An immunogenomic exome landscape of triple positive primary antiphospholipid patients.

Primary antiphospholipid syndrome is characterized by thrombosis and autoantibodies directed against phospholipids or associated proteins. The genetic etiology of PAPS remains unknown. We enrolled 21 patients with thromboembolic events associated to lupus anticoagulant, anticardiolipin and anti ß2 glycoprotein1 autoantibodies. We performed whole exome sequencing and a systematic variant-based analysis in genes associated with thrombosis, in candidate genes previously associated with APS or inborn errors of immunity. Data were compared to public databases and to a control cohort of 873 non-autoimmune patients. Variants were identified following a state-of-the-art pipeline. Enrichment analysis was performed by comparing with the control cohort. We found an absence of significant HLA bias and genetic heterogeneity in these patients, including when testing combinations of rare variants in genes encoding for proteins involved in thrombosis and of variants in genes linked with inborn errors of immunity. These results provide evidence of genetic heterogeneity in PAPS, even in a homogenous series of triple positive patients. At the individual scale, a combination of variants may participate to the breakdown of B cell tolerance and to the vessel damage.

Comparison of two peripheral mononuclear cell isolation protocols for flow cytometry crossmatching.

In clinical organ transplantation, flow cytometry crossmatching can be performed on total blood with a hemolysis step or after a preliminary mononuclear cell separation step using a standard Ficoll-Hypaque protocol. Here, we compared the Ficoll-Hypaque step with a faster technique for isolating mononuclear cells (the SepMate tube), using the same samples (collected and stored at room temperature for 0, 24, 48 or 72 hours). We found that the SepMate separation protocol is easily applied to flow cytometry crossmatching (with or without pronase treatment), provided that the samples have been stored at room temperature for 48 hours or less. Conversely, the Ficoll-Hypaque protocol should be used if the samples have been stored for more than 48 hours at room temperature.

Pronase treatment improves flow cytometry crossmatching results.

Flow cytometry crossmatching (FC-XM) is the most sensitive cell-based method for detecting donor-specific antibodies in clinical organ transplantation. Unfortunately, background FC-XM reactivity is elevated in assays with B lymphocytes-partly because of nonspecific immunoglobulin binding by Fc receptors and B-cell surface immunoglobulins. To reduce the background reactivity in a B-cell FC-XM assay, we treated lymphocytes with pronase (1 mg/mL for 30 minutes). This treatment drastically reduced the presence of kappa light chains and Fc receptors (CD32b), while the concomitant decrease in CD19, CD20 and major histocompatibility complex (MHC) I and II expression on B-cells was acceptable. Higher pronase concentrations (>2 mg/mL) started to significantly affect CD19, CD20, MHC-I and -II expression on B-cells. In subsequent prospective experiments (on 42 donor cells tested with 102 sera), we found that pronase treatment was associated with a relative increase of the sensitivity and specificity in our B-cell FC-XM assay.