Screening Assays for the Diagnosis of Lupus Anticoagulant (LA)
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1 New HemosIL TM Screening Assays for the Diagnosis of Lupus Anticoagulant (LA) Hemostasis Enhance LA Screening with a Complete Panel of Fully Automated Assays Innovation. Leadership. Commitment.
2 Screening Assays for the Diagnosis of Lupus Anticoagulant (LA) Antiphospholipid Syndrome (APS) APS is an autoimmune disorder in which antiphospholipid (apl) antibodies are associated with venous and arterial thrombosis, recurrent fetal loss, repeated unexplained spontaneous abortion or premature birth. The laboratory criteria used to support the diagnosis of APS are based on the presence of LA, Anticardiolipin antibody (acl) or anti-β 2 -Glycoprotein-I antibody. LA is a class of auto-antibodies which may be directed toward different human proteins: β 2 -glycoprotein-i and Prothrombin primarily, but also Protein C, Protein S and Annexin V. The in vitro effect of LA is based on the formation of complexes between the auto-antibodies and proteins. The formation of these complexes is enhanced in the presence of phospholipids. For this reason, LA testing is mainly based on the prolongation of clotting times of different assays such as diluted PT and APTT, drvvt or KCT. These assays use very low phospholipid concentrations, which makes them sensitive to anti-phospholipid antibodies. Due to the heterogeneity of plasma auto-antibodies and the variable sensitivity of assays, the approach to LA diagnosis is based on multiple tests. LA Testing Laboratory diagnosis of LA is a complex procedure, based on the following criteria: Prolongation of phospholipid-dependent assays Exclusion of Factor deficiencies Phospholipid-dependence No effect from Factor inhibitors To demonstrate phospholipid dependence, the most recent SSC-ISTH recommendations on LA require the use of two different phospholipiddependent clotting assays, based on different methodologies. General Screening LA may be detected by performing general screening assays, such as PT and APTT. However, PT and APTT reagents are generally not designed to be sensitive to all types of LA, and because of the variety and heterogeneity of plasma anti-phospholipid antibodies, they cannot be used alone for the diagnosis of LA. In addition, prolongation of clotting times above the normal range may have many causes, including the presence of Factor deficiencies, Factor inhibitors or anticoagulants. Samples demonstrating prolonged clotting time should be thoroughly assessed before proceeding with LA investigation. Citrated Plasma PT APTT Normal Abnormal: Warfarin? Factor deficiencies? Inhibitors to coagulation factors? Normal Abnormal: Heparin? Factor deficiencies? Inhibitors to coagulation factors? TT Abnormal: Heparin (most likely) Normal
3 Silica Clotting Time Screen (low phospholipid concentration) and Confirm (high phospholpid concentration) in the same kit Liquid formulation, easy to use, fully automated Suitable for mixing studies and oral anticoagulant-treated patient samples Sensitive to LA Sensitive to anti-β 2 -Glycoprotein-I antibodies LAC Screen and LAC Confirm Based on drvvt, is the most common Screening and Confirmatory test for LA in the laboratory Easy to use, fully automated SCT and LAC Screen/Confirm Cover the maximum spectrum of antiphospholipid antibodies Results for both are expressed as Normalized Ratio In accordance with the most recent SSC-ISTH recommendations on LA screening The combination of SCT and LAC Screen and Confirm are more informative and more likely to differentiate LA from anti-fviii inhibitors, than either test alone The combination of SCT and LAC Screen and Confirm had the highest sensitivity in the detection of LA in patients who met the clinical criteria for APS
4 Screening Algorithm for LA with HemosIL Assays The use of both SCT and LAC Screen and Confirm assays enhances the identification of LA patient samples, due to their different sensitivities to anti-phospholipid antibodies. The charts below, demonstrate the major analytical steps in the diagnosis of LA. Silica Clotting Time (SCT) Screening and confirmatory APTT-based assays performed using SCT Screen and SCT Confirm reagents, containing low and high concentrations of phospholipids respectively. LAC Screen and Confirm Screening and confirmatory drvvt-based assays performed using LAC Screen and Confirm reagents, containing low and high concentrations of phospholipids respectively. LA Investigation Platelet- Free Plasma LA Screening SCT Screen and LAC Screen LA Confirmatory LA Confirmatory SCT Confirm LAC Confirm *N.R < cut-off: SCT (-) * N.R > cut-off: SCT (+) *N.R < cut-off: LAC(-) * N.R > cut-off: LAC(+) NOTE: - Exclude the effect due to the presence of Factor deficiencies or Factor inhibitors on the Normalized Ratios. - Exclude the effect of Heparin on the Normalized Ratios. See package insert for details on Heparin interference. No LA SCT and LA Results Analysis SCT (-) and LAC (-) - Samples from oral anticoagulanttreated patients may affect the Normalized Ratios. SCT (+) and LAC (+) SCT (+) and LAC (-) If none of the conditions above apply, LA is highly probable. SCT (-) and LAC (+) *N.R.: Normalized Ratio. See package insert for instructions on calculating N.R.
