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ZusammenfassungDas Antiphospholipid-Syndrom (APS) ist eine systemische Autoimmunerkrankung, die durch arterielle und venöse Thrombosen und/oder Schwangerschaftskomplikationen in Form von Spontanaborten und/oder Fehlgeburten definiert ist. Laboranalytisch ist das APS gekennzeichnet durch den Nachweis von Antiphospholipid-Antikörpern oder einem Lupus Antikoagulans. Antiphospholipid-Antikörper repräsentieren eine heterogene Gruppe von Autoantikörpern gerichtet gegen Phospholipid-Protein-Komplexe. Lupus Antikoagulanzien stellen eine gerinnungsaktive Untergruppe dar, in der Antikörper zu einer Verlängerung von phospholipidabhängigen Gerinnungstesten führen. Die Behandlung erfolgt individualisiert und richtet sich nach dem klinischen Verlauf. Nach venösen und arteriellen Ereignissen wird in der Regel eine langfristige orale Antikoagulation durchgeführt, eine Primärprophylaxe erfolgt nicht. Eine Abortprophylaxe erfolgt mit niedermolekularem Heparin in Kombination mit Aspirin.

Background Nucleic acid-targeted pathogen inactivation technology using amustaline (S-303) and glutathione (GSH) was developed to reduce the risk of transfusion-transmitted infectious disease and transfusion-associated graft-versus-host disease with red blood cell (RBC) transfusion. Study design and methods A randomized, double-blind, controlled study was performed to assess the in vitro characteristics of amustaline-treated RBCs (test) compared with conventional (control) RBCs and to evaluate safety and efficacy of transfusion during and after cardiac surgery. The primary device efficacy endpoint was the postproduction hemoglobin (Hb) content of RBCs. Exploratory clinical outcomes included renal and hepatic failure, the 6-minute walk test (a surrogate for cardiopulmonary function), adverse events (AEs), and the immune response to amustaline-treated RBCs. Results A total of 774 RBC unis were produced. Mean treatment difference in Hb content was -2.27 g/unit (95% confidence interval, -2.61 to -1.92 g/unit), within the prespecified equivalence margins (±5 g/unit) to declare noninferiority. Amustaline-treated RBCs met European guidelines for Hb content, hematocrit, and hemolysis. Fifty-one (25 test and 26 control) patients received study RBCs. There were no significant differences in RBC usage or other clinical outcomes. Observed AEs were within the spectrum expected for patients of similar age undergoing cardiovascular surgery requiring RBCs transfusion. No patients exhibited an immune response specific to amustaline-treated RBCs. Conclusion Amustaline-treated RBCs demonstrated equivalence to control RBCs for Hb content, have appropriate characteristics for transfusion, and were well tolerated when transfused in support of acute anemia. Renal impairment was characterized as a potential efficacy endpoint for pivotal studies of RBC transfusion in cardiac surgery.

Infective endocarditis (IE) is a life-threatening disease that is associated with high morbidity and mortality. Its long-term prognosis strongly depends on a timely and optimized antibiotic treatment. Therefore, identification of the causative pathogen is crucial and currently based on blood cultures followed by characterization and susceptibility testing of the isolate. However, antibiotic treatment starting prior to blood sampling or IE caused by fastidious or intracellular microorganisms may cause negative culture results. Here we investigate the additional diagnostic value of broad-range PCR in combination with direct sequencing on resected heart tissue or swabs in patients with tissue or swab culture-negative IE in a routine clinical setting. Sensitivity, specificity, and positive and negative predictive values of broad-range PCR from diagnostic material in our patients were 33.3%, 76.9%, 90.9%, and 14.3%, respectively. We identified a total of 20 patients (21.5%) with tissue or culture-negative IE who profited by the additional application of broad-range PCR. We conclude that broad-range PCR on resected heart tissue or swabs is an important complementary diagnostic approach. It should be seen as an indispensable new tool for both the therapeutic and diagnostic management of culture-negative IE and we thus propose its possible inclusion in Duke’s diagnostic classification scheme.

