Standardization and Harmonization of Antiphospholipid Antibody Assays

“See also” article details of Recent Guidelines and Recommendations for Laboratory Detection of Lupus Anticoagulants and article details of Standardization of Antiphospholipid Antibody Testing—Historical Perspectives and Ongoing Initiatives

This issue of Seminars in Thrombosis & Hemostasis contains two articles related to the standardization and harmonization of antiphospholipid antibody (aPL) assays,[1] [2] which are important for correct diagnosis of the antiphospholipid (antibody) syndrome (APS). One of these articles deals with lupus anticoagulant (LA) testing,[1] and the other with solid phase assays,[2] most typically performed for the detection of anticardiolipin antibodies (aCL) and anti-β₂-glycoprotein-I antibodies (aβ₂GPI). We are currently at an important juncture in the improved development of standardization and harmonization of aPL assays.

For LA testing, three different testing guidelines have recently emerged.[3] [4] [5] These have been, respectively, developed by the LA Scientific Standardization Committee of the International Society on Thrombosis and Hemostasis (ISTH),[3] the British Committee for Standards in Haematology (BCSH),[4] and the Clinical and Laboratory Standards Institute (CLSI).[5] Although there is agreement between several aspects of the three guidelines, there are also notable differences. In his thorough comparative review, Moore essentially analyzes the different guidelines for these elements of similarity and disparity.[1] That the guidelines concur on several matters is not unexpected. Each guideline was developed by separate groups of experts in the areas of laboratory testing (LA) and clinical diagnosis and management (APS), and each was largely based on evidence where this existed. Several individual members of the different groups were also involved in more than one guideline development process. Conversely, that the guidelines diverge at several points, although potentially less anticipated by laboratory practitioners, should also not be totally surprising, given areas of uncertainty, differences in expert opinion, and a seemingly conflicting evidence base. The concept that consensus guidelines for aPL testing do not lead to complete consensus among experts in the field has been previously explored in this journal.[6] [7]

General agreement exists within the three LA guidelines on issues such as sample preparation, recommended use of multiple distinct assays based on different principles and including both dilute Russell viper venom time (dRVVT) and activated partial thromboplastin time (aPTT), use of normalized ratios, calculations to demonstrate phospholipid dependence and to demonstrate inhibition, as well as interpretive reporting. However, the ISTH recommendation to employ only dRVVT and aPTT is not mirrored in the BCSH and CLSI documents, which lend support for the use of additional assays as required. The use of mixing studies is encouraged by all guidelines, and the potential for false negatives in mixing tests is also acknowledged, but mixing studies are dealt with differently in each guideline, with each also providing differing perspectives on the relative importance of the process. For example, mixing studies are ascribed greater importance by the authors of the ISTH guidelines,[3] [8] [9] whereas BCSH indicates that a negative mixing test need not exclude the presence of a LA, and CLSI reprioritizes the test order from the standard “screen-mix-confirm” to “screen-confirm-mix,” with mixing studies being considered unnecessary in specific circumstances. Opinions in the guidelines also differ on setting cutoff levels (i.e., 97.5th vs. 99th percentile for normally distributed data). All guidelines cover testing of anticoagulated patients, but far more detail is given by BCSH and CLSI, in part due to the lowered word count limitation imposed on these guidelines.

Importantly, although complete agreement is not apparent among the three guidelines, these represent significant moves toward engendering common practices. It is notable that although LA tests are largely based on “simple clotting assays,” there is already reasonable harmonization of test results between methods and between laboratories.[10] [11] [12] [13] The reason for this general “concordance” may have several origins, including similar manufacturing processes for test reagents or common reagents supplied by different suppliers under different reagent names,[10] [14] the consequence of many years of LA guidelines by the ISTH group, as well as data normalization processes that ultimately reduce variability among laboratories and methods.[11] [12] [13] [14]

The situation for solid phase assays is different. These assays have largely developed from different manufacturers and research groups largely independent of each other, and also with a reduced emphasis on “standardization.” It is, therefore, both interesting and disturbing that the level of between laboratory or method concordance in solid phase assays such as aCL and aβ₂GPI is nearly an order of magnitude lower than LA assays,[11] [12] [15] despite the solid phase assays inherently representing methods with theoretically lower variance, such as enzyme linked immunosorbant assays (ELISA).

The report from Willis et al[2] represents another important and concerted attempt to redress this deficiency in hemostasis diagnostics, describing various recent initiatives to improve standardization and harmonization in the area of solid phase aPL testing. Historically, there have been many attempts to standardize solid phase aPL assays including international workshops, initiatives of the European Forum on Antiphospholipid Antibodies, and the Australasian Anticardiolipin Working Party.[16] [17] [18] [19] The authors of the current review[2] describe several newer initiatives, largely evolving from recent international congresses on aPL, such as those recently held in 2010 and 2013. For example, from the 2010 meeting, a task force composed of internationally recognized experts in the field of APS was formed to address these issues, and this resulted in several publications providing guidance and promoting standardization and harmonization of test methods and approaches.[20] [21] Willis et al[2] also highlight the importance of cutoff determination in aPL assays, along with the clinical significance of positive aPL results of varying magnitudes.

