Revisiting the Phadia/EliA cut-off values for anticardiolipin and anti-ß2-glycoprotein I antibodies: a systematic evaluation according to the guidelines.

Background Phadia/EliA fluorescence enzyme immunoassays are widely used automated assays for anticardiolipin (aCL) and anti-ß2-glycoprotein I (aß2GPI) antibodies. To date, cut-off values for these assays have not been evaluated systematically and the evidence behind manufacturer's recommended cut-off values is not clear. Objective To determine Phadia/EliA cut-off values for antiphospholipid antibodies (aPL) according to the procedures suggested by guidelines. Methods A total of 266 blood donors (135 females and 131 males) were included. The pre-handling and analysis of the samples were performed according to the International Society on Thrombosis and Hemostasis (ISTH) guideline for solid phase aPL assays. Cut-off values and corresponding 90% confidence intervals (CI) for each antibody were established and outliers were handled according to the Clinical and Laboratory Standards Institute (CLSI) guideline for reference intervals. Samples from 377 consecutive patients, referred to our thrombophilia center with evidence of thrombosis or pregnancy morbidity were included for aPL testing. Results The in-house 99th (97.5th) percentile cut-off values were 11 (8.7), 12 (6.9) 8.5 (5.0) AU/mL for aß2GPI IgG, IgM and IgA, and 21 (13) GPL-U/mL and 41 (25) MPL-U/mL for aCL IgG and IgM, respectively. The prevalence of positive results (%) defined by these cut-off values in patients with evidence of thrombosis or pregnancy morbidity was 9.5 (12.2), 1.6 (2.9), and 7.0 (9.9), and 0.8 (3.8) for aß2GPI IgG, IgM, and aCL IgG and IgM respectively. The use of in-house 99th percentile cut-off values compared to the manufacturer suggested cut-off values resulted in 1 and 39 fewer samples for aß2GPI and aCL to be classified as positive for aPL, respectively. Conclusions We present Phadia/EliA cut-off values with 90% CI for aPL determined systematically according to the ISTH and CLSI guidelines. These values are different from values previously determined, suggesting variation of aPLs in different populations. Our findings indicate the need for each laboratory to determine/validate assay specific cut-off values for aPL.

Energy availability and the female athlete triad in elite endurance athletes.

The female athlete triad (Triad), links low energy availability (EA), with menstrual dysfunction (MD), and impaired bone health. The aims of this study were to examine associations between EA/MD and energy metabolism and the prevalence of Triad-associated conditions in endurance athletes. Forty women [26.2 ± 5.5 years, body mass index (BMI) 20.6 ± 2.0 kg/m(2), body fat 20.0 ± 3.0%], exercising 11.4 ± 4.5 h/week, were recruited from national teams and competitive clubs. Protocol included gynecological examination; assessment of bone health; indirect respiratory calorimetry; diet and exercise measured 7 days to assess EA; eating disorder (ED) examination; blood analysis. Subjects with low/reduced EA (< 45 kcal/kg FFM/day), had lower resting metabolic rate (RMR) compared with those with optimal EA [28.4 ± 2.0 kcal/kg fat-free mass (FFM)/day vs 30.5 ± 2.2 kcal/kg FFM/day, P < 0.01], as did subjects with MD compared with eumenorrheic subjects (28.6 ± 2.4 kcal/kg FFM/day vs 30.2 ± 1.8 kcal/kg FFM/day, P < 0.05). 63% had low/reduced EA, 25% ED, 60% MD, 45% impaired bone health, and 23% had all three Triad conditions. 53% had low RMR, 25% hypercholesterolemia, and 38% hypoglycemia. Conclusively, athletes with low/reduced EA and/or MD had lowered RMR. Triad-associated conditions were common in this group of athletes, despite a normal BMI range. The high prevalence of ED, MD, and impaired bone health emphasizes the importance of prevention, early detection, and treatment of energy deficiency.

Hormone therapy affects plasma measures of factor VII-activating protease in younger postmenopausal women.

