The aim of the study was to determine the prevalence and clinical associations of antiphosphatidylserine/prothrombin antibodies (aPS/PT) with thrombosis and pregnancy loss in Chinese patients with antiphospholipid syndrome (APS) and seronegative APS (SNAPS).
One hundred and eighty six Chinese patients with APS (67 primary, 119 secondary), 48 with SNAPS, 176 disease controls (79 systemic lupus erythematosus [SLE], 29 Sjogren’s syndrome [SS], 30 ankylosing spondylitis [AS], 38 rheumatoid arthritis [RA]) and 90 healthy donors were examined. IgG and IgM aPS/PT, IgG/IgM/IgA anticardiolipin (aCL) and IgG/IgM/IgA anti-β2-glycoprotein I (anti-β2GPI) antibodies were tested by ELISA.
One hundred and sixty (86.0%) of APS patients were positive for at least one aPS/PT isotype. One hundred and thirty five (72.6%) were positive for IgG aPS/PT, 124/186 (66.7%) positive for IgM aPS/PT and 99 (53.2%) positive for both. Approximately half of the SNAPS patients were positive for IgG and/or IgM aPS/PT. Highly significant associations between IgG aPS/PT and venous thrombotic events (odds ratio [OR]=6.72) and IgG/IgM aPS/PT and pregnancy loss (OR=9.44) were found. Levels of IgM aPS/PT were significantly different in APS patients with thrombotic manifestations and those with fetal loss (p=0.014). The association between IgG/IgM aPS/PT and lupus anticoagulant (LAC) was highly significant (p<0.001). When both were positive, the OR for APS was 101.6. Notably, 91.95% (80/87) of LAC-positive specimens were positive for IgG and/or IgM aPS/PT, suggesting aPS/PT is an effective option when LAC testing is not available.
Anti-PS/PT antibody assays demonstrated high diagnostic performance for Chinese patients with APS, detected some APS patients negative for criteria markers and may serve as potential risk predictors for venous thrombosis and obstetric complications.
The antiphospholipid syndrome (APS) is a thrombophilic disorder characterized by clinical manifestations of vascular thrombosis and obstetric complications associated with the presence of specific antiphospholipid antibodies (aPLs). The conventional aPLs typically evaluated include anticardiolipin (aCL) antibody (IgG and IgM), anti-β2-glycoprotein I (anti-β2GPI) antibodies (IgG and IgM) and lupus anticoagulant (LAC). The persistent presence of at least one of these three biomarkers at medium to high titers is recognized as the critical laboratory diagnostic criteria for the definite classification of patients with APS by international consensus detailed in the updated 2006 Sydney modification of the original 1999 “Sapporo criteria” , .
APLs are a heterogeneous group of immunoglobulins directed against phospholipids or specific phospholipid-binding plasma proteins such as β2GPI and prothrombin (PT) , , . Antibodies to prothrombin have been investigated for years, but results have been variable and have not been included in the classification criteria. Atsumi et al.  showed that when prothrombin is complexed to phosphatidylserine however, the prothrombin-phosphatidylserine complex (PS/PT) has significantly improved performance over prothrombin alone. Numerous papers have since demonstrated the important role of aPS/PT in APS, lupus and other systemic inflammatory disorders , ,  and have confirmed the enhanced performance of aPS/PT compared with antiprothrombin (aPT) antibodies , . Anti-PS/PT antibodies are strongly correlated with risk of thrombosis and LAC, and levels are higher in “triple positive” (positive for aCL IgG and/or IgM, aβ2GPI IgG and/or IgM and LAC) patients , , , , . Anti-PS/PT antibodies were recommended as helpful to assess the risk of thrombosis and diagnosis of APS by the task force on diagnostics at the 14th International Congress on APS .
The concept of seronegative APS (SNAPS) was first introduced in 2003 by Hughes and Khamashta to describe patients with clinical manifestations highly suggestive of APS, but persistently negative for LAC, IgG/IgM aCL and IgG/IgM anti-β2GPI antibodies, the classical laboratory markers of APS . Although the Sydney modification of the original APS classification criteria officially expanded the laboratory tests criteria by inclusion of anti-β2GPI antibodies and consequently was able to classify more patients, it is clear that some patients with clinical manifestations of APS remain seronegative for the conventional assays or have laboratory results of unclear clinical significance, which in both cases can lead to diagnostic uncertainty and possible delay in obtaining timely treatment. IgA aCL and IgA β2GPI antibodies, while not part of the official laboratory criteria markers, are suggested as useful supplemental assays. New biomarkers to accurately identify SNAPS patients are critically needed since failure to treat patients with APS can lead to serious consequences. As with classical APS, patients with SNAPS can have an accelerated progression resulting in multi-organ failure, a life-threatening medical condition known as catastrophic APS .
Although the assay for aPS/PT antibodies was first reported in 2000, the test can be difficult to produce, and until recently aPS/PT antibodies were rarely detected due to lack of standardized commercial kits available to clinical laboratories . The present study was designed to evaluate the prevalence and significance of aPS/PT using a commercially available kit in a large cohort of very well-characterized patients with APS, SNAPS and other autoimmune diseases from the Shanghai area of China and to analyze the relationship of LAC, aPS/PT and other aPL antibodies with clinical manifestations.
