Seronegative antiphospholipid syndrome: Diagnostic challenges, novel biomarkers, therapeutic ambiguities, and future perspectives

Role of non-criteria aPLs

While patients with SN-APS test negative for standard aPLs (lupus anticoagulant, aCL antibodies, and anti-β2GPI antibodies), many exhibit other autoantibodies, often referred to as “non-criteria” aPLs.^[3,14] These include antibodies against phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, vimentin/cardiolipin complex, and annexin A5.^[3,14,15] Other proposed non-criteria markers include IgA isotypes of traditional aPLs and aPS/PT antibodies.^[2,5] These antibodies are believed to play a pathogenic role by either targeting similar molecular sites as conventional aPLs or by acting on distinct cellular pathways that culminate in thrombotic events similar to those seen in seropositive APS.^[15]

Molecular and cellular mechanisms

The various aPLs in SN-APS contribute to thrombosis through several interconnected mechanisms:

• Interference with hemostasis aPLs, by targeting phospholipids and/or phospholipid-binding proteins such as β2-glycoprotein I (β2-GPI), can disrupt normal hemostatic processes.^[7] The binding of aPLs to β2-GPI is crucial, as the resulting complex can activate effector cells.^[16]

• Cellular activation of aPLs engages phospholipids and phospholipid-binding proteins on the surface of endothelial cells, platelets, and leukocytes, leading to their activation.^[17] This activation promotes a prothrombotic environment by inducing the release of adhesion molecules, pro-inflammatory cytokines, and tissue factor.^[18] The resulting cellular activation shifts the balance toward coagulation and inflammation.

• Dysregulation of coagulation and fibrinolysis anti-PL antibodies can increase the risk of thrombosis by mechanisms that extend beyond simple dysregulation of coagulation pathways.^[19] They can activate platelets, monocytes, and endothelial cells, and perturb natural anticoagulant systems and fibrinolytic pathways, further contributing to a hypercoagulable state.^[20]

• Inflammation is a key pathogenic factor in APS, acting as a link between the procoagulant phenotype and the actual development of thrombi. It also plays a significant role in mediating placental injury in obstetric complications associated with the syndrome.^[20]

Thrombotic events

• Venous thrombosis: Deep vein thrombosis is a common manifestation, reported at similar frequencies in SNAPS and seropositive APS (e.g., 31.4% vs. 31.0%).^[10,21] Pulmonary embolism also occurs with similar prevalence (e.g., 23.8% vs. 28.7%).^[10,21]

• Arterial thrombosis: Patients may experience arterial events such as stroke (e.g., 14.9% vs. 17.2%) and transient ischemic attack (e.g., 11.9% vs. 10.3%).^[10,21] Other arterial events can include lower limb ischemia and myocardial infarction.^[3] Thrombophilia, a predisposition to thrombosis, may even be more frequent in the SN-APS population compared to seropositive APS.^[2]

Obstetric morbidity

• Recurrent pregnancy losses are a hallmark feature, including early spontaneous abortions (e.g., 67.1% vs. 52.1%), stillbirths (e.g., 62.5% vs. 59.4%), prematurity (e.g., 28.1% vs. 21.7%), and pre-eclampsia (e.g., 28.1% vs. 23.1%).^[10,21] Obstetric APS cases, particularly miscarriages, have been noted to be more prevalent in SN-APS patients.^[2]

Non-Criteria clinical manifestations

Beyond the core thrombotic and obstetric criteria, patients with SN-APS can present with a range of “non-criteria” manifestations, which contribute to the disease’s heterogeneity. These manifestations are also frequently observed in aPL-positive patients and include:

1. Hematologic: Thrombocytopenia and hemolytic anemia.^[22-24] Thrombocytopenia is reported to occur in the majority of aPL-positive patients.^[23]

2. Neurological: Migraine, chorea, seizures, myelitis, refractory migraine, and cognitive dysfunction.^{[10,22,24,25]} White matter lesions are also common.^[23]

