International Journal of Molecular Sciences Article Differences in Antiphospholipid Antibody Profile between Patients with Obstetric and Thrombotic Antiphospholipid Syndrome Ariadna Anunciación-Llunell 1 , Cándido Muñoz 2 , Dirk Roggenbuck 3,4 , Stefano Frasca 4 , Josep Pardos-Gea 1,5 , Enrique Esteve-Valverde 6 , Jaume Alijotas-Reig 1,5,7, * and Francesc Miró-Mur 1, * 1 Systemic Autoimmune Diseases Research Unit, Vall d’Hebron Institut de Recerca (VHIR), 08035 Barcelona, Catalonia, Spain 2 Centre for Rheumatology Research, University College of London, London WC1E 6JF, UK 3 Faculty of Health Sciences Brandenburg, Brandenburg University of Technology Cottbus-Senftenberg, 01968 Senftenberg, Germany 4 GA Generic Assays GmbH, 15827 Dahlewitz, Germany 5 Systemic Autoimmune Diseases Unit, Department of Internal Medicine, Hospital Universitari Vall d’Hebron (HUVH), 08035 Barcelona, Catalonia, Spain 6 Systemic Autoimmune Diseases Unit, Department of Internal Medicine, Hospital Universitari Parc Taulí, 08208 Sabadell, Catalonia, Spain 7 Department of Medicine, Faculty of Medicine, Universitat Autònoma de Barcelona, 08035 Barcelona, Catalonia, Spain * Correspondence: jaume.alijotas@vallhebron.cat (J.A.-R.); francesc.miro@vhir.org (F.M.-M.); Tel.: +34-93-489-4194 (J.A.-R.); +34-93-489-4047 (F.M.-M.) Abstract: Antiphospholipid syndrome (APS) is a systemic autoimmune condition characterised Citation: Anunciación-Llunell, A.; by the presence of antiphospholipid antibodies (aPL) associated with vascular thrombosis and/or Muñoz, C.; Roggenbuck, D.; Frasca, pregnancy complications. In a cohort of 74 yet diagnosed APS individuals fulfilling Sydney laboratory S.; Pardos-Gea, J.; Esteve-Valverde, E.; criteria (twice positive for lupus anticoagulant, anticardiolipin, aCL, and/or anti-β2glycoprotein I, Alijotas-Reig, J.; Miró-Mur, F. aβ2GPI), 33 out of 74 were obstetric APS (OAPS) and 41 thrombotic APS (TAPS) patients. 39% of Differences in Antiphospholipid TAPS patients were women. Although aPL detection was persistent, we observed an oscillatory Antibody Profile between Patients with Obstetric and Thrombotic aPL positivity in 56.7% and a transient seroconversion in 32.4% of APS patients at enrolment. Thus, Antiphospholipid Syndrome. Int. J. we tested their sera in a line immunoassay that simultaneously detected IgG or IgM for criteria Mol. Sci. 2022, 23, 12819. https:// (aCL and aβ2GPI) and non-criteria (anti-phosphatidylserine, aPS; anti-phosphatidic acid, aPA; anti- doi.org/10.3390/ijms232112819 phosphatidylinositol, aPI; anti-annexin 5, aA5; anti-prothrombin, aPT; anti-phosphatidylethanolamine; anti-phosphatidylglycerol, and anti-phosphatidylcholine) aPL. OAPS and TAPS patients displayed Academic Editors: Ritva Tikkanen different but overlapping clusters based on their aPL reactivities. Specifically, while OAPS patients and Joan Torras-Ambròs showed higher aPA, aPS, aA5, aβ2GPI and aPT IgM levels than TAPS patients, the latter displayed Received: 2 September 2022 higher reactivity in aCL, aPI and aA5 IgG. Eventually, with a cut-off of the 99th percentile established Accepted: 22 October 2022 from a population of 79 healthy donors, TAPS patients significantly tested more positive for aCL and Published: 24 October 2022 aA5 IgG than OAPS patients, who tested more positive for aPA, aPS and aβ2GPI IgM. Transiently Publisher’s Note: MDPI stays neutral seronegative APS patients showed non-criteria aPL positivity twice in sera obtained 3 months apart. with regard to jurisdictional claims in Overall, our data show that APS patients presented clusters of aPL that define different profiles published maps and institutional affil- between OAPS and TAPS, and persistent non-criteria aPL positivity was observed in those who are iations. transiently seronegative. Keywords: antiphospholipid antibodies; thrombotic antiphospholipid syndrome; obstetric antiphos- pholipid syndrome; non-criteria antiphospholipid antibody; line Immunoassay Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons 1. Introduction Attribution (CC BY) license (https:// Antiphospholipid syndrome (APS) is an autoimmune disease characterised by vas- creativecommons.org/licenses/by/ cular thrombosis, various obstetrical adverse events and persistent antiphospholipid anti- 4.0/). bodies (aPL). The conventionally accepted aPL in terms of classification criteria (Sydney Int. J. Mol. Sci. 2022, 23, 12819. https://doi.org/10.3390/ijms232112819 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 12819 2 of 15 criteria) include lupus anticoagulant (LA), IgG/IgM anticardiolipin antibodies (aCL) and IgG/IgM antibodies against β2-glycoprotein I (aβ2GPI) [1,2]. Patients with clinical ob- stetric and thrombotic APS features but without detectable criteria aPL are defined as seronegative or non-criteria APS patients [3]. Since then, methodological approaches to detect new antigenic targets have been developed and several non-criteria aPL can be detected in patients with clinical APS features [4,5]. The group of non-criteria aPL en- compasses anti-phosphatidylethanolamine (aPE), anti-phosphatidylserine/prothrombin (aPS/PT) complex, anti-vimentin, and anti-annexin 5 (aA5) among others [6]. The detection of non-criteria aPL aids in the serological diagnosis of seronegative APS and warrants a similar therapeutic management as seropositive APS [7]. The prevalence of these persistent non-criteria aPL is notorious in distinct thrombotic APS (TAPS) subsets, as for instance in 10–15% of APS patients with unexplained venous thrombosis [8]. In preeclampsia (PE) the occurrence of multiple aPL encompassing non-criteria aPL was associated with severe PE disease [9]. However, there are few studies investigating the link between the occurrence of aPL and obstetrical or thrombotic outcomes. As there is accumulating evidence that some non-criteria APS patients could be persis- tently negative for criteria aPL, the detection of non-criteria aPL appears to be essential for their diagnosis. Moreover, changes in aPL titres during pregnancy sometimes cause loss of aPL positivity [10]. In the last decade, several studies have reported obstetric patients suffering from seronegative-APS for whom non-criteria aPL might be present [11]. A recent retrospective study reported that seronegative APS is rather more obstetrical than thrombotic phenotypes [7]. The cumulative incidence of adverse obstetrical events was similar in seronegative and seropositive APS patients, although higher rates of intrauter- ine deaths, PE, and lower live birth term were observed in seropositive APS [7]. When comparing obstetric APS (OAPS) and non-criteria OAPS, and both receiving the same treatment, similar foetal–maternal outcomes were observed [12]. In addition, the use of methodologies that simultaneously screen for multiple criteria and non-criteria aPL help in the laboratory diagnosis of APS [13,14] as well as in defining aPL profiles [15] and clinical APS phenotypes [16]. Furthermore, signalling pathways at the intersections of coagulation and innate im- mune signalling distinct from those induced by LPS could be activated by aPL through endothelial protein C receptor (EPCR) [17]. Whether this molecular signalling depends on cellular or molecular