American Journal of Transplantation 2016; 16: 1503–1515 Wiley Periodicals Inc.
© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13606
Renal Transplantation With Final Allocation Based on the Virtual Crossmatch C. P. Johnson1,*, J. J. Schiller2, Y. R. Zhu1, S. Hariharan3,‡, A. M. Roza1, D. C. Cronin1, B. D. Shames1,† and T. M. Ellis2,# 1
Department of Surgery (Division of Transplantation), Medical College of Wisconsin, Milwaukee, WI 2 Histocompatibility and Immunogenetics, Blood Center of Wisconsin, Milwaukee, WI 3 Department of Medicine (Division of Nephrology), Medical College of Wisconsin, Milwaukee, WI † Present address: University of Connecticut Health Center, Farmington, CT # Present address: University of Wisconsin-Madison, Madison, WI ‡ Present address: University of Pittsburgh Medical Center, Pittsburgh, PA *Corresponding author: Christopher P. Johnson, [email protected] Solid phase immunoassays (SPI) are now routinely used to detect HLA antibodies. However, the flow cytometric crossmatch (FCXM) remains the established method for assessing final donor–recipient compatibility. Since 2005 we have followed a protocol whereby the final allocation decision for renal transplantation is based on SPI (not the FCXM). Here we report long-term graft outcomes for 508 consecutive kidney transplants using this protocol. All recipients were negative for donor-specific antibody by SPI. Primary outcomes are graft survival and incidence of acute rejection within 1 year (AR 80% (20% vs. 4.8%, p = 0.001). Despite these differences, 5-year actual graft survival rates are 87% and 84%, respectively. AR 80% or where multiple reactivities prevented accurate assignment of individual specificities, single antigen bead (SAB) assays were used (LabScreen; One Lambda Inc.). After July 2009 (n = 37 transplants), all patients were screened for HLA antibodies at initial workup using Flow PRA and SABs and subsequent tests were performed using either LabScreen Mixed for 0% PRA patients or Flow PRA plus SAB for patients with PRA > 0%. HLA antibody specificities were assigned using multiple criteria consistent with consensus guidelines and included patterns of epitope specificity and cross-reactivity, background levels, and individual bead performance (12,13). Mean fluorescence intensity (MFI) values for
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positive antibodies in SAB assays were >1000, with few exceptions. Exceptions included cases where antibodies may be distributed across multiple beads containing the same epitope (e.g. Bw4) or where patterns of epitope reactivity supported the presence of antibody reactivity. The VXM was defined as negative if there was an absence of detectable DSA based on results of the assay for antibody identification in use at the time of transplant. A positive VXM was defined as the presence or possible presence of DSA, the latter referring to cases where the presence or absence of DSA could not be established based on epitope reactivity patterns, MFI, assay background, and patient allosensitization history. HLAallele-specific antibodies were considered in the VXM whenever the level of resolution of HLA alleles and antibodies allowed. Historically positive donor-directed HLA antibodies (but currently negative) detected by SPI were not considered to constitute a positive VXM.
Flow cytometric crossmatches Three-color FCXM were performed as previously described using pronase-treated fresh lymphocytes collected from peripheral blood, lymph node, or spleen using a current serum sample, collected within 30 days of transplant (14). The cutoff for a positive FCXM (T or B cell) was defined as a median channel shift (MCS) that was ≥3 standard deviations of the mean MCS obtained when at least 20 different target cells were stained with normal human serum and analyzed.
VXM protocol for kidney allocation Deceased donor kidneys were allocated according to the United Network for Organ Sharing (UNOS) allocation system. As a starting point, the top 10 candidates based on the match run underwent a VXM using the most recent serum sample. If the VXM was positive (presence of any DSA) the recipient was excluded. If the VXM was negative, an updated VXM using current serum and final FCXM were performed. Candidates with high levels of anti-DPB1 antibodies were not considered for transplant if the donor HLA-DPB1 typing was unknown. Potential antibodies against donor HLA-Cw, -DPA1, and -DQA1 mismatches were not considered in the VXM for patients transplanted between January 2005 through December 2007 (n = 325 transplants). From January 2008 through June 2009, potential antibodies to DPA1 and DQA1 were not considered (n = 146). As of July 2009, there were no remaining gaps for HLA antibody detection (n = 37). A positive FCXM was not considered a contraindication for transplant (regardless of crossmatch strength as measured by MCS) if, based on results of HLA antibody specificity testing, the positive crossmatch was interpreted as not due to donor-directed HLA antibodies. For living donor transplantation, the same rationale was applied. If a recipient with a living donor was found to have a positive VXM, he or she was still potentially eligible for transplant (with desensitization or other strategies) but is not included in the current analysis. Thus, all transplant recipients included in this study were either VXM /FCXM or VXM /FCXM+.
Immunosuppressive management and measurement of transplant outcomes Nearly all transplant recipients received induction therapy (six recipients did not). In general, recipients with expected immediate function and PRA < 20% received IL-2 receptor antagonists (basiliximab or daclizumab, n = 229). Those with delayed function, PRA > 20% (or other immunologic risk factors) received lymphocyte-depleting agents: thymoglobulin (4–6 mg/kg total dose, n = 190) or Campath (during 2005–2006, 30 mg, n = 82). Maintenance immunosuppression consisted of triple therapy: tacrolimus, mycophenolate mofetil, and corticosteroids (prednisone tapered to 5 mg daily by 1 month). The definition of rejection for this study is biopsy-proven rejection (borderline changes were counted as rejection episodes). Classification and treatment of rejection was
American Journal of Transplantation 2016; 16: 1503–1515
Renal Transplantation by Virtual Crossmatch according to Banff 2007 criteria (15) and institutional protocols. Rejection episodes were categorized as acute rejection within 1 year of transplant (AR < 1 year), which includes any rejection episode (cellular or antibody mediated) and AMR < 1 year. Ascertainment of etiology for graft loss was determined by chart review (CPJ) in a blinded fashion with respect to crossmatch status. Graft losses were categorized as rejection, death with a functioning graft, and all others. Survival data were validated using data obtained from Scientific Registry for Transplant Recipients (SRTR) analysis. All recipients in this study were followed a minimum of 5 years (unless graft loss or death occurred sooner). The primary endpoints for this study are comparison of all cause and death-censored graft survival between FCXM and FCXM+ recipients and incidence of AR < 1 year.
