antagonists ICI 89.406 (beta 1-selective) and ICI 118.551 (beta 2-selective) on four different plasma membrane preparations. (1) Following density gradient centrifugation of cardiac ventricular microsomes from rats or guinea-pigs, endothelial plasma membranes migrated at slightly higher density than the sarcolemmal membranes, as verified by endothelial (angiotensin converting enzyme) and sarcolemmal markers (adenylate cyclase, [3H]ouabain binding). At the activity peak of angiotensin converting enzyme, the relative amount of beta 2-adrenoceptors in guinea-pigs and rats was 25% and 65%, respectively. (2) On sarcolemmal membranes corresponding to the activity peak of adenylate cyclase, beta-adrenoceptors consisted of the beta 1-type exclusively (guinea-pig), or to at least 90% (rat). (3) Cultures of coronary endothelial cells derived from guinea-pigs revealed only beta 2-adrenoceptors. (4) Isolated guinea-pig cardiomyocytes contained only beta 1-adrenoceptors, a finding recently established in rat myocytes as well.