A modification of the APC resistance test and its application to the study of patients on coumarin therapy.

APC resistance appears to be caused, predominantly, by a mutation in coagulation factor V (nucleotide 1691: G to A). This phenomenon is usually studied by performing APTTs in the absence and presence of added APC. We studied a modification of the assay involving dilution of the test plasma in factor V deficient plasma, to render the assay more factor V specific. This modification was applied to 76 patients with venous thrombosis on coumarin treatment and to 45 controls. Two out of 45 controls (4.4%) showed abnormal results with the modified test. They also showed loss of factor V exon 10 Mnl I restriction site, associated to APC resistance. All remaining controls, with normal functional results by the modified assay, showed normal restriction profile. We detected 9 affected patients (11.8%), one of them homozygous or double heterozygous. In conclusion, the modified assay is very sensitive for factor V dependent APC resistance, and can successfully be applied to patients on coumarin therapy.

[ELISA method for the determination of factor VII antigen]. / Método ELISA para la determinación del antígeno del factor VII.

The low plasma concentration of clotting factor VII makes it difficult to assay its antigenic fraction by the conventional methods of precipitation with specific antigens. Simple and peroxidase-conjugated antisera are currently available from commercial sources, thus allowing one to determine F VII:Ag by enzyme immunoassay. An ELISA method has been developed in this laboratory which provides sensitivity limits about 0.1% of the plasma concentration of F VII and correlates significantly with its functional activity (r = 0.603, n = 44, p less than 0.001). This technique can be highly helpful in characterising molecular variants of F VII, as well as in detecting acquired deficiencies of this factor.

[ELISA method using microplates developed with commercial antisera for measuring von Willebrand factor antigen]. / Método ELISA en microplaca desarrollado con antisueros comerciales para medir el antígeno del factor von Willebrand.

The commercial availability of free and peroxidase-conjugated anti-FvW IgG fractions makes it possible to use ELISA methods for FvW: Ag determination in the routine blood coagulation laboratory. A highly sensitive microplate ELISA method was developed in our laboratory (capable of detecting 0.02% FvW: Ag), which proved to be reproducible, quick and not expensive. No significant differences were found between the results attained on different days in 4 control samples (variance analysis), variation coefficient about 12% being observed even for samples with only 11% FvW: Ag. The standard and control sample curves were parallel, which discarded a dilution effect in the assay. The correlation with Laurell's method was r = 0.889 (n = 34, p less than 0.001) and with ristocetin cofactor it was r = 0.677 (n = 19, p less than 0.005).