3. Dermatological: Livedo reticularis and skin ulcers.^[23-25]

4. Cardiovascular: Heart valve disease (e.g., valvular thickening or vegetations like Libman–Sacks endocarditis), pulmonary hypertension, and superficial thrombophlebitis.^{[10,22,24,25]}

5. Renal: Renal microangiopathy and microangiopathic nephropathy.^[10,22,24]

These non-criteria manifestations underscore the systemic nature of APS and the wide variety of organ systems that can be affected.^[26]

The heterogeneity of SN-APS stems from several factors

• Varied antibody profiles: While conventional aPLs are negative, many SN-APS patients harbor “non-criteria” antibodies such as IgA anti-β2GPI, anti-PS/PT, anti-phosphatidylethanolamine, anti-phosphatidic acid, anti-phosphatidylserine, anti-phosphatidylinositol, anti-vimentin/cardiolipin complex, and anti-annexin A5.^{[3,5,12,14,15]} The specific profile of these underlying antibodies can influence the clinical presentation.

• Broad clinical spectrum: The disease can affect virtually any organ system or tissue, leading to a broad spectrum of clinical presentations ranging from single thrombotic events to catastrophic APS (CAPS).^[24,26]

• Diagnostic ambiguity: The diagnostic criteria for SNAPS remain a subject of ongoing debate.^[9] The term SNAPS is often used when classical tests are negative but other isolated IgA reactivity or other non-standardized tests are positive.^[10] This lack of standardized diagnostic markers and varying definitions across studies contributes to the perceived heterogeneity and makes it challenging to ascertain the exact commonality of specific manifestations.

The prevalence of “non-criteria” manifestations such as white matter lesions and thrombocytopenia can be quite high, observed in a majority (56%) of aPL-positive patients in international cohorts, and even higher in certain APS subsets (66%).^[23]

Novel biomarkers in SN-APS here are a summary of the most important novel biomarkers in Table 1.^[27-32]

Advances in laboratory testing methodologies

Improvements in laboratory techniques have greatly enhanced the sensitivity for non-criteria aPLs, thus partially fulfilling the longstanding diagnostic gap in SNAPS. The original solid-phase immunoassays were based on heterogeneous phospholipid samples, which were characterized by low inter-laboratory reproducibility and low analytical specificity. Modern immunoassays increasingly use purified phospholipid-protein complexes, such as aPS/PT, and domain-specific β2-GPI antigens, especially domain I, which improve epitope specificity and analytical signal discrimination. At the same time, the introduction of multiplex and line immunoassay formats allows for the simultaneous measurement of both criteria and non-criteria aPLs, thus improving analytical sensitivity in patients with low titer and narrowly focused autoantibody responses. These laboratory technique improvements have uncovered hidden antibody reactivity in a large proportion of patients with a high clinical index of suspicion for APS, thus providing biological plausibility for the existence of SN-APS and further validating the use of extended antibody panels as ancillary diagnostic tools.

Sensitivity, specificity, and delayed clinical adoption

The performance of non-criteria aPLs in the diagnosis of APS can vary, depending on the study and the design of the assay. While taken alone, the sensitivity of aPS/PT, IgA anti-β2-GPI, and IgA aCL for APS is modest; however, their specificity is high, especially for thrombotic and pregnancy-related manifestations. Notably, the use of combination testing approaches, including multiple non-criteria antibodies, can significantly enhance the overall diagnostic sensitivity for suspected SN-APS patients. Despite being reported in the literature for over a decade, the clinical use of these assays has remained relatively limited due to several reasons, including the lack of standardization of the assays, substantial variability between laboratories, lack of inclusion in the current APS classification criteria, and, until recently, a lack of understanding regarding their direct contribution to clinical decision-making. As a result, the use of non-criteria antibody testing has largely remained the preserve of specialized centers and research institutions, despite accumulating evidence supporting their adjunct role in the diagnosis of APS.