specificity is still elusive, but aPL signalling by only lipid-reactive antibodies or by aβ2GPI triggers the EPCR pathway both in immune cells and in tro- phoblasts [17]. Thus, different aPL could induce common molecular pathways leading to different cellular signals and activation. In contrast to patients with OAPS, only patients with thrombotic manifestations carry an increased risk of subclinical atherosclerosis [18]. Thus, distinctive pathogenic mecha- nisms may be responsible for the two outcome. In vivo models of foetal loss suggested that aPL effects could be mediated by acute placental inflammation. However, histopathological examination of APS placentae did not support a widespread inflammatory signature [19]. aPL are thought to recognise their antigens on placental tissues, inhibit the growth and differentiation of trophoblast cells and, eventually cause defective placentation on the basis of thrombogenic mechanisms [20]. However, recurrent pregnancy complications associated with aPL cannot be explained solely by thrombosis and alternative pathogenic mechanisms must be considered. Genic variants related to blood coagulation pathways and the immune responses were found to be associated with thrombotic APS and IgG purified from both types of pathologies was identified to modulate genes related to cell responses to stress, cell adhesion and extracellular matrix, MAPK signalling modulation and cell interactions [21]. These different mechanisms could be initiated by aPL having unique antigen specificity, and characterisation of the molecular basis of pathogenic mechanisms may help in the diagnosis and choice of therapy options. The current classification criteria limit the laboratory parameters to three groups of aPL: LA, aCL and aβ2GPI. However, the optimisation of laboratory diagnosis and risk Int. J. Mol. Sci. 2022, 23, 12819 3 of 15 stratification opens the field towards the so-called non-criteria aPL. In this study, we explore the differences in aPL reactivity between OAPS and TAPS patients by assessing criteria and non-criteria aPL with a multiplex line immune assay (LIA). This will provide further evidence for the presence of diverse pathological mechanisms in both clinical phenotypes of APS based on the corresponding aPL profile. 2. Results 2.1. Demographic and Laboratory Data of APS Patients A cohort of 74 diagnosed APS patients fulfilling the laboratory Sydney criteria, twice positive for LA, aCL and/or aβ2GPI, were divided according to their clinical criteria into obstetric (OAPS, n = 33) and thrombotic (TAPS, n = 41) APS patients. Differences in demographic and clinical data between both subsets, but not in laboratory category were observed (Table 1). Thrombotic patients were older than obstetric ones, and only 39% of patients were women in TAPS. The thrombotic APS patients displayed higher body mass indices and showed higher percentages of arterial hypertension or dyslipidaemia. In spite of these differences in demographic and clinical features, both subsets of APS had similar frequencies of patients belonging to one of the four laboratory categories (Table 1). However, significant differences were observed for the number of patients being persistently positive for any aPL. Whereas TAPS patients showed a higher frequency of persistent aPL positivity than OAPS patients (23/41 vs. 9/24, p = 0.018), the latter showed a more oscillatory profile (Table 1). Table 1. Demographics of obstetric antiphospholipid syndrome (OAPS) and thrombotic antiphos- pholipid syndrome (TAPS) patients. Feature OAPS TAPS p-Value Number 33 41 Age (median (IQR)) 39.96 (5.95) 53.44 (18.39) <0.0001 * Gender (Female (%)) 33 (100) 16 (39.0) <0.0001 ** BMI (median (IQR)) 22.94 (4.31) 29.79 (7.47) <0.0001 * AHT (%) 3 (9.7) 22 (53.7) <0.001 ** Dyslipidaemia (%) 0 (0) 22 (53.7) <0.0001 ** AID (%) 4 (12.1) 7 (17.1) 0.746 ** Lab cat 1 (%): 0.072 *** I 6 (18.2) 7 (17.1) IIa 18 (54.5) 30 (73.2) IIb 2 (6.1) 3 (7.3) IIc 7 (21.2) 1 (2.4) aPL persistency (%): 0.018 ** Persistent 9 (27.3) 23 (56.1) Oscillatory 24 (72.7) 18 (43.9) 1 Laboratory category: category I, triple or doble positivity for any criteria aPL; category II, single positivity for LA (IIa), aCL (IIb), or aβ2GPI (IIc). AHT, arterial hypertension; aPL, antiphospholipid antibody; BMI, body mass index; Lab cat, laboratory category; AID, other autoimmune diseases. * Mann-Whitney U test; ** Fisher’s exact test; *** Pearson’s Chi-squared test. 2.2. Criteria and Non-Criteria aPL Screening in Healthy and APS Individuals Given the lack of significant differences in the laboratory categories between the OAPS and TAPS groups and the relatively high percentage of APS patients without criteria aPL at enrolment in both subsets (TAPS: 34.1%, OAPS: 30.3%), we searched for the occurrence of non-criteria aPL in these patients. To this end, we detected 10 different aPL IgG or IgM by LIA (GA Generic Assays GmbH, Dahlewtiz, Germany) in both APS subsets and in 79 age- and gender-matched healthy donors. First, we determined individual cut-offs for each aPL using the group of healthy donors (Figure 1). The highest percentiles assessed from healthy donors for each aPL are depicted in Table 2. Some aPL, such as anti-phosphatidylcholine (aPC), showed little reactivity to both IgG and IgM, which resulted in low cut-off values. IgG or IgM by LIA (GA Generic Assays GmbH, Dahlewtiz, Germany) in both APS subsets and in 79 age- and gender-matched healthy donors. First, we determined individual cut- offs for each aPL using the group of healthy donors (Figure 1). The highest percentiles assessed from healthy donors for each aPL are depicted in Table 2. Some aPL, such as anti- phosphatidylcholine (aPC), showed little reactivity to both IgG and IgM, which resulted Int. J. Mol. Sci. 2022, 23, 12819 4 of 15 in low cut-off values. Figure 1. Antiphospholipid antibody (aPL) levels in sera of healthy individuals (blue dots) and Figure 1. Antiphospholipid antiphospholipid syndrome antibody (aPL) levels (APS) patients in sera Quantification (red dots). of healthy individuals of each(blue aPL dots) and by was run an- tiphospholipid densitometry of syndrome (APS) bands their respective patientson(red thedots). strip Quantification of each (LIA) of line immunoassay aPL was run by densi- incubated with tometry of their anti-human respective IgG (left graph) bands or IgMon the strip (right of line graph). Netimmunoassay mean density(LIA) valuesincubated withas are reported anti-human arbitrary IgG (left graph) or IgM (right graph). Net mean density values are reported as arbitrary units (AU) units (AU) as indicated in Section 4.3. of Materials and Methods. Dark lines show the mean of each as indicated in Section 4.3. of Materials and Methods. Dark lines show the mean of each aPL’s reac- aPL’s reactivity. aCL, anti-cardiolipin; aPA, anti-phosphatidic acid; aPI, anti-phosphatidylinositol; tivity. aCL, anti-cardiolipin; aPA, anti-phosphatidic acid; aPI, anti-phosphatidylinositol; aPC, anti- aPC, anti-phosphatidylcholine; phosphatidylcholine; aPE, anti-phosphatidylethanolamine; aPE, anti-phosphatidylethanolamine; aPG, aPG, anti-phosphatidylglycerol; anti-phosphatidylglycerol; aPS, aPS, anti-phosphatidylserine; aB2, anti-β2-glycoprotein-1; aA5, anti-annexin 