CLINICAL
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TRANSLATIONAL RESEARCH
Long-Term Outcomes of Kidney Transplantation Across a Positive Complement-Dependent Cytotoxicity Crossmatch Leonardo V. Riella,1 Kassem Safa,1 Jude Yagan,1 Belinda Lee,1 Jamil Azzi,1 Nader Najafian,1 Reza Abdi,1 Edgar Milford,1 Helen Mah,1 Steven Gabardi,1,2 Sayeed Malek,2 Stefan G. Tullius,2 Colm Magee,3 and Anil Chandraker1,4 Background. More than 30% of potential kidney transplant recipients have pre-existing antiYhuman leukocyte antigen antibodies. This subgroup has significantly lower transplant rates and increased mortality. Desensitization has become an important tool to overcome this immunological barrier. However, limited data is available regarding longterm outcomes, in particular for the highest risk group with a positive complement-dependent cytotoxicity crossmatch (CDC XM) before desensitization. Methods. Between 2002 and 2010, 39 patients underwent living-kidney transplantation across a positive CDC XM against their donors at our center. The desensitization protocol involved pretransplant immunosuppression, plasmapheresis, and low-dose intravenous immunoglobulinTrituximab. Measured outcomes included patient survival, graft survival, renal function, rates of rejection, infection, and malignancy. Results. The mean and median follow-up was 5.2 years. Patient survival was 95% at 1 year, 95% at 3 years, and 86% at 5 years. Death-censored graft survival was 94% at 1 year, 88% at 3 years, and 84% at 5 years. Uncensored graft survival was 87% at 1 year, 79% at 3 years, and 72% at 5 years. Twenty-four subjects (61%) developed acute antibodymediated rejection of the allograft and one patient lost her graft because of hyperacute rejection. Infectious complications included pneumonia (17%), BK nephropathy (10%), and CMV disease (5%). Skin cancer was the most prevalent malignancy in 10% of patients. There were no cases of lymphoproliferative disorder. Mean serum creatinine was 1.7T1 mg/dL in functioning grafts at 5 years after transplantation. Conclusion. Despite high rates of early rejection, desensitization in living-kidney transplantation results in acceptable 5-year patient and graft survival rates. Keywords: Desensitization, Alloantibodies, CDC crossmatch, Kidney transplantation. (Transplantation 2014;97: 1247Y1252) This work was supported by a research grant from the American Heart Association to L.V.R. The authors declare no conflicts of interest. 1 Schuster Family Transplantation Research Center, Renal Division, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, Boston, MA. 2 Division of Transplant Surgery, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA. 3 Department of Nephrology, Beaumont Hospital, Dublin, Ireland. 4 Address correspondence to: Anil Chandraker, MD, Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital Boston, 221 Longwood Ave, Boston MA 02115. E-mail: [email protected] L.V.R. and K.S. contributed equally to this paper. L.V.R. and K.S. participated in analyzing the data and writing the article. J.Y. and B.L. participated in collecting the data. C.M. participated in developing the initial desensitization protocol. L.V.R., J.A., N.N., R.A., C.M., S.G., S.G.T., S.M., and A.C. participated in patient care. H.M. and E.M. provided the tissue typing data. Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com). Received 15 March 2013. Revision requested 17 November 2013. Accepted 22 November 2013. Copyright * 2014 by Lippincott Williams & Wilkins ISSN: 0041-1337/14/9712-1247 DOI: 10.1097/01.TP.0000442782.98131.7c
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idney transplantation is the treatment of choice for end-stage renal disease patients. However, more than 30% of patients on the waiting list have been sensitized to human leukocyte antigens (HLA), creating a major barrier to kidney transplantation (1). Patients may develop anti-HLA antibodies after pregnancy, blood transfusions, or previous kidney transplant. In addition, it is believed that some infections and vaccinations might trigger an antibody response that cross-reacts with HLA antigens (2, 3). Sensitization leads to a prolonged waiting time on the deceased-donor list, limits suitable living donors, and is associated with a higher rate of rejection and graft loss posttransplant (4Y6). Historically, the presence of anti-HLA donor-specific antibodies has been a contraindication to transplantation, especially when the complement-dependent cytotoxicity (CDC) crossmatch (XM) is positive, as this has been highly associated with hyperacute rejection and immediate graft loss (7). The development of protocols to decrease the load of circulating anti-HLA antibodies has increased the opportunity for transplantation in sensitized patients (4, 5). Highdose intravenous immunoglobulin (IVIG) or a combination
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of plasmapheresis with low-dose IVIG are the two main desensitization methods used in the United States. There have been no randomized trials comparing these protocols, but some studies suggest that plasmapheresis-based protocols are associated with superior outcomes (8Y10). Paired kidney exchange is a useful initial strategy in patients with a XM-positive living donor (5, 6). The major advantage of paired kidney exchange is that the problem of HLA (or ABO) incompatibility can be circumvented without extra immunosuppression. However, paired kidney exchange will not work for a significant percentage of highly sensitized patients (5). Furthermore, sometimes, paired kidney exchange finds a ‘‘less incompatible donor’’ where some limited desensitization is still required. Thus, desensitization still is an important treatment option for sensitized patients. Our group and others have published successful shortterm outcomes using desensitization protocols (11Y15). Desensitization has been shown to significantly increase the rate of kidney transplantation among sensitized patients, albeit with a high rate of antibody-mediated rejection (AMR) and infection (5). The aim of this study was to evaluate the longterm outcomes of a single-center plasmapheresis-based desensitization protocol in a high-risk patient population with a positive CDC XM before desensitization. This is a follow-up study to the publication of Magee et al. in 2008 with additional patients and longer follow-up (12).
RESULTS Demographics Between 2002 and 2010, 39 sensitized patients underwent desensitization in our center. The mean age was 43 years old; 27 were women (69%) and 25 were Caucasian (64%). Twenty-seven patients had received a previous kidney transplant (70%) and the average mean peak panel reactive antibody (PRA) before desensitization was 47.8%T31%. The commonest cause of end-stage renal disease was glomerulonephritis (23%) followed by diabetes (13%). The majority of patients had more than two HLA mismatches against their donors (85%). Table 1 summarizes the demographics of the studied population.
