Fifth Åland Island conference on von Willebrand disease.

The fifth Åland Island meeting on von Willebrand disease (VWD) was held on the Åland Islands, Finland, from 22 to 24 September 2016-90 years after the first case of VWD was diagnosed in a patient from the Åland Islands in 1926. This meeting brought together experts in the field of VWD to share knowledge and expertise on current trends and challenges in VWD. Topics included the storage and release of von Willebrand factor (VWF), epidemiology and diagnostics in VWD, treatment of VWD, angiogenesis and VWF inhibitors.

A prospective diagnostic accuracy study evaluating rotational thromboelastometry and thromboelastography in 100 patients with von Willebrand disease.

INTRODUCTION: Rotational thromboelastometry (ROTEM® ) and thromboelastography (TEG® ) are increasingly used in the perioperative and emergency assessment of bleeding tendencies. The diagnostic value of ROTEM and TEG for von Willebrand disease (VWD) remains to be established. AIM: To investigate whether ROTEM and TEG can discriminate patients with VWD from healthy controls. METHODS: Rotational thromboelastometry and TEG whole blood coagulation profiles were compared between VWD patients (n = 100) and healthy controls (n = 89). Measures of diagnostic accuracy were calculated, including sensitivity, specificity and receiver operating characteristic (ROC) curve. RESULTS: Prolonged TEG R-time had a positive and negative predictive value (PPV, NPV) of 0.84 and 0.68 respectively. TEG clotting index (CI) had a PPV of 1.00 and an NPV of 0.60. Both R-time and CI had a high specificity and accurately discriminated VWD patients from healthy controls, with an ROC area under the curve of 0.85 and 0.99 respectively. In multivariate analysis, low FVIII levels, but not von Willebrand factor (VWF) antigen or activity, determined hypocoagulable TEG R (R2 = 0.35) and CI levels (R2 = 0.51). The ROTEM coagulation profiles of VWD patients did not differ from healthy controls. CONCLUSIONS: Thromboelastography R and CI accurately discriminated VWD patients from healthy controls, partly through the detection of low FVIII levels. The test's performance may be improved through adjustment of the test thresholds to a local reference population. Both intrinsic pathway-activated (INTEM) and tissue factor pathway-activated (EXTEM) ROTEM were of limited diagnostic value in VWD.

Studies of microparticles in patients with the antiphospholipid syndrome (APS).

OBJECTIVES: To study circulating platelet, monocyte and endothelial microparticles (PMPs, MMPs and EMPs) in patients with antiphospholipid syndrome (APS) in comparison with healthy controls. MATERIAL AND METHOD: Fifty-two patients with APS and 52 healthy controls were investigated. MPs were measured on a flow cytometer (Beckman Gallios) and defined as particles sized < 1.0 µm, negative to phalloidin, positive to lactadherin and positive to either CD42a (PMPs), CD144 (EMPs) or CD14 (MMPs). Exposure of CD142 (TF) was measured on CD144 positive MPs. RESULTS: Total number of MPs (i.e. lactadherin positive particles) was higher in APS patients versus controls (p < 0.001). An increased number of EMPs (p < 0.001), increased TF-positive EMPs (p < 0.001) and increased MMPs (p < 0.001) were also observed. PMP numbers did not differ between the groups. None of the MP types differed in numbers between obstetric and thrombotic APS patients. CONCLUSION: We observed a high number of EMPs expressing TF in APS patients. The numbers of MMPs and total EMPs were also higher as compared with healthy controls but in contrast to previous reports, the number of PMPs did not differ between groups.

Influence of female hormonal factors, in relation to autoantibodies and genetic markers, on the development of rheumatoid arthritis in northern Sweden: a case-control study.

OBJECTIVES: To study the influence of female hormonal factors on the development of rheumatoid arthritis (RA) in relation to the human leucocyte antigen (HLA)-DRB1 shared epitope (SE), the protein tyrosine phosphatase (PTPN22) 1858T variant, anti-citrullinated protein antibodies (ACPAs), and immunoglobulin (Ig)M-rheumatoid factor (IgM-RF). METHODS: A case-control study (1:4) was nested within the Medical Biobank of northern Sweden. Females who had subsequently developed RA (n = 70), median of 2.7 years before the onset of symptoms, and matched controls (n = 280) were identified from among the blood donors. A questionnaire concerning previous exposures until disease onset, including hormonal and reproductive factors, and smoking habits was distributed. RESULTS: Breastfeeding was significantly associated with the development of RA [odds ratio (OR) 4.8, 95% confidence interval (CI) 1.43-15.8]. Increasing time of breastfeeding increased the risk of RA (OR 5.7, 95% CI 1.83-17.95) for breastfeeding ≥ 17 months. In a multiple logistic regression analysis, increasing time of breastfeeding (OR 9.5, 95% CI 2.14-42.43 for ≥ 17 months), seropositivity for ACPAs (OR 19.5, 95% CI 4.47-84.81), and carriage of the PTPN22 1858T variant (OR 3.2, 95% CI 1.36-7.54) remained significant predictors of RA. Users of oral contraceptives (OC) for ≥ 7 years had a decreased risk for development of RA (OR 0.37, 95% CI 0.15-0.93). CONCLUSIONS: A longer duration of breastfeeding increased the risk of developing RA, especially among individuals seropositive for ACPA or IgM-RF or carrying the PTPN22 1858T variant. Use of OC for ≥ 7 years was associated with a decreased risk.

