Limitations of a calibrated, quantitative APC-R assay under routine conditions

Introduction The aim of this study was to evaluate the Hemoclot Quanti. V-L assay in various clinical conditions. Methods We compared the Hemoclot Quanti.V-L assay with DNA testing and with the Pefakit assay in 60 normal (no mutation) vs carriers of the factor V (FV) Leiden mutation (56 heterozygous and three homozygous). We further investigated the interference of lupus anticoagulant on test results in normal and heterozygous individuals and of direct oral anticoagulants (DOACs) at trough and peak levels. Additionally, DOAC-Remove was tested in samples containing DOACs at peak levels. We further evaluated the influence of FV deficiency on this quantitative assay. Results There was a 100% agreement between the Quant. V-L assay and DNA testing in 60 normal individuals. However, 1.85% of heterozygous and 33% of homozygous samples were falsely classified with the quantitative assay, and no misclassification was observed with the Pefakit assay. Lupus anticoagulant did not influence the test results of the quantitative assay. DOACs also interfered with test results in heterozygous patients, but this effect was prevented with the DOAC-Remove procedure. Even mild FV deficiency affected the test results of the quantitative assay in heterozygous patients leading either to misclassification or the need for subsequent PCR testing. Conclusion The quantitative FV-L assay has several limitations, especially FV deficiency and the presence of DOACs have to be ruled out before running this quantitative assay.