Desensitization and Crossmatch Conversion Eight patients had an isolated positive T-cell CDC XM, 12 had an isolated positive B-cell CDC XM, while 19 were both T- and B-cell CDC XM positive. Five patients had high T-cell titers (Q1:32), while six patients had high B-cell titers (Q1:32). The highest CDC XM titer was 1:256 (B cell). All sensitized patients were successfully transplanted after completing the desensitization protocol. The average mean PRA decreased to 34.1%T31% after desensitization. Patients received on average 5.6 plasmapheresis sessions before transplantation. The majority of patients received basiliximab as induction therapy (58%), while the remainder received ATG. Twenty-five patients also received rituximab (64%). No major complications were observed with the pretransplant desensitization protocol, with the exception of hypotension during plasmapheresis.
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TABLE 1.
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Patient demographics n=39
Age, yr Female/male Follow-up, yr Mean Median RaceVno. (%) White African American Hispanic Asian American Indian Indian ESRD etiologyVno. (%) GN (LN, IgAN, HSP, MPGN, Other) Diabetic nephropathy Polycystic kidney disease Focal segmental glomerulosclerosis Hemolytic-uremic syndrome Congenital obstructive uropathy Chronic pyelonephritis Reflux nephropathy Other/unknown HLA mismatchVno. (%) 0 1Y2 3Y4 5Y6 Previous transplants numberVno. (%) 0 1 2 3
43.1T13 27/12 5.24 5.24 25 (64.1) 8 (20.5) 3 (7.7) 1 (2.6) 1 (2.6) 1 (2.6) 9 (23) 5 (12.8) 4 (10.2) 2 (5.1) 3 (7.7) 3 (7.7) 2 (5.1) 2 (5.1) 9 (23) 0 (0) 6 (15.4) 17 (43.6) 16 (41) 12 (30.7) 20 (51.3) 6 (15.4) 1 (2.6)
GN, glomerulonephritis; LN, lupus nephritis; IgAN, IgA nephropathy; HSP, Henoch-Schonlein purpura; MPGN, membranoproliferative GN.
Rejection The mean and median follow-up was 5 years. Among the 39 sensitized recipients, 24 developed AMR after transplantation (61%) and nine patients had acute T-cellmediated rejection (23%) (Table 2). Hyperacute rejection occurred in only one patient, leading to early graft loss. The majority of cases of AMR were diagnosed in the first month posttransplant (71%). Fifteen recipients were diagnosed with chronic rejection/transplant glomerulopathy (CR/TG) upon graft biopsies on follow-up (38%). There was no statistically significant association between the type of induction immunosuppression and the occurrence of cellular or humoral rejection. The addition of rituximab to our regimen also did not seem to decrease the incidence of AMR because 18 of 24 patients who had AMR had been treated with rituximab. Mean serum creatinine levels at different time points after transplant are shown in Figure 1. Among patients followed for more than 5 years, 13 patients had a serum creatinine below 2 mg/dL and eight out of those 13 had a serum
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TABLE 2.
there were no associations that were statistically significant (data not shown).
Complications No. (%)
Immunological Acute antibody-mediated rejection G1 mo 3Y6 mo 96 mo Acute cellular rejection Chronic rejection Infectious UTI Respiratory tract infection BK viremia BK nephropathy Bacteremia CMV Neoplastic SCC/BCC Renal cell carcinoma Angiosarcoma
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24 (61.5) 17 3 4 9 (23) 15 (38.5) 6 (15.3) 7 (17.9) 7 (18) 4 (10) 5 (12.3) 2 (4.4) 4 (10.25) 1 (2.5) 1 (2.5)
creatinine of less than 1.5 mg/dL. The first desensitized patient continues to have a creatinine below 1 mg/dL and no proteinuria after 9.7 years of follow-up. Infection and Malignancy Respiratory and urinary tract infections occurred in 17% and 15% of patients, respectively (Table 2). Seven patients developed BK viremia (18%), among which four had biopsy-proven BK nephropathy (10%). Of the four patients, three subsequently lost their grafts. Five patients had bacteremia (12%) and one had endocarditis. Two patients had CMV disease (4.4%). The predominant malignancies in this cohort were skin cancers (squamous and basal cell type) affecting four patients (10%) and renal cell carcinoma (2.5%) (Table 2). There were no cases of lymphoproliferative disorders. Patient and Graft Survival Patient survival was 95% at 1 year, 95% at 3 years, and 86% at 5 years (Fig. 2A). The main cause of death was infectious respiratory failure (three patients) followed by cardiovascular events in two patients; one patient died from terminal metastatic disease and one patient’s death was of unclear etiology. Death-censored graft survival was 94% at 1 year, 88% at 3 years, and 84% at 5 years (Fig. 2B). Uncensored allograft survival was 87% at 1 year, 79% at 3 years, and 72% at 5 years (Fig. 2C). The majority of graft losses (four of six) was a result of chronic AMR. There was no association between the average number of HLA mismatches and graft failure (P=0.64). In addition, the type of positive CDC XM (T cell, B cell, or both) was not associated with death or graft loss (Table 3). We also examined the association of age, sex, race, number of previous transplants, induction type, rituximab use, and cellular- or antibodymediated rejection with graft loss or patient death, but
Baseline Donor-Specific Antibodies and Outcomes Because Luminex single-antigen bead assay was not a routine test at the beginning of this cohort in 2002, we retrospectively analyzed 29 available patients’ sera for donorspecific antibodies (DSAs). All 29 patients had confirmed positive DSAs before desensitization. The majority of patients had two or more DSAs (69%). Although there was a trend towards an association between having two or more DSAs and subsequent AMR, statistical significance was not reached (see Table S1, SDC, http://links.lww.com/TP/A926). Similarly, a trend was observed for the association of DSAs against DR and subsequent AMR development (seven of the eight patients with DR+DSAs had subsequent AMR).