Materials and methods
The study included 186 consecutive patients with APS from the APS-SH database, which was established in 2000 by expert rheumatologists and statisticians at the Shanghai Jiaotong University School of Medicine (Shanghai, China). Patients included met the criteria for the classification of APS using the Sydney criteria , . All patients had medical histories, laboratory tests, imaging studies and treatment documented and underwent a medical follow-up by expert rheumatologists. Serum samples were collected and frozen at −80°C until testing. We also recruited 48 patients with SLE and definite thrombosis or miscarriage, but negative for criteria aPL laboratory markers. These patients were classified as patients with SNAPS. Patients with other autoimmune diseases, including 79 SLE without thrombosis and miscarriage, 29 SS, 30 AS and 38 RA patients classified using standard diagnostic criteria were also recruited , , , . Finally, samples from 90 age and sex-matched healthy donors with neither autoimmune nor infectious diseases were collected as healthy controls (HC).
The study was performed in accordance with the Declaration of Helsinki and the Principles of Good Clinical Practice and approved by the Institutional Review Broad of Ruijin Hospital (ID: 2016-61), Shanghai Jiaotong University School of Medicine, Shanghai, China.
Antibodies to phosphatidylserine-prothrombin (aPS/PT)
The presence of aPS/PT IgG and IgM antibodies was analyzed using semiquantitative QUANTA Lite® ELISA kits provided by Inova Diagnostics, Inc. (San Diego, CA, USA). The suggested cutoff values for IgG and IgM aPS/PT antibodies were defined as >30 units. All samples were tested blinded in duplicate in the laboratory of Ruijin Hospital. Note throughout this paper that the nomenclature “x”/”x” indicates “and/or.”
Antibodies to cardiolipin (aCL) and β2-glycoprotein I (anti-β2GPI)
IgG/IgM aCL and IgG/IgM anti-β2GPI antibodies were measured by in-house ELISA and commercial ELISA kits (Euroimmun, Luebeck, Germany) when patients were initially enrolled. The serum samples, which had been stored at −80°C, were retested for IgG/IgM/IgA aCL and IgG/IgM/IgA anti-β2GPI antibodies by commercial ELISA kits (QUANTA Lite®, Inova Diagnostics, Inc., San Diego, CA, USA). The suggested cutoff for IgG, IgM and IgA aCL is >20 GPL/MPL and for IgG, IgM and IgA anti-β2GPI is >20 units (calculated by the 99th percentile of healthy subjects). All samples were tested blindly and in duplicate.
Lupus anticoagulant (LAC)
Plasma samples were tested for LAC when enrolled into our database according to the recommended criteria from the ISTH Subcommittee on Lupus Anticoagulant-Phospholipid-dependent antibodies , , using the Automated Coagulation laboratory 300R (Instrumentation Laboratory, Milan, Italy). Samples were screened using the dilute Russell viper venom time (dRVVT) testing and activated partial thromboplastin time (Instrumentation Laboratory). Ratios higher than 1.10 that could not be corrected by the 50:50 mixture with normal plasma were considered as suggestive of LAC and subjected to dRVVT testing. The dRVVT coagulation test was performed in all samples using Diagen Russell’s viper venom (Diagnostic Reagents Ltd, Oxon, UK) as described by Thiagarajan et al. . Both screening and confirming steps were performed. The LAC was considered positive if the ratio of dRVVT screening time/dRVVT confirming time >1.20.
Statistical analyses were performed using SPSS 23.0 (IBM, Chicago, IL, USA) or Analyze-it ver 4.6 (Analyze-it Software, LTD). T-test, Mann-Whitney U-test, κ-test, Fisher’s exact or χ2-tests were applied. Comparisons between groups were expressed as odds ratio (OR) with 95% confidence interval (95% CI). Sensitivity, specificity, positive and negative predictive values (PPV and NPV), positive and negative likelihood ratios (LR+ and LR−), OR and receiver operating characteristic (ROC) analysis of the aPL assays tested were calculated. Multivariate analysis was performed in order to identify the independent factors that are positively associated with the dependent variable and to control for possible confounding parameters. Multivariate models are controlled for age and sex. The magnitude of each factor was expressed as an OR. p-Values <0.05 were considered significant.
A total of 186 patients with APS and 48 with SNAPS were examined, 164 (88.2%) female, 22 (11.8%) male, mean age 34.2 years. Primary APS (PAPS) was present in 67 patients (36.0%), whereas APS was secondary to other diseases in 119 patients (64.0%) (Table 1), including 94 patients (79.0%) with SLE and 25 patients (21.0%) with other autoimmune diseases.
|Variables||APS (n=186)||SNAPS (n=48)||p-Value|
|Mean age (years±SD)||34.2±13.9||38.9±18.0||0.867|
|PAPS, n (%)||67 (36.0)||N/A|
|SAPS, n (%)||119 (64.0)||N/A|
|Thrombosis only, n (%)||113 (60.7)||36 (75.0)||0.096|
|AT only, n (%)||53 (28.5)||20 (41.7)||0.114|
|VT only, n (%)||68 (36.5)||12 (25.0)||0.182|
|AT+VT, n (%)a||9 (4.8)||8 (16.7)||0.012|
|Pregnancy morbidity only, n (%)||56 (30.1)||8 (16.7)||<0.01|
|Thrombosis+pregnancy morbidity, n (%)b||17 (9.1)||4 (8.3)||0.999|
APS, antiphospholipid syndromes; PAPS, primary antiphospholipid syndrome; SAPS, secondary antiphospholipid syndrome; SNAPS, seronegative APS, patients with clinical manifestations highly suggestive of APS, but with persistently negative LAC, IgG/IgM aCL and IgG/IgM anti-β2-GPI antibodies; AT, arterial thrombosis; VT, venous thrombosis. aIncluding AT+VT. bIncluding thrombosis+pregnancy morbidity.