Statistical analysis Categorical data were compared using chi-squared (or Fisher exact) tests. Continuous variables were compared using t-tests or nonparametric tests (rank sum) if data were not normally distributed. Patient and graft survival (all cause and death censored) were compared using Kaplan–Meier curves (log rank tests). Cox proportional hazards models were used to assess the impact of specific variables on graft survival (16). Variables with a p value ≤0.2 in univariate analysis were included for multivariate analysis. A p value of 0%), stratified by crossmatch results are listed in Table 1. Fifty-four of 508 recipients (11%) were FCXM positive. Of note, FCXM+ recipients exhibited a lower percentage of living donors and higher percentages of female recipients, sensitized and highly sensitized recipients (defined as PRA > 80%), retransplants, and longer duration of dialysis (prior to transplant). FCXM+ recipients were more likely to receive lymphocyte-depleting agents (thymoglobulin or Campath) for induction, compared to FCXM recipients. Deceased donor characteristics were similar for both groups. Long-term outcomes for FCXM+ versus FCXM recipients The median follow-up for the 508 recipients is 7.1 years (range 5.0–10.0 years). Five-year actual graft survival is 85%. The 3-year graft survival is 91%, which is within one percentage point of the 3-year graft outcomes reported by SRTR for the two consecutive cohorts transplanted between January 1, 2005 and December 31, 2009 (January 2005–June 2007 and July 2007–December 2009); graft survival 91.6% and 89.6%; with expected outcomes of 85.6% and 86.5% (17,18). Five-year actual graft survival rates for FCXM+ and FCXM recipients are 87% and 84%, respectively. Figure 1 demonstrates all-cause and death-censored graft survival for all recipients according to crossmatch status. Figure 2 illustrates all-cause and death-censored graft American Journal of Transplantation 2016; 16: 1503–1515
survival for sensitized recipients only (n = 118). There are no differences in overall graft or death-censored graft survival for FCXM+ versus FCXM recipients. Unsensitized recipients (0% PRA) did not show any differences in graft survival by crossmatch status either (5-year graft survival 88% for FCXM+ vs. 84% for FCXM ; data not shown). Figure 3 illustrates all-cause graft survival, specifically for sensitized recipients with a calculated PRA (cPRA) > 80 at the time of transplant. There is no difference between FCXM+ and FCXM recipients. Outcomes according to specific crossmatch results Of the 54 patients with a positive flow crossmatch, 14 were T cell–positive, B cell–negative, 20 were T cell– negative, B cell–positive, and 20 were T and B cell–positive. Only two of the 54 FCXM+ recipients had historical DSA as detected by SPI and were VXM /FCXM+ at final crossmatch. The relationship between specific crossmatch status, incidence of biopsy-proven rejection within the first year, and 5-year graft outcomes is depicted in Table 2. Long-term graft survival rates are shown in Figure 4. There were no significant differences in the incidence of rejection episodes within 1 year for FCXM+ versus FCXM recipients (13% vs. 12%). AMR occurred in 2/54 (4%) of FCXM+ recipients and 7/454 (2%) of FCXM recipients (p = 0.55). The two episodes of AMR < 1 year in FCXM+ recipients occurred at 2 months and 6 months. T cell–positive, B cell–negative FCXM+ recipients exhibited higher rates of AR < 1 year and AMR < 1 year (28% and 7%) versus FCXM recipients (12% and 2%), but these did not reach statistical significance (p = 0.15 for AR < 1 year). Sensitized T cell– positive, B cell–negative FCXM+ recipients also showed higher rates of AR < 1 year and AMR < 1 year (33% and 11%), but these did not reach statistical significance (p = 0.07 for AR < 1 year). The median MCS for positive T cell FCXMs was 75 (range 41–236). The median MCS for positive B cell FCXMs was 109 (range 53–282). Only two recipients had a T cell MCS > 150 and 3 recipients had a B cell MCS > 250. Subgroups of “strong” T and B cell FCXM+ recipients were created using arbitrary cutpoints of T cell MCS >80 and B cell MCS >120. The incidence of graft loss due to rejection < 5 years and overall graft survival rates at 5 years (all cause) were not significantly different for these subgroups of FCXM+ recipients versus FCXM recipients. For highly sensitized recipients (PRA > 80%), there were no differences in rejection rates or graft outcomes between FCXM+ and FCXM recipients. Effect of induction therapy on graft outcomes FCXM+ recipients received induction therapy with lymphocyte-depleting agents (thymoglobulin or Campath) more often than did FCXM recipients (83% vs. 50%, p < 0.001). Therefore, we performed additional analysis of graft outcomes stratified by induction agents (Table 3). For the overall population of recipients (n = 508), there were no significant effects of induction 1505
Johnson et al Table 1: Patient characteristics for positive and negative flow cytometric crossmatch recipients All recipients (n = 508) Variable Gender (female) Race (% African American) Recipient age: median (25%, 75%)1 Recipient age > 60 years Donor age: median (25%, 75%) Donor ECD (vs. SCD)2 Donor DCD (vs. brain death)2 Cold ischemia time: median (25%, 75%)2 Cold ischemia time >20 h Recipients with diabetes Living donor transplant Dialysis duration (months); median (25%, 75%) Dialysis >2 years % of recipients sensitized (PRA > 0) % of recipients with PRA > 80 0 mismatch transplants Retransplants Recipient treated with lymphocyte depletion3
Sensitized recipients (n = 118)
FCXM negative (n = 54)
FCXM positive (n = 54)
p value
FCXM negative (n = 84)
FCXM positive (n = 34)
p value
38% 27%
52% 39%
0.07 0.10
67% 29%
62% 38%
0.80 0.42