DISCUSSION In this study, we evaluated long-term outcomes of high-risk sensitized patients who underwent desensitization with a protocol that included plasmapheresis and low-dose IVIG. During a 10-year period, 39 patients with CDC XM positive before desensitization underwent successful kidney transplantation at our institution with a 5-year patient survival of 86%. A high rate of AMR was reported. Nevertheless, mean serum creatinine was 1.7T1 mg/dL in functioning grafts at 5 years after transplantation. Sensitization to HLA antigens is a major barrier for kidney transplantation, and currently there are more than 25,000 patients on the U.S. waiting list who are classified as sensitized (1). Sensitized patients have less than 8% chance of obtaining a kidney from the waiting list when compared to non-sensitized patients. Consequently, there is a high mortality among sensitized patients that remain on dialysis waiting for the promised matched kidney (16). To increase their chances for kidney transplantation, they may enroll in kidney paired exchange programs or undergo desensitization.
FIGURE 1. Serum creatinine at different time points after transplantation across a CDC XM-positive barrier. Each dot represents a patient; solid line represents the mean. Creatinine of patients with a failed allograft were omitted.
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FIGURE 2. Kaplan-Meier curves of patient survival (A), death-censored survival (B), and uncensored graft survival (C) in CDC XM-positive patients who underwent transplantation after desensitization.
Though kidney paired donation is a less costly and a more effective strategy, overall match rates for sensitized patients are low (6, 17). Therefore, these two strategies should be viewed as complementary. Montgomery et al. have recently shown that 5-year patient survival is superior in desensitized patients (80.6%) compared to matched controls (51.5%) who remained on dialysis awaiting a transplant, proving the benefits of desensitization in this subpopulation (16). Our study reinforces these findings on patient survival and provides further detail on graft function, graft survival, rejection rates, and infectious and neoplastic complications.
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The goal of desensitization is to decrease the number of circulating donor-specific antibodies and potentially reduce their production to prevent hyperacute rejection and minimize the risk of AMR. Though desensitization has proven to be effective in preventing hyperacute rejection, the prevalence of AMR in the first 6 months after transplant remains high, ranging from 20% to 55% (4, 5, 10Y15). Among studies evaluating outcomes of sensitized patients, there is considerable heterogeneity in the protocols used, differences in histocompatibility testing techniques (CDC, flow, single-antigen bead) and characteristics of donor and recipient populations, making comparisons difficult. Despite these caveats, short-term follow-up studies with desensitized patients revealed a 1-year graft survival ranging from 89% to 94% and 2-year graft survival of 81%Y89% (11, 12, 14, 18Y20), comparable to our study (94% and 92%, respectively). In a retrospective study, Stegall et al. reported that desensitization with plasmapheresis, low-dose IVIG, and rituximab was more effective than high-dose IVIG in desensitizing patients (84% vs. 36%, respectively) and was associated with lower rates of AMR (37% vs. 80%) (10). Long-term outcomes of the same group revealed a graft survival of 82% and 64.9% in CDC XM-positive patients (n=41) at 1 and 5 years posttransplant (21), respectively, slightly inferior to our cohort observed survival. Recent reports have also suggested that plasmapheresis-based protocols seem to be more effective than high-dose IVIG in reducing DSA (8, 9), although in practice, plasmapheresis protocols are difficult to use in patients on the deceaseddonor list. The difference in rates of AMR between published plasmapheresis-based protocols seems to be mostly related to the strength of DSA of the cohort studied. This has been shown by Gloor et al. who compared sensitized patients with different DSA levels and confirmed that CDC XM-positive patients had the highest incidence of AMR (50%), followed by high FCXM (9300; 37%) and then low FCXM (G300; 30%) (18). Our study solely selected sensitized patients with a CDC-positive XM, which are recognized as the highest risk group to undergo desensitization, and consequently had a high rate of AMR (61%). The incidence of AMR is particularly high in the first month after transplant (Table 2); therefore, decreasing the rate of AMR remains a challenge for sensitized recipients. Short-term results by Stegall et al. indicate that eculizumab, a complement inhibitor that blocks C5, might decrease the rate of AMR in sensitized recipients, though duration of therapy and long-term consequences are still unknown (22).
TABLE 3. Positive CDC crossmatch result across different outcomes
AMR (n=24) TCMR (n=9) CR/TG (n=15) Graft failure (n=6) Death (n=7)
Positive T-cell XM
Positive B-cell XM
Positive T- and B-cell XM
5 4 3 1 2
7 2 5 2 1
12 3 7 3 4
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Proteasome inhibitors are also being tested as an option for direct targeting antibody-producing cells (23Y25). Recent reports suggested that donor-specific class II anti-HLA antibodies might be more deleterious to the allograft than class I anti-HLA antibodies (21). In our cohort, there was a trend towards worse graft outcomes in patients with either B-cell XM or both B- and T-positive CDC XM (Table 3). Moreover, we observed a similar trend of higher AMR in patients with DSAs against HLA DR. Nonetheless, it is clear from this study and others that the current standard of anti-HLA antibody characterization is neither sensitive nor specific enough to identify those sensitized patients who will have a worse outcome. Assessing the complement binding capacity of anti-HLA antibodies may be a supplementary assay that could help risk stratify this subgroup of patients and permit prospectively to better assess the effect of different treatment strategies (26). A concern with all desensitization protocols is the need for more immunosuppression, which might lead to higher rates of infection and cancer. Infection was the leading cause of death in our cohort (three patients). Moreover, BK nephropathy occurred in 10% of our patients, likely reflecting the higher degree of immunosuppression. In general, patients tolerated the treatment well with no major bleeding or adverse events from plasmapheresis, IVIG or rituximab, or both. The incidence of malignancies, a major concern in long-term immunosuppression, was also remarkably low with no cases of lymphoproliferative disorder and an expected rate of skin malignancies (27). Identifying patients who might be at risk of severe infectious complications is still a challenging task when selecting sensitized patients to proceed with desensitization. Our study has certain limitations, including the singlecenter design, the suitability of the protocol solely for live kidney donation, and the lack of a control group. Finding controls in a single center carries a high risk of selection bias because of difficulties in matching all potentially important variables; using databases would suffer other shortcomings such as difference in immunosuppression protocols and centers’ characteristics. Lastly, we did not include patients that at baseline had a negative T-cell CDC XM but positive T-cell FCXM. These patients are known to have a higher risk of AMR compared to T-cell FCXM-negative patients (4) and may also benefit from an intervention for alloantibody removal. In conclusion, desensitization with a plasmapheresisbased protocol is a reasonable alternative for sensitized patients who have an incompatible kidney donor and have not been successful finding a suitable paired kidney in an exchange program. Our long-term outcomes are reassuring in regards to patient survival and malignant complications but raise concerns about the high rates of AMR and potential infectious complications.
