A 2 /A 2 B to B Renal Transplantation: Past, Present, and Future Directions

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1 American Journal of Transplantation 2016; 16: Wiley Periodicals Inc. Comprehensive Review C Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: /ajt A 2 /A 2 B to B Renal Transplantation: Past, Present, and Future Directions C. F. Bryan 1, *, W. S. Cherikh 2 and D. A. Sesok-Pizzini 3 1 Midwest Transplant Network, Westwood, KS 2 United Network for Organ Sharing, Richmond, VA 3 Childrens Hospital of Philadelphia, Philadelphia, PA Corresponding author: Christopher F. Bryan, cbryan@mwtn.org One component of the new national kidney allocation system (KAS) in the United States that was implemented on December 4, 2014, was the allocation of kidneys from A 2 and A 2 B (A, non-a 1 and AB, non-a 1 B) deceased donors into blood group B candidates (A 2 /A 2 B! B). In so far as this is an important component of the new KAS that has the potential to further increase the access to transplantation for blood group B candidates on the waiting list, most of whom are minority candidates, we will review the body of evidence and historical perspectives that led to its inclusion in the new KAS. This review will also describe prospects for more widespread use of A 2 /A 2 B! B transplantation and a novel mechanism of humoral immunosuppression in B patients before and after transplantation with an A 2 or A 2 B kidney. Abbreviations: A 2, A, non-a 1 ;A 2 B, AB, non-a 1 B; ABH, glycan chains to which A, B, or H epitopes are attached; AHG, anti-human globulin; DTT, dithiothreitol; HLA, human leukocyte antigen; inkt, invariant natural killer T cell; KAS, kidney allocation system; MTN, Midwest Transplant Network; OPO, organ procurement organization; OPTN, Organ Procurement and Transplantation Network; RBC, red blood cell; SLK, simultaneous liver kidney; UNOS, United Network for Organ Sharing Received 10 June 2015, revised 30 July 2015 and accepted for publication 21 August 2015 Introduction Adding the A 2 /A 2 B! B component to the U.S. kidney allocation system (KAS) on December 4, 2014, is actually the second time the national KAS has been modified in an attempt to improve access of blood group B candidates to kidney transplantation. The first was on August 29, 2001 (Organ Procurement and Transplantation Network [OPTN]/ United Network for Organ Sharing [UNOS] Policy 8), when blood group B deceased donor kidneys were directed away from AB candidates and allocated instead to blood group B candidates (except for zero HLA mismatches). A recent analysis of the OPTN data revealed that after the policy change, B candidates received 10% more kidney transplants in the 12-year period since the KAS was changed ( ) than in the 12-year period ( ) before the change went into effect (1). Despite the increase in B transplants from 11.9% to almost 13% of the total kidney transplants in those two time periods, the composition of the B waitlist in 2013 was 16%, still well above the transplant rate of B candidates, which is further rationale for adding the A 2 /A 2 B! B component to the new KAS. Blood group B candidates on the national deceased donor kidney waitlist have less access to deceased donor kidneys, resulting in a lower transplantation rate than that for candidates of the other blood groups (2). A recent analysis of OPTN data shows that the 25th percentile for days until transplantation among registrations added to the kidney waitlist between August 29, 2001, and December 3, 2014, was 1744 days for B candidates, 1554 days for O candidates, 898 days for A candidates, and 463 for AB candidates. The start date of B kidneys being diverted from AB candidates was August 29, 2001, and December 3, 2014, was the day before the new KAS was implemented. Median wait time could not be calculated by blood group because less than half of the registrations added during this period had not received a transplant. The reasons for that access disparity are multifactorial and some will be highlighted here, but reviews describing those issues in greater detail are available (3 5). The A 2 /A 2 B! B allocation component is a further attempt to improve the access of B candidates to deceased donor renal transplantation in the United States. The national blood group B waitlist in 2013 was composed of 71.4% minority candidates, compared with a 50.2% minority composition of the A waitlist, 59.7% of the AB waitlist, and 64.2% of the O waitlist. Of the 71.4% B minority candidates, 44% were black, 13.8% were Asian, and 11.6% were Hispanic, with the remaining 2% consisting of other minority candidates. Finally, black, Asian, and Hispanic candidates are much more dependent on the deceased donor waitlist for their kidney transplants than are white candidates. OPTN data from 2013 show that black, Asian, and Hispanic candidates received 82%, 71%, and 68%, respectively, of their kidney transplants from deceased donors. White candidates, in 11