Nucleic acid-targeted pathogen inactivation technology using amustaline (S-303) and glutathione (GSH) was developed to reduce the risk of transfusion-transmitted infectious disease and transfusion-associated graft-versus-host disease with red blood cell (RBC) transfusion.A randomized, double-blind, controlled study was performed to assess the in vitro characteristics of amustaline-treated RBCs (test) compared with conventional (control) RBCs and to evaluate safety and efficacy of transfusion during and after cardiac surgery. The primary device efficacy endpoint was the postproduction hemoglobin (Hb) content of RBCs. Exploratory clinical outcomes included renal and hepatic failure, the 6-minute walk test (a surrogate for cardiopulmonary function), adverse events (AEs), and the immune response to amustaline-treated RBCs.A total of 774 RBC unis were produced. Mean treatment difference in Hb content was -2.27 g/unit (95% confidence interval, -2.61 to -1.92 g/unit), within the prespecified equivalence margins (±5 g/unit) to declare noninferiority. Amustaline-treated RBCs met European guidelines for Hb content, hematocrit, and hemolysis. Fifty-one (25 test and 26 control) patients received study RBCs. There were no significant differences in RBC usage or other clinical outcomes. Observed AEs were within the spectrum expected for patients of similar age undergoing cardiovascular surgery requiring RBCs transfusion. No patients exhibited an immune response specific to amustaline-treated RBCs.Amustaline-treated RBCs demonstrated equivalence to control RBCs for Hb content, have appropriate characteristics for transfusion, and were well tolerated when transfused in support of acute anemia. Renal impairment was characterized as a potential efficacy endpoint for pivotal studies of RBC transfusion in cardiac surgery.

Background Life-threatening heparin-induced thrombocytopenia (HIT) is treated with the alternative nonheparin anticoagulants argatroban, lepirudin, or danaparoid. Frequently, the pentasaccharide fondaparinux is used off-label. Objectives The authors sought to investigate the safety and efficacy of the different anticoagulants for treating HIT. Methods In a national, multicenter registry study, hospitalized patients who were diagnosed with HIT, an at least intermediate clinical HIT-risk (4Ts score ≥4 points), and received treatment with ≥1 dose of the aforementioned anticoagulants were included. Main outcome measures were the incidences of HIT-specific complications (thromboembolic venous/arterial events, amputations, recurrent/persistent thrombocytopenia, skin lesions) and bleedings. Results Of 195 patients, 46 (23.6%), 4 (2.1%), 61 (31.3%), and 84 (43.1%) had been treated first-line with argatroban, lepirudin, danaparoid, and fondaparinux, respectively. The composite endpoint of HIT-specific complications (thromboembolic events, amputation, skin necrosis) occurred in 11.7% of patients treated with approved alternative anticoagulation and in 0.0% of fondaparinux-treated patients. The all-cause in-hospital mortality rates were 14.4% during approved alternative anticoagulation and 0.0% during fondaparinux treatment. Bleeding complications occurred in alternatively anticoagulated patients and in fondaparinux-treated patients in 6.3% and 4.8%, respectively. Post hoc analysis of clinical and laboratory features confirmed “true“ HIT in at least 74 of 195 (38.0%) patients; 35 of 74 (47.3%) were treated with fondaparinux. Conclusions Fondaparinux is effective and safe in suspected acute HIT; no HIT-specific complications occurred in the fondaparinux-treated patients, even among those with a high clinical HIT probability. Further data from randomized controlled trials are urgently needed because lepirudin was recalled from the market; danaparoid access has been limited and is not approved in the United States; and argatroban is contraindicated in patients with impaired liver function, and activated partial thromboplastin time confounding may interfere with monitoring. (Retrospective Registry of Patients With Acute Heparin-induced Thrombocytopenia Type II; NCT01304238)

