Donor-Specific Antibodies Adversely Affect Kidney Allograft Outcomes

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1 Donor-Specific Antibodies Adversely Affect Kidney Allograft Outcomes Sumit Mohan,* Amudha Palanisamy,* Demetra Tsapepas, Bekir Tanriover,* R. John Crew,* Geoffrey Dube,* Lloyd E. Ratner, David J Cohen,* and Jai Radhakrishnan* *Division of Nephrology, Department of Medicine, and Columbia University Renal Epidemiology (CURE) Group, Columbia University College of Physicians and Surgeons, New York, New York; Department of Pharmacy, New York Presbyterian Hospital, Columbia University Medical Center, New York, New York; and Department of Surgery, Columbia University College of Physicians and Surgeons, New York, New York ABSTRACT The effect of low titers of donor-specific antibodies (DSAs) detected only by sensitive solid-phase assays (SPAs) on renal transplant outcomes is unclear. We report the results of a systematic review and metaanalysis of rejection rates and graft outcomes for renal transplant recipients with such preformed DSAs, defined by positive results on SPA but negative complement-dependent cytotoxicity and flow cytometry crossmatch results. Our search identified seven retrospective cohort studies comprising a total of 1119 patients, including 145 with isolated DSA-SPA. Together, these studies suggest that the presence of DSA- SPA, despite a negative flow cytometry crossmatch result, nearly doubles the risk for antibody-mediated rejection (relative risk [RR], 1.98; 95% confidence interval [CI], ; P,0.001) and increases the risk for graft failure by 76% (RR, 1.76; 95% CI, ; P=0.01). These results suggest that donor selection should consider the presence of antibodies in the recipient, identified by the SPA, even in the presence of a negative flow cytometry crossmatch result. J Am Soc Nephrol 23: ccc ccc, doi: /ASN In 1969, Patel and Terasaki published their landmark study that definitively established the detrimental effect of preformed donor-specific antibodies (DSAs) detected by complement-dependent cytotoxicity (CDC) crossmatch on short-term allograft survival, but they noted the limited sensitivity of the technique in the detection of preformed DSAs. 1 Since that time, technological advances have led to increasingly more sensitive tests for the detection of anti-hla DSAs, including flow cytometry crossmatch and the more recent solid-phase assays (SPAs), such as Luminex. 2,3 The clinical significance of DSAs detected by sensitive SPA remains unclear, with disparate findings being reported in the literature. 4 Further, the clinical utility of these tests, particularly for prognostication or modifying immunosuppressive therapy, when used individually or in some sequential manner remains unclear. Our aim was to perform a systematic review and meta-analysis of published reports of rejection rates and graft outcomes among renal transplant recipients with preformed DSA at levels detectable only by SPA but not by CDC or flow crossmatch. RESULTS We ultimately identified seven studies that met our inclusion criteria for the primary quantitative analysis (group 1) All seven studies were retrospective cohort studies; three were from the United States and four from Europe. Our ability to compare Received July 6, Accepted September 19, S.M. and A.P. contributed equally to this work. Published online ahead of print. Publication date available at. Correspondence: Dr. Sumit Mohan, Department of Medicine, Division of Nephrology, Columbia University Medical Center, 622 West 168th Street, PH4-124 New York, NY sm2206@columbia.edu Copyright 2012 by the American Society of Nephrology J Am Soc Nephrol 23: ccc ccc, 2012 ISSN : /2312-ccc 1

2 certain patient characteristics and immunosuppression protocols across studies was limited by the variable reporting of these characteristics in the included reports. Demographic and transplant characteristics for included studies are summarized in Table 1. Notably, mean and median age of the patients included with DSA-SPA tended to be similar to the age of their comparative cohort without DSA in each individual study. The types of donors included in these studies varied; two studies included only deceased-donor transplants, 5,10 one study included only living-donor transplants, 8 three studies included both donor types, 7,9,11 and one study did not describe the type of donors in the cohort. 