Low PAI-1 activity in relation to inflammatory parameters, insulin profile and body mass index.

BACKGROUND AND OBJECTIVE: High plasminogen activator inhibitor type 1 (PAI-1) activity is associated with inflammatory reactions and insulin resistance, but it is unclear what regulates PAI-1 activity at the low end. The purpose of this study was to investigate if patients with low PAI-1 activity have a lack of inflammatory response or a low insulin level. DESIGN: Retrospective cohort study with internal controls. SUBJECTS: Sixty-three patients referred for investigation of bleeding tendency and with low PAI-1 activity were compared with 118 patients with normal or high PAI-1 activity. OUTCOME: Levels of C-peptide, proinsulin, high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6). Adjustments were made for body mass index (BMI), oral oestrogens and age. Low PAI-1 activity was defined as less than 1 U mL(-1). RESULTS: Body mass index in the low normal range, oral oestrogens, young age and low C-peptide were significantly associated with low PAI-1 activity and there was a trend for association with IL-6 in univariable analysis. The effect of age disappeared after correction for oral oestrogens and the effect of C-peptide and IL-6 disappeared after further adjustments. Low BMI remained as the strongest predictor of low PAI-1 activity. CONCLUSION: Patients with bleeding tendency and low PAI-1 activity have inflammatory and insulin profiles similar to those with normal or high PAI-1, whereas BMI seems to be the most important determinant.

Rapid determination of anticoagulating effects of dabigatran in whole blood with rotational thromboelastometry and a thrombin-based trigger.

Essentials A rapid test to detect thrombin inhibition by dabigatran would be valuable in acute situations. A thrombin-based trigger was applied in whole blood using rotation thromboelastometry. Effects of dabigatran were assessed in vitro and in samples from patients on dabigatran. The test produced data rapidly and was sensitive to dabigatran concentrations from 20 to 500 ng mL-1 . SUMMARY: Background Rapid determination of the anticoagulant effect of dabigatran is essential in emergency situations. Objective To study a viscoelastic test (rotational thromboelastometry [ROTEM]) for rapid determination of dabigatran effects in whole blood samples. Method ROTEM measurements were performed with comparison of two triggers (thrombin-based versus the commercial tissue factor-based trigger Ex-tem) in samples from 10 healthy donors spiked with dabigatran (20-500 ng mL-1 ) and in samples from 35 patients receiving dabigatran treatment; 10 healthy subjects served as controls. Clotting time (CT) and the difference in CT without versus with addition of the dabigatran antidote idarucizumab (CTdiff ) were measured. Addition of idarucizumab reveals the contribution of dabigatran to ROTEM measurements and its potential reversibility. Results In vitro studies showed that thrombin CT and thrombin CTdiff were more sensitive than Ex-tem CT and Ex-tem CTdiff in detecting dabigatran in whole blood samples. In patient samples, when thrombin CT and thrombin CTdiff were used, it was possible to detect dabigatran with a cut-off of dabigatran at 20 ng mL-1 , whereas, when Ex-tem CT and Ex-tem CTdiff were used, the method was less sensitive. Data from patient samples were obtained within 15 min of blood sampling. Conclusions ROTEM CT with a thrombin-based trigger is more sensitive to dabigatran effects than Ex-tem CT, and detects anticoagulant effects of drug concentrations in the low-very low therapeutic range. Analysis with idarucizumab (CTdiff ) reveals dabigatran-specific effects. As data are rapidly obtained, this method could, with further development and validation of its performance, be suitable for detecting clinically significant dabigatran effects in emergency situations.

Low PAI-1 activity in relation to the risk for perioperative bleeding complications in transurethral resection of the prostate.