OBJECTIVES: Current reviews indicate that hormone therapy (HT) has a protective role in coronary heart disease (CHD) in younger postmenopausal women, whereas HT contributes to CHD in older women. Factor VII-activating protease (FSAP) is a serine protease that accumulates in unstable atherosclerotic plaques. FSAP is presumably involved in plaque stability and rupture. Reduced plasma concentration of FSAP may be associated with the development and expression of atherosclerosis and may thus contribute to precipitation of CHD. Here we address the potential influence of various HT regimens on plasma measures of FSAP in postmenopausal women treated for 1 year with different HT formulations or no HT. METHODS: Six groups of postmenopausal women (n = 139) were allocated to five different HT modalities or no HT. Samples were collected at baseline and after 12 months of treatment. Prototype assays were used for the determination of FSAP antigen and FSAP activity. RESULTS: The FSAP measures were comparable at baseline. No significant changes were observed in the control group after 12 months. HT in general induced a significant increase in FSAP antigen (7.7 microg/ml at baseline and 8.0 microg/ml after 12 months, p = 0.05), FSAP activity (1.54 PEU/ml at baseline and 1.68 PEU/ml after 12 months, p < 0.001) and FSAP ratio (202 mPEU/microg at baseline and 210 mPEU/microg after 12 months, p = 0.01). CONCLUSIONS: HT increases the plasma measures of FSAP. This increase may contribute to the protective effect on CHD induced by HT in younger postmenopausal women.

The effect of continuous combined conjugated equine estrogen plus medroxyprogesterone acetate and tibolone on cardiovascular metabolic risk factors.

OBJECTIVES: Hormone treatment (HT) after the menopause affects lipid and carbohydrate metabolism and inflammation and may modify risk factors relevant for the clinical expression of the metabolic syndrome and cardiovascular disease. Tibolone has pharmacodynamic properties different from other hormone preparations. Here, we compare the effect of combined HT and tibolone on metabolic risk markers for the development of cardiovascular disease. METHODS: Postmenopausal women were randomly assigned to 1.25 or 2.5 mg/day of tibolone or oral continuous combined conjugated equine estrogen plus medroxyprogesterone acetate (CEE/MPA). Cardiovascular risk factors were determined at baseline and after 12 months of treatment. RESULTS: Body mass index and blood pressure were unaffected by the HT. HOMA-IR decreased in the CEE/MPA group (3.69 vs. 3.38; p = 0.02). Treatment with tibolone increased tissue-type plasminogen activator activity (0.87 IU/ml vs. 1.21 IU/ml; p = 0.005) and C-reactive protein (0.83 mg/l vs. 1.88 mg/l; p < 0.001), and decreased plasminogen activator inhibitor activity (6.9 IU/ml vs. 2.0 IU/ml; p < 0.001) and triglycerides (0.99 vs. 0.87 mmol/l; p = 0.004). Both treatments decreased total cholesterol significantly. CONCLUSIONS: CEE/MPA and tibolone have comparable effects on most metabolic risk factors investigated. The effect of tibolone on fibrinolysis and triglycerides suggests that tibolone has a favorable pharmacological profile on these risk factors when compared to CEE/MPA.

Genome-wide analysis of genetic determinants of circulating factor VII-activating protease (FSAP) activity.

