Bernhard Stephan

Identification of lung cancer with high sensitivity and specificity by blood testing

BackgroundLung cancer is a very frequent and lethal tumor with an identifiable risk population. Cytological analysis and chest X-ray failed to reduce mortality, and CT screenings are still controversially discussed. Recent studies provided first evidence for the potential usefulness of autoantigens as markers for lung cancer.MethodsWe used extended panels of arrayed antigens and determined autoantibody signatures of sera from patients with different kinds of lung cancer, different common non-tumor lung pathologies, and controls without any lung disease by a newly developed computer aided image analysis procedure. The resulting signatures were classified using linear kernel Support Vector Machines and 10-fold cross-validation.ResultsThe novel approach allowed for discriminating lung cancer patients from controls without any lung disease with a specificity of 97.0%, a sensitivity of 97.9%, and an accuracy of 97.6%. The classification of stage IA/IB tumors and controls yielded a specificity of 97.6%, a sensitivity of 75.9%, and an accuracy of 92.9%. The discrimination of lung cancer patients from patients with non-tumor lung pathologies reached an accuracy of 88.5%.ConclusionWe were able to separate lung cancer patients from subjects without any lung disease with high accuracy. Furthermore, lung cancer patients could be seprated from patients with other non-tumor lung diseases. These results provide clear evidence that blood-based tests open new avenues for the early diagnosis of lung cancer.

Novel autoantigens immunogenic in COPD patients.

BackgroundChronic obstructive pulmonary disease (COPD) is a respiratory inflammatory condition with autoimmune features including IgG autoantibodies. In this study we analyze the complexity of the autoantibody response and reveal the nature of the antigens that are recognized by autoantibodies in COPD patients.MethodsAn array of 1827 gridded immunogenic peptide clones was established and screened with 17 sera of COPD patients and 60 healthy controls. Protein arrays were evaluated both by visual inspection and a recently developed computer aided image analysis technique. By this computer aided image analysis technique we computed the intensity values for each peptide clone and each serum and calculated the area under the receiver operator characteristics curve (AUC) for each clone and the separation COPD sera versus control sera.ResultsBy visual evaluation we detected 381 peptide clones that reacted with autoantibodies of COPD patients including 17 clones that reacted with more than 60% of the COPD sera and seven clones that reacted with more than 90% of the COPD sera. The comparison of COPD sera and controls by the automated image analysis system identified 212 peptide clones with informative AUC values. By in silico sequence analysis we found an enrichment of sequence motives previously associated with immunogenicity.ConclusionThe identification of a rather complex humoral immune response in COPD patients supports the idea of COPD as a disease with strong autoimmune features. The identification of novel immunogenic antigens is a first step towards a better understanding of the autoimmune component of COPD.

Novel immunogenic antigens increase classification accuracy in meningioma to 93.84

There is growing evidence that simultaneous analysis of multiple autoantibody reactions can be utilized for diagnosis of neoplasms. Using a set of 57 meningioma‐associated antigens, we recently separated meningioma patients from individuals without known disease with an accuracy of 90.3%. Here, we ask whether a largely increased set of immunogenic antigens can further improve this discrimination. We used an array with 1,827 human recombinant clones and measured reactivity of serum autoantibodies against the clones by a novel automated image analysis procedure. We were able to separate meningioma sera from sera of healthy controls with a specificity of 95.62%, a sensitivity of 91.83% and an accuracy of 93.84%. Of the analyzed clones, 23 in‐frame clones were highly informative for the classification of meningioma vs. normal sera as shown by their AUC values. These results demonstrate that the accuracy of a serum‐based diagnostic can be readily and considerably improved by screening extended sets of proteins.

The use of antithrombotic agents in microvascular surgery

Despite increasing advances in microvascular free tissue transfer, flap failures, most commonly resulting from thrombosis at the anastomotic vascular site, remain a significant concern. Although several experimental and clinical studies have been carried out, no consensus has been reached so far on the efficacy, dosage and timing of anticoagulant agents available for the prevention and treatment of thrombosis in microvascular surgery. Inhibition of fibrin formation and platelet function or the use of thrombolytic agents is a common approach in the antithrombotic management. However, some agents exhibit serious side effects and all of them carry the risk of bleedings. The current literature on the use of antithrombotic agents, targeting at clinical trials in microvascular surgery, is therefore reviewed, to provide an informative basis for recommendations for an appropriate pharmacological approach.