52 (41, 59.3)
50 (44, 56.2)
0.31
47 (38, 58)
48 (41, 54)
0.97
92/454 (20%)
8/54 (15%)
0.44
12/84 (14%)
3/34 (9%)
0.62
44 (33, 52)
45 (30, 54)
0.79
42 (32, 58)
43 (31, 53)
0.98
43/224 (19%) 46/267 (17%)
8/39 (21%) 7/39 (17%)
0.65 0.90
10/62 (15%) 7/62 (11%)
4/27 (15%) 5/27 (19%)
1.00 0.50
15.0 (12, 19)
15.3 (13, 21)
0.24
16.0 (12, 21)
15.6 (13, 21)
0.53
45/267 (17%)
11/39 (28%)
0.14
15/57 (26%)
7/28 (25%)
0.97
149/454 (33%)
19/54 (35%)
0.84
23/84 (27%)
13/34 (38%)
0.35
177/454 (39%) 13.5 (0, 38)
13/54 (24%) 31 (6.3, 49)
0.05 0.01
22/84 (26%) 22 (3, 50)
7/34 (21%) 33 (14, 50)
0.68 0.33
179/454 (39%) 84/454 (19%)
32/54 (59%) 34/54 (63%)
0.008 120 only2 T cell MCS > 80 and B cell MCS > 120 For sensitized recipients, n = 118 T and B cell negative Tor B cell positive T cell neg. B cell pos. T cell pos., B cell neg. T and B cell pos. T cell MCS > 80 B cell MCS > 120 T cell MCS > 80 and B cell MCS > 120 PRA > 80% and FCXM PRA > 80% and FCXM+
AMR < l year
Grafts lost to rejection 0.3. FCXM, flow cytometric crossmatch; MCS, median channel shift; AR < 1 year, acute rejection episode within 1 year of transplant (any; includes acute cellular and/or antibody-mediated rejection); AMR < l year, antibody-mediated rejection episode within 1 year of transplant; PRA, panel-reactive antibody. 1 Actual graft survival; all recipients were followed a minimum of 5 years; all p values for graft survival >0.3. 2 T cell FCXM with MSC > 80, or any B cell FCXM with MCS > 120.
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American Journal of Transplantation 2016; 16: 1503–1515
Renal Transplantation by Virtual Crossmatch
Discussion
1.0
Survival
0.8
0.6 Negative B and T cell XM (n = 454) T cell positive, B cell negative XM (n = 14) T cell negative, B cell pos XM (n = 20)
0.4
T and B cell positive XM (n = 20)
p = 0.85
0.2
0.0 0
2
4
6
8
Years
Figure 4: All-cause graft survival according to specific T and B cell flow crossmatch status. All recipients were virtual crossmatch negative. XM, crossmatch.
group (and placed in the FCXM group), the 5-year graft survival for FCXM+ recipients would be 42/49 (86%) versus 387/458 (84%) for FCXM recipients. Four of the 54 FCXM+ recipients had a diagnosis of systemic lupus erythematosus at time of transplant. All were sensitized but none had auto-antibodies that interfered with crossmatch interpretation. Univariate and multivariate analysis of graft outcomes The results of univariate and multivariate analyses, using Cox proportional hazards models, to identify variables associated with graft loss and death-censored graft loss for all recipients (n = 508) and in sensitized recipients (n = 118) are listed in Tables 5 and 6, respectively. For all-cause graft loss in all recipients, the following variables were significant in multivariate analysis: deceased donor (vs. living), diabetes, recipient age >60, and recipient race African American (AA) (vs. non-AA). For sensitized recipients, recipient gender (female) and induction agent (lymphocyte depletion) were significant. Additional analysis, however, revealed that the population of sensitized recipients receiving IL2-R antagonists was enriched for age >60 (30% age > 60 compared with 5% who received lymphocyte-depleting agent; p = 0.003). Death with a functioning graft was responsible for 80% of graft losses in sensitized recipients treated with IL2-R antagonists. In death-censored analysis for graft loss, only donor type and recipient age (age >60 protective) were associated with graft loss. A positive FCXM did not impact graft survival in any univariate or multivariate model.
American Journal of Transplantation 2016; 16: 1503–1515
To our knowledge, this is the first study to examine longterm outcomes for kidney transplantation, when solid phase assays are used as the definitive test for assessing donor and recipient compatibility. FCXM results were used primarily to help interpret the results of solid phase testing. Using our VXM protocol, recipients who were FCXM+ but VXM were considered to have a “positive crossmatch, not due to donor-specific HLA antibodies” and were allowed to proceed to transplantation (regardless of crossmatch strength as determined by MCS). No recipient in this study underwent desensitization to achieve a negative VXM. The primary aim of this study was to monitor long-term graft outcomes for VXM / FCXM+ renal allograft recipients (which comprised 11% of our recipient population). It is worth noting that this group itself exhibits many risk factors for shortened graft survival (Table 1). Therefore, an unadjusted analysis of graft survival creates an unfavorable bias against the FCXM+ group. Despite this, long-term graft survival rates for VXM /FCXM+ recipients were not inferior to those of VXM /FCXM recipients (Figure 1). Of special interest in this study are the outcomes for sensitized recipients (PRA > 0%) who are VXM / FCXM+. We therefore performed separate analyses for this group, as well as highly sensitized recipients (PRA > 80%). We observed low rates of acute rejection (including AMR within the first year), as well as low rates of graft loss from rejection 60 years (vs. 7% receiving lymphocyte depletion agent, p = 0.003). 3 p = 0.2 for AR < l year, lymphocyte depletion versus IL2-R antagonist. 4 p = 0.11 for graft loss due to rejection, lymphocyte depletion versus IL-2R antagonist. 5 p = 0.008 for graft loss in sensitized recipients, thymoglobulin versus IL2-R antagonist. 6 p = 0.05 for AR < l year and p = 0.02 for graft loss in FCXM+, sensitized recipients, treated with thymoglobulin versus Campath. 7 p = 0.04 for graft loss in sensitized recipients treated with Campath (FCXM+ vs. FCXM ). 8 p = 0.006 for graft loss in FCXM , sensitized recipients treated with thymoglobulin versus IL2-R antagonist.