Overall challenges and gaps

• Lack of standardization: This is a recurring issue for most non-criteria aPL assays, leading to high variability between laboratories and hindering widespread clinical adoption.^[3]

• Elusive clinical utility: While many non-criteria aPLs are detected, their precise clinical utility and diagnostic value often remain elusive.^[28] More robust prospective clinical studies are needed.^[27]

• Classification criteria refinement: Current classification criteria for APS still rely on a limited panel of conventional aPLs. There is a strong call for updating these criteria to incorporate new serological markers to better identify and stratify patients.^[2]

• Unidentified cofactors: A percentage of patients with clinical APS remain seronegative even for these extended panels of non-criteria antibodies, suggesting the involvement of other unidentified cofactors in their serum reactivity.^[22,30]

Current treatment approaches

The therapeutic intervention for SN-APS primarily aims to prevent thrombotic events and manage pregnancy complications, generally following the established guidelines for seropositive APS.

Challenges in therapeutic decision-making

As noted in a 2017 review, “Although there is no consensus regarding the management of SN-APS, a pragmatic approach based on the clinical suspicion is justified.”^[44] This highlights a persistent gap where, even with strong clinical evidence, the absence of serological confirmation leaves room for therapeutic uncertainty.

Early in the understanding of APS, the focus was primarily on anticoagulation as the main treatment for thrombotic APS, often seen as an autoimmune disease rather than solely requiring immunomodulation.^[39,45] However, even in the early 2000s, “scientifically robust, evidence-based rules for the treatment of the APS” were lacking, a failure attributed partly to a scarcity of well-designed prospective studies and the syndrome’s inherent clinical complexity.^[46]

A 2015 review highlighted “areas of uncertainty regarding the management of APS exist where evidence is scarce or nonexistent,” including patients with “seronegative” APS, those not fulfilling formal classification criteria, and those with recurrent events despite optimal anticoagulation.^[47]These “difficult cases” present a “practical problem” as they often require treatment even without definitive laboratory markers.^[10] The Canadian Rheumatology Association Meeting in 2023 underscored the “challenge in providing guideline-directed therapy to patients with a history but not laboratory features of APS and the need for expanded diagnostics.”^[48]

More recent task force reports continue to emphasize the need for “further understanding of the mechanisms that lead to thrombotic and obstetric manifestations in aPL-negative patients” to guide future therapeutic strategies.^[49] This underscores that the current therapeutic approach is often extrapolative, applying treatments effective in seropositive APS to seronegative cases due to similar clinical presentations. The development of specific therapeutic guidelines for SN-APS remains a critical unmet need, as the underlying pathogenic mechanisms, while sharing similarities, may also possess unique features requiring tailored interventions.

Clinical and therapeutic implications of antibody positivity

The presence of non-criteria aPLs has significant clinical and therapeutic implications, especially in patients with high clinical probability of APS but persistently negative results in conventional tests. The presence of antibodies such as aPS/PT and IgA anti-β2-GPI has been linked to high thrombotic risk profiles, recurrent vascular events, and poor obstetric outcomes, thus aiding in risk stratification in patients with a diagnostically uncertain condition. In clinical practice, the presence of these antibodies may help in the initiation or continuation of APS-specific therapies, such as long-term anticoagulation or more aggressive antithrombotic regimens in selected patients. In obstetrics, the presence of non-criteria antibodies may help in the prophylactic or therapeutic use of low molecular weight heparin and LDA in patients with recurrent pregnancy morbidity who do not meet the laboratory criteria for classification. Although non-criteria antibodies are not currently used in the classification criteria for APS, their presence has significant clinical utility in helping in the management of patients with suspected SN-APS.

Table 3 summarizes the current challenges in therapeutic decision-making for SN-APS, integrating both current and historical perspectives.^[50,51]

Genomic and proteomic insights

High-throughput sequencing technologies, such as next-generation sequencing, are facilitating the discovery of novel biomarkers for APS, including genetic variants. These technologies allow for comprehensive analysis of the genome, transcriptome, and epigenome, moving beyond antibody levels to solve problems in APS diagnosis, prognosis, and risk assessment.^[1]

