Clin. Lab. 2009;55:XXX-XXX Copyright ORIGINAL ARTICLE Warfarin Does Not Interfere with Lupus Anticoagulant Detection by Dilute Russell s Viper Venom Time HORATIU OLTEANU 2, KATHARINE. A. DOWNES 1, JIGAR PATEL 1, DARJE PRAPROTNIK 1, RAVINDRA SARODE 2 1 Department of Pathology, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, OH, USA 2 Department of Pathology, The University of Texas Southwestern Medical Center, Dallas, TX, USA SUMMARY Background: The dilute Russell s viper venom time (DRVVT) test is part of the diagnostic armamentarium used to detect lupus anticoagulant (LA). When testing patients on warfarin therapy, there is some concern of false positive results due to their low Factor X levels. We studied the diagnostic performance of the DRVVT ratio (DRVVT-R) to confirm the presence of LA in thrombophilia patients receiving warfarin therapy, and compared those results with a control group receiving warfarin for cardiac conditions but without thrombosis. Methods: The DRVVT (screen, confirm, and ratio), Factors II and X assays, and PT/INR were performed in patients receiving warfarin for thrombosis and in patients with cardiac conditions but no thrombosis (control group). Results: Patients on warfarin in the thrombosis group (n = 22) were positive for LA by DRVVT-R (ratio >1.27 was considered positive) whereas none of the patients in the control group (n=13) were positive for LA. The median DRVVT-R was significantly higher in the thrombosis group (1.60, range 1.29-1.92) as compared to controls (1.13, range 0.79-1.23, p<0.001) even though the INRs were comparable (median 2.3 for thrombosis group versus median of 2.2 for controls, p<0.05). Similarly, FX and FII levels were not significantly different in these two groups. Conclusions: We conclude that the use of the DRVVT-R allows for diagnosis of LA in patients receiving warfarin with therapeutic INR despite their decreased Factor X levels. (Clin. Lab. 2009;55:XXX-XXX) INTRODUCTION Lupus anticoagulant (LA) was initially reported in patients with systemic lupus erythematosus (SLE) 1, but it has since been described in a host of other clinical conditions. LA comprises a group of heterogeneous autoantibodies, which are part of the larger class of acquired autoimmune antiphospholipid antibodies (apl) 2, 3. The latter are now shown to be directed against proteins with affinity for negatively charged phospholipids, such as prothrombin and β 2 -glycoprotein I (β 2 -GPI), rather than against the phospholipid per se 4. Similar antibodies found in the plasma of patients with antiphospholipid syndrome (APS) are probably targeting protein C, protein S and annexin V 4, 5. The occurrence of these antibodies in plasma has been associated with a significant risk of venous or arterial thrombotic events 6, 7 and recurrent fetal loss 8. APS is an area of active research, and the complexity of this entity is reflected in recent papers addressing evolving definition criteria 9 and management guidelines 10. Although the laboratory evaluation for LA is initiated in the context of a thrombotic clinical presentation or recurrent pregnancy loss, the antibodies have an anticoagulant effect in vitro. This property is utilized in detecting LA based on antibody interference with phospholipid-dependent coagulation tests 11, 12. For a laboratory diagnosis of LA, the International Society on Thrombosis and Haemostasis (ISTH) guidelines suggest the performance of at least two screening tests with low concentrations of phospholipid. If the screening test is prolonged, then the LA is confirmed by demonstrating correction of the prolonged clotting time by the addition of extra phospholipid to show phospholipid dependency Manuscript accepted March 16, 2009 Clin. Lab. 3+4/2009 1