MATERIALS AND METHODS Study Population From January 2002 to December 2010, a total of 39 patients underwent live-donor kidney transplantation with HLA-incompatible donors defined by a positive CDC XM against donor T or B cells, or both. The higher risk of rejection and graft loss were specifically discussed with all living donors and recipients. In cases in which multiple potential donors were available, the donor with the lowest titer XM was generally chosen. Patients who did not
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convert to CDC XM negative did not proceed to kidney transplantation, unless only the B-cell CDC XM remained weakly positive and the delta fluorescence unit (DFU) was less than 20 by flow cytometry XM (FCXM). One patient of this cohort had both HLA and ABO incompatibility with the donor (12, 28). This study was approved by the institutional review board at Brigham and Women’s Hospital, Harvard Medical School.
Desensitization Protocol Plasmapheresis was performed thrice weekly in our dialysis unit using a hollow fiber plasma filter (Plasmaflo-AP06 Asahi Medical). The baseline XM titer was used to roughly estimate the number of sessions required to obtain a negative XM, as previously described (12). Typically, a plasma volume of 50 mL per kilogram of body weight was removed and replaced with 5% albumin (2/3) and 0.9% saline (1/3). Plasmapheresis was immediately followed by hemodialysis and 10 g of IVIG during the first 1 to 2 hours of the hemodialysis session. Following transplantation, routine plasmapheresis was performed for 2 weeks with a frequency dependent on the strength of baseline XM (from two to six sessions).
Immunosuppression Tacrolimus and mycophenolate mofetil (MMF) were initiated 5 to 7 days before transplant. Target trough concentrations of tacrolimus were 8 to 12 ng/mL. MMF starting dose was 2 g daily, adjusted according to patients’ tolerance. High-dose glucocorticoids were started at the day of the transplant and then tapered to 5 to 10 mg daily by 3 months posttransplant. Induction therapy consisted of either antithymocyte globulin rabbit (ATG) or basiliximab. The protocol was briefly modified during the 10-year period of the study because of concern for alloantibody rebound with ATG (29). Valganciclovir and sulfamethoxazole-trimethoprim were prescribed as antimicrobial prophylaxis for 6 months. In cases where multiple plasmapheresis sessions (and therefore depletion of clotting factors) were anticipated, 10 mg of vitamin K was given PO at the start of desensitization unless there was a contraindication. The majority of patients were treated with at least one dose of rituximab 375 mg/m2 on the night before transplantation.
Crossmatching and Donor-Specific Anti-HLA Antibody The CDC XM against the donor was the histocompatibility test used to determine the degree of sensitization and estimate the number of plasmapheresis sessions required before transplantation (12). Briefly, T-cell crossmatches were performed by the modified Amos method (using three washes) and an antihuman globulin method to increase the sensitivity for antibody detection. B-cell crossmatches were performed by the modified Amos method (using three washes). Auto-crossmatching was performed for all donors and recipients to exclude autoantibody as a cause of any positive XM. Recipient serum was also treated with dithioerythritol to exclude IgM antibodies when appropriate. Because of the limited specificity of the B-cell CDC XM, flow panel reactive antibody testing was also used to confirm the presence of class II antibodies in the serum. Pretransplant flow cytometry XM (FCXM) was not routinely performed in all patients, as the primary goal of the protocol was to achieve a negative CDC XMVand not a negative FCXMVpretransplant. Sera were analyzed for DSA by Luminex assay single-antigen screening. As this analysis was not available at the beginning of the study, DSA data was available for 29 of the 39 patients in this cohort. When single-antigen screening was performed, a threshold of 1500 median fluorescence intensity was used to determine DSA positivity.
Posttransplant Course Outcomes measured included patient survival, graft survival, renal function (serum creatinine), rates of cellular AMR, rates of infection, and malignancy. Protocol biopsies were not routinely performed, though the threshold to perform for-cause biopsies was low (typically an increase in serum creatinine Q0.3 mg/dL). Staining for C4d by immunofluorescence was routinely performed. Renal biopsies were graded according to the Banff classification (30). Acute and borderline cellular rejections were treated with pulse methylprednisolone 500 mg IV daily for 3 days. Acute cellular rejection resistant to such therapy was treated ATG. Acute AMR was defined by
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at least two of the three following characteristics: positive FCXM against the donor, histologic changes consistent with AMR, and positive C4d staining in peritubular or glomerular capillaries (31). The FCXMVas opposed to CDC XMVwas used posttransplant for anti-HLA antibody monitoring. Acute AMR was treated with pulse methylprednisolone 500 mg IV daily for 3 days and plasmapheresis with IVIG every 24 to 48 hours until the FCXM was negative against donor T and B cells or five sessions of plasmapheresis had been completed (whichever came first). Repeat FCXMTbiopsy would then guide further therapy. In cases of severe AMR not responsive to pheresis, an additional dose of rituximab 375 mg/m2 was administered. Rituximab was introduced into the protocol in an attempt to reduce the rate of AMR observed in the beginning of the cohort.
Statistical Analysis
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11. 12. 13. 14. 15.
Kaplan-Meier plots were used to estimate patient and allograft survival. Censoring was at time of death. One-way ANOVA and Student t test were used to test for quantitative outcomes. Chi-square and Fisher exact tests were used to test for categorical outcomes.
16.
ACKNOWLEDGMENTS The authors would like to thank Brian Smith for his help in performing some of the anti-HLA antibody testing, and Sarah Conte and Christine Dyer for the coordination of this clinical research project.
18.
17.
19. 20. 21.
