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Evaluation of antiphospholipid antibody assays using latent class analysis to address the lack of a reference standard

  • Markus A. Thaler , Andreas Bietenbeck , Meng-Xin Yin , Udo Steigerwald , Andrew B. Holmes , Edelgard Lindhoff-Last and Peter B. Luppa EMAIL logo
Published/Copyright: May 26, 2016
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 54 Issue 12

Abstract

Background:

Method evaluation of new assays for the detection of antiphospholipid antibodies (aPL) such as anti-cardiolipin (aCL) or anti-β2-glycoprotein I (aβ2-GPI) is challenging, as no internationally accepted reference material is available yet. Besides a lack of standardization, unacceptable inter-laboratory comparability of established tests is regularly observed. Owing to the absence of a commonly accepted reference standard, the evaluation of two research surface plasmon resonance (SPR) biosensor assays was performed using statistical methods from latent class analysis (LCA).

Methods:

aCL and aβ2-GPI IgG and IgM were measured in sera from 63 antiphospholipid syndrome patients, fulfilling the Sydney criteria, and in 34 healthy controls with four commercial assays. LCA was performed on the results and sera were assigned to the antibody-positive or antibody-negative group. Sera were subsequently evaluated in the SPR assays for aCL and aβ2-GPI. Optimal cutoffs and diagnostic performances of the research systems were established employing the LCA-derived gold standard.

Results:

With area under the curve results of 0.96 and 0.89 for the detection of aCL and aβ2-GPI, the research SPR assays discriminated well between antibody-positive and antibody-negative sera. Their sensitivities and specificities were comparable to the investigated commercial immunoassays.

Conclusions:

SPR assays are a suitable tool for the detection of aCL and aβ2-GPI with diagnostic performances not different from currently available commercial tests. LCA enabled the calculation of sensitivities and specificities for aPL assays in absence of a reference standard.

Keywords: antiphospholipid antibodies; biosensor; latent class analysis; method evaluation; surface plasmon resonance

Acknowledgments

We thank Ms. Anita Schreiegg for excellent technical assistance, Dr. Evangeline Thaler for reviewing the manuscript, and the Australian Research Council (ARC) Discovery Project Grant (A.B.H., MXY DP1094497).

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306.10.1111/j.1538-7836.2006.01753.xSearch in Google Scholar

2. Hughes GR. Thrombosis, abortion, cerebral disease, and the lupus anticoagulant. Br Med J (Clin Res Ed) 1983;287:1088–9.10.1136/bmj.287.6399.1088Search in Google Scholar

3. Devreese K, Hoylaerts MF. Challenges in the diagnosis of the antiphospholipid syndrome. Clin Chem 2010;56:930–40.10.1373/clinchem.2009.133678Search in Google Scholar

4. Metzger J, von Landenberg P, Kehrel M, Buhl A, Lackner KJ, Luppa PB. Biosensor analysis of beta2-glycoprotein I-reactive autoantibodies: evidence for isotype-specific binding and differentiation of pathogenic from infection-induced antibodies. Clin Chem 2007;53:1137–43.10.1373/clinchem.2006.079632Search in Google Scholar

5. Schlichtiger A, Baier C, Yin MX, Holmes AB, Maruyama M, Strasser R, et al. Covalent attachment of functionalized cardiolipin on a biosensor gold surface allows repetitive measurements of anticardiolipin antibodies in serum. Anal Bioanal Chem 2013;405:275–85.10.1007/s00216-012-6467-8Search in Google Scholar

6. Favaloro EJ, Silvestrini R, Mohammed A. Clinical utility of anticardiolipin antibody assays: high inter-laboratory variation and limited consensus by participants of external quality assurance programs signals a cautious approach. Pathology 1999;31:142–7.10.1080/003130299105331Search in Google Scholar

7. Favaloro EJ, Wheatland L, Jovanovich S, Roberts-Thomson P, Wong RC. Internal quality control and external quality assurance in testing for antiphospholipid antibodies: part I – Anticardiolipin and anti-beta2-glycoprotein I antibodies. Semin Thromb Hemost 2012;38:390–403.10.1055/s-0032-1311990Search in Google Scholar

