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De Novo Donor-Specific Antibody at the Time of Kidney Transplant Biopsy Associates with Microvascular Pathology and Late Graft Failure

2009; Elsevier BV; Volume: 9; Issue: 11 Linguagem: Inglês

10.1111/j.1600-6143.2009.02800.x

1600-6143

Luis Hidalgo, Patricia Campbell, B. Sis, Gunilla Einecke, Michael Mengel, Jessica Chang, Jacobo Sellarés, J. Reeve, Philip F. Halloran,

Organ Transplantation Techniques and Outcomes

American Journal of TransplantationVolume 9, Issue 11 p. 2532-2541 Free Access De Novo Donor-Specific Antibody at the Time of Kidney Transplant Biopsy Associates with Microvascular Pathology and Late Graft Failure L. G. Hidalgo, Corresponding Author L. G. Hidalgo Laboratory Medicine and Pathology * Corresponding author: Philip F. Halloran, phil.halloran@ualberta.caSearch for more papers by this authorP. M. Campbell, P. M. Campbell Laboratory Medicine and Pathology Department of Medicine, Division of Nephrology and Transplant Immunology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, CanadaSearch for more papers by this authorB. Sis, B. Sis Laboratory Medicine and PathologySearch for more papers by this authorG. Einecke, G. Einecke Medizinische Hochschule, Hannover, GermanySearch for more papers by this authorM. Mengel, M. Mengel Laboratory Medicine and PathologySearch for more papers by this authorJ. Chang, J. Chang Department of Medicine, Division of Nephrology and Transplant Immunology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, CanadaSearch for more papers by this authorJ. Sellares, J. Sellares Nephrology Department, Hospital Universitari de Bellvitge, Barcelona, SpainSearch for more papers by this authorJ. Reeve, J. Reeve Laboratory Medicine and PathologySearch for more papers by this authorP. F. Halloran, P. F. Halloran Department of Medicine, Division of Nephrology and Transplant Immunology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, CanadaSearch for more papers by this author L. G. Hidalgo, Corresponding Author L. G. Hidalgo Laboratory Medicine and Pathology * Corresponding author: Philip F. Halloran, phil.halloran@ualberta.caSearch for more papers by this authorP. M. Campbell, P. M. Campbell Laboratory Medicine and Pathology Department of Medicine, Division of Nephrology and Transplant Immunology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, CanadaSearch for more papers by this authorB. Sis, B. Sis Laboratory Medicine and PathologySearch for more papers by this authorG. Einecke, G. Einecke Medizinische Hochschule, Hannover, GermanySearch for more papers by this authorM. Mengel, M. Mengel Laboratory Medicine and PathologySearch for more papers by this authorJ. Chang, J. Chang Department of Medicine, Division of Nephrology and Transplant Immunology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, CanadaSearch for more papers by this authorJ. Sellares, J. Sellares Nephrology Department, Hospital Universitari de Bellvitge, Barcelona, SpainSearch for more papers by this authorJ. Reeve, J. Reeve Laboratory Medicine and PathologySearch for more papers by this authorP. F. Halloran, P. F. Halloran Department of Medicine, Division of Nephrology and Transplant Immunology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, CanadaSearch for more papers by this author First published: 15 October 2009 https://doi.org/10.1111/j.1600-6143.2009.02800.xCitations: 244AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract We studied whether de novo donor-specific antibodies (DSA) in sera from patients undergoing kidney transplant biopsies associate with specific histologic lesions in the biopsy and prognosis. DSA were assessed in 145 patients at the time of biopsy between 7 days to 31 years posttransplant. DSA was detected in 54 patients (37%), of which 32 represented de novo DSA. De novo DSA was more frequent in patients having late biopsies (34%) versus early biopsies (4%), and was usually either against class II alone or class I and II but rarely against class I alone. Microcirculation inflammation (glomerulitis, capillaritis) and damage (glomuerulopathy, capillary basement membrane multilayering), and C4d staining were associated with de novo DSA. However, the degree of scarring, arterial fibrosis and tubulo-interstitial inflammation did not correlate with the presence of de novo DSA. De novo DSA correlated with reduced graft survival after the biopsy. Thus, de novo DSA at the time of a late biopsy for clinical indication is primarily against class II, and associates with microcirculation changes in the biopsy and subsequent graft failure. We propose careful assessment of de novo DSA, particularly against class II, be performed in all late kidney transplant biopsies. Abbreviations: ABMR antibody-mediated rejection TCMR T-cell-mediated rejection PRA panel- reactive antibodies