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ANA fluorescence patterns and low C4 are associated with higher antiphospholipid antibody serological intensity in routine diagnostic testing

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Published/Copyright: April 20, 2026
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM)

Abstract

Objectives

To evaluate whether antinuclear antibody (ANA) titre or HEp-2 fluorescence patterns are associated with antiphospholipid antibody (aPL) serological intensity, and to examine component-specific complement abnormalities – particularly low C4 – as laboratory correlates compatible with classical-pathway involvement.

Methods

This retrospective cross-sectional study analysed 626 consecutive diagnostic samples with same-day testing for lupus anticoagulant (LA), anticardiolipin (aCL), anti-β2-glycoprotein I (anti-β2GPI) antibodies, and ANA. aPL serological intensity was categorised from 0 to 3 (negative to triple-positive) using predefined laboratory cut-offs. ANA patterns were grouped as negative, homogeneous, speckled, or other. Complement variables included low C3, low C4, and combined hypocomplementaemia. Associations were assessed using ordered logistic regression, with Firth bias-reduced logistic regression applied for binary outcomes with sparse events.

Results

Overall, 26.5 % of samples (166/626) were aPL-positive, including 1.6 % triple-positive cases (10/626). High-titre ANA (≥1:160) was not associated with higher aPL serological intensity (OR 1.00, 95 % CI 0.51–1.96). In contrast, homogeneous (OR 1.99, 95 % CI 1.26–3.18) and speckled (OR 2.85, 95 % CI 1.66–4.91) ANA patterns were independently associated with higher aPL categories. Low C4, – but not low C3 – was associated with aPL positivity in binary analysis (Firth OR 6.84, p=0.001; low-C4 events n=10).

Conclusions

ANA fluorescence patterns, but not ANA titre, are associated with higher aPL serological intensity, indicating a distinct laboratory serological phenotype. Low C4 shows an association compatible with classical-pathway complement involvement. These findings are observational and require validation in longitudinal studies.

Keywords: antiphospholipid antibodies; lupus anticoagulant; ANA fluorescence patterns; complement C4; antiphospholipid syndrome; thrombosis
Corresponding author: Nikolaos Androulakis, Hematology Laboratory, University Hospital of Iraklion, 71500, Iraklion, Greece, E-mail:

Acknowledgments

The authors thank the staff of the Hematology Laboratory and the Laboratory of Experimental Endocrinology/Clinical Immunology of the University Hospital of Heraklion for their technical assistance and support.

  1. Research ethics: The study used fully anonymised laboratory data with no identifiable patient information. Ethical approval was obtained from the Institutional Review Board of the University Hospital of Heraklionv (Protocol No 177/02-24). The study study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

  2. Informed consent: Not applicable.

  3. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission. Conceptualization: Nikolaos E. Androulakis, Marilena Kampa. Data curation: Nikolaos E. Androulakis, Olga Striligka, Eleni Nioti. Laboratory analysis: Aikaterini Darivianaki, Fotini Nistikaki, Theodosia Iatridi. Statistical analysis: Anastasia Papadopoulou, Nikolaos E. Androulakis. Supervision: Christina Kalpadakis, Marilena Kampa. Writing – original draft: Nikolaos E. Androulakis. Writing – review and editing: All authors.

  4. Use of Large Language Models, AI and Machine Learning Tools: ChatGPT (OpenAI) was used to assist with language editing and formatting. No LLM, AI, or MLT contributed to the scientific content, analysis, or interpretation of results.

  5. Conflict of interest: The authors state no conflict of interest.

  6. Research funding: None declared.

  7. Data availability: The anonymized dataset underlying this study is available from the corresponding author upon reasonable request.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2025-1709).

Received: 2025-12-22
Accepted: 2026-03-02
Published Online: 2026-04-20

© 2026 Walter de Gruyter GmbH, Berlin/Boston

https://doi.org/10.1515/cclm-2025-1709
Keywords for this article
antiphospholipid antibodies; lupus anticoagulant; ANA fluorescence patterns; complement C4; antiphospholipid syndrome; thrombosis
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