Anti-RhD Mediates Loss of RhD Antigen Following Anti-RhD Infusion

Background: Previous studies suggest that antibody engagement of red blood cells (RBCs) can result in the actual loss of detectable target antigen in the setting of autoimmune hemolytic anemia.  However, many of these situations appear to represent complement mediated-masking of the target antigen.  Recent studies suggest that complement-independent antigen-loss can occur in a variety of murine models.  However, whether a similar form of RBC antigen loss can occur independent of complement in humans remains unknown.  As previous studies suggest that anti-RhD fails to induce significant complement activation following engagement of RhD-positive RBCs, we evaluated the potential impact of anti-RhD antibody injection on RhD antigen levels in a RhD-positive patient being treated for idiopathic thrombocytopenic purpura.

Methods: Pre- and post-anti-RhD infusion samples were obtained from the hospital blood bank. Direct antiglobulin tests (DATs) were performed and assessed via flow cytometry on pre- and post-infusion samples. RBCs from pre- and post-infusion were also treated with Warm Autoantibody Removal Medium (WARM; ImmucorGamma, Norcross, GA), a sulphydryl/enzyme reagent based on the ZZAP method, in order to remove bound antibody. Post-WARM treated cells from pre- and post-anti-RhD infusion samples were then used to perform DATs. The presence of RhD antigen was assessed in pre- and post-infusion samples with in vitro anti-RhD, from the same lot number used to treat the patient, followed by anti-IgG and anti-complement as the secondary antibody. To account for the potential impact of bound antibody on antigen detection, antigen levels were also assessed after anti-RhD removal with the WARM method. Finally, in order to determine whether antigen loss was RhD specific, cellano (k) antigen detection was tested using anti-k antibody both pre- and post-WARM treatment.   

Results: As predicted, the pre-anti-RhD infusion DAT was negative, while the post-anti-RhD injection DAT was positive (MFI 25). Post-WARM treatment, pre-and post-RhD infusion DATs showed minimal reactivity (MFI 3 and 5, respectively with background MFI of 2.7). Reduced RhD antigen levels were observed in post-anti-RhD infusion samples when compared to pre-infusion samples, while no complement could be detected in either pre- or post-anti-RhD infusion samples. The detection of antigen loss post-anti-RhD infusion was even more pronounced after RBCs were treated with WARM to remove previously bound anti-RhD antibody administered in vivo. In contrast, no difference in k antigen level could be detected pre- or post-anti-RhD infusion. As expected, post-WARM treatment, k antigen was no longer detectable pre- or post-anti-RhD infusion samples.  

Conclusion:  These results provide an example of antigen loss in the setting of anti-RhD administration. Moreover, the anti-RhD effect on the RhD-antigen appears to be antigen specific, as the RhD immune globulin did not modulate the k antigen on the same cell. Taken together, these results suggest that anti-RhD can induce the loss of detectable antigen independent of complement and may therefore influence the rate and magnitude of RBC clearance in settings of anti-RhD infusion, incompatible RBC transfusion and autoimmune hemolytic anemia.