5;5;aPT, anti-phosphatidylserine; aB2, anti-β2-glycoprotein-1; aA5, anti-annexin aPT,anti-prothrombin. anti-prothrombin. Table2.2.Percentiles Table Percentilesfor foreach eachantiphospholipid antiphospholipidantibody antibody(aPL) (aPL)assessed assessedbybyline lineimmunoassay immunoassay(LIA). (LIA). aPLaPL IgG IgG Percentile aCL aPA aPE aPG aPI aPS aA5 aβ2GPI aPT Percentile aCL aPA aPE aPG aPI aPS aA5 aβ2GPI aPT 90th 708 355 2 65 287 353 629 1016 711 90th 95th 708 880 355562 212 65 104 504287 567353 828 629 1016 1532 711 787 95th 99th 8801193 5621148 1244 104 199 688504 1029567 1421828 1532 1958 787 1006 99th 1193 1148 44 199 688 1029 1421 1958 1006 aPL IgM aCL aPA aPE aPG aPL IgM aPI aPS aA5 aβ2GPI aPT 90th aCL757 aPA 703 225 aPE 36 aPG 421 aPI 547aPS 1480aA5 1981 aβ2GPI 1231 aPT 95th 911 1054 455 72 657 767 1617 2115 1341 90th 757 703 225 36 421 547 1480 1981 1231 99th 95th 9111082 1260 1054 953 455 183 72 1073657 1198767 19431617 2375 2115 2231 1341 99th 1082 1260Antiphospholipid 953 antibodies 183 to cardiolipin 1073 (aCL), phosphatidic 1198 1943acid (aPA), phosphatidylinositol 2375 2231 (aPI), phosphatidylcholine (aPC), phosphatidylethanolamine (aPE), phosphatidylglycerol (aPG), Antiphospholipid antibodies to cardiolipin (aCL), phosphatidic acid (aPA), phosphatidylinositol (aPI), phos- phosphatidylserine phatidylcholine (aPS), β2-glycoprotein-1 (aPC), phosphatidylethanolamine (aβ2GPI), (aPE), annexin 5 (aA5), phosphatidylglycerol (aPG), and prothrombin (aPS), phosphatidylserine (aPT) were determined by LIA. Values are the mean density arbitrary units resulting from the densitom- β2-glycoprotein-1 (aβ2GPI), annexin 5 (aA5), and prothrombin (aPT) were determined by LIA. Values are the mean etry density of aPL arbitrary bands on units eachresulting from the densitometry of aPL bands on each LIA strip. LIA strip. To check for the linearity of aPL detection by LIA and the location of the cut-off on each aPL curve, we performed a serial dilution of a serum from an APS patient who was strongly positive for aCL IgG and aβ2GPI IgG by a commercial chemiluminescent assay. This APS patient also showed reactivity to other non-criteria aPL by LIA (Figure 2a). The established cut-off values (99th percentile) were in the linear parts of each corresponding aPL curve (Figure 2b, red line). These data show that the established cut-offs can readily be used to discriminate aPL positivity. To check for the linearity of aPL detection by LIA and the location of the cut-off on each aPL curve, we performed a serial dilution of a serum from an APS patient who was strongly positive for aCL IgG and aβ2GPI IgG by a commercial chemiluminescent assay. This APS patient also showed reactivity to other non-criteria aPL by LIA (Figure 2a). The established cut-off values (99th percentile) were in the linear parts of each corresponding Int. J. Mol. Sci. 2022, 23, 12819 5 of 15 aPL curve (Figure 2b, red line). These data show that the established cut-offs can readily be used to discriminate aPL positivity. Figure Figure 2. Quantification 2. Quantification of theoflinear the linear detection detection of antiphospholipid of antiphospholipid antibody antibody (aPL).(aPL). SerumSerumfrom afrom a triple-positive triple-positive patient patient with with antiphospholipid antiphospholipid syndrome syndrome (APS)(APS) was serially was serially diluted diluted to assess to assess aPLaPL levels levels by line by line immunoassay immunoassay (LIA). (LIA). (a) Levels (a) Levels of different of different aPLaPL werewere detected detected by LIA by LIA at theat indicated the indicated dilutions. dilutions. Dilution Dilution factorfactor 1 (vertical 1 (vertical greydesigned grey line) line) designed the dilution the dilution recommended recommended by the LIA byman- the LIA ufacturer (1:33). The manufacturer other (1:33). Thedilution factors factors other dilution are in are reference to thistodilution in reference factorfactor this dilution 1. (b)1.Individual (b) Individual aPL levels for anti-cardiolipin (aCL) IgG (top left), anti-phosphatidic acid (aPA) IgG (top right), anti-phosphatidylglycerol (aPG) IgG (middle left), anti-phosphatidylinositol (aPI) IgG (middle right), anti-phosphatidylserine (aPS) IgG (bottom left), and anti-prothrombin (aPT) IgG (bottom right) at their respective dilution factors. Horizontal red line denotes the 99th-percentile value for each aPL. Experiments were performed in triplicate, showing the mean values of aPL reactivity reported in arbitrary units (AU) as indicated in Section 4.3 of Materials and Methods. aPL levels for anti-cardiolipin (aCL) IgG (top left), anti-phosphatidic acid (aPA) IgG (top right), anti- phosphatidylglycerol (aPG) IgG (middle left), anti-phosphatidylinositol (aPI) IgG (middle right), anti-phosphatidylserine (aPS) IgG (bottom left), and anti-prothrombin (aPT) IgG (bottom right) at their respective dilution factors. Horizontal red line denotes the 99th-percentile value for each aPL. Experiments were performed in triplicate, showing the mean values of aPL reactivity reported in Int. J. Mol. Sci. 2022, 23, 12819 arbitrary units (AU) as indicated in Section 4.3 of Materials and Methods. 6 of 15 2.3. aPL Association in APS Patients 2.3. aPL Since weAssociation in APSdetected simultaneously Patients IgG and IgM isotypes for 10 different aPL, we in- vestigatedSince associations between we simultaneously thedetected distinct aPL IgG (Figure and IgM 3).isotypes We found forseveral clusters 10 different of we aPL, aPL with high correlations, such as the cluster for anti-phosphatidylserine (aPS) IgM, investigated associations between the distinct aPL (Figure 3). We found several clusters aCL IgM,ofand aPLanti-phosphatidic acid (aPA) with high correlations, suchIgM. as theAcluster strong for correlation was also observed anti-phosphatidylserine forIgM, (aPS) aCL aCL IgG,IgM, aPA and IgG,anti-phosphatidic anti-phosphatidylinositol acid (aPA)(aPI) IgM.IgG and aPS A strong IgG. Moreover, correlation was alsoaPE IgG for observed was aCL positively associated IgG, aPA with anti-phosphatidylglycerol IgG, anti-phosphatidylinositol (aPG) (aPI) IgG andIgG. aPS These results clearly IgG. Moreover, aPE IgG indicated distinct expression was positively associatedprofiles of several aPL in APS patients. with anti-phosphatidylglycerol (aPG) IgG. These results clearly indicated distinct expression profiles of several aPL in APS patients. Figure 3. Antiphospholipid antibody (aPL) IgG and IgM associations. Multivariate analysis performed Figureby 3. Antiphospholipid R package ggcorrplotantibody analysed (aPL) IgG and IgM the associations associations. between Multivariate the different analysis aPL. aCL, per- anti-cardiolipin; formed by R package ggcorrplot analysed the associations between the different aPL. aCL, anti- aPA, anti-phosphatidic acid; aPI, anti-phosphatidylinositol; aPC, anti-phosphatidylcholine; aPE, cardiolipin; aPA, anti-phosphatidic acid; aPI, anti-phosphatidylinositol; aPC, anti-phosphatidylcho- anti-phosphatidylethanolamine; aPG, anti-phosphatidylglycerol; aPS, anti-phosphatidylserine; aB2, line; aPE, anti-phosphatidylethanolamine; aPG, anti-phosphatidylglycerol; aPS, anti-phosphatidyl- anti-β2-glycoprotein-1; aA5, anti-annexin 5; aPT, anti-prothrombin. serine; aB2, anti-β2-glycoprotein-1; aA5, anti-annexin 5; aPT, anti-prothrombin. 