Diagnostic possibilities of specific fibrin(ogen) degradation products in relation to venous thromboembolism.

The exclusion of deep venous thrombosis (DVT) in the elderly is hampered by low specificity in clinical decision of D-dimer assays in older patients. To reduce false-positive results, we evaluated specific fibrin(ogen) degradation product assays for their contribution in the diagnosis of DVT. In a post-hoc study with outpatients suspected for DVT, we evaluated the elastase-specific fibrinogen (fibrinogen elastase degradation product, FgEDP) and D-E-specific fibrin (fibrin degradation product, FbDP) degradation product assays in relation to DVT. Results were combined with five D-dimer assays as ratio, with a specific focus on age-dependency. In 437 patients (DVT prevalence 39%), FgEDP correlated with D-dimer in DVT-negative patients (P<0.001), but not in DVT-positive patients (P > 0.55). FbDP results correlated with D-dimer in both groups (P<0.001). The values of the area under the curve of the receiver operating characteristic curve for both assays were lower than D-dimer. Using the ratios, only in the fourth age quartile D-dimer/FgEDP ratios had diagnostic value with lower number needed to test (1.8-12.7) as compared to D-dimer less than 500 µg/l alone (5.4-102). The D-dimer/FbDP ratios in DVT-negative elderly patients increased to a plateau by increasing D-dimer. In DVT-positive patients, these ratios were near constant for increasing values of D-dimer. In elderly patients, the D-dimer/FgEDP ratios may improve the number of patients in whom DVT could be excluded. The D-dimer/FbDP ratios showed differences in composition of fibrin degradation products between DVT-negative and DVT-positive patients and between young and old DVT-negative patients.

Harmonization and external quality assessment of antithrombin activity assays.

UNLABELLED: In the framework of a Dutch project named "Calibration 2000" harmonization of antithrombin activity assays was studied. The commutability of potential calibrators for antithrombin was assessed by means of a twin-study design, which is a multicentre, split-patient sample, between-field-methods protocol. The twin-study consisted of simultaneous analysis of fresh-frozen patient plasmas and three potential calibrators for antithrombin by 30 Dutch laboratories forming 15 couples. The state-of-the-art intralaboratory standard deviation (SD(SA)) was used to assess the commutability of the potential calibrators. The regression line residuals for the potential calibrators were normalized by expressing them as multiples of SD(SA). All residuals of the potential calibrators were within the 3×SD(SA) limit. One potential calibrator was used in an attempt to harmonize antithrombin assay results in a Dutch field study. The interlaboratory coefficient of variation (CV) of the antithrombin results for three test samples could be reduced from 6.9 - 13.2% (before harmonization) to 5.6 - 9.8% using the common calibrator. CONCLUSION: The potential calibrators were commutable. Limited harmonization was achieved by using a common calibrator for all participants.

Patients with deep venous thrombosis and thrombophilia risk factors have a specific prolongation of the lag time in a chromogenic thrombin generation assay.

The objective of the present study was to evaluate the influence of thrombophilia risk factors on variables of a chromogenic thrombin generation assay (ETP) in a setting with acute deep venous thrombosis (DVT) and non-DVT patients. In 152 outpatients suspected for DVT, the results of thrombophilia investigations were known. In all patients, thrombin generation parameters were determined and related to the presence of thrombophilia risk factors. The thrombin generation results were divided in quartiles to calculate the odds ratio, as a measure of the association with thrombophilia risk factors, corrected for age and sex. The groups with and without DVT were analysed independently. From the 152 patients, there were 108 patients with and 44 without DVT. In the DVT-positive group, lag time was significantly prolonged (21.9 versus 20.3 s; P=0.021) in patients with known thrombophilia risk factors (n=48). The odds ratio for thrombophilia risk factors was 3.7 (95% CI 1.1-12) uncorrected and 4.3 (95% CI 1.2-16) corrected for age and sex, both P values less than 0.05. No differences in ETP parameters were observed in patients without DVT with known thrombophilia risk factors (n=32). The relationship of thrombophilia risk factors with the occurrence of a prolonged lag time in the acute phase of DVT is a new finding. Increased coagulation activation may be the consequence of the thrombophilia risk factors with this observation. To unravel whether the test expresses increased consumption or increased release of anticoagulant factors may open new ways to develop diagnostic methods to add to further refinement of exclusion algorithms for DVT.

