The greatest risk for antibody-mediated rejection is in the sensitized patient (i.e., a patient with pre-formed antibodies against human leukocyte antigens [HLAs]). Risk factors for sensitization include pregnancy, transfusions, ventricular assist devices, or prior transplantation. Such pre-formed antibodies may cause hyperacute rejection , increase the risk of rejection post-transplantation,¹ and predispose patients to the development of cardiac allograft vasculopathy.²

Currently, the detection of anti-HLA antibodies is most commonly performed using solid phase assays. With these assays, latex beads bound with single HLAs are mixed with patient serum. Antibodies will bind to their respective antigen-coated beads, are tagged with an anti-IgG fluorescent carrier, and then detected by flow cytometry. In this manner, the identity and quantification of anti-HLA antibodies is accomplished. Quantification is important, as antibodies of greater intensity in vitro are considered to be potentially more cytotoxic in vivo. The presence of anti-HLA antibodies in high levels (usually median fluorescent intensity above 5,000) are considered potentially cytotoxic.²

However, intensity of antibodies may not be the best test of potential cytotoxicity because not all antibodies at high intensity may be detrimental to graft function. As newer studies indicate the ability of donor-specific antibodies to fix complement, a functional assay may be a better marker of their cytotoxicity.^3,4 Activation of the classical complement pathway by antibodies begins with their binding of C1q, the first component of the pathway. Once activated by C1q, the classical pathway leads to the formation of the membrane attack complex and ultimately results in cell lysis and death. Thus, one would expect that antibodies with the ability to bind C1q would be more likely to be cytotoxic. In fact, in renal transplant recipients, the presence of donor-specific antibodies with the ability to activate the complement pathway, as detected by the ability to bind C1q, is associated with a greater risk of acute rejection and allograft loss.^3,5 At the authors’ center, C1q assays are standardly used to determine which anti-HLA antibodies may require closer monitoring and potential treatment. For centers where the C1q assay is not currently available, considering only antibodies that are strong binding by MFI after a 1:8 dilution may offer comparable information.⁴

The detection of anti-HLA antibodies prior to transplantation is important since one would avoid donors who have HLA corresponding to high-level anti-HLA antibodies in the potential recipient, as this would be a risk for hyperacute rejection. In the past, the only way to assess for this was with a prospective crossmatch, in which the potential recipient’s serum was mixed with donor cells to assess for complement-dependent cytotoxicity. However, this severely geographically restricted the donor pool to hospitals near where the candidate’s serum was stored, thus reducing the number of potential donors for this patient. Currently, the virtual crossmatch has replaced the prospective crossmatch at most centers. With the virtual crossmatch, HLA corresponding to high-level anti-HLA antibodies in the transplant candidate are listed as “avoids” in the United Network of Organ Sharing database, and, thus, potential donors with such HLA are not considered. This method has proven safe and successful in heart transplantation.⁶ Ultimately, however, the major decision is which HLA to avoid, and this is an art as well as a science. For those recipients with many high-level HLA antibodies, one may choose only to avoid the HLA corresponding to the very highest intensity antibodies to allow for consideration of all potential donors. Furthermore, one could use not only the fluorescent intensity but also the ability to bind C1q to determine which antibodies are most concerning.     

Table 1 cPRA = frequencies of those antigens in the donor population. The calculated PRA computes the PRA percentage using both anti-HLA class I and class II antibody specificities, assigned to each patient as unacceptable, and knowledge of the frequency of the assigned unacceptable HLA antigens in a representative population. Table 2 Table 3

The identity and intensity of anti-HLA antibodies is useful not only in safely finding a donor organ for a sensitized recipient, but also in deciding on which sensitized patients require treatment prior to transplantation.^1,2 At our center, we use a threshold of the calculated PRA (cPRA) to decide on treatment of the sensitized patient (Table 1). The cPRA is the frequency of unacceptable HLA in the donor population.⁷ It is computed based on HLA frequencies of 12,000 kidney donors in the U.S. between 2003 and 2005. For example, if a heart transplant candidate had high-level antibodies against common HLA, the cPRA might be 90%, and, thus, only 10% of all potential donors would be compatible. On the other hand, if a heart transplant candidate had only high-level antibodies against rare HLA, the cPRA might be 10%, and, thus, 90% of all potential donors would be compatible. Therefore, the cPRA highlights the fact that not all high-level anti-HLA antibodies are created equally, and some will impact the ability to find a suitable donor heart more than others. As with deciding on which HLA to avoid when listing a patient for transplantation, deciding which HLA to include in the cPRA computation is an art as well as a science. The more antibodies that are included, the higher the cPRA will be. If the cPRA is above 50%, we will consider therapies to reduce antibody levels prior to transplantation. For this assessment, the authors will avoid only the C1q-positive anti-HLA antibodies from the most current analysis, but consider still performing a prospective crossmatch if there are multiple additional high-level but C1q-negative antibodies and their impact on potential rejection is unclear.