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TRANSLATIONAL RESEARCH
Long-Term Outcomes of Kidney Transplantation Across a Positive Complement-Dependent Cytotoxicity Crossmatch Leonardo V. Riella,1 Kassem Safa,1 Jude Yagan,1 Belinda Lee,1 Jamil Azzi,1 Nader Najafian,1 Reza Abdi,1 Edgar Milford,1 Helen Mah,1 Steven Gabardi,1,2 Sayeed Malek,2 Stefan G. Tullius,2 Colm Magee,3 and Anil Chandraker1,4 Background. More than 30% of potential kidney transplant recipients have pre-existing antiYhuman leukocyte antigen antibodies. This subgroup has significantly lower transplant rates and increased mortality. Desensitization has become an important tool to overcome this immunological barrier. However, limited data is available regarding longterm outcomes, in particular for the highest risk group with a positive complement-dependent cytotoxicity crossmatch (CDC XM) before desensitization. Methods. Between 2002 and 2010, 39 patients underwent living-kidney transplantation across a positive CDC XM against their donors at our center. The desensitization protocol involved pretransplant immunosuppression, plasmapheresis, and low-dose intravenous immunoglobulinTrituximab. Measured outcomes included patient survival, graft survival, renal function, rates of rejection, infection, and malignancy. Results. The mean and median follow-up was 5.2 years. Patient survival was 95% at 1 year, 95% at 3 years, and 86% at 5 years. Death-censored graft survival was 94% at 1 year, 88% at 3 years, and 84% at 5 years. Uncensored graft survival was 87% at 1 year, 79% at 3 years, and 72% at 5 years. Twenty-four subjects (61%) developed acute antibodymediated rejection of the allograft and one patient lost her graft because of hyperacute rejection. Infectious complications included pneumonia (17%), BK nephropathy (10%), and CMV disease (5%). Skin cancer was the most prevalent malignancy in 10% of patients. There were no cases of lymphoproliferative disorder. Mean serum creatinine was 1.7T1 mg/dL in functioning grafts at 5 years after transplantation. Conclusion. Despite high rates of early rejection, desensitization in living-kidney transplantation results in acceptable 5-year patient and graft survival rates. Keywords: Desensitization, Alloantibodies, CDC crossmatch, Kidney transplantation. (Transplantation 2014;97: 1247Y1252) This work was supported by a research grant from the American Heart Association to L.V.R. The authors declare no conflicts of interest. 1 Schuster Family Transplantation Research Center, Renal Division, Brigham and Women’s Hospital and Children’s Hospital Boston, Harvard Medical School, Boston, MA. 2 Division of Transplant Surgery, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA. 3 Department of Nephrology, Beaumont Hospital, Dublin, Ireland. 4 Address correspondence to: Anil Chandraker, MD, Transplantation Research Center, Brigham and Women’s Hospital and Children’s Hospital Boston, 221 Longwood Ave, Boston MA 02115. E-mail: [email protected] L.V.R. and K.S. contributed equally to this paper. L.V.R. and K.S. participated in analyzing the data and writing the article. J.Y. and B.L. participated in collecting the data. C.M. participated in developing the initial desensitization protocol. L.V.R., J.A., N.N., R.A., C.M., S.G., S.G.T., S.M., and A.C. participated in patient care. H.M. and E.M. provided the tissue typing data. Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com). Received 15 March 2013. Revision requested 17 November 2013. Accepted 22 November 2013. Copyright * 2014 by Lippincott Williams & Wilkins ISSN: 0041-1337/14/9712-1247 DOI: 10.1097/01.TP.0000442782.98131.7c
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idney transplantation is the treatment of choice for end-stage renal disease patients. However, more than 30% of patients on the waiting list have been sensitized to human leukocyte antigens (HLA), creating a major barrier to kidney transplantation (1). Patients may develop anti-HLA antibodies after pregnancy, blood transfusions, or previous kidney transplant. In addition, it is believed that some infections and vaccinations might trigger an antibody response that cross-reacts with HLA antigens (2, 3). Sensitization leads to a prolonged waiting time on the deceased-donor list, limits suitable living donors, and is associated with a higher rate of rejection and graft loss posttransplant (4Y6). Historically, the presence of anti-HLA donor-specific antibodies has been a contraindication to transplantation, especially when the complement-dependent cytotoxicity (CDC) crossmatch (XM) is positive, as this has been highly associated with hyperacute rejection and immediate graft loss (7). The development of protocols to decrease the load of circulating anti-HLA antibodies has increased the opportunity for transplantation in sensitized patients (4, 5). Highdose intravenous immunoglobulin (IVIG) or a combination
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of plasmapheresis with low-dose IVIG are the two main desensitization methods used in the United States. There have been no randomized trials comparing these protocols, but some studies suggest that plasmapheresis-based protocols are associated with superior outcomes (8Y10). Paired kidney exchange is a useful initial strategy in patients with a XM-positive living donor (5, 6). The major advantage of paired kidney exchange is that the problem of HLA (or ABO) incompatibility can be circumvented without extra immunosuppression. However, paired kidney exchange will not work for a significant percentage of highly sensitized patients (5). Furthermore, sometimes, paired kidney exchange finds a ‘‘less incompatible donor’’ where some limited desensitization is still required. Thus, desensitization still is an important treatment option for sensitized patients. Our group and others have published successful shortterm outcomes using desensitization protocols (11Y15). Desensitization has been shown to significantly increase the rate of kidney transplantation among sensitized patients, albeit with a high rate of antibody-mediated rejection (AMR) and infection (5). The aim of this study was to evaluate the longterm outcomes of a single-center plasmapheresis-based desensitization protocol in a high-risk patient population with a positive CDC XM before desensitization. This is a follow-up study to the publication of Magee et al. in 2008 with additional patients and longer follow-up (12).
RESULTS Demographics Between 2002 and 2010, 39 sensitized patients underwent desensitization in our center. The mean age was 43 years old; 27 were women (69%) and 25 were Caucasian (64%). Twenty-seven patients had received a previous kidney transplant (70%) and the average mean peak panel reactive antibody (PRA) before desensitization was 47.8%T31%. The commonest cause of end-stage renal disease was glomerulonephritis (23%) followed by diabetes (13%). The majority of patients had more than two HLA mismatches against their donors (85%). Table 1 summarizes the demographics of the studied population.
