Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with the persistent presence of high antiphospholipid antibodies (aPLs), which contribute significantly to the disease’s pathogenesis. Golden classification criteria for APS (Sydney criteria) required the presence of at least one clinical criteria as well as one of the laboratory criteria including lupus anticoagulant (LA), medium to high level of anti-cardiolipin (aCL), anti-β2 glycoprotein-I (aβ2GPI) immunoglobulin isotype G (IgG) or M (IgM) positivity at 12 weeks apart (1). In real-world clinical scenarios, patients may have positive laboratory results of unclear clinical significance (2), whereas others (known as seronegative APS, SNAPS) are present with clinical manifestations highly suggestive of APS but persistently negative for standard aPLs (3).

Among new biomarkers investigated for APS, non-criteria aPLs such as anti-β2GPI domain I (aDM1), anti-phosphatidylserine/prothrombin (aPS/PT), anti-phosphatidic acid (aPA), and anti- phosphatidylinositol (aPI) is increasingly recognized (4). Besides diagnosis, evaluation of non-criteria aPLs could also contribute to prognosis and risk assessment for associated clinical manifestations (5). However, due to the heterogeneity of detection methods and population, the diagnostic value of these aPLs remains controversial.

Regarding the Chinese population, previous studies indicated that aPS/PT could identify some SNAPS patients and was associated with thrombotic and obstetric complications (6, 7). Our previous work in a smaller cohort suggested that IgG or IgM antibodies of phosphatidylserine (aPS), aPI, sphingomyelin (aSM), phosphatidylcholine (aPC) and phosphatidylethanolamine (aPE) were helpful for identifying SNAPS and predicting arterial thrombosis (8). Few studies have explored all of the aforementioned extra-criteria autoantibodies, and their relations with more detailed clinical manifestations in the same patient groups. This study utilized commercial ELISA kits to test the levels of 16 aPLs in APS patients and disease or healthy controls. The diagnostic value and clinical relevance of each aPL isotype were further investigated.

In patients under the age of 50, APS contributes to a significant part of recurrent reproduction losses as well as cerebro- or cardiovascular accidents (10). It has been postulated that the pathological features of APS are driven by intracellular signaling pathways in various cellular subtypes activated by criteria and non-criteria aPLs (11). Currently, a wide spectrum of aPLs has been discovered. They could directly bind to negatively charged phospholipids (e.g., aPG, aPI) or react with phospholipid-binding proteins (e.g., aPS/PT, aDM1) (12). In this study, we explored the diagnostic power and clinical significance of 16 non-criterial aPLs in APS patients.

Our results suggest that although a single aPL isotype may not have reached comparable diagnostic performance with aCL or aβ2GPI, the combinational test of aPC IgG/M, aPE IgG/M/A, aPI IgG/M, aSM IgG/M, and aPA IgG/M compared to aCL or aβ2GPI IgG/M only could significantly increase diagnostic sensitivity and accuracy. The titer of most aPLs shown for each group was also significantly higher in APS patients compared to disease or healthy controls. The good diagnostic values of aPC and aSM were consistent with our previous findings, while PE had higher sensitivity in this study (8). Volkov et al. also reported that aPI was more prevalent among APS patients compared to healthy and diseased control subjects with sepsis, but did not report a significantly higher level of aPE positivity (13). Nevertheless, since previous works did not calculate and compare the additional value of aPLs to criteria biomarkers, the results should be cautiously interpreted. Utilizing this method of comparison, we found that the detection of aCL IgA, ab2GPI IgA, aAnxV IgG/M, and aPS/PT IgG/M provided additive power (14). The introduction of these aPLs into routine laboratory practice could accelerate APS diagnostics.

In our 9 SNAPS patients, 8 (88.9%) were positive for at least one non-criteria aPLs, of which 3 patients had 5 or more positive aPLs. The most encountered aPLs were aPS/PT IgM (in 6 patients), followed by aPI IgG (in 5 patients) and aPA IgG (in 4 patients). In a USA/UK study including 68 SNAPS patients, Zohoury et al. found that 36.8% could be identified by 11 non-criteria aPLs, of which 11.8% were positive for aPS/PT (15). Litvinova and colleagues reported that 52.9% of 17 SNAPS patients could be identified by at least one of 18 non-criteria aPLs (16). Trugliia et al. documented that 81.9% of 61 female SNAPS patients with reproductive complications were positive with at least one of 5 new aPLs (17). In all, non-criteria aPLs could also provide additive value for the identification of SNAPS patients.