On the prophylactic and therapeutic use of danaparoid sodium (Orgaran®) in patients with heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a rare but dangerous complication of heparin prophylaxis or treatment. The present laboratory tests to measure heparin-associated antibodies are not specific. The diagnosis of HIT mainly depends on the decrease in platelet count and on clinical symptoms. To evaluate clinical outcome, bleeding complications and platelet counts were evaluated in 45 patients with HIT type II (HIT II) treated prophylactically (subcutaneous injections) or therapeutically (intravenous infusion) with danaparoid. Group I included 24 patients with HIT II without thromboembolic complications who received danaparoid twice daily subcutaneously (10 IU/kg) for a mean of 16 days. Group II included 21 patients with thromboembolic complications. They were treated with intravenous danaparoid (2.6 IU/kg/h ± 1.1) for a mean of 17 days. During subcutaneous prophylaxis, mean anti-Xa levels of 0.2 U/mL and during intravenous treatment, mean anti-Xa levels of 0.4 U/mL were reached. No deaths, amputations, or serious bleeding complications occurred, and no new thromboses were observed in both patient groups.Treatment with danaparoid led to a fast normalization of the platelet counts. This normalization occurred earlier and the concentration of platelets was higher in patients treated with intravenous doses. Danaparoid with subsequent vitamin K-antagonist treatment effectively prevents thromboembolic complications in patients with HIT.

Fibrinolytic and procoagulant activity in septic and haemorrhagic shock.

Septic and haemorhagic shock carry the risk of high mortality. Failure of microcirculation secondary to alterations of haemostasis and fibrinolysis play a major role in the pathogenesis of shock. The aim of this study was to evaluate the clinical relevance of procoagulatory and fibrinolytic activities referring to survival. Therefore, 39 patients (23 to 80 yrs, 16 females, 23 males) suffering from haemorrhagic (n = 21) and septic shock (n = 18) were screened prospectively for plasmatic coagulation and fibrinolysis parameters. Thirteen patients (33.3%) developed lethal outcome. Concerning fibrinolysis, plasminogen was significantly lower in non-survivors by day 1 and plasmin-antiplasmin complex significantly higher by day 4 compared to survivors. Consecutive increase of plasminogen over day 4 and 7 was significantly stronger in survivors. Concerning haemostasis activation, thrombin-antithrombin complex was higher and D-dimers or fibrinogen levels were lower, but not significantly different, in non-survivors compared to survivors. We conclude from these data, that procoagulant activities are increased, but not significantly predictive for the clinical outcome in septic and haemorrhagic shock. By contrast, fibrinolysis, as measured by enhanced capacity and responsiveness, is clearly predictive and plays a significant role for survival, possibly due to its clearing function in microcirculation.

Comparison of the plasminogen activator inhibitor-1 4G/5G gene polymorphism in females with venous thromboembolism during pregnancy or spontaneous abortion

Genetic polymorphisms in plasminogen activator inhibitor-1 gene-675 4G/5G (PAI-1 4G/5G) are claimed to contribute to an increased risk of venous thromboembolism. Inherited thrombophilia, on the other hand, is associated with the occurrence of spontaneous abortions. The objective of this study was, to explore the significance of genetic polymorphisms of PAI-1 4G/5G with particular emphasis on 4G alleles in pregnant women suffering from venous thromboembolism or early spontaneous abortion, respectively. Therefore genetic PAI-1 4G/5G polymorphisms were studied in 108 pregnant females suffering from venous thromboembolism (n=69) or from spontaneous abortion (<20 week, n=39), respectively. Healthy volunteers (n=238) were taken as controls. The frequencies of 4G alleles (4G/4G or 4G/5G genotypes) of PAI-1 were significantly higher in venous thromboembolism (OR: 3.40, p=0.0088) and slightly higher, but not significantly, in abortions (RR: 2.33; p=0.1162) compared to controls. The incidence of 4G-carriers in females with abortion was 0.68 (-32%) compared to women suffering from venous thromboembolism alone. We conclude from these data, that the occurrence of PAI-1 4G/4G or 4G/5G genotypes, respectively, is clinically significant for the pathogenesis of venous thromboembolism in pregnancy but not for early abortion.