6 Induction and maintenance regimens also varied widely across studies. Gupta et al. reported that no induction regimen was routinely used, whereas the other studies used antithymocyte globulin, alemtuzumab, or an IL-2 inhibitor (Table 2) Two of the seven studies consistently used a steroid-free maintenance regimen, including Willicombe et al.,whomaintained patients on tacrolimus monotherapy. 10,11 Patel et al. 8 and Verghese et al. 9 maintained patients on a calcineurin inhibitor (CNI) and prednisone, whereas the Gupta, Couzi, and Higgins and colleagues used a triple-drug regimen that included a CNI, antimetabolite, and prednisone. 5 7 Although the immunosuppression protocols used varied across studies, the cohorts positive and negative for DSA-SPA within each individual study received similar induction and maintenance immunosuppression regimens. No DSA-SPA positive recipients received peri-transplant desensitization except for patients in the study by Higgins et al. 7 The seven retrospective cohort studies included in our primary analysis (group 1, flow crossmatch negative) represented a total of 1119 patients, of whom 145 were DSA-SPA positive We excluded Couzi and colleagues study from the analysis because no rejection episodes occurred in either comparison groupduringthefollow-upperiod(figure2).further,couzi et al. did not report specific graft survival data for their groups beyond stating that there was no statistical difference precluding their inclusion in the pooled analysis. 5 Although the increased relative risk (RR) of developing antibody-mediated rejection (AMR) in DSA-SPA positive patients reached statistical significance in only two of the individual studies, 10,11 the pooled incidence of AMR across the six included studies was significantly higher among patients with preformed DSA- SPA (35 of 145 [25%]) than patients without DSA (110 of 974 [11.2%]) (RR, 1.98; 95% confidence interval [CI], ; P,0.001) (Figure 2A, and Table 2). With the exception of a single study, individual studies did not show a statistically significant higher risk for graft failure in patients with preformed DSA-SPA. 9 The incidence of graft failure across all included studies was 15% (22 of 145) in the DSA-SPA positive group and 9.4% (92 of 974) in the DSA-negative group despite the relatively short (and variable) follow-up period; the pooled analysis demonstrates a significant decrease in graft survival in the presence of preformed DSA-SPA (RR, 1.76; 95% CI, ; P=0.01) (Figure 2B and Table 2). We also identified nine studies in which flow cytometry crossmatch was not performed but comparative graft outcomes data in DSA-SPA positive versus DSA-negative patients was reported with a negative T and B cell CDC crossmatch (group 2) These studies were excluded from our primary analysis. A quantitative analysis of this cohort (group 2) was limited to eight studies because adequate data could not be extracted from one of the studies identified for inclusion in this analysis. 20 As was seen in the studies included in our primary analysis (group 1, negative flow crossmatch), all eight studies in group 2 (unknown flow crossmatch) were retrospective cohort studies in which the age of patients included with DSA-SPA was similar to the age of their comparative cohort without DSA in each individual study The types of donors included in these studies varied; three studies included only deceased-donor transplants, 14,16,18 one study included only living-donor transplants, 17 and four studies included both types of donors. 12,13,15 Induction regimens varied considerably across studies. Two studies reported the absence of a standard induction regimen, 17,18 whereas the other studies variably used antithymocyte globulin or an IL-2 receptor blocker for induction therapy at the time of transplantation (Table 2). None of the studies included in group 2 consistently used a steroid-free maintenance regimen for their cohort The cohorts positive and negative for DSA-SPA within the individual studies received similar induction and maintenance regimens, and only a single study included patients with DSA-SPA who received peri-transplant desensitization. 