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Optimizing HLA-antibody Determination

Filippone, Edward J. MD1; Ravindranath, Mepur H. PhD2

doi: 10.1097/TP.0000000000002719

1 Division of Nephrology, Department of Medicine, Sidney Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA.

2 Department of Hematology and Oncology, Children’s Hospital, Los Angeles, CA.

Received 6 March 2019.

Accepted 8 March 2019.

M.H.R. received grant funding from Immucor from July, 2017 to June, 2018 that was paid to his institution but contributed to his salary. The other author declares no conflicts of interest.

Correspondence: Edward J. Filippone, MD, 2228 S Broad St, Philadelphia, PA 19145. (

Mepur H. Ravindranath, PhD. (

E.J.F. and M.H.R. participated in the writing of the letter.

We read with great interest the paper in Transplantation by Bertrand et al1 comparing the sensitivity and specificity of Labscreen (One Lambda) versus Lifecodes (Immucor) beadsets for determination of donor specific human leukocyte antigen (HLA) antibodies following kidney transplantation. Indeed, our work clarifies to some extent their findings of increased sensitivity (specifically, mean fluorescence intensity [MFI] above an arbitrary cutoff) with Labscreen beads but increased specificity with Lifecodes beads. We demonstrated previously2 (and recently validated this in a second cohort3) that Labscreen beads not only contain HLA trimers of α-chain, β-chain (β-2 microglobulin for class I), and cognate peptide as exists in vivo, but also contain dimers and monomers. Antibodies directed against epitopes exposed only in the monomers contribute to the MFI of a particular bead and may even explain the total reactivity, yet of themselves are not apparently pathogenic.4

In comparison, Lifecodes beads contain only intact trimers,2,3 and are, therefore, more likely to detect pathogenic antibodies. This would explain the greater “sensitivity” of Labscreen beads in the Bertrand et al1 paper, also without proof that the difference is the result of truly pathogenic antibodies. The greater specificity of Lifecodes beads may be explained by the restriction of antigens to only intact trimers and peptide free dimers. Accurate determination of pathogenic antibodies is critically important in defining unacceptable antigens (and the resultant calculated panel reactive antibody) and for consideration of desensitization pretransplantation, as well as for possible treatment posttransplantation. For example, we reported a patient with a calculated panel reactive antibody of 99.5% using Labscreen beads that was 0% with Lifecodes.3 Routinely, our patients had more positive alleles with Labscreen compared to Lifecodes beadsets for both class I and class II.

Additional methodologic issues with optimal determination of HLA antibodies include the nature of the detection antibody and the prozone effect. As their detection antibody, Bertrand et al1 used phycoerythrin-conjugated goat antihuman IgG, as usually specified by the manufacturer. This is somewhat of a misnomer with One Lambda, as these are not complete IgG molecules, but rather are polyclonal F(ab′)2 fragments capable of binding to multiple epitopes on the constant region of an IgG heavy chain.5 Although Immucor uses whole IgG antibodies, they are polyclonal and are also capable of biding multiple epitopes. Hence, in either case, the binding may not be 1:1, and there is the potential for significant amplification, especially with lower concentrations of primary antibody. Alternatively, an Fc-specific monoclonal IgG antibody directed against a single epitope will bind 1:1 with the Fc-domain of the primary antibody. We have shown that using such a monoclonal antibody generally results in a lower number of positive class I and class II alleles.3 Furthermore, MFIs are also usually lower with the monoclonal antibody, at least for class I alleles with lower titer antibodies,3 although not for class II alleles3 or when high titers of primary class I antibody are present2 (possibly due to primary antibody aggregation, for details, see Ravindranath et al5). Finally, Bertrand et al1 used neat serum as opposed to 1/10 or greater dilutions. This enhances chances for a prozone effect with high titer antibodies.

Nevertheless, we commend Bertrand et al1 for their work further enlightening the transplant community on potential differences between these beadsets.

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1. Bertrand D, Farce F, Laurent C, et al. Comparison of two luminex single-antigen bead flow cytometry assays for detection of donor-specific antibodies after renal transplantation. Transplantation. 2019;103:597–603.
2. Ravindranath MH, Jucaud V, Ferrone S. Monitoring native HLA-I trimer specific antibodies in luminex multiplex single antigen bead assay: evaluation of beadsets from different manufacturers. J Immunol Methods. 2017;450:73–80.
3. Ravindranath MH, Filippone EJ, Mahowald G, et al. Significance of the intraindividual variability of HLA IgG antibodies in renal disease patients observed with different beadsets monitored with two different secondary antibodies on a luminex platform. Immunol Res. 2018;66:584–604.
4. Cai J, Terasaki PI, Anderson N, et al. Intact HLA not beta2m-free heavy chain-specific HLA class I antibodies are predictive of graft failure. Transplantation. 2009;88:226–230.
5. Ravindranath MH, Jucaud V, Banuelos N, et al. Nature and clonality of the fluoresceinated secondary antibody in luminex multiplex bead assays are critical factors for reliable monitoring of serum HLA antibody levels in patients for donor organ selection, desensitization therapy, and assessment of the risk for graft loss. J Immunol. 2017;198:4524–4538.
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