2 Bryan et al contrast, received 54% of their kidney transplants from deceased donors. The A 2 /A 2 B! B allocation component to the new KAS should provide a new and robust way to further increase access for B candidates, most of whom are minority candidates, to receive a deceased donor kidney transplant. Many of these issues have resulted from the biologic impact of the phenotype frequencies of blood types of the deceased donor and candidate populations and their respective ethnicities (Table 1). In this review, we will describe the literature and relevant practical points that have been important for routine clinical and laboratory practice (allocation, anti-a titer testing, A subgrouping and transplantation) that the Midwest Transplant Network (MTN) (formerly known as Midwest Organ Bank) group has encountered during the past 26 years of performing A 2 /A 2 B! B transplantations and the 23 years of administering an OPTN/UNOS-approved kidney allocation variance to preferentially allocate A 2 and A 2 B kidneys to blood group B candidates listed on the MTN organ procurement organization (OPO) deceased donor waitlist. Those A 2 /A 2 B! B transplantations were performed at the six renal transplant centers in the MTN OPO during that period (Via Christi St. Francis Hospital, St. Luke s Hospital, Kansas University Medical Center, University of Missouri Hospitals and Clinics, Research Hospital, and The Children s Mercy Hospital). Administration of the MTN A 2 /A 2 B! B OPTN/UNOS-approved kidney allocation variance involved a yearly analysis of the data and reports sent to UNOS describing MTN s allocation, anti-a titer, and clinical outcome data. Principles established early in A 2! B and O transplantation The first report of successful transplantation of kidneys from A 2 donors was into O candidates and came from the G oteborg group (6 9). They began their A 2! O transplant protocol in 1974 based on an earlier report of successful skin grafting from A 2 donors to O recipients (10). In their series of 20 patients (9), they showed that of four grafts lost, three were due to nonimmunological reasons and only one was lost in the first month to acute rejection. They obtained posttransplantation anti-a titer levels occasionally in patients who had rejection episodes, and in those Table 1: Blood group/subgroup phenotype frequencies by ethnicity Blood group phenotype frequencies Blood group White Black Asian B 9% 19% 25% A 44% 27% 27% A 1 34% 19% 27% A 2 10% 8% 0% O 44% 49% 43% AB 3% 5% 5% From Wallace ME, Gibbs FL (eds). Blood group systems: ABH and Lewis. Arlington, VA: American Association of Blood Banks; patients, anti-a titer levels rose when rejection occurred. However, they did not report routine pretransplantation anti-a titers for their A 2! O transplant patients. The MTN group began their A 2 /A 2 B! B and O deceased and live donor transplantation protocols based on the work of the G oteborg group (6 9). MTN s first case in 1986 was an HLA-identical sibling pair the donor was A 2 and the patient was O. The transplant was successful and the kidney survived 8 years, when the patient died with a functioning graft. The first principal observation the MTN group made was that high titers of anti-a IgG present in a B or an O patient before A 2 /A 2 B! B or O transplantation was associated with poor early and late kidney function and graft survival (11). The MTN s first 25 renal transplants were performed without regard to the patient s pretransplantation level of anti-a IgG, because at that time no published evidence existed regarding outcome and pretransplantation anti-a IgG titers (6 9). The incidence of early and long-term graft function was 100% (14 of 14) in patients whose anti-a IgG titer was 4 or lower before transplantation, whereas in patients whose titer level was 8 or higher, 55% (6 of 11) of the kidneys were not functioning at 1 month and most never regained function. The Oregon group has also demonstrated that high anti-a IgG titer levels are associated with antibody-mediated rejection (AMR) and graft loss (12). The second principal observation was that the immunosuppression protocol used in our A 2 /A 2 B! B patients was center directed but was no different from that used for ABOcompatible renal transplantation, as long as transplantation was done in patients with a low anti-a IgG history (11,13 16). Further, no A 2 /A 2 B! B patient underwent plasmapheresis or splenectomy or received IVIg or rituximab therapy before transplantation specifically to lower anti-a titer levels or for induction. All MTN B recipients of A 2 or A 2 B kidneys received methylprednisolone, cyclosporine or tacrolimus, and azathioprine or mycophenolate mofetil, with or without antibody induction (monoclonal or polyclonal). The Oregon group has also shown that in B or O patients with low pretransplantation anti-a IgG titers, no additional immunosuppression is needed for successful transplantation with A 2 or A 2 B kidneys (12). The third principal observation involved frequent sequential anti-a IgG titer testing of B candidates (using the same monthly serum sample received in the laboratory for HLA antibody screening and cross-matching) for 6 months, followed by quarterly testing. Those titers were used to define an anti-a IgG titer history for each blood group B candidate s eligibility for transplantation with A 2 or A 2 B kidneys (16). After implementing the anti-a IgG titer history tenant for each eligible B candidate, MTN began to rely simply on a patient s anti-a IgG titer history, available at the time of transplantation, to decide whether he or she should receive an A 2 or A 2 B kidney. MTN then stopped anti- A IgG titer testing with a current serum sample from the patient at the time of transplantation in Blood 12 American Journal of Transplantation 2016; 16: 11 20

