1026-LB: Discrepancy between CDC-AHG Crossmatch and Flow Cytometry cross match with serum showing multiple Class I and Class II HLA Antibodies: Non-Complement Binding IgG Antibodies?

Aim To investigate the case of a potential Living Donor Renal Transplant with Negative CDC-AHG and Positive Flow cross match with diluted serum sample having high to moderate levels of multiple Class I and Class II DSAs. Methods Both Patient and living related donor were HLAtyped for A, B, C, DR, DQ, DP by reverse SSOP-PCR.ClassI and ClassII HLA specific antibodies were determined by Luminex based single antigen bead assay. Serum samples before and after plasmapheresis/IVIG were tested for antibodies with and without OTTtreatment. One of the current serum samples was also tested for HLA-Specific IgM antibodies, and Clq binding antibodies. T and Bcell cross matches were performed on pronase treated cells by Flow cytometry using serum samples with and without OTTtreatment. The CDCAHG assay was performed on serum samples with and without OTT treatment. Results The patient consistently had both Class I and Class II DSAs against A74, Cw2, Cw4, DRl3, DR1S, DRS1, and DRS2 on serum samples collected in March 2012, and July 2012. After 2 rounds of plasmapheresis and IVIG treatment in the last week of July 2012, the DSA levels remained high, although the MFI values of some of the DSAs dropped insignificantly. The flow cytometry cross match was negative with untreated serum before plasmapheresis and was positive on NHS mixed serum (which is a 1:2 dilution), and in serum diluted 1:2 using PBSindicating a slight prozone effect. The CDC-AHG and CDCAmos cross matches with T and B cells were positive on untreated pre-plasmapheresis serum sample. However, after Dn treatment, the CDC-AHG and CDC-Amos cross matches were negative for both T and B cells indicating the CDC reactivity in untreated serum sample was due to IgM antibodies. It is to be noted that this same serum sample had multiple Class I and Class II DSAs detected by the Luminex SAB assay using anti-lgG as secondary antibodies. In order to determine the complement activating function of these DSAs, the pre-plasmapheresis serum sample was tested by C1qbinding assay and it turns out that there were no DSAs that reacted in the C1q binding assay. We also tested this pre-plasmapheresis serum for IgM class of HLA Class I & II antibodies, and only 4 Class I and 2 Class II antibodies were identified-none were DSAs; however the IgM class of HLA-A23 antibody could have clones that might react with the 9p public Epitope on the Donor’s HLA-A 2. And the Cw1 antibody could cross react with the donor’s Cw4 which might have contributed for the allo CDCT and B cell cross match reaction with untreated serum. Two serum samples were tested after plasmapheresis for DSAs, T and B cell flow and CDC-AHG cross match and the reaction pattern remained the almost the same except that the Flow cytometry cross match was positive on undiluted serum sample as well and CDC-AHG was positive with B cells using untreated serum, and negative when tested with Dn treated serum. In general, it appears that the patient has high levels of DSAs likely to be of the non-complement binding or weakly complement binding class of IgG antibodies namely, IgG2/4 subtypes. The Clinical implications of these indings will be discussed.