The prevalence of major APS manifestations included 53 patients (28.5%) presenting with only arterial thrombosis (AT), 68 patients (36.5%) with only venous thrombosis (VT), 9 patients (4.8%) with both AT and VT and 56 patients (30.1%) with only fetal loss. A total number of 73 females in the pregnancy morbidity subset of APS group experienced 201 pregnancy episodes; of these, 145 had obstetric complications, including 44.3% (89/201) early fetal loss (<10 weeks), 21.4% (43/201) late fetal loss (≥10 weeks) and 18.8% (13/69) premature live births. Among the 48 SNAPS patients (female 41, male 7; mean age 38.9 years), 20 patients (41.7%) presented with only AT, 12 patients (25.0 %) with only VT, 8 patients (16.7%) with both AT and VT and 8 patients (16.7%) with only fetal loss (Table 1). In the subgroup of APS patients with thrombosis, stroke was the most frequent clinical manifestation of AT, present in 46 patients (24.7%). For VT, the most frequent clinical manifestation was lower limb DVT, present in 74 patients (39.8%).
Prevalence of aPL and aPS/PT antibodies in study groups
All APS patients were tested by APS criteria assays, as well as IgA aCL and IgA β2GPI, and IgG/IgM aPS/PT. The results are shown graphically in Figure 1A for the total cohort of APS patients and Figure 1B for the subgroup of 48 SNAPS patients. In contrast to the strong reactivity of multiple antibodies in the APS group, only IgG and IgM aPS/PT, and to a lesser extent IgA β2GPI, were detected in the SNAPS subgroup. Using the 30-unit cutoff, 160 (86.0%) of APS patients were positive for at least one aPS/PT isotype. IgG aPS/PT antibodies were positive in 134/186 patients (72.0%), IgM aPS/PT antibodies were positive in 125/186 patients (67.2%) and both IgG and IgM aPS/PT antibodies were positive in 99/186 APS patients (53.2%) (Table 2). The highest prevalence of aPS/PT was in the APS group (72.1%), with a similar percentage seen in the PAPS (70.1%) and SAPS (73.1%) subgroups. Both IgG aPS/PT and IgM aPS/PT were more frequent in the APS group than in other disease and HC groups (p<0.001). The performance of aPS/PT relative to the other aPLs assays in APS, SNAPS, SLE and HC groups is shown in Figure 2.
|No.||IgG or IgM aPS/PT, n (%)||IgG aPS/PT, n (%)||IgM aPS/PT, n (%)||Dual positivity, n (%)a|
|APS||186||160 (86.0)||134 (72.0)||125 (67.2)||99 (53.2)|
|PAPS||67||61 (91.0)||47 (70.1)||53 (79.1)||39 (58.2)|
|SAPS||119||99 (83.2)||87 (73.1)||72 (60.5)||60 (50.4)|
|SNAPS||48||25 (51.0)||14 (28.6)||17 (34.7)||6 (12.2)|
|SLE||79||30 (38.0)||10 (12.7)||26 (32.9)||6 (7.6)|
|SS||29||9 (31.0)||1 (3.4)||8 (27.6)||0 (0)|
|RA||38||0 (0)||0 (0)||0 (0)||0 (0)|
|AS||30||3 (10.0)||0 (0)||3 (10.0)||0 (0)|
|HC||90||7 (8.2)||0 (0)||7 (8.2)||0 (0)|
aPS/PT, antiphosphatidylserine/prothrombin antibody; SLE, systemic lupus erythematosus; SS, Sjogren syndrome; AS, ankylosing spondylitis; RA, rheumatoid arthritis; HC, healthy controls. aIgG and IgM aPS/PT are both positive.
Thirty (38%) of the 79 patients with SLE and no current diagnosis of APS were positive for either IgG and/or IgM PS/PT antibodies. Of the 20 patients with only IgM aPS/PT antibodies, six patients were strongly positive with values >136 units. One SLE patient had only IgG PS/PT antibody >136 units, whereas three additional SLE patients were dual positive at levels >116 units for both IgG and IgM PS/PT antibodies.
Prevalence of aPS/PT antibodies in SNAPS
IgG and/or IgM PS/PT antibodies were detected in 25 (51%) of the SNAPS patients (Table 2 and Figure 2). IgG aPS/PT was positive in 14 (28.6%), IgM aPS/PT was positive in 17 (34.7%) and both were positive in 6 (12.2%) SNAPS patients. The prevalence of IgG aPS/PT in the SNAPS group was higher than the SLE group (χ2=4.2868, p=0.038), other autoimmune diseases groups (RA, SS, AS, p<0.05) or the HC group (p<0.001). IgM aPS/PT positivity was significantly higher in the SNAPS patients compared with HC (p<0.001), but not from the SLE (χ2=0.0092, p=0.924) or SS (χ2=0.2115, p=0.646) groups.
