Naturally occurring antibodies against blood group antigens and acquired alloantibodies against donor human leukocyte antigens (HLAs; secondary to a prior kidney transplant, blood transfusions, or pregnancy) may pose major barriers to a successful renal transplantation. Because approximately 20% of renal allograft candidates may be blood group incompatible with their living donor and more than one-third will demonstrate some level of anti-HLA antibody pretransplant, understanding the unique issues pertaining to donor-specific antibody (DSA) is important for the proper management of these patients.
Over the past decade innovative strategies have been utilized to avoid incompatible transplantation, including the expansion of multicenter kidney paired donation programs and the increased priority for deceased donor allocation to highly sensitized candidates in the US. Yet evidence is mounting that a role for incompatible transplantation (ABO and/or HLA) still exists in select circumstances.
This chapter discusses the current options for transplantation in sensitized renal transplant candidates or those with ABO-incompatible living donors. Specific emphasis is placed on what is known regarding the mechanisms and treatment of both early and late antibody-mediated injury, including the results of some recent therapeutic trials. Finally, current knowledge regarding the mechanism of antibody production in the setting of renal transplantation is outlined, highlighting important gaps in current knowledge in this emerging field.
Historically, some of the first evidence for alloantibody was the retrospective study of Patel and Terasaki in 1969. This study showed that ability of the recipient’s serum to lyse donor cells in vitro was associated with allograft loss within hours of transplantation in a high percentage of cases. Thus for many years the presence of a “positive crossmatch” because of DSA was generally considered to be an absolute contraindication to kidney transplantation. However, in the late 1990s, several groups began to employ protocols aimed at overcoming the antibody barrier. In the early 2000s, an increased understanding of the histology associated with antibody and new techniques that enhanced the ability to understand the specificity and strength of DSA helped clarify the risks and outcomes of transplantation for patients with DSA. In the past decade, the development of paired living donation and increased national sharing for highly sensitized patients has provided more options for these patients, yet many still are not transplanted. Recently an increasing number of clinical trials have been instituted to test new agents that raise hopes that new therapy might further improve the transplantation rates and the long-term outcomes of highly sensitized renal transplant candidates.
To understand the therapeutic options for sensitized patients, one first must understand the various assays used to determine the presence of alloantibody ( Table 22.1 ). A more detailed description of these assays is presented elsewhere in this book (see Chapter 10 ), but some key concepts will be covered here. The sensitivity of the assay is important in determining the presence and amount of antibody. For example, complement-dependent cytotoxicity (CDC) assays in which the recipient serum is tested for its ability to lyse target lymphocytes is relatively insensitive. Antihuman globulin (T cell AHG) can be added to enhance the ability to detect class I anti-HLA antibodies, but this assay is still considered the least sensitive of antibody detection techniques. Flow cytometric crossmatch methods are semiquantitative and are more sensitive for detecting anti–class I and II antibodies.
|Panel-reactive antibody (T cell only)|
|Solid-phase bead or ELISA assay|
|Donor-Specific Alloantibody Detection Tests|
|T-cell cytotoxicity (NIH-CDC) assay||Lowest |
|T-cell AHG-CDC assay|
|T-cell flow cytometric crossmatch|
|Solid-phase bead or ELISA assay|
|Anti–Class I, II, or Both||Sensitivity|
|B-cell cytotoxicity (NIH-CDC) assay||Lowest |
|B-cell flow cytometric crossmatch|
|Solid-phase bead or ELISA assay|
Solid-phase assays have revolutionized this field because they are the most sensitive, but also provide information regarding the specificity of the HLA antibody. For this test, single HLAs are bound to microspheres or enzyme-linked immunosorbent assay (ELISA) plates. For solid-phase assays such as the commonly used LABscreen, the level of alloantibody is usually expressed as the mean fluorescence intensity (MFI). MFI levels considered to be positive vary between laboratories and transplant programs, but an MFI level of >1000 generally is accepted as positive.
Commercially available modified solid-phase assay tests have also been developed to detect complement binding (C1q or C3d). DSA with C1q and/or C3d binding positivity is associated with the development of antibody-mediated rejection (AMR) and allograft loss, and some groups routinely use these tests to distinguish the most deleterious DSA. However, it remains unclear whether C1q positivity is superior to using MFI or antibody titer to predict AMR or allograft loss, and thus testing for C1q or C3d binding is not universally performed.
