DETECTION OF ANTIBODIES AGAINST FC GAMMA RECEPTOR IIIB (FCGRIIIB) USING IMMUNOMAGNETIC NEGATIVE SELECTION FOR NEUTROPHIL ISOLATION

Abstract

The FcγRIIIb is the most immunogenic glycoprotein of the neutrophil membrane and anti-FcγRIIIb isoantibodies are formed after allogeneic exposure when the individual has a deletion of the glycoprotein and consequently has the human neutrophil antigen-1 null (HNA-1 null ) phenotype, which is uncommon in the population. We report two cases of maternal alloimmunization against fetal FcγRIIIb glycoprotein using immunomagnetic negative selection for neutrophil isolation. We report two cases of mothers that have deletion of the FCGR3B gene (FCGR3B*null) , and consequently the HNA-1-null phenotype. Neutropenia was not detected during hospitalization in both cases, however in a second case there was a considerable reduction in the number of neutrophils on the third day of the neonate's life. The isolation of neutrophils used to perform the granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (Flow-GIFT) was tested by EasySep™Direct Human Neutrophil Isolation Kit (StemCell Technologies, Inc.) and confirmed by Dextran and Ficoll (conventional technique). Furthermore, monoclonal antibody immobilization of granulocyte antigens (MAIGA) and bead-based assay also were performed. In both cases, HNA-1 genotyping of the father, mother and newborn was performed by PCR-SSP. Sera from both women contained anti-FcγRIIIb alloantibodies detected by the all applied methods. In both cases the HNA genotyping of the mothers showed absence of epitopes of the FCGR3B gene leading to the HNA-1- null phenotype. In the first case the newborn and father were genotyped as HNA-1b/1b and in the second case the newborn and father were genotyped as HNA-1b/1c, confirming the incompatibility responsible for the mothers’ alloimmunization. The isolation of neutrophils from the immunomagnetic negative selection has similar sensitivity and specificity when compared to conventional technique, however, the immunomagnetic negative selection technique is faster to be performed and has a higher cell yield. The molecular and serologic results confirm the findings of these two rare cases of HNA-1- null leading to the formation of specific FcγRIIIb alloantibodies. The isolation of neutrophils from the immunomagnetic negative selection can be used to perform the GAT and Flow-GIFT tests which are considered gold standard techniques for detecting anti-HNA antibodies.