Antibody Detection Education

Antibody screening is a process used to identify the presence of HLA-specific antibodies in a patient's blood. These antibodies can develop due to previous transplants, blood transfusions, or pregnancies and can target donor HLA antigens, leading to transplant rejection. The screening process involves testing the recipient's serum against a panel of HLA antigens to detect and characterize any existing antibodies. This helps in assessing theimmunological risk and in selecting compatible donors, thereby improving the chances of successful transplantation and reducing the risk of rejection.

Rejection of allografts may occur through either cellular or antibody-mediated immune mechanisms. In most instances, the immune response targets human leukocyte antigens (HLAs) expressed on the donor organ’s cells. Although there is no standard laboratory test to assess the cellular immune responsiveness of potential transplant recipients, the detection of HLA antibodies, especially those directed against the donor’s specific HLAs has long been central to donor–recipient histocompatibility testing since the advent of clinical transplantation.

One of the first assays introduced into routine clinical practice for transplantation was the pretransplant crossmatch complement-dependent cytotoxicity (CDC) test developed by Patel and Terasaki 1969¹ which involves testing the recipient’s serum for cytotoxic activity against the donor’s lymphocytes to determine whether preformed antibodies capable of attacking donor cells are present.

A positive CDC crossmatch indicates antibody incompatibility and high risk of hyperacute or acute rejection. In most cases, this result contraindicates transplantation.

A limitation of the CDC assay is it’s lack of sensitivity only able to detect relatively high titre complement-fixing antibodies.

The Flow Cytometry Crossmatch test 1983 by Garovoy et al., is a more sensitive alternative to the traditional CDC crossmatch. This also involves testing recipients serum against the donor lymphocytes to detect bound antibodies with a fluorescently-labelled anti-human immunoglobulin using a flow cytometer. This method is more sensitive than CDC and enables better immunologic risk stratification by being able to detect lower level DSAs.

A positive FCXM especially with a negative CDC does not necessarily contraindicate transplantation but offers a risk based approach.

The CDC assay was also used for antibody screening and identification by testing a recipient’s serum against a panel of cells with known HLA type. The reaction pattern of  positive cells bearing HLA antigens is used to determine unacceptable HLA specificities. The breadth of panel reactivity also gives Panel Reactive Antigen percentage (PRA%) score as a measure of sensitization. Higher PRA% indicates less HLA antibody compatible donors for a patient.

Since lymphocytes express Class I ( T and B cells) and Class II ( B cells) HLA phenotype, it is not always possible to clearly distinguish the which antigens are being targeted in the reaction pattern. This lowers the specificity of cell based panels.

Solid phase assays allow a specific analyte to be attached to a solid surface such as a well of a microtiter plate to increase specificity and sensitivity.

The Enzyme-Linked Immunosorbent Assay (ELISA) assay was one of the first solid phase assays used for HLA antibody screening and identification² Microtitre wells are coated with HLA antigens of varying types for screening or the same HLA antigen type for identification.

The ELISA method is more sensitive than the CDC assay for detecting HLA-specific antibodies³ but  was largely superseded  by the bead based assays that offer greater sensitivity and specificity.

Currently the most current sensitive and specific solid phase method for antibody screening and identification are microbead based assays which consist of beads with unique spectral signatures coated with antigens. Bound antibodies are detected with a fluorescently-labelled conjugate and analysed on a Luminex analyser.

The Single Antigen Bead (SAB) assay is the most sensitive and specific consisting of beads coated with a single antigen for optimum detection⁴. SAB have revolutionized HLA-specific antibody identification allowing high sensitivivity detection of HLA-specific antibodies to specific alleles and measuring their relative strengths through median fluorescence intensity (MFI). The MFI data enables a risk-based approach to managing highly sensitized patients.

Other bead-based assays coated with Class I or Class II HLA antigen phenotypes also offer higher sensitivity compared to cells due to the increased antigen density. Phenotype bead assays allow each class to be analysed separately to improve specificity, although similar to cells it is not always possible to clearly distinguish the which antigens are being targeted in the reaction pattern of phenotype beads.

Table 1 summarises common assays used for antibody screening and identification.

Table 1. Common assays used for antibody screening.