ABO antibody titer monitoring for incompatible renal transplantation

ABO blood group antigens are expressed throughout the body,1 including embryonic kidney cells,2 vascular endothelium, convoluted distal tubules, and collecting tubules.3 Naturally occurring antibodies directed against ABO antigens are key mediators of antibody-mediated rejection (AMR), preventing renal transplantation across ABO barriers.4,5 In hyperacute rejection, it is the existing circulating antibodies directed against ABO, HLA, or other alloantibody-to-donor endothelial surface antigens that usually lead to rejection within minutes to hours. The alloantibodies bind to peritubular and glomerular capillaries and activate complement.4 AMR resulting from the generation of newly formed anti-donor antibodies can occur weeks to years after transplantation and can lead to the development of transplant glomerulopathy and chronic rejection.6,7 ABO-incompatible (ABO-I) heart, liver, kidney, and marrow transplants have been performed, but ABO-I renal transplants are increasingly common. The optimal treatment of end stage renal failure is kidney transplantation, but a shortage of donors has significantly limited this practice. In 2009, there were more than 93,401 patients awaiting kidney transplantation, and more than 4858 patients died waiting for a kidney transplant (13.3 patients per day).8 The median wait time for patients of different ABO blood groups varies significantly. If registered in 2003-2004, the median wait time for patients of blood group O is 1852 days, while patients of blood group AB wait only 855 days.8 Removing the ABO barrier expands the donor pool, increases availability of organs for transplantation, and decreases time on the organ waiting list; ultimately, facilitating transplantation before patients develop comorbid conditions. ABO-I renal transplantation has the potential to significantly increase the donor pool.9,10 These transplants are occurring through a preparative regimen including the use of therapeutic plasma exchange, double-filtration plasmapheresis, or immunoadsorption and immunosuppressive therapy to reduce circulating ABO antibody titers permitting engraftment of ABO-I kidney transplants.11-14 ABO-I renal transplantation is currently an American Society for Apheresis Category II indication for therapeutic plasma exchange.15 The clinical significance of the ABO antibody titer in ABO-I kidney transplantation is not entirely clear. Although we and others use the goal of a titer of 16 or less at the anti-human globulin (AHG) phase before surgery,13,16 there are centers that use lower-titer goals17 and at least one center that does not confirm a specific preoperative titer goal.18 Minimal research has been performed to determine the optimal pretransplant titer. Some institutions have documented that high posttransplant ABO antibody titers correlate with increased graft loss.19,20 We have recently shown that the risk of AMR was significantly higher among individuals with an elevated posttransplant titer of 64 or more.21 Most individuals with AMR have an elevated titer; however, the positive predictive value of a high titer for AMR is poor.21 AMR due to ABO antibodies can also be reversed with therapeutic plasma exchange and CMVIg treatments, although it is well documented that AMR is strongly correlated with the subsequent development of transplant glomerulopathy, the latter being associated with diminished long-term graft survival.22,23 Thus, posttransplant titers should be monitored, but must be used in conjunction with other factors assessing AMR. Despite the risk of AMR after ABO-I kidney transplantation, multiple centers have documented that ABO-I kidney transplants have comparable 5-year graft survival to ABO-compatible living donor kidney transplants.12,24 ABO antibody titers have traditionally been performed by a test tube method using donor-type indicator red blood cells (RBCs) and incubation at phases that include ambient room temperature test phase, which is generally reflective of IgM antibody activity, followed by 37°C incubation and conversion to the anti-human globulin phase for optimum detection of IgG antibody. Many centers performing ABO-I kidney transplants use the antiglobulin (IgG) antibody titer endpoint as the critical titer when assessing patients before and after transplantation. Some investigators have suggested determining titers using dithiothreitol-treated serum to inactivate IgM antibodies so that the titer will correspond only to the level of IgG antibodies that are present in the sample.25,26 The test tube detection method is time-consuming and notoriously difficult to standardize, and several reports have suggested that the titers can be inaccurate.27-29 Two recent studies have shown a three- to eightfold interinstitutional difference between the antibody titers from the same samples.27,28 The inaccuracy of ABO antibody titers limits institutional sharing of patient data and also could potentially lead to hyperacute rejection if high ABO antibody titers are falsely reported as being low. In this issue of TRANSFUSION, Lindberg and colleagues30 describe an enzyme-linked immunosorbent assay (ELISA)-based method for quantifying ABO antibodies that can distinguish the immunoglobulin class and finely characterize the specificity (trisaccharide vs. tetrasaccharide binding). The results are thought-provoking and raise the prospect that the ABO antibody class and specificity may assist in further identifying individuals at highest risk of AMR. Although this article does not answer all of the questions related to the role this particular testing method may have for ABO-I patients, it raises important issues. Potentially multiple testing methods may be optimal. We may ultimately find that posttransplant monitoring for AMR requires two levels of testing: 1) an efficient, reproducible ABO antibody titer for screening, and 2) a technologically more sophisticated testing method for patients who have experienced an increase in their screening titer and are at higher risk for AMR. This second test method would ideally have a higher predictive value in identifying AMR so that the diagnosis could be made or excluded without requiring biopsy. Thus, this important area of transfusion medicine needs more research. The ideal assay for ABO antibodies should be easy to replicate and fast and simple to perform and yield reproducible results that are understandable across specialties and institutions. In addition to the ELISA, several other methods have been proposed recently. Surface plasmon resonance can measure anti-A or anti-B IgG titers accurately and can be easily standardized.31 Flow cytometry using beads carrying A or B trisaccharides has also been used to measure A and B antibody titers.32 The IgG gel microcolumn method utilizes anti-IgG gel cards, which are often used in blood banks for serology panels; this may currently be the method of choice. Cohney and colleagues33 reported that ABO antibody titers by a gel card method demonstrated better clinical correlation than standard test tube titers. We have recently demonstrated that the IgG gel microcolumn method yields results comparable to the conventional test tube method.34 In addition, the Europeans have shown that when using the IgG gel microcolumn method, the same samples and the same test RBCs at three different institutions yield titer results differing by a median of only one titer step.28 The variation of no more than one standard dilution is within acceptable limits for titration methods.35 Consequently, our laboratory has implemented ABO antibody titer determination by the IgG gel method, which has reduced our turnaround time by 50% and enabled us to provide titer values in the more timely manner for patients in the ABO-I transplantation program. Further research is still needed to optimize ABO antibody titer determinations. Using a semiquantitative titration method, the titer endpoint is affected by many variables including the RBC phenotype (e.g., group A1 or A2) and concentration of the indicator RBCs as well as incubation times and temperatures.36 Thus, reproducibility, interpretation, and comparison of anti-A or anti-B titers reported for ABO-I kidney transplant recipients are problematic. The lack of a universally accepted titer protocol makes comparative studies from different institutions difficult. Undoubtedly, the gel card method, as well as automated methods for determining ABO antibody titers, will be further explored to find the ideal method for monitoring ABO-I renal transplant patients. Additionally, research to identify the clinical relevance of titer endpoints in relationship to graft survival is needed. The ABO blood group transplantation barrier is now being crossed and ABO-I kidney transplants are becoming more common. Because of the increasing numbers of patients being followed, testing needs to be efficient and reproducible. The more labor-intensive and time-consuming test methods can only be justified if they offer significant clinical value, such as identification of patients at high risk for AMR. Further research should be performed to determine the optimal method for assessing ABO antibodies. Monitoring ABO antibodies for incompatible organ transplantation is a growing area where the reference laboratory and transfusion medicine physicians perform a critical and expanding role. The authors declare no conflicts of interest.