title: Thin-layer chromatography immunostaining in detecting anti-phospholipid antibodies in seronegative anti-phospholipid syndrome.
abstract: 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.
authors.affiliation: Reumatologia, Dipartimento di Medicina Interna e Specialità Mediche, Università di Roma, Viale del Policlinico 155, Rome, Italy. fabrizio.conti@uniroma1.it
authors.forename: F
authors.initials: F
authors.lastname: Conti
authors.forename: C
authors.initials: C
authors.lastname: Alessandri
authors.forename: M
authors.initials: M
authors.lastname: Sorice
authors.forename: A
authors.initials: A
authors.lastname: Capozzi
authors.forename: A
authors.initials: A
authors.lastname: Longo
authors.forename: T
authors.initials: T
authors.lastname: Garofalo
authors.forename: R
authors.initials: R
authors.lastname: Misasi
authors.forename: D
authors.initials: D
authors.lastname: Bompane
authors.forename: G R V
authors.initials: GR
authors.lastname: Hughes
authors.forename: M A
authors.initials: MA
authors.lastname: Khamashta
authors.forename: G
authors.initials: G
authors.lastname: Valesini
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D017152:Antibodies, Antiphospholipid / Q000097:blood*; D016736:Antiphospholipid Syndrome / Q000276:immunology*; D016022:Case-Control Studies; D002460:Cell Line; D002855:Chromatography, Thin Layer / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D013925:Thromboplastin / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis; D055815:Young Adult
chemical_list: D017152:Antibodies, Antiphospholipid; D007074:Immunoglobulin G; D016328:NF-kappa B; D019010:Vascular Cell Adhesion Molecule-1; D013925:Thromboplastin; D053592:Interleukin-1 Receptor-Associated Kinases