3 A 2 /A 2 B! B Kidney Transplantation Review group B patients whose pretransplantation titers were all 4 or lower were categorized as low and safe to transplant with an A 2 or A 2 B kidney, whereas candidates with a history of pretransplantation titers of 8 or higher were deemed prohibitively high and were not eligible to receive an A 2 or A 2 B kidney (11). Thus, the MTN group (13 16) and the centers participating in the OPTN/UNOS voluntary A 2 /A 2 B! B variance (17) have shown that both the shortand long-term outcome of B candidates, who have a low anti-a IgG titer history before transplantation, who receive A 2 or A 2 B kidneys is not significantly different from the outcome of B candidates transplanted with B kidneys. That forms the clinical foundation for implementing the A 2 /A 2 B! B allocation component into the national KAS. MTN has observed that anti-a IgG titer levels are independent of HLA sensitization. Finally, it is crucial to note that a blood group B candidate s ethnicity does not appear to influence their anti-a IgG titer level (11,13 17). MTN has reported that most (80% to 90%) B candidates have a consistently low anti-a IgG titer history (11,14,16). Current substantiation of that comes from the MTN OPO renal waitlist on January 16, 2015, showing that 95% (33 of 35) of the active B candidates waiting for their first kidney transplant are eligible to receive either an A 2 or A 2 B kidney by virtue of having consistently low (4 or less) anti-a IgG titers. A total of 250 anti-a titer tests were performed in those 35 patients, for an average of 7.1 titer tests per patient during an average time of 13 months. It should be remembered that for B candidates with a consistently high (8 or higher) anti-a titer history or with a mixture of low and high titers can receive a kidney from a B donor. In MTN s experience, about 25% of B deceased donor transplants received an A 2 or A 2 B kidney and 75% receive their kidney from a blood group B donor. The eligibility guidelines of a B candidate to receive an A 2 or A 2 B deceased donor kidney at MTN are summarized in Figure 1. With those three principal observations in place, the MTN OPO obtained approval from UNOS to implement an OPOwide A 2 /A 2 B! B kidney allocation variance in That variance was modified in 1994 to directly allocate A 2 kidneys to B candidates and no longer allocate them to A 2 candidates before B candidates. UNOS required that MTN provide a yearly document containing specific outcome and descriptive data for the B candidates transplanted with A 2 and A 2 B kidneys. Most of the data MTN published (11,13 16) originated from those reports to UNOS. This is a good example of how a local allocation variance can provide the experience necessary to translate into a national voluntary variance trial (17), which in turn ultimately became incorporated into the national KAS on December 4, Blood group A antigen expression on the kidney Breimer s group has shown that the amount of blood group A antigen expression on the vasculature of kidneys from A 1 and A 2 donors is consistent with the relative level of A antigen found on A 1 and A 2 red blood cells (RBCs) (18). They showed that in A 2 kidneys, blood group A antigen expression is consistently low in the renal cortex and the entire vascular bed endothelium. Proximal and distal tubule and glomerular epithelial staining for A antigen expression ranges from very low to not present in kidneys from A 2 donors. That low level of A antigen expression on A 2 kidneys and the low anti-a titer requirement for successful A 2 /A 2 B! B transplantation are the likely critical components that contribute to the success of these transplants. Breimer s group has also published a review of the biochemistry of blood group antigens with a special evaluation of A 2! O and B renal transplantation (19). In that Figure 1: Eligibility guidelines of a blood group B candidate for an A 2 or A 2 B deceased donor kidney (based on references 11 and using MTN titer method). American Journal of Transplantation 2016; 16:

4 Bryan et al review, they also demonstrate histologically that livers from A 2 individuals express much less A antigen in the bile duct, artery, and lymphatic capillaries than do livers from A 1 individuals (19), which may in part also explain the success of A 2! O liver (20 22) and simultaneous liver/ kidney (SLK) transplantation (23). Subgrouping of A and AB donors Subgrouping of A and AB was reported, using data from the College of American of Pathologists ABO and A subgrouping proficiency testing surveys, to have an inherently high discrepancy rate, especially for A 2 (24). That high discrepancy rate prompted UNOS to mandate that two separate A subgrouping tests occur for live (OPTN/UNOS policy 14.5) and deceased donors (OPTN/UNOS policy 2.6.B) if the organ or organs are to be transplanted into B or O candidates. If a subgroup discrepancy is observed with a donor, for patient safety, UNOS mandates that the donor not be allocated as an A 2 but rather as an A to avoid a potentially unsafe kidney transplantation into a B or an O candidate. The reasons for that high A subgrouping discrepancy rate are multifactorial, as reviewed (24), but are likely related to the fact that the anti-a lectin, Dolichos bifloris preparation is not standardized. For example, the molecular genotype of individuals who are subgrouped as A 2 may be A 2 /A 2 or A 2 /O. Greater amounts of A antigen may be present on RBC from A 2 /A 2 individuals with lesser amounts on RBC from A 2 /O individuals because of gene dose effects. Also, although we commonly refer to A subgroups as A 2, there are several other weaker A subgroups recognized (A 3,A x,a m.a el ). These subgroups are encountered infrequently and are usually apparent due to blood typing discrepancies in the red cell and serum grouping results. These weaker subgroups may be frequently non-reactive with human polyclonal anti-a reagents and can show variable reactivity with human polyclonal anti-a and anti-a, B and murine monoclonal antibodies (25). For the purpose of transplantation these are considered A 2 or non-a 1 donor kidneys. The frequency of A 2 and A 2 B donors, of blood group A or AB donors, respectively, should be 20%. After 12 years of operating the MTN OPO A 2 /A 2 B! B variance, the 2006 annual variance report to