HORATIU OLTEANU et al. 11, 12. While this algorithm benefits from guidelines published throughout the years 11-13, the laboratory detection of LAs is not always straightforward and is limited to some extent by the methodologies employed 14-16. Dilute Russell s viper venom test (DRVVT) is one of the tests routinely performed due to its higher sensitivity and specificity 17. Russell s viper venom acts on Factor X and requires PL for its action. Therefore, in presence of LA, the clotting time of DRVVT (screening test; DRVVT-S) is prolonged, but is corrected by the addition of an additional PL in the second step (confirm test; DRVVT-C). If the ratio of the DRVVT-S and DRVVT- C is greater than the ratio established by testing normal healthy volunteers, it is diagnostic of LA. Patients on warfarin have decreased vitamin K-dependent factors, including Factor X, which should be in the range of 10-30% with a therapeutic international normalized ratio (INR). Therefore, it is postulated that the DRVVT results may not be reliable (false positive) due to a lower amount of Factor X in the plasma of patients receiving warfarin. We hypothesize that since the DRVVT test result is reported as a ratio of DRVVT-S and DRVVT-C, the ratio should be unaffected by the Factor X level in patients with a therapeutic INR; therefore, there should be no false positive result. Hence, we evaluated performance characteristics of DRVVT in patients with thrombophilia receiving warfarin therapy compared to patients without thrombophilia (e.g., cardiac reasons) on warfarin to look for any interference by warfarin. MATERIALS AND METHODS Blood specimens The study was approved by the Institutional Review Board of the University Hospitals of Cleveland. Venous blood was drawn in one-tenth volume of 0.106 M sodium citrate in siliconized vacutainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) and centrifuged at 3000x g for 8 minutes to obtain platelet poor plasma, which after decanting was re-centrifuged to prepare platelet free plasma. Prothrombin time (INR) was performed, and the plasma was frozen at -70 o C, until further analysis. Prothrombin time (PT) and international normalized ratio (INR) determination PTs were performed on a CA-6000 (Sysmex North America, Inc, Mundelein, IL) using recombinant tissue thromboplastin (Innovin, ISI 1.0, Siemens Healthcare Diagnostics, Deerfield, IL); INR was then calculated. Factor II and Factor X assays Factors II and X were assayed as per manufacturer s instructions using Factor II and Factor X deficient plasmas (Siemens Healthcare Diagnostics, Deerfield, IL), respectively, with the BCS automated coagulation instrument (Siemens Healthcare Diagnostics, Deerfield, IL) to assess the anticoagulant effects of warfarin. Lupus anticoagulant test The LA was detected by DRVVT method, which was performed using the LA1 (DRVVT-screen) and LA2 (DRVVT-confirm) reagents (Siemens Healthcare Diagnostics, Deerfield, IL), on the BCS automated coagulation instrument. The LA1 reagent contains Russell s viper venom, phospholipid, calcium and a heparin neutralizer. Except for higher phospholipid content, the LA2 reagent is identical to LA1. Normal reference intervals for LA1 and LA2 and DRVVT- ratio (DRVVT- R) were established by studying 40 normal healthy volunteers (20 males and 20 females). The reference range for LA1 is 41.5 s. The diagnostic cut-off for DRVVT- R was established with mean + 2SD, which was 1.27. Thus, DRVVT-R of >1.27 was considered positive for LA. Patients with a positive LA were re-tested at intervals ranging between 3 and 5 months from the initial results, while still on warfarin. Data analysis Statistical analyses were carried out in Origin v6.0 (OriginLab Corp., Northampton, MA); a p value < 0.05 was considered statistically significant. Continuous data were processed in Microsoft Excel v11.2 and expressed as means, ranges, medians and standard deviations. RESULTS Thirty-five patients on long-term warfarin therapy were included in the study, of which 22 had a history of thrombophilia (T group) (median age 72 years, 12 females). Of these, 18 had a clinical history of at least one deep vein thrombosis and/or pulmonary embolism; 3 patients had embolic cerebral vascular accidents and deep vein thrombosis; and 1 patient had a