Clinical performance of aPL assays
IgG aPS/PT exhibited the highest sensitivity (72%) among the single aPL assays and showed high specificity (95.8%) (Table 3). IgM aPS/PT also gave a relatively good sensitivity of 67.2%, but the lowest specificity (83.1%) among the aPLs. ROC analysis for detection of APS showed anti- β2GPI IgG with an AUC of 0.874, followed closely by aPS/PT IgG and aCL IgG, both with AUCs of 0.849. LAC (AUC=0.734) showed a significantly lower AUC than aPS/PT IgG (p< 0.001) (Figure 3). Notably, aβ2GPI IgA showed higher sensitivity than aCL IgM and aβ2GPI IgM. Among the aPL assays, the combination of anti-PS/PT IgG and IgM showed the highest sensitivity of 86.0% (160/186), with a specificity of 81.2%. In comparison, LAC had a sensitivity of 46.7% (87/186) with 100% specificity. Combining LAC and aPS/PT IgG/IgM assays resulted in sensitivity of 89.9% (167/186) and a specificity of 81.2% (Table 3).
|Parameters||Sens, (%)||Spec, (%)||PPV||NPV||LR+||LR−||OR|
|aPS/PT IgG/aCL IgG/LAC||84.9||93.9||0.908||0.897||13.918||0.161||86.406|
|aPS/PT IgG/LAC/aCL IgG/aβ2GPI IgG||84.9||93.1||0.898||0.897||12.304||0.162||76.179|
|aCL IgG/aβ2GPI IgG/LAC||70.4||96.9||0.942||0.821||22.710||0.305||75.325|
|aPS/PT IgG and IgM||53.2||97.7||0.943||0.746||23.130||0.479||48.362|
|aCL IgG/M/A/aβ2GPI IgG/M/A/LAC||87.1||87.0||0.827||0.904||6.700||0.148||45.066|
|aPS/PT IgG/M/aβ2GPI IgG/M/A/aCL G/M/A/LAC||93.0||74.3||0.721||0.937||3.619||0.094||38.533|
|aPS/PT IgG/M/LAC/aβ2GPI IgG/M||90.3||78.9||0.753||0.920||4.280||0.123||34.958|
|aPS/PT IgG/M/aβ2GPI IgGM/aCL GM/LAC||90.9||77.4||0.741||0.922||4.022||0.118||34.036|
|aPS/PT IgG/M/aCL IgG/M/A||88.7||79.3||0.753||0.908||4.285||0.142||30.119|
|aPS/PT IgG/M/aβ2GPI IgG/M/A/aCL G/M/A||90.9||74.3||0.716||0.919||3.537||0.122||28.785|
|aPS/PT IgG/M/aβ2GPI IgG/M/aCL G/M||88.7||77.4||0.737||0.906||3.925||0.146||26.901|
|aPS/PT IgG/M/aβ2GPI IgG/M||87.6||78.9||0.748||0.900||4.152||0.157||26.544|
|aCL IgG/M/A/aβ2GPI IgG/M/A||72.6||87.0||0.799||0.817||5.585||0.315||17.673|
aSNAPS patients excluded from calculations, “/”= and/or.
Relationship of aPS/PT with LAC
The association between IgG or IgM aPS/PT antibodies and LAC was highly significant (OR=14.32, 95% CI=7.82–27, p<0.001; OR=7.84, 95% CI=4.45–14.16, p<0.001). Of the 135 patients positive for IgG aPS/PT, 69 (51.1%) were LAC positive, whereas 69/87 (80.2%) LAC-positive patients were IgG aPS/PT positive (Figure 4). Similarly, of the 125 patients with IgM aPS/PT positivity, 65/125 (52.0%) had LAC positivity, whereas of the 87 patients with positive LAC, 66/87 (75.9%) had positive IgM aPS/PT. Of the total 160 aPS/PT IgG/IgM-positive patients, 49.4% (79/160) were LAC positive. By contrast, of the 87 LAC positive sera, 90.8% (79/87) were PS/PT IgG and/or IgM positive. The combination of LAC and aPS/PT IgG yielded an OR of 101.6 (Table 3).
Clinical associations of aPS/PT
Finally, we investigated aPS/PT in subgroups of patients divided by disease manifestations: thrombosis, AT, VT and pregnancy loss. APS patients with thrombotic manifestations only displayed significantly lower IgM aPS/PT titers (median titer=45.63 units) compared with patients with fetal loss only (median titer=91.60 units) (p=0.014). By contrast, there was no significant difference in the median IgG aPS/PT titers among the three thrombosis groups. We then investigated the relationship between IgG and IgM aPS/PT and APS-related clinical manifestations (Table 4). Using multivariate analysis, the most significant associations were aPS/PT IgG (OR=10.50, 95% CI=6.54–17.23, p<0.001) with total thrombosis events, aPS/PT IgG and/or IgM with arterial thrombosis events (OR=6.30, 95% CI=3.58–11.57, p<0.001), aPS/PT IgG (OR=6.42, 95% CI=3.91–10.76, p<0.001) with venous thrombotic events, and aPS/PT IgG and/or IgM with fetal loss (OR=10.41, 95% CI=5.47–21.63, p<0.001).
