Pre-operative plasma levels of soluble fibrin polymers correlate with the development of deep vein thrombosis after elective neurosurgery.

The role of blood tests in identifying patients at high risk for post-operative venous thromboembolism is undefined. The aim of this study was to evaluate the correlation between pre-operative plasma levels of soluble fibrin polymers (SFP), as determined by a recently developed enzyme-linked immunosorbent assay (ELISA) assay (TpP), and the incidence of deep vein thrombosis (DVT) after elective neurosurgery. Blood samples for SFP assay were withdrawn on the day before surgery from 157 consecutive patients undergoing elective neurosurgery for brain or spinal tumour. Patients were randomized to subcutaneous enoxaparin (40 mg once daily) or placebo given for at least 7 days. All patients wore compression stockings. DVT was assessed by bilateral venography, performed on day 8 +/- 1. Thirty-four patients (21.7%) were found to have a DVT, proximal in 11 (7%) and isolated distal in 23. Patients with and without DVT had a plasma pre-operative SFP levels of 6.2 +/- 4.6 and 1.9 +/- 1.5 mg/ml respectively (mean +/- SD) (P < 0.001). SFP levels in patients with proximal and isolated distal DVT were 7.6 +/- 5.1 and 5.5 +/- 4.4 microg/ml, respectively (P = 0.22). SFP cut-off levels categorized patients into three classes of DVT incidence. The incidence of DVT was 7.4% (6 of 81) for SFP levels < 2 microg/ml, 20.4% (11 of 54) for levels between 2 and 4.5 microg/ml, and 77.3% (17 of 22) for levels > 4.5 microg/ml (P= 0.001, Cochran-Mantel-Haenszel test). We conclude that pre-operative SFP levels correlate with post-operative DVT in elective neurosurgery patients. Further studies are required to define whether pre-operative SFP measurement could be useful in patient management.

The utility and cost-effectiveness of D-dimer measurements in the diagnosis of deep vein thrombosis.

BACKGROUND AND OBJECTIVE: The potential utility of D-dimer measurements for the diagnosis of deep vein thrombosis became evident soon after the development of reliable commercial assays. The purpose of this review is to outline some critical aspects affecting cost-effectiveness of D-dimer measurements in the diagnosis of deep vein thrombosis (DVT). METHODS: The authors have been working in this field contributing original papers whose data have been used for this study. In addition, the material analyzed in this article includes papers published in the journals covered by the Science Citation Index and Medline. RESULTS: D-dimer levels are very sensitive to the process of fibrin formation/dissolution occurring with ongoing thrombosis. However, they may not be highly specific for venous thromboembolism as they are influenced by the presence of comorbid conditions potentially elevating plasma D-dimer (cancer, surgery, infectious diseases). In addition, commercially available ELISA assays, although quantitative and reproducible, cannot be used under emergency conditions because they are time-consuming and suited for batch-processing of plasma samples. Recently, new assays have been introduced which permit fast and quantitative D-dimer estimations in individual patients. We have evaluated the utility of two new rapid assays (LPIA D-dimer. Mitsubishi, and VIDAS D-DIMER, bio-Merieux) in combination with compression real-time-B-mode ultrasonography for the detection of deep vein thrombosis in asymptomatic patients following elective hip replacement and in patients with clinically suspected deep vein thrombosis. In both settings, we identified cut-off values with optimal sensitivity which allow exclusion of deep vein thrombosis in a considerable percentage of patients, with substantial sparing of economic resources. In fact, based on a cost-effectiveness analysis, a diagnostic algorithm combining D-dimers measurement and compression ultrasonography would result in cost-savings ranging from 5% to 55% in patients with high or low clinical pretest probability respectively. However, the specificity of D-dimer measurements for deep vein thrombosis was much higher in symptomatic than in asymptomatic patients. Choice of the cut-off value proved to be dependent on the method as well as on the patient populations studied. CONCLUSIONS: The cost-effectiveness of D-dimers measurement in the diagnosis of asymptomatic DVT remains questionable. Conversely, our data strongly support the utility of D-dimers determinations in the diagnosis of symptomatic DVT. In terms of sparing economic resources, the introduction in the clinical laboratory of the rapid quantitative assays would be highly convenient, because they avoid a source of bias in the interpretation of D-dimers results, are easy to perform and do not require dedicated personnel or instrumentation. Prospective management studies validating the utility of D-dimer measurement in the diagnosis of deep vein thrombosis are urgently needed.