DSA donor-specific antibodies Introduction Late loss of kidney transplants with return to dialysis remains common and largely unexplained. One of the principal associations of late graft loss is the presence of HLA antibodies in the recipient (1). As prevention of T-cell-mediated rejection (TCMR) has improved with current immunosuppressive regimens, the impact of HLA antibodies is increasingly recognized as a risk factor for kidney transplant failure (2, 3). Historic studies performing panel-reactive antibodies (PRA) identified HLA antibodies to a panel of cells expressing different HLA antigens (4). However, identifying HLA specificities of sera was difficult with those tests. The emergence of methodologies for identifying antibody specificities using single antigen bead technology (5-7) provides an opportunity to define the significance of these antibodies in patients presenting with clinical problems. A key to understanding the effects of antibody-mediated graft damage is to define the relationship between donor-specific antibody (DSA) in the recipients' sera and the histological lesions in their biopsies. According to the current Banff consensus criteria, the diagnosis of antibody-mediated rejection (ABMR) relies on the presence of DSA and of certain histological lesions (8). However, with the advent of new technologies the diagnostic criteria continue to evolve (9). One key issue is the existence of C4d-negative cases with histological features of ABMR but lying outside the current diagnostic rules and thus are called suspicious for ABMR. The important relationship of antibody is unlikely to be with the evolving diagnostic labels but rather with the individual pathology lesions mediated by antibody on the allograft. The key lesions are probably those reflecting inflammation and deterioration of the microcirculation, which associate with one another and with the presence of HLA antibodies (Sis et al., submitted). We recently studied the phenotype of late kidney graft failure in a prospective study of unselected kidney transplant biopsies taken for clinical indications (Einecke et al. (AJT, in press)). Patients undergoing a late biopsy (>1-year posttransplant) frequently displayed DSA in their sera and microcirculation inflammation and damage with or without C4d staining in their biopsies. Grafts biopsied late also often progressed to failure. The major cause of late kidney transplant failure was found to be antibody-mediated microcirculation injury. This paper is a detailed analysis of the frequency and features of DSA, particularly de novo DSA, in our prospective study of patients undergoing a biopsy for clinical indications. The population included all consenting patients undergoing biopsies between 2004 and 2007, regardless of time posttransplant, with complete DSA studies at the time of biopsy. Our center is the exclusive provider of renal transplant follow-up in a large region, and biopsies all transplants with indications (dysfunction and/or proteinuria). We sought to define the frequency of de novo DSA in patients presenting with clinical problems, and the associations of these findings with histopathology lesions and the subsequent course of the graft. Materials and Methods Patients and sample collection The study was approved by the institutional review board of the University of Alberta (issue 5299). Written informed consent was obtained from all study patients. All consenting renal transplant patients undergoing a transplant biopsy for clinical indication (deterioration in function, proteinuria, stable impaired function) as standard of care between September, 2004 and March, 2007 were included. Biopsies were obtained under ultrasound guidance by spring-loaded needles (ASAP Automatic Biopsy, Microvasive, Watertown, MA). Graft failure was defined as return to dialysis. Death-censored graft survival analysis was performed using the Kaplan–Meier method (GraphPad Prism 5, GraphPad Software Inc., San Diego, CA). Patients were censored for the end of study (October 21st, 2008), death with functioning graft (n = 5), or lost to follow-up (n = 1). Significance was assessed by the log-rank test. Only one biopsy—the last biopsy available—for each patient (n = 145) was included in the analysis of HLA antibody status, and histologic lesions. Analyses assessing the effects on graft survival were limited to late biopsies (n = 89) since HLA antibodies were rare in patients undergoing an early biopsy. The number of biopsies included in each analysis is specified in the figures and tables. Histopathology Paraffin sections were graded according to Banff criteria by a renal pathologist (BS) (Table S1) (10). Interstitial inflammation was scored both in nonscarred and scarred cortical parenchyma. C4d staining was performed on frozen sections using a monoclonal anti-C4d antibody (Quidel, San Diego, CA) by indirect immunofluorescence. Diffuse linear C4d staining (>50% of biopsy area) was interpreted as positive. Peritubular capillary basement membrane multilayering (ptcml) was examined by electron microscopy (available for biopsies >3 months posttransplant) and the highest number of basement membrane layers found on the overall peritubular capillary circumference was recorded (11). HLA antibody screening Antibody specificities of patient sera were determined by FlowPRA® specific class I and or II and/or FlowPRA® single antigen I and II beads (One Lambda, Canoga Park, CA). Manufacturer's instructions for staining and acquiring were followed. Beads were analyzed on a BD FACSCalibur™ cytometer (Becton Dickinson Biosciences, Mississauga, Ontario, Canada). Antibody screening was performed using FlowPRA® beads. These beads have HLA-A, -B, -Cw, -DR, -DQ and -DP antigens represented. Further testing for specificities was only done if the screen was positive (≥5% PRA or clear pattern of reactivity with screening beads). Single antigen beads were used to test for antibodies against HLA-A, B, DRB1, DRB3, 4 and 5, DQB1 and DP. We did not test for specificities to Cw. Donor typing for DP was not performed and therefore DSA were not attributed to DP. De novo DSA was defined as new DSA detected by single antigen bead technology and/or a donor-specific flow crossmatch that was negative pretransplant and positive at the time of biopsy. Flow T- and B-cell crossmatches were performed as previously described (12). HLA typing Low-to medium-resolution HLA class I and II typing was performed using the One Lambda Micro SSP assay as previously shown (12). Manufacturer's instructions for amplification and electrophoresis were followed. Data analysis Data analyses were performed using GraphPad Prism 5 statistical software package, Bioconductor version 2.4, R version 2.9 (13). For comparisons between groups (early vs. late biopsies, failed vs. functioning grafts) t-tests were used for means, while chi-square or Fisher's exact tests were used for count data. Results Patient demographics and diagnostic classification We prospectively studied 145 patients undergoing transplant biopsies for clinical indication (unexplained renal dysfunction, and/or proteinuria) between 2004 and 2007, ranging 6 days to 31 years posttransplant (median 17 months). The biopsies are the subset from the larger study (Einecke et al., in press) that have complete HLA antibodies data. There was no selection of biopsies for disease states. There were 56 early biopsies ( 1-year posttransplant). The median time posttransplant of early biopsies was 2.9 months, and of late biopsies was 68.1 months. The demographics of the patients biopsied 1-year posttransplant were similar except for some differences in primary diseases (Table 1). No transplants were ABO incompatible. Only one patient had undergone desensitization with a negative flow cytometry crossmatch at the time of transplant. Table 1. Patient demographics Patient demographics [n = 145] All patients (n = 145) Patients biopsied 1 year (n = 89) p-Value (early vs. late) Recipient gender (% male) [n = 145] 92 (63%) 38 (68%) 54 (61%) 0.38 Race [n = 145] Caucasian 92 (63%) 33 (59%) 59 (66%) 0.37 Black 3 (2%) 1 (2%) 2 (2%) 1.00 Other 50 (34%) 22 (39%) 28 (31%) 0.33 Primary disease [n = 145] Diabetic nephropathy 21 (14%) 12 (21%) 9 (10%) 0.06 Hypertension/large vessel disease 8 (6%) 6 (11%) 2 (2%) 0.06 Glomerulonephritis/vasculitis 68 (47%) 20 (35%) 48 (54%) 0.03 Interstitial nephritis/pyelonephritis 12 (8%) 5 (9%) 7 (8%) 1.00 Polycystic kidney disease 23 (16%) 10 (18%) 13 (15%) 0.60 Others 6 (4%) 1 (2%) 5 (5%) 0.41 Unknown etiology 7 (5%) 2 (4%) 5 (5%) 0.71 Donor gender (% male) [ n = 144] 64 (44%) 26 (46%) 38 (43%) 0.66 Donor type (% deceased donor transplants) [n = 144] 83 (57%) 33 (59%) 50 (56%) 0.74 Clinical characteristics at time of biopsy [n = 145] All biopsies (n = 145) Biopsies taken 1 year (n = 89) p-Value (early vs. late) Indication for biopsy Delayed graft function 3 (2%) 3 (5%) 0 (0%) 0.06 Rapid deterioration of graft function 32 (22%) 9 (16%) 23 (26%) 0.17 Slow deterioration of graft function 51 (35%) 19 (34%) 32 (36%) 0.80 Stable impaired graft function 15 (10%) 9 (16%) 6 (7%) 0.07 Investigate proteinuria 21 (14%) 7 (13%) 14 (16%) 0.59 Follow-up from previous biopsy 10 (7%) 5 (9%) 5 (6%) 0.51 Others 4 (3%) 1 (2%) 3 (3%) 1.00 Indication unknown 9 (6%) 3 (5%) 6 (7%) 1.00 Of 145 biopsies, 22 biopsies were diagnosed as TCMR, 26 borderline, 12 ABMR, three mixed ABMR plus TCMR, three BK nephropathy and 79 other, according to the Banff criteria. De novoDSA at the time of biopsies for clinical indications Patient sera drawn at the time of biopsy were screened for HLA antibodies, and the results were expressed as % PRA. PRA+ (≥5%) sera were then tested for DSA (Figure 