Eine 51-jahrige Patientin mit valvularer Kardiomyopathie seit dem 34. Lebensjahr wurde wegen progredienter Herzinsuffizienz zur Evaluation fur eine Herztransplantation vorgestellt. Im Rahmen der Diagnostik fielen Anti-Phospholipid-Antikorper auf. Die daraufhin veranlasste rheumatologische Evaluation erbrachte die Diagnosen eines bisher unentdeckt gebliebenen systemischen Lupus erythematodes (SLE) mit fuhrender kardialer Manifestation und eines Anti-Phospholipid-Antikorper-Syndroms. Trotz des fortgeschrittenen Stadiums der Herzinsuffizienz konnte ein Ansprechen auf Azathioprin erzielt werden, das jedoch noch nicht ausreicht, um auf eine Herztransplantation ganz verzichten zu konnen. Der atypische Verlauf des SLE hat die Diagnose erheblich erschwert. Eine fruhere Diagnose ware in der Ruckschau moglich gewesen. Wenngleich selten, ist der SLE eine wichtige Differentialdiagnose bei Auftreten eines schweren Klappenvitiums und einer Kardiomyopathie, insbesondere bei jungen Frauen.

The etiology of complex regional pain syndrome (CRPS) is unclear yet. Recently autoantibodies and antecedent viral infections have been discussed to be involved in the pathogenesis of CRPS. We investigated sera from 39 CRPS patients and healthy controls for parvovirus B19 IgG and the occurrence of antiendothelial autoantibodies (AECA). CRPS patients showed a higher seroprevalence of parvovirus B19 IgG than controls (p < 0.01). All CRPS 2 patients were positive. 10.2% of the CRPS patients and 10.0% of the controls had AECA (n.s.) and AECA were not associated with parvovirus B19 seropositivity. Our findings suggest the involvement of parvovirus B19, but not autoantibody-mediated endothelial cell damage, in the pathogenesis of CRPS.

Objectives: We investigated the diagnostic value of a new system, the Innotrac Aio!™ immunoassays for troponin, myoglobin and CK-MB, in 270 samples from patients with ACS, after bypass surgery (CABG) or with stable heart failure in comparison to the respective Roche assays. Results: The values of the cardiac markers assessed by the respective assays correlated (cTnT/cTnI Rho = 0.94, myoglobin Rho = 0.87, CK-MB Rho = 0.84). If values were dichotomised, we found a high concordance of test positive and negative classified patients by troponins with the respective assays. Conclusion: There is strong evidence that the Innotrac Aio!™ system for cTnI measurement can be used reliably.

Background: Genetic analysis of platelet mRNA may facilitate the diagnosis of disorders affecting the megakaryocytic-platelet lineage. Its use, however, is limited by the exceptionally small yield of platelet mRNA and the risk of leukocyte contamination during platelet preparation. Methods: We depleted platelet suspensions of leukocytes by filtration and used a PCR-based RNA amplification step [switching mechanism at the 5′ end of RNA templates (SMART)]. We tested the reliability and precision of the RNA amplification procedure by use of real-time PCR to measure quantities of specific transcripts: von Willebrand factor (vWF), A-subunit of coagulation factor XIII (F13A), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Microarray analysis was performed on platelet RNA with and without amplification. Results: Microgram quantities of platelet-specific cDNAs were produced from as little as 50 ng of total platelet RNA or 40 mL of whole blood. At cycle numbers Conclusion: The proposed protocol makes extremely small amounts of platelet RNA available for gene expression analysis in single patients.

Cerebral venous thrombosis (CVT) associated with vasculitis has been described only in the context of small vessel angiitis so far. We report on a 30-year-old woman who developed CVT which was followed by progressive occlusion of large extracranial arteries. Cerebrospinal fluid (CSF) showed evidence for inflammation with pleocytosis and positive oligoclonal bands. Angiography revealed no abnormalities of the aortic arch, but three remaining criteria for Takayasu arteritis were fulfilled. Antiphospholipid antibodies were elevated or within borderline values, possibly indicating the beginning of lupus erythematosus. Because of additional protein S deficiency and increased factor VIII activity, the patient was orally anticoagulated. Immunosuppressive therapy was started with azathioprine. This was followed by a normalization of cell count in CSF. Immunosuppression was changed to methotrexate after acute progression of the occlusive disease. As far as we know this is the first case report with a combination of sinus thrombosis and large vessel arteritis.

