The Journal of Heart and Lung Transplantation
Volume 29, Issue 11 , Pages 1207-1209, November 2010

On solid-phase antibody assays

  • Octavio E. Pajaro, MD, PhD

      Affiliations

    • Corresponding Author InformationReprint requests: Octavio E. Pajaro, MD, PhD, Division of Cardiothoracic Surgery, University of Alabama at Birmingham, LHRB 780, 1530 3rd Ave S, Birmingham, AL 35294. Telephone: 205-996-9290. Fax: 205-996-6638
    • ,
  • James F. George, PhD

published online 02 September 2010.

Article Outline

There is considerable evidence that pre-operative and post-operative anti-human leukocyte antigen (anti-HLA) antibodies are deleterious in thoracic transplantation. While debate continues in heart and lung transplantation on the role of and the diagnosis and treatment of antibody-mediated rejection (AMR), central to the discussion is our ability to detect anti-HLA antibodies. This perspective outlines the concerns elicited by new technology for detection of anti-HLA antibodies using solid-phase assays, and highlights the need for functional assays to further understand the clinical significance of these antibodies.

Key Words: solid-phase assays, antibody-mediated rejection, anti-HLA antibody, virtual crossmatch, panel reactive antibodies

 

Outcomes in thoracic organ transplantation have significantly improved during the last 30 years, but immune-mediated events continue to limit short-term and long-term survival. Although initial efforts in immunosuppressive therapy were directed primarily against the cellular response, the humoral antibody response can also threaten organ and patient survival. Recent retrospective studies by Nwakanma et al1 (2007) and Shah et al2 (2008) have shown that an elevated panel-reactive antibody screen (PRA) before thoracic transplantation is still a significant risk factor, even in the current era of improved immunosuppressive therapies. Data from these studies implicitly support the concept that antibody-mediated rejection (AMR) plays a role in both heart and lung transplantation.3 Central to the diagnosis of AMR is the detection of donor-specific antibodies.4, 5 Thus, efforts to monitor the antibody response before and after transplant and to improve the post-operative diagnosis of AMR6, 7, 8 are clearly justified and necessary.

Methods for the detection of anti-human leukocyte antigen (HLA) antibodies have undergone significant improvements with the development of solid-phase assays (SPAs). Allelic HLA molecules can be bound to a solid matrix, such as a microbead, and therefore can be used to determine the specificity of antibodies existing within serum, plasma, or other fluids with high accuracy and sensitivity (Figures 1 and 2). However, these new assays have generated considerable debate about how to interpret and apply the results clinically. The debate is far from simply academic, because incorrect interpretations can lead to a patient's death. Rigid exclusion of recipients with positive results can lead to highly selective and limited donor organ acceptance and, thus, potentially lost opportunities for patients awaiting transplant. On the other hand, failure to properly consider anti-HLA antibodies could lead to acute or chronic AMR.

  • View full-size image.
  • Figure 1. 

    Detection of anti-human leukocyte antigen (HLA) antibodies has traditionally involved the use of peripheral blood leukocytes or cell lines that constitutively express HLA on the cell membrane (upper flow diagram). Anti-HLA antibodies are detected by the serum's ability to lyse the cells in the presence of complement (complement-dependent cytotoxicity [CDC]) or by fluorescence-based techniques using flow cytometry. Recently (lower flow diagram), purified HLA molecules bound onto a solid matrix (e.g., microbeads) are used as the substrate and the antibodies are detected by either enzyme-linked immunosorbent assays (ELISA) or again by flow cytometry. A recent consensus conference organized by the International Society for Heart and Lung Transplantation9 reported that 78% of centers performing thoracic organ transplantation used Luminex assays (solid-phase HLA microbead assays) for the detection of circulating antibodies.

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  • Figure 2. 

