A review of laboratory considerations in thrombophilia testing

Published:October 11, 2022DOI:


      Venous thromboembolism is a multifactorial disease with interacting genetic and acquired predisposing factors. Thrombophilia screening is utilised in specific individuals when the test result is likely to influence management decisions, rather than universal screening in all patients with thrombosis. When thrombophilia testing is undertaken, the results must be considered in the context of pre-analytical, analytical and post-analytical variables to minimise misinterpretation. Clinical indications for thrombophilia testing have been covered elsewhere, and the focus of this review will be the laboratory considerations in thrombophilia testing, highlighting potential interferences when investigating for factor V Leiden, prothrombin gene mutation, protein C deficiency, protein S deficiency, antithrombin deficiency and antiphospholipid antibodies.

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        • Connors J.M.
        Thrombophilia testing and venous thrombosis.
        N Engl J Med. 2017; 377: 1177-1187
        • Baglin T.
        • Gray E.
        • Greaves M.
        • et al.
        Clinical guidelines for testing for heritable thrombophilia.
        Br J Haematol. 2010; 149: 209-220
        • Gosselin R.C.
        • Marlar R.A.
        Preanalytical variables in coagulation testing: setting the stage for accurate results.
        Semin Thromb Hemost. 2019; 45: 433-448
        • Kitchen S.
        • Adcock D.M.
        • Dauer R.
        • et al.
        International Council for Standardization in Haematology (ICSH) recommendations for processing of blood samples for coagulation testing.
        Int J Lab Hematol. 2021; 43: 1272-1283
        • Svensson P.J.
        • Dahlback B.
        Resistance to activated protein C as a basis for venous thrombosis.
        N Engl J Med. 1994; 330: 517-522
        • Zehnder J.L.
        • Benson R.C.
        Sensitivity and specificity of the APC resistance assay in detection of individuals with factor V Leiden.
        Am J Clin Pathol. 1996; 106: 107-111
        • JuanI Jorquera
        • JoséM Montoro
        • Fernández M.A.
        • et al.
        Modified test for activated protein C resistance.
        Lancet. 1994; 344: 1162-1163
        • Kadauke S.
        • Khor B.
        • van Cott E.M.
        Activated protein C resistance testing for factor V Leiden.
        Am J Hematol. 2014; 89: 1147-1150
        • Moore G.W.
        • van Cott E.M.
        • Cutler J.A.
        • et al.
        Recommendations for clinical laboratory testing of activated protein C resistance; communication from the SSC of the ISTH.
        J Thromb Haemost. 2019; 17: 1555-1561
        • Quehenberger P.
        • Handler S.
        • Mannhalter C.
        • et al.
        The factor V (Leiden) test: evaluation of an assay based on dilute Russell viper venom time for the detection of the factor V Leiden mutation.
        Thromb Res. 1999; 96: 125-133
        • Wilmer M.
        • Stocker C.
        • Bühler B.
        • et al.
        Improved distinction of factor V wild-type and factor V Leiden using a novel prothrombin-based activated protein C resistance assay.
        Am J Clin Pathol. 2004; 122: 836-842
        • Douxfils J.
        • Ageno W.
        • Samama C.-M.
        • et al.
        Laboratory testing in patients treated with direct oral anticoagulants: a practical guide for clinicians.
        J Thromb Haemost. 2018; 16: 209-219
        • Cooper P.C.
        • Rezende S.M.
        An overview of methods for detection of factor V Leiden and the prothrombin G20210A mutations.
        Int J Lab Hematol. 2007; 29: 153-162
        • Norstrøm E.
        • Thorelli E.
        • Dahlbäck B.
        Functional characterization of recombinant FV Hong Kong and FV Cambridge.
        Blood. 2002; 100: 524-530
        • Poort S.
        • Rosendaal F.
        • Reitsma P.
        • et al.
        A common genetic variation in the 3’-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis.
        Blood. 1996; 88: 3698-3703
        • Soria J.M.
        • Almasy L.
        • Souto J.C.
        • et al.
        Linkage analysis demonstrates that the prothrombin G20210A mutation jointly influences plasma prothrombin levels and risk of thrombosis.
        Blood. 2000; 95: 2780-2785
        • Warshawsky I.
        • Hren C.
        • Sercia L.
        • et al.
        Detection of a novel point mutation of the prothrombin gene at position 20209.