|aPL assay||Thrombosis||Arterial thrombosis||Venous thrombosis||Pregnancy loss|
|OR (95% CI)||p-Value||OR (95% CI)||p-Value||OR (95% CI)||p-Value||OR (95% CI)||p-Value|
|aCL IgG/M (≥40 PL)||7.72 (4.42–14.05)||<0.001||3.76 (2.13–6.66)||<0.001||3.78 (2.23–6.44)||<0.001||3.74 (2.10–6.67)||<0.001|
|aCL IgG (≥40 GPL)||9.49 (5.15–18.61)||<0.001||3.98 (2.22–7.15)||<0.001||4.18 (2.41–7.26)||<0.001||3.23 (1.76–5.91)||<0.001|
|aCL IgM (≥40 MPL)||3.93 (1.61–10.55)||0.004||1.38(0.44–3.72)||0..546||3.27 (1.32–7.92)||<0.001||4.26 (1.70–10.75)||<0.001|
|aβ2GPI IgG/M||6.21 (3.80–10.37)||<0.001||3.16 (1.85–5.39)||<0.001||3.96 (2.40–6.54)||<0.001||4.36 (2.54–7.49)||<0.001|
|aβ2GPI IgG||7.50 (4.24 13.84)||<0.001||3.95 (2.21–7.06)||<0.001||3.61 (2.09–6.20)||<0.001||3.37 (1.86–6.06)||<0.001|
|aβ2GPI IgM||3.22 (1.72– 6.24)||<0.001||1.62 (0.76–3.27)||0.192||2.68 (1.38–5.11)||<0.001||4.87 (2.48–9.60)||<0.001|
|LAC||7.85 (4.50–14.23)||<0.001||2.72 (1.53–4.78)||<0.001||4.56 (2.70–7.74)||<0.001||5.13 (2.92–9.07)||<0.001|
|aPS/PT IgG/M||9.10 (5.73–14.81)||<0.001||6.30 (3.58–11.57)||<0.001||5.58 (3.27–9.94)||<0.001||10.41 (5.47–21.63)||<0.001|
|aPS/PT IgG||10.50 (6.54–17.23)||<0.001||4.62 (2.76–7.88)||<0.001||6.42 (3.91–10.76)||<0.001||5.66 (3.35–9.75)||<0.001|
|aPS/PT IgM||4.24 (2.78–6.56)||<0.001||3.12 (1.89–5.23)||<0.001||3.24 (2.02–5.29)||<0.001||4.71 (2.79–8.12)||<0.001|
|aCL IgG+aβ2GPI IgG||9.36 (4.81–19.79)||<0.001||4.07 (2.18–7.58)||<0.001||4.09 (2.27–7.36)||<0.001||3.33(1.73–6.37)||<0.001|
|aCL IgG+aβ2GPI IgG+LAC||6.33 (2.79–16.27)||<0.001||3.99 (1.83–8.58)||<0.001||2.33 (1.07–4.88)||0.011||4.06 (1.77–9.35)||<0.001|
|aCL IgG+aβ2GPI IgG+aPS/PT IgG||8.90 (4.44–19.49)||<0.001||3.77 (1.97–7.19)||<0.001||4.27 (2.32–7.89)||<0.001||3.31 (1.67–6.51)||<0.001|
|Double (aCL+aβ2GPI)||6.93 (3.87–12.98)||<0.001||3.55 (1.96–6.40)||<0.001||3.68 (2.12–6.39)||<0.001||4.31 (2.36–7.88)||<0.001|
|Double (aCL+LAC)||6.38 (3.14–14.11)||<0.001||3.96 (1.99–7.87)||<0.001||2.36 (1.20–4.56)||0.002||3.97 (1.94–8.14)||<0.001|
|Double (aCL+PS/PT)||7.05 (4.03–12.85)||<0.001||4.03 (2.27–7.16)||<0.001||3.28 (1.91–5.60)||<0.001||3.79 (2.11–6.81)||<0.001|
|Double (aβ2GPI+LAC)||7.11 (3.52–15.67)||<0.001||3.12 (1.57–6.10)||<0.001||3.21 (1.68–6.06)||<0.001||4.47 (2.23–9.03)||<0.001|
|Double (aβ2GPI+PS/PT)||7.05 (4.21–12.15)||<0.001||3.72 (2.16–6.45)||<0.001||3.84 (2.31–6.40)||<0.001||4.95 (2.86–8.63)||<0.001|
|Double (PS/PT+LAC)||7.07 (4.02–12.94)||<0.001||3.22 (1.80–5.76)||<0.001||3.79 (2.21–6.49)||<0.001||4.90 (2.75–8.76)||<0.001|
|Triple (aCL+aβ2GPI+LAC) “Triple Pos”||5.32 (2.58–11.85)||<0.001||3.63 (1.77–7.36)||<0.001||2.21 (1.08–4.38)||0.025||5.29 (2.50–11.35)||<0.001|
|Triple (aPS/PT+aβ2GPI+LAC)||6.54 (3.22–14.43)||<0.001||3.32 (1.67–6.57)||<0.001||2.82 (1.45–5.38)||<0.001||4.48 (2.20–9.20)||<0.001|
|Triple (aCL+aβ2GPI+aPS/PT)||6.30 (3.51–11.81)||<0.001||3.83 (2.11–6.97)||<0.001||3.16 (1.79–5.53)||<0.001||4.42 (2.39–8.18)||<0.001|
|Quadruple(aCL+aβ2GPI+aPS/PT+LAC)||4.83 (2.33–10.80)||<0.001||3.93 (1.90–8.09)||<0.001||1.86 (0.88–3.78)||0.092||5.39 (2.50–11.84)||<0.001|
Multivariate models are controlled for age and sex.
The association of aPS/PT antibodies with stroke and transient ischemic attack (TIA) has been reported previously. In the present cohort, 62 patients (46 with APS, 15 with SNAPS and 1 with SLE) had a history of stroke, and 88.7% (55/62) of these patients were positive for aPS/PT IgG and/or IgM. Of the 46 patients with APS and stroke, 35 (76.1%) were positive for aPS/PT IgG, 31 (67.4%) for aPS/PT IgM and 44 (95.6%) for aPS/PT IgG and/or IgM. In comparison, LAC was positive in 24/46 (52.2%) of the APS patients with a history of stroke. For the 15 SNAPS patients with a history of stroke, 10 (66.7%) were positive for aPS/PT IgG and/or IgM (4 IgG only, 4 IgM only and 2 both IgG and IgM). By definition, all SNAPS were LAC negative. Notably, all positive aPS/PT values for the SNAPS patients were >50 units.
