Presence Of Donor-specific Alloantibodies Research Articles

Long-term Outcomes of Kidney Transplantation in Patients With High Levels of Preformed DSA: The Necker High-Risk Transplant Program.

There is an increasing number of anti-HLA sensitized and highly sensitized renal transplant candidates on waiting lists, and the presence of donor-specific alloantibodies (DSAs) at the time of transplantation leads to acute and chronic antibody-mediated rejection (AMR). Acceptable short-term outcomes have been described, notably because of desensitization protocols, but mid- and long-term data are still required. Our high immunologic risk program included 95 patients with high peak or day 0 DSA levels (mean fluorescence intensity [MFI] > 3000) with a complement-dependent cytotoxicity-negative crossmatch, who received a posttransplant desensitization protocol starting at day 0 with high-dose intravenous immunoglobulin, plasma exchanges, and eventually rituximab. Their characteristics were compared with a control group including 39 patients with a lower immunologic risk (MFI between 500 and 3000 at day 0) who received the same posttransplant desensitization. The median MFI of the immunodominant class I or II DSA in the peak or day 0 serum was 9421 (interquartile range, 4959-12 610). An AMR occurred during the first posttransplant year in 31 patients (32.6%), and at one year, the rate of chronic AMR was 39.5%. The 1-, 3-, 5- and 7-year death-censored allograft survival rates were 98%, 91%, 86%, and 78%, respectively, with concomitant recipient survival rates of 97%, 93%, 85%, and 79%, respectively. These results suggest that DSA-sensitized patients with high MFI levels can receive transplantation across the HLA-barrier, with the use of an intensified posttransplant immunosuppressive therapy starting at day 0 combined with close clinical, immunologic, and histologic monitoring.

0113: Cardiac magnetic resonance T1 mapping pre and post contrast in heart transplant patients with clinical antibody-mediated rejection: a preliminary experience

Antibody-mediated rejection (AMR) is characterized by histopathological and immunophenotypic findings such as activated endothelial cells, intravascular macrophages and evidence of capillary C4d deposition. This inflammatory reaction could be followed by diffuse fibrosis. Cardiac magnetic resonance (CMR) with recently T1 mapping is a promising technique to identify diffuse myocardial fibrosis. The purpose of this study was to assess T1 mapping in patients with AMR. 2 patients with clinical AMR (histopathological and immunophenotypic findings, presence of donor-specific allo antibodies and allograft dysfunction) performed a CMR study one week (for the first patient) and 3 weeks (for the second patient) after the treatment of AMR (plasmapheresis, IV Immunoglobulins and Rituximab). Images were acquired on a 1.5 Tesla scanner (Siemens) including T1 mapping using a shortened modified look-locker inversion-recovery sequence and T2 mapping in a matched mid-ventricular short axis slice using a black- blood single shot fast spin echo pulse sequence. Segmental and global T1 values were measured before and 15 minutes after administration of 0.2 mmol/kg of Gadoteric acid and compared to our cohort of 17 controls. Mean non contrast T1 values were significantly higher in heart transplants patients compared to controls (1100±5ms vs 947±29ms, P<0.001). Segmental T1 values were significantly higher in the 6 regions of interest compared to controls (P <0.001 in all segments). Mean post contrast T1 values were not significantly different in patients and controls. Mean T2 value was higher in patients compared to controls (73±13 vs 50± 4ms), suggesting the presence of global edema. Heart transplant patients with clinical antibody-mediated rejection show a significant increased global and segmental non contrast T1 values suggesting the presence of diffuse myocardial fibrosis. Further studies are required to confirm these data.

Transplantation: Molecular diagnosis of kidney transplant rejection.

Acute or chronic antibody-mediated rejection (ABMR) of kidney allografts is currently diagnosed by the presence of donor-specific alloantibodies and distinct pathological findings in biopsy samples. A new study highlights the potential of molecular diagnostics incorporated into standard criteria for acute ABMR to help identify patients at risk of graft loss.