        Diagn Mol Pathol. 2002; 11: 152-156
        • Khor B.
        • Van Cott E.M.
        Laboratory tests for protein C deficiency.
        Am J Hematol. 2010; 85: 440-442
        • Cooper P.C.
        • Pavlova A.
        • Moore G.W.
        • et al.
        Recommendations for clinical laboratory testing for protein C deficiency, for the subcommittee on plasma coagulation inhibitors of the ISTH.
        J Thromb Haemost. 2020; 18: 271-277
        • Cooper P.C.
        • Cooper S.M.
        • Goodfellow K.J.
        • et al.
        Evaluation of a new venom-based clotting assay of protein C.
        Int J Lab Hematol. 2008; 30: 437-443
        • Meijer P.
        • Kluft C.
        • Haverkate F.
        • et al.
        The long-term within- and between-laboratory variability for assay of antithrombin, and proteins C and S: results derived from the external quality assessment program for thrombophilia screening of the ECAT Foundation.
        J Thromb Haemost. 2003; 1: 748-753
        • Pabinger I.
        • Kyrle P.A.
        • Speiser W.
        • et al.
        Diagnosis of protein C deficiency in patients on oral anticoagulant treatment: comparison of three different functional protein C assays.
        Thromb Haemost. 1990; 63: 407-412
        • Malm J.
        • Laurell M.
        • Dahlbäck B.
        Changes in the plasma levels of vitamin K-dependent proteins C and S and of C4b-binding protein during pregnancy and oral contraception.
        Br J Haematol. 1988; 68: 437-443
        • Monagle P.
        • Barnes C.
        • Ignjatovic V.
        • et al.
        Developmental haemostasis.
        Thromb Haemost. 2006; 95: 362-372
        • Reitsma P.H.
        Protein C deficiency: from gene defects to disease.
        Thromb Haemost. 1997; 78: 344-350
        • Dahlbäck B.
        C4b-binding protein: a forgotten factor in thrombosis and hemostasis.
        Semin Thromb Hemost. 2011; 37: 355-361
        • Marlar R.A.
        • Gausman J.N.
        Protein S abnormalities: a diagnostic nightmare.
        Am J Hematol. 2011; 86: 418-421
        • Marlar R.A.
        • Gausman J.N.
        • Tsuda H.
        • et al.
        Recommendations for clinical laboratory testing for protein S deficiency: communication from the SSC committee plasma coagulation inhibitors of the ISTH.
        J Thromb Haemost. 2021; 19: 68-74
        • Marlar R.A.
        • Potts R.M.
        • Welsh C.
        Accuracy of diagnosis of protein S deficiency by protein S activity and antigen assays.
        J Clin Ligand. 2005; 28: 130-136
        • Mackie I.
        • Cooper P.
        • Lawrie A.
        • et al.
        Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis.
        Int J Lab Hematol. 2013; 35: 1-13
        • Tate J.
        • Ward G.
        Interferences in immunoassay.
        Clin Biochem Rev. 2004; 25: 105-120
        • Dykes A.C.
        • Walker I.D.
        • McMahon A.D.
        • et al.
        A study of Protein S antigen levels in 3788 healthy volunteers: influence of age, sex and hormone use, and estimate for prevalence of deficiency state.
        Br J Haematol. 2001; 113: 636-641
        • Liberti G.
        • Bertina R.M.
        • Rosendaal F.R.
        Hormonal state rather than age influences cut-off values of protein S: reevaluation of the thrombotic risk associated with protein S deficiency.
        Thromb Haemost. 1999; 82: 1093-1096
        • Gandrille S.
        • Borgel D.
        • Sala N.
        • et al.
        Protein S deficiency: a database of mutations--summary of the first update.
        Thromb Haemost. 2000; 84: 918
        • Picard V.
        • Nowak-Göttl U.
        • Biron-Andreani C.
        • et al.
        Molecular bases of antithrombin deficiency: twenty-two novel mutations in the antithrombin gene.
        Hum Mutat. 2006; 27: 600
        • Rossi E.
        • Chiusolo P.
        • Za T.
        • et al.
        Report of a novel kindred with antithrombin heparin-binding site variant (47 Arg to His): demand for an automated progressive antithrombin assay to detect molecular variants with low thrombotic risk.
        Thromb Haemost. 2007; 98: 695-697
        • Van Cott E.M.
        • Orlando C.
        • Moore G.W.
        • et al.