Delayed diagnosis of APS can have severe consequences for patients such as repeated pregnancy loss and/or thrombosis. New biomarkers are needed to improve diagnosis, prognosis and treatment. In addition to the conventional “criteria” markers (cardiolipin, β2GPI and LAC), numerous markers have been proposed and evaluated. The definition of “aPL positive”, however, is still controversial. On the one hand, the lack of standardization among laboratories often makes it difficult for physicians to consistently identify “aPL positive” patients with APS. On the other hand, standard testing with current criteria aPLs may miss some APS patients with significant thrombosis and/or obstetric problems, but who are repeatedly negative for aCL, β2GPI and LAC of the SNAPS patients. While these patients are “seronegative” for the current criteria markers, they are seropositive for other aPL antibodies. Effective new markers could help decrease the number of seronegative patients and help more patients receive timely medical attention before devastating potential consequences.
Autoantibodies directed towards prothrombin and/or phosphatidylserine/prothrombin complexes have been extensively investigated for evaluation of patients with suspected APS . Our study clearly demonstrated a higher prevalence of IgG/IgM aPS/PT in APS and SNAPS groups compared with other autoimmune disease groups. Furthermore, we demonstrated a strong association of aPS/PT with thrombosis and obstetric complications.
Evaluation of APS and SNAPS demographics suggested that the SNAPS group may have experienced fewer obstetric complications than the APS group (p<0.01). The prevalence and level of IgG aPS/PT in SNAPS group was significantly higher than in the SLE group in our study, suggesting IgG aPS/PT may help the diagnosis of APS lacking positive “criteria” aPL. A follow-up study of IgG aPS/PT positive patients in our SLE group will evaluate patient outcome, specifically assessing the utility of aPS/PT antibodies for risk assessment of future thrombotic events. In addition to diagnostic value, studies have shown that aPS/PT antibodies may be strongly related to ischemic/ thrombotic cerebrovascular events. In a prospective study of 167 patients with TIA, Mullen et al.  showed by multivariate analysis adjusted by ABCD2 risk score, that aPS/PT IgG antibodies were independently associated with stroke or death in patients with TIA (OR=15.7, 95% CI=2.0–125.6, p=0.009). Akhter et al.  demonstrated that aPS/PT (IgG and IgG/IgM) were significantly associated with venous thrombosis (OR=2.1, 95% CI=1.2–3.8, p=0.015) in a cohort of 326 SLE patients. The current study provides additional support of these observations. Among the 62 patients with a history of stroke (46 APS, 15 SNAPS, 1 SLE), IgG and/or IgM aPS/PT antibodies were positive in 88.7% (55/62) of the APS, SNAPS and SLE patients with a history of stroke. By contrast, LAC was positive in only 38.7% (24/62) of these patients.
Our study demonstrated the significant value aPS/PT IgG testing for evaluation of patients suspected of APS. Notably, 28.6% of the SNAPS group was found to have IgG aPS/PT antibodies. Whether these cases possibly belong to a different subset of APS patients merits further study. We found that 12.7% of SLE patients without thrombosis were positive for IgG aPS/PT. Of particular interest was the finding that 10 of the SLE patients had very high levels of IgG and/or IgM aPS/PT antibodies (116–199 units). Since patients with SLE can develop APS (and vice versa), the presence of the high aPS/PT antibody levels may indicate the future development of APS or may indicate unrecognized APS in these patients. Close follow-up of these patients will continue to determine if the high PS/PT value portended the development of clinical APS.
Our results showed a higher prevalence of aPS/PT compared with similar studies previously reported , , , perhaps reflecting the specific make-up of our very well-characterized clinical cohort. Overall, the IgG isotype showed higher specificity and prevalence than IgM for both criteria aPL antibodies and aPS/PT antibodies. We found low positivity (8.2%) for aPS/PT IgM in HC group (6 of 7 positives <50 units). Interestingly, the HC group showed more reactivity than the RA and AS disease groups. The same lot of kits were used for all testing. The HC group had no known morbidities, and we do not currently have an explanation for this observation. Our study showed aβ2GPI IgA displayed better performance than aCL IgM and aβ2GPI IgM in APS, SNAPS and SLE groups, consistent with other studies, indicating that IgA aβ2GPI might be more useful than measuring aCL IgM and aβ2GPI IgM , , .
The association of aPS/PT (IgG or IgM) antibodies with clinical manifestations of APS was statistically significant. In the multivariate analysis, AT was associated with the presence of aPS/PT, IgG aCL, IgG aβ2GPI and their combinations. Venous thrombosis and pregnancy loss were associated with the presence of IgG/IgM aPS/PT, IgG aPS/PT and LAC. Previous studies reported similar data. For example, Vlagea et al.  reported that the presence of IgG/IgM aPS/PT, specifically the IgG isotype, had a strong association with VT, and their study confirmed aPS/PT as an independent risk factor for such an event. Moreover, in our study, LAC demonstrated lower diagnostic performance in comparison with aPS/PT antibodies in APS group by ROC analysis. IgG aPS/PT and LAC were significantly associated with both vascular thrombosis and pregnancy morbidity. Compared with LAC, IgG aPS/PT was shown to be an increased risk factor for thrombosis and pregnant morbidity alone or when combined with “criteria” aPLs such as anticardiolipin and/or β2GPI antibody.
The strong correlation between aPS/PT and LAC activity has been reported previously , . Despite efforts to improve standardization of LAC assays, accurate detection and intralaboratory reproducibility is still not fully achieved. Because the aPS/PT assay is not affected by anti-coagulant treatment, it represents an important alternative to LAC, especially for patients who have already been anticoagulated when the serum is drawn or in those with unknown or uncertain anticoagulation history. During the preparation of our manuscript, the results of a study conducted in Beijing was published with similar observations and which agreed with our conclusion that aPS/PT antibodies could play an important role in the diagnosis of Chinese APS patients .