A thrombin generation assay may reduce the need for compression ultrasonography for the exclusion of deep venous thrombosis in the elderly.

INTRODUCTION: The exclusion of deep venous thrombosis (DVT) by pre-test probability (PTP) and D-dimer in the elderly is safe, but has a low specificity. There is increasing interest in the diagnostic role of thrombin generation in patients with thrombosis. We evaluated the value of thrombin generation in the exclusion of DVT, especially in elderly patients. MATERIAL AND METHODS: All thrombin generation parameters of the Endogenous Thrombin Potential assay were tested retrospectively in a cohort of 443 patients suspected for DVT. The performance of the assay was calculated as AUC of the ROC curve and association with DVT as odds ratio (OR). The lag time value was combined with results of PTP and Innovance D-dimer assay. RESULTS: All thrombin generation parameters had a low AUC. In the 4th age quartile, the corrected OR of lag time increased to 16 and the AUC of lag time for the combination PTP < 2 and D-dimers ≥ 500 µg/L was 0.96 with a cut-off value of 23.0 sec. The exclusion of DVT in patients ≥ 75 years (n = 30), based on this combination and lag time values ≤ 23.0 sec had a NPV of 100% and a specificity of 96.0% in this subgroup of patients and decreased the number needed to test from 10 to 1.1. CONCLUSION: Thrombin generation parameters alone are inappropriate for the exclusion of DVT. However, in the elderly, the current algorithm of exclusion of DVT may improve markedly by the addition of the lag time results of the ETP.

An age-adapted approach for the use of D-dimers in the exclusion of deep venous thrombosis.

A normal D-dimer (DD) concentration for the exclusion of deep venous thrombosis (DVT) has a low specificity in older patients and compression ultrasonography is often required. Three D-dimer assays, STA Liatest, Tina-quant, and Innovance, are evaluated in symptomatic outpatients suspected for DVT with emphasis on its performance in older patients by using different cut-off levels. This study includes 466 outpatients suspected for having DVT. The diagnostic accuracy, measured as sensitivity and area under the curve of the receiver operation characteristic curve is good for all DD assays. The specificity of the DD assays combined with a low pretest probability varies from 42.6 to 51.5%. The specificity of the three DD assays in patients > or = 60 years varies, however, between 24.6 and 40.9%. Several cut-off values in different age-subgroups are studied. For patients < 60 years, the most accurate cut-off value is 500 microg/L for all DD assays. For patients > or = 60 years, a threshold of 750 microg/L has the best results with NPV of 100% for all assays and specificity of 48.5% (STA Liatest), 60.6% (Tina-quant), and 49.2% (Innovance), respectively. For the three assays, the number needed to test (NNT) decreases in both subgroups of patients compared to the standard algorithm. A cut-off level of 750 microg/L for patients > or = 60 years improves the clinical performance of DD assays in combination with the PTP score without the loss of NPV. The NNT improves substantially with an age-adapted algorithm.

Myocardial oxidative stress, and cell injury comparing three different techniques for coronary artery bypass grafting.

OBJECTIVE: Oxidative stress as a result of reperfusion injury is a known causative factor of cardiac muscle injury. In the peripheral blood as well in the coronary sinus, oxidative stress parameters and cardiac biomarkers were measured to investigate the different levels of oxidative stress during three different CABG techniques; MCABG (with minimal prime volume and warm blood cardioplegia) that was newly introduced in our hospital, versus OPCAB, versus our current standard, conventional CABG (CCABG, consisting of high volume prime and cold crystalloid cardioplegia). Concomitantly, cardiac biomarkers were measured to detect myocardial cell injury. METHODS: Thirty patients scheduled for CABG with the intention to treat three-vessel disease were randomly assigned for CCABG, MCABG or OPCAB. Perioperatively, plasma levels of malondialdehyde (MDA) as a marker of oxidative stress, and the allantoin/uric acid ratio (A/U ratio) as a marker of antioxidant activity were measured in the ascending aorta (Aa), and in the coronary sinus (Cs), simultaneously. Additionally peripheral (Aa) blood levels of heart fatty acid binding protein (HFABP), troponin T, CPK and CKMB as markers of myocardial injury were obtained. RESULTS: The MCABG group had significantly lower MDA levels in the Cs compared to the CCABG group, respectively, to the OPCAB group (p=0.04 and p=0.03). At all time points the A/U ratio in the CCABG group remained significantly higher in the Cs as well in the Aa samples compared to the MCABG and the OPCAB group (p<0.001, respectively, p<0.001, for both groups). HFABP and troponin T showed consistent curves compared to the CPK figure over time in all groups. CONCLUSION: In this study coronary sinus blood levels of oxidative stress parameters were consistently higher compared to peripheral blood levels. The levels were lowest in the MCABG study group. In this group also the lowest levels cardiac biomarkers of myocardial injury were found.