8. Tincani A, Allegri F, Sanmarco M, Cinquini M, Taglietti M, Balestrieri G, et al. Anticardiolipin antibody assay: a methodological analysis for a better consensus in routine determinations – a cooperative project of the European Antiphospholipid Forum. Thromb Haemost 2001;86:575–83.10.1055/s-0037-1616089Search in Google Scholar

9. Reber G, Schousboe I, Tincani A, Sanmarco M, Kveder T, de Moerloose P, et al. Inter-laboratory variability of anti-beta2-glycoprotein I measurement. A collaborative study in the frame of the European Forum on Antiphospholipid Antibodies Standardization Group. Thromb Haemost 2002;88:66–73.10.1055/s-0037-1613156Search in Google Scholar

10. Harris EN, Gharavi AE, Patel SP, Hughes GR. Evaluation of the anti-cardiolipin antibody test: report of an international workshop held 4 April 1986. Clin Exp Immunol 1987;68:215–22.Search in Google Scholar

11. Ichikawa K, Tsutsumi A, Atsumi T, Matsuura E, Kobayashi S, Hughes GR, et al. A chimeric antibody with the human gamma1 constant region as a putative standard for assays to detect IgG beta2-glycoprotein I-dependent anticardiolipin and anti-beta2-glycoprotein I antibodies. Arthritis Rheum 1999;42:2461–70.10.1002/1529-0131(199911)42:11<2461::AID-ANR25>3.0.CO;2-OSearch in Google Scholar

12. Ichikawa K, Khamashta MA, Koike T, Matsuura E, Hughes GR. β2-Glycoprotein I reactivity of monoclonal anticardiolipin antibodies from patients with the antiphospholipid syndrome. Arthritis Rheum 1994;37:1453–61.10.1002/art.1780371008Search in Google Scholar PubMed

13. Willis R, Grossi C, Orietta Borghi M, Martos-Sevilla G, Zegers I, Sheldon J, et al. International standards for IgG and IgM anti-beta2glycoprotein antibody measurement. Lupus 2014;23:1317–9.10.1177/0961203314544535Search in Google Scholar PubMed

14. Meroni PL, Biggioggero M, Pierangeli SS, Sheldon J, Zegers I, Borghi MO. Standardization of autoantibody testing: a paradigm for serology in rheumatic diseases. Nat Rev Rheumatol 2014;10:35–43.10.1038/nrrheum.2013.180Search in Google Scholar PubMed

15. Pierangeli SS, Favaloro EJ, Lakos G, Meroni PL, Tincani A, Wong RC, et al. Standards and reference materials for the anticardiolipin and anti-beta2glycoprotein I assays: a report of recommendations from the APL Task Force at the 13th International Congress on Antiphospholipid Antibodies. Clin Chim Acta 2012;413:358–60.10.1016/j.cca.2011.09.048Search in Google Scholar PubMed

16. Lakos G, Favaloro EJ, Harris EN, Meroni PL, Tincani A, Wong RC, et al. International consensus guidelines on anticardiolipin and anti-beta2-glycoprotein I testing: report from the 13th International Congress on Antiphospholipid Antibodies. Arthritis Rheum 2012;64:1–10.10.1002/art.33349Search in Google Scholar PubMed

17. Bertolaccini ML, Amengual O, Andreoli L, Atsumi T, Chighizola CB, Forastiero R, et al. 14th International Congress on Antiphospholipid Antibodies Task Force. Report on antiphospholipid syndrome laboratory diagnostics and trends. Autoimmun Rev 2014;13:917–30.10.1016/j.autrev.2014.05.001Search in Google Scholar PubMed

18. de Groot PG, Derksen RH, de Laat B. Twenty-two years of failure to set up undisputed assays to detect patients with the antiphospholipid syndrome. Semin Thromb Hemost 2008;34:347–55.10.1055/s-0028-1085477Search in Google Scholar PubMed

19. Devreese KM. Standardization of antiphospholipid antibody assays. Where do we stand? Lupus 2012;21:718–21.10.1177/0961203312439335Search in Google Scholar PubMed