In practice, a solid-phase assay is commonly used as an initial screening tool to determine the presence of alloantibody at the time the transplant candidate is being evaluated. This test is used to determine which HLA antigens to avoid at the time of transplantation and can be used to determine the breadth of sensitization against HLA by calculating the panel-reactive antibody (cPRA). The cPRA provides information regarding the probability of finding a donor against whom the recipient has no DSA, but does not give information about the level of alloantibody. Currently, the cPRA can be used as a factor in deceased donor allocation, as will be discussed later. The PRA was historically determined when the transplant candidate’s serum was added to a panel of cells that represented the donor pool (hence the name “panel-reactive antibody”), but that method is now infrequently used.
When a potential donor is identified, both solid-phase and crossmatch assays are performed and interpreted together in context (i.e., sensitization history). However, as the breadth of antibodies detectable by the solid-phase assay has expanded, some experts support the use of solid-phase assays alone for a virtual crossmatch.
Despite the technologic advances in HLA detection, there remains a considerable lack of standardization of practice among transplant centers regarding the method, interpretation, and reporting of the various tests. This lack of standardization, combined with the small number of patients who undergo incompatible transplantation at individual centers, limits the ability to understand immunologic risk in various settings. Major efforts are underway internationally to standardize practices, and our understanding of the immunologic risk of incompatible transplantation has advanced.
Clinical Approaches to Sensitized Patients
Ideally alloantibody would be avoided at the time of transplantation, but that is not always possible. The next section describes the options for patients with preformed anti-HLA antibodies before transplantation.
Deceased Donor Transplantation
If sensitized patients do not have any prospective living donors, their only transplant option is to be placed on the deceased donor waiting list. Approximately 30,000 patients on the Organ Procurement and Transplantation Network/United Network for Organ Sharing (UNOS) cadaver donor kidney waiting list are “sensitized” and currently about 7000 patients have a cPRA of 99% or 100% in the in US.
In 2014 the deceased donor kidney allocation system (KAS) changed, and it now offers renewed hope for sensitized transplant candidates. This system increases the allocation priority for sensitized candidates on a sliding scale starting with a cPRA of 20%. Although all patients with a cPRA of greater than 80% received allocation points in the old US system, now patients with a cPRA of 80% to 84% receive 2.5 points and those with a cPRA of 100% receive 202 points. Patients with 99% and 100% cPRA have even greater access to the available donor pool because they also now have regional and national priority. Indeed, these changes have increased the number of sensitized patients who have been transplanted. Within the first year of implementation, nearly 1000 patients with cPRA of 99% or 100% received a transplant.
The Acceptable Mismatch Program of Eurotransplant is another approach to transplanting sensitized candidates with a deceased donor. In this program, anti-HLA antibodies are identified using a CDC assay. Highly sensitized patients (PRA >85%) are placed at the top of the kidney match run and organs are allocated based on the absence of DSA against the donor HLA (i.e., “acceptable mismatch” for HLA). In this program, approximately 60% of the highly sensitized patients are transplanted within 2 years after inclusion in this acceptable mismatch program and the short-term graft survival appears similar to that of unsensitized patients. Low levels of DSA that are present at the time of transplant are not considered at the time of allocation, and it is unclear what the long-term outcomes will be in this program.
Kidney Paired Donation
If a sensitized candidate has potential living donors, these donors should be HLA-typed to find a crossmatch-negative donor. If no such donor can be found, sensitized candidates may opt to enter into one of the growing number of paired living donor programs. These “exchange” schemes have been shown to increase the transplantation rate of ABO-incompatible and sensitized patients ( Fig. 22.1 ). Although these programs increase the number of potential donors for sensitized individuals, patients with antibodies against a wide variety of HLA types still may not be able to find a crossmatch-negative donor, even if the donor pool is very large. One of the central questions in paired donation is: How long should a sensitized patient wait for a crossmatch-negative donor versus proceeding to a transplant using a donor against whom the recipient has DSA? Because many sensitized patients may not find a DSA-negative donor, most paired donor programs employ “optimization” protocols that seek to identify donors for sensitized patients with lower levels of DSA than their original donor. Thus transplantation of a sensitized patient in this setting might employ both paired donation and living donor incompatible transplant protocols.
