Donor-specific Antibody Strength Research Articles

Abstract 12052: Non-complement-Binding Donor Specific Antibodies and Cardiac-Allograft Vasculopathy in Pediatric Heart Transplant Recipients

Aim: We hypothesized that the non-complement binding of de novo donor specific antibodies (DSA) detected after heart transplant (HT) confer a lower risk for acute rejections and coronary artery vasculopathy (CAV). The purpose of this study was to compare the clinical outcomes between pediatric HT recipients with C1q+ and C1q- fixing de novo DSA Methods: 127 patients from January 2005 to December 2014 were analyzed in this study to evaluate the outcomes including acute rejections and CAV of pediatric HT recipients who had C1q+ DSA compared to those with C1q- DSA. Serum samples from patients with circulating DSA were analyzed for C1q fixing according to the manufacturer’s protocol (C1qScreen, One Lambda). Results: Out of 127 patients transplanted between 2005 and 2014, 59 (46.4%) patients developed de novo DSA, of those 37 (62.7%) had C1q+ DSA. No difference in baseline characteristics: age at HT, gender, pre-HT diagnosis of CHD, ischemic time, number of HLA mismatch and pre-HT PRA >10. We found that DSA strength is significantly associated with C1q+ (C-statistics 0.89 and p <0.05) and at cut off 7000 MFI has 80% sensitivity and 80% specificity. C1q positive rate varied according to DSA class type: Class I (25% C1q+), class II (66% C1q +) and Class I and II (67% C1q+). C1q+ DSA patients had 2.4 times higher rate of acute rejection episodes compared to those with C1q- DSA. C1q+ DSA patients had significantly higher rates of CAV at 10-yr post-HT compared to C1q- DSA patients (72% vs 28%) (Figure) (P<0.001). C1q+ DSA patients had a trend towards poor allograft survival at 10-yr post-HT compared to C1q-DSA patients (70% vs 52%) (p =0.15). Conclusions: De novo DSA which are C1q+ have significantly greater deleterious effect on long-term allograft outcome in pediatric heart transplant recipients. Thus, measuring DSA and determining their complement binding capacity is helpful in assessing the risk of cardiac allograft vasculopathy. Figure:

Determining donor-specific antibody C1q-binding ability improves the prediction of antibody-mediated rejection in human leucocyte antigen-incompatible kidney transplantation.

Detrimental impact of preformed donor-specific antibodies (DSAs) against human leucocyte antigens on outcomes after kidney transplantation are well documented, however, the value of their capacity to bind complement for predicting antibody-mediated rejection (AMR) and graft survival still needs to be confirmed. We aimed to study DSA characteristics (strength and C1q binding) that might distinguish harmful DSA from clinically irrelevant ones. We retrospectively studied 60 kidney-transplanted patients with preformed DSA detected by single antigen bead (SAB) assays (IgG and C1q kits), from a cohort of 517 kidney graft recipients (124 with detectable anti-HLA antibodies). Patients were divided into DSA strength (MFI < vs. ≥ 15 000) and C1q-binding ability. AMR frequency was high (30%) and it increased with DSA strength (P = 0.002) and C1q+ DSA (P < 0.001). The performance of DSA C1q-binding ability as a predictor of AMR was better than DSA strength (diagnostic odds ratio 16.3 vs. 6.4, respectively). Furthermore, a multivariable logistic regression showed that C1q+ DSA was a risk factor for AMR (OR = 16.80, P = 0.001), while high MFI DSAs were not. Graft survival was lower in high MFI C1q+ DSA in comparison with patients with C1q- high or low MFI DSA (at 6 years, 38%, 83% and 80%, respectively; P = 0.001). Both DSA strength and C1q-binding ability assessment seem valuable for improving pretransplant risk assessment. Since DSA C1q-binding ability was a better predictor of AMR and correlated with graft survival, C1q-SAB may be a particularly useful tool.

OR7 The UCLA virtual crossmatch exchange

The sensitivity and specificity of solid phase assays for HLA antibody identification has given rise to the Virtual Crossmatch (VXM). By providing a means to detect and assess the strength of donor specific antibodies (DSA), solid phase assays have the potential to increase the accuracy of VXM predictions. A Virtual Paper Challenge was offered through the UCLA Flow Crossmatch Exchange in 2015. Initially, participants predicted the T/B XM outcomes for 4 virtual donors against 4 sera based on single antigen bead results and donor specific HLA profiles. In the 1st exchange, 10 laboratories worldwide provided virtual predictions, calculated %PRA, class I/II DSA, and renal transplant risk assessment for each donor–patient pair. In the 2nd exchange, actual cells from 1 original virtual donor were sent (O) and tested against 1 repeat serum (S), allowing comparisons between VXM and actual Flow Cytometric crossmatch (FXM) results. A total of 24 T and 24 B-cell predictions were submitted over 2 exchanges. For the T cell VXM, consensus >80% agreement) was achieved for 22/24 (92%) predictions when DSA strength was strong (MFI > 9000) or multiple DSA were present. For the B cell VXM, 16/24 (67%) predictions reached consensus. Variability in VXM predictions was observed when DSA strength was low to moderate (MFI < 6200) especially when class II DSA was absent but class I DSA were weak. Seven labs reported both VXM and FXM results over the 2 consecutive exchanges for donor–patient pair OS. Comparison of the results showed T-cell XMs were 100% concordant positive/positive for VXM/FXM respectively; whereas, B-cell XMs were 71% concordant with 5 labs reporting positive/positive and 2 labs reporting negative/positive for VXM/FXM, respectively. All judged the renal transplant risk as ‘unacceptable’ for this OS pair from an immunological point of view. The data suggests that virtual crossmatch predictions may vary among laboratories and that the variability may be due to factors such as DSA strength and center practice. Thus, educational activities remain an important tool in ensuring quality control standards for crossmatch testing methods.