20. Lieby P, Soley A, Levallois H, Hugel B, Freyssinet JM, Cerutti M, et al. The clonal analysis of anticardiolipin antibodies in a single patient with primary antiphospholipid syndrome reveals an extreme antibody heterogeneity. Blood 2001;97:3820–8.10.1182/blood.V97.12.3820Search in Google Scholar PubMed

21. Reber G, Boehlen F, de Moerloose P. Technical aspects in laboratory testing for antiphospholipid antibodies: is standardization an impossible dream? Semin Thromb Hemost 2008;34:340–6.10.1055/s-0028-1085476Search in Google Scholar PubMed

22. Andreoli L, Rizzini S, Allegri F, Meroni P, Tincani A. Are the current attempts at standardization of antiphospholipid antibodies still useful? Emerging technologies signal a shift in direction. Semin Thromb Hemost 2008;34:356–60.10.1055/s-0028-1085478Search in Google Scholar PubMed

23. Lazarsfeld PF, Henry NW. Latent structure analysis. Boston: Houghton Mill, 1968.Search in Google Scholar

24. Formann AK, Kohlmann T. Latent class analysis in medical research. Stat Methods Med Res 1996;5:179–211.10.1177/096228029600500205Search in Google Scholar PubMed

25. Goetghebeur E, Liinev J, Boelaert M, Van der Stuyft P. Diagnostic test analyses in search of their gold standard: latent class analyses with random effects. Stat Methods Med Res 2000;9:231–48.10.1177/096228020000900304Search in Google Scholar PubMed

26. Pepe MS, Janes H. Insights into latent class analysis of diagnostic test performance. Biostatistics 2007;8:474–84.10.1093/biostatistics/kxl038Search in Google Scholar PubMed

27. Taylor WJ, Marchesoni A, Arreghini M, Sokoll K, Helliwell PS. A comparison of the performance characteristics of classification criteria for the diagnosis of psoriatic arthritis. Semin Arthritis Rheum 2004;34:575–84.10.1016/j.semarthrit.2004.05.001Search in Google Scholar PubMed

28. Grayson PC, Maksimowicz-McKinnon K, Clark TM, Tomasson G, Cuthbertson D, Carette S, et al. Distribution of arterial lesions in Takayasu’s arteritis and giant cell arteritis. Ann Rheum Dis 2012;71:1329–34.10.1136/annrheumdis-2011-200795Search in Google Scholar PubMed PubMed Central

29. Johns MK, Yin MX, Conway SJ, Robinson DE, Wong LS, Bamert R, et al. Synthesis and biological evaluation of a novel cardiolipin affinity matrix. Org Biomol Chem 2009;7:3691–7.10.1039/b909306kSearch in Google Scholar PubMed

30. Menten J, Boelaert M, Lesaffre E. Bayesian latent class models with conditionally dependent diagnostic tests: a case study. Stat Med 2008;27:4469–88.10.1002/sim.3317Search in Google Scholar PubMed

31. Qu Y, Tan M, Kutner MH. Random effects models in latent class analysis for evaluating accuracy of diagnostic tests. Biometrics 1996;52:797–810.10.2307/2533043Search in Google Scholar

32. Beath K. randomLCA: Random effects latent class analysis. R package version 1.0-1 2011 [cited 2014]. Available from: http://CRAN.R-project.org/package=randomLCA.Search in Google Scholar

33. Müller C, Schlichtiger A, Balling G, Steigerwald U, Luppa PB, Thaler M. Standardized antigen preparation to achieve comparability of anti-beta2-glycoprotein I assays. Thromb Res 2010;126:e102–9.10.1016/j.thromres.2010.05.022Search in Google Scholar PubMed

34. Cucnik S, Kveder T, Ulcova-Gallova Z, Swadzba J, Musial J, Valesini G, et al. The avidity of anti-beta2-glycoprotein I antibodies in patients with or without antiphospholipid syndrome: a collaborative study in the frame of the European forum on antiphospholipid antibodies. Lupus 2011;20:1166–71.10.1177/0961203311406308Search in Google Scholar PubMed