The influence on INRs and coagulation factors of the time span between blood sample collection and intake of phenprocoumon or acenocoumarol: consequences for the assessment of the dose.

Managing treatment with vitamin K antagonists, the prothrombin time (PT), expressed as international normalized ratio (INR), may not represent the INR during the entire 24 hour (h) period, and this variation may be different between long-acting phenprocoumon and short-acting acenocoumarol. For both drugs we investigated the variation in 24 h of the PT/INR, the consequencesfor the assessment of the doses and which vitamin K-dependent factor causes the daily variation. Patients on self-management took their medication at 6 p.m. and determined their INRs for eight weeks, once a week and three times daily (8.30 a.m., 6 p.m. and 11 p.m., thus 14.5 h, 24 h and 29 h after taking the medication, respectively). Acenocoumarol showed a significant variation in INRs over the 24 h period, with 22 out of 80 INRs >20% lower at 11 p.m. versus 8.30 a.m. Phenprocoumon showed only few variations. Patients managed by the anticoagulation clinic took their medication at 6 p.m. for four weeks and then at 8 a.m. for four weeks, 15 h and 25 h, respectively, before the weekly blood collection. PT/INR and coagulation factors VII, X and II were determined. With acenocoumarol, taken 25 h before blood collection, the INRs were significantly different compared to 15 h, especially attributed to plasma levels of factor VII. Those on phenprocoumon were equal. These variations of INRs during 24 h may have major effects on the prescribed dose of short-acting vitamin K antagonists, such as acenocoumarol, especially for INRs at the limits of the therapeutic ranges.

The role of fibrin monomers in optimizing the diagnostic work-up of deep vein thrombosis.

Despite the use of a clinical score and D-dimers to exclude deep vein thrombosis (DVT), the majority of patients still need repeated ultrasound (US). The aim of the study was to investigate whether fibrin monomers (FMs), as markers of thrombin generation, have additional value in the diagnosis of DVT. This is a posthoc analysis of 464 outpatients, participants in a management study using D-dimers (Tina-Quant and a clinical score in the exclusion of DVT. Two new FM assays (Auto LIA-FM and IATRO SF, Japan) were performed. Overall sensitivity, negative predictive value (NPV) and specificity of the D-dimer test were 98%, 98% and 42%. The optimal cut-off point for the Auto LIA-FM test was

Harmonisation of factor VIII:C assay results: study within the framework of the Dutch project 'Calibration 2000'.

In a Dutch project for harmonisation of factor VIII coagulant activity (FVIII:C) assays, the commutability of potential calibrators for FVIII:C was assessed by means of a 'twin-study design', which is in essence a multi-centre, split-patient sample, between-field-methods protocol. Commutability was defined as the degree to which a material yielded the same numerical relationships between results of measurements by a given set of measurement procedures as those between the expectations of the relationships for the same procedures applied to those types of material for which the procedures were intended. The study consisted of the simultaneous analysis of fresh frozen patient plasmas and three potential calibrators for FVIII:C by 16 Dutch laboratories forming eight couples. The state-of-the-art intra-laboratory standard deviation was used to assess the commutability of the potential calibrators. One potential calibrator was used to harmonise FVIII:C assay results in a Dutch field study. The inter-laboratory coefficient of variation of two test samples could be reduced significantly, but no significant effect was observed with three other test samples. We recommend that at least three different sample dilutions be used in each FVIII:C assay, in agreement with previous recommendations.