2.4. Detection of aPL Profiles in TAPS and OAPS Patients 2.4. Detection of aPL Profiles in TAPS and OAPS Patients In order to identify differences in the aPL profiles between TAPS and OAPS patients, In order we to identify performed differencesanalysis a multivariate in the aPL by profiles between principal TAPSanalysis component and OAPS patients, (PCA) from the we performed data of aPLareactivity. multivariate We analysis sought toby principal identify component unique analysis aPL profiles (PCA) aPL and which fromreactivity the datacontributed of aPL reactivity. We most to sought this to identify analysis. uniqueaPS, Accordingly, aPLaPA, profiles aCL,and aPIwhich aPLIgG and aPG reactivity reactivities contributed most to this to mainly contributed analysis. Accordingly, Dimension 1, whereasaPS, aPA, aCL,2aPI Dimension wasand aPG IgG reactivi- determined by aPS, aCL, ties mainly contributed to Dimension 1, whereas Dimension 2 was determined aPA and aβ2GPI IgM reactivities (Figure 4a). PCA clearly distinguished IgG from by aPS, IgM aPL aCL,(Figure aPA and 4b)aβ2GPI IgM reactivities and showed (Figure a different but 4a). PCA overlapping clearly distinguished clustering IgG from of OAPS and TAPS patients IgM (Figure aPL (Figure 4c). 4b) and showed a different but overlapping clustering of OAPS and TAPS patients (Figure 4c). 2.5. Individual aPL Reactivity in TAPS and OAPS Our data suggest that OAPS and TAPS patients are characterised by different aPL profiles. To identify which aPL were differently expressed in TAPS and in OAPS patients, we compared the aPL levels in both phenotypes (Figure 5a). We observed that aPA, aPS, aA5, aβ2GPI and aPT IgM levels were significantly higher in OAPS patients, whereas aCL, aPI and aA5 IgG levels were higher in TAPS patients (Figure 5b). Thus, the different aPL profiles displayed by OAPS and TAPS patients encompassed both criteria and non- criteria aPL. Int. J. Mol. Sci. 2022, 23, 12819 7 of 16 Int. J. Mol. Sci. 2022, 23, 12819 7 of 15 Figure4.4.Antiphospholipid Figure Antiphospholipidantibody antibody(aPL) (aPL)clustering clusteringfor for obstetric obstetric (OAPS) (OAPS) and and thrombotic thrombotic antiphos- antiphos- pholipidsyndrome pholipid syndrome (TAPS) (TAPS) patients. AA principal principalcomponent componentanalysis analysis(PCA) (PCA)ofof logistic regression logistic by regression using the R packages FactoMineR and factoextra was run. (a) The histograms show by using the R packages FactoMineR and factoextra was run. (a) The histograms show the selected the selected variablesand variables andtheir theirvalue valueof ofcontribution contribution toto define defineDimension Dimension 11 (x-axis) (x-axis) and and Dimension Dimension 22 (y-axis). (y-axis). (b) PCA bidimensional reduction resulting from using the defined Dimensions 1 and 2 and the de- (b) PCA bidimensional reduction resulting from using the defined Dimensions 1 and 2 and the degree gree of correlation (cos2) between the variables. (c) OAPS and TAPS patients plotted on this PCA of correlation (cos2) between the variables. (c) OAPS and TAPS patients plotted on this PCA based based on their aPL levels. CL, anti-cardiolipin; PA, anti-phosphatidic acid; PI, anti-phosphatidylin- on theirPC, ositol; aPLanti-phosphatidylcholine; levels. CL, anti-cardiolipin; PE,PA, anti-phosphatidic acid; PI, anti-phosphatidylinositol; anti-phosphatidylethanolamine; PG, anti-phosphatidyl- PC, anti-phosphatidylcholine; glycerol; PE, anti-phosphatidylethanolamine; PS, anti-phosphatidylserine; B2GPI, anti-β2-glycoprotein-1; PG,A5, anti-phosphatidylglycerol; anti-annexin 5; PT, anti- PS, anti-phosphatidylserine; B2GPI, anti-β2-glycoprotein-1; A5, anti-annexin 5; PT, anti-prothrombin. prothrombin. 2.6. aPL Positivity in TAPS and OAPS Patients 2.5. Individual aPL Reactivity in TAPS and OAPS. Whether this aPL reactivity could be translated to a different aPL positivity between Our data suggest that OAPS and TAPS patients are characterised by different aPL TAPS and OAPS patients was assessed thanks to the cut-off value of the 99th percentile profiles. To identify which aPL were differently expressed in TAPS and in OAPS patients, described in Table 2. TAPS patients significantly tested more positive for aCL and aA5 IgG we compared the aPL levels in both phenotypes (Figure 5a). We observed that aPA, aPS, than OAPS patients, who tested more positive for aPA, aPS and aβ2GPI IgM (Table 3). We aA5, aβ2GPI and aPT IgM levels were significantly higher in OAPS patients, whereas aCL, can conclude that OAPS patients presented a set of aPL positivity that was different from aPI and aA5 IgG levels were higher in TAPS patients (Figure 5b). Thus, the different aPL that displayed by TAPS patients. profiles displayed by OAPS and TAPS patients encompassed both criteria and non-crite- ria aPL. Int. J. Mol. Sci. 2022, 23, 12819 8 of 16 Int. J. Mol. Sci. 2022, 23, 12819 8 of 15 Figure 5. Different antiphospholipid antibody (aPL) levels between obstetric (OAPS) and thrombotic Figure 5. Differentsyndrome antiphospholipid antiphospholipid antibody (a) (TAPS) patients. (aPL) levels Radar between plots obstetricaPL of individual (OAPS) levels.and throm- Each aPL boticnormalised was antiphospholipid syndromeof(TAPS) to its maximum patients.reported aPL reactivity (a) Radar in plots of individual arbitrary units (AU)aPL levels. Each as indicated in aPL was normalised to its maximum of aPL reactivity reported in arbitrary units (AU) as indicated Section 4.3 of Materials and Methods. (b) aPL that showed significant differences in the aPL levels in Section 4.3 of Materials and Methods. (b) aPL that showed significant differences in the aPL levels (t-test, p < 0.05) between OAPS (blue dots) and TAPS (yellow dots) patients were selected. IgM of anti- (t-test, p < 0.05) between OAPS (blue dots) and TAPS (yellow dots) patients were selected. IgM of phosphatidic anti-phosphatidic acid (aPA), anti-phosphatidylserine acid (aPA), (aPS),(aPS), anti-phosphatidylserine anti-annexin 5 (aA5), anti-annexin anti-β2 5 (aA5), glycoprotein anti-β2 glycopro-I (aβ2GPI) and anti-prothrombin (aPT) displayed higher levels in OAPS than in TAPS tein I (aβ2GPI) and anti-prothrombin (aPT) displayed higher levels in OAPS than in TAPS patients. patients. IgG of aCL, anti-phosphatidylinositol (aPI) and aA5 presented higher levels in TAPS than IgG of aCL, anti-phosphatidylinositol (aPI) and aA5 presented higher levels in TAPS than in OAPS in OAPS patients. *patients. p < 0.05,***p p< < 0.05, ** *** 0.01, p 95th percentile) or high (>99th percentile) titers for the same non-criteria aPL was two (20.0%) in OAPS and eight (57.1%) in TAPS. There were no differences in the positivity of non-criteria aPL between seronegative OAPS and TAPS (Table 4). The non-criteria antibodies that tested twice positive in the seronegative OAPS were aA5, aPE, aPG, and aPA. We found an enlarged list of the non-criteria aPL that tested twice positive in seronegative TAPS: aA5, aPE, aPG, aPA, aPI, aPS and aPT. Table 4. Percentage of currently seronegative patients in obstetric antiphospholipid syndrome (OAPS) and thrombotic antiphospholipid syndrome (TAPS) who tested positive for the non-criteria antiphospholipid antibody (nc aPL). aPL OAPS TAPS p-Value * Seronegative (%)1 10 (30.3) 14 (34.1) 0.806 nc aPL pos 2 (%) 6 (60.0) 9 (64.3) 1 Twice nc aPL pos 3 (%) 2 (20.0) 8 (57.1) 0.104 1APS diagnosed patients who at the moment of enrolment were seronegative in criteria aPL. 2 APS patients with positive results in any of the non-criteria antiphospholipid used in the line immunoassay. 