The ISCCA flow protocol for the monitoring of anti-CD20 therapies in autoimmune disorders.

BACKGROUND Anti-CD20 monoclonals (MoAbs) are used in a variety of autoimmune disorders. The aim is to eliminate memory B cells sustaining the tissue damage and the production of pathogenic autoantibodies, while preserving naive cells. The disappearance of memory B cells and the repopulation by naive cells correlate with good clinical response, while the reappearance of memory B cells and plasmablasts correlates with relapse or resistance to therapy. Anti-CD20 induce extremely low B cell levels, requiring high-resolution techniques. The immune monitoring protocol developed by ISCCA is described and validated, to provide a standardized method for the clinical decision-making process during anti-CD20 therapies in autoimmune diseases. METHODS A 10-marker, 8-color staining panel (CD20-V450, CD45-V500c, CD4-FITC + sIgM-FITC, CD38-PE, CD3-PerCP Cy5.5, CD19-PE-Cy7, CD27-APC, CD8-APC H7 + sIgG-APC-H7) is used to identify B cells, plasma cells/blasts, naive and memory B cells, sIgM+ and sIgG-switched memory B cells, T and NK cells, with high-sensitivity analysis (>106 CD45+ cells). RESULTS After an anti-CD20 dose, the B cell level is about zero in most patients. If B cells remain virtually absent ( 0.3-0.5/μl, subsetting is possible and informative, acquiring >1.0-1.5 × 106 CD45+ events. Further testings can follow the quality of B cell repopulation. If B cells become detectable (>1/μl), the prevalence of memory B cells indicates non-responsiveness or a possible relapse. CONCLUSIONS The ISCCA Protocol is proposed for a standardized prospective monitoring of patients with autoimmune disorders, to assist the safe and rational usage of anti-CD20 therapies.