Malondialdehyde in plasma, a biomarker of global oxidative stress during mini-CABG compared to on- and off-pump CABG surgery: a pilot study.

In contrast to conventional on-pump coronary artery bypass grafting only mild increase of parameters of oxidative stress is reported during and after off-pump coronary artery bypass grafting. In an attempt to reduce the side effects of extra corporeal circulation the mini- extra corporeal circulation concept was introduced. In this study peroperative oxidative stress biomarkers were compared using three different techniques for CABG (conventional, mini and off-pump). It concerns a prospective randomized pilot study of 60 aged patients (70+ years) divided over 3 study groups. During the peroperative time points there was a significant increase in the mean concentration of uric acid for the CCABG group. On arrival at the intensive care unit the mean concentrations decreased significantly. During the per-operative period all groups showed significant increase in the concentration of malondialdehyde, however, this increase was the steepest for the CCABG group. On arrival at the intensive care unit the mean concentration decreased significantly for all groups. We found only mild organ ischemia/reperfusion injury and oxidative stress in the OPCAB group and the MCABG group with respect to the CCABG group.

DPSS yellow-green 561-nm lasers for improved fluorochrome detection by flow cytometry.

INTRODUCTION: Blue-green 488-nm laser sources are widespread in flow cytometry but suffer some drawbacks for cell analysis, including their excitation of endogenous proteins (resulting in high cellular autofluorescence) and their less-than-optimal coincidence with the excitation maxima of commonly used fluorochromes, including the phycoerythrins (PE). Longer wavelength lasers such as green helium-neons and, more recently, diode-pumped solid state (DPSS) 532-nm sources have previously been employed to overcome these difficulties and improve overall sensitivity for PE. In this study, we evaluate an even longer wavelength DPSS 561-nm for its ability to improve PE and DsRed fluorescent protein detection sensitivity. METHODS: A DPSS 561-nm laser emitting at 10 mW was mounted onto a BD LSR II. Mouse thymoma cells labeled with cell surface marker antibodies conjugated to the R- and B-forms of PE were analyzed and compared with conventional 488-nm excitation using the same bandpass filters and signal travel distances. A similar analysis was carried out with cell lines expressing the red fluorescent protein DsRed, several green-yellow excited low molecular weight fluorochromes, and a rhodamine-based caspase substrate. Additionally, cells labeled with PE and co-labeled with fluorescein or simultaneously expressing green fluorescent protein (GFP) were analyzed to determine if PE excitation at 561 nm with simultaneous fluorescein/GFP detection was feasible. RESULTS: The DPSS 561-nm laser gave a several-fold improvement in the fluorochrome to autofluorescence ratios between PE-labeled cells and unlabeled controls. Analysis of cells expressing the fluorescent protein DsRed with the DPSS 561-nm source gave a 6-7-fold improvement in sensitivity over 488-nm excitation, and gave excellent excitation of yellow-green excited fluorochromes and rhodamine-based physiological probes. Yellow-green laser light also caused virtually no impingement on the spatially separated fluorescein/GFP detector, a significant problem with green laser sources, and also allowed simultaneous analysis of GFP and PE with virtually no signal overlap or requirement for color compensation. CONCLUSIONS: DPSS 561-nm laser excitation gave significantly improved sensitivity for both PE-labeled and DsRed expressing cells, with little contamination of a typical fluorescein/GFP detector.

Validation of a new method to measure hydrogen peroxide in exhaled breath condensate.

Inflammatory processes in the lung can or will elicit oxidative stress. The degree of oxidative stress can be determined by measuring hydrogen peroxide (H(2)O(2)) concentration in exhaled breath condensate (EBC) but the methods to measure H(2)O(2) are all rather time consuming and only reliable and/or accurate in the hand of skilled technicians in a dedicated laboratory. We tested a new commercial device (Ecocheck), developed to offer a less time-consuming method to measure H(2)O(2). We validated this new method according the NCCLS EP10-A2 protocol. The validation shows that the imprecision in the low range is high (28.4%) and declines with higher concentrations of H(2)O(2) (up to 6.6%). The Ecocheck is "an easy to use" measuring device for routine measurements getting quick results without the need for skilled technicians to determine H(2)O(2) concentrations.

