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The Cutting Edge of HLA Diagnostics

Reed, Elaine F. PhD1; Nickerson, Peter W. PhD2

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doi: 10.1097/TP.0000000000001865
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Since the 1950s, landmark twin transplant studies at the Peter Bent Brigham Hospital in Boston, Massachusetts, the critical role of histocompatibility has been evident.1 Nevertheless, 2 pivotal advances in the 1960s were required to make transplantation across HLA barriers available to all patients with end-stage organ failure: (1) the identification of effective immunosuppressive combinations; and (2) the development of a diagnostic assay to detect preexisting recipient humoral memory to the donor, which if crossed, would lead to hyperacute allograft rejection.2,3 Thereafter, the transplant community has seen continuous advances in both of these fields of study that have directly impacted patients by improving outcomes.

While advances were made between the 1960s and 1990s in HLA diagnostics, it was not until the advent of molecular biology technologies that the field of histocompatibility underwent a modern renaissance. Indeed, solid-phase microbeads, coated with purified HLA proteins, have allowed for novel mechanistic insights into the role donor-specific HLA antibodies (DSA) play in limiting allograft survival both pretransplant and posttransplant and refocused the attention of the community on the humoral arm of the alloimmune response. Characterization of antibody features that are critical for effector functions may help to identify HLA antibodies that are more likely to cause rejection. Several recent modifications proposed to these solid-phase assays explore whether the attributes of donor specific HLA antibodies hold diagnostic or prognostic utility in patient care. It is in this context that the current supplement has been conceived.

Liwski and Gebel4 provide a comprehensive overview of the evolution in HLA diagnostics. They lay out the strengths, limitations and pitfalls of both cell-based and solid phase assays. Further, they highlight key considerations when interpreting HLA diagnostics and their requirement to be integrated with the clinical context. Most importantly, they emphasize the critical requirement to have excellent communication between the laboratory and the clinical teams.

Valenzuela and Schaub5 provide an excellent review in our understanding of the basic biology leading to antibody generation—focusing on the highly ordered process of IgG subclass switching and affinity maturation. Importantly, they highlight the complexity of functions that are attributable to each IgG subclass and remind us that one cannot treat subclasses as occurring in isolation nor does each possess a black and white effector function (eg, IgG2 is capable of complement fixation under the right conditions). Limitations in the current literature are discussed, as are the critical assay elements that need to be considered when trying to assess a given subclass.

The last several years have seen a rapid growth in the literature describing the utility of complement-dependent solid phase assays as prognostic biomarkers. Lan and Tinckam6 provide a critical review of this body of work detailing the strengths, weaknesses, and gaps. They highlight the debate regarding the ability of these assays to truly reflect complement activating capacity independent of the antibody titer. Finally, they discuss the variation in assay methods and study designs used to date, leading them to conclude that there are insufficient data available to make a strong recommendation for their application in routine clinical care at present.

Increasingly, it is apparent that antibody quantity is a primary determinant of clinical outcome. This begs the question: “How to best quantitate donor specific antibody?” Solid-phase assays, although Food and Drug Administration–approved for qualitative detection of HLA antibody, never claimed to quantitate antibody levels using mean fluorescence intensity readouts. Tambur and Wiebe7 highlight the pitfalls of mean fluorescence intensity and discuss the value of antibody titration to provide this quantitative assessment. Specifically, they discuss the utility of antibody titration in the context of pretransplant and posttransplant assessment.

It should be clear from all of these articles that although major advances have occurred in HLA diagnostics since the 1960s and more recently in the last decade, we are at best as, Winston Churchill stated, “[at] the end of the beginning.” The effector functions of HLA antibodies, including complement activation, FcγR-dependent macrophage and Natural Killer cell functions, and endothelial cell activation synergize to mediate allograft damage. Key features regulating antibody effector function such as antibody subclass, Fc glycosylation, and FcγR polymorphisms are likely important elements of HLA-antibody pathogenicity and recipient risk of antibody-mediated rejection. Therefore, characterization of the patient’s DSAs and knowledge of how they mediate graft damage should enable more precise patient risk stratification and treatment for antibody-mediated rejection. To accomplish this objective, further basic and clinical research is needed to truly understand how to optimize and evolve the current HLA diagnostic tools. In parallel, as evident in this supplement, the field requires improvements in assay quality, standardization of assay performance, and careful study design with meticulous patient characterization—too often studies are not comparable to one another because of lack of investment in these foundational parameters, making it difficult to make strong recommendations. Finally, the histocompatibility community throughout its history has been marked by collaboration to advance the science. Today, we need to continue this legacy to advance our knowledge and optimize the utility of HLA diagnostics in maximizing patient care.


1. Murray JE, Merrill JP, Harrison JH. Renal homotransplantation in identical twins. Surg Forum. 1956;6:432–436.
2. Starzl TE, Marchioro TL, Rifkind D, et al. Factors in successful renal transplantation. Surgery. 1964;56:296–318.
3. Patel R, Terasaki PI. Significance of the positive crossmatch test in kidney transplantation. N Engl J Med. 1969;280:735–739.
4. Liwski R, Gebel HM. Of cells and microparticles: assets and liabilities of HLA antibody detection. Transplantation. 2018;1(Suppl 1):S1–S6.
5. Valenzuela N, Schaub S. The biology of IgG subclasses and their clinical relevance to transplantation. Transplantation. 2018;1(Suppl 1):S7–S13.
6. Lan JH, Tinckam K. Clinical utility of complement dependent assays in kidney transplantation. Transplantation. 2018;1(Suppl 1):S14–S22.
7. Tambur AR, Wiebe C. HLA diagnostics: evaluating DSA strength by titration. Transplantation. 2018;1(Suppl 1):S23–S30.
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