Red blood cell (RBC) antibodies of the IgG class can lead to hemolysis in patients depending on many factors, including the ability to activate the complement cascade, the Fc glycosylation, the antigen specificity, and the antibody avidity as well as the IgG subclass. IgG1 and IgG3-coated RBCs are quickly recognized for phagocytosis leading to a greater potential for RBC destruction. Patients presenting with antibodies directed to high frequency RBC antigens or multiple antibodies may urgently require blood transfusion, limiting the provision of rare RBC units. In those cases, in vitro accessing the biological relevance of the RBC antibodies may be an important tool for meeting the transfusion needs.

The goal of the present study was to map the biological relevance of RBC antibodies of different specificities using in vitro phagocytosis tests and to verify if the presence of IgG1 or IgG3 subclasses correlates with the cell assay results.

Patients presenting with antibodies directed to high frequency antigens or low frequency antigens were tested for the presence of IgG1 or IgG3 subclasses using IgG1/IgG3 gel cards designed for direct antiglobulin test and sensitized incompatible RBCs, named here modified IgG1 and IgG3 IAT. A monocyte monolayer assay (MMA) was tested in parallel, and the results were expressed in monocyte index (MI). A positive MMA result, meaning risk of in vivo hemolysis, was considered in cases of MI > 5%.

A total of 17 patients presenting antibodies against high frequency antigens (anti-Ge2 / n = 7; anti-Joa / n = 2; anti-Yta / n = 2 and anti-Vel; anti-hrS; anti-Jra and anti-Lwa, all with n = 1) or low frequency antigens (anti-VS / n = 1) were included. Of these, 64% (n = 11/17) tested positive for IgG1 or IgG3 and 62.5% (n = 10/16) were associated with MI>5% in MMA. Anti-Vel; -VS; -hrS; -Jra antibodies were all considered relevant in MMA. 42.9% of the anti-Ge2 cases tested positive in MMA and 71.4% were of IgG1 or IgG3. Half of the anti-Yta and anti-Joa cases tested positive in the MMA. The presence of IgG1 or IgG3 correlated with the MMA results, with sensitivity of 78% (95%CI 0.4-0.96) and positive predicted value (PPV) of 77%. If the samples reacting very weakly (w or less) in the IgG control column were excluded, then the sensitivity of the IgG1 and IgG3 test for detecting relevant antibodies increased to 100% (CI95% 0.56-1) with negative predicted value (NPV) of 100%.

1) The biological relevance of RBC antibodies vary and in vitro assessing the harmfulness of certain antibodies might be an interesting tool for difficult cases in urgent need of transfusion; 2) Anti-Ge2 is of reported in literature as non-harmful, but the present in vitro analysis of theses antibodies contradicts this hypothesis 2) Even though MMA is the gold standard for predicting the clinical relevance of RBC antibodies, detecting the presence of IgG1 or IgG3 subclasses has high accuracy for identifying positive MMA cases.