        Recommendations for clinical laboratory testing for antithrombin deficiency; Communication from the SSC of the ISTH.
        J Thromb Haemost. 2020; 18: 17-22
        • Kim Y.A.
        • Gosselin R.
        • Van Cott E.M.
        The effects of dabigatran on lupus anticoagulant, diluted plasma thrombin time, and other specialized coagulation assays.
        Int J Lab Hematol. 2015; 37: e81-e84
        • Gosselin R.
        • Grant R.P.
        • Adcock D.M.
        Comparison of the effect of the anti-Xa direct oral anticoagulants apixaban, edoxaban, and rivaroxaban on coagulation assays.
        Int J Lab Hematol. 2016; 38: 505-513
        • Khor B.
        • Van Cott E.M.
        Laboratory tests for antithrombin deficiency.
        Am J Hematol. 2010; 85: 947-950
        • Liebman H.A.
        • Wada J.K.
        • Patch M.J.
        • et al.
        Depression of functional and antigenic plasma antithrombin III (AT-III) due to therapy with L-asparaginase.
        Cancer. 1982; 50: 451-456
        • Rao A.K.
        • Niewiarowski S.
        • Guzzo J.
        • et al.
        Antithrombin III levels during heparin therapy.
        Thromb Res. 1981; 24: 181-186
        • James A.H.
        • Rhee E.
        • Thames B.
        • et al.
        Characterization of antithrombin levels in pregnancy.
        Thromb Res. 2014; 134: 648-651
        • Andrew M.
        • Paes B.
        • Milner R.
        • et al.
        Development of the human coagulation system in the full-term infant.
        Blood. 1987; 70: 165-172
        • Gindele R.
        • Oláh Z.
        • Ilonczai P.
        • et al.
        Founder effect is responsible for the p.Leu131Phe heparin-binding-site antithrombin mutation common in Hungary: phenotype analysis in a large cohort.
        J Thromb Haemost. 2016; 14: 704-715
        • Puurunen M.
        • Salo P.
        • Engelbarth S.
        • et al.
        Type II antithrombin deficiency caused by a founder mutation Pro73Leu in the Finnish population: clinical picture.
        J Thromb Haemost. 2013; 11: 1844-1849
        • Miyakis S.
        • Lockshin M.D.
        • Atsumi T.
        • et al.
        International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).
        J Thromb Haemost. 2006; 4: 295-306
        • Pengo V.
        • Ruffatti A.
        • Legnani C.
        • et al.
        Clinical course of high-risk patients diagnosed with antiphospholipid syndrome.
        J Thromb Haemost. 2010; 8: 237-242
        • Devreese K.M.J.
        • Groot P.G.
        • Laat B.
        • et al.
        Guidance from the Scientific and Standardization Committee for Lupus Anticoagulant/Antiphospholipid Antibodies of the International Society on Thrombosis and Haemostasis.
        J Thromb Haemost. 2020; 18: 2828-2839
        • Cohen H.
        • Mackie I.J.
        • Devreese K.M.J.
        Clinical and laboratory practice for lupus anticoagulant testing: an International Society of Thrombosis and Haemostasis Scientific and Standardization Committee survey.
        J Thromb Haemost. 2019; 17: 1715-1732
        • Pengo V.
        • Tripodi A.
        • Reber G.
        • et al.
        Update of the guidelines for lupus anticoagulant detection.
        J Thromb Haemost. 2009; 7: 1737-1740
        • Smock K.J.
        • Rodgers G.M.
        Laboratory identification of lupus anticoagulants.
        Am J Hematol. 2009; 84: 440-442
        • Topping J.
        • Quenby S.
        • Farquharson R.
        • et al.
        Marked variation in antiphospholipid antibodies during pregnancy: relationships to pregnancy outcome.
        Hum Reprod. 1999; 14: 224-228
        • De Kesel P.M.M.
        • Devreese K.M.J.
        The effect of unfractionated heparin, enoxaparin, and danaparoid on lupus anticoagulant testing: can activated carbon eliminate false-positive results?.
        Res Pract Thromb Haemost. 2019; 4: 161-168
        • Favaloro E.
        • Kershaw G.
        • Mohammed S.
        • et al.
        How to optimize activated partial thromboplastin time (APTT) testing: solutions to establishing and verifying normal reference intervals and assessing APTT reagents for sensitivity to heparin, lupus anticoagulant, and clotting factors.