Diagnostic value of compression ultrasonography and fibrinogen-related parameters for the detection of postoperative deep vein thrombosis following elective hip replacement: a pilot study.

To determine their ability to diagnose postoperative deep vein thrombosis (DVT) D-dimer - by three methods -, fibrinogen degradation products (FgDP) and fibrinogen levels were measured in 68 consecutive patients before elective surgery for hip replacement and on postoperative day 1, 3, 6, and 10. All patients received prophylaxis and underwent compression real-time B-mode ultrasonography (C-US) on postoperative day 5 and 9, and bilateral ascending venography on day 10. Twenty-two out of 68 patients developed asymptomatic postoperative DVT, which was limited to the calf veins in 14 and involved the proximal veins in 8 patients. C-US was negative in all patients on day 5. On day 9, C-US sensitivity and specificity for proximal DVT were 63% (95% confidence interval: 26%-90% and 98% (89%-100%) respectively. Postoperative changes in the laboratory parameters evaluated were not different in patients with or without DVT until day 10. On day 10, mean D-dimer, FgDP and fibrinogen levels were significantly higher in patients with DVT than in those without DVT (p values between 0.006 and 0.032), but only D-dimer was higher with DVT involving two or more venous segments than with thrombosis involving one venous segment only (p < 0.05). Stepwise logistic regression analysis identified D-dimer and fibrinogen on day 10 as predictors of postoperative DVT. In a receiver operator curve and after weighing for the coefficients generated by logistic regression analysis, the combination of a latex photometric immuno-assay and of PT-derived fibrinogen yielded-at a cut-off value of 7.0 a sensitivity of 100% (73%-100%) and a specificity of 58% (39%-75%) for DVT, with a negative predictive value of 100% (78%-100%), a positive predictive value of 52% (32%-71%) and an overall accuracy of 71% (55%-83%). These results suggest that two simple, fast and reproducible tests may permit the identification of patients at low risk of having postoperative DVT and that a combination of sensitive laboratory assays and of the highly specific C-US may select patients requiring anticoagulant treatment. Efficacy and cost-effectiveness of this approach should be evaluated in large clinical management studies.

Central retinal vein and branch artery occlusion associated with inherited plasminogen deficiency and high lipoprotein(a) levels: a case report.

We describe a case of central retinal vein and branch artery occlusion associated with inherited type I plasminogen deficiency (68%) and permanent elevation of Lp(a) (460 mg/l, S2 phenotype) in a 45 year old white woman with no associated local or systemic risk factors. Pedigree analysis revealed inheritance of plasminogen deficiency from the deceased father and of high Lp(a) levels from the mother. Both the patient's sons had plasminogen deficiency, but they had normal Lp(a) levels. In a series of 40 consecutive patients with central retinal vein occlusion we previously reported the observation of high Lp(a) levels--consistently associated with the S2 phenotype--in 30% of the patients as compared to a 10% incidence in controls. This case emphasizes the importance of screening patients with occlusion of the retinal vessels and no associated risk factors for coagulation abnormalities predisposing to thrombosis.

Severe autoimmune protein S deficiency in a boy with idiopathic purpura fulminans.

Idiopathic purpura fulminans usually occurs in young children and is frequently preceded by a preparatory viral or bacterial infection. Following a severe streptococcal pharyngitis, an 8-year-old boy developed purpura fulminans with disseminated intravascular coagulation and severe protein S deficiency (total antigen < 0.05 u/ml). Despite generous plasma infusions, skin necrosis progressed rapidly into compartment syndrome which required fasciotomy and skin grafting and resulted in the loss of three digits of the right foot. Total protein S remained low for over a month despite plasma supplementation and complete normalization of protein C levels. A polyclonal anti-protein S IgG was demonstrated in the patient's plasma, which decreased to 25% of baseline titre after 1 month and was undetectable 6 months after purpura fulminans, when plasma protein S had returned to normal. Transient, isolated and severe deficiencies of protein S have been reported in patients with idiopathic purpura fulminans and a previous preparatory infection. Autoimmune protein S deficiency may play a key role in the aetiopathogenesis of idiopathic purpura fulminans.