1), and the results were expressed as positive or negative. If DSA was present, the serum drawn immediately pretransplant was examined to determine whether DSA existed at the time of transplant or developed de novo. Figure 1Open in figure viewerPowerPoint Flow chart of HLA antibodies analysis for 145 patients showing the number of serum samples and percent of samples with respect to the previous group. Of 145 patient sera tested, 84 (58%) were PRA+ and 61 (42%) were PRA negative (Figure 1). Within the 84 PRA+ patients, 54 (64% of PRA+) were DSA+ and 30 (36% of PRA+) were DSA negative. Most DSA+ cases (32/54 (59%)) displayed de novo DSA; the remaining 22 (41%) showed preexisting DSA. PRA and DSA were predominantly found in sera from patients having late biopsies In other studies, we have observed an excess of PRA and DSA in sera from patients presenting late (>1-year posttransplant) with biopsy indications (Einecke et. al., in press). We now examined in more detail the relationship of HLA antibodies to time of the biopsy posttransplant (Figure 2). A marked difference was observed at approximately 1 year. PRA was more frequent in patients having late biopsies, but within these late biopsies there was no apparent relationship between time and PRA I or PRA II (Spearman r = 0.107 (p = 0.318), r =−0.015 (p = 0.889), for %PRA I and II, respectively). Figure 2Open in figure viewerPowerPoint Scatter plot of %PRA values over time posttransplant for sera from patients undergoing a biopsy. Each dot represents a single %PRA value against an HLA class I panel (upper panel) or HLA class II panel (lower panel). We examined the incidence and specificity of preexisting and de novo DSA to class I, class II or both in patients undergoing a late or an early biopsy. Sera from patients presenting for late biopsies were frequently DSA+ (49/89) (Figure 3A). The majority of DSA in patients with late biopsies was de novo (30/49), with 19/49 preexisting and was primarily DSA II for both preexisting and de novo DSA. In contrast, DSA was detectable in only 5/56 patients biopsied early: three preexisting and two de novo (Figure 3B). The number of PRA+ DSA negative patients was similar between patients undergoing an early or late biopsy. However, the term non-DSA used in this manuscript is an assumption given that some PRA+ non-DSA sera may contain DSA with specificities to Cw and DP. Antibodies to Cw were not always tested for and DP typing was not available in this cohort. Thus patients presenting for late biopsies were more likely to have de novo DSA against class II compared to patients undergoing an early biopsy. Figure 3Open in figure viewerPowerPoint DSA distribution in patients undergoing early or late biopsies for clinical indication. (A) Stacked bar graph denotes the number of patients who underwent a late (>1-year posttransplant) biopsies and the type of HLA antibodies detected. (B) Stacked bar graph denotes the number of patients who underwent an early (<1-year posttransplant) biopsies and the type of HLA antibodies detected. Associations ofde novoDSA with histopathology lesions in late biopsies We examined the association of DSA at the time of biopsy with the histopathology findings in the biopsy. The strength of preexisting DSA and de novo DSA was not quantified in this study. Given the strong association of DSA with late biopsies, we restricted this analysis to late biopsies. Lesions and their abbreviations are explained in Table S1. Patients were split according to their DSA status; PRA negative, PRA+ DSA negative, preexisting DSA+ and de novo DSA+. Mean scores for lesions representing microvascular inflammation (g, ptc), microvascular damage (cg, mm, ptcml) and intimal arteritis (v) differed across the DSA subgroups (p < 0.05, one-way ANOVA) (Table 2). In contrast, scarring lesions (interstitial fibrosis (ci), tubular atrophy (ct), arterial hyalinosis (ah)) were not different across the DSA subgroups. Thus in late biopsies, de novo DSA is associated with microvascular lesions but not with scarring lesions. Table 2. Mean lesion scores for Banff lesions that differ among DSA subgroups based on one-way ANOVA analysis for patients undergoing a late biopsy Banff lesion Lesion category PRA neg (n = 25) PRA + DSA neg (n = 16) Pre-ex DSA (n = 19) De novo DSA (n = 30) ANOVA p-Value1 ptc Microvascular inflammation 0.04 0.50 0.83 1.28 0.0001 ptcml Microvascular deterioration 3.46 2.83 4.06 7.13 0.0006 g Microvascular inflammation 0.12 0.38 0.37 0.72 0.0066 mm Microvascular deterioration 0.64 0.50 1.16 1.28 0.0081 cg Microvascular deterioration 0.52 0.25 0.89 1.24 0.0121 v Inflammation 0.00 0.00 0.06 0.25 0.0431 ci Scarring 1.72 1.19 1.74 1.79 0.2311 cv Scarring 1.76 1.27 1.50 1.54 0.4384 ct Scarring 1.64 1.31 1.63 1.76 0.4547 i Inflammation 1.12 1.25 1.42 1.38 0.5341 t Inflammation 0.52 1.00 0.74 