Desensitization and Crossmatch Conversion Eight patients had an isolated positive T-cell CDC XM, 12 had an isolated positive B-cell CDC XM, while 19 were both T- and B-cell CDC XM positive. Five patients had high T-cell titers (Q1:32), while six patients had high B-cell titers (Q1:32). The highest CDC XM titer was 1:256 (B cell). All sensitized patients were successfully transplanted after completing the desensitization protocol. The average mean PRA decreased to 34.1%T31% after desensitization. Patients received on average 5.6 plasmapheresis sessions before transplantation. The majority of patients received basiliximab as induction therapy (58%), while the remainder received ATG. Twenty-five patients also received rituximab (64%). No major complications were observed with the pretransplant desensitization protocol, with the exception of hypotension during plasmapheresis.
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TABLE 1.
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Patient demographics n=39
Age, yr Female/male Follow-up, yr Mean Median RaceVno. (%) White African American Hispanic Asian American Indian Indian ESRD etiologyVno. (%) GN (LN, IgAN, HSP, MPGN, Other) Diabetic nephropathy Polycystic kidney disease Focal segmental glomerulosclerosis Hemolytic-uremic syndrome Congenital obstructive uropathy Chronic pyelonephritis Reflux nephropathy Other/unknown HLA mismatchVno. (%) 0 1Y2 3Y4 5Y6 Previous transplants numberVno. (%) 0 1 2 3
43.1T13 27/12 5.24 5.24 25 (64.1) 8 (20.5) 3 (7.7) 1 (2.6) 1 (2.6) 1 (2.6) 9 (23) 5 (12.8) 4 (10.2) 2 (5.1) 3 (7.7) 3 (7.7) 2 (5.1) 2 (5.1) 9 (23) 0 (0) 6 (15.4) 17 (43.6) 16 (41) 12 (30.7) 20 (51.3) 6 (15.4) 1 (2.6)
GN, glomerulonephritis; LN, lupus nephritis; IgAN, IgA nephropathy; HSP, Henoch-Schonlein purpura; MPGN, membranoproliferative GN.
Rejection The mean and median follow-up was 5 years. Among the 39 sensitized recipients, 24 developed AMR after transplantation (61%) and nine patients had acute T-cellmediated rejection (23%) (Table 2). Hyperacute rejection occurred in only one patient, leading to early graft loss. The majority of cases of AMR were diagnosed in the first month posttransplant (71%). Fifteen recipients were diagnosed with chronic rejection/transplant glomerulopathy (CR/TG) upon graft biopsies on follow-up (38%). There was no statistically significant association between the type of induction immunosuppression and the occurrence of cellular or humoral rejection. The addition of rituximab to our regimen also did not seem to decrease the incidence of AMR because 18 of 24 patients who had AMR had been treated with rituximab. Mean serum creatinine levels at different time points after transplant are shown in Figure 1. Among patients followed for more than 5 years, 13 patients had a serum creatinine below 2 mg/dL and eight out of those 13 had a serum
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TABLE 2.
there were no associations that were statistically significant (data not shown).
Complications No. (%)
Immunological Acute antibody-mediated rejection G1 mo 3Y6 mo 96 mo Acute cellular rejection Chronic rejection Infectious UTI Respiratory tract infection BK viremia BK nephropathy Bacteremia CMV Neoplastic SCC/BCC Renal cell carcinoma Angiosarcoma
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24 (61.5) 17 3 4 9 (23) 15 (38.5) 6 (15.3) 7 (17.9) 7 (18) 4 (10) 5 (12.3) 2 (4.4) 4 (10.25) 1 (2.5) 1 (2.5)
creatinine of less than 1.5 mg/dL. The first desensitized patient continues to have a creatinine below 1 mg/dL and no proteinuria after 9.7 years of follow-up. Infection and Malignancy Respiratory and urinary tract infections occurred in 17% and 15% of patients, respectively (Table 2). Seven patients developed BK viremia (18%), among which four had biopsy-proven BK nephropathy (10%). Of the four patients, three subsequently lost their grafts. Five patients had bacteremia (12%) and one had endocarditis. Two patients had CMV disease (4.4%). The predominant malignancies in this cohort were skin cancers (squamous and basal cell type) affecting four patients (10%) and renal cell carcinoma (2.5%) (Table 2). There were no cases of lymphoproliferative disorders. Patient and Graft Survival Patient survival was 95% at 1 year, 95% at 3 years, and 86% at 5 years (Fig. 2A). The main cause of death was infectious respiratory failure (three patients) followed by cardiovascular events in two patients; one patient died from terminal metastatic disease and one patient’s death was of unclear etiology. Death-censored graft survival was 94% at 1 year, 88% at 3 years, and 84% at 5 years (Fig. 2B). Uncensored allograft survival was 87% at 1 year, 79% at 3 years, and 72% at 5 years (Fig. 2C). The majority of graft losses (four of six) was a result of chronic AMR. There was no association between the average number of HLA mismatches and graft failure (P=0.64). In addition, the type of positive CDC XM (T cell, B cell, or both) was not associated with death or graft loss (Table 3). We also examined the association of age, sex, race, number of previous transplants, induction type, rituximab use, and cellular- or antibodymediated rejection with graft loss or patient death, but
Baseline Donor-Specific Antibodies and Outcomes Because Luminex single-antigen bead assay was not a routine test at the beginning of this cohort in 2002, we retrospectively analyzed 29 available patients’ sera for donorspecific antibodies (DSAs). All 29 patients had confirmed positive DSAs before desensitization. The majority of patients had two or more DSAs (69%). Although there was a trend towards an association between having two or more DSAs and subsequent AMR, statistical significance was not reached (see Table S1, SDC, http://links.lww.com/TP/A926). Similarly, a trend was observed for the association of DSAs against DR and subsequent AMR development (seven of the eight patients with DR+DSAs had subsequent AMR).