35. Cucnik S, Kveder T, Artenjak A, Ulcova Gallova Z, Swadzba J, Musial J, et al. Avidity of anti-beta2-glycoprotein I antibodies in patients with antiphospholipid syndrome. Lupus 2012;21:764–5.10.1177/0961203312440057Search in Google Scholar PubMed

36. Harris EN, Pierangeli SS. Primary, secondary, and catastrophic antiphospholipid syndrome: what’s in a name? Semin Thromb Hemost 2008;34:219–26.10.1055/s-0028-1082265Search in Google Scholar PubMed

37. Favaloro EJ, Wong RC. The antiphospholipid syndrome: a large elephant with many parts or an elusive chameleon disguised by many colours? Auto Immun Highlights 2010;1:5–14.10.1007/s13317-010-0003-7Search in Google Scholar PubMed PubMed Central

38. de Moerloose P, Reber G, Musial J, Arnout J. Analytical and clinical performance of a new, automated assay panel for the diagnosis of antiphospholipid syndrome. J Thromb Haemost 2010;8:1540–6.10.1111/j.1538-7836.2010.03857.xSearch in Google Scholar PubMed

39. Forastiero R, Papalardo E, Watkins M, Nguyen H, Quirbach C, Jaskal K, et al. Evaluation of different immunoassays for the detection of antiphospholipid antibodies: report of a wet workshop during the 13th International Congress on Antiphospholipid Antibodies. Clin Chim Acta 2014;428:99–105.10.1016/j.cca.2013.11.009Search in Google Scholar PubMed

40. Noubouossie D, Valsamis J, Corazza F, Rozen L, Debaugnies F, Demulder A. An automated chemiluminescence immunoassay may detect mostly relevant IgG anticardiolipin antibodies according to revised Sydney criteria. Acta Clin Belg 2012;67:184–9.Search in Google Scholar

41. Vikerfors A, Johansson AB, Gustafsson JT, Jonsen A, Leonard D, Zickert A, et al. Clinical manifestations and anti-phospholipid antibodies in 712 patients with systemic lupus erythematosus: evaluation of two diagnostic assays. Rheumatology (Oxford) 2013;52:501–9.10.1093/rheumatology/kes252Search in Google Scholar PubMed

42. Decavele AS, Schouwers S, Devreese KM. Evaluation of three commercial ELISA kits for anticardiolipin and anti-beta2-glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome. Int J Lab Hematol 2011;33:97–108.10.1111/j.1751-553X.2010.01259.xSearch in Google Scholar PubMed

43. Chung Y, Kim JE, Lim HS, Kim HK. Clinical performance of anticardiolipin and antibeta2 glycoprotein I antibodies using a new automated chemiluminescent assay: superior thrombotic prediction of combined results measured by two different methods. Blood Coagul Fibrinolysis 2014;25:10–5.10.1097/MBC.0b013e32836466b5Search in Google Scholar PubMed

44. Li R, Daguzan M, Vandermijnsbrugge F, Gyling M, Cantinieaux B. Both IgG and IgM anti-beta2 glycoprotein I antibodies assays are clinically useful to the antiphospholipid syndrome diagnosis. Acta Clin Belg 2014;69:433–8.10.1179/2295333714Y.0000000060Search in Google Scholar PubMed

45. Persijn L, Decavele AS, Schouwers S, Devreese K. Evaluation of a new set of automated chemiluminescense assays for anticardiolipin and anti-beta2-glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome. Thromb Res 2011;128:565–9.10.1016/j.thromres.2011.04.004Search in Google Scholar PubMed

46. Van Hoecke F, Persijn L, Decavele AS, Devreese K. Performance of two new, automated chemiluminescence assay panels for anticardiolipin and anti-beta2-glycoprotein I antibodies in the laboratory diagnosis of the antiphospholipid syndrome. Int J Lab Hematol 2012;34:630–40.10.1111/j.1751-553X.2012.01448.xSearch in Google Scholar PubMed

47. Villalta D, Alessio MG, Tampoia M, Da Re A, Stella S, Da Re M, et al. Accuracy of the first fully automated method for anti-cardiolipin and anti-beta2 glycoprotein I antibody detection for the diagnosis of antiphospholipid syndrome. Ann NY Acad Sci 2009;1173:21–7.10.1111/j.1749-6632.2009.04659.xSearch in Google Scholar PubMed