UNOS reported that the frequency of A 2 donors was 14% (94 of 674) of all A donors, short of the expected 20%. However, 21% (11 of 52) of the AB donors were A 2 B, much nearer the expected frequency of 20%. MTN speculated that the likely reason the frequency was not closer to 20% for A 2 donors was that they were not consistently performing the A subgrouping on a blood sample drawn from the deceased donor before blood transfusions were given. The fourth and final principal observation is that A subgrouping of deceased donors should be performed on pretransfusion blood. Transfusion would not usually occur with live donors. The reasoning was that if an A 2 /A 2 B donor was transfused, it was likely that most blood given (approximately 80%) would be from A 1 or A 1 B donors and the subgroup typing of the A 2 donor after transfusion could change to A 1 or A 1 B. Thus, it is important that both A subgrouping tests (UNOS mandates two subgroupings of A 2 /A 2 B donors in Policy 2.6.B for an A/AB organ to be transplanted into non-a candidate) be done before any transfusions are given. Anti-A titer testing method(s) Anumberofdifferentmethodsareavailabletoperform anti-a titers. The method used by the MTN laboratory (test tube and non anti-human globulin [AHG] method) was used throughout MTN s clinical transplantation experience and was correlated with outcome data from their A 2 /A 2 B! B and O transplants (11,13 16). MTN has made changes to that methodology over the years such that it now just uses A 1 RBCs (titers are not done using A 2 RBCs), and rather than performing total (IgG and IgM) anti-a testing, MTN just uses a serum that has been treated with dithiothreitol (DTT) to yield an estimate of the IgG anti-a titer. MTN stopped performing total (IgG and IgM) several years ago because so few total anti-a titers were greater than IgG (DTT-treated) titers. Only a small quantity of IgG anti-a is detected in group B individuals. This is in contrast to group O serum, where IgG is the major isotype for anti-a and anti-b. The new KAS system allows each transplant center to use an anti-a titer method of their choosing and select the titer they consider appropriate to rule out B candidates from receiving an A 2 or A 2 B kidney. As an example of how to perform such a comparison, the MTN laboratory compared their DTT tube method to the AHG method used by the Mayo Clinic (26). The Mayo Clinic s titers were higher than those achieved with the less-sensitive method used by the MTN laboratory. The Mayo Clinic has also published their clinical experience with A 2 /A 2 B! B live donor renal transplantation (27). Another example of a correlative study was provided by the Wisconsin Donor Network in comparing their chosen method (gel testing with IgG AHG) with the DTT method (Dr Anand Padmanabhan, personal communication, Wisconsin Blood Center). One universal concern with determining an anti-a titer is the high degree of variability among laboratories and among methods, which affects being able to compare titers between transplant centers. One recent study tried to minimize such method variation but was only able to reduce the variation in titer testing for the AHG phase determination with the tube technique (28). Importantly, Kumlein et al (29) found that the gel hemagglutination technique significantly decreased inter transplant center titer variation compared with the tube method. Bentall et al (30) recommended a reference method for titration with an antibody standard as a way to improve comparability of titers between transplant programs. One way the MTN attempts to minimize variation in anti-a titer testing is that 14 American Journal of Transplantation 2016; 16: 11 20

5 A 2 /A 2 B! B Kidney Transplantation Review for more than 20 years, we have used the same three medical technologists, who rotate in performing the assay. For quality control in each assay, the MTN laboratory uses a control that has a high anti-a IgG titer with little IgM that will not be reduced by DTT and one with mostly IgM activity that is reduced with DTT. Table 2 provides a summary of the literature that examined the relationship of anti-a titers with A 2 /A 2! B or O renal transplant outcome for deceased and live donors, liver and SLK transplant outcomes, possible ways to reduce titer variation, and how titers can be more comparable between transplant centers. Beyond the issue of what anti-a titer method should be used in A 2 /A 2 B! B transplantation, the more clinically relevant issue is that we should be measuring anti-a titers not only with RBCs, which most laboratories now use, but also with an assay that allows detection of anti-a or anti-b epitopes that are attached to the tissue-specific blood group antigen glycan precursor chains, termed ABH chains. Table 2: Literature summary surrounding A 2 /A 2 B!B/O renal transplantation and anti-a/b titer testing Reference Central message as it relates to A 2 /A 2 B! B/O renal transplantation Anti-A/B titration method Kidney transplantation: deceased donor Bryan et al (11,13 16) Low anti-a IgG titer history (all titers 4 pretransplantation) portend that A 2 /A 2 B! B outcome is same as B! B Williams et al (17) Low anti-a IgG titer history (all titers 4 pretransplantation) portend that A 2 /A 2 B! B outcome is same as B! B Alkhunaizi et al (12) Low titers (4) associated with good graft function Kidney transplantation: living donor Gloor et al (26) Alkhunaizi et al (12) Bryan et al (35) Sorensen et al (34) Tierney et al (36) Hanto et al (32) Liver and SLK transplantation Kluger et al (22) Skogsberg et al (20) Fishbein et al (21) Bryan et al (23) Reducing anti-a/b titer variation and method used for transplantation AuBuchon et al (27) Kumlien et al (28) Bentall et al (29) High anti-a/b IgG (>32) is best predictor of antibody-mediated rejection in A 2! non-a patients High anti-a IgG titer (16) and IgM (64) associated with hyperacute rejection of A 2! O kidney Good graft outcome with anti-a IgG titers that were 8 Good graft outcome with anti-a IgG titers that were 16; no association of high (8 to 254; n ¼ 10) IgM with rejection or outcome High (8) IgM anti-a titer is associated with early acute rejection/thrombotic microangiopathy in A 2! O transplantation High anti-a 2 IgM titer (64) associated with accelerated acute rejection in A 2! O live donor kidney; titers increase after transplant No titer data, but liver survival of A 2! O is same as O! O using UNOS data Anti-A/B titers do not play a significant role in liver rejection using A 2 and B donors A 2! O liver transplantation outcome is not associated with anti-a titer level A 2! O SLK transplant; kidney (and liver) graft survival was successful despite a high (512) anti-a IgG titer A uniform method for titration (anti-a/b) read at the AHG phase in a tube-based method reduced interlaboratory variability The gel hemagglutination technique significantly decreases intercenter variation of titers compared with tube method A refererence technique and antibody standard for anti-a/b titer is a worthy goal to allow between center titer comparison AHG tube Non-AHG tube Non-AHG tube No titer data AHG gel card Tube at 378C (IgG) Multiple methods AHG gel and tube Gel and tube American Journal of Transplantation 2016; 16:

6 Bryan et al However, such an assay is not readily available in most laboratories. That issue was elegantly and recently framed by Jeyakanthan et al, who used such an assay to describe the differential distribution of the four ABH precursor chains that express the respective A and B epitopes on cardiac tissue versus RBCs (31). One important point they made is that RBCs express each of the four ABH precursor glycan chains, but cardiac vascular endothelium expresses only the type II ABH glycan chain. By measuring titers with RBCs, they could not distinguish which of the four ABH chains on the RBCs was reacting, but their new assay could determine that the antibody was directed specifically to the type II chain. Such information is clearly relevant to know whether treatment of ABO-incompatible heart transplant candidates is necessary. The G oteburg group has also published and reviewed similar ABH precursor glycan expression data for kidney tissue (19). An advancement sorely needed for ABO-incompatible renal transplantation, including A 2 /A 2 B! B, is an assay (perhaps similar to the Luminex platform we now use for detecting donor-specific HLA antibodies) that distinguishes the anti-a and anti-b antibodies that actually bind to the four tissue-specific ABH antigens in the kidney tissue. Until such an assay is widely available, we will continue to wonder which of the antibody levels that we measure with RBCs is the clinically relevant antibody that reacts with the kidney endothelium and causes AMR. An important shortcoming of many published studies and from personal communication from other groups regularly performing A 2 /A 2 B! B transplantation (personal communication from Dr Paul Warner, Seattle, and Dr Cathi Murphey, San Antonio) is that anti-a titer levels are seldom performed after transplantation in the current era of transplantation. The Portland group has shown that anti- A 1 titers rose in five of their 10 patients, but AMR was seen in only two of those patients (12). The G oteburg group only occasionally performed anti-a titers after transplantation and usually did so in patients who experienced a rejection; all their patients were blood group O (6 9). Therefore, it is not known if anti-a IgG titer levels in B patients who receive an A 2 or A 2 B kidney after transplantation specifically increase in response to AMR caused by anti-a. An important control group for such an evaluation should include B! B transplants to evaluate the natural history of anti-a IgG titer changes after transplantation in B patients who receive a B kidney. For example, MTN recently had a B patient whose anti-a titer levels were high (five titers were 8) before transplantation and received a transplant from a B donor. Ten 10 years later, after the transplant failed, the patient came back on the list with consistent titers of 2. That case suggests that transplantation, immunosuppression, aging, or a combination of those factors can reduce a patient s titer levels. Finally, as mentioned, many of these titer issues and discrepancies need to be reexamined with an assay like that Jeyakanthan et al described (31). Finally, biopsy data evaluating the histology of A 2 /A 2 Bkidneysin blood group B candidates have not been reported. Long-term success of A 2 /A 2 B! B renal transplantation, follow-up care, and transfusion support The first group to demonstrate that the long-term (7-year) outcome of B candidates who received A 2 or A 2 B kidneys was not significantly different from that of B candidates who received a B kidney (15) was the MTN. The OPTN/ UNOS voluntary A 2 /A 2 B! B variance is the only prospective study to date with follow-up to 41 months from 101 A 2 /A 2 B! B patients (17). That study shows that A 2 /A 2 B! B is a robust way to improve the access of B candidates to transplantation with no decrement in their renal graft function and graft or patient survival. It is important to point out that in the MTN experience (11,13 16) and the OPTN/UNOS voluntary variance (17), the immunosuppression protocol used for A 2 /A 2 B! B patients was center directed but was no different from that used for ABO-compatible renal