A Memory B Cell Crossmatch Assay for Quantification of Donor‐Specific Memory B Cells in the Peripheral Blood of HLA ‐Immunized Individuals

Humoral responses against mismatched donor HLA are routinely measured as serum HLA antibodies, which are mainly produced by bone marrow–residing plasma cells. Individuals with a history of alloimmunization but lacking serum antibodies may harbor circulating dormant memory B cells, which may rapidly become plasma cells on antigen reencounter. Currently available methods to detect HLA ‐specific memory B cells are scarce and insufficient in quantifying the complete donor‐specific memory B cell response due to their dependence on synthetic HLA molecules. We present a highly sensitive and specific tool for quantifying donor‐specific memory B cells in peripheral blood of individuals using cell lysates covering the complete HLA class I and class II repertoire of an individual. Using this enzyme‐linked immunospot ( ELISpot) assay, we found a median frequency of 31 HLA class I and 89 HLA class II –specific memory B cells per million IgG‐producing cells directed at paternal HLA in peripheral blood samples from women (n = 22) with a history of pregnancy, using cell lysates from spouses. The donor‐specific memory B cell ELISpot can be used in HLA diagnostic laboratories as a cross‐match assay to quantify donor‐specific memory B cells in patients with a history of sensitizing events.