Elsevier

Human Immunology

Volume 76, Issue 9, September 2015, Pages 651-656
Human Immunology

Evaluation of the iBeads assay as a tool for identifying class I HLA antibodies

https://doi.org/10.1016/j.humimm.2015.09.012Get rights and content

Abstract

In addition to antibodies targeting native class I human leukocyte antigens (HLA), the single antigen flow beads assay (SAFB) detects antibodies recognizing denatured forms (anti-dHLA). Acid treated SAFB and the modified SAFB reagent named iBeads are expected to distinguish anti-native (anti-nHLA) from anti-dHLA. Sera from 280 class I HLA-sensitized SAFB-positive kidney transplant candidates were retested with acid-treated SAFB and iBeads. Concordance between SAFB and iBeads, taking into account acid-treatment results, was described at global and locus levels. T-lymphocyte flow cytometry crossmatches (FCXM) were performed to identify an accurate iBeads MFI threshold allowing predicting FCXM results. Concordance between acid-treatment and iBeads assays was observed for 86.9% of alleles. The iBeads MFI were lower than for classical SAFB, especially for HLA-B and C alleles. Anti-dHLA identified with acid-treated SAFB were more frequently negative with iBeads for HLA-B and -C alleles. An iBeads MFI threshold of 1000 allowed predicting positive FCXM with 95.6% sensitivity, 91.6% negative predictive value and 0.08 negative likelihood ratio. The iBeads assay still has limitations, but might represent an invaluable alternative to SAFB for virtual crossmatch strategies in organ transplant allocation programs.

Introduction

Nowadays, the single antigen flow bead (SAFB) assay has become the most used technique to define acceptable class I and class II HLA antigens in organ allocation programs. Indeed, it allows anticipating the pretransplant crossmatch results with a virtual crossmatch strategy, thanks to its unprecedented high sensitivity and resolution [1], [2]. For the same reasons, it occupies a central place in diagnosis and monitoring of antibody-mediated rejection after transplantation [3], [4], [5].

However, it has been recently discovered that SAFB display not only native class I HLA (nHLA), consisting of correctly folded heterotrimers of class I heavy chain, beta 2-microglobulin (B2m) and peptide, but also denatured class I HLA (dHLA), i.e. free class I HLA heavy chain (FHC) dissociated from B2m and peptide, and that dHLA can be targeted by serum antibodies [6], [7]. Differentiating anti-nHLA from anti-dHLA antibodies has been rendered possible with the concomitant use of acid-treated SAFB (D-SAFB) and classical, non-treated SAFB (N-SAFB) [6], [7], [8], [9], [10], which recently allowed us describing the prevalence and allele/antigen distribution of anti-dHLA in a large cohort of kidney awaiting recipients [11]. For this purpose, we established a local threshold for anti-dHLA definition, relying on the ratio between SAFB mean fluorescence intensity (MFI) with D-SAFB and N-SAFB equal to or higher than 1.2 (i.e. anti-dHLA and anti-nHLA displaying D  1.2N and D < 1.2N, respectively) [11]. The accuracy of this threshold was confirmed with a T-cell flow cytometry crossmatch (FCXM) assay, as anti-dHLA are not expected to recognize HLA molecules expressed by normal cells [11]. By associating a large cohort of patients and a cell-based assay, our results strengthened the scarce previously published data and mostly relying on case reports that antigens targeted by anti-dHLA should not be taken for granted as unacceptable in most cases.

A modified assay largely devoid of denatured class I HLA through enzymatic treatments of the SAFB (iBeads, One Lambda Inc., Canoga Park, CA) has recently been developed, and was suggested to distinguish clinically relevant DSA (i.e. iBeads-positive) from clinically irrelevant ones (i.e. iBeads-negative) [9]. Accordingly, we demonstrated that acid-treatment assay and iBeads at least in part identified the same antibodies recognizing denatured HLA molecules, and we have shown that iBeads were more selective toward antibodies able to trigger FCXM positivity than SAFB [11]. However, iBeads appeared not strictly specific for anti-nHLA, as they could still detect FCXM-negative anti-HLA antibodies identified as anti-dHLA, and some FCXM-positive anti-nHLA were iBeads-negative. Noteworthy, locus specificity of these drawbacks has not been evaluated yet.

The objectives of the present study were therefore to, (1) describe in a larger cohort of class I HLA-sensitized patients awaiting a kidney transplant the concordance between SAFB and iBeads assays at a global level, taking into account results from acid-treatment assay, (2) perform the same analysis at the locus level, (3) define for iBeads a MFI threshold allowing accurate prediction of FCXM results for the purpose of a routine use.

Section snippets

Patients, donors, and sera

All the 336 class I HLA-sensitized patients enrolled on our adult kidney waiting list between January 2000 and March 1st 2011 were eligible, but 24 (7.1%) were excluded because of high background in SAFB assay and/or iBeads assays [22 (6.5%) with both assays, 1 (0.3%) with iBeads only and 1 (0.3%) with SAFB only], and 32 (9.5%) because no serum was available anymore. The final study group included 140 women and 140 men, among whom 251 [90%, with 135 (96%) women and 116 (83%) men] had faced at

Description of class I anti-HLA antibodies defined with acid-treatment and iBeads assays

Among the 27,160 alleles tested with the 280 sera included in this study, 7831 (28.8%) were positive (MFI  1000) with N-SAFB and/or with iBeads [7553 (27.8%) positive with N-SAFB and 6593 (24.3%) with iBeads] (Fig. 1). According to the MFI ratio between the D-SAFB and N-SAFB assays, 469 (6.2%) alleles were defined as recognized by anti-dHLA (D  1.2N) and 7084 (93.8%) by anti-nHLA (D < 1.2N). Among the anti-dHLA, 109 (23.2%) and 360 (76.8%) were positive and negative with iBeads, respectively, while

Discussion

In this study, we confirmed with a larger cohort the findings from our previous work, i.e. that most of the anti-dHLA defined with acid-treatment of SAFB were iBeads negative [11], with about 87% of concordance. However, the existence of discrepancies between the two assays, i.e. iBeads-positive anti-dHLA and iBeads-negative anti-nHLA, raised the hypothesis that one of the two assays, or both, could fail in identifying such antibodies.

A fraction of authentic anti-dHLA was indeed unexpectedly

Author contributions

J.T. and J.V.: study design. J.V., G.G., T.N., J.M., P.M., L.C., J.L. and J.T.: performance of the research. J.V. and J.T.: data analysis. J.V. and J.T.: writing of the article. G.G., J.M., P.M., L.C. and J.L.: critical revision of the manuscript.

Conflict of interests

T.N. and J.L. are employees of One Lambda Inc. No specific funding was obtained for this study. The class I SAFB assays in acid-denatured condition for the patients with DSA in non-treated condition, and the iBeads assays were performed in One Lambda’s research department as a scientific collaboration, with reagents and equipments provided by One Lambda Inc and by staff people employed by One Lambda Inc.

The other authors of this manuscript have no conflicts of interest to disclose.

Acknowledgments

We acknowledge Catherine Rio, transplant coordinator nurse, for her valued assistance. We are grateful to the technicians of the immunology laboratory, for their technical expertise.

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