Pre-transplant donor specific anti-HLA antibody is associated with antibody-mediated rejection, progressive graft dysfunction and patient death.

BACKGROUND: The long term effect of donor specific antibodies (DSA) detected by Luminex Single Antigen Bead (SAB) assay in the absence of a positive complement-dependant cytotoxicity (CDC) crossmatch is unclear. DSA at the time of transplant were determined retrospectively in 258 renal transplant recipients from 2003 to 2007 and their relationship with rejection and graft function prospectively evaluated. After a median of 5.6 years follow-up 9% of patients had antibody mediated rejection (AMR) (DSA 11/37 (30%), DSA-Neg 13/221 (6%), HR 6.6, p<0.001). Patients with anti-HLA class II (HR 6.1) or both class I+II (HR 10.1) DSA had the greatest risk for AMR. The Mean Fluorescent Intensity (MFI) of the DSA was significantly higher in patients with AMR than those with no rejection (p=0.006). Moreover, the strength of the antibody was shown to be important, with the risk of AMR significantly greater in those with DSA >8000 MFI than those with DSA <8000 MFI (HR 23, p<0.001). eGFR progressively declined in patients with DSA but was stable in those without DSA (35.7 ± 20.4 mls/min vs 48.5 ± 22.7) and composite patient and graft survival was significantly worse in those with class II (HR 2.9) or both class I+II (HR 3.7) but not class I DSA. Class II DSA alone, or in combination with class I DSA had the strongest association with graft loss and patient death. Patients with DSA not only have increased rates of acute AMR, but also chronic graft dysfunction, graft loss and death. Antibody burden quantified by SAB assay may identify patients at highest immunological risk and therefore influence patient management and improve long-term patient outcome.

HLA typing using bead-based methods.

The LABType(®) SSO (One Lambda, Inc) and Gen-Probe LIFECODES HLA-SSO HLA Typing tests are rapid and efficient assays for determining human leukocyte antigens (HLAs). The principle of these assays involves the hybridization of reverse sequence-specific oligonucleotide probes each attached to a unique colour coded microsphere to identify HLA class I and class II alleles. Target DNA is polymerase chain reaction (PCR) amplified using group-specific primers and then biotinylated which allows it to be detected using R-Phycoerythrin-conjugated Streptavidin. The PCR product is then denatured and allowed to hybridise to complementary DNA probes conjugated to fluorescently code microsperes. The Luminex(®) Flow Analyser achieves detection by determining the fluorescent intensity of PE on each microsphere. The assignment of HLA alleles is based on the reaction pattern of the various beads compared to patterns with known HLA alleles.

Separation and cryopreservation of lymphocytes from spleen and lymph node.

Spleen and lymph node retrieved post-mortem from deceased organ donors are a rich source of lymphocytes. Storage of lymphocytes separated from these sources can be valuable where post-transplant testing (crossmatching) is required. DNA extraction from stored lymphocytes also allows further genetic testing where required, for example additional HLA typing not performed at the time of transplant for donor-specific antibody monitoring. Methods for the isolation and freezing of such cells is described.

Crossmatching by complement-dependent lymphocytotoxicity.

The presence of preformed donor-specific HLA antibodies detected by Complement-dependent cyto-toxicity (CDC) crossmatch assay is associated with a high incidence of hyperacute or accelerated rejection and remains one of the gold standard tests pre-transplant. The standard CDC crossmatch detects IgG1, IgG3, and IgM antibody, i.e. complement fixing, bound to the native viable cell surface of lymphocytes. The crossmatch can be enhanced with the addition of anti-human-globulin to detect non-complement fixing antibodies (IgG2 and IgG4), and sensitivity can be improved with prolonged incubation times.