Essentials Knowledge of genetic regulators of plasma factor VII activating protease (FSAP) levels is limited. We performed a genome-wide analysis of variants influencing FSAP activity in Scandinavian cohorts. We replicated an association for Marburg-1 and identified an association for a HABP2 stop variant. We identified a novel locus near ADCY2 as a potential additional regulator of FSAP activity. SUMMARY: Background Factor VII-activating protease (FSAP) has roles in both coagulation and fibrinolysis. Recent data indicate its involvement in several other processes, such as vascular remodeling and inflammation. Plasma FSAP activity is highly variable among healthy individuals and, apart from the low-frequency missense variant Marburg-I (rs7080536) in the FSAP-encoding gene HABP2, determinants of this variation are unclear. Objectives To identify novel genetic variants within and outside of the HABP2 locus that influence circulating FSAP activity. Patients/Methods We performed an exploratory genome-wide association study (GWAS) on plasma FSAP activity amongst 3230 Swedish subjects. Directly genotyped rare variants were also analyzed with gene-based tests. Using GWAS, we confirmed the strong association between the Marburg-I variant and FSAP activity. HABP2 was also significant in the gene-based analysis, and remained significant after exclusion of Marburg-I carriers. This was attributable to a rare HABP2 stop variant (rs41292628). Carriers of this stop variant showed a similar reduction in FSAP activity as Marburg-I carriers, and this finding was replicated. A secondary genome-wide significant locus was identified at a 5p15 locus (rs35510613), and this finding requires future replication. This common variant is located upstream of ADCY2, which encodes a protein catalyzing the formation of cAMP. Results and Conclusions This study verified the Marburg-I variant to be a strong regulator of FSAP activity, and identified an HABP2 stop variant with a similar impact on FSAP activity. A novel locus near ADCY2 was identified as a potential additional regulator of FSAP activity.

Calibrated kallikrein generation in human plasma.

OBJECTIVES: The physiological role of the contact system remains inconclusive. No obvious clinical complications have been observed for factor XII (FXII), prekallikrein (PK), or high molecular weight kininogen deficiencies even though the contact system in vitro is associated with coagulation, fibrinolysis, and inflammation. A global generation assay measuring the initial phase of the contact system could be a valuable tool for studies of its physiological role. DESIGN AND METHODS: We investigated whether such a method could be developed using the principle of the Calibrated Automated Thrombin generation method as a template. RESULTS: A suitable kallikrein specific fluorogenic substrate was identified (KM=0.91mM, kcat=19s-1), and kallikrein generation could be measured in undiluted plasma when silica was added as activator. Disturbing effects, including substrate depletion and the inner-filter effect, however, affected the signal. These problems were corrected for by external calibration with α2-macroglobulin-kallikrein complexes. Selectivity studies of the substrate, experiments with FXII and PK depleted plasmas, and plasma with high or low complement C1-esterase inhibitor activity indicated that the obtained and calibrated signal predominantly was related to FXII-dependent kallikrein activity. CONCLUSIONS: The findings described show that establishment of a kallikrein generation method is possible. Potentially, this setup could be used for clinical studies of the contact system.

Individual levels of plasma alpha 2-antiplasmin and alpha 2-macroglobulin during the normal menstrual cycle and in women on oral contraceptives low in oestrogen.

Determinations of alpha 2-antiplasmin and alpha 2-macroglobulin were made in plasma samples collected during one normal or hormone induced cycle in 15 normal women and 11 women using oral contraceptives containing 30 micrograms ethinyl oestradiol and 150 micrograms levo-norgestrel. The immediate plasmin inhibition test for determination of alpha 2-antiplasmin was modified for application on a centrifugal analyser using the chromogenic peptide substrate Chromozym PL. alpha 2-Macroglobulin concentration was determined by radial-immunodiffusion. There were no differences between the two groups in the mean concentrations of alpha 2-antiplasmin and alpha 2-macroglobulin, but during the cycle a slight, and statistically significant fall occurred in the alpha 2-antiplasmin concentration in both groups, while the fluctuations of alpha 2-macroglobulin were small and insignificant. Distinctly individual levels of both inhibitors were found to exist. The variations in these during the periods of the cycle fluctuated within a much narrower range than the variation of the total average. The results stress the importance of determining the fluctuations in individual levels in longitudinal studies. These findings exclude variations in the concentrations of these inhibitors as possible sources of a change in the antithrombotic potential caused by oral contraceptives.

Does low protein concentration of tissue-type plasminogen activator predict a low risk of spontaneous deep vein thrombosis?