16 The eight retrospective cohort studies in group 2 represented 1967 patients, of whom 306 (15.6%) were DSA-SPA positive (Table 1). Only three of these studies reported data on rejection episodes, and the pooled incidence of AMR across these three studies was significantly higher in patients with preformed DSA-SPA (62 of 154 [40%]) than patients without DSA (23 of 695 [3.3%]) (RR, 7.81; 95% CI, ; P,0.001) (Figure 3A and Supplemental Table 1A). Of the three studies that reported rejection episodes, two showed a significantly higher risk for graft failure among patients with preformed DSA- SPA. 12,14 Among the eight studies in group 2, two 13,17 were excluded from the pooled analysis for graft failure because the rate of graft failure was not reported. Graft failure across the studies in group 2 was significantly more frequent in the DSA-SPA positive group (56 of 306 [18.3%]) than in the DSA-negative group (174 of 1661 [10.5%]) (RR, 1.74; 95% CI, , P,0.001) (Figure 3B and Supplemental Table 1B). Although no significant heterogeneity was noted when studies in group 1 and group 2 were pooled for analysis of risk for graft failure (I 2 = 0%; P=0.847, Figure 3B), heterogeneity was significant in the combined pooled analysis of risk for biopsy-proven AMR (I 2 = 72.2%; P,0.001, Figure 3A). Finally, no significant publication bias was detected according to the Peters or the Begg test for studies that reported AMR (P=0.520 and 0.851, respectively, for group 1) and graft failure (P=0.937 and 0.573, respectively, for group 1). The funnel plot for publication bias is shown for studies that 2 Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc ccc, 2012

3 CLINICAL RESEARCH Table 1. Characteristics of patient cohorts in the individual studies included in the analysis Study, Year Patients (n) Age (yr) Women (%) Black (%) First Transplant (%) Follow-up Period (mo) Patients with Living Donors (%) Group 1 Patel et al., DSA a NA 8.2 a DSA a NA 11.1 a 100 Gupta et al., DSA b 60 NA 79 NA 95 DSA d NA 91 d 12 and 60 c NA Vlad et al., DSA+ 265 DSA2 52 b 38% NA 80% 48 0 Couzi et al., DSA+ 39 DSA b b 0 Willicombe et al., DSA b b DSA b 32.4 d d b 45.3 Verghese et al., DSA b b DSA b b 43.1 Higgins et al., DSA b NA b DSA b NA Group 2 Gibney et al., DSA+ 35 DSA b,e 62 F e 9 e 75 e 6 b 39 e van den Berg-Loonen 13 DSA+ 51 a 62 NA f 0 et al., DSA a 64 NA 36 0 Aubert et al., DSA b 73 NA NA 12 f DSA b 26 NA NA 40 Amico et al., DSA+ 47 b 57 NA DSA2 52 a 60 mo f and 200 d g 31 NA Mahmoud et al., DSA b NA DSA b 3 f NA Lefaucheur et al., DSA+ NA NA NA NA 51.4 b ;96 f DSA2 NA NA NA NA 96 f 0 Dunn et al., DSA b i a DSA b a 70 Caro-Orleas et al., DSA+ 47 a NA a DSA2 48 a 37 NA a 0 Group 3 Bielmann et al., DSA+ 48 a 67 NA DSA2 53 a 36 NA Billen et al., DSA NA a 133 DSA NA a Morris et al., DSA b h 134 DSA2 100 Group 4 Vlad et al., DSA+ 52 b,e 0 38% NA Majority DSA2 0 Higgins et al., DSA b 75 NA DSA b 39.6 b NA NA, not available. a Median. b Mean. c Time intervals at which data are reported. d Estimate of percent female. e Data for entire cohort reported. f Maximum duration of follow-up for allograft survival. g Maximum duration of follow-up for AMR. h Minimum follow-up period. i Nonwhite. J Am Soc Nephrol 23: ccc ccc, 2012 Effect of Donor-Specific Antibodies 3

4 Table 2. Transplant-specific characteristics for patients included in the analysis Study, Year Patients (n) Desensitized MFI Cutoff Positive DSA Induction Maintenance Steroid-Free No DSA Group Includes Pts with Other HLA Ab Patel et al., DSA+ No NA 20% none, 55% ratg, 85% prednisone, Some ND DSA2 15% CD25 monoclonal 15% CSA, 85% FK 1.1 antibody, 10% alemtuzumab Gupta et al., DSA+ No NA None CSA, azathioprine, No Yes DSA2 prednisone 1.77 Vlad et al., DSA+ No ratg (6 mg/kg) FK, MMF, steroids Yes ND NA 265 DSA2 tapered on day 5 Renal Function at Follow-up Couzi et al., DSA+ No.500 ratg or daclizumab MMF + CNI + steroids No ND 1 yr egfr, DSA2 1 yr egfr,66 Willicombe et al., DSA2 3 yr Cr, DSA+ No NA Alemtuzumab FK alone Yes ND 3 yr Cr, 2.67 Verghese 10 DSA+ No NA Daclizumab majority; et al., DSA2 alemtuzumab small minority Higgins CNI/prednisone or some with FK + (MMF or rapamycin) Some ND NA et al., DSA2 3 yr egfr, DSA+ Yes.500 basiliximab FK + MMF + prednisone No ND 3 yr egfr, 46.9 Gibney 20 DSA+ No NA 52% ratg, 15% 70% FK/rapamycin/prednisone, et al., DSA2 CD25 monoclonal 30% CNI + (rapamycin or MMF) antibody + prednisone entire cohort van den 13 DSA+ No NA None CNI + (azathioprine or MMF or Berg-Loonen 14 DSA2 rapamycin) + prednisone et al., No ND NA No ND NA Aubert 11 DSA+ No.500 Basiliximab: first kidney 67 DSA+ No % none, 39% FK + MMF + prednisone No Yes (44 NA et al., DSA2 transplant; or ratg: patients) retransplant or panel-reactive antibody.50% Amico et al., No ND NA DSA2 basiliximab, 9% daclizumab Mahmoud 16 DSA+ No NA None Steroid + azathioprine No Yes 3 yr Cr, 2.5 et al., DSA2 + (CNI or rapamycin) 3 yr Cr, 1.5 Lefaucheur et al., DSA2 76 DSA+ Yes.300 (normalized) ratg CNI + MMF + steroids No Yes NA Dunn et al., DSA+ No NA ratg (4 6 mg/kg) CNI + MPA Yes Yes NA 541 DSA2.500 (baseline adjusted) 4 Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc ccc, 2012

