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Time Course of Pathologic Changes in Kidney Allografts of Positive Crossmatch HLA-Incompatible Transplant Recipients Serena M. Bagnasco,1,4 Andrea A. Zachary,2 Lorraine C. Racusen,1 Lois J. Arend,1 Naima Carter-Monroe,1 Nada Alachkar,2 Susanna M. Nazarian,3 Bonnie E. Lonze,3 Robert A. Montgomery,3 and Edward S. Kraus2 Background. Recipients of incompatible allografts are at increased risk of graft loss. We hypothesized that analysis of sequential biopsies from these grafts could define progression of graft lesions and identify features predictive of progression. Methods. We studied the time course of histologic injury in 745 kidney graft biopsies from 129 patients transplanted with a positive crossmatch human leukocyte antigenYincompatible kidney between 2000 and 2010 (follow-up of 1Y9 years). Results. Graft survival was 98% at 1 year and 80% at 5 years after transplantation. Throughout follow-up, 70% of patients experienced rejection, with 52% showing subclinical rejection in the first year. Cell-mediated rejection was more frequent than antibody-mediated rejection throughout follow-up. Transplant glomerulopathy (TxGN; cgQ1) developed in 47% of patients over the period of the study, as early as 3 months in a few patients. TxGN was preceded by glomerulitis in more than 90% of cases, with a median time interval of 12 months. Glomerulitis and detectable posttransplantation donor-specific antibodies were risk factors for TxGN (PG0.0001 and PG0.05). C4d-negative antibody-mediated rejection manifesting as capillaritis (gQ1 and ptcQ1) with detectable donor-specific antibodies was observed in some recipients (G20%). There was progressively higher average tubulointerstitial scarring (ci+ct) from 3 to 6 to 12 months (PG0.001). Conclusions. Despite good graft survival, a significant incidence of biopsy-proven rejection occurred in this subset of closely monitored human leukocyte antigenYincompatible recipients throughout follow-up. Microcirculation inflammation, particularly glomerulitis, irrespective of C4d, is associated with a high risk of development of TxGN at 1 year. Keywords: Kidney, Transplant, Rejection, Glomerulopathy, Antibody-mediated rejection. (Transplantation 2014;97: 440Y445)
ecent progress in the development of desensitization protocols for recipients with antibodies to donor antigens has made possible transplantation across human leukocyte antigen (HLA) and ABO barriers (1Y4). However, recipients of incompatible allografts remain at risk of harboring persistent donor-specific antibodies (DSA) and developing antibody-mediated rejection (AMR) after transplantation (5), with increased risk of graft loss when compared with compatible transplants (6). As more is known about the mechanisms of graft injury, defining early and late morphologic manifestations of rejection, particularly antibody-
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Support provided by grant RC1 DK098431 from the National Institute of Diabetes and Digestive and Kidney Diseases and by the Charles T. Bauer Foundation (R.A.M.). Part of the results described in this study were presented in poster format at the 2012 Renal Week in San Diego, CA, and in platform presentation at the 2013 USCAP meeting in Baltimore, MD. 1 Department of Pathology, Johns Hopkins University, Baltimore, MD. 2 Department of Medicine, Johns Hopkins University, Baltimore, MD. 3 Department of Surgery, Johns Hopkins University, Baltimore, MD. 4 Address correspondence to: Serena M. Bagnasco, M.D., Department of Pathology, Johns Hopkins University, Ross 632, 720 Rutland Avenue, Baltimore, MD 21205. E-mail: [email protected]
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mediated damage, can offer better insight into the progression of graft deterioration (7, 8). The time course in which pathologic changes develop in living donor recipients undergoing desensitization for preformed DSA (HLA-incompatible) has not been analyzed systematically, and often in outcome analyses, correlations are made using only a single time point for posttransplantation biopsies. The goal of this study was to define the temporal manifestation of pathologic changes associated with rejection and development of chronic injury in serial protocol and indication allograft biopsies in a group of transplant recipients, S.M.B. designed the study and wrote the article. A.A.Z. contributed data and participated in the writing of the article. L.C.R., L.J.A., and N.C.-M. reviewed the biopsies. N.A., S.M.N., and B.E.L. followed up the patients and performed the biopsies. R.A.M. contributed to the writing of the article, performed the transplants, and followed up the patients. E.D.K. maintained the database and participated in the design and writing of the article. Received 26 June 2013. Revision requested 12 July 2013. Accepted 6 September 2013. Copyright * 2013 by Lippincott Williams & Wilkins ISSN: 0041-1337/14/9704-440 DOI: 10.1097/01.TP.0000437177.40551.f4
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requiring pretransplantation desensitization for anti-HLA DSAs. We hypothesize that biopsy findings in sequential graft biopsies, including protocol biopsies, could aid in defining progression of allograft lesions and identifying features predictive of progression in these high-risk patients.
RESULTS Demographics The study cohort consisted of 129 recipients of livedonor kidneys who were desensitized for anti-HLA DSA (HLA-incompatible) (3), who met the criteria for inclusion in the study as described in Patients and Methods. Whereas all patients had DSA at the start of desensitization, at the time of transplantation, 41 patients had no DSA (DSA-), 42 had low-level DSA insufficient to yield a positive flow cytometric crossmatch (DSA+/FCXM-), 27 had DSA sufficient to yield positive FCXM (DSA+/FCXM+), and 19 had DSA sufficient to yield positive cytotoxicity crossmatch (DSA+/CDXM+). The follow-up ranged from a minimum of 1 up to 9 years after transplantation (median: 3.2) and included protocol graft biopsies at 1, 3, 6, and 12 months as well as indication graft biopsies. All patients received kidneys from living donors. The median age of the cohort was 41 years, 68% were female, 81% were white, and 50.4% were receiving a first transplant (Table 1). The median calculated panel-reactive antibody was 93%, and most patients (88%) had both class I and II HLA antibodies before transplantation. Over the follow-up period, five patients died, four with a functioning graft. Death-censored graft survival was 98% at 1 year and 80% at 5 years after transplantation; graft loss occurred in 15 recipients. Graft function based on estimated glomerular filtration rate (eGFR; mL/min/1.73 m2) was 63T19 at 3 months, 54T17 at 1 year (PG0.0001), and 48T24 at 4 years. Pathologic Changes A total of 745 biopsies from 129 patients were available, of which 650 were performed in the first year and included 380 protocol biopsies. The average number of biopsies per patients during the entire follow-up period was 6 (median: 6; range: 3Y12). After the first year, 62 patients required indication biopsies. Incidence and Type of Biopsy-Proven Rejection Throughout follow-up, one or more episodes of biopsyproven rejection were detected in 90 (70%) patients. In the first year, 86 (66%) patients developed rejection (clinical and subclinical, all types), most frequently in the first month (n=62 [66%]), declining below 30% later during the first year, with 52% subclinical rejections (Fig. 1A). Cell-mediated rejection (CMR) was observed more frequently than AMR during the first year after transplantation (Fig. 1B,C), with subclinical CMR in 46 (36%) patients and subclinical AMR in 17 (13%) patients. CMR was also the predominant type of rejection after the first year, detected in 20 patients, whereas AMR occurred in 12 patients. Borderline inflammation was observed in 18 patients. Polyomavirus nephropathy occurred in 9 patients, and thrombotic microangiopathy occurred in 2 patients.