(292) - Acute Fibrinous and Organizing Pneumonia is a Common Histological Pattern of Acute Lung Injury in Patients Developing Restrictive Allograft Syndrome

identify the histologic features of AMR in allograft biopsies and correlate them with the presence of serologic alloantibodies in lung transplant patients. Methods: We reviewed a consecutive series of lung allograft biopsies stained with hematoxylin-eosin, anti-CD68, and anti-C4d, blinded to serologic data. Findings of ACR were classified by ISHLT criteria. Complement deposition was considered positive if there was capillary endothelial positivity, excluding stromal and larger vessel staining. Macrophages were considered increased if there was linear granular staining in capillary septa. Results: There were 628 biopsies from 111 patients. Panel reactive alloantibodies (PRA) and donor specific antibodies (DSA) were available in 103 and 100 patients, respectively. There were 73 men (64 ± 13 years) and 38 women (60 ± 12 years). PRA was positive in 38% of patients, and DSA in 30%. 24 patients had ≥ 1 biopsy with positive complement staining and 26 patients had increased macrophage density. 65 patients had ≥ 1 episode of ACR (31 A only, 25 AB, and 6 B; all were grade 1 or 2). There was a positive correlation between ACR and PRA to type II HLA antigens (p= .02), any DSA (p= .0016), the number of DSA (p= .0008) as well as the presence of DSA to type II HLA antigens (p= .003). Additionally, there was a positive correlation between increased macrophages and PRA to type II HLA antigens (p= .04), presence of DSA (p= .009) and the number of DSA type II HLA antibodies (p= .02). There was a positive correlation between complement staining and the presence of any DSA (p= .05). Other correlations were not significant. Conclusion: There is a correlation between multiple histologic findings suggestive of rejection and the serologic presence of donor specific alloantibodies, specifically against type II HLA antigens. The correlation is strongest for ACR, followed by the presence of increased capillary septal macrophages, and weakest for complement staining for C4d.

Cardiac magnetic resonance T1 mapping pre and post contrast in heart transplant patients with clinical antibody-mediated rejection: a preliminary experience

Background Antibody-mediated rejection (AMR) is characterized by histopathological and immunophenotypic findings such as activated endothelial cells, intravascular macrophages and evidence of capillary C4d deposition. This inflammatory reaction could be followed by diffuse fibrosis. Cardiac magnetic resonance (CMR) with recently T1 mapping is a promising technique to identify diffuse myocardial fibrosis. The purpose of this study was to assess T1 mapping in patients with AMR. Methods Two patients with clinical AMR (histopathological and immunophenotypic findings, presence of donor-specific allo antibodies and allograft dysfunction) performed a CMR study one week (for the first patient) and 3 weeks (for the second patient) after the treatment of AMR (plasmapheresis, IV Immunoglobulins and Rituximab). Images were acquired on a 1.5 Tesla scanner (Siemens) including T1 mapping using a shortened modified look-locker inversion-recovery sequence and T2 mapping in a matched mid-ventricular short axis slice using a black- blood single shot fast spin echo pulse sequence. Segmental and global T1 values were measured before and 15 minutes after administration of 0.2 mmol/kg of Gadoteric acid and compared to our cohort of 17 controls. Results Mean non contrast T1 values were significantly higher in heart transplants patients compared to controls (1100 ± 5 ms vs 947 ± 29 ms, P < 0.001). Segmental T1 values were significantly higher in the 6 regions of interest compared to controls (P < 0.001 in all segments). Mean post contrast T1 values were not significantly different in patients and controls. Mean T2 value was higher in patients compared to controls (73 ± 13 vs 50 ± 4 ms), suggesting the presence of global edema. Conclusions Heart transplant patients with clinical antibodymediated rejection show a significant increased global and segmental non contrast T1 values suggesting the presence of diffuse myocardial fibrosis. Further studies are required to confirm these data. Funding

Pathology of C4d-negative antibody-mediated rejection in renal allografts

To summarize current evidence supporting the existence of C4d-negative antibody-mediated rejection (AMR) in renal allografts, its potential to cause chronic graft injury, and whether histopathologic features of C4d-negative AMR differ from those of C4d-positive AMR. Recently published molecular, clinicopathologic, and ultrastructural studies provide strong evidence that microvascular injury in the presence of donor-specific alloantibodies (DSA) has the potential to cause interstitial fibrosis/tubular atrophy, transplant glomerulopathy, and graft loss, whether or not peritubular capillary (PTC) C4d is present. Although C4d-positive AMR may represent a more severe form of AMR, recent studies have found that in patients with DSA, microvascular injury (glomerulitis, peritubular capillaritis) is more strongly associated with graft loss than C4d deposition. Our data suggest that C4d-positive and C4d-negative AMR show similar degrees of glomerulitis and peritubular capillaritis, similar frequencies of concurrent cell-mediated rejection, and that both may occur early or late posttransplantation. In renal allografts, microvascular injury in the presence of DSA but with negative C4d staining in PTC nonetheless is indicative of humorally mediated graft injury that has the potential to cause tubular atrophy/interstitial fibrosis, transplant glomerulopathy, and graft loss. Prompt treatment for AMR may prevent or at least delay subsequent development of transplant glomerulopathy.

