Leucoreduction helps to preserve activity of antioxidant barrier enzymes in stored red blood cell concentrates.

BACKGROUND: Oxidoreductive imbalance is a major cause of excessive haemolysis in in vitro conditions. Leucocytes and blood platelets present in red blood cell concentrates (RBCs) are one of the sources of free radicals, which have a significant effect on the status of stored erythrocytes. The study objective was to assess the effect of leucoreduction on the intensity of lipid peroxidation and the activity of antioxidant barrier enzymes in RBC. STUDY DESIGN AND METHODS: Red blood cell concentrates units obtained from 10 whole-blood units were split into two equal units, one of which was leucoreduced on the day of donation. Both units were stored for 35 days. The following markers of oxidoreductive balance were measured on day 0 (donation day) and on storage days 7, 14, 21 and 35: concentration of malondialdehyde (MDA) and activities of antioxidant barrier components, that is superoxide dismutase, glutathione peroxidase and glutathione reductase. RESULTS: Lipid peroxidation in leucodepleted units (LRBC) was slower than that in non-leucodepleted ones. The analysis of LRBC revealed statistically significant decrease in concentrations of MDA. The activities of superoxide dismutase, glutathione peroxidase and glutathione reductase were higher throughout the storage period as compared to non-leucoreduced RBC. Statistically significant differences between RBC and LRBC units were noted throughout the storage in the activity of lactate dehydrogenase, and concentrations of K(+) ions and free haemoglobin. CONCLUSIONS: Leucoreduction of RBC before storage helps to preserve the activity of antioxidant barrier enzymes in stored RBCs and significantly improves the quality of stored red blood cell components.

Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion-Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. MATERIAL AND METHODS: Four Bacteria References (Staphylococcus epidermidis PEI-B-06, Streptococcus pyogenes PEI-B-20, Klebsiella pneumoniae PEI-B-08 and Escherichia coli PEI-B-19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. RESULTS: Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19-1·32 × 10(7) CFU/ml, S. pyogenes: 0·58-0·69 × 10(7) CFU/ml, K. pneumoniae: 18·71-20·26 × 10(7) CFU/ml and E. coli: 1·78-2·10 × 10(7) CFU/ml. CONCLUSION: The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low-titre spiking of blood components, (ii) the property of donor-independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion-Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.

Decreased levels of PAI-1 and alpha 2-antiplasmin contribute to enhanced fibrinolytic activity in divers.

BACKGROUND: There are a number of reported cases of decompression sickness (DCS) with haemorrhages. These cases have not been sufficiently investigated and thus bleeding complications could not be directly correlated to the enhanced fibrinolysis. OBJECTIVES: The effect of hyperbaric exposition and decompression on the main components of fibrinolytic system has been measured. METHODS: Two groups of 25 male divers each were subjected to hyperbaric exposures to the pressure of either 400 kPa - group I - or 700 kPa - group II followed by a staged decompression. The divers were monitored for clinical symptoms of DCS and checked for Doppler-detected venous gas bubbles. Venous blood was drawn from divers before exposition and 15 min after decompression. The concentrations and activities of t-PA and PAI-1 as well as concentrations of PAP and alpha2-antiplasmin and activity of factor XIIa were measured. RESULTS: In all groups of divers no cases of DCS as well as detectable gas bubbles were noted. We observed elevated concentration of PAP, decreased concentration of alpha2-AP, decreased PAI-1 concentration and activity. There were no significant changes in factor XIIa activity as well as of t-PA concentration and activity. CONCLUSIONS: Hyperbaric exposition and decompression induce activation of fibrinolysis, even in the absence of detectable gas bubbles. Fibrinolytic activity increases mainly due to decrease of PAI-1 concentration and activity. Further clinical trials are necessary for the estimation of the importance of activation of fibrinolysis with decreased level of PAI-1 and alpha2-AP as a possible risk factor for bleeding in divers.

Ozonation of human blood increases sphingosine-1-phosphate in plasma.

Ozonated blood therapy is used in the treatment of several diseases, including superficial infections, burns, dental and intestinal conditions. Except that, the possibility of using ozone to sterilize blood supplies is under promising investigation. However, still little is known regarding the impact of blood ozonation, especially on biologically active serum sphinoglipids. In the present work we sought to investigate the contents of sphingolipids, such as sphingosine, sphingosine-1-phosphate (S-1-P), sphinganine, and ceramide (CER) in the plasma, after immediate and prolonged (1 h) ozonation of human whole blood. For the measurements liquid chromatography hyphenated with the mass spectrometry was applied. We demonstrated that only the content of sphingosine-1-phosphate in the plasma was increased significantly, possibly exerting its beneficial effect for various physiological and clinical events.