3 APS patients with two positive results for the same non-criteria antiphospholipid used in the line immunoassay in serum samples drawn >12 weeks apart. * Fisher’s exact test. Since three TAPS patients showed twice aPS and/or aPT positivity, we decided to test all the seronegative APS patients for aPS/PT by ELISA (Aeskulisa Serine-Prothrombin- GM, AESKU.DIAGNOSTICS, Wendelsheim, Germany). We observed that none of the seronegative OAPS patients yielded positive for aPS/PT. The two seronegative TAPS patients who were positive for aPT by LIA also tested positive for aPS/PT by ELISA. A Int. J. Mol. Sci. 2022, 23, 12819 third TAPS patient with twice aPS positivity resulted in medium positivity by10aPS/PT of 16 (Figure 6). Figure 6. Antiphosphatidylserine/prothrombin (aPS/PT) IgG levels in transient seronegative ob- Figure 6. Antiphosphatidylserine/prothrombin (aPS/PT) IgG levels in transient seronegative obstet- ricstetric (OAPS) (OAPS) and thrombotic and thrombotic antiphospholipid antiphospholipid syndromesyndrome (TAPS)(TAPS) patients. patients. Sera fromSera from transient transient sero- seronegative OAPS and TAPS patients were isolated twice at least 12 weeks negative OAPS and TAPS patients were isolated twice at least 12 weeks apart (E1 = extractionapart (E1 = extraction 1, E2 1, E2 = extraction = extraction 2) and2)the andIgG the aPS/PT IgG aPS/PTlevelslevels werewere measured measured by ELISA. by ELISA. Dotted Dotted lines lines represen represen both both ex-ex- tractions of each patient. Horizontal red line denotes the threshold for aPS/PT positivity (>18 tractions of each patient. Horizontal red line denotes the threshold for aPS/PT positivity (>18 U/mL). U/mL). Horizontal Horizontal blue line blue denotes line thethe denotes limit forfor limit low-middle low-middle aPS/PT aPS/PTlevels (>14 levels U/mL (>14 U/mLandand <18<18 U/mL). U/mL). 3. 3. Discussion Discussion Our Our results results pointed pointed outout the the factfact thatthat a distinctive a distinctive set ofset aPLof isaPL is detected detected in OAPS in OAPS and and TAPS patients, suggesting a different profile of aPL in both clinical phenotypes. TAPS patients, suggesting a different profile of aPL in both clinical phenotypes. The diag- The diagnosis of APS relies on the detection of heterogeneous aPL and currently nosis of APS relies on the detection of heterogeneous aPL and currently only criteria aPL only criteria are being accepted for APS classification and, subsequently, for diagnosis. Moreover, in the interpretation of aPL results, antibody profiles help in identifying patients at risk of thrombosis [22]. The non-criteria aPL may be useful in patients with incomplete antibody profiles to confirm or exclude the increased risk profile [23]. The cumulative incidence of adverse obstetrical events was significantly improved in treated versus untreated sero- Int. J. Mol. Sci. 2022, 23, 12819 10 of 15 aPL are being accepted for APS classification and, subsequently, for diagnosis. Moreover, in the interpretation of aPL results, antibody profiles help in identifying patients at risk of thrombosis [22]. The non-criteria aPL may be useful in patients with incomplete antibody profiles to confirm or exclude the increased risk profile [23]. The cumulative incidence of adverse obstetrical events was significantly improved in treated versus untreated seronega- tive APS. These results suggest that we could improve our clinical practice with a better understanding of non-criteria APS patients. Large-scale histological studies found that placental infarction, impaired spiral artery remodelling, decidua inflammation, and the deposition of complement split products were the most common features in the placentas of aPL-positive women. These pathological manifestations suggest the role of angiogenic and inflammatory factors in the pathological process of the disease [24]. aPLs can target β2GPI expressed on the cell surface of trophoblasts, leading to the enhancement of tro- phoblast secretion of pro-inflammatory cytokines and chemokines via the activation of the Toll-like Receptor 4 (TLR4)/MyD88 pathway. The presence of other aPL could induce alternative molecular pathways resulting in similar pathological mechanisms. Evidence of the existence of alternative pathways could be found in the TNF signalling. TNF-α is a critical effector in aPL-related placental injury and further miscarriage [25]. Although these OAPS patients are treated with the standard of care, some of them are refractory to the treatment and additional therapies are required to improve the aPL-related poor obstetric outcomes [26]. Although the Sydney criteria were developed for classification purposes, the labora- tory parameters are used as diagnostic criteria. However, clinical outcomes of APS patients are often not related to criteria aPL detection, suggesting the need for improvement in reli- able aPL assays. Non-criteria aPL could help to further define these diagnostic laboratory criteria that are still prone to discussion and need for regular updates. In this study, we have used the LIA technique manufactured by Generic Assays to report the presence of either criteria and non-criteria aPL in obstetric and thrombotic APS patients. Thus, we are describing the profile of aPL (including criteria and non-criteria) in both phenotypes of APS patients rather than searching for non-criteria aPL in seronegative patients. All APS patients included in this study were diagnosed by fitting the laboratory and the clinical Sydney criteria [1]. Nevertheless, aPL titres are not permanently stable, and their levels and even the aPL positivity could change. We observed that around 32% of APS patients were seronegative for the conventional aPL by the time that the sera for the LIA screening was collected. In agreement with [27], indiscriminate testing is strongly discouraged to avoid incidental findings and, consequently, we only tested for aPL in the context of APS. A limitation of our study is that the LIA did not include the detection of the outmost non-criteria aPL such as the anti-domain I of β2GPI protein and the aPS/PT complex antibodies. aPS/PT have been recently detected in patients with either thrombotic events or pregnancy complications [28], and the anti-domain I of β2GPI was described as pathogenic in APS-related manifestations [29,30]. Common pathways of aPL signalling in OAPS and TAPS would be the complement activation. Studies on murine models of APS have shown that C3 and C5 complement components are crucial for mediating aPL-induced thrombosis in mice [31]; in humans, the placenta from OAPS women exhibited high levels of C4a and C3b deposition, which indicated the possibility of complement activation in the pathogenesis of pregnancy