Karel DeCeulaer

A unique antiphospholipid assay recognizing phospholipid mixture compared with criteria antiphospholipid immunoassays in lupus patients

Background While essential for the classification of antiphospholipid syndrome (APS), anticardiolipin (aCL) assays lack specificity and anti-β2glycoproteinI (anti-β2GPI) assays lack sensitivity in this regard. Our aim was to perform a comparative analysis of the APhL ELISA assay (IgG/IgM) and criteria antiphospholipid (aPL) immunoassays in identifying APS-related clinical manifestations in a large group of patients with systemic lupus erythematosus (SLE). Methods Serum samples from 1178 patients from the Hopkins (n = 543), LUMINA (n = 588) and Jamaican SLE cohorts (n = 47) were examined for IgG/IgM positivity in aCL (in-house), anti-β2GPI (two commercial kits) and APhL (Louisville APL) ELISA assays. Correlation of assay positivity with clinical manifestations and sensitivity, specificity, positive and negative predictive values and likelihood ratios were evaluated. A case series analysis was also performed in patients for whom there was isolated positivity in the specific aPL assays. Results The prevalence of aCL positivity was 34.9%, anti-β2GPI kit A was 22.6%, APhL was 11.5% and anti-β2GPI kit B was 7.6% in the study population. Anti-β2GPI kit B, aCL and APhL assays were correlated with venous thrombosis, while only APhL was significantly correlated with arterial thrombosis and consistently correlated with pregnancy-related morbidity. No significant correlations were noted for anti-β2GPI kit A. Sensitivity was greatest for aCL assays followed by anti-β2GPI kit A, APhL and anti-β2GPI kit B, while specificity was greatest and equal for anti-β2GPI kit B and APhL assays. Conclusions Overall, APhL antibodies, especially IgG, represent a promising biomarker for the classification of APS patients in the context of autoimmunity and in risk assessment with regards to pregnancy morbidity and thrombotic manifestations.

Clinical associations of proinflammatory cytokines, oxidative biomarkers and vitamin D levels in systemic lupus erythematosus:

Background The abnormal biological activity of cytokines plays an important role in the pathophysiology of both systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). Several studies have highlighted the association of vitamin D and certain pro-inflammatory cytokines with disease activity in SLE. However, there are limited data on the association of vitamin D and antiphospholipid antibodies (aPL) with various proinflammatory biomarkers in these patients and their relative impact on clinical outcomes. Methods The serum levels of several aPL, 25-hydroxy-vitamin D, pro-inflammatory cytokines including IFNα, IL-1β, IL-6, IL-8, IP10, sCD40L, TNFα and VEGF were measured in 312 SLE patients from the Jamaican (n = 45) and Hopkins (n = 267) lupus cohorts using commercial Milliplex and ELISA assays. Oxidized LDL/β2glycoprotein antigenic complexes (oxLβ2Ag) and their associated antibodies were also measured in the Jamaican cohort. Healthy controls for oxidative marker and cytokine testing were used. Results Abnormally low vitamin D levels were present in 61.4% and 73.3% of Hopkins and Jamaican SLE patients, respectively. Median concentrations of IP10, TNFα, sCD40L and VEGF were elevated in both cohorts, oxLβ2Ag and IL-6 were elevated in the Jamaican cohort, and IFNα, IL-1β and IL-8 were the same or lower in both cohorts compared to controls. IP10 and VEGF were independent predictors of disease activity, aPL, IP10 and IL-6 were independent predictors of thrombosis and IL-8, and low vitamin D were independent predictors of pregnancy morbidity despite there being no association of vitamin D with pro-inflammatory cytokines. Conclusions Our results indicate that aPL-mediated pro-inflammatory cytokine production is likely a major mechanism of thrombus development in SLE patients. We provide presumptive evidence of the role IL-8 and hypovitaminosis D play in obstetric pathology in SLE but further studies are required to characterize the subtle complexities of vitamin D’s relationship with cytokine production and disease activity in these patients.

Antinuclear antibodies and HLA class II alleles in Jamaican patients with systemic lupus erythematosus

OBJECTIVE The relationship between human leukocyte antigens class II (HLA) and antinuclear antibodies was investigated in Jamaican patients with Systemic Lupus Erythematosus (SLE). METHODS Samples of blood of 82 patients with SLE and 75 healthy controls were tested for antinuclear antibodies using the fluorescent antinuclear antibody (FANA) test, counterimmunoelectrophoresis (CIEP) and the Crithidia luciliae immunofluorescence test (CL-IFT). A DNA-based HLA typing method was used to determine the frequencies of alleles of HLA-DRB1, DRB3, DRB4 and DRB5 in patients and healthy controls. RESULTS The FANA test was positive in all of the sera from patients with SLE. Anti-dsDNA antibodies were present in 49% (40/82), anti-Sm/RNP 44% (36/82) and anti-Ro/La 43% (35/82) of the sera from SLE patients. The frequency of HLA-DR4 was significantly lower in SLE patients than in healthy controls (2/82, 2% vs 15/75, 20%; RR = 0.12; p = 0.0004; CP = 0.005) but no other HLA-DRB1 SLE associations were found. A positive HLA-DR3 anti-Ro/La antibody association was found in the patients with SLE (9/21, 43% vs 5/55, 9%; odds ratio (OR) = 7.5; CP = 0.01). In contrast, possession of HLA-DR6 was negatively associated with the absence of anti-dsDNA antibodies (9/32, 28% vs 27/44, 61%; OR = 0.2; CP = 0.05). CONCLUSION The HLA-DR6 allele is associated with the absence of antinuclear antibodies and HLA-DR3 with the presence of anti-Ro/La antibodies in Jamaican patients with SLE. However, these results and those of previous studies of Jamaican patients suggest that the HLA-DR3 association with the development of SLE reported in other populations might in fact reflect the association of HLA-DR3 with anti-Ro/La antibodies. Further investigations are needed to determine whether HLA-DRB antinuclear antibody associations define clinical subsets of SLE in Jamaican patients.

Performance Evaluation and Clinical Associations of Immunoassays That Detect Antibodies to Negatively Charged Phospholipids Other Than Cardiolipin

Objectives We evaluate the performance characteristics of antiphosphatidylserine (anti-PS), antiphosphatidylinositol (anti-PI), and antiphospholipid mixture (APhL) enzyme-linked immunosorbent assays (ELISAs) compared with anticardiolipin (aCL) and anti-β2 glycoprotein I (anti-β2GPI) in a large group of patients with antiphospholipid (aPL)-related diseases. Methods Serum samples from 548 patients from the Hopkins and Jamaican systemic lupus erythematosus cohorts, the PROMISSE cohort, and the Antiphospholipid Standardization Laboratory were examined for immunoglobulin G (IgG)/immunoglobulin M (IgM) positivity in aCL, anti-β2GPI, anti-PS, anti-PI, and APhL ELISA assays. Results All IgG assays were associated with one or more thrombotic and/or obstetric manifestations, with an increased risk associated with higher antibody titers. Analytical performance was similar among assays, but IgG assays performed better than IgM counterparts. Conclusions Increasing titers of APhL, anti-PS, and anti-PI antibodies could indicate an increased risk of thrombotic and/or obstetric aPL-related manifestations. These assays may be promising biomarkers for particular APS manifestations.