Impact of Different Levels of Preformed Anti-HLA Donor Specific Antibodies Detected by Single Antigen Luminex Assay on Kidney Transplants Performed with Negative Complement Dependent Cytotoxicity T and B Crossmatches

Abstract

This study aimed to evaluate the clinical relevance of different levels of preformed anti-HLA donor specific antibodies (DSA), revealed by Luminex single antigen beads assay (SAB) in kidney transplants performed with negative complement dependent cytotoxicity (CDC) crossmatches against donor T (CDC-anti-human globulin) and B lymphocytes. The study comprised 298 recipients of first (N=256) or second (N=42) kidney grafts, transplanted in a single center, between 2001 and 2006. At the time of transplantation no information regarding Luminex-detected antibodies was available. In this study, stored pre-transplant sera were screened for anti-HLA antibodies (Labscreen® Mixed, One Lambda) and the positive ones were further analyzed with the SAB assay (Labscreen® Single Antigen, One Lambda). In the SAB assay, all reactions with normalized mean fluorescence intensity (nMFI) >300 were classified as positive and DSA were defined as antibodies against HLA-A, B or DR mismatches. Based on these results, the recipients were divided into three groups: PRA+,DSA+, PRA+,DSA-, and PRA-. Females, non-white patients, retransplants, and ATG induction therapy were significantly (p< 0.001) more frequent among PRA+ recipients. The proportion of transplants with deceased donors was the same in the three groups (42, 43 and 36%, in PRA+,DSA+, PRA+,DSA-, and PRA- recipients, respectively). The mean time of post-transplant follow-up was 57.8, 59.0 and 54.4 months for PRA+,DSA+, PRA+,DSA-, and PRA- recipients, respectively. Graft survival curves were constructed with Kaplan-Meier method and compared with the log-rank test; Cox regression analysis was used to calculate risk of graft loss. Graft survival did not differ between the three above mentioned groups of recipients. We then sub-divided the PRA+,DSA+ recipients into the following DSA nMFI ranges: 300-1,500, >1,500-3,000, >3,000-6,000 and >6,000. No difference in graft survival was observed among PRA+,DSA+ with nMFIs 300-1,500, PRA+,DSA-, and PRA- recipients. On the other hand, no graft survival difference was observed among recipients with DSA with nMFIs >1,500-3,000, >3,000-6,000 or >6,000. Therefore, further analyses were conducted considering only two groups of recipients: one comprising all recipients without DSA or with DSA < 1500 nMFI (N=228), and the other comprising recipients with DSA >1,500 MFI (N=70). Graft survival was significantly lower (p< 0.001) in recipients with DSA >1,500 MFI (one-year graft survival: 78.6% vs 92.1%; 5-year graft survival: 64.3% vs 78.9%). DSA >1500 MFI conferred a relative risk of 4.4 (95% CI 1.7-11.1) for graft loss at one year and of 3.0 (95% CI 1.4-6.4) for late graft loss in recipients with functional kidneys at one year. Recipients transplanted with DSA >1500 nMFI also presented lower estimated glomerular filtration rates (Cockroft-Gault formula), at the first month (43.0±19.9 vs. 56.0±20.3 ml/min, p< 0.001) and first year (56.6±17.2 vs. 65.0±21.2 ml/min, p< 0.01). In conclusion, the results of this study showed that DSAs directed to HLA-A, B and DR mismatches with nMFIs from 300 to 1,500 did not have any influence on the transplant outcome, whereas DSAs with nMFIs >1,500, although not associated with immediate graft losses, conferred a risk for graft dysfunction already at the end of the first month, and a risk for long-term graft loss.