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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 63  |  Issue : 2  |  Page : 247-250
Comparative analysis of “APTT vs RVVT” based activated protein C resistance assay in the diagnosis of Factor V Leiden mutation


Department of Pathology, BLDE's Shri B.M. Patil Medical College Hospital and Research Centre, Bijapur, Karnataka, India

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Date of Web Publication18-Apr-2020
 

   Abstract 


Background: Thrombophilia is a hypercoagulable state characterized by increased venous thrombosis. The most common cause of heritable thrombophilia is Factor V Leiden (FVR506Q) homozygous state, with a relative risk of 10–80 times as compared to normal individuals and Lupus anticoagulant is the most common cause of acquired thrombophilia. The main objective of this study is to compare the sensitivity of activated partial thromboplastin time (APTT) vs dilute Russell viper venom test (DRVVT) based APCR assays with predilution in Factor V-deficient plasma for diagnosis of Factor V Leiden mutation. Materials And Methods: The coagulometer used for APCR test was Sysmex CS-5100. APTT reagent used is Pathrombin SL supplied by Siemens. All data were expressed as mean ± SD. Statistical analysis was done using unpaired students t-test and a P value <0.05 was considered as statistical significance. Results: A total of 300 cases of APCR (200 cases of Factor V Leiden mutation was confirmed by PCR and 100 acquired) were studied. The sensitivity of screening APTT-based APCR for detection of Factor V Leiden mutation is 67% and for the noncarrier state, it is 62%. The sensitivity of modified APTT and DRVVT with predilution in FV-deficient plasma for detection of Factor V Leiden mutation is 82% and 84%, respectively and for acquired causes, it is 48% and 86%, respectivelyConclusion: Screening APTT test has increased in activated protein C resistance (APCR) due to Factor V Leiden mutation as well as acquired causes such as patients on direct-acting oral anticoagulants, warfarin, lupus anticoagulants, and oral contraceptive pills which are independent risk factors of venous thrombosis. Modified DRVVT with predilution in FV-deficient plasma is more sensitive than screening and modified APTT-based APCR test in the diagnosis of Factor V Leiden mutation and the former test can distinguish homozygous and heterozygous states from normal individuals.

Keywords: Activated protein C resistance, activated partial thromboplastin time, Factor V Leiden mutation, lupus anticoagulant

How to cite this article:
Baig MA. Comparative analysis of “APTT vs RVVT” based activated protein C resistance assay in the diagnosis of Factor V Leiden mutation. Indian J Pathol Microbiol 2020;63:247-50

How to cite this URL:
Baig MA. Comparative analysis of “APTT vs RVVT” based activated protein C resistance assay in the diagnosis of Factor V Leiden mutation. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Aug 10];63:247-50. Available from: http://www.ijpmonline.org/text.asp?2020/63/2/247/282720





   Introduction Top


Activated protein C resistance (APCR) is a hemostatic disorder associated with increased risk of deep venous thrombosis and pulmonary embolism. Factor V Leiden mutation accounts for 95% of cases of APCR and remainders 5% are acquired causes such as intake of Vitamin K antagonists, oral contraceptive pills, direct oral acting anticoagulants (DOACs), lupus anticoagulant (LAC,) and conditions associated with an increase in Factor VIII like pregnancy.[1]

Protein C Proteolysis APC -- degrades FVa and FVIIIa - Inhibits coagulation-- prolongs APTT.

In APC-R – no degradation of Factor V – increases coagulation – APTT is not prolonged,[2],[3]

APCR was first reported in 1995 in approximately 95% of cases due to the Factor V Leiden [Figure 1].
Figure 1: Missense Mutation.[2],[3] [FVL] mutation – a G1691a missense mutation at Arginine 506 resulting in its replacement by a glutamine [R506Q] and the abolition of an APC inactivation cleavage site in Factor Va[1]

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The incidence of Factor V Leiden mutation in patients with venous thrombosis is approximately 20–40% and it is the most common hereditary cause of the increased risk of venous thrombosis (3–7% of Caucasian). Patients who are heterozygous for Factor V Leiden mutation have 5 to 8 times increased risk of venous thrombosis as compared to the 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 rethrombosis than individuals with DVT but normal Factor V.[4]

The first generation assay for screening APCR was initially described by Dahlback (1995) and is based on APTT assay and its prolongation by addition of APC. Second generation tests are modified APTT and modified DRVVT-based APCR assays for differentiating normal individuals from homozygous and heterozygous state of Factor V Leiden mutation.[1]

APTT based screening test for APCR 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 protein 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 such as 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 and specific for factor V Leiden mutation.[1],[3] Modified DRVVT with predilution in FV-deficient plasma offers extra-advantage in detecting APCR due to LAC cases.