Reduced efficacy of clinical probability score and D-dimer assay in elderly subjects suspected of having deep vein thrombosis.

The combined strategy of a pretest clinical probability (PCP) score and D-dimer has shown to be of value in the diagnosis of deep vein thrombosis (DVT). As D-dimer concentrations increase with age, the effect of age on the usefulness of this strategy was retrospectively investigated in outpatients suspected of having DVT. In all patients, participants of a prospective management trial, a PCP score and D-dimer (Tina-quant) were performed. In a total of 812 patients, 317 (39%) had thrombosis. Patients were divided into quartiles according to their age. Sensitivity and negative predictive value of a low/moderate PCP score and a normal D-dimer were 98-100% and did not differ between the different age quartiles. Specificity in the highest quartile was 17.4% compared with 49.2% in the youngest (P < 0.000001). The proportion of patients with a low/moderate PCP score and a normal D-dimer decreased with age: 12% in the highest quartile (>73.8 years) versus 25% in younger patients (P = 0.00005). We therefore conclude that the combined strategy of a low/moderate PCP score with a normal D-dimer test is safe for excluding DVT in all age groups, but is less useful in the elderly.

Elevated levels of D-dimer and fragment 1+2 upon central venous catheter insertion and factor V Leiden predict subclavian vein thrombosis.

BACKGROUND AND OBJECTIVES: Subclavian vein thrombosis is a well-recognized complication following central venous catheter insertion. We studied whether the determination of D-dimer levels, fragment 1+2 levels and factor V Leiden can identify patients at high risk of developing subclavian vein thrombosis. DESIGN AND METHODS: The presence of central venous catheter associated thrombosis was analyzed in 235 patients undergoing allogeneic bone marrow transplantation, of whom 30 (13%) developed thrombosis. A case-control study was performed with 30 patients matched for age, gender, and type of transplantation who did not develop thrombosis. Blood was sampled 3-5 days after catheter insertion. D-dimer levels were determined using a latex microparticle assay and an enzyme linked immunosorbent assay (ELISA). An ELISA was used to determine fragment 1+2 levels. The factor V genotype was determined by polymerase chain reaction. RESULTS: The levels of D-dimer and fragment 1+2 were significantly elevated in the patients who developed thrombosis. Five patients tested positive for factor V Leiden and all 5 developed subclavian vein thrombosis. Patients with high D-dimer levels (> 1300 microg/L measured by latex agglutination and >350 microg/L measured by ELISA) had a 7.0 and 6.0 times higher risk of developing subclavian vein thrombosis, respectively. A 5.5-fold increased risk of thrombosis was observed in patients with a fragment 1+2 level higher than 1.300 nmol/L. This resulted in positive predictive values of 0.78, 0.80 and 0.83 for the fragment 1+2, D-dimer and D-dimer latex agglutination assays, respectively. The accompanying negative predictive values were 0.39, 0.40 and 0.42, respectively. INTERPRETATION AND CONCLUSIONS: We conclude that the measurement of D-dimer and fragment 1+2 levels after central venous catheter insertion, as well as factor V Leiden determination, can be used to identify patients at high risk of developing symptomatic subclavian vein thrombosis.

The predictive value of D-dimer measurement for cancer in patients with deep vein thrombosis.