• Genomics and epigenetics: Research is exploring genetic and epigenetic changes (like microRNAs) related to the clinical profile of APS patients and how these might be modulated by specific therapies.^[52,53] For instance, certain microRNAs (e.g., miR-19b/miR-20a) have been identified as potential modulators of tissue factor, a key player in thrombosis development in APS. aPLs can induce changes in miRNA biogenesis proteins in leukocytes, leading to an altered miRNA profile and affecting protein targets related to thrombosis and atherosclerosis. This suggests that a specific signature of circulating microRNAs could serve as potential biomarkers for clinical features in APS.^[53]

• Proteomics: Proteomic approaches involve studying proteins to discover novel biomarkers based on changes in their concentration levels or post-translational modifications. These techniques are being applied to better understand the pathogenic mechanisms of thrombosis in APS.^[54] For example, an unbiased proteomic screen of aPL-positive patients has been conducted to define the inflammatory signature of APS across different clinical phenotypes.^[55] This type of research helps in exploring the pathogenesis, clinical phenotype, and biomarkers of APS from multiple dimensions.^[1]

Advanced immunological profiling

This involves techniques like flow cytometry and single-cell RNA sequencing, providing a high-resolution view of the immune landscape in SN-APS.

• Immune cell profiling: These methods aim to characterize the overall features of cells and molecules related to APS patients, offering higher resolution to distinguish heterogeneity between APS patients and healthy individuals.^[15] By analyzing immune cell subsets, their activation states, and cytokine profiles, researchers hope to uncover unique immunological signatures specific to SN-APS.

Future possibilities

• Improved diagnosis and risk stratification expanding the laboratory panel beyond enzyme-linked immunosorbent assay-based testing for criteria aPLs, utilizing techniques like thin-layer chromatography immunostaining, has already shown that many SN-APS patients possess detectable aPL/cofactor antibodies.^[10,22,30] This can significantly improve diagnostic accuracy and enable better risk stratification, especially by identifying “non-criteria” antibodies such as aPS/PT, IgA aCL, IgA anti-β2GPI, and antibodies against various phospholipids.^[2,3,56]

• Personalized medicine: By identifying specific genetic predispositions, unique protein signatures, or distinct immune cell profiles, future treatments could be tailored to individual patients.^[32]

• Deeper understanding of pathogenesis: Omics data can reveal intricate molecular pathways and cellular interactions that drive SN-APS, offering new targets for therapeutic intervention.

• Updated classification criteria: The detailed insights gained from these technologies can inform the development of more comprehensive and inclusive classification criteria for APS, moving beyond the current reliance on a limited panel of conventional aPLs.^[28]

Table 4 summarizes the emerging omics and immunophenotyping approaches^[53-55]

Refining diagnostic criteria and biomarker discovery

• Expanded antibody panels: Continued investigation into “non-criteria” aPLs is critical. The aim is to transition these markers into routine clinical practice through standardization of assays and clinical utility validation.

• Omics and immunophenotyping integration

• Updated classification criteria: The 2023 American College of Rheumatology/European Alliance of Associations for Rheumatology APS classification criteria are expected to support a future research agenda aimed at a better definition and subphenotyping of APS.^[32]

Advancements in therapeutic strategies

The therapeutic ambiguity in SN-APS necessitates the development of targeted and evidence-based treatments.^[10]

• Pathophysiology-driven therapies: This includes

  • Novel drug development

  • Targeted immunomodulation

• Risk-stratified management

• Long-term management: Future research must specifically address comorbidities, optimize pregnancy protocols, and mitigate bleeding risks associated with anticoagulation in SN-APS patients.^[5]

Translational research opportunities

Translational research is crucial to bridge the gap between scientific discoveries and clinical implementation.

• Multidisciplinary collaboration

• International registries and cohorts: Establishing and maintaining large, international multicenter studies and registries, such as the APS ACTION registry, is essential to improve the quality of evidence, validate biomarkers, and assess therapeutic interventions in diverse patient populations.^[57-59] For instance, a European Platform for the registry will be disseminated to all healthcare providers.^[60]

• Outcome measures development: There is a need to focus on devising suitable outcome measures, including a disease activity index, an optimal damage index, and a specific quality of life index, for meaningful advances in clinical management.^[57]