High Prevalence of Antiphospholipid Antibodies in Children with Non-Transfusion Dependent Thalassemia and Possible Correlations with Microparticles

Antiphospholipid antibodies (APAs) are developed antibodies to the phospholipid surfaces or to the proteins binding to phospholipids. Increased PS-expressed red blood cells (RBC) may be a risk factor in APAs development. There were only a few studies reported on APAs in thalassemia, and their associations to PS-expressing microparticles (MPs), remain unknown. The presence of antiphospholipid antibodies (APAs) in pediatric thalassemia patients and their associations with phosphatidylserine (PS)- expressing microparticles (MPs), a previously unexplored relationship, were the aims of this report. We identified that positive APA rates were highest in non-transfusion-dependent thalassemia (NTDT) subjects, followed by transfusion-dependent thalassemia (TDT) subjects and controls (29.5%, 18.2% and 17.9%, respectively).  Subgroup analysis demonstrated that positive anti- ?2 glycoprotein I (a?2-GPI)- IgG rates in NTDT subjects were significantly greater than in controls and that the percentage of MPs was also significantly elevated in thalassemia subjects. More specifically, there were significant correlations found between a?2GPI-IgG and anticardiolipin (aCL)-IgM APAs, and MPs.

groups: 55 subjects with TDT, 44 subjects with NTDT, and 62 controls. TDT subjects had received regular RBC transfusions (every 3-4 weeks) to maintain a pretransfusion mean±SD of Hb at 9.0±1.2 g/dL. After receiving RBC transfusion, their mean Hb level was 12.3 g/dL. They required mean±SD RBC transfusions of around 145.0±49.3 ml/kg/year. NTDT group who required only occasional RBC transfusion of around 4.0±11.7 ml/kg/year. As a result of regular RBC transfusion, the Hb levels in TDT and NTDT subjects in the study were similar, with higher mean corpuscular volume present in TDT than in NTDT subjects ( Table  1).
The positive APA rate in all thalassemia patients as a group (23.0%) was higher than in controls (17.9%). The positive APA rate was highest in NTDT subjects (29.5%), and similar levels were shown between TDT (18.2%) and control groups (17.9%), although no significant differences were demonstrated. The LA test was positive in 14.5% of TDT subjects, 20.5% of NTDT subjects, and 12.8% of controls. When using the level cut-off of the 99 th percentile in controls to determine the positivity of aCL (IgM = 9.99 U/mL; IgG = 8.46 U/mL) and aβ2-GPI (IgM = 23.45 U/mL, IgG = 3.44 U/mL) respectively, the aCL-IgM test was positive in 1.8% of TDT subjects and 1.6% of controls. The aCL-IgG test was positive in 4.5% of NTDT subjects and 3.2% of controls. The aβ2GPI-IgM and aβ2GPI-IgG were positive in 1.8% and 5.4% respectively in TDT subjects, 4.5%, and 11.4% in NTDT subjects, and 1.6% for both IgM and IgG in controls. The prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) values in thalassemia subjects, when compared to the values in controls, can be attributed to the patients with positive APAs present in the thalassemia groups, as the APTT and PT values were higher in thalassemia patients who had positive APA when compared to negative APA. It is noted that a significant difference was demonstrated only in PT values (34.1±4.2 vs. 30.9±3.7 Table 1. Demographic data and laboratory parameters of transfusion-dependent thalassemia (TDT) subjects, non-transfusion-dependent thalassemia (NTDT) subjects, and controls.  Percentages of RBC, platelet, endothelial, and leucocyte MPs in the TDT and NTDT groups were significantly higher than those in the controls (Table 2). There were no significant differences in MPs percentages between the TDT and NTDT groups except for platelet MPs, which were significantly higher in TDT subjects than NTDT subjects ( Table 2). Aβ2GPI-IgG level significantly correlated with leucocyte (CD11b) MPs. ACL-IgM level significantly correlated with endothelial (CD31) MPs (Table 3).
Previous studies have reported a positive rate of LA in β thalassemia of 1.5-16.0%, aCL-IgG of 13.0-42.7%, Table 2. Percent red blood cell; RBC (Glycophorin A; GPA), platelet (CD41), endothelium (CD31, 144) and white blood cell (CD11b, CD45) microparticles (MPs) in transfusion-dependent thalassemia (TDT) subjects, non-transfusion-dependent thalassemia (NTDT) subjects, and controls.  Table 3. Correlation between antiphospholipid antibodies and percentages of microparticles. and aCL-IgM of 6.0%. 9,10,12 Our study demonstrated that subjects with positive LA in the TDT group consisted of 14.5% and aCL-IgM of 1.8%. The differences between the positivity rates in APAs may be due to several factors, including the diversity of the population among the studies, the disease severity, treatment plan (e.g., regular RBC transfusions), antibody detection methods, and differences in cut-off value for positivity. The other types of APAs -aβ2GPI-IgM and IgG -were also included in the present study but did not feature any previous studies. 9,10,12 The rates of positive aβ2GPI-IgM and IgG in the TDT group were 1.8% and 5.4%, respectively, which were higher than the aCL-IgM and IgG positivity rates. In the NTDT group, the rates of all positive APAs (29.5%) were higher than those in the TDT group (18.1%), although there was no statistically significant difference demonstrated. Higher APA positivity rates in NTDT subjects were also found for individual antibodies, including LA, aCL-IgG, aβ2GPI-IgM, and IgG antibodies. To our knowledge, there have been very few studies that have reported on positive APAs in NTDT subjects, particularly in children.

