Re‐visiting Fc‐receptor blocking maneuvers in man

SOME problems will just not go away, especially if they challenge both dogma and patchy practice. A particularly “sticky” issue in flow cytometry (FCM) experiments involves the nonspecific binding of monoclonal antibodies (mAbs) to leukocyte Fc-receptors. The Andersen et al. report in this issue (page 1001; DOI: 10.1002/cyto.a.22995 ) describes an accessible framework for exploring these issues, providing some clarity and illuminating the remaining challenges. Like cellular autofluorescence, non-specific antibody binding introduces background noise, thereby limiting resolution and impairing the ability to identify cells with low marker expression. Various solutions have been proposed for dealing with non-specific binding, including the use of (i) isotype controls for gating, (ii) serum to broadly compete for the receptors involved in non-specific binding, or (iii) purified IgG to specifically block Fc receptors. Fc receptor proteins are ubiquitous, which is not surprising given their important role in immune responses. Cells expressing these receptors include neutrophils, eosinophils, basophils, B lymphocytes, follicular dendritic cells, NK cells, human platelets, macrophages, and mast cells; however, Fc receptor levels differ by cell type, and there is substantial variation in turnover and re-synthesis dynamics. Moreover, the results of the Andersen et al. study suggest that there is substantial donor-to-donor variation in Fc expression. This heterogeneity has conspired to confuse the cytometry community, leaving us to question, “should there be a standard or consensus for dealing with non-specific binding? ” The Andersen et al. report begins to answer this question by providing foundational knowledge. They demonstrate the limits of isotype controls, and then explore the value of Fc receptor blocking. The details of Fc receptor blocking are important, particularly in mononuclear cells (MNCs) and monocyte-derived macrophages (MDMs), as these cells have abundant Fc-receptors. Multiple types of blocking reagents are available commercially. While employing mAbs specific for Fc receptors as isotype controls remains a debated practice, it does present a knowledge-based rationale for improving signal-over-noise in FCM analyses. Alternative protocols typically involve blocking Fc receptors broadly with serum, specifically with purified IgG, or even by removing Fc molecules by using enzymatic digestion (e.g., with pronase). Digestion has been successful, but clearly risks the non-specific digestion of different cell surface proteins. The more recently introduced recombinant protein or peptide-based Fc receptor blocking reagents have the advantages of increased uniformity and offering bio-safe alternatives to human-derived reagents. The contribution of the Andersen et al. study is its focus on evaluating commonly used blocking reagents for nonspecific binding while pointing to a preferred approach. In their study the authors reported on the use of control reagents (kappa-chain isotype antibodies for IgG1, IgG2a, and IgG2b) and blocking reagents comprising: human BD Fc Block (BD Biosciences), heat-inactivated serum (human AB and mouse), and purified IgG (human and mouse). They employed a somewhat idealized system comprising isolated MNCs and MDMs, and therefore the results do not address the context of staining whole blood. By extension, the degree to which one will find variation in non-specific binding across different donors would be an important future contribution.