0.62 0.5872 ah Scarring 1.84 1.69 1.90 1.52 0.6151 1Kruskal–Wallis test. Lesions that differed across the DSA subgroups in late biopsies were examined further using a post hoc test (Dunn's Multiple Comparison) (Figure 4). Biopsies from patients with de novo DSA had higher lesion scores for microvascular inflammation (g, ptc) and damage (cg, mm, ptcml) and intimal arteritis (v) compared to biopsies from PRA negative patients (p < 0.05). Biopsies from patients with preexisting DSA showed higher ptc scores than biopsies from PRA negative patients (p < 0.05). Biopsies from PRA+ DSA negative patients were similar to biopsies from PRA negative patients for all lesions. Thus de novo DSA in serum at the time of a late biopsy for clinical indication increases the probability that lesions of microcirculation inflammation and damage will be present in the biopsy. Given that the majority of the de novo DSA in this cohort is DSA II, these findings are in agreement with other studies showing associations of HLA class II antibodies and cg lesions (14). The significance of preexisting DSA is less clear, being only associated with peritubular capillaritis but not significantly with other microcirculation changes (glomerulitis or microvascular damage). Figure 4Open in figure viewerPowerPoint Mean Banff lesion scores for all late biopsies for the lesions whose scores were found to differ across DSA subgroups detected in the patient sera at the time of biopsy. Bar graphs denote mean lesion score ± SE. The p-values represent the results of the posttest (Dunns multiple comparison) that compared biopsies of each DSA subgroup to PRA negative patients. De novoDSA and detection of C4d deposition in late biopsies C4d staining was available in 138/145 biopsies for which frozen sections were available. A total of 15 biopsies showed C4d positivity (diffuse peritubular capillary staining) by Banff criteria (8). C4d positivity was not detected in any early biopsies. In late biopsies, C4d positivity was primarily found in DSA positive patients, either de novo (10/15) or preexisting (3/15) (Figure 5). Interestingly, 67% of biopsies from patients with de novo DSA did not show C4d deposition, in agreement with the uncommon concurrence of C4d and DSA described (15, 16). C4d deposition was detected in two late biopsies from patients lacking DSA: one PRA negative and one with PRA but no DSA. Thus in patients presenting for late biopsies, absence of DSA makes C4d positivity unlikely (2/84) in agreement with other studies (15, 17). Figure 5Open in figure viewerPowerPoint Detection of C4d deposition in late biopsies according to DSA subgroup of the sera for the respective biopsy. Stacked bar graph denotes the number of biopsies scored as C4d+ (>50% diffuse linear staining) in grey and C4d negative or focal C4d staining in white. Number in parenthesis is the number of biopsies as a percentage of all biopsies in the study. Effects of de novo DSA on graft survival in patients undergoing late biopsies Death-censored allograft survival after a late biopsy was analyzed according to the DSA status of the patient at the time of biopsy. Death-censored graft failure (return to dialysis) occurred in 22 of 145 patients and five patients died with functioning grafts. Kidney allograft survival in PRA negative patients was similar to that of PRA+ DSA negative patients and patients with preexisting DSA (p > 0.05, log-rank test) (Figure 6). Grafts from patients with de novo DSA showed the poorest graft survival (47.6% survival, 13 losses), significantly lower than PRA negative cases (p = 0.001, log-rank test). With the predominance of DSA II in the de novo DSA+ sera, our findings corroborate other studies showing association of anti-class II with poor allograft outcome (18, 19). Figure 6Open in figure viewerPowerPoint Probability of graft survival curves in patients who underwent a late biopsy and were assessed for DSA. Probability of graft survival in patients in each DSA subgroup was compared to patients with no detectable HLA antibodies (PRA neg). N depicts the number of patients in each group and F; the number of failed grafts. *p = 0.001 (log-rank test). These conclusions were robust even when glomerulonephritis cases were analyzed as a separate group. Glomerulonephritis in a biopsy carries a risk of subsequent graft loss (20). After excluding glomerulonephritis cases, 18 kidneys progressed to graft loss after a late biopsy, of which 14 were DSA positive (12 de novo DSA, two preexisting DSA) (Figure S1). Two of the four cases of glomerulonephritis that were lost had de novo DSA but were negative for C4d, raising the possibility that some cases labeled glomerulonephritis may be undergoing antibody-mediated injury. Discussion This study indicates that de novo DSA is frequent in patients presenting with indications for a late renal