DIAGNOSIS STRATEGIES IN ACTIVATED PROTEIN-C RESISTANCE - IS GENOTYPING STILL NECESSARY

Resistance to activated protein C (APC) is due, in most cases, to a G to A mutation at nucleotide 1691 of factor V (FV) gene (the Leiden mut ation). This inherited abnormality is now considered to be the major h ereditary cause associated with an elevated risk of thrombosis. For th is reason, laboratories are faced with an increasing number of samples referred for APC resistance diagnosis. This could have serious econom ic consequences and a comprehensive laboratory screening strategy for APC resistance is necessary. An original DNA assay based on denaturing gradient gel electrophoresis (DGGE) was designed in our laboratory. D uring a first period we systematically performed DNA analysis and comp ared the results with phenotypic assays. Using the modified functional test with a 1:5 predilution of plasmas, the cut-off value for APC res istance ratio was 2.6 in our sample. Among 94 consecutive patients ref erred to our laboratory we found a clear cut-off between the APC resis tance ratio obtained for normal and abnormal individuals. The modified test had a predictive value of 1.0 found by a cut-off less than or eq ual to 2.6 for the heterozygote FV Leiden. This obviates the necessity of genotyping subjects with a normal phenotype. Among patients with a n abnormal phenotype we were able to fully discriminate between homozy gous and heterozygous patients using a cut-off value of 1.5. Neverthel ess, our results demonstrate that, because of false-positive results s uch as lupus anticoagulant, genotyping is still indicated for patients with an abnormal ratio determined with the modified APC resistance te st. The strategy described here allows us to safely lower the number o f samples analysed by DGGE.