Es wird von 6 Patienten mit einer Heparin-induzierten Thrombozytopenie und koronarer Herzkrankheit oder Aortenklappenstenose berichtet, die erfolgreich am Herzen operiert werden konnten, wobei rekombinantes (r) Hirudin als spezifischer und Cofaktoren-unabhangiger Thrombininhibitor fur den kardiopulmonalen Bypass (CPB) verwendet wurde. Ein intravenoser r-Hirudin-Bolus von 0,25 mg/kg Korpergewicht und eine Dosis von 0,2 mg/kg Korpergewicht ins Priming der Herz-Lungen-Maschine erwiesen sich als ausreichend fur eine effektive Antikoagulation wahrend CPB. Zusatzliche r-Hirudin-Boli von 5 mg (Gesamtdosis: 0,1–0,8 mg/kg Korpergewicht) wurden verabreicht, sobald der r-Hirudin-Plasmaspiegel unter 2,5 μg/ml abfiel. Bei 3 Patienten wurden koronare Bypassoperationen, bei den anderen 3 Patienten jeweils ein Aortenklappenersatz durchgefuhrt. Zum Monitoring der Antikoagulation wurden die Ecarinzeit (ECT) und die aktivierte partielle Thromboplastinzeit (aPTT) in Abstanden von 10 min bestimmt. ECT und aPTT-Werte korrelierten zuverlassig mit dem r-Hirudin-Plasmaspiegel. Die Perfusionszeit lag zwischen 83 und 180 min, die Aortenklemmzeit zwischen 0 und 83 min. Wahrend der Operation gab es keine Blutungs- oder thromboembolischen Komplikationen und der Einlasdruck am Oxygenator blieb konstant. Infolge der normalen Nierenfunktion kam es bei allen Patienten nach Ende des CPB zum zugigen Abfall des r-Hirudin-Plasmaspiegels und zur Normalisierung der Gerinnung. Bei einem Patienten konnte die r-Hirudin-Elimination mittels einer Hamofiltrationsphase von 15 min gegen Ende des CPB beschleunigt werden. Der postoperative Verlauf war bei allen Patienten unauffallig bis auf eine Rethorakotomie bei einem Patienten 6 h postoperativ wegen vermehrter Fordermenge aus den Thoraxdrainagen. Es fand sich eine chirurgische retrosternale Blutungsquelle, die durch Diathermie koaguliert wurde. Die ADP- und kollagen-induzierte Plattchenaggregation als Parameter fur die Thrombozytenfunktion war bei allen Patienten nach 60 min CPB-Zeit noch relativ gut erhalten. Die Ergebnisse zeigen, das r-Hirudin bei Patienten mit einer HIT erfolgreich als alternatives Antikoagulans wahrend CPB verwendet werden kann.

SummaryThe use of recombinant ® hirudin as an anticoagulant in performing extracorporeal circulation systems including cardiopulmonary bypass (CPB) devices requires a specific and easy to handle monitoring system. The usefulness of the celite-induced activated clotting time (ACT) and the activated partial thromboplastin time (APTT) for r-hirudin monitoring has been tested on ex vivo blood samples obtained from eight patients treated with r-hirudin during open heart surgery. The very poor relationship between the prolongation of the ACT and APTT values and the concentration of r-hirudin as measured using a chromogenic factor Ila assay indicates that both assays are not suitable to monitor r-hirudin anticoagulation. As an alternative approach a whole blood clotting assay based on the prothrombin-activating snake venom ecarin has been tested. In vitro experiments using r-hirudin- spiked whole blood samples showed a linear relationship between the concentration of hirudin added and the prolongation of the clotting times up to a concentration of r-hirudin of 4.0 µg/ml. Interassay coefficients (CV) of variation between 2.1% and 5.4% demonstrate the accuracy of the ecarin clotting time (ECT) assay. Differences in the interindividual responsiveness to r-hirudin were analyzed on r-hirudin- spiked blood samples obtained from 50 healthy blood donors. CV- values between 1.8% and 6% measured at r-hirudin concentrations between 0.5 and 4 µg/ml indicate remarkably slight differences in r-hirudin responsiveness. ECT assay results of the ex vivo blood samples linearily correlate (r = 0.79) to the concentration of r-hirudin. Moreover, assay results were not influenced by treatment with aprotinin or heparin. These findings together with the short measuring time with less than 120 seconds warrant the whole blood ECT to be a suitable assay for monitoring of r-hirudin anticoagulation in cardiac surgery.