The main objective of this study is to compare the sensitivity of Screening and modified APTT vs RVVT based APCR test for diagnosis of Factor V Leiden mutation.


   Materials and Methods Top


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



Inclusion criteria

  1. APCR positive homozygous and heterozygous Factor V Leiden mutation cases
  2. Acquired APCR causes such as OCPs, DOACs, and LAC).


Exclusion criteria

  1. Anti-thrombin III Deficiency
  2. Protein S and C deficiency
  3. Nephrotic syndrome
  4. Pregnancy.



Test Procedure (Summary) – Practical Hemostasis

Factor V Leiden (FVR506Q) mutation was detected by Real-time-polymerase chain reaction (RT-PCR). PCR technique amplify genomic DNA followed by identification of presence or absence of the missense mutation e.g., restriction enzyme digestion and mapping. Homozygous and heterozygous state of Factor V Leiden mutation were determined. All steps of PCR like amplification, heating were employed.


   Results Top


The present study includes a total of 300 APCR positive cases Of these, 200 cases are of FV Leiden mutation confirmed by PT-PCR 100 APCR positive cases (APTT/modified APTT based) with history of venous thrombosis and noncarrier of FV Leiden mutation.

  • 70 LAC + Cases confirmed dilute Russell viper venom test (DRVVT)
  • 20 cases on warfarin
  • 10 cases on OCPs.


Modified DRVVT with predilution in FV-deficient plasma (1:4) is more sensitive than Screening and modified APTT-based APCR test in the diagnosis of Factor V Leiden mutation and the former test can distinguish homozygous and heterozygous states from normal individuals [Table 3].
Table 3: Sensitivity of Modified DRVVT-based APCR test

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However, in contrast to modified test, screening APTT is increased in APCR due to Factor V Leiden mutation as well as acquired causes such as patients on DOACs, LAC, and OCPs which are independent risk factors of venous thrombosis.


   Discussion Top


APCR is a hemostatic disorder characterized by an increased risk of venous thrombosis, including deep vein thrombosis and pulmonary embolism. FV Leiden mutation accounts for 95% of APCR cases and the remainder are acquired like Vitamin K antagonists, DOACs, LAC, and OCPs.[1]

APCR can be tested by

  1. APTT based screening test
  2. Modified APTT with predilution in FV-deficient plasma
  3. DRVVT based
  4. Chromogenic assay.


In this study, 3 types of assays were performed

  1. Routine APTT test
  2. Modified APTT
  3. Modified DRVVT test.


The Gold standard for diagnosis of Factor 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],[9],[10]

Overall Sensitivity of Screening APTT-based APCR test for detection of FV Leiden mutation is 67%, for heterozygous state sensitivity is 69%, and for the homozygous state, it is 64%. The sensitivity of screening APTT-based APCR test for the detection of acquired causes such as LAC, DOACs, and OCPs therapy is 62% [Table 1].
Table 1: Sensitivity of Screening APTT-based APCR

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Studies by Elizabeth et al.[11] also concludes that APC-R assays that dilute patient plasma into Factor V-deficient plasma are 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 V Leiden[4] [Table 4].
Table 4: Comparative Analysis of various studies in FV Leiden mutation

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Devreese et al. showed the effect of DOACs such as Dabigatran, Rivaroxaban, and Apixaban on APCR tests and the results are in correlation with present studies.

Results of the present study suggest that modified APTT with predilution 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 and Fristma et al.,[8] Juliana et al.,[9] and Pruller et al.[10] shows similar results.

The overall sensitivity of modified APTT with predilution in FV-deficient plasma (1:4), based APCR test for detection of FV Leiden mutation is 82%, for heterozygous state sensitivity is 84% and for the homozygous state is 77%. The sensitivity of modified APTT-based APCR test for the detection of acquired causes such as LAC, warfarin, and OCPs therapy is 48% [Table 2].
Table 2: Sensitivity of Modified APTT-based APCR

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In the present study overall sensitivity of modified DRVVT with predilution in FV-deficient plasma (1:4), based APCR test for detection of FV Leiden mutation is 84%, for heterozygous state sensitivity is 86%, and for Homozygous state, it is 80%. The sensitivity of modified DRVVT-based APCR test for detection of acquired causes like LAC, warfarin, and OCPs therapy is 86%.