transplant biopsy and is associated with microcirculation lesions and subsequent graft loss. The PRA status predicted the de novo DSA status: when PRA II was present, de novo DSA was common, either DSA II alone or DSA I/II. PRA I alone was rarely associated with de novo DSA. De novo DSA was associated with microcirculation changes characteristic of ABMR (microcirculation inflammation (glomerulitis (g), peritubular capillaritis (ptc)) and microcirculation deterioration (transplant glomerulopathy (cg), mesangial matrix increase (mm), peritubular capillary basement membrane multilayering (ptcml-score)) and with diffuse C4d positivity. Intimal arteritis (v), which is associated with ABMR but also with TCMR (8, 21), was weakly associated with de novo DSA. De novo DSA was not associated with lesions typical of TCMR (tubulitis (t), interstitial inflammation (i)). Surprisingly, de novo DSA was not associated with higher degrees of scarring or arterial fibrosis, which are the lesions common in late biopsies, when compared with PRA negative cases. Thus de novo DSA at the time of a late biopsy for clinical indications increases the probability that the biopsy will demonstrate ABMR features and subsequent graft deterioration. The significance of preexisting DSA was unclear and requires further study. The present late biopsy population allows us to study why kidneys fail, indicating that many do so because they develop a disease in which de novo DSA stresses their microcirculation, causing them to present with clinical indications for biopsy at a median of about 6 years posttransplant. This unique patient population differs from other studies, offering a cross-section of kidney transplants at risk for deteriorating. This prospective study of biopsies of consenting patients in 2004–2007 sampled the troubled transplants in the regional transplant population, transplanted over many decades and finds de novo DSA associated with reduced graft survival. In patients followed for shorter periods posttransplant, de novo DSA detected at the time of rejection is also associated with reduced graft survival regardless of C4d deposition (22), suggesting that this phenotype leads to poor graft outcome regardless of time posttransplant. What this study does not (and cannot) address is two major questions: when (and why) do patients start to make de novo DSA, and when (and why) do those with de novo DSA develop evidence of renal injury. Future studies are needed to answer these questions. DSA is mainly detectable at the time of late biopsies within this study population arguing that time must be taken into account to properly identify antibody-mediated lesions. Limiting the analysis of lesions associated with de novo DSA to late biopsies differentiates lesions associated with antibody-mediated damage from those associated with time. Fibrosis, atrophy, and arterial fibrous intimal thickening are commonly found in late biopsies (23) but are also included in the diagnostic criteria for the diagnosis of chronic active ABMR (10), suggestive of an association with antibody-mediated injury. However, this study shows that scarring in biopsies from patients with de novo DSA was similar to those from PRA negative patients in a population of late biopsies. Thus scarring acts a nonspecific, time-dependent feature rather than a feature of antibody-mediated injury. In other words, de novo DSA and scarring lesions are time associated variables and their association should not be directly extrapolated to a mechanistic link between the two. The biopsies from patients with preexisting DSA were inconclusive but suspicious, and preclude concluding that preexisting DSA is benign. In this study, preexisting DSA primarily indicates patients transplanted from earlier eras, before current crossmatching methods and practices were developed (24). These biopsies showed higher scores for peritubular capillaritis compared to those from PRA negative patients, but most of the other analyses were not significant. The strength of the antibodies in the preexisting DSA and de novo DSA may play a role but this was not a parameter examined in this study. It is equally as likely that it is not the strength of the antibody at the time of biopsy but rather the trend of the antibody response that determines the histopathology lesions. However, we lack serial sera samples from the patients in this cohort to examine trends of the HLA antibody response but this should be a parameter to be considered in future studies. The trends observed with preexisting DSA warrant further study and may show significance in a study when a larger number of cases with preexisting DSA is examined. The low frequency of HLA antibodies in patients requiring an early biopsy for clinical indication, and of early ABMR probably reflects the effectiveness of current antibody scree