Antiplatelet and anticoagulant effects of "HN-11 500," a selective thromboxane receptor antagonist.

The antiplatelet and anticoagulant effect of a thromboxane receptor (TX receptor) antagonist developed by Nycomed (Linz) has been studied in a placebo-controlled double-blind phase I study. Sixteen healthy male volunteers received different single oral doses of "HN-11 500" (C(14)H(15)NO(5)S(2); 1, 10, 100, 200, and 400 mg). Eight volunteers received placebo. The washout period between each dosage applied was at least 12 days. Platelet aggregation induced by the thromboxane mimetic "U 46 619" (C(21)H(34)0(4)) and platelet adhesion to siliconized glass were significantly and dose-dependently inhibited. The effect lasted between 3 and 4 h (10 mg) and 8 h (400 mg), respectively, and correlated well with the pharmacokinetic data. Platelet aggregation seems to be more sensitive to monitor the effects of HN-11 500 on platelet function than platelet adhesion. Plasma levels of 300 ng/ml HN-11 500 probably leads to >90% inhibition of platelet aggregation. The template bleeding time slightly increased but did not exceed the normal range. Furthermore, there was a wide variation of results. There were no significant changes in platelet counts, platelet-induced thrombin generation time (PITT), and blood coagulation parameters. All doses of HN-11 500 were well tolerated. HN-11 500 is a potent TX receptor antagonist (TXRA), which inhibits either platelet aggregation or platelet adhesion, which has not yet been described. In clinical routine, TXRAs have to demonstrate the effectiveness in large clinical trials for different clinical indications and to compete with single or combined administrations of cyclooxygenase (COX) inhibitors, thienovridines, thromboxane synthase inhibitors, and GIIb/IIIa inhibitors.

Platelet activation and its role in thrombin generation in platelet-induced thrombin generation time.

Platelet-induced thrombin generation time (PITT) is a newly developed global coagulation assay in which a small amount of partially anticoagulated platelet-rich plasma (PRP) is rotated in a disc-shaped cuvette within the light beam of a photometer. The time intervals from onset of rotation until aggregation and coagulation of the sample are registered. The aim of our study was to compare platelet activation with generation of thrombin during rotation of PRP in PITT system. Aliquots of PRP were taken before, 1, 3, and 8 min after the onset of rotation as well as at the beginning of aggregation and shortly before coagulation. Thrombin activity was measured with chromogenic substrate S-2238. We have also measured the level of generated prothrombin activation fragment 1+2 (F1+2), which reflects the concentration of liberated thrombin. Platelet activation was assayed by means of platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) concentration and registration of the aggregation. The concentrations of the F1+2, PF4, beta-TG increased very slowly from the beginning of the test until aggregation occurred. From the start of aggregation, the levels of F1+2 rose rapidly. In contrast to the F1+2 measurements, thrombin activity has not been detected from onset of rotation until the end of the test. Only trace thrombin activity was detectable just after the plasma sample had been clotted in the cuvette. Our results demonstrate that there exists a close relationship between platelet activation and thrombin generation. Viable platelets, which adhered to the cuvette walls, form an active template on which thrombin can be generated from prothrombin.

Laboratory monitoring of new antithrombotic drugs.

This article examines laboratory methods that are used or may be used to monitor newly developed heparins, low-dose vitamin K antagonists, new thrombin inhibitors such as recombinant hirudin, and oligopeptide thrombin inhibitors (some of which it is hoped will be orally active), thromboxane receptor antagonists, glycoprotein IIb-IIIa inhibitors, and ticlopidine-like structures.

Changes in the extrinsic and intrinsic coagulation pathways in humans after decompression following saturation diving.