Protein S and protein C anticoagulant activity in acute and chronic cardiac ischemic syndromes. Relationship to inflammation, complement activation and in vivo thrombin activity.

Protein S (PS) and protein C (PC) anticoagulant activities and thrombin-antithrombin complex (TAT) were measured in 20 patients with AIS, 25 patients with chronic stable angina (CSA) and a control group (C). Although plasma levels of TAT were significantly elevated in patients with CSA (p < 0.01 vs C), they were much higher in patients with AIS (p < 0.001 vs CSA). PC anticoagulant activity was similar in patients and controls. At variance, PS anticoagulant activity was lower in patients with AIS than in those with CSA and controls (p < 0.05), reflecting differences in total PS and C4B-binding protein (C4B-BP) antigen possibly resulting from involvement in the mechanisms of inflammation, complement activation and acute-phase response. The ratios of anticoagulant PS and PC to procoagulant vitamin K-dependent factors IX and II were reduced in AIS patients (0.05 > p > 0.005 vs C). In addition, the ratios of anticoagulant PC and PS to factor IX were lower in patients with AIS than in those with CSA (p < 0.05). These results indicate that in patients with acute ischemic cardiac syndromes the markedly increased in vivo thrombin generation is associated with an unbalance between coagulant and anticoagulant vitamin K-dependent factors.

Prevention of postoperative deep vein thrombosis in cancer patients. A randomized trial with low molecular weight heparin (CY 216).

Sixty-one consecutive patients were enrolled in a randomized, controlled trial of thromboprophylaxis with a low molecular weight heparin (Seleparina, CY 216) in major abdominal oncological surgery. Thirty patients received 2 x 3,825 anti-Xa international units of CY 216 subcutaneously on the day of surgery followed by a single daily 3,825 anti-Xa international units injection for 7 days; thirty-one patients did not receive any form of prophylaxis. The occurrence of deep vein thrombosis (DVT) was detected by 125I-labelled fibrinogen leg scan. Postoperative DVT developed in 2 patients in the CY 216 group and in 11 patients in the control group (6.8% vs 35.4%, p < 0.01). Although there was a higher postoperative transfusional requirement in the group receiving CY216 (p < 0.05), the total number of patients transfused was similar in the two groups (14 vs 13). On day 1 after surgery, the two patients who later developed DVT in the CY216 group had plasma anti-Xa activity significantly lower (p < 0.01) than the remaining patients. As a good relationship was found between plasma anti-Xa activity and body weight, adoption of a personalized dosage schedule might improve efficacy of CY 216 prophylaxis.

Total plasma homocysteine: influence of some common physiological variables.

The purpose of this study was to investigate H(e) concentration in plasma from 80 healthy donors in relation to age (6 newborns are also included), sex, daily variation (9, 11 a.m.; 2, 6, 12 p.m.) and a period of 5 subsequent months. A significant correlation (r = 0.63, p < 0.001) was observed between plasma H(e) and age and a statistical difference (p < 0.05) was found between female and male. No circadian rhythm or significant variations over 5 months were found.

Monoclonal antibody-based enzyme-linked immunosorbent assays (ELISA) for the measurement of vitamin K-dependent protein S: the effect of antibody immunoreactivity on plasma protein S antigen determinations.

Two monoclonal antibodies (Mabs) specifically directed to human protein S (PS) - named 5E9E9 and 3B10.25 - were produced and their properties compared to those of 2 previously characterized anti-PS-Mabs (HPS-2 and S10). 3B10.25, similar to S10, was directed to the calcium-free conformation of PS and had virtually identical affinity for free and C4b-binding protein (C4b-BP)-bound PS; 5E9E9 similar to HPS-2, had no calcium-dependency and was selectively directed to free PS. All Mabs were equally reactive to freshly purified and thrombin-cleaved PS. To evaluate the influence of C4b-BP bound PS on PS antigen determinations, ELISA systems employing the four Mabs individually as capture antibody (Ab) and peroxidase-conjugated polyclonal anti-PS IgG as detecting Ab were developed and compared to immunoelectrophoresis (EIA) and to an ELISA employing polyclonal anti-PS IgG as capture and detecting Ab, in the determination of PS in purified systems and in plasma. With all the ELISAs there was parallelism of dilution curves obtained with normal plasma and purified PS; however, supplementation of plasma with purified C4b-BP resulted in loss of parallelism when employing the Mabs directed to free PS as capture Ab. Influence of high C4b-BP on PS antigen determinations was confirmed in a series of plasma samples from patients with C4b-BP levels ranging from 70% to over 200%. Compared to the values obtained with the S10- or 3B10.25 - based ELISAs - which were similar despite a 10-fold difference in sample dilution - plasma PS was underestimated by the ELISAs employing 5E9E9 or HPS-2 while it was overestimated by EIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of clot-detection methods and reagents on activated partial thromboplastin time (APTT). Implications in heparin monitoring by APTT.