INTRODUCTION: Low levels of plasminogen activator inhibitor type 1 (PAI-1) have been associated with increased risk for perioperative bleeding in some case reports. The aim of this study was to investigate prospectively whether low PAI-1 activity increases the risk for perioperative bleeding in patients undergoing transurethral resection of prostate, an organ with high fibrinolytic activity. PATIENTS AND METHODS: 62 patients with benign prostatic hyperplasia planned for transurethral resection were included. Blood samples for PAI-1 were taken together with other routine preoperative blood samples 1week before surgery but analyzed after the hospitalization. The intraoperative blood loss was determined by measuring the amount of hemoglobin in the irrigating fluid. The postoperative blood loss was estimated from calculations of hemoglobin mass (Hb mass), which is a product of hemoglobin concentration and blood volume. Hb mass was calculated before surgery and on the day of discharge, and was adjusted for intraoperative blood loss and transfused Hb mass. Bleeding complications were defined as re-operation due to bleeding, more than 40ml intraoperative bleeding/g resected prostatic tissue or postoperative blood loss corresponding to more than 100g of hemoglobin. RESULTS: Bleeding complications were observed in 3 of 4 (75%) patients with low PAI-1 levels, defined as <1U/ml, and in 16 of 58 (28%) patients with PAI-1 levels >1U/ml (P=0.082). After adjustment for resection time, resected prostatic mass and systolic blood pressure this became borderline significant (odds ratio 11.8; 95% confidence interval 1.00-139; P=0.05). CONCLUSION: Low PAI-1 activity may contribute to the risk of bleeding after transurethral resection of the prostate.

Evaluation of low PAI-1 activity as a risk factor for hemorrhagic diathesis.

BACKGROUND: Prospective studies of the epidemiology and clinical significance of low plasminogen activator inhibitor type 1 (PAI-1) activity are lacking. OBJECTIVE: To evaluate the prevalence of low PAI-1 activity in patients with a bleeding tendency in comparison with a normal population. METHODS: In 586 consecutive patients, referred because of bleeding symptoms, we added analyses of PAI-1 activity and tissue plasminogen activator complex with PAI-1 (t-PA-PAI-1) to the routine investigation, consisting of platelet count, bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen, factor VIII, von Willebrand factor activity, and antigen. Controls were 100 blood donors and 100 age- and sex-matched healthy individuals. The latter were also evaluated regarding the previous bleeding episodes. The bleeding history was classified as clinically significant or not, and the criteria were fulfilled in 75% of the patients and 18% of the healthy controls. RESULTS: The routine laboratory investigation of the patients was negative in 57%. Low PAI-1 activity, defined as <1.0 U mL(-1), was found in 23% of the patients and in 13% and 10% of the blood donors and healthy controls, respectively (odds ratio and 95% CI, 2.04; 1.11-3.77 and 2.75; 1.39-5.42, respectively). The difference remained statistically significant after the adjustment for body mass index, use of estrogens, sex and age (odds ratio for patients vs. healthy controls 3.23; 95% CI, 1.22-8.56, P = 0.019). The distribution of the 4G/5G genotypes in the patients was not different from that of two control populations. No specific symptom predicted for low PAI-1, which did not aggravate the clinical picture in association with the other hemostatic defects. Low tPA-PAI-1 was not associated with the increased bleeding tendency. CONCLUSION: Low PAI-1 activity is common in patients with a bleeding diathesis, but it is a risk factor of minor clinical importance and not associated with specific bleeding manifestations.

Effects of metabolic inhibitors on the regulation of pancreatic glucagon release.

The effects of metabolic inhibition on the kinetics of glucagon release by pancreatic islets from normal guinea pigs have been studied in a perfusion system. All three metabolic inhibitors, malonate, iodoacetate and 2,4-dinitrophenol, induced a marked stimulation of glucagon secretion, each displaying its own characteristic pattern of response. This was accompanied by a rapid and marked decrease in the ATP concentration of the A2-cell as evidenced by measurements performed in A2-cell rich islets, isolated from guinea pigs treated with streptozotocin. The ATP levels were reduced by about 90% following iodoacetate and 2,4-dinitrophenol exposure and by about 60% after exposure to malonate. The data support the hypothesis that the inhibition of glucagon release from the A2-cell is regulated via an intracellular, energy-dependent mechanism.

Species-specific autoantibodies in type 1 diabetes.