Coagulation and complement system in critically ill patients

Activation of coagulation and inflammatory response including the complement system play a major role in the pathogenesis of critical illness. However, only limited data are available addressing the relationship of both pathways and its assessment of a predictive value for the clinical outcome in intense care medicine. Therefore, parameters of the coagulation and complement system were studied in patients with septicaemia and multiple trauma regarded as being exemplary for critical illness. 34 patients (mean age: 51.38 years (±16.57), 15 females, 19 males) were investigated at day 1 of admittance to the intensive care unit (ICU). Leukocytes, complement factors C3a and C5a were significantly (p <  0.0500) higher in sepsis than in trauma, whereas platelet count and plasma fibrinogen were significantly lower in multiple trauma. Activation markers of coagulation were elevated in both groups, however, thrombin-antithrombin-complex was significantly higher in multiple trauma. DIC scores of 5 were not exceeded in any of the two groups. Analysing the influences on mortality (11/34; 32.35% ), which was not different in both groups, non-survivors were significantly older, had significantly higher multiple organ failure (MOF) scores, lactate, abnormal prothrombin times and lower C1-inhibitor activities, even more pronounced in early deaths, than survivors. In septic non-survivors protein C was significantly lower than in trauma. We conclude from these data that activation of the complement system as part of the inflammatory response is a significant mechanism in septicaemia, whereas loss and consumption of blood components including parts of the coagulation and complement system is more characteristic for multiple trauma. Protein C in case of severe reduction might be of special concern for surviving in sepsis. Activation of haemostasis was occurring in both diseases, however, overt DIC was not confirmed in this study to be a leading mechanism in critically ill patients. MOF score, lactate, C1-inhibitor and prothrombin time have been the only statistically significant predictors for lethal outcome suggesting that organ function, microcirculation, haemostasis and inflammatory response are essential elements of the pathomechanism and clinical course of diseases among critically ill patients.

Diagnostic and Prognostic Value of Factor VIII Binding Antibodies in Acquired Hemophilia A: Data from the GTH‐AH 01/2010 Study

Essentials Factor VIII (FVIII) binding IgG detected by ELISA could be an alternative to the Bethesda assay. We studied the performance of anti‐FVIII IgG ELISA in patients with acquired hemophilia and controls. Anti‐FVIII IgG > 99th percentile of controls was highly sensitive and specific. Patients with high anti‐FVIII IgG have a lower chance of achieving remission.

Influence of bleeding on haemorheology and haemostasis in surgery

Changes in haemorheology and haemostasis may contribute to bleeding or thrombosis, which is of concern particularly in surgery. Blood loss itself has a major influence on both parameters being closely involved in the clinical outcome. In order to analyze the underlying interrelations, a prospective study with 122 patients (64 females, 58 males) aged between 18 and 83 years (mean: 51.8 years) was conducted. All patients were electively submitted to orthopaedic surgery. Haemorheological parameters included measurements of plasma viscosity, red body cell (RBC) and platelet aggregation index preoperatively, as well as by day 1 and day 7 after surgery. Additionally hematological and haemostaseological parameters including leukocyte and platelet counts, haematocrit and fibrinogen were investigated. Bleeding was defined as high (>500 ml) or low blood loss (≤500 ml) according to the drainage volume. High but not low blood loss was associated with an increase of RBC aggregation by day 1 and 7 after surgery. Plasma viscosity decreased significantly by day 1, returning to normal 7 days after surgery. Platelet count decreased significantly, concurrent with the haematocrit, by day 1 postoperatively, whereas by day 7 a significant increase was observed, being more distinct in high blood loss. Platelet aggregation index did not change under the influence of blood loss. Plasma fibrinogen, clearly corresponding to the extend of blood loss, showed a continuous postoperative increase, which was significantly higher at day 7. Leukocytes increased moderately but significantly in particular in high blood loss. In conclusion, the postoperative decrease of plasma viscosity and of platelet counts, concurrent with the haematocrit, provides evidence of being clearly dependent on blood loss which is regarded as a dilution effect corresponding with the haemorrhagic risk. The increase of RBC aggregation at the early postoperative stage is solely observed in high blood loss and is esteemed as a result of volume therapy. The marked increase of platelet counts and plasma fibrinogen at the late postoperative stage, being more pronounced in high blood loss, might contribute to an elevated prothrombotic risk and is ascribed to an inflammatory response to surgery. In summary, it is concluded, that bleeding tendency corresponding with haemorheologic parameters is enhanced in the early, whereas the prothrombotic risk, well correlating with haemostaseologic parameters, is elevated in the later stage after surgery.