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.

Keywords: CDC; ELISA; HLA antibody; Luminex; beads; transplantation.

Figure 1

The figure represents the principles underlying the Luminex bead assay. Each bead has one or more different types of human leukocyte antigen (HLA) molecules attached depending on the level of testing being performed. If the test serum contains an HLA antibody it will bind to the appropriate HLA molecule. This binding can be detected by the use of a second phycoerythrin (PE)-labeled anti-human IgG. Each bead gives a specific signal when excited by one of the lasers built into the Luminex instrument due to the unique intensity of fluorophore embedded in the bead. A second laser detects the fluorescent excitation produced by the PE on the second antibody. The combination of the two signals indicates first the presence (PE fluorescence) and second the specificity (bead fluorescence) of the HLA antibody in the test serum.

Figure 2

The figure outlines the technical steps involved in the assay. The test serum and beads are incubated together at room temperature for 30 min and then washed three times with buffer prior to adding the second antibody. A second incubation period of 30 min at room temperature is followed by two further washes with buffer and then the mixture is resuspended in phosphate buffered saline for reading in the Luminex instrument.

Figure 3

The top panel shows the Luminex instrument. There are two lasers in the Luminex instrument (bottom panel). The red laser excites the fluorophore in the bead which provides a unique signal thereby identifying the HLA molecule attached. The green laser excites the phycoerythrin bound to the second anti-human IgG antibody indicating IgG antibody in the test serum has bound to the appropriate HLA molecule attached to the bead. (Modified from a figure provided by Serologicals Corporation.)