GAD65 autoantibodies (GAD65Ab) are important markers for type 1 (insulin-dependent) diabetes mellitus. Although most patients have GAD65Ab at the time of clinical diagnosis, there are also GAD65Ab-positive individuals in the population at low risk of developing type 1 diabetes. The aim of this study was to test the hypothesis that the GAD65Ab reactivity to GAD65 cloned from human, mouse, and rat in newly diagnosed type 1 diabetic patients differ from antibody-positive healthy individuals. Sera from 254 new-onset 0- to 34-yr-old type 1 diabetic patients and 270 controls were assayed for their reactivity to human, mouse, and rat GAD65. Among the type 1 diabetic patients there was a significant better binding of human GAD65 compared to either mouse (P = 0.03) or rat GAD65 (P = 0.0005). The preference for human GAD65 increased with increasing age at onset (P = 0.0002). This differentiation was not observed in 88 GAD65Ab-positive control subjects. Our data indicate that recognition of epitopes by GAD65Ab in type 1 diabetes is different from that in nontype 1 diabetes, GAD65Ab-positive individuals.

A new method which investigates lysosomal sensitivity to superoxide with endocytotic, site-specific, chemiluminescent probes.

The end result of cellular autophagocytosis and lysosomal digestion is incompletely understood in regard to the roles of the superoxide anion radical (O2-.). Cultured glial cells sequestered endocytotically two probes that were site-specific to the lysosome vacuome and sensitive to free-radical activities. The Sepharose-4B-polyisoluminol probe emitted chemiluminescent light in proportion to externally injected O2(-).. Bioluminescence measurements of beta-glucuronidase activity in intact glial cell lysosomes was achieved with the second site-specific, enzyme-specific Sepharose-4B-(dodecanate)5'-luciferinylglucuronate probe. Considerable activity was found in the lysosomal vacuome. In conclusion, we suggest that the use of site-specific, chemiluminescent probes can be of importance in the study of free radicals.

Application of chemiluminescent probes in investigating lysosomal sensitivity to superoxide versus suspected radical scavengers.

The role of the superoxide anion radical (O2(-).) relative to catalytic/inhibitory substances in lysosomes is poorly understood. Cultured glial cells sequestered endocytotically two probes that were site-specific to the lysosome vacuome and sensitive to radical activities. The Sepharose-4B-isoluminol probe emitted chemiluminescent light in proportion to externally injected O2(-). and was inhibited or catalysed by various radical scavengers, transition metals and other substances which may affect lysosomal metabolism and lipofuscin formation. We conclude that lysosomal radical activities may be inhibited by butylated hydroxytoluene, hydrocortisone, ACF, RNA, alpha-tocopherol, and, in special circumstances, with fully metabolized iron. Choline and oxidized copper and iron cations in overload concentrations in incubated freshly in lysosomes may catalyse radical activities, and they may be important factors in lipofuscin formation and its role in aging.

Optimization of uncomplicated control for head and neck tumors.

Almost 200 patients have been treated for head and neck tumors at two different dose levels. Based on the clinically observed probabilities for tumor control and fatal normal tissue complications at the two dose levels, the dose giving maximum uncomplicated control has retrospectively been calculated and compared with the clinical data. A Poisson statistical model for control and complications has been used including a correlation parameter, delta, to describe the fraction of patients where control and complications are statistically independent. The clinically observed probability of uncomplicated tumor control, P+, is consistent with only a small fraction of the patients treated being statistically independent (delta = 0.2 or 20%). Customarily, 100% of the patients are assumed to be statistically independent with regard to tumor control and normal tissue complications. More precisely, the clinical data are consistent, with almost 20% of the patients being significantly more sensitive to radiation since they gain local tumor control but simultaneously suffer fatal complications. An even larger fraction of the patients (almost 30%) seemed to be more resistant to radiation, showing neither serious treatment complications nor control of the local tumor growth. It is suggested that if these patient groups could be identified by a predictive assay for the radiation sensitivity of their normal tissues and preferably also for their tumors, the uncomplicated tumor control could be increased by about 20%. This figure is based on the actuarial survival of the patients and has been corrected for the inevitable uncertainty in dose delivery. It is also pointed out that about 20% of the patients can never be saved by a predictive assay because of the considerable statistical variance associated with the Poisson process and the eradication of the last clonogenic tumor cell. Finally, note that the possible existence of radiation sensitive and resistant patient groups is consistent with known genetic deficiencies such as ataxia telangiectasia for the sensitive patients and the existence of repair efficient head and neck tumors that are unusually efficient in repairing double strand breaks. If such sensitive and resistant patient groups do exist, it should be sufficient to perform a predictive assay on normal tissues alone avoiding the often impossible task of sampling the most radiation resistant tumor cell line.