Up-regulation of tissue factor (TF) expression in circulating blood cells, especially monocytes, plays a key role in the pathogenesis of various thromboembolic diseases (1)(2)(3)(4). Measurement of TF expression in monocytes therefore might be helpful in the diagnosis of a hypercoagulable state (5)(6)(7). Monocytic TF expression can be measured on both the protein and RNA levels. Testing on the RNA level seems to be more appropriate than antigen testing to detect a procoagulant state because monocytes that already express functionally active TF on their membrane surfaces are rapidly cleared from the circulation (8). The aim of the present study was to develop a protocol for quantitative determination of TF mRNA transcripts in monocytes and other circulating blood cells. The method should be rapid, robust, and applicable in whole blood without the need for cell isolation. We developed a one-step quantitative reverse transcription (qRT)-PCR assay based on the real-time TaqMan® technology and evaluated the preanalytical conditions required for the use of TF mRNA measurements on a routine clinical basis as well as several normalization strategies, including normalization based on CD14 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference gene expression. Finally, we measured baseline TF expression in healthy individuals and in thrombophilic patients. RNA calibrators for quantification were prepared by in vitro transcription of DNA hybrids that combined the T7 promoter sequence with the following sequences of TF, CD14, or GAPDH cDNA: for TF, bp 265–848 (GenBank accession no. M16553); for CD14, bp 18–564 (GenBank accession no. M86511); and for GAPDH, bp 8–525 (GenBank accession no. M33197). Generated RNAs were treated with DNase I (Roche) to remove the added DNA templates and purified by use of the RNeasy Mini Kit (Qiagen). RNA was quantified by photometric measurement ( A 260 reading of 1 = 44 …

Evaluation of a novel approach to eliminate air microemboli from extracorporeal circulation via ultrasonic destruction.In vitro proof-of-concept study.Research laboratory.None.None.An extracorporeal circulation device was filled with human blood circulating at 3 L/min. Air bubbles were injected into the system. For bubble destruction, the blood in the tubing system was repeatedly insonated for 3 minutes using a therapeutic 60-kHz device, with variation of intensity and duty cycle settings, ranging from 0.2 W/cm² to 1.0 W/cm² and from duty cycle 60% to continuous wave (CW). Number and diameter of air microemboli were counted upstream and downstream of the ultrasound device by a 2-channel microemboli Doppler detector. For safety assessment, circulating blood was insonated continuously for 2 hours at 0.8 W/cm² CW and compared with circulation without insonation; and standard blood parameters were analyzed. Without treatment, 1,313 to 1,580 emboli were detected upstream, diameter ranging between 10 and 130 μm. Ultrasound treatment eliminated up to 87% of all detected bubbles in cw application (p0.01) and showed comparable effects at intensities from 0.4 W/cm² to 1.0 W/cm² cw. Bubbles sized15 μm almost were eliminated completely (p0.001). Pulsed wave application rendered inferior results (p0.05). No relevant changes of blood parameters were observed compared with control circulation.Ultrasound destruction of air emboli is a very efficient method to reduce number and size of emboli. Within the limits of safety assessment, the authors could not detect relevant side effects on standard blood parameters.