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TABLE 1. Characteristics of the recipients of HLA-incompatible kidney transplant in this study Patient number Gender, male/female Median (range) age at transplantation Ethnicity, white/black/other First transplant Cause of end-stage renal disease Diabetes Hypertension Focal segmental glomerulosclerosis Glomerulonephritis Congenital, Familial Other Unknown Median (range) years follow-up after transplantation Median (range) calculated panel-reactive antibody Median (range) number of HLA mismatches Total Class I Class II DSA present at transplanta No DSAb HLA class I only HLA class II only HLA class I and II Patients with HLA and ABO mismatches a b
129 41/88 41 (19Y71) 105/17/7 65 13 6 8 33 39 20 10 3.2 (1Y9) 93 (0Y100)
5 (1Y9) 2 (1Y5) 2 (1Y5) 40 33 30 25 11
One case had uninterpretable test. Postdesensitization on the day of transplantation.
Fourteen patients never showed biopsy-proven rejection or other pathologic changes on biopsy. Arteritis was detected in the biopsies of 81 patients: in 72 patients in the first year and later in 9 patients, with Banff score of v1 in almost all biopsies, v2 in 6 biopsies, and v3 in 2 biopsies. DSA at time of biopsy was documented in 16 patients with arteritis, with low level (below positive FCXM strength) in 13 and moderate (sufficient for positive FCXM) in 3. The DSA level was unknown at the time of biopsy in the majority of patients with arteritis, and for this reason, it is not possible to determine which proportion of vascular lesions was a manifestation of cell-mediated or antibody-mediated injury. C4d-Negative AMR In 25 recipients, single or multiple biopsies showed C4d-negative capillaritis (gQ1, ptcQ1, C4d0) with no evidence of CMR and separated by more than 30 days from treatment and resolution of biopsy-documented episodes of rejection (CMR or C4d-positive AMR). Of these patients, 16 had evidence of DSA at time of biopsy (8 with sufficient strength to yield positive FCXM and 8 with DSA strength not sufficient to yield positive FCXM). Four patients had no DSA at time of biopsy, and DSA were not tested at time of biopsy in five patients. Thus, C4d-negative AMR (without even minimal or
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P=0.007) and 12-month eGFR (59T16 vs. 50T17 mL/min/ 1.73 m2; P=0.005). Transplant glomerulopathy (TxGN), defined as cgQ1, was detected in the biopsy of 61 (47%) recipients throughout follow-up (hepatitis C and thrombotic microangiopathy were excluded as possible causes of TxGN-like glomerular histologic changes). In those patients who underwent indication biopsies after the first year, the cg scores were, on average, higher than those measured at 1 year (Fig. 2B). There was no significant difference in the 12-month eGFR between patients with or without TxGN. However, at last follow-up (median: 34.1 months), the average last known eGFR in patients with TxGN (36.93T20.57) was below the average last known eGFR of all recipients (46.26T21.88; PG0.01), and a moderate but significant inverse correlation was detected between the degree of TxGN and the last known eGFR value in individual recipients (r=j0.3999; PG0.0001). We examined the time of the first appearance of glomerulitis and the time of first onset of TxGN. The earliest detection of glomerulitis occurred most frequently in the
FIGURE 1. Patients with biopsy-proven rejection during the first year after transplantation. Indication and protocol biopsies are included and grouped according to the time after transplantation. A, patients who experienced any type of rejection. Patients with rejections detected in protocol biopsies, representing subclinical rejection, are shown in gray. Patients with rejections detected in for cause biopsies are shown in black. All remaining patients are shown in white. B, patients who experienced CMR, including patients with subclinical CMR. C, patients who experienced AMR, including patients with subclinical AMR.
focal staining for C4d) probably occurred, but in less than 20% of patients. Glomerulitis and Transplant Glomerulopathy Glomerulitis (gQ1) was detected in a total of 434 biopsies from 94 patients. Glomerulitis was most frequent in the first month and was detected in some protocol biopsies in the absence of diagnostic signs of rejection (Fig. 2A). Glomerulitis was associated with leukocyte margination in peritubular capillaries (ptcQ1) in more than 80% of biopsies and with positive C4d in 141 biopsies. Sixty-nine patients with glomerulitis (gQ1) in biopsies at 1 month compared with those without glomerulitis (g=0) had significantly lower 6-month eGFR (60T16 vs. 53T13 mL/min/1.73 m2;
FIGURE 2. A, patients with glomerulitis (gQ1) all in serial biopsies in the first year and patients in whom glomerulitis was present without diagnostic evidence of rejection in protocol biopsies. B, TxGN (cg score) in graft biopsies of individual patients. Data are meanTSD for biopsies taken at the indicated time after transplantation, with difference among groups tested by ANOVA.