the subgroup of FCXM+, sensitized retransplants, which appears to have experienced worse outcomes compared to FCXM , sensitized retransplants. In our study, these groups comprise very small subsets of recipients, so proper risk adjustment for outcomes was not possible. Of note, the group of FCXM , sensitized retransplants had unexpectedly high 5-year graft survival rates (100%), which further distorts comparison with FCXM+ recipients. Sensitized retransplants would be an important group of recipients to examine carefully in future studies. A positive crossmatch in this group of recipients (without DSA by solid phase assays) could indicate pre-existing sensitization to minor histocompatibility antigens, which could very well be important in this transplantation scenario. A VXM /FCXM+ result would not reflect sensitization to various non-HLA targets such as MHC class I–related chain A antigens or endothelium-restricted antigens (angiotensin II type 1 receptor and vimentin), since these antigens are not expressed on the surface of lymphocytes used for the FCXM (23). 1510
It should be noted that the virtual crossmatches in the early part of our study were based on incomplete recipient and donor HLA typing and did not account for donorspecific DQA1, DPB1, or DPA1 antibodies. Unfortunately, more detailed retrospective analyses to further characterize VXM /FCXM+ cases could not be performed as most early samples were no longer available for testing. Additionally, heavy reliance on low-resolution phenotypic assays to identify antibody specificities (i.e. LabScreen PRA) limited our ability to resolve antibodies against donor HLA-Cw. The impact of these limitations would be the inaccurate categorization of crossmatch results as VXM /FCXM+ when, in fact, the positive crossmatch might have resulted from antibodies against these untyped loci. This is consistent with what appears to be a higher incidence of AR < 1 year and AMR < 1 year in our sensitized FCXM+ recipients transplanted during the 2005–2007 period, compared to 2008–2009. To address the impact of incomplete donor HLA typing on the accuracy of virtual crossmatching, the UNOS has American Journal of Transplantation 2016; 16: 1503–1515
Renal Transplantation by Virtual Crossmatch Table 4: Summary of HLA antibody detection methodology, potential undetected DSA, and outcomes for FCXM+ recipients (n = 54)
Number of transplants Sensitized recipients (PRA > 0) Number of positive crossmatches (%) Potential undetected HLA DSA Methodology for HLA DSA detection Antibody screen Antibody specificity Recipients with potential undetected DSA1 Cw (in recipients with class I PRA > 0) DPB1, DPA1 and/or DQA1 (in recipients with class II PRA > 0) Total AR < 1 year in recipients with potential undetected DSA AMR < 1 year in recipients with potential undetected DSA 5-year graft survival in recipients with potential undetected DSA
January 2005– December 2007
January 2008–June 2009
July 2009–December 2009
325 71 (22%) 22/325 (7%)
146 35 (24%) 26/146 (18%)
37 12 (32%) 6/37 (16%)
Cw, DPB1, DPA1 and DQA1
DPA1 and DQA1
None
Flow PRA Beads
Flow PRA Beads
LabScreen PRA Beads 1
LabScreen PRA Beads or Luminex SAB2
Flow PRA and LabScreen Mixed Beads Luminex SAB3
18/22 8/22
0/26 8/26
0/6 0/6
18/22 4/18 (22%)4
8/26 2/8
0/6 0/6
2/18 (11%)4
0/8
0/6
13/18 (72%)
6/8 (75%)
0/6
DSA, donor-specific antibody; FCXM+, positive flow cytometric crossmatch; PRA, panel-reactive antibody; AR < 1 year, biopsy proven acute rejection episode (cellular or antibody mediated) < 1 year from transplant date; AMR < 1 year, biopsy proven antibody mediated rejection < 1 year from date of transplant; SAB, single antigen beads. 1 HLA Cw antibodies not detectable due to inability to resolve using Flow PRA Beads; HLA DPB1, DPA1, and DQA1 antibodies not detectable due to lack of donor typing for these loci. Recipients with PRA = 0, using flowPRA phenotypic beads are considered not at risk for undetected DSAs at time of transplant. Recipients with PRA > 0, using flowPRA phenotypic beads are considered at risk for undetected DSAs at time of transplant. 2 Luminex testing was performed in all patients with PRAs > 80% or if specificities could not be resolved using LabScreen PRA beads. 3 All patients initially screened using Flow PRA and Luminex SAB; subsequent samples on 0% PRA patients were screened using LabScreen Mixed beads. 4 For AR < 1 year, p = 0.26 versus sensitized, FCXM recipients; for AMR < 1 year, p = 0.03 versus sensitized, FCXM recipients.
expanded donor typing requirements, although surprisingly these requirements still do not include HLA-DQA1, DPB1, and DPA1. It is notable that the frequency of VXM /FCXM+ cases did not decrease with the implementation of extended donor typing and higher-resolution antibody testing, as one might expect. The frequency actually increased—an observation that might relate to the implementation in 2008 of a new flow cytometer with increased sensitivity. We examined the effect of different induction agents used during the study on graft outcomes. Unfortunately, there is significant selection bias involved in administering induction therapy. Lymphocyte-depleting agents are generally given to sensitized recipients and other subsets of recipients considered to be at increased risk of rejection. In our study, sensitized recipients who received thymoglobulin or Campath actually experienced higher rates of rejection and graft loss due to rejection, compared to American Journal of Transplantation 2016; 16: 1503–1515
recipients receiving IL2-R antagonists. The latter group, perhaps as expected since it was enriched with older recipients, experienced lower rates of rejection and higher rates of death with a functioning graft (compared to recipients receiving lymphocyte depletion). Since only five of 34 sensitized, FCXM+ recipients received IL2-R antagonists, it is not possible to make any definitive statements about use of induction agents for these patients. At present our practice is in general to use thymoglobulin for sensitized VXM /FCXM+ recipients, reserving IL2-R antagonists for unsensitized VXM / FCXM+ recipients. Of note, FCXM+ recipients who received Campath exhibited higher rates of AMR and graft loss than those who received thymoglobulin. This is consistent with previous reports in the literature (24,25). While our findings support the use of virtual crossmatching over the flow crossmatch as the primary measure of recipient/donor compatibility, there remain significant 1511
Johnson et al Table 5: Univariate and multivariate analysis for variables associated with all-cause graft survival Multivariate analysis1
Univariate analysis Risk factor Recipient age: >60 (vs. 2 years (vs. 60 (vs. 2 years (vs. 80 (vs.
© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13606
Renal Transplantation With Final Allocation Based on the Virtual Crossmatch C. P. Johnson1,*, J. J. Schiller2, Y. R. Zhu1, S. Hariharan3,‡, A. M. Roza1, D. C. Cronin1, B. D. Shames1,† and T. M. Ellis2,# 1
Department of Surgery (Division of Transplantation), Medical College of Wisconsin, Milwaukee, WI 2 Histocompatibility and Immunogenetics, Blood Center of Wisconsin, Milwaukee, WI 3 Department of Medicine (Division of Nephrology), Medical College of Wisconsin, Milwaukee, WI † Present address: University of Connecticut Health Center, Farmington, CT # Present address: University of Wisconsin-Madison, Madison, WI ‡ Present address: University of Pittsburgh Medical Center, Pittsburgh, PA *Corresponding author: Christopher P. Johnson, [email protected] Solid phase immunoassays (SPI) are now routinely used to detect HLA antibodies. However, the flow cytometric crossmatch (FCXM) remains the established method for assessing final donor–recipient compatibility. Since 2005 we have followed a protocol whereby the final allocation decision for renal transplantation is based on SPI (not the FCXM). Here we report long-term graft outcomes for 508 consecutive kidney transplants using this protocol. All recipients were negative for donor-specific antibody by SPI. Primary outcomes are graft survival and incidence of acute rejection within 1 year (AR 80% (20% vs. 4.8%, p = 0.001). Despite these differences, 5-year actual graft survival rates are 87% and 84%, respectively. AR 80% or where multiple reactivities prevented accurate assignment of individual specificities, single antigen bead (SAB) assays were used (LabScreen; One Lambda Inc.). After July 2009 (n = 37 transplants), all patients were screened for HLA antibodies at initial workup using Flow PRA and SABs and subsequent tests were performed using either LabScreen Mixed for 0% PRA patients or Flow PRA plus SAB for patients with PRA > 0%. HLA antibody specificities were assigned using multiple criteria consistent with consensus guidelines and included patterns of epitope specificity and cross-reactivity, background levels, and individual bead performance (12,13). Mean fluorescence intensity (MFI) values for
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positive antibodies in SAB assays were >1000, with few exceptions. Exceptions included cases where antibodies may be distributed across multiple beads containing the same epitope (e.g. Bw4) or where patterns of epitope reactivity supported the presence of antibody reactivity. The VXM was defined as negative if there was an absence of detectable DSA based on results of the assay for antibody identification in use at the time of transplant. A positive VXM was defined as the presence or possible presence of DSA, the latter referring to cases where the presence or absence of DSA could not be established based on epitope reactivity patterns, MFI, assay background, and patient allosensitization history. HLAallele-specific antibodies were considered in the VXM whenever the level of resolution of HLA alleles and antibodies allowed. Historically positive donor-directed HLA antibodies (but currently negative) detected by SPI were not considered to constitute a positive VXM.
Flow cytometric crossmatches Three-color FCXM were performed as previously described using pronase-treated fresh lymphocytes collected from peripheral blood, lymph node, or spleen using a current serum sample, collected within 30 days of transplant (14). The cutoff for a positive FCXM (T or B cell) was defined as a median channel shift (MCS) that was ≥3 standard deviations of the mean MCS obtained when at least 20 different target cells were stained with normal human serum and analyzed.
VXM protocol for kidney allocation Deceased donor kidneys were allocated according to the United Network for Organ Sharing (UNOS) allocation system. As a starting point, the top 10 candidates based on the match run underwent a VXM using the most recent serum sample. If the VXM was positive (presence of any DSA) the recipient was excluded. If the VXM was negative, an updated VXM using current serum and final FCXM were performed. Candidates with high levels of anti-DPB1 antibodies were not considered for transplant if the donor HLA-DPB1 typing was unknown. Potential antibodies against donor HLA-Cw, -DPA1, and -DQA1 mismatches were not considered in the VXM for patients transplanted between January 2005 through December 2007 (n = 325 transplants). From January 2008 through June 2009, potential antibodies to DPA1 and DQA1 were not considered (n = 146). As of July 2009, there were no remaining gaps for HLA antibody detection (n = 37). A positive FCXM was not considered a contraindication for transplant (regardless of crossmatch strength as measured by MCS) if, based on results of HLA antibody specificity testing, the positive crossmatch was interpreted as not due to donor-directed HLA antibodies. For living donor transplantation, the same rationale was applied. If a recipient with a living donor was found to have a positive VXM, he or she was still potentially eligible for transplant (with desensitization or other strategies) but is not included in the current analysis. Thus, all transplant recipients included in this study were either VXM /FCXM or VXM /FCXM+.
Immunosuppressive management and measurement of transplant outcomes Nearly all transplant recipients received induction therapy (six recipients did not). In general, recipients with expected immediate function and PRA < 20% received IL-2 receptor antagonists (basiliximab or daclizumab, n = 229). Those with delayed function, PRA > 20% (or other immunologic risk factors) received lymphocyte-depleting agents: thymoglobulin (4–6 mg/kg total dose, n = 190) or Campath (during 2005–2006, 30 mg, n = 82). Maintenance immunosuppression consisted of triple therapy: tacrolimus, mycophenolate mofetil, and corticosteroids (prednisone tapered to 5 mg daily by 1 month). The definition of rejection for this study is biopsy-proven rejection (borderline changes were counted as rejection episodes). Classification and treatment of rejection was
American Journal of Transplantation 2016; 16: 1503–1515
Renal Transplantation by Virtual Crossmatch according to Banff 2007 criteria (15) and institutional protocols. Rejection episodes were categorized as acute rejection within 1 year of transplant (AR < 1 year), which includes any rejection episode (cellular or antibody mediated) and AMR < 1 year. Ascertainment of etiology for graft loss was determined by chart review (CPJ) in a blinded fashion with respect to crossmatch status. Graft losses were categorized as rejection, death with a functioning graft, and all others. Survival data were validated using data obtained from Scientific Registry for Transplant Recipients (SRTR) analysis. All recipients in this study were followed a minimum of 5 years (unless graft loss or death occurred sooner). The primary endpoints for this study are comparison of all cause and death-censored graft survival between FCXM and FCXM+ recipients and incidence of AR < 1 year.