5 CLINICAL RESEARCH Table 2. Continued Study, Year Patients (n) Desensitized MFI Cutoff Positive DSA Caro-Orleas 103 DSA+ No.1500 (raw) , ratg; et al., Bielmann 780 DSA2 Induction Maintenance Steroid-Free 1997 onward: for high immunologic risk, ratg; for moderate immunologic risk, daclizumab or basiliximab CNI + (azathioprine or MMF) + steroids No DSA Group Includes Pts with Other HLA Ab Renal Function at Follow-up No Yes NA et al., DSA2 Basiliximab Yes DSA+ No NA ratg FK + MMF + steroids No Yes 1.1 Billen et al., DSA+ No.1000 No induction; DSA2 received basiliximab 50% CNI (FK majority) + prednisolone; 50% receiving CNI + prednisolone + azathioprine/mmf/rapamycin No ND NA Morris et al., DSA+ No NA ratg MMF, FK, prednisone No Yes NA 134 DSA2 Vlad et al., DSA+ No ratg (6 mg/kg) FK, MMF, steroids Yes ND NA 265 DSA2 tapered on day 5 Higgins 44 DSA+ Yes.500 Basiliximab FK + MMF + prednisone No ND 3 yr egfr, 54.2 et al., DSA2 3 yr egfr, 56.7 Renal function reported as creatinine mg/dl unless otherwise stated; estimated GFR reported as ml/min. MFI, mean fluorescence intensity; NA, not applicable; CSA, cyclosporine A; ND, not discussed; ratg, rabbit antithymocyte globulin; FK, tacrolimus; MMF, mycophenolate mofetil; CNI, calcineurin inhibitor; egfr, estimated GFR; Cr, creatinine. J Am Soc Nephrol 23: ccc ccc, 2012 Effect of Donor-Specific Antibodies 5

6 Figure 1. Flow diagram of search results and identification of studies included in the analysis. reported AMR and/or graft failure in the various groups (Figure 4A and 4B). DISCUSSION The CDC crossmatch has definitively been shown to be a specific test for the detection of DSA and to be associated with immediate poor allograft outcomes. 1 However, to date, the long-term prognostic value of the highly sensitive SPA for DSA has remained inconclusive. 4 Our pooled analysis of the seven retrospective cohort studies comparing groups that were positive and negative for DSA-SPA in the setting of a negative CDC and negative flow crossmatch result demonstrates a statistically significantly increased risk for biopsy-proven AMR and allograft failure in the DSA-SPA positive group, despite the relatively short follow-up period of most studies included in the analysis (Table 1). Our results suggest that preformed DSA-SPA are clinically significant even in the absence of a positive flow cytometery crossmatch and indicate an immunologically higher-risk transplant recipient. As a result, these patients should be monitored closely for development of early AMR and appear to be at risk for worse graft outcomes in the short to intermediate term. The meta-analysis of eight retrospective cohort studies (group 2) comparing DSA-SPA positive and negative patients by SPA in the setting of a negative CDC crossmatch result but with unknown results on flow crossmatch (because it was not performed) showed an even greater significant increase in the likelihood of developing a biopsy-proven AMR episode and allograft failure in the DSA-SPA positive group. These studies were excluded from the group 1 cohort because they probably include patients who are flow crossmatch positive (if they were to be tested) and as a result reflect a group at heterogeneous immunologic risk group compared with recipients who have a definite negative flow crossmatch result. This cohort (group 2) demonstrated a much higher risk for AMR (RR, 7.81; 95% CI, ; P,0.001) consistent with our hypothesis that this group includes some higher-immunologicrisk patients. In addition, the fact that significant statistical heterogeneity was detected when we attempted to combine studies in group 1 and 2 but no significant heterogeneity was found in group 1 or group 2 individually supported our aprioristratification of patient cohorts based on the results (or absence thereof) of the flow