An early evaluation of the HISTO SPOT® AB ID Class I & II test in cardiothoracic transplant patients.

The HISTO SPOT® AB ID assay (BAG Diagnostics GmbH) is a novel single antigen HLA Class I & II antibody definition test used with the MR.SPOT® processor. We compared this assay with Luminex® -based assays to assess its potential application in defining unacceptable antigens for transplantation in patients awaiting transplants with cardiothoracic organs. A cohort of 40 sensitized cardiothoracic patients were identified, and one sample was selected from each patient. The required screening was based on the patients' antibody profiles (Class I, n = 17, Class II, n = 11, Class I & II, n = 12). Samples were screened with LABScreen™ Single Antigen (SAg), LIFECODES® LSA™, HISTO SPOT® AB ID, and an acid modified LABScreen™ SAg test for detecting antibodies against denatured HLA. Results indicated that HISTO SPOT® AB ID had reduced sensitivity (68% for Class I; 69% for Class II). When compared to LABScreen™ and LIFECODES® , HISTO SPOT® AB ID failed to detect Luminex® -defined antibodies with median fluorescence intensity (MFI) ranging from 1114 to 24,489. The HISTO SPOT® AB ID panel used in the study had reduced antigen representation compared with Luminex® -based assays which further compromised its capacity for antibody detection and definition. Further work is needed to evaluate the clinical relevance of these differences between the performance of HISTO SPOT® and Luminex® -based methods.

Low incidence of IgA isotype of HLA antibodies in alloantigen exposed individuals.

Human leukocyte antigen (HLA) antibodies are induced by pregnancy, transfusion, or transplantation. Serum from transplant recipients is regularly screened for IgG HLA antibodies because of their clinical relevance for transplant outcome. While other isotypes of HLA antibodies, such as IgA may also contribute to the alloimmune response, validated detection assays for IgA HLA antibody detection are lacking. Therefore, we modified the commonly used luminex screening assay for IgG HLA antibody detection (IgG-LMX) into an IgA HLA antibody screening assay (IgA-LMX). Optimization and validation was performed with IgG, IgA1, and IgA2 isotype variants of HLA-specific human recombinant monoclonal antibodies (mAbs). Reactivity patterns of IgA1 and IgA2 isotype HLA-specific mAbs in IgA-LMX were identical to those of the IgG isotype. Cross-reactivity with IgG and IgM antibodies and nonspecific binding to the beads were excluded. Further assay validation showed the absence of IgA HLA antibodies in serum from individuals without alloantigen exposure (n = 18). When the IgA-LMX assay was applied to sera from 289 individuals with known alloantigen exposure through pregnancy (n = 91) or kidney transplantation (n = 198), IgA HLA antibodies were detected in 3.5% of individuals; eight patients on the kidney retransplant waitlist and two women immunized through pregnancy. The majority (90%) of IgA HLA antibodies were directed against HLA class II and were always present in conjunction with IgG HLA antibodies. Results of this study show that this validated IgA-LMX method can serve as a screening assay for IgA HLA antibodies and that the incidence of IgA HLA antibodies in alloantigen exposed individuals is low.

The good, the bad, and the ugly of luminex donor-specific crossmatch.

The presence of donor-specific antibodies directed against human leukocyte antigen significantly influences renal transplant because of antibody-mediated rejection. We performed the screening of pre-renal transplant patients for preformed anti-HLA antibodies using anti-human globulin augmented-complement-dependent lymphocytotoxicity crossmatch (AHG-CDCXm), luminex donor-specific crossmatch (LumXm) and HLA antibody screening. Seven hundred and fifty-four patients were assessed for LumXm. HLA antibody screening was possible in 325 out of 754 cases. All the three investigations viz. CDCXm, HLA antibody screening and LumXm was performed in 325 patients. All CDCXm positive patients (10/325, 3.08%) were also positive with LumXm and HLA antibody screen whereas 14 cases (4.31%) with CDCXm negative were positive with luminex-based assays. LumXm and HLA antibody screening were both positive in 24 (7.38%) cases, LumXm and HLA antibody screening were both negative in 275 (84.63%) cases and LumXm negative and HLA antibody screening was positive in 22 (6.76%) cases. However, there were four cases (1.23%) which were positive in LumXm in spite of being negative in HLA antibody testing. Single Antigen Bead (SAB) assay was performed in all patients positive for HLA antibody test. We suggest that LumXm is a useful and sensitive technique for the detection of anti-HLA antibodies in pre-transplant renal patients. However, other measures such as luminex antibody screen, SAB assay, history of the donor, and the class of antibodies involved should be taken into consideration for pre-transplant work up of renal patients.