From the perspective of clinical relevance, we found that certain non-criteria aPLs are associated with both criteria and extra-criteria clinical manifestations. For diagnostic manifestations, our correlation analysis indicated that aSM IgG and IgM were positively associated with pregnancy morbidities, among which pre-eclampsia or premature birth was significantly associated with aSM IgG. Antibodies against SM were less reported in previous literature, which reside in the outer leaflet of the plasma membrane (12). Additionally, aPI IgG correlated with pre-eclampsia or premature birth, and it was also significantly correlated with stroke. Castanon et al. observed a high sensitivity for aPI (41.2%-59.2%) across manifestations including thrombotic and obstetric events (18). Our previous results also demonstrated a correlation between aPI IgG and thrombosis (8).

For other arterial events, we found that IgG of aPC, aPE, aPG, and aPA were associated with lower limb artery occlusion. Both PC and PE are major neutral components of the phospholipid, and their antibodies have been observed in pediatric patients with cerebral infarction (19). Several studies have reported that aPE was significantly associated with major clinical events including fetal loss and/or thrombosis, especially in the absence of laboratory APS tests (20). For venous events, we calculated the correlation of aPLs with pulmonary embolism, portal thrombosis, and cerebral and deep venous thrombosis. Only aPS/PT IgG positivity was associated with DVT. Several studies have supported that aPS/PT IgG was a strong indicator of the risk of thrombosis or obstetric complications and could be used as a confirmatory diagnostic marker (6, 7, 16). In addition, we did not find any clinical association for aPG or aPE IgA, which also had low sensitivity in APS diagnosis. IgA isotypes of non-criteria aPLs have hardly been investigated in previous studies, and more experience must be accumulated to further evaluate their ability in diagnosis.

Extra-criteria APS features could be associated with an increased risk of relapse and the need for additional therapies (21). Thus, the predictive value of non-criteria aPLs for these manifestations was explored. For microvasculopathy, aPC IgG and aPG IgG showed a significantly increased risk. Diffuse thrombotic microvasculopathy was a critical characteristic of catastrophic APS (CAPS) (22). Although we did not include these patients, the positivity of aPG IgG has been observed in 2 of 3 CAPS subjects previously (16). For other extra-criteria manifestations, many of these new aPLs presented a significant association with hematological disorders (thrombocytopenia or hemolytic anemia). One potential mechanism of thrombocytopenia was due to increased activation and destruction of platelets by aPLs (23). Activation of the complement pathway and coagulation system by aPLs was also hypothesized to be the underlying cause of hemolytic anemia (24).

Another interesting finding was a strong positive association between aPE IgM and livedo reticularis, which was observed to be linked with thrombosis and heart valve disease (25, 26). Additionally, heart valve lesions were positively associated with aSM IgM (by Fisher’s test), and APS nephropathy was positively associated with aPC IgG. While SM was found to be correlated with atherosclerosis and thrombosis (27), more subjects should be added to consolidate our findings. In summary, examination of the broad spectrum of non-criteria aPLs may allow better characterization of APS pathophysiology and multifaceted clinical phenotypes, which could shed light on the early diagnosis and better management of the disease.

This study has its limitations. Since most subjects were recruited from the outpatient clinic, the number of CAPS or severe patients could be unbalanced. Other promising biomarkers such as the anti-first domain (DI) of β2GPI were not explored here. The follow-up information of included patients was not sufficient for the surveillance of recurrent events, especially for thrombosis. Currently, the lack of standardized detection systems and their cut-off values may reduce the reliability of the findings.

In conclusion, additional detection of non-criteria aPLs including aPC IgG/M, aPE IgG/M/A, aPI IgG/M, aSM IgG/M, and aPA IgG/M could assist in APS diagnosis. The positivity of certain aPLs was statistically associated with both criteria and extra-criteria APS clinical manifestations.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethics Committee of Peking Union Medical College Hospital. The patients/participants provided their written informed consent to participate in this study.

All authors were involved in the design of this study. SL, YB, JM, QW, XT, ML, XZ, JZ, and CH contributed to the collection of blood samples and other experimental procedures. SL, YB, and JM were involved in data collection and pre-processing. SL and CH analyzed the data and wrote the manuscript. QW, XT, ML, XZ, and JZ contributed to the recruitment of patients and the evaluation of clinical data. All authors contributed to the article and approved the submitted version.