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* 2013 Lippincott Williams & Wilkins
first 3 months after transplantation (mean: 3.8T5.2 months; median: 1.2 month) (Fig. 3A), whereas the average time of onset of TxGN was 17.6T16.7 months, with a median first detection at 12 months (Fig. 3A). Fifty-eight patients, 61% of those with glomerulitis (gQ1), developed TxGN (cgQ1). Of the 61 patients who developed TxGN, 58 (95%) had previous evidence of glomerulitis. In this group, the time interval between detection of glomerulitis and first appearance of TxGN was 15T15 months, with a median time interval of 12 months (Fig. 3B). There was no significant difference in this time interval between patients in whom the preceding glomerulitis was associated with positive C4d (n=38; 14T18 months) and those in whom the preceding glomerulitis was associated with negative C4d (n=20; 18T18 months; P=0.37). We estimate that glomerulitis detected in the first month may confer a significant risk of developing TxGN (cgQ1) by the first year after transplantation (PG0.0001; odds ratio [OR], 8.33; 95% confidence interval [CI], 2.3Y29.6). Patients with any DSA in the posttransplantation period had a significantly higher probability of developing TxGN compared with those patients where DSA were not detected at 1 year (PG0.05, Fisher’s exact test; OR, 3.100; 95% CI, 0.96Y9.924) and over the entire follow-up (PG0.02, Fisher’s exact test; OR, 2.745; 95% CI, 3.48Y16.40). The impact of posttransplantation DSA specificity and strength on development of TxGN could not be determined, because such information was not available at the time of biopsy in several patients. DSA present after desensitization at the time of transplantation did not seem to be associated with increased probability to develop TxGN (P=0.88).
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Tubulointerstitial Scarring The average tubulointerstitial scarring increased significantly from 3 to 6 to 12 months within the first year (Fig. 4), with 41% of recipients showing ci+ctQ1 at 3 months and 71% of recipients showing ci+ctQ1 at 1 year. There was a small but significant inverse correlation between the degree of tubulointerstitial scarring at last follow-up and the last known eGFR value in individual recipients (r=j0.2992; PG0.0006). Episodes of CMR in the first year did not result in a higher ci+ct score at 12 month and were not associated with decreased graft function compared to the whole group.
DISCUSSION This study examines the patterns and time course of histologic allograft features in serial graft biopsies in individual HLA-incompatible transplant recipients with pretransplantation DSA. In these high-risk transplant recipients, CMR occurred more frequently than AMR. The overall observed incidence of CMR was higher than AMR and remained such throughout follow-up. However, patients with CMR did not show worse tubulointerstitial scarring or decreased graft function over time, consistent with previous observations that the acute lesions of CMR are not usually associated with increased risk of graft loss (9, 10), although, if not promptly eradicated, they may contribute to tubulointerstitial scarring. Further insights into the relative roles of cellular and antibody-mediated pathways resulting in chronic injury need to be pursued, as the contribution of CMR may be underappreciated in the pathophysiology of vascular lesions, particularly late vasculitis.
FIGURE 3. A, time of first detection of glomerulitis (gQ1; 3.88T0.73 months; n=94) and TxGN (cgQ1; 17.55T2.11 months; n=61) throughout follow-up (one data point is beyond 72 months). B, time interval between appearance of glomerulitis gQ1 and TxGN cgQ1 in the biopsy of 58 patients who developed TxGN after glomerulitis. The mean is indicated by a vertical bar.
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FIGURE 4. Tubulointerstitial scarring (ci+ct score) in graft biopsies of individual patients. Data are meanTSD for biopsies taken at the indicated time after transplantation, with difference among groups tested by ANOVA.
In sequential analysis, the highest incidence of CMR and AMR rejection was detected within the first month after transplantation, consistent with a previous study on a much smaller number of HLA-incompatible recipients from our center (8). This study confirms a significant incidence of subclinical rejection (51% for all types of rejection in the first year), similar to previous findings in HLA-incompatible transplant recipients with stable graft function (8). Overall, subclinical AMR was observed in a relatively small proportion of patients (13%), much lower than the 34% reported by Loupy et al. in recipients of deceased-donor kidneys with DSA (11). Recent studies suggest that microcirculation inflammation in glomeruli and peritubular capillaries may be a more reliable indicator of early injury from circulating DSA than C4d deposition in peritubular capillaries detected in allograft biopsies (12, 13) and may evolve into TxGN. However, the time of first appearance and the time interval between early and chronic endothelial lesions has remained so far undefined. This study provides new information in this respect, showing prevalent onset of glomerulitis in the first 3 months, and showing that 61% of the patients with glomerulitis develop TxGN within an average of 15 months. This observation warrants further study with a view toward earlier therapeutic intervention. The finding of correlation between the presence of DSA after transplantation and development of TxGN supports and expands previous observations of significant relationship between early posttransplantation DSA and acute histologic lesions of AMR in positive crossmatch kidney transplants (14). There are some potential limitations in this study. Given the necessity of sufficient number of biopsies in the first year after transplantation to analyze pathologic findings over time, the inclusion criteria were met by part (but still a substantial number) of all HLA-incompatible recipients transplanted from 2000 to 2010. Information on DSA measurement after transplantation was not available in sufficient detail for all biopsies in all patients to analyze the specificity of DSA relative to the development of specific pathologic lesions in the graft and will have to be addressed in further study. We were not
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able to determine which proportion of the arteritis (v1 to v3) detected in the graft biopsies could be categorized as antibodymediated vascular injury recently proposed by Lefaucheur (15) or as ‘‘v1 isolated lesion’’ as proposed in the last Banff transplant conference (16). Further investigation will be necessary to define the outcome of those HLA-incompatible recipients in whom the nature of the vascular injury can be attributed to antibody-mediated mechanisms. Our study defines the prevalent time course of pathologic changes associated with rejection in serial graft biopsies of HLA-incompatible recipients. Our findings emphasize the negative impact of antibody-mediated lesions, despite acute CMR being the more frequent type of rejection throughout follow-up. TxGN occurred in a significant number of HLA-incompatible recipients with previous glomerulitis. We show that, in the majority of cases, TxGN developed in 1 year or less from detection of glomerulitis, which has not been described previously. These findings underscore the importance of protocol biopsies in detecting early microcirculation inflammation.
MATERIALS AND METHODS Patients To define early and later posttransplantation histologic features in the graft biopsies in the first year, a minimum of at least two protocol biopsies, of which one was performed at 1 or 3 months and one at 6 or 12 months after transplantation, were considered necessary. This requisite was met by 129 recipients out of a total of 218 patients who received a positive crossmatch HLA-incompatible kidney transplant between 2000 and 2010 at The Johns Hopkins Hospital. Protocol biopsies (1, 3, 6, and 12 months) and indication biopsies were examined for all the patients during the entire posttransplantation follow-up. DSAs were present in all patients at the time desensitization was initiated, detected by solid-phase immunoassay, and/or reactivity in a cytotoxic or flow cytometric assay. Eleven of these patients received grafts from donors who were both HLA and ABO incompatible, and the pathologic and clinical findings in these patients did not appear different from the rest of the group to warrant exclusion. Indication biopsies were performed for an increase in serum creatinine Q20% from baseline. Graft failure was defined by irreversible deterioration of graft function requiring initiation of dialysis or retransplantation. The study was approved by the Johns Hopkins Institutional Review Board (protocol NA_00001141).
