Comparative Analysis of “ Screening vs Modified ” APTT based activated protein C resistance (APCR) assay in the diagnosis of Factor V leiden mutation

Introduction: APCR is a hemostatic disorder characterized by increased risk of deep vein thrombosis and pulmonary embolism. Factor V Leiden mutation accounts for 95% of APCR cases & the remainder are due to acquired causes like patients on vitamin k antagonist therapy, direct oral acting anticoagulants, Lupus anticoagulant & oral contraceptive pills. T he main objective of this study is to compare the sensitivity of APTT based APCR test vs Modified APTT with pre -dilution in F actor V-deficient plasma for diagnosis of Factor V leiden mutation & to formulate a systematic diagnostic algorithm for interpretation of APCR tests. Materials and Methods: The Coagulometer used for APCR test is Sysmex CS-5100. APTT reagent used is Pathrombin SL supplied by seimens. All data were expressed as Mean ± SD. Statistical analysis was done using unpaired students t test & a P value <0.05 is considered as statistical significance. Results : A total of 150 cases of APCR (100 cases of factor V Leiden mutation confirmed by PCR & 50 non carrier /acquired cases) were studied retrospectively. Sensitivity of screening APTT base d APCR for detection of factor V Leiden mutation is 78% & for non carrier state it is 82%. Sensitivity of modified APTT with predilution in FV-deficient plasmafor detection of factor V Leiden mutation is 93% & for noncarrier state (acquired) is 34%. Conclusion: Screening APTT test is increased in Activated protein C resistance due to factor V Leiden mutation as well as acquired causes like patients on direct acting oral anticoagulants, warfarin, lupus anticoagulants and oral contraceptive pills which are independent risk factors of venous thrombosis. Modified APTT with predilution in FV-deficient plasma (1:4) is more sensitive than Screening APTT based APCR test in diagnosis of Factor VLeiden mutation & this test can distinguish homozygous & heterozygous states from normal individuals. © 2020 Published by Innovative Publication. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by/4.0/)


Introduction
Activated protein C resistance (APCR) is a hemostatic disorder characterized by increased risk of venous thrombosis, including deep vein thrombosis and pulmonary embolism. APCR can be 1.
Hereditary -Factor V Leiden mutation in approximately. 95% of cases 2. Acquired -Vit K antagonists, DOACs, LAC, increased FVIII (pregnancy) 1 In physiological conditions Activated Protein C degrades Factor Va and VIIIa. This leads to inhibition of Coagulation cascade and prolongs APTT In APC-R -no degradation of factor V -increases coagulation -APTT is not prolonged, 2,3 APCR was first reported in 1995 i n approximately 95% of cases due to the Factor V Leiden [FVL] mutation -a G1691a missense mutation at Arginine 506 resulting in its replacement by a glutamine [R506Q] ( Figure 1) and the abolition of an APC inactivation cleavage site in Factor Va 1 The incidence of factor V Leiden mutation in patients with venous thrombosis is approximately 20-40% & it is the mos t common hereditary cause of increased risk of https://doi.org/10.18231/j.jdpo.2020.010 2581-3714/© 2020 Innovative Publication, All rights reserved. 48 Fig. 1: Missense mutation venous thrombosis (3-7% of Caucasian). Patients who are heterozygous for factor V Leiden mutation are 5 to 8 times increased risk of venous thrombosis as compared to general population but only 10% of these develop thrombosis during their lifetime .Individuals who are homozygous have a 30-140-fold risk. Following venous thrombosis, they have a higher risk of re-thrombosis than individuals with DVT but normal factor V. 4 APTT based screening test for APCR test is sensitive for factor V Leiden mutation but it has certain limitations 1. Requires a normal baseline APTT 2. There is considerable overlap between healthy individuals and heterozygotes 3. Low pro tein S will also skew the ratio 5 Screening APTT test is increased in Activated protein C resistance due to factor V Leiden mutation as well as acquired causes like patients on direct acting oral anticoagulants, warfarin, lupus anticoagulants and oral contraceptive pills which are independent risk factors of venous thrombosis. Modified APTT with predilution in FVdeficient plasma is independent of these confounding factors & specific for factor V leiden mutation. 1,3 The main objective of this study is to compare the sensitivity of APTT based APCR test Vs Modified APTT with predilution in FV-deficient plasma in diagnosis of factor V Leiden mutation & to formulate a systematic diagnostic algorithm for interpretation of APCR tests

Materials and Methods
This is a Retrospective study of 1 year duration (from July 2018 to June 2019) carried out in a tertiary care hospital, medical college & research centre. The Coagulometer used for APCR test is Sysmex CS-5100 with Pathrombin SL APTT reagent supplied by seimens. All data were expressed as Mean ± SD. Statistical analysis was done using unpaired students t -test & a P Value<0.05 is considered as statistical significance.

If the APCR normalized ratio is <2 then What test next?
Individuals with a low APC ratio should confirm F5 gene for the factor V Leiden mutation by PCR. However, it should be remembered that although most cases of APC resistance are due to the factor V Leiden mutation, testing with the original APTT-based APC resistance assay may be useful in detecting independent risk factors for venous thrombosis including pregnancy, oral contraceptive, LAC. 8,9 The Gold standard for diagnosis of F actor V Leiden mutation is by PCR technique but this is not cost effective. The cost evaluated per test is $36.38 for Modified APCR test and $83.77 for RT-PCR(Mayo special coagulation lab). 8,10 Overall Sensitivity of Screening APTT based APCR test for detection of FV Leiden mutation is 78%, for This is a modification of the original APTT screening test in which a pre-dilution [1 : 4] of patient plasma with factor V-deficient plasma is made before the addition of APC and calcium. The modified assay reduces the number of exogenous confounding factors that might affect the APTT e.g. high FVIII levels and makes the test specific for mutations within FV. However, the presence of lupus anticoagulants, by competing for phospholipid, can prolong PTT measurements in these assays, and are a major source of false-positive results if the test is used as a screening test for FVL. {It is important to remember that this modified assay is specific only for mutations within FV whereas the original APTT assay without factor V-deficient plasma pre-dilution, measures APC resistance from any cause.}   Results of present study suggests that Modified APTT with pre-dilution in Factor V-deficient plasma is more sensitive than screening APTT based test for diagnosis of Factor V leiden mutation. Studies conducted by Stephan et al, 4 Taylor & Fristma et al, 8 Juliana et al, 9 Pruller et al, 10

Conflict of interest
None.