Statistical analysis Categorical data were compared using chi-squared (or Fisher exact) tests. Continuous variables were compared using t-tests or nonparametric tests (rank sum) if data were not normally distributed. Patient and graft survival (all cause and death censored) were compared using Kaplan–Meier curves (log rank tests). Cox proportional hazards models were used to assess the impact of specific variables on graft survival (16). Variables with a p value ≤0.2 in univariate analysis were included for multivariate analysis. A p value of 0%), stratified by crossmatch results are listed in Table 1. Fifty-four of 508 recipients (11%) were FCXM positive. Of note, FCXM+ recipients exhibited a lower percentage of living donors and higher percentages of female recipients, sensitized and highly sensitized recipients (defined as PRA > 80%), retransplants, and longer duration of dialysis (prior to transplant). FCXM+ recipients were more likely to receive lymphocyte-depleting agents (thymoglobulin or Campath) for induction, compared to FCXM recipients. Deceased donor characteristics were similar for both groups. Long-term outcomes for FCXM+ versus FCXM recipients The median follow-up for the 508 recipients is 7.1 years (range 5.0–10.0 years). Five-year actual graft survival is 85%. The 3-year graft survival is 91%, which is within one percentage point of the 3-year graft outcomes reported by SRTR for the two consecutive cohorts transplanted between January 1, 2005 and December 31, 2009 (January 2005–June 2007 and July 2007–December 2009); graft survival 91.6% and 89.6%; with expected outcomes of 85.6% and 86.5% (17,18). Five-year actual graft survival rates for FCXM+ and FCXM recipients are 87% and 84%, respectively. Figure 1 demonstrates all-cause and death-censored graft survival for all recipients according to crossmatch status. Figure 2 illustrates all-cause and death-censored graft American Journal of Transplantation 2016; 16: 1503–1515
survival for sensitized recipients only (n = 118). There are no differences in overall graft or death-censored graft survival for FCXM+ versus FCXM recipients. Unsensitized recipients (0% PRA) did not show any differences in graft survival by crossmatch status either (5-year graft survival 88% for FCXM+ vs. 84% for FCXM ; data not shown). Figure 3 illustrates all-cause graft survival, specifically for sensitized recipients with a calculated PRA (cPRA) > 80 at the time of transplant. There is no difference between FCXM+ and FCXM recipients. Outcomes according to specific crossmatch results Of the 54 patients with a positive flow crossmatch, 14 were T cell–positive, B cell–negative, 20 were T cell– negative, B cell–positive, and 20 were T and B cell–positive. Only two of the 54 FCXM+ recipients had historical DSA as detected by SPI and were VXM /FCXM+ at final crossmatch. The relationship between specific crossmatch status, incidence of biopsy-proven rejection within the first year, and 5-year graft outcomes is depicted in Table 2. Long-term graft survival rates are shown in Figure 4. There were no significant differences in the incidence of rejection episodes within 1 year for FCXM+ versus FCXM recipients (13% vs. 12%). AMR occurred in 2/54 (4%) of FCXM+ recipients and 7/454 (2%) of FCXM recipients (p = 0.55). The two episodes of AMR < 1 year in FCXM+ recipients occurred at 2 months and 6 months. T cell–positive, B cell–negative FCXM+ recipients exhibited higher rates of AR < 1 year and AMR < 1 year (28% and 7%) versus FCXM recipients (12% and 2%), but these did not reach statistical significance (p = 0.15 for AR < 1 year). Sensitized T cell– positive, B cell–negative FCXM+ recipients also showed higher rates of AR < 1 year and AMR < 1 year (33% and 11%), but these did not reach statistical significance (p = 0.07 for AR < 1 year). The median MCS for positive T cell FCXMs was 75 (range 41–236). The median MCS for positive B cell FCXMs was 109 (range 53–282). Only two recipients had a T cell MCS > 150 and 3 recipients had a B cell MCS > 250. Subgroups of “strong” T and B cell FCXM+ recipients were created using arbitrary cutpoints of T cell MCS >80 and B cell MCS >120. The incidence of graft loss due to rejection < 5 years and overall graft survival rates at 5 years (all cause) were not significantly different for these subgroups of FCXM+ recipients versus FCXM recipients. For highly sensitized recipients (PRA > 80%), there were no differences in rejection rates or graft outcomes between FCXM+ and FCXM recipients. Effect of induction therapy on graft outcomes FCXM+ recipients received induction therapy with lymphocyte-depleting agents (thymoglobulin or Campath) more often than did FCXM recipients (83% vs. 50%, p < 0.001). Therefore, we performed additional analysis of graft outcomes stratified by induction agents (Table 3). For the overall population of recipients (n = 508), there were no significant effects of induction 1505
Johnson et al Table 1: Patient characteristics for positive and negative flow cytometric crossmatch recipients All recipients (n = 508) Variable Gender (female) Race (% African American) Recipient age: median (25%, 75%)1 Recipient age > 60 years Donor age: median (25%, 75%) Donor ECD (vs. SCD)2 Donor DCD (vs. brain death)2 Cold ischemia time: median (25%, 75%)2 Cold ischemia time >20 h Recipients with diabetes Living donor transplant Dialysis duration (months); median (25%, 75%) Dialysis >2 years % of recipients sensitized (PRA > 0) % of recipients with PRA > 80 0 mismatch transplants Retransplants Recipient treated with lymphocyte depletion3
Sensitized recipients (n = 118)
FCXM negative (n = 54)
FCXM positive (n = 54)
p value
FCXM negative (n = 84)
FCXM positive (n = 34)
p value
38% 27%
52% 39%
0.07 0.10