5 HemosIL Silica Clotting Time and LAC Screen and Confirm Silica Clotting Time (SCT) LAC Screen and Confirm Part number and Stability 2-8 C 20 Days 48 Hours Stability on-board 5 Days 3 Days Results Screen Ratio Normalized SCT Ratio = Normalized Confirm Ratio Screen Ratio LAC Ratio = Confirm Ratio Precision Mean CV% CV% Mean CV% CV% (N. Ratio) Within run Total (N. Ratio) Within run Total Normal ~1.5 <2.5 <3.0 ~1.0 <2.0 <2.5 Low ~2.5 <4.5 <6.0 ~1.5 <2.0 <3.0 High ~3.5 <5.5 <6.0 ~2.0 <4.0 <5.0 Expected values Normalized SCT Ratio Normalized LAC Ratio ACL Advance/Futura ACL 8/9/ In published clinical studies, HemosIL SCT demonstrated a very high sensitivity to Antiphospholipid antibodies in patients with Antiphospholipid Syndrome. The association of total thrombotic events, and specifically of venous thrombosis, with LA detected by the combination of SCT and drvvt was significantly higher than with LA detected by the single tests alone or by the combination of APTT and drvvt. (13) Summary HemosIL SCT was the most sensitive test with APS patients identified by the Sapporo criteria. When used in conjunction with the drvvt, the sensitivity increased further with the APS population. Incorporating multiple assays, based on different methodologies, into the analytical test profile, increases the probability of detecting LA.
6 New HemosIL TM Screening Assays for the Diagnosis of Lupus Anticoagulant (LA) Reagent Part Kit Number Configuration Silica Clotting Time (SCT) SCT Screen 3 x 5 ml SCT Confirm 3 x 5 ml SCT CaCl 2 3 x 10 ml LAC Screen x 2 ml LAC Confirm x 2 ml References Europe, America and Pacific Rin 1. Horbach DA, Van Oort E, Donders RCJM, Derksen RHWM, De Groot PG. Lupus anticoagulant is the strongest risk factor for both venous and arterial thrombosis in patients with systemic lupus erythematosus Comparison between different assays for the detection of antiphospholipid antibodies. Thromb Haemost 1996; 76: Whal DG, Guillemin F, de Maistre E, Perret C, Lecompted T, Thibaut G. Risk of venous thrombosis related to antiphospholipid antibodies in systemic lupus erythematosus. Lupus 1997; 6: Galli M. Which antiphospholipid antibodies should be measured in the antiphospholipid syndrome? Haemostasis 2000; 30 (Suppl. 2) Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC, Brey R, Derksen R, Harris EN, Hughes GR, Triplett DA, Khamashta MA. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of international workshop. Arthritis Rheum 1999; 42: Brandt JT, Triplett DA, Alving B, Sharrer I. Criteria for the diagnosis of lupus anticoagulants: an update. Thromb Haemost 1995; 74: Arnout J. Antiphospholipid syndrome: diagnostic aspects of lupus anticoagulants. Thromb Haemost 2001; 86: Chantarangkul V, Tripodi A, Arbini A, Mannucci PM. Silica Clotting Time (SCT) as a screening and confirmatory test for detection of lupus anticoagulants. Thromb Res 1992; 67: Chantarangkul V, Tripodi A, Clerici M, Bressi C, Mannucci PM. Laboratory diagnosis of lupus anticoagulants. Thromb Haemost 2002; 87: Dragoni F, Minotti C, Palumbo G, Faillace F, Redi R, Bongarzoni V, Avvisati G. As compared to kaolin clotting time, silica clotting time is a specific and sensitive method for detecting lupus anticoagulant. Thromb Res 2001; 101: Galli M. Dlott J, Norbis F, Ruggeri L, Cler L, Triplett DA, Barbui T. Lupus anticoagulants and thrombosis: clinical association of different coagulation and immunological tests. Thromb Haemost 2000; 84: Montaruli B, Vaccarino A, Foli C, Rus C, Agnes C, Saitta M, Bazzan M. Lupus Anticoagulant: Performance of a New, Fully Automated Commercial Screening and Confirmation Assay. Clin Chem 2005; 6: Tripodi A, Mancuso ME, Chantarangkul V, Clerici M, Bader R, Meroni PL, Santagustino E, Mannuci PM. Lupus Anticoagulants and their Relationship with the Inhibitors against Coagulation FVIII: Considerations on the differentiation between the 2 Circulating Anticoagulants. Clin Chem 2005; 10: Grypiotis P, Ruffati A, Pengo V, Tonello M, Biasiolo A, Zamboni D, Cavazzana A, Todesco S. Use of a New Silica Clotting Time for Diagnosing Lupus Anticoagulant in Patients Who Meet the Clinical Criteria for Antiphospholipid Syndrome. Journal of Clinical Laboratory Analysis 2006; 20: International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost Feb;4(2): Worldwide Locations Instrumentation Laboratory Corporate Headquarters Barcelona, Spain Tel US, Canada, Latin America Headquarters Lexington, MA, U.S.A. Tel Mexico Col. Granada Tel USA Lexington, MA Tel Pacific Headquarters Minato-ku, Tokyo, Japan Tel Hong Kong Hong Kong Tel Japan Minato-ku, Tokyo Tel Europe, Middle East, Africa Headquarters Milano, Italy Tel Austria Wien Tel Belgium Zaventem Tel Czech Praha Tel France Paris Tel Germany Kirchheim bei München Tel Hungary Budapest Tel or 11 Italy Milano Tel Lithuania Kaunas Tel Applications/tests listed may not yet be approved by the regulatory authorities in every country. IL product specifications are subject to modification to assure the highest quality performance. Some of the IL sites may still be in the process of completing ISO. HemosIL and ACL are trademarks of Instrumentation Laboratory. HemosIL reagents are not available in all countries Instrumentation Laboratory 2006 Poland Warszawa Tel Russia Moscow Tel The Netherlands Breda Tel United Kingdom Warrington, Cheshire Tel p/n EU Rev. 0 All rights reserved - Printed in Italy - Grafica Briantea - 06/06 A CH Werfen Company
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