Establishment of ELISA-comparable moderate and high thresholds for anticardiolipin and anti-β2 glycoprotein I chemiluminescent immunoassays according to the 2023 ACR/EULAR APS classification criteria and evaluation of their diagnostic performance

Clin Chem Lab Med. 2024 Jul 24;63(1):164-176. doi: 10.1515/cclm-2024-0570. Print 2025 Jan 29.

Abstract

Objectives: Recently published 2023 ACR/EULAR APS classification criteria emphasize the importance of quantifying single-, double-, and triple-antiphospholipid antibody positivity, distinguishing between IgG and IgM isotypes, and delineating moderate/high levels of anticardiolipin (aCL) and anti-β2 glycoprotein I (anti-β2GPI) antibodies. We aimed to establish clinically important moderate/high thresholds for aCL and anti-β2GPI IgG/IgM chemiluminescent immunoassays (CLIA), in particular QUANTA Flash, comparable to our in-house ELISAs used for over two decades, and to evaluate their diagnostic performance.

Methods: QUANTA Flash CLIA and in-house ELISAs were used to measure aCL and anti-β2GPI IgG/IgM. Moderate thresholds for QUANTA Flash CLIA were determined using a non-parametric approach, calculating a 99th percentile on serum samples from 139 blood donors, and by mirroring the diagnostic performance of in-house ELISA on 159 patient samples.

Results: Thresholds for QUANTA Flash CLIA achieving diagnostic performance equivalent to in-house ELISAs were 40 CU for moderate and 80 CU for high levels for aCL and anti-β2GPI IgG and IgM. The assays showed good qualitative agreement, ranging from 76.10 to 91.19 %. When considering in-house ELISA results, 14 out of 80 (17.5 %) patients did not fulfill the new ACR/EULAR laboratory classification criteria, while 27 out of 80 (33.8 %) did not when considering QUANTA Flash CLIA results.

Conclusions: We determined moderate and high thresholds for aCL and anti-β2GPI IgG and IgM detected with QUANTA Flash CLIA, aligning with long-established in-house ELISA thresholds. These thresholds are crucial for seamlessly integrating of the new 2023 ACR/EULAR classification criteria into future observational clinical studies and trials.

Keywords: anti-β2GPI; anticardiolipin; antiphospholipid syndrome; classification criteria.

MeSH terms

  • Antibodies, Anticardiolipin* / blood
  • Antiphospholipid Syndrome / blood
  • Antiphospholipid Syndrome / classification
  • Antiphospholipid Syndrome / diagnosis
  • Antiphospholipid Syndrome / immunology
  • Enzyme-Linked Immunosorbent Assay* / methods
  • Female
  • Humans
  • Immunoassay / methods
  • Immunoglobulin G / blood
  • Immunoglobulin G / classification
  • Immunoglobulin M* / blood
  • Immunoglobulin M* / immunology
  • Luminescent Measurements* / methods
  • Male
  • beta 2-Glycoprotein I* / blood
  • beta 2-Glycoprotein I* / immunology

Substances

  • beta 2-Glycoprotein I
  • Antibodies, Anticardiolipin
  • Immunoglobulin M
  • Immunoglobulin G