Two automatic coagulometers, ACL 810 (Instrumentation Laboratory), a laser-nephelometric centrifugal analyzer, and KoaguLab 40 A (Ortho Diagnostics), an optical automatic coagulometer, were compared with the manual tilt-tube method for the performance of activated partial thromboplastin time (APTT). Seven commercial APTT reagents were used for duplicate determinations in 30 normal controls, 26 patients with liver disease, and 33 patients on full-dose heparin treatment. Clotting times were longer with the manual method than with ACL 810 and, to a lesser extent, with KoaguLab 40 A. Average imprecision of duplicate determinations (coefficient of variation [CV]) was less with ACL 810 (less than 1.5%) than with KoaguLab 40 A (2.9%) and the manual method (2.4%). Differences in slope of the regression curves of clotting times obtained with the coagulometers over the tilt-tube method were observed with all the reagents tested (P less than 0.01). Transformation of clotting times of controls, patients with liver disease, and patients on heparin therapy to APTT ratios did not eliminate the bias resulting from the different reagents (P less than 0.001) and clot-detection methods (P less than 0.001); in controls, significant (P less than 0.001) reagent-method interaction was also observed. The in vitro heparin sensitivity differed with the APTT reagents evaluated and was influenced by the clot-detection method used. Transformation of APTT ratios of anticoagulated patients to apparent plasma heparin levels--as derived from in vitro dose-response curves--effectively eliminated the bias resulting from the different clot-detection methods but had no effect on the bias resulting from the different APTT reagents. In vitro heparin activity curves thus have little, if any, relevance for the ex vivo monitoring of heparin treatment.

Association of lupus anticoagulant with polyarteritis nodosa: report of a case.

A patient with a documented diagnosis of polyarteritis nodosa and laboratory evidence of a circulating lupus anticoagulant is described. Additional clinical features suggestive of the antiphospholipid antibody syndrome were found. The patient underwent amputation of the first two digits of the foot due to ischemic necrosis. Steroid and immunosuppressive treatment resulted in clinical improvement and disappearance of the circulating anticoagulant, without necessitating additional treatment with oral anticoagulants. The presence of the lupus anticoagulant might have worsened the vascular damage done by polyarteritis nodosa in this patient.

Familial protein S deficiency presenting as deep vein thrombosis occurring during pregnancy.

Subjects with congenital deficiency of protein S, a natural anticoagulant which serves as a co-factor for the antithrombotic activity of activated protein C, are at risk of thrombosis. We describe a family in which deep vein thrombosis-occurring during pregnancy in three members with abnormally low protein S activity - was the only manifestation of congenital heterozygous protein S deficiency. Early diagnosis and active treatment of protein S deficiency should be pursued in any woman with a family history of thrombosis, who is planning pregnancy.

Evaluation of coagulometric assays in the assessment of protein C anticoagulant activity; variable sensitivity of commercial APTT reagents to the cofactor effect of protein S.

