P117 : MASKING OF HLA IGG ANTIBODIES BY HLA IGM ANTIBODIES IN SINGLE ANTIGEN BEAD TESTING

Abstract

Listen

Translate article

Aim On postoperative day (POD) 17 after deceased donor transplant, an 18 month old kidney recipient experienced acute humoral and cellular rejection with high titer HLA class 1 & 2 donor specific antibodies (DSA) present. The recipient was exposed to transfusions, but showed only DPB ∗ 04:01 DSA at an MFI of 792 in pretransplant sera. This was determined with the Luminex Single Antigen Bead (SAB) assay for IgG HLA antibodies after heat inactivation (HI) of serum. Pronased flow crossmatch produced negative results with peak PRA and current sera. The speed that DSA appeared post transplant (POD 9) suggested undetected DSA was present pretransplant. Further methods were explored in an attempt to detect the hidden antibodies. Methods HI day-of-transplant serum was tested with Flow PRA Specific Beads, and found to have both IgG and IgM HLA DSA. The serum was dialyzed to precipitate IgM euglobulin. The IgM depleted fraction was tested by the same methods. Luminex SAB testing for IgG and IgM were performed as separate assays, using PE labeled anti-IgG or anti-IgM. Flow PRA Specific Bead testing was done as a single combined test using FITC labeled anti-IgG and APC labeled anti-IgM. Results After HI, IgM antibody was detectable by Luminex SAB and Flow PRA. Dialysis was able to remove IgM antibodies. Flow PRA efficiently detected IgG and IgM in a single assay. IgG DSA that was not found by Luminex SAB was detected by Flow PRA. When IgM was removed by dialysis, multiple IgG DSA antibodies were unmasked in Luminex SAB. Adding back the re-dissolved IgM euglobulin fraction to Luminex IgG SAB decreased DSA MFI values. Conclusion Patients frequently make both IgG and IgM HLA antibodies. Heat inactivation does not effectively remove IgM antibodies. High epitope density of Luminex SAB increases sensitivity to IgG anti-HLA antibodies. It also increases the possibility of competitive inhibition from IgM anti-HLA antibodies. IgG Luminex SAB testing alone may not be sufficient for detecting all HLA antibodies that may have clinical relevance. DSA results for day of transplant serum. Donor HLA type Luminex Single Antigen IgG Luminex Single Antigen IgG Dialyzed Luminex Single Antigen IgG Dialyzed Luminex Single Antigen IgM Flow PRA Single Antigen IgG Flow PRA Single Antigen IgG Dialyzed Flow PRA Single Antigen IgG Dialyzed Flow PRA Single Antigen IgM A 01, 02 – – 01 – – 02 – – B 08, 57 – 57 8, 57 – – 57 57 – C 06, 07 – – 06, 07 – – – – – DR 07, 17 – – 07 – – 07 – – DQ 02, 09 – 02 09 – – 02 – – B345 B3 ∗ 01 – – – – – – – – DP 0101, 0401 0401 – 0101, 0401 – – 0401 – – “–” indicates no DSA present. Other antibody specificities may be present.