transplantation, as long as transplantation was done in patients with a low anti-a IgG history. One shortcoming of the MTN and voluntary variance studies (11,13 17) is that AMR was not specifically reported; rather, in those studies, only treated rejection was reported. AMR should be reported in future studies. Another large study evaluated 238 live and deceased donor A 2! BandO and A 2 B! B transplants from the United States Renal Data System (32). It should be noted that many of the patients reported in that study are likely from the two previously described studies (15,17). They showed that graft and patient survival was not significantly different from that of ABO-compatible transplants. Finally, it is crucial not to forget that A 2 /A 2 B! B and O transplants are ABO incompatible because in cases where anti-a titer levels are high, hyperacute or accelerated acute AMR can result (12,33). Clinicians who provide follow-up care to renal transplant patients need to be aware of the following clinical scenario in blood group B or O patients who have received an A 2 (B or O patients) or A 2 B kidney (B patients) and are treated for AMR with plasmapheresis. Patients treated for AMR with plasmapheresis may need to receive plasma infused back. If plasma from O or B donors has high anti-a titers and that plasma is infused back into the patient AMR may be worsened. The patient who receives massive transfusion exchanges, as well as patients who receive less plasma, should be supported with plasma that is compatible not only with the patient (O or B) but also with the donor (A 2 or A 2 B). AB plasma would be appropriate in such circumstances (personal communication, Dr. Shaffer, Vanderbilt University, and Dr. Padmanabhan, Wisconsin Blood Center) or A plasma if the patient is an O and the donor is an A 2. That would also be true for pretransplantation conditioning protocols that involve plasmapheresis (34). Finally, AMR in B or O patients transplanted with A 2 or A 2 B kidneys may be caused by high levels of anti-a, donor-specific anti-hla antibodies or both but clinical treatment currently is not different depending on the causative antibody. 16 American Journal of Transplantation 2016; 16: 11 20

7 A 2 /A 2 B! B Kidney Transplantation Review Effect of A 2 /A 2 B! B allocation on B, A, and AB transplantation rate Two groups have directly addressed the question of how much the A 2 /A 2 B! B allocation component is likely to increase the transplant rate of the blood group B waitlist. The MTN data have shown that between 23% (13) and 34% (14) of their blood group B deceased donor transplant recipients receive an A 2 or A 2 B kidney. The results from the OPTN/UNOS voluntary variance showed that in the nine OPOs that participated, 9.4% more B candidates were transplanted by receiving an A 2 ora 2 B kidney (17). Thus, the national blood group B transplantation rate may improve from 9% (17) to 34% (14). Because the A 2 /A 2 B! B allocation system increases access of B candidates to deceased donor renal transplantation by diverting a portion of the A and AB kidneys from the A and AB candidate pool, it is crucial to know how much of an effect that diversion had on the transplantation rate of either A or AB candidates (15,17). We evaluated the speculative impact of this allocation algorithm on A and AB transplants by using 2013 OPTN data along with the actual transplant rate data generated by the OPTN/UNOS A 2 /A 2 B! B voluntary variance (17). Those data showed that B transplants increased by 9.4%, while A and AB transplants decreased by 7.4% and 5.5%, respectively. The redistribution would have lowered the proportion of A transplants in 2013 from 36.2% to 33.5% (a 7.4% reduction) and AB transplants from 5.3% to 5.0% (a 5.5% decrease). Importantly, the waitlist composition in 2013 was 29.4% for A candidates and 3.1% for AB candidates, yielding a transplant rate for A and AB candidates that was higher than the waitlist composition of each blood group. A 2! O SLK transplantation In view of the poor kidney outcome experienced with A 2 /A 2 B! B and O renal transplantation when pretransplantation anti-a titers are high (8 or greater) (11), one area of concern is kidney outcome after A 2! O SLK transplantation. Liver transplantation outcomes, unlike kidney transplant outcomes, are not influenced by high anti-a titers (20,21). Furthermore, an OPTN database study showed that graft survival of 358 A 2! O liver transplant patients was not significantly different from the outcome of 43,335 O! O liver transplants (22). OPTN liver allocation policy permits A 2! O liver as well as SLK allocation and transplantation (Policy 9.5.B). An informative case of an A 2! O SLK transplant in a patient with a high level of anti-a IgG of 512 has been reported. The kidney functioned well immediately after transplantation and continues to function well over 1 year after SLK transplantation (23). The anti-a, as well as anti-b, titers in that O SLK transplant patient fell after transplantation with the anti-a titer falling to 2 on day 184 after transplantation with a creatinine of 0.9 mg/dl. That is consistent with the notion that the liver may have initially protected the kidney from high titers of anti-a present at transplantation. Further evidence that the liver may be protective comes from an analysis of 14 A 2! O SLK transplants using OPTN data (23). The graft outcome of those 14 A 2! O SLK transplants was not significantly different from outcome of the ABOcompatible SLK transplants evaluated. One shortcoming of those 14 SLK transplants is that the OPTN database did not collect anti-a titer data outside of the A 2 /A 2 B! Bnational voluntary kidney variance, so the association of outcome