Analysis of factors contributing to the formation of mononuclear cell aggregates (“escapees”) in flow cytometric immunophenotyping

• Published in: Cytometry (New York, N.Y.) N.Y.), 1997-11, Vol.29 (3), p.250-260
• Main Authors: Gratama, Jan W., van der Linden, Reinier, van der Holt, Bronno, Bolhuis, Reinder L. H., van de Winkel, Jan G. J.
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - immunology; Antigens, CD - analysis; Antigens, CD - genetics; cell aggregates; Cell Aggregation; Cell Separation - methods; Flow Cytometry - methods; Humans; IgG Fc receptors; Immunoglobulin Fab Fragments - immunology; immunophenotyping; Immunophenotyping - methods; Light; monoclonal antibodies; Monocytes - cytology; polymorphism; Polymorphism, Genetic; Receptors, IgG - analysis; Receptors, IgG - genetics; Scattering, Radiation
• ISSN: 0196-4763; 1097-0320
• DOI: 10.1002/(SICI)1097-0320(19971101)29:3<250::AID-CYTO8>3.0.CO;2-F

During immunostaining, human lymphocytes may form aggregates with activated platelets and monocytes, resulting in increased forward (FSC) and sideward (SSC) light scatter signals. Consequently, aggregated cells “escape” from the standard FSC–SSC analysis gate, thereby producing erroneous results. We observed that the frequency of aggregate formation in peripheral blood mononuclear cell (PBMC) suspensions depended on cell donor, murine (m) monoclonal antibody (mAb) specificity and IgG subclass, type of fluorochrome conjugated to the mAb, amount of mAb used for immunostaining, time lapse between fixation of PBMC and flow cytometry, and other, as yet unidentified, factors. Platelets, monocytes, and granulocytes express the polymorphic class IIa IgG receptor (FcγRIIa; CD32). A single amino acid difference, either arginine (R) or histidine (H) at amino acid position 131, underlies differential interaction with mIgG1. Because the FcγRIIa‐R131 allotypic form binds mIgG1 well in contrast to FcγRIIa‐H131, we studied the frequency of aggregate formation in PBMC suspensions from apparently healthy individuals allotyped for FcγRIIa. The FcγRIIa polymorphism contributed significantly to the frequency of mIgG1‐induced cell aggregates, which was highest in FcγRIIa‐R/R131 individuals, intermediate in FcγRIIa‐R/H131 individuals, and lowest in FcγRIIa‐H/H131 individuals. The role of mIgG1‐FcγRIIa interactions in aggregate formation was confirmed by blocking FcγRIIa by using F(ab′)2 fragments of CD32 mAb. These data document the role of mIgG1 mAb binding by human class IIa IgG receptors in the formation of cell aggregates and show that inhibition of this interaction reduces this technical problem in flow cytometric immunophenotyping. Cytometry 29:250–260, 1997. © 1997 Wiley‐Liss, Inc.