One limitation of our study is that the cohort of patients in our study were very well-characterized APS patients who had been identified and followed by clinical experts in APS. The cohort is therefore more homogeneous than would typically be encountered in a “real-life” patient population, and the high performance achieved by the assays in this study may not be so striking in a more heterogenous general population. Nevertheless, we believe that the strong performance achieved by the aPL assays tested, in particularly aPS/PT, provides support for the value of these assays to identify patients with APS, both those meeting and not meeting the current laboratory criteria for classification of APS. Additional prospective studies will help clarify and validate the utility of the antibody profiles examined in our study.
In summary, the present study supports aPS/PT as a promising biomarker for APS in the Chinese population. The aPS/PT assays showed high diagnostic efficiency for APS, detected patients negative for conventional “criteria markers” (SNAPS patients) who might not receive timely treatment with conventional testing schemes and supported previous studies, indicating that measurement of aPS/PT antibodies could serve as additional serological markers for thrombosis and obstetric complications. Although additional studies are needed, our results add strong support to recommendations for inclusion of aPS/PT antibodies as a new laboratory criteria biomarker for the formal classification of APS .
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: This work was supported by the National Natural Science Foundation of China (81373211, 81671589, 81601400), the Shanghai Sailing Program (16YF1407000) and the Prairie fire program (LYJH-358).
Employment or leadership: Gary L. Norman is an employee of Inova Diagnostics, and Jin-Feng Zhou is an employee of Werfen China.
Honorarium: Chengde Yang has received honorarium from Inova Diagnostics.
Competing interests: The funding organizations played no role in the study design, in the collection, analysis, and interpretation of data; the writing of the report; or in the decision to submit the report for publication.
1. Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306.10.1111/j.1538-7836.2006.01753.xSearch in Google Scholar
2. Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC, et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop. Arthritis Rheum 1999;42:1309–11.10.1002/1529-0131(199907)42:7<1309::AID-ANR1>3.0.CO;2-FSearch in Google Scholar
3. Arvieux J, Darnige L, Caron C, Reber G, Bensa JC, Colomb MG. Development of an ELISA for autoantibodies to prothrombin showing their prevalence in patients with lupus anticoagulants. Thromb Haemost 1995;74:1120–5.10.1055/s-0038-1649891Search in Google Scholar
5. Galli M, Comfurius P, Maassen C, Hemker HC, de Baets MH, van Breda-Vriesman PJ, et al. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990;335:1544–7.10.1016/0140-6736(90)91374-JSearch in Google Scholar
6. Atsumi T, Ieko M, Bertolaccini ML, Ichikawa K, Tsutsumi A, Matsuura E, et al. Association of autoantibodies against the phosphatidylserine-prothrombin complex with manifestations of the antiphospholipid syndrome and with the presence of lupus anticoagulant. Arthritis Rheum 2000;43:1982–93.10.1002/1529-0131(200009)43:9<1982::AID-ANR9>3.0.CO;2-2Search in Google Scholar
7. Hoxha A, Ruffatti A, Mattia E, Meneghel L, Tonello M, Salvan E, et al. Relationship between antiphosphatidylserine/prothrombin and conventional antiphospholipid antibodies in primary antiphospholipid syndrome. Clin Chem Lab Med 2015;53: 1265–70.10.1515/cclm-2014-1129Search in Google Scholar
8. Syuto T, Shimizu A, Takeuchi Y, Tanaka S, Hasegawa M, Nagai Y, et al. Association of antiphosphatidylserine/prothrombin antibodies with neuropsychiatric systemic lupus erythematosus. Clin Rheumatol 2009;28:841–5.10.1007/s10067-009-1123-1Search in Google Scholar
9. Kawakami T, Yamazaki M, Mizoguchi M, Soma Y. High titer of anti-phosphatidylserine-prothrombin complex antibodies in patients with cutaneous polyarteritis nodosa. Arthritis Rheum 2007;57:1507–13.10.1002/art.23081Search in Google Scholar
10. Sciascia S, Sanna G, Murru V, Roccatello D, Khamashta MA, Bertolaccini ML. Anti-prothrombin (aPT) and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies and the risk of thrombosis in the antiphospholipid syndrome. A systematic review. Thromb Haemost 2014;111:354–64.10.1160/TH13-06-0509Search in Google Scholar
11. Jaskowski TD, Wilson AR, Hill HR, Branch WD, Tebo AE. Autoantibodies against phosphatidylserine, prothrombin and phosphatidylserine-prothrombin complex: identical or distinct diagnostic tools for antiphospholipid syndrome? Clin Chim Acta 2009;410:19–24.10.1016/j.cca.2009.09.006Search in Google Scholar PubMed
12. Vlagea A, Gil A, Cuesta MV, Arribas F, Diez J, Lavilla P, et al. Antiphosphatidylserine/prothrombin antibodies (aPS/PT) as potential markers of antiphospholipid syndrome. Clin Appl Thromb Hemost 2013;19:289–96.10.1177/1076029612437578Search in Google Scholar PubMed
13. Zigon P, Cucnik S, Ambrozic A, Kveder T, Semrl SS, Rozman B, et al. Detection of antiphosphatidylserine/prothrombin antibodies and their potential diagnostic value. Clin Dev Immunol 2013;2013:724592.10.1155/2013/724592Search in Google Scholar PubMed PubMed Central
