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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-beta 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)-B 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-B 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.

Thin-layer chromatography immunostaining in detecting anti-phospholipid antibodies in seronegative anti-phospholipid syndrome / Conti, Fabrizio; Alessandri, Cristiano; Sorice, Maurizio; Capozzi, Antonella; Longo, Agostina; Garofalo, Tina; Misasi, Roberta; D., Bompane; G. R. V., Hughes; M. A., Khamashta; Valesini, Guido. - In: CLINICAL AND EXPERIMENTAL IMMUNOLOGY. - ISSN 0009-9104. - STAMPA. - 167:3(2012), pp. 429-437. [10.1111/j.1365-2249.2011.04532.x]

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