67% 29%
62% 38%
0.80 0.42
52 (41, 59.3)
50 (44, 56.2)
0.31
47 (38, 58)
48 (41, 54)
0.97
92/454 (20%)
8/54 (15%)
0.44
12/84 (14%)
3/34 (9%)
0.62
44 (33, 52)
45 (30, 54)
0.79
42 (32, 58)
43 (31, 53)
0.98
43/224 (19%) 46/267 (17%)
8/39 (21%) 7/39 (17%)
0.65 0.90
10/62 (15%) 7/62 (11%)
4/27 (15%) 5/27 (19%)
1.00 0.50
15.0 (12, 19)
15.3 (13, 21)
0.24
16.0 (12, 21)
15.6 (13, 21)
0.53
45/267 (17%)
11/39 (28%)
0.14
15/57 (26%)
7/28 (25%)
0.97
149/454 (33%)
19/54 (35%)
0.84
23/84 (27%)
13/34 (38%)
0.35
177/454 (39%) 13.5 (0, 38)
13/54 (24%) 31 (6.3, 49)
0.05 0.01
22/84 (26%) 22 (3, 50)
7/34 (21%) 33 (14, 50)
0.68 0.33
179/454 (39%) 84/454 (19%)
32/54 (59%) 34/54 (63%)
0.008 120 only2 T cell MCS > 80 and B cell MCS > 120 For sensitized recipients, n = 118 T and B cell negative Tor B cell positive T cell neg. B cell pos. T cell pos., B cell neg. T and B cell pos. T cell MCS > 80 B cell MCS > 120 T cell MCS > 80 and B cell MCS > 120 PRA > 80% and FCXM PRA > 80% and FCXM+
AMR < l year
Grafts lost to rejection 0.3. FCXM, flow cytometric crossmatch; MCS, median channel shift; AR < 1 year, acute rejection episode within 1 year of transplant (any; includes acute cellular and/or antibody-mediated rejection); AMR < l year, antibody-mediated rejection episode within 1 year of transplant; PRA, panel-reactive antibody. 1 Actual graft survival; all recipients were followed a minimum of 5 years; all p values for graft survival >0.3. 2 T cell FCXM with MSC > 80, or any B cell FCXM with MCS > 120.
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Renal Transplantation by Virtual Crossmatch
Discussion
1.0
Survival
0.8
0.6 Negative B and T cell XM (n = 454) T cell positive, B cell negative XM (n = 14) T cell negative, B cell pos XM (n = 20)
0.4
T and B cell positive XM (n = 20)
p = 0.85
0.2
0.0 0
2
4
6
8
Years
Figure 4: All-cause graft survival according to specific T and B cell flow crossmatch status. All recipients were virtual crossmatch negative. XM, crossmatch.
group (and placed in the FCXM group), the 5-year graft survival for FCXM+ recipients would be 42/49 (86%) versus 387/458 (84%) for FCXM recipients. Four of the 54 FCXM+ recipients had a diagnosis of systemic lupus erythematosus at time of transplant. All were sensitized but none had auto-antibodies that interfered with crossmatch interpretation. Univariate and multivariate analysis of graft outcomes The results of univariate and multivariate analyses, using Cox proportional hazards models, to identify variables associated with graft loss and death-censored graft loss for all recipients (n = 508) and in sensitized recipients (n = 118) are listed in Tables 5 and 6, respectively. For all-cause graft loss in all recipients, the following variables were significant in multivariate analysis: deceased donor (vs. living), diabetes, recipient age >60, and recipient race African American (AA) (vs. non-AA). For sensitized recipients, recipient gender (female) and induction agent (lymphocyte depletion) were significant. Additional analysis, however, revealed that the population of sensitized recipients receiving IL2-R antagonists was enriched for age >60 (30% age > 60 compared with 5% who received lymphocyte-depleting agent; p = 0.003). Death with a functioning graft was responsible for 80% of graft losses in sensitized recipients treated with IL2-R antagonists. In death-censored analysis for graft loss, only donor type and recipient age (age >60 protective) were associated with graft loss. A positive FCXM did not impact graft survival in any univariate or multivariate model.
American Journal of Transplantation 2016; 16: 1503–1515
To our knowledge, this is the first study to examine longterm outcomes for kidney transplantation, when solid phase assays are used as the definitive test for assessing donor and recipient compatibility. FCXM results were used primarily to help interpret the results of solid phase testing. Using our VXM protocol, recipients who were FCXM+ but VXM were considered to have a “positive crossmatch, not due to donor-specific HLA antibodies” and were allowed to proceed to transplantation (regardless of crossmatch strength as determined by MCS). No recipient in this study underwent desensitization to achieve a negative VXM. The primary aim of this study was to monitor long-term graft outcomes for VXM / FCXM+ renal allograft recipients (which comprised 11% of our recipient population). It is worth noting that this group itself exhibits many risk factors for shortened graft survival (Table 1). Therefore, an unadjusted analysis of graft survival creates an unfavorable bias against the FCXM+ group. Despite this, long-term graft survival rates for VXM /FCXM+ recipients were not inferior to those of VXM /FCXM recipients (Figure 1). Of special interest in this study are the outcomes for sensitized recipients (PRA > 0%) who are VXM / FCXM+. We therefore performed separate analyses for this group, as well as highly sensitized recipients (PRA > 80%). We observed low rates of acute rejection (including AMR within the first year), as well as low rates of graft loss from rejection 60 years (vs. 7% receiving lymphocyte depletion agent, p = 0.003). 3 p = 0.2 for AR < l year, lymphocyte depletion versus IL2-R antagonist. 4 p = 0.11 for graft loss due to rejection, lymphocyte depletion versus IL-2R antagonist. 5 p = 0.008 for graft loss in sensitized recipients, thymoglobulin versus IL2-R antagonist. 6 p = 0.05 for AR < l year and p = 0.02 for graft loss in FCXM+, sensitized recipients, treated with thymoglobulin versus Campath. 7 p = 0.04 for graft loss in sensitized recipients treated with Campath (FCXM+ vs. FCXM ). 8 p = 0.006 for graft loss in FCXM , sensitized recipients treated with thymoglobulin versus IL2-R antagonist.