Adenylate kinase activity in various organs and tissues of mice with the obese-hyperglycemic syndrome (gene symbol Ob/Ob).

The activities of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in lyophilized cryostat sections of various organs from mice with the obese-hyperglycemic syndrome (gene symbol ob/ob) were measured fluorometrically. Skeletal and heart muscle tissues had the highest enzyme activities, i.e. 258 and 155 mmoles substrate converted per kg dry weight and per hr (MKH), respectively. Pancreatic islet specimens, which were composed mainly of B-cells, possessed the highest activitty of all parenchymatous cells studied, with 18 MKH as compared to 16 MKH for the cortical tubules of the kidney and 7 and 6 for the exocrine pancreas and liver cells, respectively. The granular layer of the cerebellum had enzyme activity of 31 MKH. The adenylate kinase activity in the B-cells was apparently not influenced by hyperglycemia since it was similar both in obese-hyperglycemic mice of different ages and in lean mice, although the blood sugar concentrations in such animals are much different.

Tumour and normal tissue responses to fractionated non-uniform dose delivery.

The dose-volume response of tumours and normal tissues is discussed in terms of 'parallelity' and 'seriality'. The volume dependence of the radiation response of a tumour depends primarily on the eradication of all its clonogenic cells and the tumour has a parallel organization. The response of heterogeneous tumours is examined, and it is shown that a small resistant clonogen population may cause a low dose-response gradient, gamma. Injury to normal tissue is a much more complex and gradual process. It depends on earlier effects induced long before depletion of stem cells or differentiated cells that in addition may have a complex structural and functional organization. The volume dependence of the dose-response relation of normal tissues is therefore described here by a new parameter, the 'relative seriality', s, of the infrastructure of the organ. The model is compared with clinical and experimental data on normal tissue response, and shows good agreement both with regard to the shape of dose-response relation and the volume dependence of the isoeffect dose. For example, the spinal cord has a high and the lung a low 'relative seriality', which is reasonable with regard to the organization of these tissues. The response of tumours and normal tissues to non-uniform dose delivery is quantified for fractionated therapy using the linear quadratic cell survival parameters alpha and beta. The steepness, gamma, and the 50% response dose, D50, of the dose-response relationship are derived both for a constant dose per fraction and a constant number of dose fractions.

Photokinetic microassay of adenylate kinase using the firefly luciferase reaction.

A new rapid photokinetic method is described for determining the activity of adenylate kinase (ATP:AMP phosphotranspherase, EC 2.7.4.3) in 0.1--5.0 micrograms of freeze-dried tissue. This represents a weight range far below that obtainable by fine-needle biopsy. The reaction 2 ADP in equilibrium with AMP + ATP was employed and the ATP formed assayed with firefly luciferase as light yielder. The light emission was recorded on a multi-channel scaler. The adenylate kinase activities found in tissues of mice were in the same range as previously described in a study using fluorometric microassay.

Potentialities of bioluminescence analyses in research on the pancreatic islets.

Progress in bioluminescence assay permits not only determinations of nucleotide and substrate concentrations, but also estimation of concentration shifts. The analyses can be extended to comprise Ca2+ since the Aequorea system is sensitive enough for applications in islet research. By connecting the bioluminometer to a microprocessor with a suitable readout device, it is possible to collect and evaluate large amounts of data which may be required in studies of concentration shifts. Thus, blanks, samples and standards can be processed completely within short time periods so that the light-yielding solutions remain stable.

Fluorometric microassays of adenylate kinase, an enzyme important in energy metabolism.

The adenylate kinase system offers a mechanism for the rapid provision of energy by catalysing the production of ATP from ADP. Fluormetric micromethods were developed for determination of the activity of this enzyme using either formation of ADP or ATP, in each case measured by coupling to suitable dehydrogenase reactions. Both procedures yielded results in good agreement, but when ADP formation was measured an interfering phosphatase splitting of ATP had to be corrected for. Therefore, ADP was preferred as the substrate and its conversion to ATP was determined in a coupled hexokinase-glucose-6-phosphate dehydrogenase reaction yielding stoichiometric amounts of NADPH which were measured by the native fluorescence of this form of the nucleotide. The sensitivity and reproducibility of our micro-method permitted assay of small samples (50-500 ng) such as a layer of cerebellar cortical nerve cells and of insulin producing cells from the islets of Langerhans. Although not reaching the high values in muscle, these cells showed significantly higher activities than parenchymatous cells from the liver and the exocrine pancreas. The sensitivity attained is more than required for assay of clinical fine needle biopsies and is quite satisfactory for detection and estimation of adenylate kinase contaminants in enzyme preparations.