Sinusoidal C4d deposits in liver allografts indicate an antibody-mediated response: Diagnostic considerations in the evaluation of liver allografts

There is a paucity of data concerning the correlation of complement component 4d (C4d) staining in liver allografts and antibody-mediated rejection. Data about the location and character of C4d deposits in native and allograft liver tissues are inconsistent. We performed C4d immunofluorescence (IF) on 141 fresh-frozen liver allograft biopsy samples and native livers, documented the pattern of C4d IF staining, and correlated the findings with the presence of donor-specific alloantibodies (DSAs). A linear/granular sinusoidal pattern of C4d IF was noted in 18 of 28 biopsy samples obtained after transplantation from patients with positive crossmatch and detectable donor-specific alloantibody (pos-XM/DSA) findings. None of the 59 tested biopsy samples from patients with negative crossmatch and detectable donor-specific alloantibody (neg-XM/DSA) findings were C4d-positive (P < 0.001). No significant association was found between pos-XM/DSA and C4d IF staining in other nonsinusoidal liver compartments. To compare the results of sinusoidal C4d staining with IF and 2 immunohistochemistry (IHC) techniques, C4d IHC was performed on 19 liver allograft biopsy samples in which a sinusoidal pattern of C4d IF had been noted. Sinusoidal C4d IHC findings were negative for 17 of the 19 biopsy samples; 2 showed weak and focal staining, and both patients had pos-XM/DSA findings. Portal vein endothelium staining was present in only 1 IF-stained biopsy sample (pos-XM/DSA) but in 11 IHC-stained biopsy samples (2 of the 11 samples had neg-XM/DSA findings). We conclude that sinusoidal C4d deposits detected by IF in frozen tissue samples from liver allograft recipients correlate with the presence of DSAs and an antibody-mediated alloresponse. These observations are similar to findings reported for other solid organ transplants and can provide relevant information for patient management. Further validation of IHC techniques for C4d detection in liver allograft tissue is required.

Abstract 18156: Donor Specific Alloantibodies to HLA II Antigens Result in Accelerated Cardiac Allograft Vasculopathy Assessed by 3d Volumetric Ivus

BACKGROUND: The immunologic risk associated with preformed donor-specific allo-antibodies (DSA) against human leukocyte antigens (HLA) in heart transplant (HTX) recipients is unclear. The goal of this study was to evaluate potential role of DSA on the progression of cardiac allograft vasculopathy (CAV) in HTX recipients. METHODS: The presence of DSA against HLA was evaluated using solid phase Single Antigen Bead assay (One Lambda, Inc., Canoga Park, CA; Luminex platform) before transplant in 35 HTX recipients. Using alloantibody specification analysis, 11% (4/35) and 20% (7/35) had DSA against donor Class I and II HLA respectively including the one patient who had both (DSA I &amp; II+) . 71% (25/35) of patients had no demonstrable DSA (DSA-). All patients had negative cytotoxicity cross match. Baseline three-dimensional intravascular ultrasound studies (3D IVUS) were performed at 1.0 [0.18, 1.4] year after transplant. Follow up 3D IVUS exams were performed 0.92 [0.85, 1.04] years after the baseline exam. RESULTS: The increase in mean plaque (media and intima) volume (PV) was accelerated in the DSA II+ group as compared to the DSA- group (0.87±1.9 mm (3)/mm vs. 0.16±1.8 mm (3)/mm) but the vessel volume index (follow up vessel volume/ baseline vessel volume) was similar between groups (1.08±0.5 vs. 1.09± 0.4; p=0.9). Plaque index (PI) (PV/vessel volume x 100 [%]) increased significantly in the DSA II+ group (17.9±6.3%) but not in the DSA- group (-13.6±13.2%, P=0.02). None of these values were significant in the DSA I+ only group. Treatment with Sirolimus did not affect PI progression between the DSA II+ and DSA- groups (PI progression: 15.9±8.7% vs. -11.7±7.1%, P=0.03). Age, gender, lipid and uric acid levels, prevalence of diabetes, ischemic time, time between IVUS examinations, the use of steroids, mycophenolate vs. azathioprine or calcineurin inhibitors vs. sirolimus were not different between the groups (P&gt;0.4 for all). CONCLUSIONS: Heart transplant recipients with clearly defined anti-Class II DSA are at risk for accelerated cardiac allograft vasculopathy. Whether interventions to minimize the risk of DSA such as virtual crossmatch, desensitization and complement inhibition will retard CAV remains to be proven.