    This schematic illustrates the basic steps involved in flow cytometric antibody detection. (1) Incubation of recipient serum with microbeads coated with purified human leukocyte antigen (HLA) molecules. If the serum contains anti-HLA antibodies specific to the HLA on the beads, the antibodies will remain bound to the HLA-bead complex. (2) Incubation of the microbeads with fluorescent-tagged anti-human globulin. (3) Detection of the fluorescent tagged anti-human globulin using a flow cytometer.

Arguments in favor of strict avoidance of potential antigens determined by the presence of antibodies detected by the SPAs come from several perspectives:

1.Antibody titres do not necessarily determine the clinical significance of an antibody.10 As a consequence, a “weakly-positive” result theoretically could be as relevant as a “strongly-positive” result. This view is directly supported by data from Rose and Smith10 and Smith et al.11 Low antibody titres can increase dramatically within a few days of antigen restimulation due to an anamnestic response. Even antibodies that remain low-titre can eventually have significant deleterious effects, and even a history of anti-HLA antibodies directed against donor antigens is a risk factor for poor outcomes.12

2.Currently, no accurate and standardized method is clinically available to determine the functional characteristics of the antibodies detected by the SPAs. Rose and Smith10 show convincing data that the ability of pre-transplant antibodies to activate the complement cascade is an important determinant of poor outcomes in heart transplant recipients, and that even non-HLA immunoglobulin M antibodies that fix complement can have deleterious effects.

Attempts at clinical validation in thoracic transplantation have come only from few retrospective analyses comparing the results of traditional complement-dependent cytotoxicity (CDC) crossmatch (enhanced with antihuman globulin (AHG)) with the virtual crossmatch.10, 13 The virtual crossmatch is a pre-transplant comparison of the donor HLA genotype (using results from low-resolution DNA testing) with the gene families represented by the beads that bound antibody from the potential recipient. A positive virtual crossmatch is defined as concordance between the gene family represented by the bead and the gene family of the donor (Figure 3). The recent study by Stehlik et al13 estimated the positive predictive value of an incompatible class I virtual crossmatch as 79%. Thus, ignoring a positive virtual crossmatch without a prospective CDC-AHG crossmatch could lead to poor outcomes in approximately 79% of the positive recipients.

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  • Figure 3. 

    Simplified example, using only 2 class I human leukocyte antigen (LCA) loci of a positive and negative virtual crossmatch (VXM). The sensitized imaginary recipient has had antibodies detected against A1, A11, and B7 by Luminex single-antigen beads. The first donor offered to the recipient has been typed as A1, A25; B55 and B57. Because the class I antibody (anti-A1) detected by the SPA corresponds to the donor typing as A1, the VXM is considered positive. The second donor does not express a class I allele that corresponds to the antibodies detected in this recipient. Thus, the VXM is negative in the second example.

Until we have a way to detect potentially suitable compatible donors, it can be argued that the prudent approach would be to refuse non-local donors based on a positive virtual crossmatch and to insist on a prospective CDC-AHG crossmatch when logistically possible. In fact, Ross et al12 have proposed this approach in conjunction with the use of a continuous-flow mechanical circulatory support as a bridge to a negative virtual crossmatch. They and we have also suggested a priority allocation system for sensitized patients awaiting thoracic transplant.14

On the other hand, those who are unconvinced about how to make critical decisions using SPAs express legitimate concerns:

1.There is no standardized method of determining a positive or negative result. Thus, the raw data from these assays may yield different interpretations from center to center. This inconsistency partly reflects the void in our understanding of the functional implications of the antibodies detected by fluorescence. Ultimately, the definition of a positive result will have to be made with some knowledge of the functional consequences of the antibody that binds to the beads, as proposed by Smith et al11 in 2006.

2.The observation by centers that patients can have a “positive PRA” with no history of a specific sensitizing event, neither while on the waiting list nor before evaluation for transplant (our transplant center experience and others),15, 16 can raise significant doubt about the clinical relevance of these antibodies and makes the definition of a positive result even more difficult by blurring the threshold for positivity.

