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#26-OR
HETEROGENEITY OF IGG ISOTYPES IN HLA ALLOANTISERA: SIGNIFICANCE IN FLOW POSITIVE/SEROLOGY NEGATIVE CROSSMATCHES.
Melissa B. Warden , Karen L. Pierce , Michael C. Janasik , Howard M. Gebel, PhD , Robert A. Bray, PhD and Thomas M. Ellis, PhD . Milwaukee WI, the Blood Center of Southeastern Wisconsin, 53201, Histocompatibility and Immunogenetics and Atlanta GA, Emory University School of Medicine, 30322, Department of Pathology .

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Despite the quantitative capabilities of flow cytometry for HLA antibody detection, the channel shift of a flow cytometric crossmatch rarely corresponds to or predicts the outcome of the serologic crossmatch. This could reflect the increased sensitivity of flow cytometry, or its lack of discrimination between IgG isotypes that fix complement poorly or not at all (IgG2, IgG4) and those that do (IgG1, IgG3). To determine whether the presence of IgG2 and IgG4 isotypes contribute to this dissociation of flow cytometric and serologic crossmatch results, the relative abundance of IgG1, IgG2, IgG3, and IgG4 HLA antibodies was determined in 13 PRA+ (>85%) serum samples by flow cytometry using FlowPRA screening or specificity beads, stained with FITC-conjugated anti-human IgG1, 2, 3 or 4. Donor-specific HLA antibody isotypes were determined either by flow cytometric crossmatches or by identification of donor-specific HLA antibodies using specificity beads. The relative abundance of each isotype was determined by percent reactivity and the adjusted fluorescence intensity. While IgG1 was the predominant isotype of HLA antibodies in all individuals tested, the relative abundance of IgG2 and IgG4 isotypes varied considerably among study subjects. However, of the 3 sera tested that were strongly FCXM+ but serologic (AHG) crossmatch negative, all contained relatively high levels of IgG2 and IgG4 HLA antibodies. In contrast, (7/10) sera that produced positive AHG crossmatches exhibited lower levels of IgG2 and IgG4 HLA antibodies compared to IgG1. These results are consistent with the premise that the outcome of a serologic crossmatch might be influenced by the relative amounts of IgG2 and/or IgG4 HLA antibodies.