crossmatch. The presence of DSA-SPA for studies included in group 2 was also associated with relatively early graft failure (RR, 1.737; 95% CI, ; P,0.001) but appeared to be similar to the pooled analysis of group 1. The absence of statistical heterogeneity upon pooling studies for risk of graft failure suggests that although positive flow crossmatch results are more useful in identifying patients at high risk of AMR, they do not appear to improve the risk stratification that occurs with the identification of DSA-SPA for overall graft survival in the short term. These results demonstrate the relative value of these two testing modalities in the stratification of immunologic risk; the addition of a flow cytometry crossmatch test improves the specificity for identifying patients at risk of developing AMR. However, the risk identified by flow crossmatch improves but does not appear to help refine the risk of graft failure in the short term. As a result, we suggest that an effective means of risk stratification at the time of transplant would be the following sequence of immunologic testing: CDC crossmatch followed by SPA for DSA identification and then followed by flow crossmatch testing only if further stratification for risk of AMR is desired. Unfortunately, our pooled analysis was unable to discern trends in risk associated with specific HLA antibodies because these were identified relatively infrequently in the individual studies. In addition, when mentioned, there was little uniformity in the description of the class of antigen directed against or the mean fluorescence intensity. To our knowledge, our study represents the first attempt to systematically review the literature for the influence of preformed DSA-SPA on the risk of rejection and renal allograft outcomes. Careful risk stratification of our included studies by the reported flow cytometry crossmatch results allowed us to pool patients of similar immunologic risk into groups and demonstrates that with adequate power, the detrimental effects of DSA-SPA can be observed. This stratification also allows us to have greater confidence that the relatively similar (low) concentrations (mean fluorescence intensity) of DSA in the flownegative studies (group 1) were similar. These results refute the assertion by some authors that low DSA levels with a negative 6 Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc ccc, 2012

7 CLINICAL RESEARCH Figure 2. Forest plots for risk stratified by flow crossmatch results for group 1. (A) Risk of AMR with and without DSA. (B) Risk of graft failure with and without DSA. For all forest plots, the solid line represents the null effect. The dotted line represents the summary effect for the studies included in the analysis. The shaded boxes are proportional to the relative weight of the studies used in each analysis. The center dot within the box represent the point estimate for that study and the horizontal line passing through each square represents the confidence interval of each study point estimate. The center of the diamond represents the summary effect and the lateral tips of the diamond represent the confidence intervals of the summary estimate. flow cytometry crossmatch result at the time of transplantation do not suggest a high-immunologic-risk transplant recipient with a greater risk of earlier failure. Our results also support the suspicion that negative studies in the literature were underpowered or, in the case of graft outcomes, had follow-up durations inadequate for demonstrating a difference. We noted large variations in the duration of follow-up in the individual studies that reported graft outcomes, and this is likely to have a direct effect on the graft failure rates reported. This is an important limitation to note in terms of interpreting our point estimate for the relative risk of graft failure in the presence of DSAs. Our analysis has several limitations related to the reviewed literature. Immunosuppression protocols with respect to both induction agents and maintenance immunosuppressive protocols varied considerably across the included studies and in some cases within individual studies (Table 1). The considerable variation noted precludes us from comparing the risk of AMR or failure with any specific protocol. Further J Am Soc Nephrol 23: ccc ccc, 2012 Effect of Donor-Specific Antibodies 7