compli- cations in OAPS women [32]. aPL could trigger the activation of complement, generating the active components C3a, C3b, and C5a and ultimately leading to the generation of the membrane attack complex (MAC) [33]. In addition, the binding of these ligands to their receptors on target cells could induce TF release by those cells and results in the formation of thrombosis [34]. Nevertheless, as to which aPL induce MAC or TF should be explored. New technical approaches to aPL profiling have been reported, which elucidate the possibility of aPL profiling for the diagnosis of APS [15]. Besides the LIA, bead- based multiplex techniques to detect several aPL in the same analysis might represent an improvement in the quality of routine laboratory measurements. The different aPL Int. J. Mol. Sci. 2022, 23, 12819 11 of 15 profiles described by our study in OAPS and TAPS patients indicates a more striking underlying immune response against phospholipid structures or corresponding cofactors in these patients, consistent with the conclusion that activation of the immune system is different in those patients who experience obstetrical morbidities compared to those patients who experience thrombosis as clinical manifestations of APS. aPL profiling assists in the differentiation of clinical phenotypes in APS patients. Different studies have reported that the LIA could distinguish APS apart from infectious non-APS patients and could also predict the thrombotic risk in APS patients [14,22]. The clinical meaning of non-criteria antibodies is still being debated, because in the APS diagnosis, triple-positive consensus criteria aPL were more powerful to predict APS early than single-positive or non-criteria aPL positivity [35]. However, the dynamics of aPL levels is also a matter of debate and many APS patients showed an oscillatory aPL positivity. Our data described that thrombotic APS patients who transiently tested seronegative for criteria aPL could be detected as being twice positive for aPS and/or aPT by two different techniques (aPS, aPT by LIA, and aPS/PT by ELISA). Consequently, the reliability of APS diagnosis would improve with testing more aPL. Principal component analysis based on aPL reactivity clearly discriminated IgG from IgM aPL. We observed that TAPS patients overlaid on IgG area whereas OAPS did on IgM. This observation associated with the finding that whereas thrombotic APS displayed more IgG aPL reactivities than obstetric APS, IgM aPL reactivities were more prevalent in OAPS. The idea that IgM isotypes are more prevalent in gestational than thrombotic APS, and vice versa, was also observed recently in a study describing the prevalence of circulating β2GPI-aβ2GPI immune complexes [36]. A limitation of the study is the relatively small number of OAPS and TAPS patients. Nevertheless, we tested multiplex LIA in a total of 153 individuals, where 79 were healthy donors and 74 were APS patients. Data from healthy donors were used to define the cut-offs for each aPL in the multiplex assay. APS patients were encompassed by 33 OAPS and 41 TAPS patients carefully selected for fulfilling the clinical and laboratory Sydney criteria [1] for APS classification. The APS patient enrolment was done in only one centre (Vall d’Hebron Hospital, Barcelona) because the use of a non-standardised methodology and design demanded data acquisition and analysis to be uniformly performed. In addition to describing a set of aPL that is unique for each clinical phenotype of APS, we found for those transiently seronegative OAPS and TAPS patients a positivity for non-criteria aPL detected by LIA and by ELISA. Moreover, this different set of aPL positivity suggests that distinct molecular mechanisms are involved in the pathology of APS and would require future research studies. Different aPL positivity due to gender and age is also a factor that requires attention. Our two groups of APS patients (OAPS and TAPS) presented a bias in gender and age composition and future research must take into account a balanced design. In our study, the group of healthy donors was gender- and age-matched against the whole APS patients. 4. Materials and Methods 4.1. Patients Patients already diagnosed with APS (n = 74, 33 with OAPS and 41 with TAPS) were enrolled. Variable age was considered at enrolment. Blood collected by venipuncture was processed for serum isolation. A second blood sample was obtained from those patients attending medical consultation three months after the first withdrawal. Sera from age- and gender-matched healthy donors (n = 79) were obtained from the Catalan Biobank (Banc de Sang i Teixits, BST). Sera were collected from gel clot tubes (vacutainer, BD) after centrifugation for 15 min at 1500× g. Serum aliquots were stored at −80 ◦ C. Clinical history and laboratory data of these APS patients were summarised in a codified and anonymised database and stored in our institutional repository (Hospital Universitari Vall d’Hebron- Institut de Recerca, HUVH-VHIR). All participants signed an informed consent form. This Int. J. Mol. Sci. 2022, 23, 12819 12 of 15 informed consent together with the research proposal project was approved by the local (HUVH-VHIR) ethics committee (PR(AG)83/2020). 4.2. Laboratory Clinics Arterial hypertension was defined as blood pressure measure in the clinic ≥ 140/90 mm Hg in two or more assessments or when patient was taking oral anti-hypertensive medica- tion. Serum total cholesterol levels were determined with standardised enzymatic methods by the clinical biochemistry laboratory of HUVH-VHIR and dyslipidaemia was defined as total cholesterol values ≥ 240 mg/dL. LA was measured at the clinical haematology laboratory of HUVH-VHIR by using standardised protocols of diluted Russell viper venom time (dRVVT) and silica clotting time (SCT). A patient was considered positive for LA when any test reached values ≥ 1.2. aCL IgG/IgM and aβ2GPI IgG/IgM were measured at the clinical immunology laboratory of HUVH-VHIR. They were assessed by commercial chemiluminescence assays (CLIA; QUANTA Flash, Werfen, Spain) with the BIO-FLASH system (Werfen, Spain). A patient was considered positive for aCL IgG/IgM when aCL CLIA values tested ≥ 40 GPL/MPL and positive for aβ2GPI IgG/IgM when aβ2GPI CLIA values were ≥40 AU. A patient fulfilling clinical criteria for APS according to Sydney criteria [1] and with any aPL positivity in two blood samples collected at least 12 weeks apart was considered as an APS patient. Depending on their aPL positivity, APS patients were classified by laboratory categories: category I, triple (positive for LA and aCL and aβ2GPI) or double (any combination of 2 positives) positivity; category II, single positivity for LA (IIa), aCL (IIb) or aβ2GPI (IIc). 