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The ISCCA flow protocol for the monitoring of anti‐CD20 therapies in autoimmune disorders
Author(s) -
Gatti Arianna,
Buccisano Francesco,
Scupoli Maria T.,
Brando Bruno
Publication year - 2021
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.21930
Subject(s) - medicine , protocol (science) , pathology , alternative medicine
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 naïve cells. The disappearance of memory B cells and the repopulation by naïve 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, naïve and memory B cells, sIgM+ and sIgG‐switched memory B cells, T and NK cells, with high‐sensitivity analysis (>10 6 CD45+ cells). Results After an anti‐CD20 dose, the B cell level is about zero in most patients. If B cells remain virtually absent (<0.1/μl), subsetting is not reliable nor meaningful. If B cells raise >0.3–0.5/μl, subsetting is possible and informative, acquiring >1.0–1.5 × 10 6 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.