Biopsies Allograft biopsies were performed using an 18-gauge spring-loaded biopsy needle under real-time ultrasound guidance and processed as described previously (17). Staining for C4d was performed on frozen tissue by indirect immunofluorescence using anti-human C4d antibody (Quidel, San Diego, CA) at 1:40 dilution followed by secondary antibody (fluorescein isothiocyanateYconjugated goat anti-mouse IgG; Jackson Immunoresearch Laboratories, West Grove, PA) or on paraffin-embedded tissue sections using a rabbit polyclonal anti-human antibody (American, Pfungstadt, Germany) at a 1:50 dilution, coupled with a biotin-free polymer detection system (Leica, Wetzlar, Germany). Biopsies were evaluated for CMR and AMR based on update Banff ‘97 criteria (18, 19). C4d staining was considered positive if present in 50% or more of the peritubular capillaries with intensity Q1+ (C4d2-3). The Banff histologic parameters were used to score biopsies for tubulointerstitial scarring as ci (interstitial fibrosis)+ct (tubular atrophy), presence of glomerulitis as gQ1, capillaritis in peritubular capillaries as ptcQ1, and chronic TxGN as cgQ1. Biopsies where evidence of immune complex glomerulonephritis was detected by immunofluorescence and electron microscopy were excluded from the analysis of glomerulitis and TxGN.
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Immunosuppression To remove DSA before transplantation, patients were treated with alternateday plasmapheresis and 100 mg/kg intravenous hyperimmunoglobulin (CMVIg: Cytogam or Medimmune, Gaithersburg, MD). The immunosuppressive regimen included induction with either daclizumab or antithymocyte globulin, tacrolimus, mycophenolate, and steroids. Episodes of cellular rejection (both clinical and subclinical) with Banff grades of 1A or 1B were treated with a 3-day pulse of dexamethasone 100 mg per day followed by a taper. If the Banff score was 2A, 2B, or 3, patients received a 7-day course of antithymocyte globulin. Both clinical and subclinical C4d-positive AMR in the presence of DSA was treated with plasmapheresis/intravenous immunoglobulin. Therapy was discontinued when the flow crossmatch converted to negative and/or AMR resolved on histologic examination. However, C4d-negative histopathologic lesions (e.g., isolated peritubular capillaritis or glomerulitis) were not treated.
Compatibility Testing and DSA Monitoring HLA typing was performed by sequence-specific primer amplification (HLA-DR, DQ; MicroSSP, LABType; One Lambda, Canoga Park, CA) or by reverse sequence-specific oligonucleotide probe hybridization. Typing was performed for donors and recipients for allele groups encoded by HLA-A, -B, -C, -DRB1, -DRB3-5, and -DQB1 loci. Lymphocyte crossmatch was performed by complement-dependent cytotoxicity (20) and flow cytometry as described previously (21, 22). Antibody characterization was performed in tests of phenotype panels (Lifecodes ID; Gen-Probe Life Sciences, Stanford, CT) and single antigen panels (Single Antigen Beads, One Lambda) on the Luminex platform. HLA-specific antibody levels were determined by actual crossmatch or virtual crossmatch (23).
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Outcome Variables Collected demographic information included age, gender, and race. Clinical information included primary diagnosis and cause of end-stage renal disease before transplantation, number of transplants, mismatches among class I and II HLA antigens, and DSA measurement closest (within 1 week) to the time of biopsy. The eGFR was calculated by the Modified Diet in Renal Disease formula (24).
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Statistical Analysis All values are expressed as meanTSD, median, OR, 95% CI, and P values as appropriate. Comparisons among groups were performed by analysis of variance (ANOVA) with Tukey’s or Bonferroni’s post-test analysis. Graft survival was evaluated by KaplanYMeier method. Correlations were analyzed with Pearson’s test. Categorical variables were analyzed with chi-square statistics and Fisher’s test. PG0.05 (two tailed) was considered indicative of statistical significance.
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Glotz D, Antoine C, Julia P, et al. Desensitization and subsequent kidney transplantation of patients using intravenous immunoglobulins (IVIg). Am J Transplant 2002; 2: 758. Montgomery RA, Locke JE, King KE, et al. ABO incompatible renal transplantation: a paradigm ready for implementation. Transplantation 2009; 87: 1246. Montgomery RA, Lonze BE, King KE, et al. Desensitization in HLAincompatible kidney recipients and survival. N Engl J Med 2011; 365: 318. Stegall MD, Gloor J, Winters JL, et al. A comparison of plasmapheresis versus high-dose IVIG desensitization in renal allograft recipients
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with high levels of donor specific alloantibody. Am J Transplant 2006; 6: 346. Mauiyyedi S, Crespo M, Collins AB, et al. Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification. J Am Soc Nephrol 2002; 13: 779. Bentall A, Cornell LD, Gloor JM, et al. Five-year outcomes in living donor kidney transplants with a positive crossmatch. Am J Transplant 2013; 13: 76. Gloor JM, Cosio FG, Rea DJ, et al. Histologic findings one year after positive crossmatch or ABO blood group incompatible living donor kidney transplantation. Am J Transplant 2006; 6: 1841. Kraus ES, Parekh RS, Oberai P, et al. Subclinical rejection in stable positive crossmatch kidney transplant patients: incidence and correlations. Am J Transplant 2009; 9: 1826. Einecke G, Sis B, Reeve J, et al. Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure. Am J Transplant 2009; 9: 2520. Naesens M, Kuypers DRJ, De Vusser K, et al. Chronic histological damage in early indication biopsies is an independent risk factor for late renal allograft failure. Am J Transplant 2013; 13: 86. Loupy A, Suberbielle-Boissel C, Hill GS, et al. Outcome of subclinical antibody-mediated rejection in kidney transplant recipients with preformed donor-specific antibodies. Am J Transplant 2009; 9: 2561. Loupy A, Hill GS, Suberbielle C, et al. Significance of C4d Banff scores in early protocol biopsies of kidney transplant recipients with preformed donor-specific antibodies (DSA). Am J Transplant 2011; 11: 56. Sis B, Jhangri GS, Riopel J, et al. A new diagnostic algorithm for antibody-mediated microcirculation inflammation in kidney transplants. Am J Transplant 2012; 12: 1168. Burns JM, Cornell LD, Perry DK, et al. Alloantibody levels and acute humoral rejection early after positive crossmatch kidney transplantation. Am J Transplant 2008; 8: 2684. Lefaucheur C, Loupy A, Vernerey D, et al. Antibody-mediated vascular rejection of kidney allografts: a population-based study. Lancet 2013; 381: 313. Mengel M, Sis B, Haas M, et al. Banff 2011 meeting report: new concepts in antibody-mediated rejection. Am J Transplant 2012; 12: 563. Bagnasco SM, Tsai W, Rahman MH, et al. CD20-positive infiltrates in renal allograft biopsies with acute cellular rejection are not associated with worse graft survival. Am J Transplant 2007; 7: 1968. Racusen LC, Colvin RB, Solez K, et al. Antibody-mediated rejection criteriaVan addition to the Banff ‘97 classification of renal allograft rejection. Am J Transplant 2003; 3: 708. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant 2008; 8: 753. Hopkins KA. The Basic Lymphocyte Microcytoxicity Test: Standard and AHG Enhancement. 4th ed.Hahn AB, Land GA, Strothman RM, editors. New York: American Society for Histocompatibility and Immunogenetics; 2001. Lucas D, Paparounis M, Myers L, et al. Detection of HLA class Ispecific antibodies by the QuikScreen enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol 1997; 4: 252. Hetrick SJ, Schillinger KP, Zachary AA, et al. Impact of pronase on flow cytometric crossmatch outcome. Hum Immunol 2011; 72: 330. Zachary AA, Sholander JT, Houp JA, et al. Using real data for a virtual crossmatch. Hum Immunol 2009; 70: 574. Levey AS, Bosch JP, Lewis JB, et al. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Ann Intern Med 1999; 130: 461.
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TRANSLATIONAL RESEARCH
Time Course of Pathologic Changes in Kidney Allografts of Positive Crossmatch HLA-Incompatible Transplant Recipients Serena M. Bagnasco,1,4 Andrea A. Zachary,2 Lorraine C. Racusen,1 Lois J. Arend,1 Naima Carter-Monroe,1 Nada Alachkar,2 Susanna M. Nazarian,3 Bonnie E. Lonze,3 Robert A. Montgomery,3 and Edward S. Kraus2 Background. Recipients of incompatible allografts are at increased risk of graft loss. We hypothesized that analysis of sequential biopsies from these grafts could define progression of graft lesions and identify features predictive of progression. Methods. We studied the time course of histologic injury in 745 kidney graft biopsies from 129 patients transplanted with a positive crossmatch human leukocyte antigenYincompatible kidney between 2000 and 2010 (follow-up of 1Y9 years). Results. Graft survival was 98% at 1 year and 80% at 5 years after transplantation. Throughout follow-up, 70% of patients experienced rejection, with 52% showing subclinical rejection in the first year. Cell-mediated rejection was more frequent than antibody-mediated rejection throughout follow-up. Transplant glomerulopathy (TxGN; cgQ1) developed in 47% of patients over the period of the study, as early as 3 months in a few patients. TxGN was preceded by glomerulitis in more than 90% of cases, with a median time interval of 12 months. Glomerulitis and detectable posttransplantation donor-specific antibodies were risk factors for TxGN (PG0.0001 and PG0.05). C4d-negative antibody-mediated rejection manifesting as capillaritis (gQ1 and ptcQ1) with detectable donor-specific antibodies was observed in some recipients (G20%). There was progressively higher average tubulointerstitial scarring (ci+ct) from 3 to 6 to 12 months (PG0.001). Conclusions. Despite good graft survival, a significant incidence of biopsy-proven rejection occurred in this subset of closely monitored human leukocyte antigenYincompatible recipients throughout follow-up. Microcirculation inflammation, particularly glomerulitis, irrespective of C4d, is associated with a high risk of development of TxGN at 1 year. Keywords: Kidney, Transplant, Rejection, Glomerulopathy, Antibody-mediated rejection. (Transplantation 2014;97: 440Y445)
ecent progress in the development of desensitization protocols for recipients with antibodies to donor antigens has made possible transplantation across human leukocyte antigen (HLA) and ABO barriers (1Y4). However, recipients of incompatible allografts remain at risk of harboring persistent donor-specific antibodies (DSA) and developing antibody-mediated rejection (AMR) after transplantation (5), with increased risk of graft loss when compared with compatible transplants (6). As more is known about the mechanisms of graft injury, defining early and late morphologic manifestations of rejection, particularly antibody-
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Support provided by grant RC1 DK098431 from the National Institute of Diabetes and Digestive and Kidney Diseases and by the Charles T. Bauer Foundation (R.A.M.). Part of the results described in this study were presented in poster format at the 2012 Renal Week in San Diego, CA, and in platform presentation at the 2013 USCAP meeting in Baltimore, MD. 1 Department of Pathology, Johns Hopkins University, Baltimore, MD. 2 Department of Medicine, Johns Hopkins University, Baltimore, MD. 3 Department of Surgery, Johns Hopkins University, Baltimore, MD. 4 Address correspondence to: Serena M. Bagnasco, M.D., Department of Pathology, Johns Hopkins University, Ross 632, 720 Rutland Avenue, Baltimore, MD 21205. E-mail: [email protected]
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mediated damage, can offer better insight into the progression of graft deterioration (7, 8). The time course in which pathologic changes develop in living donor recipients undergoing desensitization for preformed DSA (HLA-incompatible) has not been analyzed systematically, and often in outcome analyses, correlations are made using only a single time point for posttransplantation biopsies. The goal of this study was to define the temporal manifestation of pathologic changes associated with rejection and development of chronic injury in serial protocol and indication allograft biopsies in a group of transplant recipients, S.M.B. designed the study and wrote the article. A.A.Z. contributed data and participated in the writing of the article. L.C.R., L.J.A., and N.C.-M. reviewed the biopsies. N.A., S.M.N., and B.E.L. followed up the patients and performed the biopsies. R.A.M. contributed to the writing of the article, performed the transplants, and followed up the patients. E.D.K. maintained the database and participated in the design and writing of the article. Received 26 June 2013. Revision requested 12 July 2013. Accepted 6 September 2013. Copyright * 2013 by Lippincott Williams & Wilkins ISSN: 0041-1337/14/9704-440 DOI: 10.1097/01.TP.0000437177.40551.f4
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requiring pretransplantation desensitization for anti-HLA DSAs. We hypothesize that biopsy findings in sequential graft biopsies, including protocol biopsies, could aid in defining progression of allograft lesions and identifying features predictive of progression in these high-risk patients.