Many reports have demonstrated an abnormal fibrinolysis in a subset of patients with deep vein thrombosis. We have studied systemic global fibrinolytic activity and protein concentrations of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in plasma of 25 young patients with a previous instance of spontaneous deep vein thrombosis documented by phlebography and in 50 healthy controls. The two populations were comparable with respect to a number of base-line variables (age, height, weight, etc.), while the patients had significantly lower fibrinolytic activity (p < 0.02), and significantly higher protein concentrations of t-PA (p < 0.0001) and PAI-1 (p < 0.0006). We used probit scale plots to identify the consequence of different cut-off points to separate patients from controls. Reasonable separation could be obtained for t-PA with a cut-off point of 5.2 ng/ml and for PAI-1 18 ng/ml. The sensitivity and specificity for these cut-off points were for t-PA 73% (95% confidence interval 63%-84%) and for PAI-1 67% (confidence interval 55%-77%). The negative predictive value with a cut-off point t-PA concentration of 5.2 ng/ml was 85% (95% confidence interval 70%-94%). We observed a significantly negative association between concentration of t-PA and fibrinolytic activity (rs = -0.47; p < 0.005) and also between PAI-1 and fibrinolytic activity (rs = -0.78; p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Multicentre evaluation of commercial kit methods: plasminogen activator inhibitor activity.

In order to study the analytical performance of different commercial kits for determination of plasminogen activator inhibitor (PAI) activity we distributed eight selected split samples to 11 European laboratories experienced with haemostasis testing. Three different laboratories were involved in the production of data from each of the commercial kits tested. A considerable variation of PAI activity results reported from the laboratories testing the same commercial kits was observed. The range of reported results could in individual samples exceed the median value indicating an interlaboratory variation of more than 100%. When we harmonized the results reported from different kits in different laboratories by means of an international standard from National Institute for Biological Standards and Control (NIBSC) we still observed that the results produced by some kits deviated systematically from results produced by other kits. Also, the harmonized results were used to estimate the overall coefficient of variation (CV) of PAI activity determined in various laboratories by different kits. We observed an inverse correlation between the PAI activity level and the CV with a CV of about 100% for low PAI activity levels and a CV of about 16% for high PAI activity levels. The high imprecision of the kits in the low concentration range of PAI activity indicates that unspecific factors in plasma may interfere with determination of active PAI. This was confirmed by the evaluation of the results from one of the plasma samples, which was PAI-1 depleted. The laboratories involved in the testing reported for this sample a mean value of 6.1 IU/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of endothelial function during oral contraception in women with insulin-dependent diabetes mellitus.

The effects of contraceptive steroids on the expression of endothelial homeostasis were examined by direct and indirect measures in women with insulin-dependent diabetes mellitus (IDDM) in a prospective nonrandomized controlled study. Study subjects were 13 women with uncomplicated IDDM treated with a monophasic combination of 30 micrograms ethinyl estradiol and 75 micrograms gestodene for 12 consecutive cycles and 13 women of comparable diabetic status as control. During the study period, none of the participants developed increased renal albumin excretion, which was used as a direct measure of endothelial function. In the indirect assessment of endothelial function, we found a proportionate increase in plasma levels of thrombin-antithrombin III (TAT) complexes and D-dimer during treatment. Hormonal intake was followed by decreased antigen concentrations of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (type 1 [PAI-1]), whereas the activities of t-PA and PAI-1 were unchanged. Plasma levels of plasminogen and histidine-rich glycoprotein (HRG) increased and decreased, respectively, whereas an increase in von Willebrand factor was observed in the treatment group. No significant changes in direct or indirect measures were observed in the control group during the observation period of 12 months. In conclusion, no adverse effect on endothelial function was demonstrated by direct measures, but our findings suggest that a procoagulant state, compensated by enhanced activity of the fibrinolytic system, is induced by hormonal treatment. Clinical and metabolic monitoring is recommended if the use of oral contraceptives in women with IDDM is extended.

The effect of chemical anti-inhibitors on fibrinolytic enzymes and inhibitors.