In vivo expression of protein C activity is dependent on the availability of the activated protein C (APC) cofactor protein S. In the clinical laboratory, measurement of protein C anticoagulant activity is mostly performed in modified APTT assays. We have evaluated 13 commercial APTT reagents for their sensitivity to the cofactor effect of protein S by comparing APC-dependent clotting time prolongations in normal plasma and in protein S depleted plasma. In normal plasma, the sensitivities of the APTT reagents to the anticoagulant effect of APC were markedly different and correlated with the sensitivity of reagents to factor V and VIII. Reagents containing soy phosphatides appeared more sensitive than reagents containing phospholipid of animal origin. Analysis of dose-response curves obtained in normal plasma distinguished one group of reagents showing clotting time prolongations linearly related to the APC concentrations, a second group showing a log-linear relationship and a third group showing a log-log relationship. In protein S depleted plasma, sensitivity of APTT reagents to APC was in general proportional to that observed in normal plasma. However, for some reagents dose-response curves were qualitatively different in normal and in protein S depleted plasma. With all the APTT reagents, APC-dependent clotting time prolongations corresponding to 30-80% of APC anticoagulant activity observed in normal plasma, were observed in protein S depleted plasma. At variance, in a modified Xa one-stage clotting assay, negligible clotting time prolongations were observed in protein S depleted plasma, indicating that over 90% of the APC anticoagulant activity was protein S dependent in this assay system. Dilution of a relative insensitive APTT reagent effectively increased its sensitivity to the cofactor effect of protein S, suggesting that different phospholipid content and/or composition might be responsible for the different sensitivity of APTT reagents to protein S. These results question the validity of APTT based assays for the identification of qualitative protein C abnormalities with defective interaction with protein S.

Comparison of two automated coagulometers and the manual tilt-tube method for the determination of prothrombin time.

Two automatic coagulometers--ACL 810 (Instrumentation Laboratory), a laser-nephelometric centrifugal analyzer, and KoaguLab 40 A (Ortho Diagnostics), an optical automatic coagulometer--were compared with the manual tilt-tube method for the performance of prothrombin time (PT). Seven ISI- (International Sensitivity Index) calibrated commercial thromboplastin reagents were used for duplicate determinations in 30 normal subjects, 30 patients with liver disease, and 30 patients receiving stabilized oral anticoagulation. Clotting times were longer with the manual method than with ACL 810 and, to a lesser extent, with KoaguLab 40 A. Average imprecision of duplicate determinations (CV) was less than 1% with ACL 810; KoaguLab 40 A and the manual method had similarly higher imprecisions (2.8% and 2.7%). Differences in origin and slope of the regression curves of clotting times obtained with the coagulometers over the tilt-tube method were observed with all the reagents tested. Transformation of clotting times to PT ratios did not eliminate the bias resulting from the different clot-detection methods. A higher percentage of patients with liver disease had abnormal PT ratios when their plasma was tested with the coagulometers than with the manual method. Transformation of PT ratios to International Normalized Ratios effectively eliminated the bias resulting from the different thromboplastin reagents but had no effect on the bias resulting from the different clot-detection methods. A significant proportion of patients appeared excessively anticoagulated (INR greater than 4.5) with the coagulometers but not with the manual method. These results highlight the need for standardization of both instrumentations and reagents to improve monitoring of oral anticoagulant treatment.

Protein S is a cofactor for activated protein C neutralization of an inhibitor of plasminogen activation released from platelets.

Platelets stimulated with thrombin release an inhibitor of plasminogen activator (PAI), which has been shown previously to be neutralized by activated protein C (APC). The requirements for optimal neutralization of PAI activity were investigated. The releasate of gel-filtered human platelets stimulated with thrombin served as a source of PAI. When 6 X 10(8) platelets/mL were incubated with thrombin (1 IU/mL), the releasate contained 18 to 26 ng/mL PAI as determined by incubation of the releasate with urokinase and measurement of residual urokinase activity on plasminogen (S2251). Preincubation of PAI with up to 4 micrograms/mL APC for two hours yielded less than 20% neutralization of PAI activity. In the presence of protein S, phospholipid, and Ca2+, neutralization of PAI activity was time-dependent with 50% neutralization occurring in two hours with 1 microgram/mL APC. The cofactor effects of protein S and phospholipid were concentration-dependent with half-maximal acceleration at approximately 3 micrograms/mL protein S and 10 micrograms/mL phospholipid when the experiments were performed at 1 microgram/mL APC. Diisopropylfluorophosphate-inactivated APC, gla-domainless APC, and thrombin-cleaved protein S had no effect on PAI activity, indicating requirement for preservation of the APC active site and of the Ca2+ binding ability of both APC and protein S. These results suggest coordinate binding of APC and protein S onto phospholipid membrane as a prerequisite for optimal expression of PAI neutralized by APC.