Of note, the most recent aPL initiatives were largely headed by Silvia Pierangeli, who recently and sadly passed away.[22] Nevertheless, we hope that these initiatives continue to lead to improved diagnostic practice related to the identification, diagnosis, and management of conditions associated with aPL, thus including APS.

• References

• 1 Moore GW. Recent guidelines and recommendations for laboratory detection of lupus anticoagulants [In Focus Article]. Semin Thromb Hemost 2014; 40 (2) 163-171
• 2 Willis R, Lakos G, Harris EN. Standardization of antiphospholipid antibody testing—historical perspectives and ongoing initiatives [In Focus Article]. Semin Thromb Hemost 2014; 40 (2) 172-177
• 3 Pengo V, Tripodi A, Reber G , et al; Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. Update of the guidelines for lupus anticoagulant detection. J Thromb Haemost 2009; 7 (10) 1737-1740
• 4 Keeling D, Mackie I, Moore GW, Greer IA, Greaves M ; British Committee for Standards in Haematology. Guidelines on the investigation and management of antiphospholipid syndrome. Br J Haematol 2012; 157 (1) 47-58
• 5 CLSI. Laboratory Testing for the Lupus Anticoagulant; Approved Guideline. CLSI document H60-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2014
• 6 Wong RC, Adelstein S, Gillis D, Favaloro EJ. Development of consensus guidelines for anticardiolipin and lupus anticoagulant testing. Semin Thromb Hemost 2005; 31 (1) 39-48
• 7 Favaloro EJ, Wong RCW. Laboratory testing and identification of antiphospholipid antibodies and the antiphospholipid syndrome: a potpourri of problems, a compilation of possible solutions. Semin Thromb Hemost 2008; 34 (4) 389-410
• 8 Favaloro EJ, Bonar R, Zebeljan D, Kershaw G, Marsden K. Laboratory investigation of lupus anticoagulants: mixing studies are sometimes required. J Thromb Haemost 2010; 8 (12) 2828-2831
• 9 Tripodi A. To mix or not to mix in lupus anticoagulant testing? That is the question. Semin Thromb Hemost 2012; 38 (4) 385-389
• 10 McGlasson DL, Fritsma GA. Comparison of six dilute russell viper venom time lupus anticoagulant screen/confirm assay kits. Semin Thromb Hemost 2013; 39 (3) 315-319
• 11 Favaloro EJ, Bonar R, Marsden K. Internal quality control and external quality assurance in testing for antiphospholipid antibodies: part II—lupus anticoagulant. Semin Thromb Hemost 2012; 38 (4) 404-411
• 12 Favaloro EJ. Trials and tribulations in lupus anticoagulant testing. Clin Chem Lab Med 2013; 51 (2) 253-256
• 13 Pradella P, Azzarini G, Santarossa L , et al. Cooperation experience in a multicentre study to define the upper limits in a normal population for the diagnostic assessment of the functional lupus anticoagulant assays. Clin Chem Lab Med 2013; 51 (2) 379-385
• 14 Favaloro EJ, Plebani M, Lippi G. Regulation in hemostasis and thrombosis: part I-in vitro diagnostics. Semin Thromb Hemost 2013; 39 (3) 235-249
• 15 Favaloro EJ, Wheatland L, Jovanovich S, Roberts-Thomson P, Wong RCW. Internal quality control and external quality assurance in testing for antiphospholipid antibodies: part I—anticardiolipin and anti-β2-glycoprotein I antibodies. Semin Thromb Hemost 2012; 38 (4) 390-403
• 16 Tincani A, Allegri F, Balestrieri G , et al. Minimal requirements for antiphospholipid antibodies ELISAs proposed by the European Forum on antiphospholipid antibodies. Thromb Res 2004; 114 (5-6) 553-558
• 17 Reber G, Tincani A, Sanmarco M, de Moerloose P, Boffa MC ; Standardization group of the European Forum on Antiphospholipid Antibodies. Proposals for the measurement of anti-β2-glycoprotein I antibodies. Standardization group of the European Forum on Antiphospholipid Antibodies. J Thromb Haemost 2004; 2 (10) 1860-1862
• 18 Wong RCW, Gillis D, Adelstein S , et al. Consensus guidelines on anti-cardiolipin antibody testing and reporting. Pathology 2004; 36 (1) 63-68
• 19 Wong RC, Favaloro EJ, Adelstein S , et al. Consensus guidelines on anti-beta 2 glycoprotein I testing and reporting. Pathology 2008; 40 (1) 58-63
• 20 Pierangeli SS, de Groot PG, Dlott J , et al. 'Criteria' aPL tests: report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, Texas, April 2010. Lupus 2011; 20 (2) 182-190
• 21 Pierangeli SS, Favaloro EJ, Lakos G , et al. Standards and reference materials for the anticardiolipin and anti-β2glycoprotein I assays: a report of recommendations from the APL Task Force at the 13th International Congress on Antiphospholipid Antibodies. Clin Chim Acta 2012; 413 (1-2) 358-360
• 22 Harris EN, Willis R. In memoriam. Silvia Pierangeli, PhD (1955–2013). Semin Thromb Hemost 2014; 40 (2) 137-139