Evaluating RhD Assessment By Automated Methodology: A Potential ‘Blind Spot’ For RhD Variant Identification

Abstract Background Automated blood typing platforms are ideal for blood donor centers, given their broad recognition of RhD antigen variants, resulting in products from such donors to be labeled as RhD positive. However, this ‘blind spot’ for variants may be problematic for hospital-based transfusion services, since RhD variants may require provision of RhD- RBC units and Rh immune globulin. The extent to which automated methods accurately characterize variant D antigen expression is not known. As such, the objective of our study was to assess D typing results, as well as RHD genotyping, among two automated testing platforms at our facility. Methods The study site is a single tertiary care center with analysis done from 12/2020-1/2024. At our facility, all females <60 years of age with RhD antigen testing results of ≤2+ agglutination (0-4+ scale) via automated methods [solid phase testing 12/20-4/22 (Neo, Immucor, Norcross, GA) and gel testing 5/22-1/24 (Erytra, Grifols, Emeryville, CA)] are assessed by a tube RhD antigen test (Grifols reagents). If tube testing confirms a ≤2+ result (0-4+ scale), molecular variant D analysis is performed (Versiti, Milwaukee, WI). Patients typed as Rh negative with anti-D antibodies, or those with inconsistent D typing, are also subjected to genetic D testing. The Wilcoxon matched pairs signed rank test was employed for comparative data (P< 0.05 was deemed significant). Results Amongst tested patients, 73 demonstrated ≤2+ agglutination via an automated typing method, leading to variant D genetic analysis. Of these, 71% (n=52) harbored variant D antigens. Within this subgroup, 29 (56%) subjects exhibited variants associated with potential allo-anti-D development (n=5 partial D variants; n=24 weak D variants). Notably, 4 patients with partial D and 1 with weak D demonstrated allo-anti-D antibodies. Of 23 patients with variant D who underwent automated gel testing, 20 concurrent tube testing results were available. Gel testing revealed significantly stronger agglutination compared to tube testing (p < 0.001). In contrast, for paired solid phase and tube testing (n=11), there was no statistically significant difference in agglutination strength (p = 0.28). Conclusion Automated testing, particularly gel-based platforms, exhibits reduced sensitivity to RhD antigen variant identification when compared to tube testing, placing subsets of patients potentially at risk for forming allo-anti-D. Implementing protocols for verification of D status could enhance the sensitivity of variant D detection for facilities that largely rely on automated methods for antigen typing.