GPA MPs
MPs were higher in the TDT and NTDT groups when compared to those levels in controls. All MPs levels (except for platelet MPs) between TDT and NTDT groups were not significantly different. The similar MPs levels in TDT subjects, despite more severe symptoms, may be related to the regular RBC transfusions received by TDT subjects, and that can reduce the amount of abnormal PS surfaces exposed. This hypothesis is supported by a study by Atichartakarn et al. 13 The study enrolled severe splenectomized thalassemia with pulmonary hypertension subjects. After receiving RBC transfusions, the amount of PS surface exposing RBC was reduced in those subjects. The report suggested that the reduction of erythropoiesis and PS exposing cells' dilution was due to RBC transfusion. 13 In addition, platelet activation was reduced after regular RBC transfusion.
Our study also demonstrated statistically significant correlations between aβ2GPI-IgG and leucocyte (CD11b) MPs and aCL-IgM level and endothelial (CD31) MPs, although the correlations were not strong. These findings point to the likelihood that APAs in thalassemia subjects may be related to PS's expression. The sites of PS expressed surfaces are where glycoproteins, such as β2GPI, bind to anionic PS surfaces. After binding to PS surfaces, β2GPI changes the conformation of the molecule and induces antibody formation. 8 Even stronger correlations may be demonstrated in older subject age groups because the antibody formation may require time after exposure to the PS expressed apoptotic cells. 14 In this study, the lower positive APA rate in the TDT group compared to the NTDT group may be related to the regular RBC transfusion, which may reduce the exposure to PS expressed MPs, especially early after transfusion. In addition to regular RBC transfusions, deferiprone has been reported to improve immunological response, possibly from the iron chelator's direct action and the reduction of free iron radicles. 15 All TDT patients in the present study received iron chelation, which was started when serum ferritin levels reached more than 1,000 ng/mL. Deferiprone was the most prescribed medication in the present study, accounting for 76.4% of TDT subjects. The strengths of the present study were that it demonstrated a high prevalence rate of APAs, especially in thalassemia patients who received an occasional transfusion, and to our knowledge, the correlations of APAs to MPs was first demonstrated.
In summary, high APA positive rates, associated with high MPs, were demonstrated in a pediatric population with thalassemia disease, especially NTDT. This suggests that MPs may play a role in APA development. Further larger cohort and basic research studies are required to confirm these results, better understand the occurrence of APAs in this population, and demonstrate the risk of TE-linked APA presence in thalassemia subjects.