P152 Prozone phenomena may skew the strength of Donor Specific Antibody (DSA)

Aim Positive Complement Dependent Cytotoxicity (CDC) and Flow Cytometry Crossmatch (FCXM) usually correlate with high titer of Donor Specific Antibodies (DSA). Now a days assignment of relevant DSA is based on solid phase assay (SPA), in which false low/negative results can be observed due to prozone effect. Here we present three cases underwent a workup for living related kidney transplant that their crossmatch data does not correlate with the single antigen (SA) Median Fluorescent Intensity (MFI) value. Method B cell and/or T cell AHG-CDC, FCXM and antibody identification by SPA were performed for three living related pairs. Results Two patients show positive T cell AHG-CDC crossmatch and one shows positive B cell CDC crossmatch with 8 reaction strength at 1:8 dilution and positive FCXM with very high Median Channel Shift (MCS). However the DSA result was negative for one patient and the other two patients showed A2 (891 MFI) and DQ6 (1299 MFI) respectively. The SA testing indicated possible skewed MFI value, therefore all serum were diluted 1:4 to overcome a prozone effect if present. Conclusion MFI value of the SPA was not correlated with the high MCS and positive CDC crossmatch in undiluted serum due prozone phenomena of high antibody concentration. Therefore serum dilution should be considered if strong crossmatch reactivity observed.

Donor-specific antibodies present at the time of kidney transplantation in immunologically unmodified patients increase the risk of acute rejection

BackgroundHuman leukocyte antigens (HLA) class II donor-specific antibodies (DSAs) are associated with microcirculation inflammation, transplant glomerulopathy and ultimately graft loss. There is however no data on allograft outcomes in deceased donor kidney transplant recipients who have not received any desensitization prior to transplantation. MethodsWe prospectively evaluated the association of HLA DR and DQ DSAs on rejection and short-term graft survival in patients who did not receive desensitization prior to transplantation. On the basis of their cumulative strength of HLA DR and/or DQ DSA, the patients were dichotomized into: 1) median fluorescence intensity (MFI)<1000 and 2) MFI≥1000. ResultsIn the two year study period, 50 consecutive patients with HLA DR and/or DQ sensitization were transplanted in our two centers. Post-transplantation, the incidence of acute rejection was significantly greater in the MFI≥1000 group (35%; 8/22) compared to the MFI<1000 group (7%; 2/28) (p<0.001). There were two graft losses, both in the MFI≥1000 group. ConclusionThe strength of DR and/or DQ DSA at the time of renal transplantation influences the risk of rejection in non-desensitized recipients with HLA class II DSA.

Clinically-relevant threshold of preformed donor-specific anti-HLA antibodies in kidney transplantation

BackgroundPretransplant anti-HLA donor-specific antibodies (DSA) are recognized as a risk factor for acute antibody-mediated rejection (AMR) in kidney transplantation. The predictive value of C4d-fixing capability by DSA or of IgG DSA subclasses for acute AMR in the pretransplant setting has been recently studied. In addition DSA strength assessed by mean fluorescence intensity (MFI) may improve risk stratification. We aimed to analyze the relevance of preformed DSA and of DSA MFI values. Methods280 consecutive patients with negative complement-dependent cytotoxicity crossmatches received a kidney transplant between 01/2008 and 03/2014. Sera were screened for the presence of DSA with a solid-phase assays on a Luminex flow analyzer, and the results were correlated with biopsy-proven acute AMR in the first year and survival. ResultsPretransplant anti-HLA antibodies were present in 72 patients (25.7%) and 24 (8.6%) had DSA. There were 46 (16.4%) acute rejection episodes, 32 (11.4%) being cellular and 14 (5.0%) AMR. The incidence of acute AMR was higher in patients with pretransplant DSA (41.7%) than in those without (1.6%) (p<0.001). The median cumulative MFI (cMFI) of the group DSA+/AMR+ was 5680 vs 2208 in DSA+/AMR− (p=0.058). With univariate logistic regression a threshold value of 5280 cMFI was predictive for acute AMR. DSA cMFI’s ability to predict AMR was also explored by ROC analysis. AUC was 0.728 and the best threshold was a cMFI of 4340. Importantly pretransplant DSA>5280 cMFI had a detrimental effect on 5-year graft survival. ConclusionsPreformed DSA cMFI values were clinically-relevant for the prediction of acute AMR and graft survival in kidney transplantation. A threshold of 4300–5300 cMFI was a significant outcome predictor.