A 51-year-old woman diagnosed as having valvular cardiomyopathy since age 34 was admitted for an evaluation for a heart transplant because of progressive congestive heart failure. When antiphospholipid antibodies were detected, the diagnosis of a thus far undetected systemic lupus erythematosus (SLE) was confirmed, manifesting primarily by cardiac involvement and an antiphospholipid antibody syndrome. Despite an advanced stage of heart failure, the patient responded well to azathioprine. Nevertheless, the potential necessity of a heart transplant remained. Its atypical presentation impeded a timely diagnosis of SLE significantly, however, in retrospect the correct diagnosis would have been possible at an earlier time point.Though rare, SLE represents an important differential diagnosis in cases of severe valvular disease and cardiomyopathy, particularly in young women.

Several case reports have linked iron deficiency anemia with the occurrence of cerebral venous thrombosis (CVT) or stroke, yet, it is unclear whether this is a chance association. In a case-control design data of whole blood count and screening for thrombophilic coagulation abnormalities of 121 prospectively identified patients with CVT and 120 healthy controls were compared. Anemia was defined as a hemoglobin (Hb) concentration of

To the Editor: Patients with heparin-induced thrombocytopenia are at high risk for thromboembolic complications. Heparin-induced thrombocytopenia is caused by heparin-related and platelet-activating antibodies.1 These antibodies, referred to as HIT antibodies, are usually undetectable 100 days after the cessation of heparin therapy.2 Avoidance of a secondary immune response and the use of alternative anticoagulants are strongly recommended in patients who have had heparin-induced thrombocytopenia to avoid another bout. If such patients require cardiopulmonary bypass, danaparoid and hirudin are available.3,4 Both treatments, however, carry the risk of potentially life-threatening hemorrhage, because their anticoagulant actions cannot be neutralized at the end of . . .

Assay conditions of a plasma-based ecarin clotting time (ECT) were evaluated and the precision of the ECT in monitoring plasma levels of r-hirudin assessed. The snake venom enzyme ecarin converts prothrombin to meizothrombin possessing only weak coagulant but strong esterase activity. In r-hirudin containing plasma samples, meizo thrombin is rapidly neutralized by r-hirudin resulting in a dose-dependent prolongation of the clotting times. Among the different assay conditions tested, addition of 50 microliters of ecarin (4 U/ml) to 100 microliters of undiluted citrate-anticoagulated plasma gave optimal results with respect to precision and reproducibility. The measuring range of the ECT performed in this way is about 0.02-5.0 micrograms/ml r-hirudin. In vitro studies performed on r-hirudin-spiked plasma samples of 50 healthy individuals demonstrate remarkably low interindividual differences in r-hirudin responsiveness as indicated by CV-values below 5% and 7% at r-hirudin concentrations between 0-3 micrograms/ml and 4-5 micrograms/ml, respectively. The specificity for r-hirudin of the ECT is further demonstrated by the strong correlation (r = 0.94) between the results of a chromogenic assay and the ECT-measured r-hirudin concentrations obtained on 67 ex vivo blood samples. Depending on the concentration of r-hirudin the ECT is sensitive to plasma levels of prothrombin. In the absence of r-hirudin the critical prothrombin concentration was found to be 20% but increasing to 60% in the presence of 2.0 micrograms/ml r-hirudin. A fibrinogen concentration of 50 mg/dl was found to be the minimal concentration independent of the r-hirudin concentration. The precision of the ECT in measuring plasma levels of r-hirudin is not influenced by treatment with heparin, aprotinin or oral anticoagulants. The data demonstrate that the ECT is a rapid and easily perfor mable clotting assay which allows accurate measurement of r-hirudin plasma levels. ECT-monitored hirudin treatment will help to establish optimal dose regimens that are more efficacious but still as safe as heparin.

We present a patient with coronary heart disease and a heparin- induced thrombocytopenia , who was successfully treated by coronary ar- tery bypass grafting (CABG) using recombinant hirudin as an anticoagu- lant for cardiopulmonary bypass (CPB) instead of heparin. (Eur J Car- dio-thorac Surg (1996) 10: 386-388)

Application of the TaqMan-PCR for Genotyping of the Prothrombin G20210A Mutation and of the Thermolabile Methylenetetrahydrofolate Reductase Mutation