8 Figure 3. Forest plots for risk stratified by flow crossmatch results for groups 1 and 2. (A) Risk of AMR with and without DSA. (B) Risk of graft failure with and without DSA. For all forest plots, the solid line represents the null effect. The dotted line represents the summary effect for the studies included in the analysis. The shaded boxes are proportional to the relative weight of the studies used in each analysis. The center dot within the box represent the point estimate for that study and the horizontal line passing through each square represents the confidence interval of each study point estimate. The center of the diamond represents the summary effect and the lateral tips of the diamond represent the confidence intervals of the summary estimate. work is needed to determine whether a characteristic or threshold level of DSA detected by SPA alone is most clinically predictive of the development of AMR and/or allograft failure. Standardization of assays, which would ensure greater uniformity in how results in the field are reported, would allow the improvement or development of an evidence base to inform 8 Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc ccc, 2012

9 CLINICAL RESEARCH Figure 4. Funnel plot to assess the influence of publication bias on the pooled analysis. OR, odds ratio; XM, crossmatch. Table 3. RRs and tests of heterogeneity for the pooled analysis for AMR and graft failure rates for group 1 Study, Year RR (95% CI) Weight (%) zstatistic PValue I 2 Statistic (%) P Value AMR Patel et al., ( ) 2.78 Gupta et al., ( ) Vlad et al., ( ) 4.53 Willicombe et al., ( ) Verghese et al., ( ) 7.84 Higgins et al., ( ) Couzi et al., Excluded Group 1 pooled RR ( ) , Graft failure rates Patel et al., ( ) 3.28 Gupta et al., ( ) Vlad et al., ( ) Willicombe et al., ( ) Verghese et al., ( ) Higgins et al., ( ) 8.99 Group 1 pooled RR ( ) Bold indicates pooled estimates. clinical care. Given the widespread use of survival analysis in reporting outcomes after transplantation, uniform implementation of the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines and greater transparency in the results with the use of reporting tools for such outcomes as at-risk tables with survival curves, tabular presentation of the Kaplan-Meier estimates, and the use of life tables with standard time points should be encouraged if not required. 21,22 Finally, the risk of graft failure at least partly depends on how individuals with AMR were treated; inadequate information about treatment and response to treatment in the pooled studies limits our ability to comment on this. In conclusion, our pooled analysis demonstrates the significant elevated risk for early acute AMR and inferior graft outcomes associated with the presence of DSA-SPA despite negative flow crossmatch results. These findings suggest the need for increased vigilance, perhaps with protocol biopsies, consideration of desensitization, and augmented immunosuppression induction/maintenance regimens even in patients with DSA detected by SPA with a negative crossmatch and perhaps more so in patients with DSAs detected by SPA at centers that have forgone flow crossmatch testing. CONCISE METHODS Search Strategy We searched PubMed using the following search phrase (antibodies OR solid-phase assay OR LuminexÒ OR antibody mediated rejection) AND kidney AND crossmatch. A similar search was performed in the Cochrane database. In addition, we hand-searched archived abstracts presented at the American Society of Nephrology and the American Transplant Congress meetings between 2005 and 2010 for reported studies that were not subsequently published as full J Am Soc Nephrol 23: ccc ccc, 2012 Effect of Donor-Specific Antibodies 9

10 manuscripts in the peer reviewed literature. For completeness, we also reviewed all references listed in the full-text publications included in our meta-analysis that were not identified by our search criteria. Study Selection and Data Extraction In the first step, all abstracts of the 458 potentially relevant citations identified by our search were reviewed (Figure 1). We excluded unrelated articles. We reviewed the full text of the 54 remaining citations that possibly met our inclusion criteria. Any study that compared data between DSA-positive and -negative groups according to the Luminex solid-phase assay (DSA-SPA) on biopsy-proven (antibody-mediated) rejection episodes