Fibrinolytic enzyme inhibitors hamper the determination of the specific fibrinolytic serine protease activity. Reportedly, chemical anti-inhibitors eliminate the influence of fibrinolytic inhibitors, but it remains unclear to what extent they change the specific activity of fibrinolytic serine proteases. We studied the influence of chemical anti-inhibitors (chloramine T, flufenamate, sodium lauryl sulfate, and methylamine) on fibrinolytic serine proteases and fibrinolytic enzyme inhibitors using the physiological substrate fibrin as plasmin substrate. Low concentrations of chloramine T (0.01 mmol/l) prevent the inhibition of plasminogen activators. Higher concentrations (1 mmol/l) reduce the inhibition of plasmin, but simultaneously quench the plasminogen activator activity. Flufenamate eliminates most fibrinolytic enzyme inhibitors, but increases the activity of plasmin (apparent recovery 140%) and plasminogen activators (apparent recovery > 200%). Sodium lauryl sulfate eliminates the major fibrinolytic enzyme inhibitors, but increases the activity of plasmin (apparent recovery > 200%) and plasminogen activator, urokinase type (apparent recovery 130%). Methylamine affects only plasmin inhibition. We conclude that chemical anti-inhibitors may invalidate the analytical specificity of methods for the determination of fibrinolytic serine protease activity.

Effects of inhaled plasminogen activator on the balance between coagulation and fibrinolysis in traumatized pigs.

A profibrinolytic state is normal in the alveoli, but this may change as a result of trauma, possibly leading to fibrin deposition, a characteristic of acute lung injury/acute respiratory distress syndrome. Therefore, the present study investigated in a double-blind, placebo-controlled manner the effect of severe trauma on the alveolar fibrinolytic/coagulation balance, and the effect here-upon of inhalation of single-chain urokinase plasminogen activator (scu-PA) in pigs. The study shows an increased concentration of scu-PA in the bronchoalveolar lavage fluid of the treated animals in association with an increased plasmin-dependent fibrinolytic activity without increased systemic fibrinolytic activity, the transient increase in the concentration of scu-PA in the plasma being minimal. In conclusion, the study shows that activatable scu-PA can be nebulized to the lower respiratory tract and can increase the alveolar fibrinolysis without any significant systemic effects.

[Hemostatic balance during treatment with the newest contraceptives]. / Den haemostatiske balance under behandling med nyere p-pillepraeparater.

Thirty-four healthy young women were allocated to 12 consecutive cycles of treatment with monophasic combinations of: 20 micrograms ethinyl estradiol and 150 micrograms desogestrel (n = 15) or 30 micrograms ethinyl estradiol and 75 micrograms gestodene (n = 19). In both groups plasma levels of fibrinogen and factor VII increased while the capacity of coagulation inhibition was affected by increased protein C and decreased protein S levels. Increased fibrinolytic capacity was indicated by elevated activity and reduced antigen levels of tissue plasminogen activator and reduced activity and concentration of tissue plasminogen activator inhibitor. The ratio between thrombin-antithrombin-III-complexes and fibrin degradation products were unchanged signifying no effect of hormonal intake on the balance between thrombin formation and fibrin resolution. In conclusion, the dynamic balance between generation and resolution of fibrin was undisturbed during treatment with both hormonal compounds and our findings do not provide evidence for increased risk of thrombosis in normal women.