DISCUSSION In this study, we evaluated long-term outcomes of high-risk sensitized patients who underwent desensitization with a protocol that included plasmapheresis and low-dose IVIG. During a 10-year period, 39 patients with CDC XM positive before desensitization underwent successful kidney transplantation at our institution with a 5-year patient survival of 86%. A high rate of AMR was reported. Nevertheless, mean serum creatinine was 1.7T1 mg/dL in functioning grafts at 5 years after transplantation. Sensitization to HLA antigens is a major barrier for kidney transplantation, and currently there are more than 25,000 patients on the U.S. waiting list who are classified as sensitized (1). Sensitized patients have less than 8% chance of obtaining a kidney from the waiting list when compared to non-sensitized patients. Consequently, there is a high mortality among sensitized patients that remain on dialysis waiting for the promised matched kidney (16). To increase their chances for kidney transplantation, they may enroll in kidney paired exchange programs or undergo desensitization.
FIGURE 1. Serum creatinine at different time points after transplantation across a CDC XM-positive barrier. Each dot represents a patient; solid line represents the mean. Creatinine of patients with a failed allograft were omitted.
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FIGURE 2. Kaplan-Meier curves of patient survival (A), death-censored survival (B), and uncensored graft survival (C) in CDC XM-positive patients who underwent transplantation after desensitization.
Though kidney paired donation is a less costly and a more effective strategy, overall match rates for sensitized patients are low (6, 17). Therefore, these two strategies should be viewed as complementary. Montgomery et al. have recently shown that 5-year patient survival is superior in desensitized patients (80.6%) compared to matched controls (51.5%) who remained on dialysis awaiting a transplant, proving the benefits of desensitization in this subpopulation (16). Our study reinforces these findings on patient survival and provides further detail on graft function, graft survival, rejection rates, and infectious and neoplastic complications.
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The goal of desensitization is to decrease the number of circulating donor-specific antibodies and potentially reduce their production to prevent hyperacute rejection and minimize the risk of AMR. Though desensitization has proven to be effective in preventing hyperacute rejection, the prevalence of AMR in the first 6 months after transplant remains high, ranging from 20% to 55% (4, 5, 10Y15). Among studies evaluating outcomes of sensitized patients, there is considerable heterogeneity in the protocols used, differences in histocompatibility testing techniques (CDC, flow, single-antigen bead) and characteristics of donor and recipient populations, making comparisons difficult. Despite these caveats, short-term follow-up studies with desensitized patients revealed a 1-year graft survival ranging from 89% to 94% and 2-year graft survival of 81%Y89% (11, 12, 14, 18Y20), comparable to our study (94% and 92%, respectively). In a retrospective study, Stegall et al. reported that desensitization with plasmapheresis, low-dose IVIG, and rituximab was more effective than high-dose IVIG in desensitizing patients (84% vs. 36%, respectively) and was associated with lower rates of AMR (37% vs. 80%) (10). Long-term outcomes of the same group revealed a graft survival of 82% and 64.9% in CDC XM-positive patients (n=41) at 1 and 5 years posttransplant (21), respectively, slightly inferior to our cohort observed survival. Recent reports have also suggested that plasmapheresis-based protocols seem to be more effective than high-dose IVIG in reducing DSA (8, 9), although in practice, plasmapheresis protocols are difficult to use in patients on the deceaseddonor list. The difference in rates of AMR between published plasmapheresis-based protocols seems to be mostly related to the strength of DSA of the cohort studied. This has been shown by Gloor et al. who compared sensitized patients with different DSA levels and confirmed that CDC XM-positive patients had the highest incidence of AMR (50%), followed by high FCXM (9300; 37%) and then low FCXM (G300; 30%) (18). Our study solely selected sensitized patients with a CDC-positive XM, which are recognized as the highest risk group to undergo desensitization, and consequently had a high rate of AMR (61%). The incidence of AMR is particularly high in the first month after transplant (Table 2); therefore, decreasing the rate of AMR remains a challenge for sensitized recipients. Short-term results by Stegall et al. indicate that eculizumab, a complement inhibitor that blocks C5, might decrease the rate of AMR in sensitized recipients, though duration of therapy and long-term consequences are still unknown (22).
TABLE 3. Positive CDC crossmatch result across different outcomes
AMR (n=24) TCMR (n=9) CR/TG (n=15) Graft failure (n=6) Death (n=7)
Positive T-cell XM
Positive B-cell XM
Positive T- and B-cell XM
5 4 3 1 2
7 2 5 2 1
12 3 7 3 4
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Proteasome inhibitors are also being tested as an option for direct targeting antibody-producing cells (23Y25). Recent reports suggested that donor-specific class II anti-HLA antibodies might be more deleterious to the allograft than class I anti-HLA antibodies (21). In our cohort, there was a trend towards worse graft outcomes in patients with either B-cell XM or both B- and T-positive CDC XM (Table 3). Moreover, we observed a similar trend of higher AMR in patients with DSAs against HLA DR. Nonetheless, it is clear from this study and others that the current standard of anti-HLA antibody characterization is neither sensitive nor specific enough to identify those sensitized patients who will have a worse outcome. Assessing the complement binding capacity of anti-HLA antibodies may be a supplementary assay that could help risk stratify this subgroup of patients and permit prospectively to better assess the effect of different treatment strategies (26). A concern with all desensitization protocols is the need for more immunosuppression, which might lead to higher rates of infection and cancer. Infection was the leading cause of death in our cohort (three patients). Moreover, BK nephropathy occurred in 10% of our patients, likely reflecting the higher degree of immunosuppression. In general, patients tolerated the treatment well with no major bleeding or adverse events from plasmapheresis, IVIG or rituximab, or both. The incidence of malignancies, a major concern in long-term immunosuppression, was also remarkably low with no cases of lymphoproliferative disorder and an expected rate of skin malignancies (27). Identifying patients who might be at risk of severe infectious complications is still a challenging task when selecting sensitized patients to proceed with desensitization. Our study has certain limitations, including the singlecenter design, the suitability of the protocol solely for live kidney donation, and the lack of a control group. Finding controls in a single center carries a high risk of selection bias because of difficulties in matching all potentially important variables; using databases would suffer other shortcomings such as difference in immunosuppression protocols and centers’ characteristics. Lastly, we did not include patients that at baseline had a negative T-cell CDC XM but positive T-cell FCXM. These patients are known to have a higher risk of AMR compared to T-cell FCXM-negative patients (4) and may also benefit from an intervention for alloantibody removal. In conclusion, desensitization with a plasmapheresisbased protocol is a reasonable alternative for sensitized patients who have an incompatible kidney donor and have not been successful finding a suitable paired kidney in an exchange program. Our long-term outcomes are reassuring in regards to patient survival and malignant complications but raise concerns about the high rates of AMR and potential infectious complications.
