BACKGROUND: Luminex-based single-antigen human leukocyte antigen (HLA) antibody detection beads (SAB) are a major advance for the characterization of HLA-specific antibodies but their clinical utility is limited unless the analysis is performed and interpreted optimally. Here, we identify problems encountered in routine monitoring of antibody levels that may give rise to misleading results, and describe how these can be overcome to provide more meaningful clinical information. METHODS AND RESULTS: HLA class I specific antibody-binding levels were determined using SAB in the sera of 42 highly sensitized patients awaiting renal transplantation. Normalization of the results against the HLA class I specific monoclonal antibody W6/32 overcame the problems caused by variation in antigen density on SAB and also suggested the presence of alloantibodies directed against multiple HLA class I epitopes of a given specificity. Routine analysis using undiluted sera gave an incomplete assessment of antibody levels. On serum dilution, three patterns of antibody binding became apparent: most sera showed a sequential reduction in immunoglobulin G (IgG) binding levels but several sera displayed antibody binding which remained unchanged (suggesting antigen saturation) or increased IgG binding on serum dilution (suggesting inhibition of IgG binding using neat serum). The presence of immunoglobulin M (IgM) HLA-specific antibodies in sera correlated with inhibition of IgG antibody binding for the corresponding specificity and treatment of sera with dithiothreitol to eliminate IgM HLA-specific blocking antibodies restored maximum IgG antibody binding levels. CONCLUSION: When using SAB to monitor HLA-specific antibody binding levels, sera should be pretreated with dithiothreitol to eliminate blocking IgM HLA-specific antibodies that may mask clinically relevant allosensitization.
BACKGROUND: Luminex-based single-antigen human leukocyte antigen (HLA) antibody detection beads (SAB) are a major advance for the characterization of HLA-specific antibodies but their clinical utility is limited unless the analysis is performed and interpreted optimally. Here, we identify problems encountered in routine monitoring of antibody levels that may give rise to misleading results, and describe how these can be overcome to provide more meaningful clinical information. METHODS AND RESULTS: HLA class I specific antibody-binding levels were determined using SAB in the sera of 42 highly sensitized patients awaiting renal transplantation. Normalization of the results against the HLA class I specific monoclonal antibody W6/32 overcame the problems caused by variation in antigen density on SAB and also suggested the presence of alloantibodies directed against multiple HLA class I epitopes of a given specificity. Routine analysis using undiluted sera gave an incomplete assessment of antibody levels. On serum dilution, three patterns of antibody binding became apparent: most sera showed a sequential reduction in immunoglobulin G (IgG) binding levels but several sera displayed antibody binding which remained unchanged (suggesting antigen saturation) or increased IgG binding on serum dilution (suggesting inhibition of IgG binding using neat serum). The presence of immunoglobulin M (IgM) HLA-specific antibodies in sera correlated with inhibition of IgG antibody binding for the corresponding specificity and treatment of sera with dithiothreitol to eliminate IgM HLA-specific blocking antibodies restored maximum IgG antibody binding levels. CONCLUSION: When using SAB to monitor HLA-specific antibody binding levels, sera should be pretreated with dithiothreitol to eliminate blocking IgM HLA-specific antibodies that may mask clinically relevant allosensitization.
Authors: Annelieke W M Paantjens; Ed A van de Graaf; Johanna M Kwakkel-van Erp; Tineke Hoefnagel; Walter G J van Ginkel; Farzia Fakhry; Diana A van Kessel; Jules M M van den Bosch; Henny G Otten Journal: Pulm Med Date: 2011-08-29