3.The overall excellent results obtained in thoracic transplantation before the use of SPAs make it difficult to adopt a new method that, at first glance, appears to deny thoracic transplantation to many potential recipients.

4.The unclear significance of class II antibodies in the pre-transplant setting for heart and lung transplantation adds further fuel for debate.

Further studies, including prospective clinical studies, are needed to help define the significance of the antibodies detected by the bead assays. These studies would help standardize the interpretation of the fluorescence data by establishing rational and clinically-relevant definitions of positivity and negativity. Furthermore, they would establish whether interpretation of the fluorescence data differs before and after transplant or differs in certain clinical scenarios, such as in patients with no known sensitizing events. In addition, studies are necessary to define the functional properties of the antibodies, specifically their ability to bind and activate complement.

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Disclosure statement 

None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.

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References 

  1. Nwakanma LU, Williams JA, Weiss ES, Russell SD, Baumgartner WA, Conte JV. Influence of pretransplant panel-reactive antibody on outcomes in 8,160 heart transplant recipients in recent era. Ann Thorac Surg. 2007;84:1556–1562
  2. Shah AS, Nwakanma L, Simpkins C, Williams J, Chang DC, Conte JV. Pretransplant panel reactive antibodies in human lung transplantation: an analysis of over 10,000 patients. Ann Thorac Surg. 2008;85:1919–1924
  3. Glanville AR. Antibody-mediated rejection in lung transplantation: myth or reality?. J Heart Lung Transplant. 2010;29:395–400
  4. Golocheikine A, Nath DS, Basha HI, et al. Increased erythrocyte C4D is associated with known alloantibody and autoantibody markers of antibody-mediated rejection in human lung transplant recipients. J Heart Lung Transplant. 2010;29:410–416
  5. Takemoto SK, Zeevi A, Feng S, et al. National conference to assess antibody-mediated rejection in solid organ transplantation. Am J Transplant. 2004;4:1033–1041
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  8. Kfoury AG, Renlund DG, Snow GL, et al. A clinical correlation study of severity of antibody-mediated rejection and cardiovascular mortality in heart transplantation. J Heart Lung Transplant. 2009;28:51–57
  9. Kobashigawa J, Mehra M, West L, et al. Report from a consensus conference on the sensitized patient awaiting heart transplantation. J Heart Lung Transplant. 2009;28:213–225
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  11. Smith JD, Hamour IM, Banner NR, Rose ML. C4d fixing, luminex binding antibodies—a new tool for prediction of graft failure after heart transplantation. Am J Transplant. 2007;7:2809–2815
  12. Ross H, Tinckam K, Rao V, West LJ. In praise of ventricular assist devices-mechanical bridge to virtual crossmatch for the sensitized patient. J Heart Lung Transplant. 2010;[E-pub doi: 10.1016/j.healun.2010.02.006]
  13. Stehlik J, Islam N, Hurst D, et al. Utility of virtual crossmatch in sensitized patients awaiting heart transplantation. J Heart Lung Transplant. 2009;28:1129–1134
  14. Pajaro OE, Kirklin JK, George JF. Computational analysis of the single antigen beads used in solid phase assays for the detection of anti-HLA antibodies. J Heart Lung Transplant. 2010;29:S10–S11
  15. Claas FH, Doxiadis II. Human leukocyte antigen antibody detection and kidney allocation within Eurotransplant. Hum Immunol. 2009;70:636–639
  16. Poli F, Cardillo M, Scalamogna M. Clinical relevance of human leukocyte antigen antibodies in kidney transplantation from deceased donors: the North Italy Transplant program approach. Hum Immunol. 2009;70:631–635

PII: S1053-2498(10)00441-9

doi:10.1016/j.healun.2010.06.016

The Journal of Heart and Lung Transplantation
Volume 29, Issue 11 , Pages 1207-1209, November 2010

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