history of ischemic stroke and peripheral arterial thrombosis. The remaining 13 patients had no history of thrombophilia (NT group) and were receiving warfarin for various cardiac conditions (median age 68 years, 11 females). Lupus anticoagulant All thrombophilic patients (T group) had a prolonged DRVVT-S, and the DRVVT-C showed correction of long clotting times; all subjects had a DRVVT-R > 1.27 (Figure 1), thus displaying a phospholipid-dependent nature. The median DRVVT-R was 1.60 (mean ± SD; 1.59 ± 0.18). In the NT control group, 10/13 (77%) had a prolonged DRVVT-S, which did not correct in DRVVT-C, thus indicating that the prolongation was due to low Factor X levels only and not due to LA. All of the subjects in the NT control group had a DRVVT-R < 1.27 (Figure 1) and were negative for LA. The DRVV T-R (median 1.1, mean ± SD; 1.1 ± 0.12) was signifycantly lower than the T group, which had a positive LA (p<0.001). 2 Clin. Lab. 3+4/2009
DRVVT RATIO AND LUPUS ANTICOAGULANT Figure 1: Comparison of DRVVT normalized ratio (DRVVT-R) in patients with ( ) and without ( ) lupus anticoagulant on warfarin therapy. The dashed line is at the cut-off value of 1.27 for DRVVT-R. Straight horizontal lines within each group mark the mean value for DRVVT-R. Table 1: Comparison between thrombophilia and non-thrombophilia groups on warfarin for detection of lupus anticoagulant. T group (n=22) NT group (n=13) p value DRVVT-S (sec) Mean ± SD 72.5 ± 13.7 44.8 ± 5.1 < 0.001 Median (Range) 70.5 (45.6 99.6) 43.4 (37.8 54.3) DRVVT-C (sec) Mean ± SD 45.4 ± 5.4 40.8 ± 5.6 0.02 Median (Range) 45.6 (35.4 54.3) 41.3 (33.5 54.5) DRVVT-R Mean ± SD 1.59 ± 0.18 1.10 ± 0.12 < 0.001 Median (Range) 1.60 (1.29 1.92) 1.13 (0.79 1.23) PT (sec) Mean ± SD 29.3 ± 12.8 25.6 ± 6.1 > 0.05 Median (Range) 24.3 (21.6 56.0) 24.1 (16.2 46.5) INR Mean ± SD 2.74 ± 1.2 2.39 ± 0.57 > 0.05 Median (Range) 2.27 (2.02 5.23) 2.25 (1.51 4.35) Factor II (%) Mean ± SD 35.8 ± 12.5 47.0 ± 22.8 > 0.05 Median (Range) 32.2 (14.5 70.3) 40.2 (19.8 93.0) Factor X (%) Mean ± SD 22.0 ± 12.9 32.7 ± 18.6 > 0.05 Median (Range) 19.1 (6.7 55.2) 26.1 (11.0 76.3) T group (thrombophilia); NT group (non-thrombophilia); DRVVT (Dilute Russell s Viper Venom Time); DRVVT-S (Dilute Russell s Viper Venom Time-screen); DRVVT-C (Dilute Russell s Viper Venom Time-confirm); DRVVT-R (Dilute Russell s Viper Venom Time-ratio); PT (prothrombin time); INR (International Normalized Ratio). Clin. Lab. 3+4/2009 3
HORATIU OLTEANU et al. Anticoagulation was considered adequate (INR 2-3.5) in all cases except for 2 patients in the T group, although Factors II and X reflected antithrombotic levels. There was no significant difference in INR, Factor II and Factor X between the two groups (Table 1). DISCUSSION The propensity for thrombosis in patients with APS underscores the clinical significance of LA 9, 10. The resulting increased morbidity and mortality mandates prompt and accurate detection, in order to insure adequate therapeutic intervention. The DRVVT was initially described two decades ago as a sensitive and reproducible assay in the identification of LA 17. The method is extensively used for LA detection 14, 18 and is one of the laboratory tests recommended by the International Society on Thrombosis and Hemostasis 12. In a study comparing abnormalities of either DRVVT or kaolin clotting time (KCT), it was shown that patients with a DRVVT profile had a higher thrombotic risk than those with a KCT profile 19. Reasons for potentially false positive and false negative reports 16, 18, 20 can usually be traced to biological venom heterogeneity and/or to differences in the various manufacturing processes. It is important to mention that an isolated positive DRVVT has relatively low clinical significance 21 and there are risk (such as life-threatening bleeding) associated with the anticoagulant therapy administered to patients based on this laboratory test 9. While no method is 100% sensitive and/or specific for the diagnosis of LA 14, 20, the DRVVT assay is