with anti-a titer cannot be ascertained. However, because most O renal candidates have high anti-a titers (16), many of those 14 SLK transplant patients should have had high levels of anti-a. One potential mechanism for the success is that livers from A 2 donors have much less blood group A antigen than do livers from A 1 donors in the bile ducts, arteries, and lymphatic capillaries (19) and higher anti-a titers do not have a detrimental effect. A 2 /A 2 B! B and O live donor renal transplantation Although this review is predominantly about deceased donor A 2 /A 2 B! B transplantation, we believe it is important to point out that A 2 /A 2 B! B and O live donor renal transplantation is also successful in the context of the patient s anti-a IgG titer history (12,26,35,36). The anti-a IgG titer cutoff used for deceased donors (11), which was 8 for the MTN group, may not be the same for live donors. The Oregon group reported that hyperacute rejection occurred in a living donor related A 2! O transplant when the anti-a titer was 16 and their routine therapeutic effort to lower the titer with plasmapheresis was not performed (12). MTN reported that two A 2! O live donor transplants were successfully done in patients whose pretransplantation anti-a titer was 8 (36). Transplant programs must be also be mindful that when evaluating blood group B or O candidates for live donor transplantation and the potential donor is blood group A (for B and O candidates) or AB (for B candidates), subgrouping should be a first response. If the potential donor is an A 2 or A 2 B, the transplant center must subgroup the donor a second time per OPTN/UNOS Policy 14.5.B. It is important to perform those two A or AB subgroupings early in the transplant evaluation process so that if a discrepancy occurs between the subgroup results, the family and transplant center knows early on that donation as an A 2! O/B or A 2 B! Bis ruled out. If the donor is A 2 or A 2 B, the next step would be to perform anti-a titer testing on the candidate. It is important to establish a clear and current anti-a titer history for the patient and give the transplant program confidence that anti-a titer levels are consistently low for transplantation to proceed. The reason for having a history of several anti-a titers is that most of these transplants are into O candidates (12,35,36) and most O candidates have anti-a titer histories that are high or variable (16) because IgG is the major isotype for anti-a. Importantly, A 2 /A 2 B! B and O live donor renal transplantation has the potential to increase the live donor pool for B and O candidates. A recent study from the Vanderbilt group (37) raised the possibility that in addition to IgG anti-a titers, IgM anti-a American Journal of Transplantation 2016; 16:

8 Bryan et al titers may also be related to acute early rejection or thrombotic microangiopathy in live donor A 2 to O transplants. Although this study included only seven patients, four of those patients with low IgG but high IgM titers (8 or greater) experienced early allograft dysfunction. In contrast, Sorensen et al (35) showed that in 10 A 2! O or B live donor transplants, each with a high IgM anti-a titer (8 to 256), a high IgM anti-a titer had no detrimental effect on graft outcome. Clearly, further studies are warranted to substantiate whether high IgM anti-a titers are detrimental to kidney function and may raise the question of the role of IgM anti-a titers in deceased donor A 2 /A 2 B to B transplantation. Future Directions Several directions can be taken to expand A 2! B renal transplantation. The first is simultaneous pancreas and kidney transplantation. Four successful cases of A 2! B simultaneous pancreas and kidney transplantation have been reported (14). A change in OPTN/UNOS allocation policy would be needed for this to occur. Second, kidney paired donation should be considered when an O or a B candidate is being evaluated and potential donors are either A (for O and B candidates) or AB (for just B candidates). All blood group A and AB donors should be subgrouped so allocation may occur to O and B candidates with low anti-a titers. The University of Kansas kidney transplant program has performed two A 2! O paired kidney donor transplants (personal communication from Dr. Amna Ilahi). Both patients had a low anti-a IgG titer history before transplantation (one had four titer results and the other had five titer results) and the follow-up of the patients at 7 and 3 months shows good kidney function (creatinine levels of 1.4 and 1.5, respectively) with no rejection episodes. SLK transplantation in A 2! O situations appears as successful as ABO compatible SLK transplantations (23). One SLK transplant patient who had an anti-a titer of 512 had good renal function throughout follow-up and titers gradually lowered to a value of 2. The OPTN database did not collect anti-a titer data outside of the A 2 /A 2 B! B national kidney variance, so the effect of titers could not be evaluated on the 14 SLK transplant patients from the OPTN database. Because MTN has seen that about two-thirds of O candidates have high anti-a IgG titer levels, taken together those data and the fact that blood group A antigen is expressed much less on A 2 livers (19) suggest that high anti-a titers may not be detrimental to renal function in the setting of SLK transplantation but certainly more cases with titer data are needed. Pediatric renal transplantation of A 2 /A 2 B! B and A 2! O candidates with deceased or live donors is an uncommon and seldom reported practice. Only one registry report consisting of 11 cases is published (38). Although a high incidence of rejection was reported, it was noted that