the subgroup of FCXM+, sensitized retransplants, which appears to have experienced worse outcomes compared to FCXM , sensitized retransplants. In our study, these groups comprise very small subsets of recipients, so proper risk adjustment for outcomes was not possible. Of note, the group of FCXM , sensitized retransplants had unexpectedly high 5-year graft survival rates (100%), which further distorts comparison with FCXM+ recipients. Sensitized retransplants would be an important group of recipients to examine carefully in future studies. A positive crossmatch in this group of recipients (without DSA by solid phase assays) could indicate pre-existing sensitization to minor histocompatibility antigens, which could very well be important in this transplantation scenario. A VXM /FCXM+ result would not reflect sensitization to various non-HLA targets such as MHC class I–related chain A antigens or endothelium-restricted antigens (angiotensin II type 1 receptor and vimentin), since these antigens are not expressed on the surface of lymphocytes used for the FCXM (23). 1510
It should be noted that the virtual crossmatches in the early part of our study were based on incomplete recipient and donor HLA typing and did not account for donorspecific DQA1, DPB1, or DPA1 antibodies. Unfortunately, more detailed retrospective analyses to further characterize VXM /FCXM+ cases could not be performed as most early samples were no longer available for testing. Additionally, heavy reliance on low-resolution phenotypic assays to identify antibody specificities (i.e. LabScreen PRA) limited our ability to resolve antibodies against donor HLA-Cw. The impact of these limitations would be the inaccurate categorization of crossmatch results as VXM /FCXM+ when, in fact, the positive crossmatch might have resulted from antibodies against these untyped loci. This is consistent with what appears to be a higher incidence of AR < 1 year and AMR < 1 year in our sensitized FCXM+ recipients transplanted during the 2005–2007 period, compared to 2008–2009. To address the impact of incomplete donor HLA typing on the accuracy of virtual crossmatching, the UNOS has American Journal of Transplantation 2016; 16: 1503–1515
Renal Transplantation by Virtual Crossmatch Table 4: Summary of HLA antibody detection methodology, potential undetected DSA, and outcomes for FCXM+ recipients (n = 54)
Number of transplants Sensitized recipients (PRA > 0) Number of positive crossmatches (%) Potential undetected HLA DSA Methodology for HLA DSA detection Antibody screen Antibody specificity Recipients with potential undetected DSA1 Cw (in recipients with class I PRA > 0) DPB1, DPA1 and/or DQA1 (in recipients with class II PRA > 0) Total AR < 1 year in recipients with potential undetected DSA AMR < 1 year in recipients with potential undetected DSA 5-year graft survival in recipients with potential undetected DSA
January 2005– December 2007
January 2008–June 2009
July 2009–December 2009
325 71 (22%) 22/325 (7%)
146 35 (24%) 26/146 (18%)
37 12 (32%) 6/37 (16%)
Cw, DPB1, DPA1 and DQA1
DPA1 and DQA1
None
Flow PRA Beads
Flow PRA Beads
LabScreen PRA Beads 1
LabScreen PRA Beads or Luminex SAB2
Flow PRA and LabScreen Mixed Beads Luminex SAB3
18/22 8/22
0/26 8/26
0/6 0/6
18/22 4/18 (22%)4
8/26 2/8
0/6 0/6
2/18 (11%)4
0/8
0/6
13/18 (72%)
6/8 (75%)
0/6
DSA, donor-specific antibody; FCXM+, positive flow cytometric crossmatch; PRA, panel-reactive antibody; AR < 1 year, biopsy proven acute rejection episode (cellular or antibody mediated) < 1 year from transplant date; AMR < 1 year, biopsy proven antibody mediated rejection < 1 year from date of transplant; SAB, single antigen beads. 1 HLA Cw antibodies not detectable due to inability to resolve using Flow PRA Beads; HLA DPB1, DPA1, and DQA1 antibodies not detectable due to lack of donor typing for these loci. Recipients with PRA = 0, using flowPRA phenotypic beads are considered not at risk for undetected DSAs at time of transplant. Recipients with PRA > 0, using flowPRA phenotypic beads are considered at risk for undetected DSAs at time of transplant. 2 Luminex testing was performed in all patients with PRAs > 80% or if specificities could not be resolved using LabScreen PRA beads. 3 All patients initially screened using Flow PRA and Luminex SAB; subsequent samples on 0% PRA patients were screened using LabScreen Mixed beads. 4 For AR < 1 year, p = 0.26 versus sensitized, FCXM recipients; for AMR < 1 year, p = 0.03 versus sensitized, FCXM recipients.
expanded donor typing requirements, although surprisingly these requirements still do not include HLA-DQA1, DPB1, and DPA1. It is notable that the frequency of VXM /FCXM+ cases did not decrease with the implementation of extended donor typing and higher-resolution antibody testing, as one might expect. The frequency actually increased—an observation that might relate to the implementation in 2008 of a new flow cytometer with increased sensitivity. We examined the effect of different induction agents used during the study on graft outcomes. Unfortunately, there is significant selection bias involved in administering induction therapy. Lymphocyte-depleting agents are generally given to sensitized recipients and other subsets of recipients considered to be at increased risk of rejection. In our study, sensitized recipients who received thymoglobulin or Campath actually experienced higher rates of rejection and graft loss due to rejection, compared to American Journal of Transplantation 2016; 16: 1503–1515
recipients receiving IL2-R antagonists. The latter group, perhaps as expected since it was enriched with older recipients, experienced lower rates of rejection and higher rates of death with a functioning graft (compared to recipients receiving lymphocyte depletion). Since only five of 34 sensitized, FCXM+ recipients received IL2-R antagonists, it is not possible to make any definitive statements about use of induction agents for these patients. At present our practice is in general to use thymoglobulin for sensitized VXM /FCXM+ recipients, reserving IL2-R antagonists for unsensitized VXM / FCXM+ recipients. Of note, FCXM+ recipients who received Campath exhibited higher rates of AMR and graft loss than those who received thymoglobulin. This is consistent with previous reports in the literature (24,25). While our findings support the use of virtual crossmatching over the flow crossmatch as the primary measure of recipient/donor compatibility, there remain significant 1511
Johnson et al Table 5: Univariate and multivariate analysis for variables associated with all-cause graft survival Multivariate analysis1
Univariate analysis Risk factor Recipient age: >60 (vs. 2 years (vs. 60 (vs. 2 years (vs. 80 (vs.