14. Pengo V, Ruffatti A, Legnani C, Testa S, Fierro T, Marongiu F, et al. Incidence of a first thromboembolic event in asymptomatic carriers of high-risk antiphospholipid antibody profile: a multicenter prospective study. Blood 2011;118:4714–8.10.1182/blood-2011-03-340232Search in Google Scholar PubMed
15. Bertolaccini ML, Amengual O, Andreoli L, Atsumi T, Chighizola CB, Forastiero R, et al. 14th International Congress on Antiphospholipid Antibodies Task Force. Report on antiphospholipid syndrome laboratory diagnostics and trends. Autoimmun Rev 2014;13:917–30.10.1016/j.autrev.2014.05.001Search in Google Scholar PubMed
17. Kazzaz NM, McCune WJ, Knight JS. Treatment of catastrophic antiphospholipid syndrome. Curr Opin Rheumatol 2016;28: 218–27.10.1097/BOR.0000000000000269Search in Google Scholar PubMed PubMed Central
18. Amengual O, Forastiero R, Sugiura-Ogasawara M, Otomo K, Oku K, Favas C, et al. Evaluation of phosphatidylserine-dependent antiprothrombin antibody testing for the diagnosis of antiphospholipid syndrome: results of an international multicentre study. Lupus 2017;26:266–76.10.1177/0961203316660203Search in Google Scholar PubMed
19. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315.10.1002/art.1780310302Search in Google Scholar PubMed
20. Hochberg MC. For the Diagnostic and Therapeutic Criteria Committee of the American College of Rheumatology. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40:1725.10.1002/art.1780400928Search in Google Scholar PubMed
21. Vitali C, Bombardieri S, Moutsopoulos HM, Balestrieri G, Bencivelli W, Bernstein RM, et al. Preliminary criteria for the classification of Sjogren’s syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum 1993;36:340–7.10.1002/art.1780360309Search in Google Scholar PubMed
22. van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria. Arthritis Rheum 1984;27:361–8.10.1002/art.1780270401Search in Google Scholar PubMed
23. Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, et al. Update of the guidelines for lupus anticoagulant detection. Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis. J Thromb Haemost 2009;7:1737–40.10.1111/j.1538-7836.2009.03555.xSearch in Google Scholar PubMed
24. Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: an update. On behalf of the Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the ISTH. Thromb Haemost 1995;74:1185–90.10.1055/s-0038-1649901Search in Google Scholar
25. Thiagarajan P, Pengo V, Shapiro SS. The use of the dilute Russell viper venom time for the diagnosis of lupus anticoagulants. Blood 1986;68:869–74.10.1182/blood.V68.4.869.869Search in Google Scholar
26. Bertolaccini ML, Amengual O, Atsumi T, Binder WL, de Laat B, Forastiero R, et al. ‘Non-criteria’ aPL tests: report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, TX, USA, April 2010. Lupus 2011;20:191–205.10.1177/0961203310397082Search in Google Scholar PubMed
27. Mullen MT, Messe SR, Kasner SE, Sansing L, Husain MR, Norman GL, et al. Anti-phosphatidylserine-prothrombin antibodies are associated with outcome in a TIA cohort. Front Neurol 2012;3:137.10.3389/fneur.2012.00137Search in Google Scholar PubMed PubMed Central
28. Akhter E, Shums Z, Norman GL, Binder W, Fang H, Petri M. Utility of antiphosphatidylserine/prothrombin and IgA antiphospholipid assays in systemic lupus erythematosus. J Rheumatol 2013;40:282–6.10.3899/jrheum.120084Search in Google Scholar PubMed PubMed Central
30. Bertolaccini ML, Gomez S, Pareja JF, Theodoridou A, Sanna G, Hughes GR, et al. Antiphospholipid antibody tests: spreading the net. Ann Rheum Dis 2005;64:1639–43.10.1136/ard.2005.035824Search in Google Scholar PubMed PubMed Central
31. Nojima J, Iwatani Y, Suehisa E, Kuratsune H, Kanakura Y. The presence of anti-phosphatidylserine/prothrombin antibodies as risk factor for both arterial and venous thrombosis in patients with systemic lupus erythematosus. Haematologica 2006;91:699–702.Search in Google Scholar
32. Pericleous C, Ferreira I, Borghi O, Pregnolato F, McDonnell T, Garza-Garcia A, et al. Measuring IgA anti-beta2-glycoprotein I and IgG/IgA anti-domain I antibodies adds value to current serological assays for the antiphospholipid syndrome. PLoS One 2016;11:e0156407.10.1371/journal.pone.0156407Search in Google Scholar PubMed PubMed Central
33. Murthy V, Willis R, Romay-Penabad Z, Ruiz-Limon P, Martinez-Martinez LA, Jatwani S, et al. Value of isolated IgA anti-beta2 -glycoprotein I positivity in the diagnosis of the antiphospholipid syndrome. Arthritis Rheum 2013;65:3186–93.10.1002/art.38131Search in Google Scholar PubMed PubMed Central
34. Pregnolato F, Chighizola CB, Encabo S, Shums Z, Norman GL, Tripodi A, et al. Anti-phosphatidylserine/prothrombin antibodies: an additional diagnostic marker for APS? Immunol Res 2013;56:432–8.10.1007/s12026-013-8421-zSearch in Google Scholar PubMed
35. Zhu L, Li C, Liu N, Yang X, Jia RL, Mu R, et al. Diagnostic value of antibodies to phosphatidylserine/prothrombin complex for antiphospholipid syndrome in Chinese patients. Clin Rheumatol 2017;36:401–6.10.1007/s10067-016-3498-0Search in Google Scholar PubMed
©2018 Hui Shi et al., published by De Gruyter, Berlin/Boston
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.