considered an excellent method when reproducibility, cost and automation are factored into the assessment. The DRVVT is based on Factor X activation by the venom and the subsequent generation of thrombin in the presence of calcium and phospholipids 17. The coagulation process is slowed down in the presence of LA, thus leading to an abnormal prolongation of the clotting time. Oral anticoagulation therapy decreases Factor X; thus, the DRVVT screen may be abnormal and test results may erroneously be called positive 20. A confirmatory test must be used to assess the presence of LAs in patients on warfarin. The DRVVT assay performed very well in a study for patients on warfarin that compared the diagnostic proficiency of the DRVVT with the hexagonal phospholipid neutralization test Staclot LA (Diagnostica Stago, Asnieres, France), designated as gold standard 22. We studied two patient groups receiving warfarin, one with and one without history of thrombophilia. Of the 35 patients studied, 32 (91.4%) had a prolonged clotting time with the DRVVT-S. In the T group, 22/22 (100%) of the patients that tested positive for LA while on warfarin, had an abnormal DRVVT-S and a DRVVT-R of >1.27 (mean 1.59). In the NT group, 10/13 (77%) had abnormal prolongation of DRVVT-S, and all had DRVVT-R of <1.27 (mean 1.1). Thus, by performing the DRVVT-C and calculating the DRVVT-R, we were able to clearly identify patients on warfarin as LA positive or LA negative. While our study did not employ an alternate assay to confirm the diagnosis, the methodology is nevertheless supported by professional guidelines 11, 12, and the performance is similar to other studies evaluating DRVVT 22 or other venom-based assays 23. As compared to the previous report on DRVVT 22, we used a commercially available test kit rather than a test developed in-house, and we employed the ratio rather than a percent correction for data analysis. There is no consensus on the optimal method for calculating the correction in DRVVT, and any one of three methods (percentage correction of ratio, percentage correction of clotting time and test-confirm ratio) can be used 11. Furthermore, the quantitation of Factor II and Factor X levels in our study provides additional evidence of the efficacy of the anticoagulant therapy. In conclusion, we found no false-positive results when employing DRVVT-R in the detection of LA in a limited cohort of patients receiving warfarin therapy (INR < 5.0), a condition that often poses diagnostic difficultties. References: 1. Conley C, Hartmann R. A hemorrhagic disorder caused by circulating anticoagulants in patiens with disseminated lupus erythematosus. J Clin Invest 1952;31:621-2. 2. Triplett DA. Antiphospholipid-protein antibodies: laboratory detection and clinical relevance. Thromb Res 1995;78(1):1-31. 3. Arnout J. Antiphospholipid syndrome: diagnostic aspects of lupus anticoagulants. Thromb Haemost 2001;86(1):83-91. 4. Arnout J, Vermylen J. Current status and implications of autoimmune antiphospholipid antibodies in relation to thrombotic disease. J Thromb Haemost 2003;1(5):931-42. 5. Roubey RA. Antiphospholipid antibodies: immunological aspects. Clin Immunol 2004;112(2):127-8. 6. Wahl DG, Guillemin F, de Maistre E, Perret-Guillaume C, Lecompte T, Thibaut G. Meta-analysis of the risk of venous thrombosis in individuals with antiphospholipid antibodies without underlying autoimmune disease or previous thrombosis. Lupus 1998;7(1):15-22. 7. Wahl DG, Guillemin F, de Maistre E, Perret C, Lecompte T, Thibaut G. Risk for venous thrombosis related to antiphospholipid antibodies in systemic lupus erythematosus-a meta-analysis. Lupus 1997;6(5):467-73. 8. Lockshin MD. Pregnancy loss and antiphospholipid antibodies. Lupus 1998;7 Suppl 2:S86-9. 9. Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4(2):295-306. 10. Lim W, Crowther MA, Eikelboom JW. Management of antiphospholipid antibody syndrome: a systematic review. JAMA 2006;295(9):1050-7. 4 Clin. 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