the registry patients lacked a field for anti-a titer levels and thus could not be assessed. The authors recommend that this is an area worthy of pursuit giving careful attention to low anti-a titer levels. Recent experience at Children s Mercy Hospital transplant program in Kansas City (Dr. Bradley A. Warady, personal communication) is that two children, one 7 the other a 10-year-old received an A 2! B deceased and live donor transplant, respectively since August Both had low (4) anti-a IgG titers (using the MTN titer method) before transplantation. Follow-up at 16 and 23 months after transplantation revealed that the creatinine levels were 0.79 and 0.74 mg/dl, respectively and neither patient had experienced a rejection episode. Taken together those data warrant expanding A 2 /A 2 B! B transplantation to include pediatric recipients with low anti-a IgG titers. A new and potentially important area to explore in A 2 /A 2 B! B transplantation is the critical role of invariant NKT (inkt) cells in A isoagglutinin production. The Tazawa group used anti-cd1a treatment to down regulate A isoagglutinin production by B cells in a humanized mouse model (lymphocytes from a blood group O individual that produced anti-a and anti-b were transplanted into a mouse) (39). They demonstrated that CD1d-restricted inkt cells are required to induce anti-a antibody production, presumably through interacting with CD1d expressed on B cells. The anti-cd1a effect was mediated by inhibiting interleukin-5 production from those cells. This has clinical promise in A 2 /A 2 B! B transplantation, as well as ABO fully incompatible transplantation, for reducing anti-a titer levels before transplantation and controlling humoral rejection when AMR is suspected specifically due to anti-a production as opposed to AMR caused by HLA donor-specific antibodies. Summary Performing an A 2 or A 2 B donor transplant into a blood group B candidate is now a part of the new kidney allocation system in the United States. A high titer of blood group A isoaggultinins is a contraindication to successful A 2 /A 2 B! B transplantation. A 2 /A 2 B! B transplants have the potential to improve access for blood group B candidates to deceased donor transplantation. Diversion of A 2 and A 2 B kidneys from the A and AB candidate pool still leaves A and AB candidates a transplant rate well above their respective waiting list compositions. A 2 /A 2 B! B transplantation is important because B candidates are at a distinct disadvantage in getting transplanted in part because the national B waitlist is composed of more than 70% minority patients, the highest level for any of the other blood groups. Finally, new immunosuppressive modalities such as inkt cell therapy with an anti-cd1d antibody may prove useful for treatment of AMR due to anti-a antibodies or even to lower anti-a levels before transplantation. A recent online report from OPTN/UNOS titled KAS out-ofthe-gate monitoring was released on May 22, 2015, 18 American Journal of Transplantation 2016; 16: 11 20

9 A 2 /A 2 B! B Kidney Transplantation Review describing metrics revealing performance of the new KAS with just over 5 months of operation. Of note were the data showing that of the 11,181 B candidates on the active renal waitlist, 436 (3.9%) have been indicated as willing to accept an A 2 or A 2 B kidney. Also, 1775 (15.9%) were reported as ineligible, while 80% (8940) still have an unknown status making them in effect not eligible for an A 2 or A 2 B kidney. The transplant centers in the MTN DSA routinely inform blood group B patients of their option to have access to A 2 or A 2 B kidneys in their routine patient education session, by a transplant physician and/or coordinator, and the patient signs a consent form. That high number with unknown eligibility status may just mean that it is taking time for each transplant center to get all the B patients consented or an anti-a titer method developed and/or patient s tested. To illustrate the possibility that it is simply taking time for centers to get their B candidates eligible, the Inland Northwest Blood Center HLA Laboratory and Providence Sacred Heart Medical Center and Children s Hospital kidney transplant programs have just completed performing anti-a titers and are waiting legal review of their consent form before they make their B candidates eligible (Dr. Ellen Klohe, personal communication). On June 30, 2015, their first B candidate was listed as eligible in UNet and on July 9 received an A 2 deceased donor kidney! Despite the small percent of B candidates indicated to be eligible to receive A 2 /A 2 B kidneys, there were 38 A 2 /A 2 B! B transplants in the 5 months after the new KAS was implemented compared with six done during the 5 months before the KAS. It is important that most B candidates be entered as eligible so they can also have increased access to A 2 /A 2 B kidneys, a crucial advantage available for B minority candidates as indicated by the UNOS/OPTN A 2 /A 2 B! B voluntary variance study (17). Otherwise the A 2 /A 2 B! B allocation component cannot fully work for the B minority and nonminority candidates as it was intended. Acknowledgments C.F.B. would like to dedicate this article to the memory of his mentor, Dr. Jerry Caldwell. The authors thank Stacia S. Jackson for her administrative help. Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. References 1. Bryan CF, Cherikh WS, Debroy MA, et al. Increased access for blood group B candidates in the U.S. after a policy change to not allocate B kidneys to AB candidates. Am J Transplant 2014; S3: Stegall MD, Dean PG, Gloor JM. ABO-incompatible kidney transplantation. 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