        Semin Thromb Hemost. 2019; 45: 22-35
        • Tripodi A.
        • Cohen H.
        • Devreese K.M.J.
        Lupus anticoagulant detection in anticoagulated patients. Guidance from the Scientific and Standardization Committee for Lupus Anticoagulant/Antiphospholipid Antibodies of the International Society on Thrombosis and Haemostasis.
        J Thromb Haemost. 2020; 18: 1569-1575
        • Favaloro E.J.
        • Pasalic L.
        Lupus anticoagulant testing during anticoagulation, including direct oral anticoagulants.
        Res Pract Thromb Haemost. 2022; 6e12676
        • Devreese K.M.J.
        • de Laat B.
        Mixing studies in lupus anticoagulant testing are required at least in some type of samples.
        J Thromb Haemost. 2015; 13: 1475-1478
        • Ratzinger F.
        • Lang M.
        • Belik S.
        • et al.
        Lupus-anticoagulant testing at NOAC trough levels.
        Thromb Haemost. 2016; 116: 235-240
        • Favaloro E.J.
        • Mohammed S.
        • Curnow J.
        • et al.
        Laboratory testing for lupus anticoagulant (LA) in patients taking direct oral anticoagulants (DOACs): potential for false positives and false negatives.
        Pathology. 2019; 51: 292-300
        • Favaloro E.J.
        • Gilmore G.
        • Arunachalam S.
        • et al.
        Neutralising rivaroxaban induced interference in laboratory testing for lupus anticoagulant (LA): a comparative study using DOAC Stop and andexanet alfa.
        Thromb Res. 2019; 180: 10-19
        • Frans G.
        • Meeus P.
        • Bailleul E.
        Resolving DOAC interference on aPTT, PT, and lupus anticoagulant testing by the use of activated carbon.
        J Thromb Haemost. 2019; 17: 1354-1362
        • Favresse J.
        • Lardinois B.
        • Sabor L.
        • et al.
        Evaluation of the DOAC-Stop® procedure to overcome the effect of DOACs on several thrombophilia screening tests.
        TH Open. 2018; 2: e202-e209
        • Jacquemin M.
        • Toelen J.
        • Schoeters J.
        • et al.
        The addition of idarucizumab to plasma samples containing dabigatran allows the use of routine coagulation assays for the diagnosis of hemostasis disorders.
        J Thromb Haemost. 2015; 13: 2087-2092
        • Keeling D.
        • Mackie I.
        • Moore G.W.
        • et al.
        Guidelines on the investigation and management of antiphospholipid syndrome.
        Br J Haematol. 2012; 157: 47-58
        • Zhou J.
        • Hou X.
        • Zhang H.
        • et al.
        The clinical performance of a new chemiluminescent immunoassay in measuring anti-β2 glycoprotein 1 and anti-cardiolipin antibodies.
        Med Sci Monit. 2018; 24: 6816-6822
        • Devreese K.M.J.
        Antiphospholipid antibody testing and standardization.
        Int J Lab Hematol. 2014; 36: 352-363
        • Lakos G.
        Interference in antiphospholipid antibody assays.
        Semin Thromb Hemost. 2012; 38: 353-359
        • Ridker P.M.
        • Hennekens C.H.
        • Lindpaintner K.
        • et al.
        Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men.
        N Engl J Med. 1995; 332: 912-917
        • Ridker P.M.
        • Hennekens C.H.
        • Miletich J.P.
        G20210A mutation in prothrombin gene and risk of myocardial infarction, stroke, and venous thrombosis in a large cohort of US men.
        Circulation. 1999; 99: 999-1004
        • Koster T.
        • Rosendaal F.R.
        • Briët E.
        • et al.
        Protein C deficiency in a controlled series of unselected outpatients: an infrequent but clear risk factor for venous thrombosis (Leiden Thrombophilia Study).
        Blood. 1995; 85: 2756-2761
        • de Groot P.G.
        • Lutters B.
        • Derksen R.H.W.M.
        • et al.
        Lupus anticoagulants and the risk of a first episode of deep venous thrombosis.
        J Thromb Haemost. 2005; 3: 1993-1997
        • Naess I.A.
        • Christiansen S.C.
        • Cannegieter S.C.
        • et al.
        A prospective study of anticardiolipin antibodies as a risk factor for venous thrombosis in a general population (the HUNT study).
        J Thromb Haemost. 2006; 4: 44-49