In a similar study conducted by Aboud MR et al.[11] both modified assays (APTT and DRVVT) demonstrated a sensitivity and specificity of 100% for FV Leiden, thrombophilic patients and patients on oral anticoagulants, with the modified RVVT-based assay giving better separation between normals and FV Leiden. Inhibition of phospholipid-dependent coagulation by LAC antibodies rendered the APTT-based system less suitable than the phospholipid-rich RVVT-based one, and as nine of the 20 LAC-positive patients were on warfarin, showed only the modified RVVT assay to be a reliable predictor of factor V Leiden in this patient group.[11]

The present study showed that Modified DRVVT is more sensitive than screening and modified APTT-based APCR assay in the diagnosis of Factor V Leiden mutation patients presenting with features of venous thrombosis and pulmonary embolism as well as in asymptomatic individuals. Modified DRVVT offers extra-advantage of identifying LAC cases (sensitivity = 90%), patients on warfarin and is independent of factor VIII, IX, XI, and XII levels.


   Conclusion Top


Screening APTT detects APC-R due to Factor V Leiden mutation and acquired causes like LAC cases, patients on OCPs and DOAC cases, presenting with features of venous thrombosis. Modified APTT with predilution in FV-deficient plasma is independent of these confounding factors and specific for Factor V Leiden mutation.

Modified DRVVT is more sensitive than screening and modified APTT based APCR assay in the diagnosis of Factor V Leiden mutation and offers extra-advantage of identifying LAC cases and can distinguish normal cases from homozygous and heterozygous state of Factor V Leiden mutation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Dahlback B. Inherited deficiency of Activated protein C, A major cause of venous thrombosis is due to mutation in factor V gene. Hemostasis 1994;24:139-51.  Back to cited text no. 1
    
2.
Mohammed S, Favaloro EJ. Laboratory testing for activated protein C resistance (APCR). Methods Mol Biol 2017;1646:137-43.  Back to cited text no. 2
    
3.
Jorquera HI, Fernandez MA. Modified test for APCR. Lancet 1994;344:1162-3.  Back to cited text no. 3
    
4.
Kadauke S, Khor B, Van Cott EM. Activated protein C resistance testing for factor V Leiden. Am J Hematol 2014;89:1147-50.  Back to cited text no. 4
    
5.
Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance). Blood 1995;85:1504-8.  Back to cited text no. 5
    
6.
Ronde H, Bertina RM. Laboratory diagnosis of APC-resistance: A critical evaluation of the test and the development of diagnostic criteria. Thromb Haemost 1994;72:880-6.  Back to cited text no. 6
    
7.
Brandt G, Gruppo R, Glueck CJ, Stroop D, Becker A, Pillow A, et al. Sensitivity, specificity and predictive value of modified assays for activated protein C resistance in children. Thromb Haemost 1998;79:567-70.  Back to cited text no. 7
    
8.
Taylor LJ, Oster RA, Fritsma GA, Tichenor PH, Reed CE, Eiland BM, et al. Screening with the activated protein C resistance assay yields significant savings in a patient population with low prevalence of factor V Leiden. Am J Clin Pathol 2008;129:494-9.  Back to cited text no. 8
    
9.
Perez Botero J, Majerus JA, Strege AK, Johnson RD, Chen D, Pruthi RK. Diagnostic testing approaches for APCR and factor V Leiden. A comparison of Institutional and national provider practices. Am J Clin Pathol 2017;147:604-10.  Back to cited text no. 9
    
10.
Prüller F, Weiss EC, Raggam RB, Cervar-Zivkovic M, Renner W, Wagner J, et al. Activated protein C resistance assay and factor V Leiden. N Engl J Med 2014;371:685-6.  Back to cited text no. 10
    
11.
Aboud MR, Ma DD. A comparison between two activated protein C resistance methods as routine diagnostic tests for factor V Leiden mutation. Br J Haematol 1997;97:798-803.  Back to cited text no. 11
    

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Correspondence Address:
Mirza Asif Baig
Former Asstt. Prof. SBMPMC, Bijapur, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_899_19

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