Circulating microparticles in pregnant patients with primary anti-phospholipid syndrome: an exploratory study

The discovery of novel prognostic markers could help to establish a clear and definitive risk-stratification strategy and personalized therapy for pregnant women with antiphospholipid syndrome (APS) (1–5).Microparticles (MPs) have shown promising results as potential biomarkers of thrombotic risk in hypercoagulable conditions including pregnancy (6). We aimed to evaluate the trend of circulating MPs in pregnant women with APS. Eleven consecutive pregnant women affected by primary APS (1) and 15 healthy pregnant women matched for age and body mass index (BMI) were enrolled between January 2013 and July 2016. Upon obtaining informed consent, venous blood samples were longitudinally collected during the first [8th gestational week (GW)], second (20th GW), and third (33rd GW) trimesters of pregnancy. Women with hereditary thrombophilia, acute infections, cardiovascular diseases, chronic hypertension, diabetes mellitus, cancer, or renal or hepatic failure were excluded. The women were divided into high-risk [i.e. triple anti-phospholipid antibody (aPL) positivity plus a history of vascular thrombosis and/or history of severe pregnancy complications/placental insufficiency] and low-risk (i.e. history of pregnancy morbidity or vascular thrombosis without triple aPL positivity) APS patients. High-risk women were treated with therapeutic heparin plus low-dose aspirin (LDA) and additional treatments [weekly plasma exchange (PE) plus fortnightly 1 g/kg intravenous immunoglobulins (IVIGs)], whereas low-risk APS women received therapeutic or prophylactic heparin plus LDA. The rationale underlying the additional therapy is based on the potential benefit of PE in removing some of the aPLs from the maternal circulation, and IVIGs further increase aPL clearance (4). Samples were collected on the day before PE to preserve the endothelium and standardize the measurements as much as possible. Immunoglobulin G/immunoglobulin M anti-cardiolipin and anti-β2-glycoprotein I antibody enzyme-linked immunosorbent assays were performed using a home-made method following the European Forum recommendations (7). Cut-off values were calculated as > 99th percentile using sera from 120 healthy blood donors, and LAPL-GM-200 international calibrators (Louisville Diagnostics, Seabrook, TX, USA) were used to make calibration curves. Preanalytical and analytical MP conditions have been described previously (8). In brief, the MP gate was set via preliminary standardization experiments using fluorescent beads 0.5, 0.9 and 3 μm in diameter (Megamix; BioCytex, Diagnostica Stago, Marseille, France) on a Cytomics FC500 flow-cytometer (Beckman Coulter, Miami, FL, USA). MPs were identified by size and annexin V– fluorescein isothiocyanate (Bender MedSystems, Vienna, Austria) labelling [phosphatidylserine-positive (PS+) MPs]. Annexin V-negative MPs were also recorded [phosphatidylserine-negative (PS− MPs)]. Subpopulations of MPs were identified by double staining with annexin V and a second monoclonal antibody. Differences between cases and controls were evaluated using the non-parametric Mann–Whitney U-test. For multiple comparisons, the Kruskal–Wallis test was used with post-hoc Bonferroni–Dunn test. Clinical and laboratory characteristics of the APS patients are reported in Table 1. Six patients had a preterm delivery and two newborns had respiratory complications; however, pregnancy outcomes were favourable in all cases. Healthy pregnant subjects had a mean ± SD age of 31.1 ± 5.2 years (range 24–41 years) and a mean BMI of 23.1 ± 4.3 kg/m. Women with APS had significantly higher PS+ MPs (p < 0.001), PS− MPs (p < 0.001), endothelium-derived MPs (p = 0.02), and endoglin+ MPs (p < 0.001) in the first and second trimesters. In the third trimester, APS patients showed significantly higher levels of all MPs considered (Figure 1). High-risk APS patients had higher PS+ MPs (p < 0.001), platelet-derived (p < 0.001), and endoglin + MPs (p < 0.0001) in all three trimesters. Low-risk APS patients had higher PS+ MPs (p < 0.001) in all three trimesters and endoglin+ MPs in the second and third trimesters (p = 0.018 and p < 0.01, respectively). High-risk APS patients had higher PS+ MPs in the first and third trimesters (p = 0.02 and 0.01, respectively), and higher platelet-derived (p < 0.01), Scand J Rheumatol 2018;47:501–504 501