Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay

Abstract

This review outlines the development of human leukocyte antigen (HLA) antibody detection assays and their use in organ transplantation in both antibody screening and crossmatching. The development of sensitive solid phase assays such as the enzyme-linked immunosorbent assay technique, and in particular the bead-based technology has revolutionized this field over the last 10–15 years. This revolution however has created a new paradigm in clinical decision making with respect to the detection of low level pretransplant HLA sensitization and its clinical relevance. The relative sensitivities of the assays used are discussed and the relevance of conflicting inter-assay results. Each assay has its advantages and disadvantages and these are discussed. Over the last decade, the bead-based assay utilizing the Luminex® fluorocytometer instrument has become established as the “gold standard” for HLA antibody testing. However, there are still unresolved issues surrounding this technique, such as the presence of denatured HLA molecules on the beads which reveal cryptic epitopes and the issue of appropriate fluorescence cut off values for positivity. The assay has been modified to detect complement binding (CB) in addition to non-complement binding (NCB) HLA antibodies although the clinical relevance of the CB and NCB IgG isotypes is not fully resolved. The increase sensitivity of the Luminex® bead assay over the complement-dependent cytotoxicity crossmatch has permitted the concept of the “virtual crossmatch” whereby the crossmatch is predicted to a high degree of accuracy based on the HLA antibody specificities detected by the solid phase assay. Dialog between clinicians and laboratory staff on an individual patient basis is essential for correct clinical decision making based on HLA antibody results obtained by the various techniques.

Current state of pediatric cardiac transplantation

“…More recently, solid-phase immunoassay has superseded the CDC test for the detection and characterization of HLA antibodies, which is a much more sensitive technique but has resulted in new challenges with respect to the interpretation of donor-specific antibodies (DSA). This test is able to detect HLA antibodies with a high sensitivity but applying this knowledge clinically has proved difficult as the presence of the antibody in and of itself does not mean that it is detrimental to the graft (24).…”

Nature and Clonality of the Fluoresceinated Secondary Antibody in Luminex Multiplex Bead Assays Are Critical Factors for Reliable Monitoring of Serum HLA Antibody Levels in Patients for Donor Organ Selection, Desensitization Therapy, and Assessment of the Risk for Graft Loss

“…The MFIs of anti-HLA Abs also remained higher with IgHPolyFab in the normal sera and in IVIg, but the reverse was true when the autologous and allogeneic IgG concentrations were augmented in allograft recipients. T he development of Luminex Multiplex Single-Antigen Bead (SAB) Assays was a significant step toward sensitive and specific detection of Abs against multiple Ags simultaneously in sera of patients with autoimmune diseases (1)(2)(3)(4), cancer (5), transplantation (6,7), and other disease conditions (8). The Luminex SAB assays involve Ags immobilized onto spherical beads in suspension and the use of these immobilized beads to capture serum Abs against the coated Ags.…”

“…The levels of Abs are assessed as mean fluorescent intensity (MFI). MFI has become a critical guide for monitoring anti-HLA-IgG Abs in patients waiting for donor organ selection, desensitization therapy, and to assess the risk for acute and chronic humoral rejection (6,7). Because titration of HLA Abs with Luminex SAB assay is cost prohibitive, the MFI derived from the fluorescent signal of sera at a particular dilution is considered suitable to predict the level of primary-Abs bound to Ags on the beads (6,7,9,10).…”

An Update on Crossmatch Techniques in Transplantation

“…This method while effective in detecting sensitization in transplant candidates has been replaced by bead technology [11].…”

“…The beads are washed and incubated with a second antibody, usually anti-human IgG labeled with phycoerythrin. The result is that a conglomerate of heterogenous antibodies can be tested and specific HLA antigens can be identified [11].…”