Development and Evaluation of a Novel Flow Cytometric Assay for Determination of Fcγ Receptor IIIa-158 V/F Dimorphism.

Abstract

A recently described dimorphism at position 559 of FCGR3A gene results in two allotypes of Fcγ receptor IIIa (Fcγ RIIIa) with phenylalanine (F) or valine (V) at amino acid position 158. It has been shown that Fcγ RIIIa-VV homozygous patients with follicular lymphoma respond better to Rituximab as a single agent than F carriers. However, there is evidence that this disadvantage in F carriers can be overcome by higher Rituximab concentrations or by the use of defucosylated therapeutic antibodies. Therefore rapid, widely applicable, and cost-effective methods for the determination of this Fcγ RIIIa dimorphism are necessary. Currently available methods to determine this dimorphism are based on PCR techniques. To simplify the determination of Fcγ RIIIa-V/F dimorphism we developed a novel flow cytometric approach using known differences in epitope recognition of monoclonal antibodies (moabs) to Fcγ RIIIa. The moab MEM-154 recognizes Fcγ RIIIa-V only, whereas moab 3G8 recognizes Fcγ RIIIa irrespective of the dimorphism. We determined the expression level of epitopes recognized by 3G8 and MEM-154 in NK cells of 35 healthy donors (FF 14; V/F 17; VV 4) by three color flow cytometry and secondary immunofluorescence. Results were compared to genotypes determined by a TaqMan assay using allele specific fluorochrome labelled probes. Donors genotyped as Fcγ RIIIa FF, V/F, and VV demonstrated overlapping immunofluorescence levels detected by both 3G8 and MEM-154. However, the ratio of fluorescence measured using MEM-154 divided by the immunofluorescence measured using 3G8 was 100% accurate for predicting genotypes. Ratios below 0.05 were measured in Fcγ RIIIa FF individuals, ratios between 0.1 and 0.5 were measured in heterozygotes, whereas ratios higher than 0.6 were found in Fcγ RIIIa VV individuals only. Quantitative flow cytometry demonstrated a great variation in Fcγ RIIIa expression on NK cells between individuals with identical Fcγ R IIIa genotype explaining the failure to predict the genotype by a single Fcγ R IIIa moab only. This novel flow cytometric assay is cost-efficient, easy to implement, reliable and uses standard flow cytometric techniques. Compared to known methods to determine the dimorphism it is faster and applicable in laboratories without sophisticated PCR technology.