Diagnosis of antibody-mediated rejection in cardiac transplantation: a call for standardization

Currently clinical antibody-mediated rejection (AMR) requires demonstration of histopathologic changes, presence of donor-specific allo-antibodies and allograft dysfunction to establish the diagnosis. Pathology practice patterns in the immunopathologic evaluation and interpretation of cardiac transplant biopsies vary. Specific recommendations for post-transplant allo-antibody monitoring are lacking. Recently, the occurrence of surveillance biopsies being positive for immunopathologic markers of AMR without concomitant graft dysfunction is increasingly recognized. This review focuses on issues of standardization in the diagnosis of AMR and the need for updated criteria. Concomitant use of C4d and C3d proved to be strongly predictive of the presence of circulating allo-antibodies and allograft dysfunction in heart transplant recipients. Asymptomatic patients with histopathologic evidence of AMR in their biopsies appear to have increased risk for cardiac allograft vasculopathy and cardiovascular mortality. The role of nonhuman leukocyte antigen antibodies in AMR has not been adequately addressed. A consensus on the frequency of AMR screening, antibody panels, interpretation and reporting of stains will enhance standardization of the diagnosis of AMR. Further studies are needed to define asymptomatic or subclinical AMR and to determine the long-term outcome of pathologic evidence of complement activation without allograft dysfunction.

Desensitization Protocol in Highly HLA-Sensitized and ABO-Incompatible High Titer Kidney Transplantation

Background A positive crossmatch indicates the presence of donor-specific alloantibodies and is associated with a graft loss rate of >80%; anti-ABO blood group antibodies develop in response to exposure to foreign blood groups, resulting in immediate graft loss. However, a desensitization protocol for highly HLA-sensitized and ABO-incompatible high-titer kidney transplantation has not yet been established. Methods We treated 6 patients with high (≥1:512) anti-A/B antibody titers and 2 highly HLA-sensitized patients. Our immunosuppression protocol was initiated 1 month before surgery and included mycophenolate mofetil (1 g/d) and/or low-dose steroid (methylprednisolone 8 mg/d). Two doses of the anti-CD20 antibody rituximab (150 mg/m 2) were administered 2 weeks before and on the day of transplantation. We performed antibody removal with 6–12 sessions of plasmapheresis (plasma exchange or double-filtration plasmapheresis) before transplantation. Splenectomy was also performed on the day of transplantation. Postoperative immunosuppression followed the same regimen as ABO-compatible cases, in which calcineurin inhibitors were initiated 3 days before transplantation, combined with 2 doses of basiliximab. Results Of the 8 patients, 7 subsequently underwent successful living-donor kidney transplantation. Follow-up of our recipients showed that the patient and graft survival rates were 100%. Acute cellular rejection and antibody-mediated rejection episodes occurred in 1 of the 7 recipients. Conclusions These findings suggest that our immunosuppression regimen consisting of rituximab infusions, splenectomy, plasmapheresis, and pharmacologic immunosuppression may prove to be effective as a desensitization protocol for highly HLA-sensitized and ABO-incompatible high-titer kidney transplantation.

Immunologic study of the donor-receptor couple

The objective of the study is to evaluate the risk of graft failure. The presence of donor specific alloantibodies and the HLA incompatibilities between donor and receptor must be identified. There are several methods to identify alloandibodies that has different sensitivity and different Prognostic Value. Some define a high risk of hyperacute rejection, others an increase in the risk to loss the graft in defined subgroups. First steps of the pretransplant study identify: a) HLA typing of the receptor and available donors; b) alloantibodies by Complement Dependent Cytotoxicity against Panel (PRA-CDC) and screening of alloantibodies against HLA by Solid Phase; c) in sensitized receptors it can be useful to identify acceptable incompatibilities using Single Antigen Solid Phase technique and to evaluate the &laquo;Virtual Crossmatch&raquo;. Pretransplant study (10 days): a) crossmatch by Citotoxicity (CM-CDC) between receptor and donor; b) crossmatch by Flow-Cytometry (FCCM) between receptor and donor specially indicated in the retransplant. Useful also to discard IgM auto-antibodies. Receptors desensitization: the necessity and success probability of desensitization should be evaluated before treatment. Post-Transplant Monitoring: identify alloantibodies for: a) the differential diagnostic of corticorresistant rejection episodes with humoral component, and b) as a marker of long term reduced graft survival probability in the long term. Final remarks: Evaluation should consider the allosensibilization history of the receptor. The cytotoxicity crossmatch indicates a high risk of hyperacute rejection and is considered a contraindication. The Flow Cytometry crossmatch indicates an increase in the probability to loss the graft in the first year that is low for first transplants (>10%) but higher for retransplantation (>30%). The virtual crossmatch by solid phase indicates an increase in the probability to have an antibody mediated rejection (from 5% to 55%) but did not contraindicate always the transplant.