RESULTS Demographics The study cohort consisted of 129 recipients of livedonor kidneys who were desensitized for anti-HLA DSA (HLA-incompatible) (3), who met the criteria for inclusion in the study as described in Patients and Methods. Whereas all patients had DSA at the start of desensitization, at the time of transplantation, 41 patients had no DSA (DSA-), 42 had low-level DSA insufficient to yield a positive flow cytometric crossmatch (DSA+/FCXM-), 27 had DSA sufficient to yield positive FCXM (DSA+/FCXM+), and 19 had DSA sufficient to yield positive cytotoxicity crossmatch (DSA+/CDXM+). The follow-up ranged from a minimum of 1 up to 9 years after transplantation (median: 3.2) and included protocol graft biopsies at 1, 3, 6, and 12 months as well as indication graft biopsies. All patients received kidneys from living donors. The median age of the cohort was 41 years, 68% were female, 81% were white, and 50.4% were receiving a first transplant (Table 1). The median calculated panel-reactive antibody was 93%, and most patients (88%) had both class I and II HLA antibodies before transplantation. Over the follow-up period, five patients died, four with a functioning graft. Death-censored graft survival was 98% at 1 year and 80% at 5 years after transplantation; graft loss occurred in 15 recipients. Graft function based on estimated glomerular filtration rate (eGFR; mL/min/1.73 m2) was 63T19 at 3 months, 54T17 at 1 year (PG0.0001), and 48T24 at 4 years. Pathologic Changes A total of 745 biopsies from 129 patients were available, of which 650 were performed in the first year and included 380 protocol biopsies. The average number of biopsies per patients during the entire follow-up period was 6 (median: 6; range: 3Y12). After the first year, 62 patients required indication biopsies. Incidence and Type of Biopsy-Proven Rejection Throughout follow-up, one or more episodes of biopsyproven rejection were detected in 90 (70%) patients. In the first year, 86 (66%) patients developed rejection (clinical and subclinical, all types), most frequently in the first month (n=62 [66%]), declining below 30% later during the first year, with 52% subclinical rejections (Fig. 1A). Cell-mediated rejection (CMR) was observed more frequently than AMR during the first year after transplantation (Fig. 1B,C), with subclinical CMR in 46 (36%) patients and subclinical AMR in 17 (13%) patients. CMR was also the predominant type of rejection after the first year, detected in 20 patients, whereas AMR occurred in 12 patients. Borderline inflammation was observed in 18 patients. Polyomavirus nephropathy occurred in 9 patients, and thrombotic microangiopathy occurred in 2 patients.
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TABLE 1. Characteristics of the recipients of HLA-incompatible kidney transplant in this study Patient number Gender, male/female Median (range) age at transplantation Ethnicity, white/black/other First transplant Cause of end-stage renal disease Diabetes Hypertension Focal segmental glomerulosclerosis Glomerulonephritis Congenital, Familial Other Unknown Median (range) years follow-up after transplantation Median (range) calculated panel-reactive antibody Median (range) number of HLA mismatches Total Class I Class II DSA present at transplanta No DSAb HLA class I only HLA class II only HLA class I and II Patients with HLA and ABO mismatches a b
129 41/88 41 (19Y71) 105/17/7 65 13 6 8 33 39 20 10 3.2 (1Y9) 93 (0Y100)
5 (1Y9) 2 (1Y5) 2 (1Y5) 40 33 30 25 11
One case had uninterpretable test. Postdesensitization on the day of transplantation.
Fourteen patients never showed biopsy-proven rejection or other pathologic changes on biopsy. Arteritis was detected in the biopsies of 81 patients: in 72 patients in the first year and later in 9 patients, with Banff score of v1 in almost all biopsies, v2 in 6 biopsies, and v3 in 2 biopsies. DSA at time of biopsy was documented in 16 patients with arteritis, with low level (below positive FCXM strength) in 13 and moderate (sufficient for positive FCXM) in 3. The DSA level was unknown at the time of biopsy in the majority of patients with arteritis, and for this reason, it is not possible to determine which proportion of vascular lesions was a manifestation of cell-mediated or antibody-mediated injury. C4d-Negative AMR In 25 recipients, single or multiple biopsies showed C4d-negative capillaritis (gQ1, ptcQ1, C4d0) with no evidence of CMR and separated by more than 30 days from treatment and resolution of biopsy-documented episodes of rejection (CMR or C4d-positive AMR). Of these patients, 16 had evidence of DSA at time of biopsy (8 with sufficient strength to yield positive FCXM and 8 with DSA strength not sufficient to yield positive FCXM). Four patients had no DSA at time of biopsy, and DSA were not tested at time of biopsy in five patients. Thus, C4d-negative AMR (without even minimal or
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P=0.007) and 12-month eGFR (59T16 vs. 50T17 mL/min/ 1.73 m2; P=0.005). Transplant glomerulopathy (TxGN), defined as cgQ1, was detected in the biopsy of 61 (47%) recipients throughout follow-up (hepatitis C and thrombotic microangiopathy were excluded as possible causes of TxGN-like glomerular histologic changes). In those patients who underwent indication biopsies after the first year, the cg scores were, on average, higher than those measured at 1 year (Fig. 2B). There was no significant difference in the 12-month eGFR between patients with or without TxGN. However, at last follow-up (median: 34.1 months), the average last known eGFR in patients with TxGN (36.93T20.57) was below the average last known eGFR of all recipients (46.26T21.88; PG0.01), and a moderate but significant inverse correlation was detected between the degree of TxGN and the last known eGFR value in individual recipients (r=j0.3999; PG0.0001). We examined the time of the first appearance of glomerulitis and the time of first onset of TxGN. The earliest detection of glomerulitis occurred most frequently in the
FIGURE 1. Patients with biopsy-proven rejection during the first year after transplantation. Indication and protocol biopsies are included and grouped according to the time after transplantation. A, patients who experienced any type of rejection. Patients with rejections detected in protocol biopsies, representing subclinical rejection, are shown in gray. Patients with rejections detected in for cause biopsies are shown in black. All remaining patients are shown in white. B, patients who experienced CMR, including patients with subclinical CMR. C, patients who experienced AMR, including patients with subclinical AMR.
