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,3, 2
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 Va1
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 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
Requires a normal baseline APTT
There is considerable overlap between healthy individuals and heterozygotes
Low pro tein S will also skew the ratio5
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 is independent of these confounding factors & specific for factor V leiden mutation.3, 1
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.
Test Procedure (Summary) – Practical Hemostasis
Table 1
2.3. Test Procedure (Summary) – Practical Hemostasis
Results
Present study includes a total of 150 APCR positive cases chosen retrospectively.
Of these, 100 cases are of FVleiden mutation confirmed by RT -PCR
50 APCR positive cases (APTT/ modified APTT based) with history of venous thrombosis & non carrier of FV ladeinmutation.
20 LAC cases confirmed by dilute Russell viper venom test DRVVT
20 cases on warfarin 10 cases on OCPs
Table 2
Sensitivity of Screening APTT based APCR
Overall Sensitivity of Screening APTT based APCR test for detection of carrier FV Leiden mutation is 78%, for Heterozygous state sensitivity is 77% & for Homozygous state it is 80%. Sensitivity of Screening APTT based APCR test for detection of non carrier FVleiden cases like LAC, warfarin & OCPs therapy is 82% (Table 2)
Table 3
Sensitivity of Modified APTT based APCR
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.9, 8
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).10, 8
Overall Sensitivity of Screening APTT based APCR test for detection of FV Leiden mutation is 78%, for heterozygous state sensitivity is 77% & for homozygous state it is 80%.
Sensitivity of Screening APTT based APCR test for de tection of non carrier FV leiden cases like LAC, DOACs & OCPs therapy is 82%.
Studies by Elizabeth et al also concludes that APC-R assays that dilute patient plasma into factor V-deficient plasmaare much more accurate for detecting FV Leiden than other assays.
APC-R assays are advantageous as they are easily automated, cost effective and may detect rare causes of APC resistance other than Factor VLeiden.4
Table 4
| Test | Mayo et al9 | Optum lab database9 | Present study |
| APCR | 1256 | 5395 | 150 |
| FV Leiden mutation | 268 | 78525 | 100 |
| APCR / FV Leiden | 1 : 0.2 | 1:15 | 1.5 : 1 |
Comparative Analysis of various studies in FV Leiden mutation
Overall Sensitivity of Modified APTT with predilution in FV-deficient plasma (1:4), based APCR test for detection of F V Leiden mutation is 93%, for Heterozygous state sensitivity is 94% & for Homozygous state it is 92%.Sensitivity of Screening APTT based APCR test for detection of non carrier FV ladein cases like LAC, warfarin & OCPs therapy is 34%.
These results shows that 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.(Table 3)
However in contrast to Modified test, Screening APTT is increased in APCR due to factor V Leiden mutation as well as acquired causes like patients on DOACs, LAC & OCPs which are independent risk factors of venous thrombosis.
Discussion
APCR is a hemostatic disorder characterized by increased risk of venous thrombosis, including deep vein thrombosis and pulmonary embolism. Factor V Leiden mutation accounts for 95% of APCR cases & the remainder are due to acquired causes like intake of warfarin,OCPs, DOACs & LAC.1
APCR can be tested by
APTT based screening test
Modified APTT with predilution in FV-deficient plasma
DRVVT based
Chromogenic assay
The discrepancy in APCR/FV Leiden ratio in Mayo et al and present study is because the former study was conducted on a large population and duration of study was longer
Devreese et al showed the effect of DOACs like Dabigatran, Rivaroxaban, Apixaban on APCR tests & the results are in correlation with present studies
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 shows similar results.
APTT based Screening test For APCR
If Normalized ratio = < 1.7
Conclusion
Screening APTT based APC-R test is increased in Factor V Leiden mutation & in LAC, OCPs & DOAC intake.
Modified APTT with predilution in FV-deficient plasma (1:4) is more sensitive than Screening APTT test in diagnosis of Factor V Leiden mutation & can distinguish homozygous & heterozygous states from normal individuals.
Abbreviations
DOACs - direct acting oral anticoagulants, LAC - lupus anticoagulant, OCPs -oral contraceptive pills,
FVLeiden - Factor V Leiden, APCR - Activated protein C resistance. DOACs - direct acting oral anticoagulants, LAC - lupus anticoagulant, OCPs -oral contraceptive pills,
OCPs -oral contraceptive pills
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