Kidney Transplantation in the Presence of DSA
A viable option for sensitized candidates is to perform a kidney transplant despite the presence of DSA. Overall, data suggests that patient survival may be improved after incompatible transplant compared with remaining on dialysis ( Fig. 22.2 ). This is despite reduced allograft survival as will be discussed in the next section. In a multicenter trial involving 22 centers in the US, the survival of 1025 patients who received an incompatible living donor kidney transplant were matched with controls who remained on the waitlist or received a deceased donor transplant. At 1, 3, and 5 years patients who received an incompatible kidney transplant had improved survival (see Fig. 22.2 ). This patient survival benefit was significant at 8 years across all levels of incompatibility (DSA positive only and positive flow cytometric crossmatch). Specifically, at 5 years the patient survival was 86.0% in those patients who received an incompatible living donor transplant, 74.4% in those who received a deceased donor transplant and/or remained on the waitlist, and 59.2% in those patients who remained on the waitlist and did not receive a transplant.
The survival advantage of an incompatible transplant compared with being on the waiting list in the US was not found in a recent study. A matched cohort analysis was performed in the United Kingdom comparing 213 patients who underwent positive crossmatch transplantation and 852 matched controls. The patient survival was similar among patients who received a positive crossmatch transplant or remained on dialysis at 3 and 7 years. In this cohort, the 5-year patient survival after incompatible transplant was 68% after HLA-incompatible transplantation, 89% after compatible living donor transplant, and 77% for compatible deceased donor transplant. The differences in the results between the US and United Kingdom cohort are likely related to the increased mortality on dialysis in the US and high-risk transplant group from the United Kingdom (all patients received positive crossmatch transplants in the United Kingdom whereas 185 patients in the US cohort had positive DSA by solid-phase single-antigen bead assay, but had a negative crossmatch).
Regardless of the patient survival advantage after incompatible transplantation, allograft survival is reduced. These transplants are also associated with higher rates of hospital readmission in the first year posttransplant and are more expensive. The potential disadvantages of incompatible transplantation should not be used to discourage this practice, but should be used for clinical decision making.
Whereas avoiding DSA is ideal, many highly sensitized patients remain on the waiting list despite paired donor programs and increased priority for deceased donors. When considering a transplant in the setting of DSA, the major risk to consider is that of graft loss. Some incompatible transplants are relatively low risk whereas others are prohibitively high risk for early graft loss. Thus before we discuss the various options for incompatible transplantation, we need to examine the concept of immunologic risk associated with DSA.
Clinicians now have the ability to semiquantitatively estimate the level DSA from very low to very high. Although DSA detected with solid-phase assays and/or crossmatch testing is no longer considered an absolute contraindication to transplant, it continues to represent an immunologic risk. This concept of immunologic risk has emerged as one of the core principles in the transplantation of sensitized patients. Quantifying this risk is an important aspect of designing protocols to enable successful kidney transplantation in sensitized patients. A combination of the various previously described assays allows clinicians to better determine the risk of antibody-mediated graft damage in sensitized patients. However, current assays cannot completely determine the entire immunologic risk of all patients. Even when all DSA testing is negative, sensitized patients are at increased risk for T cell mediated rejection and or may possess antibodies against antigens not detected by current assays.
AMR can be categorized broadly as hyperacute rejection, early active AMR, acute active AMR, and chronic active AMR.
The primary goal of “desensitization” is the avoidance of the catastrophic occurrence of hyperacute rejection, which occurs minutes to hours posttransplant leading to almost immediate allograft loss. The incidence is related to the level of DSA at the time of transplantation. The exact level of DSA that leads to this complication is unclear, but most programs consider a positive T cell AHG crossmatch a high risk for hyperacute rejection. Indeed, early in our experience, we transplanted 10 patients who, despite multiple plasmapheresis treatments (mean of 10 treatments), were unable to achieve a negative T cell AHG crossmatch. Given that these highly sensitized patients had almost no other option for transplant at the time, we performed the transplant despite the persistence of a low titer T cell AHG crossmatch (undiluted to 1:8) on the day of transplantation. Of these 10 patients, two developed hyperacute rejection. The incidence of early active AMR in this group was 70% and the 1-year allograft survival was only 50%. In today’s transplant environment, almost no kidney transplants are performed in the setting of high levels of DSA as defined by a positive T cell AHG crossmatch or MFI >10,000.
Early Active Antibody-Mediated Rejection
Even if hyperacute rejection is avoided, patients with DSA still have a high incidence of active AMR within the first few weeks after transplantation typically during an amnestic response. Although the incidence of hyperacute rejection has decreased, active AMR that occurs early posttransplant has emerged as one of the major complications when transplanting highly sensitized recipients.