BACKGROUND AND OBJECTIVES: Venous thromboembolism can be related to malignancy, but routine screening for cancer in patients with deep vein thrombosis (DVT) is not a recommended practice. The aim of this study was to evaluate the value of D-dimer concentration in predicting cancer in patients with DVT. DESIGN AND METHODS: D-dimer levels were measured in outpatients presenting with DVT. In a proportion of patients, D-dimer levels were measured daily for 4 days. The occurrence of malignancy was documented. RESULTS: Patients were followed for a median of 34 months. Fifty (23%) of 218 patients with thrombosis had cancer in the study period including 14 who developed cancer during the follow-up. High initial D-dimer levels (levels > 4000 mg/L) were associated with more cancer during follow-up than were lower D-dimer levels: 13% versus 4% (p=0.048). High D-dimer levels after 4 days of treatment were associated with a 15% prevalence of cancer whereas the prevalence in patients with lower D-dimer levels was 5% (p=0.1). The total cancer prevalence (including cancer diagnosed before thrombosis) in patients with initial D-dimer levels < 4000 mg/L was 16% compared to 32% in patients with higher levels (p=0.009, RR=2.0). After 4 days of treatment, total cancer prevalences were 14% and 46%, respectively (p=0.02, RR=3.4). In patients aged < 60 years, initial D-dimer levels of < 4000 mg/L were associated with a cancer prevalence of 3% whereas higher levels were associated with a prevalence of 23% (p=0.001, RR=8.6). After 4 days of treatment, the prevalences associated with lower and higher levels of D-dimer were 0% and 100%, respectively (p=0.003). There was no difference in older patients. INTERPRETATION AND CONCLUSIONS: High D-dimer concentrations at presentation or during the first days of treatment are indicators of an increased probability of overt or occult forms of cancer, especially in patients under 60 years old.

Markers of inflammation and oxidative stress in exacerbated chronic obstructive pulmonary disease patients.

COPD is characterised by damage to small airways due to an inflammatory process as well as an imbalance between oxidants and antioxidants. Several cytokines and cell adhesion molecules enhancing a mainly neutrophilic inflammation have been associated with COPD. The aim of the study was to investigate whether inflammation or oxidative markers gave an indication of the course of COPD during an exacerbation. Fourteen patients with moderate to severe COPD admitted to the St. Antonius Hospital because of an exacerbation have been monitored during treatment with prednisolone 50 mg intravenously during 24 h at admission, reduced to 25 mg at day 3 and tapered off with oral prednisolone at day 7. On three separate occasions, day 1, 3 and 7, H2O2 in exhaled air, IL-8 and the soluble cell adhesion molecule sICAM and sE-selectin in serum were measured. We compared the patients at day 1 with healthy controls (in both non-smokers and smokers). Furthermore, we examined the changes from the study group in time during therapy. At admission all the markers were raised in comparison with the control groups. During treatment H2O2 concentrations in breath condensate declined significantly (P<0.001) as well as IL-8 and sICAM in serum (P=0.002, respectively, P<0.001). There was no significant change in sE-selectin (P=0.132). No significant improvement has been found in spirometry. These data suggest that the markers H2O2 in exhaled air, IL-8 and sICAM in serum are suitable markers in monitoring exacerbated COPD.

Low molecular weight heparin (dalteparin) is equally effective as unfractionated heparin in reducing coagulation activity and perfusion abnormalities during the early treatment of pulmonary embolism.

Little is known about the differences between unfractionated heparin (UFH) and low molecular weight heparin (LMWH) with regard to their effects on coagulation activity during treatment for pulmonary embolism. The objective of this study was to compare UFH and LMWH (dalteparin) in the early treatment of pulmonary embolism in terms of control of coagulation markers and perfusion abnormalities. Thirty-seven patients with acute pulmonary embolism were randomized to receive intravenous UFH or subcutaneous dalteparin, each accompanied by acenocoumarol. Daily blood samples were obtained for the measurement of thrombin generation (fragments 1 and 2 [F1+2], thrombin-antithrombin (TAT) complexes and fibrin monomers [FMs]) and fibrinolysis (d-dimer concentrations and clot-lysis times). Ventilation-perfusion scintigraphies were performed, and with the data they yielded, percentage of vascular obstruction scores (PVOs) were calculated on days 0 and 5. The international normalized ratio was within the therapeutic range in both groups on day 3. F1+2 and TAT complexes rapidly normalized, without differences between the groups (P =.5 and.4, respectively). FM levels did not decrease and, in fact, showed an increase in the UFH group from day 3 on (P <.05 between groups). d-Dimer levels decreased over time, with no differences between groups (P =.6). Clot-lysis times were shorter in the UFH group (P <.05). PVOs on days 0 and 5 were not different (P =.5 and.8, respectively), but the decrease in PVOs over time was greater in the dalteparin group (P =.04). These results show that dalteparin is at least as effective as UFH in reducing coagulation activity and perfusion abnormalities in the early treatment of pulmonary embolism.