and/or allograft survival rates among adult renal transplant patients was included in our analysis. Studies that did not explicitly report a negative current T and B cell complement dependent cytotoxicity and T cell flow cytometry crossmatch (n=47) for their cohort were excluded from our primary cohort (group 1) for analysis, resulting in a final group of seven studies (Figure 2, A and B, and Table 3). For reports that did not include the requisite data in the published manuscript, we attempted to contact corresponding authors to obtain additional information (Figure 1). An additional 14 studies reporting comparative graft outcomes data in DSA-SPA positive versus DSA-negative patients with negative current T and B cell CDC crossmatch were identified. Although these were not part of our group 1 analysis, they were included for additional analyses. An additional study was brought to our attention by an expert. 19 These additional studies were grouped into one of the following categories: (1) flow cytometry crossmatch not performed (group 2); (2) flow crossmatch performed, with reported results that did not differentiate patients by the results of the flow crossmatch (group 3); and (3) flow performed selectively in high-risk patients, with negative results (group 4). We have included pooled analyses of cohorts that were included in group 1 and group 2 for AMR and graft loss (Figure 3, A and B, and Supplemental Table 1, A and B). Analysis of data extracted from studies included in groups 2 through 4 are provided in the supplemental material. Data extracted from the studies, including patient demographic characteristics, allograft characteristics, induction and maintenance regimens, rates of AMR episodes, and graft survival when available, are shown in Tables 1 and 2. Outcome Measures We identified twoprimaryoutcomes apriorifor our analysis: the RR of developing (1) an AMR episode and (2) graft failure during the follow-up period. Quantitative Data Synthesis Treatment effects were summarized as RRs with 95% CIs using the metan function in Stata software, version 11.2 (Stata Corp., College Station, TX). The heterogeneity statistic (Q) andi 2 values were also estimated by the metan function on the basis of the work of Higgins and Thompson, and, when appropriate, stratification of data was used to try to assess the cause for heterogeneity. For the purposes of our analysis, we used both the Mantel-Haenszel fixed-effects method as well as the DerSimonian and Laird random-effects method (1986), which incorporates an estimate of between-study variation (i.e., heterogeneity). Given the very similar results, we have reported the results of the Mantel-Haenszel fixed-effects method that resulted in slightly more conservative pooled ratios than the random-effects model (1.984 versus for risk of AMR and versus for risk of graft failure) for our primary analysis. Publication bias was tested separately for reports of AMR and graft overall graft outcomes according to use of both the Begg adjusted rank correlation test and the Peter test; in addition, these were depicted graphically using a funnel plot (Figure 4) Given the variability of the study sizes, a sensitivity analysis was also performed to ensure that the results of our analysis were not being influenced by any one trial (data not shown). ACKNOWLEDGMENTS Parts of this analysis were presented at the 2011 meeting of the American Society of Nephrology. DISCLOSURES None. REFERENCES 1. Patel R, Terasaki PI: Significance of the positive crossmatch test in kidney transplantation. NEnglJMed280: , Karpinski M, Rush D, Jeffery J, Exner M, Regele H, Dancea S, Pochinco D, Birk P, Nickerson P: Flow cytometric crossmatching in primary renal transplant recipients with a negative anti-human globulin enhanced cytotoxicity crossmatch. JAmSocNephrol12: , VaidyaS,PartlowD,SusskindB,NoorM,BarnesT,GugliuzzaK:Prediction of crossmatch outcome of highly sensitized patients by single and/or multiple antigen bead luminex assay. Transplantation 82: , Segev D, Wang G, Gloor J, Stegall M, Kapur S, Dunn T, Pelletier R, SinghP,PosnerM,ShapiroRE-A:JM,Light,J,Marsh,C,Melancon,J, Lipkowitz, G, Wellen, J, Oberholzer, J, Montgomery, R: Risks of HLA incompatible kidney transplants by antibody strength: Initial results from a National study of 603 patients [Abstract]. 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