Management of the sensitized patients involves protocols to target antibodies by inactivation (intravenous immune globulin⁸), removal (plasmapheresis), and decreased production (rituximab⁸ and bortezomib⁹) (Tables 2 and 3). Production of intravenous immune globulin begins with pooled human plasma from several thousand screened volunteer donors, from which highly purified polyvalent IgG is derived. While the mechanisms of action are incompletely understood, intravenous immune globulin suppresses inflammatory and immune-mediated processes. Rituximab is a monoclonal antibody directed against the CD20 antigen on B-lymphocytes. It is most commonly used for B-cell lymphoma but, in conjunction with intravenous immune globulin, also reduces HLA antibodies in patients awaiting kidney transplantation.⁸ If the protocol of intravenous immune globulin and rituximab is ineffective in reducing the cPRA below 50%, or if a patient requires rapid desensitization (i.e., they are listed Status 1A), then we will use bortezomib, a proteasome inhibitor against plasma cells. It is most commonly used for the treatment of multiple myeloma but also reduces HLA antibodies in patients awaiting heart transplantation.⁹ To increase effectiveness, the protocol at our center combines bortezomib with plasmapheresis over a two-week cycle with plasmapheresis on the day before and day of bortezomib administration (days 0, 1, 3, 4, 7, 8, 10, and 11).⁹

References:

1. 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;28:213-25.
2. Kobashigawa J, Crespo-Leiro MG, Ensminger SM, et al. Report from a consensus conference on antibody-mediated rejection in heart transplantation. J Heart Lung Transplant 2011;30:252-69.
3. Loupy A, Lefaucheur C, Vernerey D, et al. Complement-binding anti-HLA antibodies and kidney-allograft survival. TN Engl J Med 2013;369:1215-26.
4. Zeevi A, Lunz J, Feingold B, et al. Persistent strong anti-HLA antibody at high titer is complement binding and associated with increased risk of antibody-mediated rejection in heart transplant recipients. J Heart Lung Transplant 2013;32:98-105.
5. Sutherland SM, Chen G, Sequeira FA, Lou CD, Alexander SR, Tyan DB. Complement-fixing donor-specific antibodies identified by a novel C1q assay are associated with allograft loss. Ped Transplant 2012;16:12-7.
6. Stehlik J, Islam N, Hurst D, et al. Utility of virtual crossmatch in sensitized patients awaiting heart transplantation. J Heart Lung Transplant 2009;28:1129-34.
7. Cecka JM. Calculated PRA (CPRA): the new measure of sensitization for transplant candidates. Am J Transplant 2010;10:26-9.
8. Vo AA, Lukovsky M, Toyoda M, et al. Rituximab and intravenous immune globulin for desensitization during renal transplantation. N Engl J Med 2008;359:242-51.
9. Patel J, Everly M, Chang D, Kittleson M, Reed E, Kobashigawa J. Reduction of alloantibodies via proteosome inhibition in cardiac transplantation. J Heart Lung Transplant 2011;30:1320-6.

Keywords: Allografts, Antibodies, Anti-Idiotypic, Antibodies, Monoclonal, Murine-Derived, Antigens, CD20, B-Lymphocytes, Boronic Acids, Complement Membrane Attack Complex, Complement Pathway, Classical, Flow Cytometry, Heart Transplantation, Heart-Assist Devices, HLA Antigens, Immunoglobulins, Intravenous, Kidney Transplantation, Microspheres, Plasma Cells, Plasmapheresis, Proteasome Inhibitors, Pyrazines, Risk Factors

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