From a histologic perspective the kidney biopsy usually shows evidence of C4d deposition in the peritubular capillaries and microvascular inflammation (peritubular and/or glomerular capillaritis). Depending on the severity, thrombosis and evidence of acute tubular injury may also be present. Usually these rejections are associated with an abrupt reduction in glomerular filtration rate (GFR), but these rejections can also be subclinical.
The reported incidence of early active AMR is based largely on single-center reports and ranges from as low as approximately 1% in patients with preformed DSA and negative flow cytometric crossmatch, to up to approximately 40% in patients with preformed DSA and high positive flow cytometric crossmatch. In a series from Hôpital St. Louis in Paris, the incidence of AMR was 36.4% with a baseline DSA MFI of 3001 to 6000 and 51.3% with a baseline DSA MFI of >6000.
The incidence of early active AMR appears to correlate with the development of high levels of DSA after transplantation. Our group examined the natural history of DSA early after transplantation and its relationship with early active AMR in 70 positive crossmatch kidney transplant recipients. The overall incidence of AMR was 36% with the mean time to diagnosis of approximately 10 days, and all episodes occurred within 1 month of transplantation. All but one AMR episode were associated with allograft dysfunction (defined as an increase in serum creatinine over nadir >3.0 mg/dL in the first month), but in many instances evidence of histologic injury preceded the increase in serum creatinine.
The time course of changes in DSA posttransplant correlated well with the development of AMR ( Fig. 22.3 ). Overall, mean DSA levels decreased by day 4 and remained low in patients who did not develop AMR. By day 10, however, DSA levels increased in patients developing AMR, with 92% (23/25) of patients with a flow cytometric crossmatch of >359 (molecules of equivalent soluble fluorochrome units of approximately 34,000) developing AMR. The B flow cytometric crossmatch and the total DSA measured by single-antigen beads with the solid-phase assay correlated well across a wide spectrum, suggesting that either could be used for monitoring. Importantly, when allografts demonstrated the cluster indicative of AMR (high DSA, histologic injury, and graft dysfunction), all but one (24/25) were also C4d-positive.
Late Antibody-Mediated Rejection: Active and Chronic AMR
Although classification systems have been developed to differentiate between active and chronic active AMR, these histologic lesions represent a continuum of injury. Histologically, active AMR usually presents with glomerulitis and peritubular capillaritis with or without C4d positivity (whether it occurs early or late posttransplant). The timing and clinical relevance of active AMR is variable. As described previously, patients can present with an active AMR that presents early within the first few weeks posttransplant. If the early active AMR is averted, many patients with DSA present at the time of transplantation develop active AMR at a later time point. The onset is often insidious, subclinical, and unrecognized without surveillance biopsy protocols.
When the microvascular inflammation (glomerulitis and peritubular capillaritis) and/or C4d positivity is accompanied by duplication of glomerular basement membranes or transplant glomerulopathy, criteria for chronic active AMR has been reached. Chronic antibody-mediated allograft injury has become increasingly implicated as a major cause of late renal allograft loss. This is especially common in patients with DSA at the time of transplantation, but actually most of the chronic active AMR that is encountered in clinical practice occurs in the setting of de novo DSA.
Chronic active AMR generally carries one of the worst prognoses of any histologic lesion found on surveillance biopsy. In one study, 60% of grafts with chronic active AMR on a 1-year biopsy either failed or lost >50% of their function by 5 years after transplantation. Many patients with chronic active AMR have minimal or no interstitial fibrosis suggesting that it is its own distinct process separate from other forms of chronic injury.
Like early acute, active AMR, the incidence of chronic active AMR depends on the DSA and crossmatch status at the time of transplantation. We performed a retrospective observational study to examine the allograft histology and allograft survival after transplantation in the setting of various levels of DSA and flow cytometric crossmatch. We found that the incidence of chronic active AMR correlated with increasing DSA at baseline ( Fig. 22.4 ) and was as follows at a mean follow-up of 4.1 ± 1.9 years: 8.2% in patients who had negative DSA and negative flow cytometric crossmatch at the time of transplant (−DSA/−XM), 17.0% in patients who were positive for DSA by solid-phase testing but negative with flow cytometric crossmatch tests (+DSA/-XM), 30.6% in patients with positive DSA based on solid-phase tests and low positive flow cytometric crossmatch testing (+DSA/low+XM), and 51.2% in patients with positive solid-phase tests and high positive flow cytometric crossmatch testing (+DSA/high+XM) (see Fig. 22.4 ).