Thin‐layer chromatography immunostaining in detecting anti‐phospholipid antibodies in seronegative anti‐phospholipid syndrome

Summary In clinical practice it is possible to find patients with clinical signs suggestive of anti‐phospholipid syndrome (APS) who are persistently negative for the routinely used anti‐phospholipid antibodies (aPL). Therefore, the term proposed for these cases was seronegative APS (SN‐APS). We investigated the clinical usefulness of thin‐layer chromatography (TLC) immunostaining in detecting serum aPL in patients presenting clinical features of SN‐APS. Sera from 36 patients with SN‐APS, 19 patients with APS, 18 patients with systemic lupus erythematosus (SLE), 20 anti‐hepatitis C virus (HCV)‐positive subjects and 32 healthy controls were examined for aPL using TLC immunostaining. Anti‐β 2 ‐glycoprotein‐I, anti‐annexin II, anti‐annexin V and anti‐prothrombin antibodies were tested by enzyme‐linked immunosorbent assays (ELISA). Eahy926, a human‐derived endothelial cell line, was incubated with immunoglobulin (Ig)G fraction from SN‐APS patients and analysis of phospho‐interleukin (IL)‐1 receptor‐associated kinase (IRAK) and phospho‐nuclear factor (NF)‐κB was performed by Western blot, vascular cell adhesion molecule 1 (VCAM‐1) expression by cytofluorimetric analysis and supernatants tissue factor (TF) levels by ELISA. TLC immunostaining showed aPL in 58·3% of SN‐APS patients: anti‐cardiolipin in 47·2%, anti‐lyso(bis)phosphatidic acid in 41·7% and anti‐phosphatidylethanolamine in 30·5%. Six of 36 patients showed anti‐annexin II. Incubation of Eahy926 cells with IgG from SN‐APS induced IRAK phosphorylation, NF‐κB activation, VCAM‐1 surface expression and TF cell release. TLC immunostaining could identify the presence of aPL in patients with SN‐APS. Moreover, the results suggest the proinflammatory and procoagulant effects in vitro of these antibodies.