Tissue Typing
HLA tissue typing is performed using molecular biological techniques. Typing for HLA class I and class II alleles uses DNA amplification with sequence-specific primers to assign genetic variants for the HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5 and HLA-DQB1 loci.
Antibody detection and analysis
Detection of antibody to HLA antigens uses solid phase assays or immunoflourescense techniques using flow cytometry. Flow cytometry is the most sensitive test for detection of HLA antibodies that are not normally detected by lymphocytotoxicity.
Panel Reactive Antibody (PRA)
Patient serum is tested against a panel of HLA class I and class II antigens. Results are expressed as the percentage of the panel that reacted positively with the serum.
Specificity analysis using Luminex flourescence bead assay / PRA testing with solid phase flourescence beads
Antibodies to individual HLA antigens are identified using flourescence bead-based assays. These assays utilize color-coded micro spheres coated with human leukocyte antigens (HLA) to identify both complement-binding and non-complement-binding HLA class I and II antibodies in recipient and candidate sera.
Crossmatching
Prior to transplant, patient serum is cross matched with the donor lymphocytes in order to ascertain the presence of donor specific antibodies. Many sera have non-specific non-donor specific antibodies. By using pronase-treated lymphocytes true positive donor specific antibodies and false positive cross match tests are elucidated.
Deceased donor testing using pronase and CD20 antibody to facilitate cross matching in Rituximab desensitized patients
Rituximab binds to B cells, often rendering false-positive cross matches. Pronase removes CD20 from B cells, allowing the detection of class I or class II antibodies on treated B cells.
Virtual Crossmatch
Crossmatch results can now be predicted accurately by using single HLA antigen beads. A positive crossmatch can be predicted when the donor HLA type is known. This may be a benefit to recipients who are disadvantaged by sensitization.
Using Luminex for post-transplant monitoring of donor specific antibody (DSA) for prognostic value in transplant survival
Luminex micro sphere-based assays are used to analyze the significance of antibodies detected during post-transplant monitoring.