MATERIALS AND METHODS Study Population From January 2002 to December 2010, a total of 39 patients underwent live-donor kidney transplantation with HLA-incompatible donors defined by a positive CDC XM against donor T or B cells, or both. The higher risk of rejection and graft loss were specifically discussed with all living donors and recipients. In cases in which multiple potential donors were available, the donor with the lowest titer XM was generally chosen. Patients who did not
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convert to CDC XM negative did not proceed to kidney transplantation, unless only the B-cell CDC XM remained weakly positive and the delta fluorescence unit (DFU) was less than 20 by flow cytometry XM (FCXM). One patient of this cohort had both HLA and ABO incompatibility with the donor (12, 28). This study was approved by the institutional review board at Brigham and Women’s Hospital, Harvard Medical School.
Desensitization Protocol Plasmapheresis was performed thrice weekly in our dialysis unit using a hollow fiber plasma filter (Plasmaflo-AP06 Asahi Medical). The baseline XM titer was used to roughly estimate the number of sessions required to obtain a negative XM, as previously described (12). Typically, a plasma volume of 50 mL per kilogram of body weight was removed and replaced with 5% albumin (2/3) and 0.9% saline (1/3). Plasmapheresis was immediately followed by hemodialysis and 10 g of IVIG during the first 1 to 2 hours of the hemodialysis session. Following transplantation, routine plasmapheresis was performed for 2 weeks with a frequency dependent on the strength of baseline XM (from two to six sessions).
Immunosuppression Tacrolimus and mycophenolate mofetil (MMF) were initiated 5 to 7 days before transplant. Target trough concentrations of tacrolimus were 8 to 12 ng/mL. MMF starting dose was 2 g daily, adjusted according to patients’ tolerance. High-dose glucocorticoids were started at the day of the transplant and then tapered to 5 to 10 mg daily by 3 months posttransplant. Induction therapy consisted of either antithymocyte globulin rabbit (ATG) or basiliximab. The protocol was briefly modified during the 10-year period of the study because of concern for alloantibody rebound with ATG (29). Valganciclovir and sulfamethoxazole-trimethoprim were prescribed as antimicrobial prophylaxis for 6 months. In cases where multiple plasmapheresis sessions (and therefore depletion of clotting factors) were anticipated, 10 mg of vitamin K was given PO at the start of desensitization unless there was a contraindication. The majority of patients were treated with at least one dose of rituximab 375 mg/m2 on the night before transplantation.
Crossmatching and Donor-Specific Anti-HLA Antibody The CDC XM against the donor was the histocompatibility test used to determine the degree of sensitization and estimate the number of plasmapheresis sessions required before transplantation (12). Briefly, T-cell crossmatches were performed by the modified Amos method (using three washes) and an antihuman globulin method to increase the sensitivity for antibody detection. B-cell crossmatches were performed by the modified Amos method (using three washes). Auto-crossmatching was performed for all donors and recipients to exclude autoantibody as a cause of any positive XM. Recipient serum was also treated with dithioerythritol to exclude IgM antibodies when appropriate. Because of the limited specificity of the B-cell CDC XM, flow panel reactive antibody testing was also used to confirm the presence of class II antibodies in the serum. Pretransplant flow cytometry XM (FCXM) was not routinely performed in all patients, as the primary goal of the protocol was to achieve a negative CDC XMVand not a negative FCXMVpretransplant. Sera were analyzed for DSA by Luminex assay single-antigen screening. As this analysis was not available at the beginning of the study, DSA data was available for 29 of the 39 patients in this cohort. When single-antigen screening was performed, a threshold of 1500 median fluorescence intensity was used to determine DSA positivity.
Posttransplant Course Outcomes measured included patient survival, graft survival, renal function (serum creatinine), rates of cellular AMR, rates of infection, and malignancy. Protocol biopsies were not routinely performed, though the threshold to perform for-cause biopsies was low (typically an increase in serum creatinine Q0.3 mg/dL). Staining for C4d by immunofluorescence was routinely performed. Renal biopsies were graded according to the Banff classification (30). Acute and borderline cellular rejections were treated with pulse methylprednisolone 500 mg IV daily for 3 days. Acute cellular rejection resistant to such therapy was treated ATG. Acute AMR was defined by
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at least two of the three following characteristics: positive FCXM against the donor, histologic changes consistent with AMR, and positive C4d staining in peritubular or glomerular capillaries (31). The FCXMVas opposed to CDC XMVwas used posttransplant for anti-HLA antibody monitoring. Acute AMR was treated with pulse methylprednisolone 500 mg IV daily for 3 days and plasmapheresis with IVIG every 24 to 48 hours until the FCXM was negative against donor T and B cells or five sessions of plasmapheresis had been completed (whichever came first). Repeat FCXMTbiopsy would then guide further therapy. In cases of severe AMR not responsive to pheresis, an additional dose of rituximab 375 mg/m2 was administered. Rituximab was introduced into the protocol in an attempt to reduce the rate of AMR observed in the beginning of the cohort.
Statistical Analysis
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11. 12. 13. 14. 15.
Kaplan-Meier plots were used to estimate patient and allograft survival. Censoring was at time of death. One-way ANOVA and Student t test were used to test for quantitative outcomes. Chi-square and Fisher exact tests were used to test for categorical outcomes.
16.
ACKNOWLEDGMENTS The authors would like to thank Brian Smith for his help in performing some of the anti-HLA antibody testing, and Sarah Conte and Christine Dyer for the coordination of this clinical research project.
18.
17.
19. 20. 21.
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