We have investigated the effect of simulated saturation diving on the activation of intrinsic and extrinsic coagulation pathways. Thirty-one male divers divided into two groups were tested in decompression habitat LSH-200. The first group of 16 divers was subjected to hyperbaric exposure at pressure of 180 kPa with air as a breathing mixture, and the second group of 15 divers, exposed to a pressure of 400 kPa with a heliox breathing mixture (helium-oxygen mixture: pO2, 40 kPa; pN2, 40 kPa; pHe, 420 kPa). The concentrations of tissue factor, tissue factor pathway inhibitor, factors XII, X, VII, and I, prothrombin fragment F1 + 2, and thrombin-antithrombin complex as well as platelet count, prothrombin time, activated partial thromboplastin time, plasmin-antiplasmin complex (PAP) and D-dimers were measured. We did not detect activation of the extrinsic coagulation pathway after decompression. There was a statistically significant decrease in platelet counts and factor I, XII and X concentrations after air-diving, and a potent and statistically significant increase of PAP concentration in both groups of divers. We suggest that saturated air or heliox diving followed by decompression have little if any effect on thrombin generation. Saturated air diving, however, may induce a decrease in platelet count and factor XII concentration. The observed elevation of PAP concentrations in both groups of divers suggests possible activation of fibrinolysis. The exact effect of diving and decompression on fibrinolytic system has to be further investigated.

Experimental studies on the anticoagulant and antithrombotic effects of sodium and calcium pentosan polysulphate.

In the present study we have compared the antithrombotic and anticoagulant properties of sodium and calcium derivatives of pentosan polysulphate (Na-PPS, Ca-PPS). The antithrombotic effect of these agents have been investigated in an experimental thrombosis model in which rat mesenteric venules diameter of 20-30 microm were injured by well defined Argon laser lesions. Furthermore, the in vivo and in vitro anticoagulant activities (aPTT, Heptest) of these agents have been studied. Thrombus formation was significantly inhibited after s.c. injection of Na-PPS and Ca-PPS in doses above 10 mg/kg. The duration of the antithrombotic effect lasted 8 h for Na-PPS and 12 h for Ca-PPS. After oral administration of Na-PPS an antithrombotic effect was not observed. Oral application of Ca-PPS in doses higher than 20 mg/kg significantly inhibited thrombus formation. Na-PPS and Ca-PPS markedly prolonged clotting time in aPTT and Heptest in concentrations ranging from 0.01 to 0.2 mg/ml rat PTT. Two h after s.c. administration of these agents in a dose 10 mg/kg, the aPTT increased 3-fold and Heptest 2.5-fold compared to controls. After oral application of 50 mg/kg Na-PPS and Ca-PPS no effect on coagulation test could be measured.

Von Willebrand factor antigen in assessment of vasculitis in patients with connective tissue diseases.

Von Willebrand factor antigen (vWF:Ag) is synthesized and secreted by endothelial cells. In the present study we tried to assess the relationship between plasma level of vWF:Ag and vascular damage in patients with vasculitis. The study was carried out on 59 patients with connective tissue diseases. Vasculitis was diagnosed by biopsies of the skin. The patients with vasculitis had a significantly elevated level of vWF:Ag; however, no significant correlation between the amount of plasma vWF:Ag and the degree of vasculitis was found. The obtained results show that the plasma level of vWF:Ag may reflect the presence of vascular, especially endothelial, damage in patients with connective tissue diseases.

Effects of aprosulate, a novel synthetic glycosaminoglycan, on coagulation and platelet function parameters: a prospective, randomized phase I study.

In a phase I clinical trial the effect of the highly sulfated polyanion "Aprosulate" was studied in healthy volunteers using different coagulation and platelet function parameters. Eighteen healthy volunteers aged 21 to 30 years received two single subcutaneous doses of aprosulate (0.5 mg/kg body weight; 1.0 mg/kg body weight), or unfractionated heparin (Calciparin 7,500 IU). The washout period between the different drugs/doses was at least 7 days. Coagulation and platelet function parameters (activated partial thromboplastin time, Heptest, fibrinogen, von Willebrand factor, ristocetin cofactor, platelet adhesion to siliconized glass, and platelet-induced thrombin generation time [a new method for measuring thrombin generation in platelet-rich plasma in the presence of platelets]) were assessed during 24 hours after each injection. Aprosulate led to a significant and dose-dependent prolongation of activated partial thromboplastin time and Heptest. This effect lasted for 4 hours (activated partial thromboplastin time) to 8 hours (Heptest). Activated partial thromboplastin time was not prolonged after the injection of unfractionated heparin (7,500 IU). Fibrinogen, von Willebrand factor, and ristocetin cofactor remained unchanged with both drugs. Platelet induced thrombin generation time was slightly prolonged and platelet adhesion was slightly diminished up to 2 hours using 0.5 mg/kg aprosulate, and up to 4 hours using 1.0 mg/kg aprosulate while the platelet induced thrombin generation time system was not influenced by the subcutaneous injection (7,500 IU) of unfractionated heparin. Both drugs and doses were well tolerated. Plasma transaminase concentrations alanin aminotransferase and aspartate aminotransferase serum values were slightly increased in some volunteers but returned to normal during or after the study (< 4 weeks). Further clinical trials will have to establish whether aprosulate is an effective drug for the prophylaxis of deep venous thrombosis.

