HLA and non-HLA antibody testing assesses an individual’s sensitization status and identifies the antigens specifically targeted by those antibodies. The tests used to identify anti-HLA antibodies assess reactions of antibodies with well-characterized panels of individual lymphocytes (using T and B Cell Cytotoxic Antibody Screen), or with purified or recombinant HLA antigens coupled to specialized microparticles (Panel Reactive Antibody Screen) or HLA Single Antigen Class I and Class II Luminex Bead Test. Reactivity of the patient’s serum with a specific donor’s antigens is assessed in the T and B cell Complement-Dependent Cytotoxicity and Flow Crossmatches. Each of these assays has varying sensitivity and specificity for HLA antibody identification (Table 2).
Non-HLA Antibodies reactive with donor endothelial cell antigens have been implicated in rejection and graft loss. The MICA Single Antigen Antibody Screen detects IgG antibodies against the most common MICA alleles and allows high throughput antibody screening and identification using Luminex technology. An ELISA based platform is used to assess antibodies to angiotensin type II receptor 1 (AT1R). The Endothelial Cell Crossmatch detects IgG antibodies to surrogate endothelial cell lines.
|Table 2. HLA and Non-HLA Antibody Testing and Crossmatching|
|T and B Cell Complement-Dependent Cytotoxic Crossmatch||Cell Based||IgG, Class I and Class II||Positive or Negative|
|HLA Single Antigen-C1q Screen||Solid Phase||IgG, Class I and Class II||Positive HLA Specificities|
|T and B Cell Flow Cytometric Crossmatch (w/ or w/o Pronase)||Cell Based||IgG, Class I and Class II *||Median Channel Shift (MCS)|
|Luminex Panel Reactive Antibody Screen||Solid Phase||IgG, Class I and Class II||%PRA and Positive HLA Specificities **|
|HLA Single Antigen Class I and II Luminex Bead Test||Solid Phase||IgG, Class I and Class II||Positive HLA Specificities and Strength (MFI)|
|Antibody Specificity Profile||Solid Phase||IgG, Class I||Positive HLA Specificities|
|MICA Single Antigen Anitbody Screen||Solid Phase||IgG, MICA||Positive HLA Specificities|
|AT1R ELISA||Solid Phase||IgG, AT1R||U/ml ***|
|Endothelial Cell Crossmatch||Cell Based||IgG||Median Channel Shift (MCS)|
|* should be paired with HLA Single Antigen Bead Test
** HLA specificities can be identified up to 70% PRA
*** >10 U/ml is associated with endothelial cell dysfunction
Luminex Panel Reactive Antibody Screen
Antibodies in sera can be identified by the Luminex based Panel Reactive Antibody Screen. Microspheres are coated with two haplotypes of HLA Class I or II antigens isolated from the surface of cell lines in their native (natural) conformation. The beads, loaded with varying amounts of florescent dye, are then incubated with patient serum. HLA displayed by the panel of microspheres is representative of the donor pool. HLA antibodies in the serum bind their specific antigen targets, and are identified by Luminex multiplex technology. The result is the percentage of microspheres on the panel that are identified to react with HLA antibodies in the serum, termed percentage panel reactive antibody (%PRA) as well as positive antibody specificities. This solid phase assay is more sensitive than the cell based Cytotoxic Antibody Screen and less sensitive and specific than the HLA Single Antigen Class I and Class II Luminex Bead Test. This assay does not identify antibodies to low expression antigens such as HLA C, DP or DR51/52/53.
The single antigen bead test is the most sensitive and specific solid phase test for identifying HLA antibodies in patient serum. Similar to the Luminex Panel Reactive Antibody Screen, single antigen bead panels are made up of solid phase microspheres loaded with varying amounts of florescent dye. In contrast, however, each bead only displays one single recombinantly generated HLA antigen. Sera are DTT treated to remove interfering substances and then incubated with the HLA Class I or Class II single antigen bead panels. HLA antibodies in the sera bind their specific targets, and are identified by Luminex multiplex technology. Antibodies to antigen specificities are reported with the strength of the antibody (MFI). The data can be used to calculate the percentage of donors to which the patient is sensitized in the UNOS donor pool (cPRA). Sera can also be diluted to assay for the effects of prozone, or to determine the response to desensitization therapy.
