Development of a new modified Bethesda method for coagulation inhibitors: the Osaka modified Bethesda method.

The Nijmegen assay for the factor VIII (F-VIII) inhibitor is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee. However, due to cumbersome and complicated preprocessing, it is presently difficult to introduce this assay into hospital laboratories. We used buffered plasma that was made by addition of 1 volume of 1 mol/l HEPES buffer at pH 7.35 to 9 volumes of plasma to form the test samples. The inhibitor titer was calculated by the remaining rate of F-VIII coagulation activity (F-VIII:C), using the ratio of actual value to the theoretical value. Five hundred microliters of the buffered test plasma and the control (30 mmol/l HEPES buffered saline at pH 7.35) were each mixed with equal volumes (500 µl) of normal pooled plasma in a test tube (11 mm internal diameter and 6.5 ml volume capacity), and incubated at 37°C for 2 h. In our modified Bethesda method, there were no significant changes in pH and F-VIII:C of control and test mixtures after incubation tests for stability. With the modified method, the inhibitor titers (mean, SD) from examining three hemophilia A plasma samples (F-VIII:C, <1-3%) and 40 normal samples (F-VIII:C, 34.5-168.3%) were 0.032, 0.057 and -0.009, 0.057, respectively. By our method, the F-VIII inhibitor titer of type I inhibitor-positive samples was higher than the Nijmegen method, and for type II inhibitor-positive samples, the titer was similar. We believe that our method can be applied to not only the type I inhibitor, but also to assays of type II inhibitor, without cumbersome and complicated preprocessing.

Low level of factor V is associated with development of deep-vein thrombosis in Japanese patients.

BACKGROUND: Factor V, having two functions (procoagulant and anticoagulant), is a key factor in blood coagulation, and low plasma levels of factor V may be a risk factor for thrombosis. OBJECTIVE: The levels of plasma factor V antigen (FV:Ag), and the phospholipid binding capability of Factor V (FV:PL-bound) were evaluated in patients with deep-vein thrombosis (DVT). METHODS: Levels of FV:Ag, and FV:PL-bound were expressed as a percentage of the normal level found in pooled plasma from control subjects. One hundred and twenty-three Japanese patients with deep-vein thrombosis (DVT) were included, with 100 age and sex-matched healthy control subjects. RESULTS: The FV:Ag, and FV:PL-bound values were significantly lower in DVT patients than in healthy subjects (p<0.05 and p<0.005, respectively). Among the 123 patients, 30 for FV:Ag (24.4%), and 32 for FV:PL (26%) had less than the arbitrary cutoff point (set at the 5th percentile of the value for FV:Ag and FV:PL-bound from healthy subjects), and the odds ratios (ORs) were 6.1 (95% confidence interval [CI], 2.3-16.5) and 6.7 (95%CI, 2.5-17.9), respectively. When patients with a deficiency of natural anticoagulants (antithrombin, protein C, and protein S) were excluded from the analysis, the ORs increased for all patients (6.6 for FV:Ag (95%CI, 2.4-18.3) and 7.4 for FV:PL-bound (95%CI, 2.7-20.3). Moreover, twenty-one (17%) of the 123 DVT patients, and 1 (1%) of 100 control subjects had values below the cutoff points for both FV:Ag and FV:PL-bound, and the OR was 21.6 (95%CI, 2.85-163.1). CONCLUSIONS: These results suggest that low levels of factor V are associated with development of DVT, and may be a predictor for DVT.

Frequencies of mild factor V, VII and X deficiencies in a Japanese population.

We investigated the frequencies of coagulation factor deficiencies in a Japanese population. We measured factor II, V, VII and X activity in 100 healthy individuals. A specific factor deficiency was determined according to the factor activity and the ratio of the factor activity to that of other coagulation factors. Seven samples showed factor activity less than the mean -2SD of standardized factor activity (factor II: three; factor V: one; factor VII: one; factor X: one and factor V+factor VII: one). The samples with low factor II and factor VII activity were determined not to be due to deficiency because the ratios of these factor activities to other factor activities were within the range of the mean +/- 2SD. We measured activity ratios in the remaining 97 samples and identified one sample with factor V deficiency and two with factor VII deficiency. Thus, six samples with coagulation factor deficiency were identified (factor X: one; factor V: two; factor VII: two and factor V + factor VII: one). These results suggest that the Japanese population has relatively high frequencies of mild factor V, factor VII and factor X deficiencies, in which activity is reduced to approximately 50% (36-64%) of normal plasma.

Impaired binding of thrombin to thrombomodulin is associated with risk of deep vein thrombosis.

The complex of thrombin and thrombomodulin (TM) activates protein C, and impaired binding of thrombin to TM may be a risk factor for thrombosis. In this study, we evaluated the reactivity of thrombin to TM by determining the TM-bound thrombin (TMBTh) to total thrombin generation (t-Th) ratio (TMBT ratio). We also examined whether a decreased TMBT ratio is associated with increased risk of thrombosis. TMBTh was measured on TM-coated plates. Thrombin was generated by addition of prothrombin time reagent to plasma. Levels of t-Th and TMBTh were expressed as percentages of the levels in pooled normal plasma. The study included 124 patients with deep vein thrombosis and 150 age- and sex-matched healthy subjects. The TMBT ratio (TMBTh/t-Th) was significantly lower in patients than in control subjects (p<0.05). Among the 124 patients, 43 (34.7%) showed TMBT ratios below the 5th percentile value of control subjects, and the odds ratio (OR) for development of deep vein thrombosis was 9.4 (95% confidence interval [CI], 4.6-19.1). When patients with a deficiency of natural anticoagulant (antithrombin III, protein C, or protein S) were excluded from analysis, the TMBT ratio in 37 (42.5%) of the remaining 87 patients was below this cutoff point, and the OR (13.1; 95% CI, 6.4-26.9) was increased compared to that in the total patient group. These results suggest that it is possible to evaluate the reactivity of thrombin to TM by determining the TMBT ratio, and this ratio may be a predictor of deep vein thrombosis.