The incidence of HLA DRB1*0103 in ulcerative colitis patients in north-eastern Poland.

BACKGROUND/AIMS: Susceptibility to inflammatory bowel disease is partially genetically determined and the HLA (human leukocyte antigen) alloantigens and genes located in the HLA region have been studied over the course of many years as the candidate genes responsible for ulcerative colitis. Improvements in molecular genotyping have allowed disease association with HLA to be narrowed down to specific subtypes. For class II antigens, increasing phenotype frequency of DRB1*0103, DRB1*1502 is observed and positive correlation to disease susceptibility is proposed. We investigated the incidence of HLA DRB1*0103 in ulcerative colitis patients in North-Eastern Poland and possible association with overall disease susceptibility and clinical course of the disease. METHODOLOGY: 41 patients and 45 healthy control blood donors were included in this study. All subjects were Polish. RESULTS: The incidence of HLA DRB1*0103 was low (2.44%), but was associated with fulminant course of the disease (pancolitis with megacolon toxicum). None of the ethnically matched healthy control blood donors possessed the HLA DRB1*0103 allele (0.00%). CONCLUSIONS: The results gained in the presented study confirm, that in the Polish population HLA DRB1*0103 allele is uncommon and it would not be a useful marker of disease susceptibility.

[Alloimmune neonatal thrombocytopenia caused by serologic incompatibility of HPA-1a platelet antigen--case report]. / Alloimmunologiczna maloplytkowosc noworodka wywolana konfliktem serologicznym w zakresie antygenu plytkowego HPA-1 a--opis przypadku.

A case of newborn of the 7th pregnancy; 3rd delivery with NAIT is presented. We focus on the necessity of performing serological diagnostics of platelet antigens and antibodies if in the former neonate the thrombocytopenia of unknown etiology was observed or previous pregnancies resulted in natural abortion of unknown origin.

The role of MAP kinases in the induction of iNOS expression in neutrophils exposed to NDMA: the involvement transcription factors.

PURPOSE: The role of MAP kinases in the activation of AP-1 (c-Jun, c-Fos) and NF-κB p65 engaged in the regulation of iNOS expression in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was analyzed in the study. MATERIAL AND METHODS: The study included a group of 20 healthy individuals. Isolated human PMN were incubated in the presence of NDMA. Selective MAP kinases inhibitors were used. The expression of proteins in the cytoplasmic and nuclear fractions was assessed using Western blot method. RESULTS: The results show that NDMA intensifies iNOS, c-Jun, NF-κB p65 and IκB-α expression in the analyzed PMNs. The blocking of the p38 pathway led to lower iNOS expression, and higher expression of c-Jun and c-Fos in the cytoplasmic fraction, and also lower c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. A decrease in iNOS expression in the cytoplasmic fraction, and also c-Jun in both fractions of the examined cells, was observed as a result of JNK pathway inhibition. The blocking of the ERK5 pathway led to higher iNOS, c-Jun and c-Fos expression in the cytoplasmic fraction, and higher c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. The study also demonstrated that blocking of the p38 and JNK pathways resulted in higher expression of NF-κB p65 and IκB-α in the cytoplasmic fraction and their lower expression in the nuclear fraction of these cells. CONCLUSION: Our data indicate the role of MAP kinases p38 and JNK in the activation of c-Jun and NF-κB p65 transcription factors engaged in the regulation of iNOS expression in human neutrophils exposed to NDMA. However ERK5 kinase is not involved in the regulation of iNOS and NO production by those cells.

Trimix instead of air, decreases the effect of short-term hyperbaric exposures on platelet and fibrinolysis activation.