PIP: 34 healthy women aged 21-30 years were assigned to 12 consecutive menstrual cycles of treatment with monophasic combinations. 15 women with a median age of 24 years received 20 mcg ethinyl estradiol (EE) and 150 mcg desogestrel (DSG) and 19 women with a median age of 23 years were treated with 30 mcg EE and 75 mcg gestodene (GST). Three women from the EE+DSG group and four women from the EE+GST group quit after six months because of personal reasons. Two more women from the EE+GST group quit after six months because of mammary tension and weight gain. Two women in each group smoked between one and ten cigarettes daily, the rest were nonsmokers. The evaluation of the hemostatic system was carried out in the luteal phase before the treatment began and within the last ten days in the third, sixth, and twelfth treatment cycle. In both groups plasma levels of fibrinogen (7.2 mcmol/l pretreatment to 8.7 mcmol/l posttreatment) and factor VIIc (80% pretreatment to 126% posttreatment) increased significantly under treatment, while the capacity of coagulation inhibition was affected after three months by increased protein C concentrations (15% in the EE+DSG group and 14% in the EE+GST group) and significantly decreased levels of protein C's cofactor, protein S levels by 11% and 15%, respectively. Increased fibrinolytic capacity was indicated by elevated activity and reduced antigen levels of tissue plasminogen activator and reduced activity and concentration of tissue plasminogen activator inhibitor. The ratio between thrombin antithrombin-III-complexes (TAT) and fibrin degradation products were unchanged, signifying no effect of hormonal intake on the balance between thrombin formation and fibrinolysis. The dynamic balance between coagulation and fibrinolysis was undisturbed during treatment with both hormonal compounds, and findings do not provide evidence for increased risk of thrombosis in normal women.

Lupus anticoagulant is significantly associated with inflammatory reactions in patients with suspected deep vein thrombosis.

OBJECTIVE: Lupus anticoagulant (LA) and antiphospholipid antibodies (aPL) are suggested as risk factors for development of deep vein thrombosis (DVT) among patients without systemic lupus erythematosus (SLE). Other conditions, e.g. inflammation, are reported to induce LA and it is uncertain whether the association between LA and DVT is causal. In this study the associations between aPL, LA and inflammation were investigated in 170 consecutive patients without SLE, but with a tentative diagnosis of DVT. MATERIAL AND METHODS: DVT was diagnosed in 64 patients. LA was determined according to the criteria of the International Society of Thrombosis and Haemostasis. The concentration of anticardiolipin (aCL) and beta(2)-glycoprotein I (anti-beta(2)-GPI) antibodies as well as C-reactive protein (CRP) was determined with sensitive and precise methods. RESULTS: LA was demonstrated in 8 patients with DVT and in 10 patients without DVT, relative risk 1.33 (CI: 0.55-3.18). No significant association was observed between aCL or anti-beta(2)-GPI and DVT. Patients suffering from DVT had significantly higher concentrations of CRP than patients without DVT. However, CRP was also significantly higher in patients positive for LA than in patients without LA irrespective of the presence of DVT (p<0.001). CONCLUSIONS: The present study supports a strong association between inflammatory reactions and development of LA in patients with suspected DVT, whereas no significant association was demonstrated between LA or aPL and DVT.

Relationship of glucose concentrations with PAI-1 and t-PA in subjects with normal glucose tolerance.

AIM: To study metabolic risk factors for the development of cardiovascular disease (CVD), including markers of the fibrinolytic system in relation to blood glucose levels in subjects with normal glucose tolerance and fasting blood glucose levels below 5.6 mmol/l. METHODS: Cross-sectional, community-based study from a primary health-care centre of adult subjects with normal glucose tolerance. Analysis of fasting and 2-h post-load blood glucose concentrations were centralized and related to anthropometric characteristics, metabolic variables, inflammatory markers, and coagulation and fibrinolytic variables. RESULTS: Increasing fasting blood glucose concentrations within the normal range in subjects with normal glucose tolerance were associated with increasing age, body mass index, and waist circumference, and with increasing concentrations of metabolic risk factors for development of CVD. After adjustment for gender, age, body mass index (BMI), and fasting insulin, levels of plasmin activator inhibitor (PAI-1) and tissue type plasminogen activator (t-PA) increased significantly with increasing levels of fasting glucose within the normal range (P = 0.012 and P < 0.0001, respectively). CONCLUSIONS: We found risk factors for CVD, specifically key components of the fibrinolytic system, PAI-1 and t-PA, increased with increasing fasting glucose levels even in subjects with normal glucose tolerance. This observation may help to explain the increased risk of CVD with increasing values of fasting glucose in the normal range.