4.3. LIA for the Detection aPL Simultaneous detection of the lipid reactive antibodies aCL, aPE, aPS, aPA, aPC, aPG, aPI, as well as the phospholipid binding protein antibodies aβ2GPI, aA5, and aPT was performed in diluted (30 µL in 1 mL) serum samples following the recommendations of the manufacturer (GA Generic Assays GmbH, Dahlewtiz, Germany). LIA strips were scanned with the GelDoc XR and GelDoc XRplus (Bio-Rad, Hercules, CA, USA) instruments and densitometrically analysed with the Quantity One and Image Lab (Bio-Rad, Hercules, CA, USA) software. Background was subtracted for each sample. The resulting mean density value was considered as the aPL reactivity for each respective aPL and reported in arbitrary units (AU). aPL reactivity can also be deemed as aPL level. High positivity was considered when aPL reactivity reached the 99th percentile, assessed from the aPL reactivity of 79 serum samples from healthy donors. Medium positivity was considered when aPL reactivity reached the 95th percentile assessed as above. 4.4. Quantitative Linear Detection by LIA Technique To study the linearity in the detection of aPL by LIA, serum diluted 1 in 33 (30 µL in 1 mL) from an aPL-positive donor showing positivity for aCL IgG (1584 GPL), aβ2GPI IgG (>6100 AU) and LA (dRVVTS = 3.96; dRVTTC = 2.44) was further serially diluted 0.5, 2, 4, 10, 50, 100 and 500 times. aPL detection was performed as described above. 4.5. Anti-Phosphatidylserine/Prothrombin ELISA aPS/PT IgG and IgM were measured in 96 well microplates by ELISA (Aeskulisa Serine-Prothrombin-GM, AESKU.DIAGNOSTICS, Wendelsheim, Germany) following the procedures indicated by the manufacturer. Optical density was obtained by reading the microplates at 450 nm and 620 nm, for background subtraction, in a microplate reader (Bio Tek ELx800, Agilent, Santa Clara, CA, USA). Sera from 79 healthy donors were used to setup the cut-off at 99th percentile for aPS/PT IgG and IgM, which resulted in a value of 17 U/mL, nearly equal to the 18 U/mL established by the manufacturers. Patients above 18 U/mL were deemed as positive for aPS/PT. Those patients between 14 U/mL and 18 U/mL were considered as low–middle positive. Int. J. Mol. Sci. 2022, 23, 12819 13 of 15 4.6. Statistical Analysis Data were analysed using R software (version 4.2.1) and packages dplyr, tidyverse and R-stats. Data were grouped by OAPS versus TAPS. Continuous variables were sum- marised as means and standard deviations (SD) or medians and interquartile ranges (IQR). Categorical variables are presented as absolute number and relative frequency. Bivari- ate analyses were done by an unpaired t-test for continuous parametric variables or the Mann–Whitney U test for non-parametric variables. Assumption of normal distribution was proven with Shapiro–Wilk tests (p > 0.05 normally distributed data assumed) and Q-Q plots. Fisher’s exact test or the Chi-squared test were used for categorical variables. A two-sided α-level of 0.05 was deemed statistically significant. Multivariate analyses for principal component analysis and correlation were done in R with the packages FactoMineR and factoextra. Multivariate analyses of association between continuous variables were done in R package ggcorrplot. 5. Conclusions A different profile of aPL is displayed by OAPS and TAPS patients by means of multiplexed LIA that combined criteria and non-criteria aPL. It suggests different molecular signalling elicited by the aPL would be the underlying pathological mechanisms for each APS phenotype. The dynamics of aPL positivity were observed in APS patients and rendered some of these patients as transiently seronegative APS patients. Non-criteria aPL positivity would help in the diagnosis of APS and the assessment of these aPL would prevent the underestimation of these patients. Author Contributions: Conceptualization, J.A.-R., A.A.-L. and F.M.-M.; methodology, A.A.-L. and F.M.- M.; formal analysis, F.M.-M., D.R. and S.F.; enrolment of patients, J.A.-R., J.P.-G. and E.E.-V.; collection of data patients, A.A.-L. and C.M.; writing—original draft preparation, F.M.-M.; writing—review and editing, F.M.-M. and D.R.; supervision, J.A.-R.; funding acquisition, J.A.-R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Fundació Ona Futura (ONA-JAR-2021; Port d’Alcudia, Balearic Islands, Spain); and Gravida Fertilitat Avançada (GRA-JAR-2021; Barcelona, Spain). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Vall d’Hebron University Hospital (CEIC- HUVH; PR(AG)83-2020, 4 February 2020). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data used for this study are stored in our institutional repository and will be available upon reasonable request to the corresponding authors. Acknowledgments: The authors thank Laura Donaire Lancharro for the administrative and technical support. Conflicts of Interest: A.A.-L., C.M., J.P.-G., E.E.-V., J.A.-R. and F.M.-M. declare no conflicts of interest. D.R. and S.F. are employees of GA Generic Assays GmbH and D.R. holds shares of the company. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. References 1. Miyakis, S.; Lockshin, M.D.; Atsumi, T.; Branch, D.W.; Brey, R.L.; Cervera, R.; Derksen, R.H.W.M.; De Groot, P.G.; Koike, T.; Meroni, P.L.; et al. International Consensus Statement on an Update of the Classification Criteria for Definite Antiphospholipid Syndrome (APS). J. Thromb. Haemost. 2006, 4, 295–306. [CrossRef] [PubMed] 2. Garcia, D.; Erkan, D. Diagnosis and Management of the Antiphospholipid Syndrome. N. Engl. J. Med. 2018, 378, 2010–2021. [CrossRef] [PubMed] 3. Sanmarco, M. Clinical Significance of Antiphosphatidylethanolamine Antibodies in the So-Called “Seronegative Antiphospholipid Syndrome”. Autoimmun. Rev. 2009, 9, 90–92. [CrossRef] [PubMed] Int. J. Mol. Sci. 2022, 23, 12819 14 of 15 4. Misasi, R.; Capozzi, A.; Longo, A.; Recalchi, S.; Lococo, E.; Alessandri, C.; Conti, F.; Valesini, G.; Sorice, M. New Antigenic Targets and Methodological Approaches for Refining Laboratory Diagnosis of Antiphospholipid Syndrome. J. Immunol. Res. 2015, 2015, 858542. [CrossRef] 5. Egerer, K.; Roggenbuck, D.; Büttner, T.; Lehmann, B.; Kohn, A.; von Landenberg, P.; Hiemann, R.; Feist, E.; Burmester, G.R.; Dörner, T. Single-Step Autoantibody Profiling in Antiphospholipid Syndrome Using a Multi-Line Dot Assay. Arthritis Res. Ther. 2011, 13, R118. [CrossRef] [PubMed] 6. Sciascia, S.; Amigo, M.C.; Roccatello, D.; Khamashta, M. Diagnosing Antiphospholipid Syndrome: “extra-Criteria” Manifestations and Technical Advances. Nat. Rev. Rheumatol. 2017, 13, 548–560. [CrossRef] 7. Abisror, N.; Nguyen, Y.; Marozio, L.; Esteve Valverde, E.; Udry, S.; Pleguezuelo, D.E.; Billoir, P.; Mayer-Pickel, K.; Urbanski, G.; Zigon, P.; et al. Obstetrical Outcome and Treatments in Seronegative Primary APS: Data from European Retrospective Study. RMD Open 2020, 6, e001340. [CrossRef] 8. Cervera, R.; Conti, F.; Doria, A.; Iaccarino, L.; Valesini, G. Does Seronegative Antiphospholipid Syndrome Really Exist? Autoimmun. Rev. 2012, 11, 581–584. [CrossRef] 9. Ferrer-Oliveras, R.; Llurba, E.; Cabero-Roura, L.; Alijotas-Reig, J. Prevalence and Clinical Usefulness of Antiphospholipid and Anticofactor Antibodies in Different Spanish Preeclampsia Subsets. Lupus 2012, 21, 257–263. [CrossRef] 10. Yelnik, C.M.; Porter, T.F.; Branch, D.W.; Laskin, C.A.; Merrill, J.T.; Guerra, M.M.; Lockshin, M.D.; Buyon, J.P.; Petri, M.; Sammaritano, L.R.; et al. Brief Report: Changes in Antiphospholipid Antibody Titers During Pregnancy: Effects on Pregnancy Outcomes. Arthritis Rheumatol. 