48. Reitsma JB, Rutjes AW, Khan KS, Coomarasamy A, Bossuyt PM. A review of solutions for diagnostic accuracy studies with an imperfect or missing reference standard. J Clin Epidemiol 2009;62:797–806.10.1016/j.jclinepi.2009.02.005Search in Google Scholar PubMed

49. Forastiero R. Multiple antiphospholipid antibodies positivity and antiphospholipid syndrome criteria re-evaluation. Lupus 2014;23:1252–4.10.1177/0961203314531635Search in Google Scholar PubMed

Supplemental Material:

The online version of this article (DOI: 10.1515/cclm-2016-0116) offers supplementary material, available to authorized users.

Received: 2016-2-12
Accepted: 2016-4-14
Published Online: 2016-5-26
Published in Print: 2016-12-1

©2016 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2016-0116
Keywords for this article
antiphospholipid antibodies; biosensor; latent class analysis; method evaluation; surface plasmon resonance

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Biomarkers, inflammation and cancer: where to go?
  4. Review
  5. Analysis, detection and quantitation of mixed cryoglobulins in HCV infection: brief review and case examples
  6. Mini Reviews
  7. Calcitonin measurement and immunoassay interference: a case report and literature review
  8. Exosomal non-coding RNAs: a promising cancer biomarker
  9. Opinion Paper
  10. Towards a new paradigm in laboratory medicine: the five rights
  11. EFLM Recommendation
  12. Recommendation for the review of biological reference intervals in medical laboratories
  13. IFCC Position Paper
  14. Assuring the quality of interpretative comments in clinical chemistry
  15. General Clinical Chemistry and Laboratory Medicine
  16. The effect of centrifugation speed and time on pre-analytical platelet activation
  17. An assessment of clinical laboratory performance for the determination of manganese in blood and urine
  18. Evaluation of antiphospholipid antibody assays using latent class analysis to address the lack of a reference standard
  19. Evaluation of the hypochromic erythrocyte and reticulocyte hemoglobin content provided by the Sysmex XE-5000 analyzer in diagnosis of iron deficiency erythropoiesis
  20. Cancer Diagnosis
  21. Identifying risk in the use of tumor markers to improve patient safety
  22. Association between Echinococcus granulosus infection and cancer risk – a pilot study in Cyprus
  23. Dynamic change of the systemic immune inflammation index predicts the prognosis of patients with hepatocellular carcinoma after curative resection
  24. Aberrant methylation of tumour suppressor genes WT1, GATA5 and PAX5 in hepatocellular carcinoma
  25. Cardiovascular Diseases
  26. High homocysteine and low folate plasma concentrations are associated with cardiovascular events but not bleeding during warfarin treatment
  27. Hemolysis and Coagulation
  28. Hemolysis rates in blood samples: differences between blood collected by clinicians and nurses and the effect of phlebotomy training
  29. Letters to the Editor
  30. Harmonisation of the laboratory testing process: need for a coordinated approach
  31. Pseudohyperkalemia due to severe leukocytosis: case presentation
  32. When obtaining a blood sample from the right arm was not the right thing to do: a case of elevated parathyroid hormone levels 27 years after thyroidectomy
  33. Raising awareness of assay compatibility with heparinized plasma
  34. Improved protocol for extraction of genomic DNA from formalin-fixed paraffin-embedded tissue samples without the use of xylene
  35. Effect of refrigeration, centrifugation, acidification, heat treatment and storage on urine calcium, magnesium and phosphate
  36. Letter in response to: Identifying risk in the use of tumor markers to improve patient safety
  37. Letter to the Editor in reply to Dayyani and Morgenstern’s comment on the article “Identifying risk in the use of tumor markers to improve patient safety”
  38. Level of red cell distribution width is affected by various factors
  39. Red blood cell distribution: an index without additional cost in estimating the prognosis of acute pancreatitis
  40. Testing of total 25(OH)vitamin D: agreement and discrepant cases between Cobas® 8000 and Liaison® XL methods
  41. Expression profiling and ontology analysis of circulating long non-coding RNAs in septic acute kidney injury patients
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