focal staining for C4d) probably occurred, but in less than 20% of patients. Glomerulitis and Transplant Glomerulopathy Glomerulitis (gQ1) was detected in a total of 434 biopsies from 94 patients. Glomerulitis was most frequent in the first month and was detected in some protocol biopsies in the absence of diagnostic signs of rejection (Fig. 2A). Glomerulitis was associated with leukocyte margination in peritubular capillaries (ptcQ1) in more than 80% of biopsies and with positive C4d in 141 biopsies. Sixty-nine patients with glomerulitis (gQ1) in biopsies at 1 month compared with those without glomerulitis (g=0) had significantly lower 6-month eGFR (60T16 vs. 53T13 mL/min/1.73 m2;
FIGURE 2. A, patients with glomerulitis (gQ1) all in serial biopsies in the first year and patients in whom glomerulitis was present without diagnostic evidence of rejection in protocol biopsies. B, TxGN (cg score) in graft biopsies of individual patients. Data are meanTSD for biopsies taken at the indicated time after transplantation, with difference among groups tested by ANOVA.
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first 3 months after transplantation (mean: 3.8T5.2 months; median: 1.2 month) (Fig. 3A), whereas the average time of onset of TxGN was 17.6T16.7 months, with a median first detection at 12 months (Fig. 3A). Fifty-eight patients, 61% of those with glomerulitis (gQ1), developed TxGN (cgQ1). Of the 61 patients who developed TxGN, 58 (95%) had previous evidence of glomerulitis. In this group, the time interval between detection of glomerulitis and first appearance of TxGN was 15T15 months, with a median time interval of 12 months (Fig. 3B). There was no significant difference in this time interval between patients in whom the preceding glomerulitis was associated with positive C4d (n=38; 14T18 months) and those in whom the preceding glomerulitis was associated with negative C4d (n=20; 18T18 months; P=0.37). We estimate that glomerulitis detected in the first month may confer a significant risk of developing TxGN (cgQ1) by the first year after transplantation (PG0.0001; odds ratio [OR], 8.33; 95% confidence interval [CI], 2.3Y29.6). Patients with any DSA in the posttransplantation period had a significantly higher probability of developing TxGN compared with those patients where DSA were not detected at 1 year (PG0.05, Fisher’s exact test; OR, 3.100; 95% CI, 0.96Y9.924) and over the entire follow-up (PG0.02, Fisher’s exact test; OR, 2.745; 95% CI, 3.48Y16.40). The impact of posttransplantation DSA specificity and strength on development of TxGN could not be determined, because such information was not available at the time of biopsy in several patients. DSA present after desensitization at the time of transplantation did not seem to be associated with increased probability to develop TxGN (P=0.88).
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Tubulointerstitial Scarring The average tubulointerstitial scarring increased significantly from 3 to 6 to 12 months within the first year (Fig. 4), with 41% of recipients showing ci+ctQ1 at 3 months and 71% of recipients showing ci+ctQ1 at 1 year. There was a small but significant inverse correlation between the degree of tubulointerstitial scarring at last follow-up and the last known eGFR value in individual recipients (r=j0.2992; PG0.0006). Episodes of CMR in the first year did not result in a higher ci+ct score at 12 month and were not associated with decreased graft function compared to the whole group.
DISCUSSION This study examines the patterns and time course of histologic allograft features in serial graft biopsies in individual HLA-incompatible transplant recipients with pretransplantation DSA. In these high-risk transplant recipients, CMR occurred more frequently than AMR. The overall observed incidence of CMR was higher than AMR and remained such throughout follow-up. However, patients with CMR did not show worse tubulointerstitial scarring or decreased graft function over time, consistent with previous observations that the acute lesions of CMR are not usually associated with increased risk of graft loss (9, 10), although, if not promptly eradicated, they may contribute to tubulointerstitial scarring. Further insights into the relative roles of cellular and antibody-mediated pathways resulting in chronic injury need to be pursued, as the contribution of CMR may be underappreciated in the pathophysiology of vascular lesions, particularly late vasculitis.
FIGURE 3. A, time of first detection of glomerulitis (gQ1; 3.88T0.73 months; n=94) and TxGN (cgQ1; 17.55T2.11 months; n=61) throughout follow-up (one data point is beyond 72 months). B, time interval between appearance of glomerulitis gQ1 and TxGN cgQ1 in the biopsy of 58 patients who developed TxGN after glomerulitis. The mean is indicated by a vertical bar.
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FIGURE 4. Tubulointerstitial scarring (ci+ct score) in graft biopsies of individual patients. Data are meanTSD for biopsies taken at the indicated time after transplantation, with difference among groups tested by ANOVA.
In sequential analysis, the highest incidence of CMR and AMR rejection was detected within the first month after transplantation, consistent with a previous study on a much smaller number of HLA-incompatible recipients from our center (8). This study confirms a significant incidence of subclinical rejection (51% for all types of rejection in the first year), similar to previous findings in HLA-incompatible transplant recipients with stable graft function (8). Overall, subclinical AMR was observed in a relatively small proportion of patients (13%), much lower than the 34% reported by Loupy et al. in recipients of deceased-donor kidneys with DSA (11). Recent studies suggest that microcirculation inflammation in glomeruli and peritubular capillaries may be a more reliable indicator of early injury from circulating DSA than C4d deposition in peritubular capillaries detected in allograft biopsies (12, 13) and may evolve into TxGN. However, the time of first appearance and the time interval between early and chronic endothelial lesions has remained so far undefined. This study provides new information in this respect, showing prevalent onset of glomerulitis in the first 3 months, and showing that 61% of the patients with glomerulitis develop TxGN within an average of 15 months. This observation warrants further study with a view toward earlier therapeutic intervention. The finding of correlation between the presence of DSA after transplantation and development of TxGN supports and expands previous observations of significant relationship between early posttransplantation DSA and acute histologic lesions of AMR in positive crossmatch kidney transplants (14). There are some potential limitations in this study. Given the necessity of sufficient number of biopsies in the first year after transplantation to analyze pathologic findings over time, the inclusion criteria were met by part (but still a substantial number) of all HLA-incompatible recipients transplanted from 2000 to 2010. Information on DSA measurement after transplantation was not available in sufficient detail for all biopsies in all patients to analyze the specificity of DSA relative to the development of specific pathologic lesions in the graft and will have to be addressed in further study. We were not
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able to determine which proportion of the arteritis (v1 to v3) detected in the graft biopsies could be categorized as antibodymediated vascular injury recently proposed by Lefaucheur (15) or as ‘‘v1 isolated lesion’’ as proposed in the last Banff transplant conference (16). Further investigation will be necessary to define the outcome of those HLA-incompatible recipients in whom the nature of the vascular injury can be attributed to antibody-mediated mechanisms. Our study defines the prevalent time course of pathologic changes associated with rejection in serial graft biopsies of HLA-incompatible recipients. Our findings emphasize the negative impact of antibody-mediated lesions, despite acute CMR being the more frequent type of rejection throughout follow-up. TxGN occurred in a significant number of HLA-incompatible recipients with previous glomerulitis. We show that, in the majority of cases, TxGN developed in 1 year or less from detection of glomerulitis, which has not been described previously. These findings underscore the importance of protocol biopsies in detecting early microcirculation inflammation.