2016, 68, 1964–1969. [CrossRef] 11. Conti, F.; Andreoli, L.; Crisafulli, F.; Mancuso, S.; Truglia, S.; Tektonidou, M.G. Does Seronegative Obstetric APS Exist? “Pro” and “Cons”. Autoimmun. Rev. 2019, 18, 102407. [CrossRef] [PubMed] 12. Alijotas-Reig, J.; Esteve-Valverde, E.; Ferrer-Oliveras, R.; Sáez-Comet, L.; Lefkou, E.; Mekinian, A.; Belizna, C.; Ruffatti, A.; Hoxha, A.; Tincani, A.; et al. Comparative Study of Obstetric Antiphospholipid Syndrome (OAPS) and Non-Criteria Obstetric APS (NC-OAPS): Report of 1640 Cases from the EUROAPS Registry. Rheumatology 2020, 59, 1306–1314. [CrossRef] [PubMed] 13. Nalli, C.; Somma, V.; Andreoli, L.; Büttner, T.; Schierack, P.; Mahler, M.; Roggenbuck, D.; Tincani, A. Anti-Phospholipid IgG Antibodies Detected by Line Immunoassay Differentiate Patients with Anti-Phospholipid Syndrome and Other Autoimmune Diseases. Autoimmun. Highlights 2018, 9, 6. [CrossRef] 14. Roggenbuck, D.; Borghi, M.O.; Somma, V.; Büttner, T.; Schierack, P.; Hanack, K.; Grossi, C.; Bodio, C.; Macor, P.; von Landenberg, P.; et al. Antiphospholipid Antibodies Detected by Line Immunoassay Differentiate among Patients with An- tiphospholipid Syndrome, with Infections and Asymptomatic Carriers. Arthritis Res. Ther. 2016, 18, 111. [CrossRef] [PubMed] 15. Roggenbuck, D.; Egerer, K.; von Landenberg, P.; Hiemann, R.; Feist, E.; Burmester, G.R.; Dörner, T. Antiphospholipid Antibody Profiling—Time for a New Technical Approach? Autoimmun. Rev. 2012, 11, 821–826. [CrossRef] [PubMed] 16. Roggenbuck, D.; Egerer, K.; Feist, E.; Burmester, G.R.; Dörner, T. Antiphospholipid Antibody Profiling: Association with the Clinical Phenotype of Antiphospholipid Syndrome? Comment on the Article by Otomo et Al. Arthritis Rheum. 2012, 64, 2807–2808. [CrossRef] 17. Müller-Calleja, N.; Hollerbach, A.; Royce, J.; Ritter, S.; Pedrosa, D.; Madhusudhan, T.; Teifel, S.; Meineck, M.; Häuser, F.; Canisius, A.; et al. Lipid Presentation by the Protein C Receptor Links Coagulation with Autoimmunity. Science 2021, 371, eabc0956. [CrossRef] 18. Bettiol, A.; Emmi, G.; Finocchi, M.; Silvestri, E.; Urban, M.L.; Mattioli, I.; Scalera, A.; Lupoli, R.; Vannacci, A.; Di Minno, M.N.D.; et al. Obstetric Antiphospholipid Syndrome Is Not Associated with an Increased Risk of Subclinical Atherosclerosis. Rheumatology 2021, 59, 3709–3716. [CrossRef] 19. Meroni, P.L.; Borghi, M.O.; Raschi, E.; Tedesco, F. Pathogenesis of Antiphospholipid Syndrome: Understanding the Antibodies. Nat. Rev. Rheumatol. 2011, 7, 330–339. [CrossRef] 20. Alijotas-Reig, J.; Esteve-Valverde, E.; Anunciación-Llunell, A.; Marques-Soares, J.; Pardos-Gea, J.; Miró-Mur, F. Pathogenesis, Diagnosis and Management of Obstetric Antiphospholipid Syndrome: A Comprehensive Review. J. Clin. Med. 2022, 11, 675. [CrossRef] 21. Ripoll, V.M.; Lambrianides, A.; Pierangeli, S.S.; Poulton, K.; Ioannou, Y.; Heywood, W.E.; Mills, K.; Latchman, D.S.; Isenberg, D.A.; Rahman, A.; et al. Changes in Regulation of Human Monocyte Proteins in Response to IgG from Patients with Antiphospholipid Syndrome. Blood 2014, 124, 3808–3816. [CrossRef] [PubMed] 22. Park, H.S.; Gu, J.Y.; Jung, H.S.; Kim, H.K. Thrombotic Risk of Non-Criteria Anti-Phospholipid Antibodies Measured by Line Immunoassay: Superiority of Anti-Phosphatidylserine and Anti-Phosphatidic Acid Antibodies. Clin. Lab. 2019, 65, 207–213. [CrossRef] [PubMed] 23. Devreese, K.M.J.; Zuily, S.; Meroni, P.L. Role of Antiphospholipid Antibodies in the Diagnosis of Antiphospholipid Syndrome. J. Transl. Autoimmun. 2021, 4, 100134. [CrossRef] [PubMed] 24. Viall, C.A.; Chamley, L.W. Histopathology in the Placentae of Women with Antiphospholipid Antibodies: A Systematic Review of the Literature. Autoimmun. Rev. 2015, 14, 446–471. [CrossRef] 25. Berman, J.; Girardi, G.; Salmon, J.E. TNF-α Is a Critical Effector and a Target for Therapy in Antiphospholipid Antibody-Induced Pregnancy Loss. J. Immunol. 2005, 174, 485–490. [CrossRef] 26. Alijotas-Reig, J.; Esteve-Valverde, E.; Llurba, E.; Gris, J.M. Treatment of Refractory Poor APL-Related Obstetric Outcomes with TNF-Alpha Blockers: Maternal-Fetal Outcomes in a Series of 18 Cases. Semin. Arthritis Rheum. 2019, 49, 314–318. [CrossRef] Int. J. Mol. Sci. 2022, 23, 12819 15 of 15 27. Devreese, K.M.J.; de Groot, P.G.; de Laat, B.; Erkan, D.; Favaloro, E.J.; Mackie, I.; Martinuzzo, M.; Ortel, T.L.; Pengo, V.; Rand, J.H.; et al. Guidance from the Scientific and Standardization Committee for Lupus Anticoagulant/Antiphospholipid Antibodies of the International Society on Thrombosis and Haemostasis: Update of the Guidelines for Lupus Anticoagulant Detection and Interpretation. J. Thromb. Haemost. 2020, 18, 2828–2839. [CrossRef] 28. Radin, M.; Foddai, S.G.; Cecchi, I.; Rubini, E.; Schreiber, K.; Roccatello, D.; Bertolaccini, M.L.; Sciascia, S. Antiphosphatidylser- ine/Prothrombin Antibodies: An Update on Their Association with Clinical Manifestations of Antiphospholipid Syndrome. Thromb. Haemost. 2020, 120, 592–598. [CrossRef] 29. Chighizola, C.B.; Pregnolato, F.; Andreoli, L.; Bodio, C.; Cesana, L.; Comerio, C.; Gerosa, M.; Grossi, C.; Kumar, R.; Lazzaroni, M.G.; et al. Beyond Thrombosis: Anti-B2GPI Domain 1 Antibodies Identify Late Pregnancy Morbidity in Anti- Phospholipid Syndrome. J. Autoimmun. 2018, 90, 76–83. [CrossRef] 30. Yin, D.; Chayoua, W.; Kelchtermans, H.; de Groot, P.G.; Moore, G.W.; Gris, J.C.; Zuily, S.; Musial, J.; de Laat, B.; Devreese, K.M.J. Detection of Anti-Domain I Antibodies by Chemiluminescence Enables the Identification of High-Risk Antiphospholipid Syndrome Patients: A Multicenter Multiplatform Study. J. Thromb. Haemost. 2020, 18, 463–478. [CrossRef] 31. Thurman, J.M.; Kraus, D.M.; Girardi, G.; Hourcade, D.; Kang, H.J.; Royer, P.A.; Mitchell, L.M.; Giclas, P.C.; Salmon, J.; Gilkeson, G.; et al. A Novel Inhibitor of the Alternative Complement Pathway Prevents Antiphospholipid Antibody-Induced Pregnancy Loss in Mice. Mol. Immunol. 2005, 42, 87–97. [CrossRef] [PubMed] 32. Shamonki, J.M.; Salmon, J.E.; Hyjek, E.; Baergen, R.N. Excessive Complement Activation Is Associated with Placental Injury in Patients with Antiphospholipid Antibodies. Am. J. Obstet. Gynecol. 2007, 196, 167.e1. [CrossRef] 33. Velásquez, M.; Rojas, M.; Abrahams, V.M.; Escudero, C.; Cadavid, Á.P. Mechanisms of Endothelial Dysfunction in Antiphospho- lipid Syndrome: Association with Clinical Manifestations. Front. Physiol. 2018, 9, 1840. [CrossRef] [PubMed] 34. Chaturvedi, S.; Braunstein, E.M.; Yuan, X.; Yu, J.; Alexander, A.; Chen, H.; Gavriilaki, E.; Alluri, R.; Streiff, M.B.; Petri, M.; et al. Complement Activity and Complement Regulatory Gene Mutations Are Associated with Thrombosis in APS and CAPS. Blood 2020, 135, 239–251. [CrossRef] [PubMed] 35. Chayoua, W.; Yin, D.M.; Kelchtermans, H.; Moore, G.W.; Gris, J.C.; Musiał, J.; Zuily, S.; Ten Cate, H.; De Laat, B.; Devreese, K.M.J. Is There an Additional Value in Detecting Anticardiolipin and Anti-B2 Glycoprotein i IgA Antibodies in the Antiphospholipid Syndrome? Thromb. Haemost. 2020, 120, 1557–1568. [CrossRef] [PubMed] 36. Naranjo, L.; Stojanovich, L.; Djokovic, A.; Andreoli, L.; Tincani, A.; Maślińska, M.; Sciascia, S.; Infantino, M.; Garcinuño, S.; Kostyra-Grabczak, K.; et al. Circulating Immune-Complexes of IgG/IgM Bound to B2-Glycoprotein-I Associated with Complement Consumption and Thrombocytopenia in Antiphospholipid Syndrome. Front. Immunol. 2022, 13, 957201. [CrossRef] [PubMed]