For research and clinical trials, HLA antibody testing results are available in a batched electronic file output, that can be tailored to the investigators’ requirements.
The HLA Single Antigen-C1qScreen is a modification of the HLA Single Antigen Class I and Class II Luminex Bead Test that detects the fixation of C1q, the first component of the classical complement cascade, to anti-HLA antibodies bound to Luminex beads. This test enables characterization of potentially high titer or complement activating antibodies. The specificities of C1q binding antibodies are reported.
The Antibody Specificity Profile assays for antibodies to HLA Class I antigens using the HLA Single Antigen Class I Luminex Bead Test. This test is used to assess a patient’s sensitization to platelet donor HLA. Antibodies to antigen specificities are identified and reported as positive or negative.
The Complement-Dependent Cytotoxic Crossmatch identifies the most important antibodies in the crossmatch test - those responsible for hyperacute rejection of grafts. Donor T and B lymphocytes are incubated with patient serum. Complement is added, and cell death is visually assessed via microscopy. A positive crossmatch due to IgG antibodies directed against HLA-A, -B, -C, -DR and -DQ antigens is a clear contraindication to transplantation because of the high risk of hyperacute rejection.
The T and B Flow cytometric crossmatch identifies IgG HLA antibodies that bind to the surface of the T and/or B cell. This test is more sensitive than the T and B Cell Complement-Dependent Cytotoxic Crossmatch. Patient sera is incubated with donor T and B cells. HLA antibodies in the patient sera bind to HLA antigens on the surface of the lymphocytes and are detected flow cytometrically. The results are reported as the median channel shift (MCS) over the negative control sera. A positive result is >50 MCS for the T cell and for the B cell crossmatch- >100 MCS for crossmatches with cells isolated from living potential donors, and >120 MCS for crossmatches with cells isolated from deceased potential donors. The crossmatch can also be performed after pronase treatment of the lymphocytes. Pronase treatment enzymatically removes Fc receptors and the CD20 molecule leaving HLA molecules intact. The treatment reduces false positive reactions due to non-specific antibody:FC receptor interactions, and rituxan binding to CD20 in patients undergoing desensitization therapy.
The MICA Single Antigen Antibody Screen detects antibodies against a panel of the most common MICA antigens coupled to Luminex beads. Antibodies to MICA antigen specificities are reported with the strength of the antibody (MFI).
Angiotensin II type I receptor II (AT1R) is a G protein-coupled receptor that mediates angiotensin effects and causes vasoconstriction in vascular smooth muscle cells, aldosterone secretion by the adrenal cortex and sodium reabsorption in proximal tubules. Binding of antibodies to AT1R mimics angiotensin II binding, results in agonistic activation of AT1R signaling pathways, and contributes to hypertension. Recent studies found a significant association of anti-AT1R antibodies, both pre- and post- transplantation, with graft rejection and failure in kidney and heart transplantation. Antibodies to AT1R are identified in an ELISA based platform. At a serum dilution of 1:40 a result >10 U/ml indicates risk of endothelial cell dysfunction. A sera with AT1R levels >40U/ml can be diluted to determine the titer of the antibody.
Endothelial cells constitute the first point of contact between the transplanted organ and the recipient's immune system. Antibodies reactive with donor endothelial cell antigens have been implicated in cases of humoral rejection in the absence HLA antibodies. The Endothelial Cell crossmatch (ECXM) identifies antibodies to non-HLA antigens expressed on endothelial cells. The results are reported as the median channel shift (MCS) over the negative control sera. A positive result is >50 MCS.