PURPOSE: Short-term and saturated simulated dives followed by decompression with air, cause a decrease in platelet count and increased activation of fibrinolysis. The aim of this study was to determine whether short-term dives with trimix as a breathing mixture induce the activation of platelets, and/or fibrinolysis. MATERIAL AND METHODS: 30 male divers were subjected to short-term hyperbaric exposures to 0.7 MPa. Thirty divers used air and then the same divers used trimix as a breathing mixture. RESULTS: The mean platelet count dropped significantly after decompression only in the group breathing air. The number of CD62P positive platelets and the amount of platelet-derived micro particles were statistically significant higher after decompression in both exposures. The number of CD61 positive platelets increased significantly only in the group breathing air. We observed a significant decrease of factor XII and fibrinogen concentrations after decompression only in the group breathing air. A significant increase in the concentration of plasminantiplasmin complex in both groups was detected. CONCLUSIONS: Short-term hyperbaric exposure and decompression performed according to current safety standards activates platelets and the fibrinolytic system. Trimix protects divers from a reduction in the amount of platelets, fibrinogen and factor XII in the course of these exposures.

Does smoking affect thrombocytopoiesis and platelet activation in women and men?

PURPOSE: Smoking is a significant risk factor of cardiac ischaemia. Changes in platelet count, morphology and platelet activation enhance the risk. MATERIAL AND METHODS: The objective of the study was to assess platelet parameters in smoking healthy subjects with reference to sex. In the group of women, 27% were smokers, in the group of men--49%. All the subjects were tested for platelet count (PLT), mean platelet volume (MPV), percentage of large platelets (L(PLT)), concentrations of beta-thromboglobulin, sP-selectin (soluble) and thrombopoietin, percentage of reticulated platelets (RP) and absolute count of reticulated platelet. RESULTS: Lower platelet count (237.00 +/- 39.52 vs 258.34 +/- 40.81 x 10(9)/l, p = 0.0002), higher percentage of reticulated platelets (1.39 +/- 0 .66 vs 1.04 +/- 0.35%, p = 0.04) and higher concentration of sP-selectin (52.66 +/- 18.54 vs 43.94 +/- 17.14 ng/ml, p = 0.03) were observed only in the group of smoking women, compared to non-smokers. In neither of the sexes smoking had an effect on the following parameters: mean platelet volume, percentage of large platelets, concentration of thrombopoietin, absolute count of reticulated platelet and concentration of beta1 -thromboglobulin. CONCLUSIONS: The results allow the hypothesis that women are more sensitive to smoking than men. Platelets in male smokers are less sensitive to smoking--the study showed no significant changes in the parameters.

The effect of continuous-flow automated plateletpheresis on fibrinolytic activity of donor plasma.

Blood circulating in extracorporeal circuit of the apheresis sets has a contact with an artificial surface. The data on the influence of plateletpheresis on fibrinolytic activity are very limited and difficult to interpret. The aim of our study was to estimate the effect of plateletpheresis on the activation of fibrinolysis. Plateletpheresis was performed in 17 healthy blood donors using continuous-flow cell separator COM.TEC (Fresenius, Bad Homburg, Germany). Before and after plateletpheresis, blood samples were taken and markers of fibrinolysis (PAP, t-PA, PAI-1) as well as factor XII activity have been measured. We observed statistically significant decrease in t-PA and factor XII activities after plateletpheresis. There were no significant changes in concentrations of t-PA, PAI-1 and PAP as well as PAI-1 activity after plateletpheresis. Plateletpheresis performed by COM.TEC cell separator has very little, if any, effect on the activation of fibrinolysis. The mechanism of the inhibition of t-PA activity needs further investigations.

Erythropoietin (r-HuEPO) in the treatment of anemia of nodular sclerosis type Hodgkin's disease--a preliminary report.

The purpose of the study was to evaluate the effectiveness of recombinant human erythropoetin (r-HuEPO) treatment in patients suffering from anemia in the course of Hodgkin's disease (HD). 6 patients suffering from HD (4 of nodular sclerosis type II (NS II) and 2 of nodular sclerosis type I (NS I)) were treated with r-HuEPO for 10 weeks. All patients suffering from the NS II of HD exhibited an increase in the level of Hb by more than 2 g/dl after 10 weeks of r-HuEPO therapy whereas patients suffering from the NS I subtype of HD did not benefit from such treatment.