MATERIALS AND METHODS Patients To define early and later posttransplantation histologic features in the graft biopsies in the first year, a minimum of at least two protocol biopsies, of which one was performed at 1 or 3 months and one at 6 or 12 months after transplantation, were considered necessary. This requisite was met by 129 recipients out of a total of 218 patients who received a positive crossmatch HLA-incompatible kidney transplant between 2000 and 2010 at The Johns Hopkins Hospital. Protocol biopsies (1, 3, 6, and 12 months) and indication biopsies were examined for all the patients during the entire posttransplantation follow-up. DSAs were present in all patients at the time desensitization was initiated, detected by solid-phase immunoassay, and/or reactivity in a cytotoxic or flow cytometric assay. Eleven of these patients received grafts from donors who were both HLA and ABO incompatible, and the pathologic and clinical findings in these patients did not appear different from the rest of the group to warrant exclusion. Indication biopsies were performed for an increase in serum creatinine Q20% from baseline. Graft failure was defined by irreversible deterioration of graft function requiring initiation of dialysis or retransplantation. The study was approved by the Johns Hopkins Institutional Review Board (protocol NA_00001141).
Biopsies Allograft biopsies were performed using an 18-gauge spring-loaded biopsy needle under real-time ultrasound guidance and processed as described previously (17). Staining for C4d was performed on frozen tissue by indirect immunofluorescence using anti-human C4d antibody (Quidel, San Diego, CA) at 1:40 dilution followed by secondary antibody (fluorescein isothiocyanateYconjugated goat anti-mouse IgG; Jackson Immunoresearch Laboratories, West Grove, PA) or on paraffin-embedded tissue sections using a rabbit polyclonal anti-human antibody (American, Pfungstadt, Germany) at a 1:50 dilution, coupled with a biotin-free polymer detection system (Leica, Wetzlar, Germany). Biopsies were evaluated for CMR and AMR based on update Banff ‘97 criteria (18, 19). C4d staining was considered positive if present in 50% or more of the peritubular capillaries with intensity Q1+ (C4d2-3). The Banff histologic parameters were used to score biopsies for tubulointerstitial scarring as ci (interstitial fibrosis)+ct (tubular atrophy), presence of glomerulitis as gQ1, capillaritis in peritubular capillaries as ptcQ1, and chronic TxGN as cgQ1. Biopsies where evidence of immune complex glomerulonephritis was detected by immunofluorescence and electron microscopy were excluded from the analysis of glomerulitis and TxGN.
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Immunosuppression To remove DSA before transplantation, patients were treated with alternateday plasmapheresis and 100 mg/kg intravenous hyperimmunoglobulin (CMVIg: Cytogam or Medimmune, Gaithersburg, MD). The immunosuppressive regimen included induction with either daclizumab or antithymocyte globulin, tacrolimus, mycophenolate, and steroids. Episodes of cellular rejection (both clinical and subclinical) with Banff grades of 1A or 1B were treated with a 3-day pulse of dexamethasone 100 mg per day followed by a taper. If the Banff score was 2A, 2B, or 3, patients received a 7-day course of antithymocyte globulin. Both clinical and subclinical C4d-positive AMR in the presence of DSA was treated with plasmapheresis/intravenous immunoglobulin. Therapy was discontinued when the flow crossmatch converted to negative and/or AMR resolved on histologic examination. However, C4d-negative histopathologic lesions (e.g., isolated peritubular capillaritis or glomerulitis) were not treated.
Compatibility Testing and DSA Monitoring HLA typing was performed by sequence-specific primer amplification (HLA-DR, DQ; MicroSSP, LABType; One Lambda, Canoga Park, CA) or by reverse sequence-specific oligonucleotide probe hybridization. Typing was performed for donors and recipients for allele groups encoded by HLA-A, -B, -C, -DRB1, -DRB3-5, and -DQB1 loci. Lymphocyte crossmatch was performed by complement-dependent cytotoxicity (20) and flow cytometry as described previously (21, 22). Antibody characterization was performed in tests of phenotype panels (Lifecodes ID; Gen-Probe Life Sciences, Stanford, CT) and single antigen panels (Single Antigen Beads, One Lambda) on the Luminex platform. HLA-specific antibody levels were determined by actual crossmatch or virtual crossmatch (23).
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Outcome Variables Collected demographic information included age, gender, and race. Clinical information included primary diagnosis and cause of end-stage renal disease before transplantation, number of transplants, mismatches among class I and II HLA antigens, and DSA measurement closest (within 1 week) to the time of biopsy. The eGFR was calculated by the Modified Diet in Renal Disease formula (24).
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16. 17.
Statistical Analysis All values are expressed as meanTSD, median, OR, 95% CI, and P values as appropriate. Comparisons among groups were performed by analysis of variance (ANOVA) with Tukey’s or Bonferroni’s post-test analysis. Graft survival was evaluated by KaplanYMeier method. Correlations were analyzed with Pearson’s test. Categorical variables were analyzed with chi-square statistics and Fisher’s test. PG0.05 (two tailed) was considered indicative of statistical significance.
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