Impact of preformed donor-specific antibodies against HLA class I on kidney graft outcomes: Comparative analysis of exclusively anti-Cw vs anti-A and/or -B antibodies.

AIMTo analyze the clinical impact of preformed antiHLA-Cw vs antiHLA-A and/or -B donor-specific antibodies (DSA) in kidney transplantation.METHODSRetrospective study, comparing 12 patients transplanted with DSA exclusively antiHLA-Cw with 23 patients with preformed DSA antiHLA-A and/or B.RESULTSOne year after transplantation there were no differences in terms of acute rejection between the two groups (3 and 6 cases, respectively in the DSA-Cw and the DSA-A-B groups; P = 1). At one year, eGFR was not significantly different between groups (median 59 mL/min in DSA-Cw group, compared to median 51 mL/min in DSA-A-B group, P = 0.192). Moreover, kidney graft survival was similar between groups at 5-years (100% in DSA-Cw group vs 91% in DSA-A-B group, P = 0.528). The sole independent predictor of antibody mediated rejection (AMR) incidence was DSA strength (HR = 1.07 per 1000 increase in MFI, P = 0.034). AMR was associated with shortened graft survival at 5-years, with 75% and 100% grafts surviving in patients with or without AMR, respectively (Log-rank P = 0.005).CONCLUSIONOur data indicate that DSA-Cw are associated with an identical risk of AMR and impact on graft function in comparison with “classical” class I DSA.

The importance of donor-specific anti-HLA antibodies (DSA) identification in renal transplant patients with C4d-negative biopsies

Two index cases of living-related donor renal allografts patients developed C4d-negative rejection. Both cases had negative cytotoxic crossmatch and negative flow crossmatch before transplantation. The serum creatinine levels were tabulated. Both cases experienced augmented anti-T cell therapy (intravenous methyl prednisolone) at the time of rejection, which failed to improve renal function. Meantime, our HLA lab identified circulating anti-class I and/or II HLA antibodies towards donor mismatched antigens by Luminex multiplex bead array. Additional therapy included high-dose IVIg and plasma exchange. The renal function improved significantly. Furthermore, the donor-specific antibody strength decreased after combined plasmapheresis and IVIG therapy. These cases highlight the importance of donor-specific antibody detection by sensitive solid phase assays in the context of C4d-negative ABMR.

OR34 Flow and virtual cross match exchange – A new component of the International Cell Exchange

Aim Single antigen bead assays provide investigators with a means to accurately identify the presence and relative strength of HLA antibodies through the use of MFI (Mean Fluorescence Intensity) values. However, inter-laboratory variation to the interpretation of MFI values creates the potential for discordant results. The aim of this study was to provide a medium to compare and correlate MFI data together with flow crossmatch outcomes. Methods In 2014, the International Cell Exchange program introduced the UCLA Flow and Virtual Cross Match Exchange. In three exchanges, a total of 6 cells and 12 sera were tested by 17 laboratories around the world. T and B flow cytometric crossmatch results, along with class I and class II single antigen data were reported and analyzed. Fifteen laboratories used a Luminex single antigen bead assay (LSAB) and 2 used a flow single antigen bead assay (FSAB). To further investigate the correlation between MFI values and positive flow crossmatch results, the strength of donor-specific antibodies were compared with their crossmatch outcomes. In addition, coefficients of variations (%CV) were calculated using the MFI reported for each DSA and by each laboratory. We also examined the prediction of positive T/B virtual crossmatches in relationship to the strength of DSAs identified. Results The mean class I and class II DSA MFI of the positive T and B flow crossmatches were 15355 and 14641, respectively. Among labs reporting LSAB MFI, the mean %CV was 26%. Overall, 16 (67%) T-cell and 8 (33%) B-cell flow crossmatch challenges were reported among the labs without discordance. When restricting analysis to DSA MFI greater than mean values, the T flow concordance rate improved to 79%, while B flow concordance remained poor at 31%. In contrast, high concordance rates were observed for virtual crossmatch predictions: 92% (22/24) for T- and 88% (21/24) for B-cell crossmatches. Conclusions The data suggests that the variability of cell based assays is higher than that observed in solid phase SAB test. It also suggests that variability exists in the sensitivity of flow crossmatch testing among laboratories and that this variability is correlated with DSA strength. As such, educational activities should continue to provide standards for quality control of antibody and crossmatch testing methods.

Strength of Donor-Specific Antibodies With the Use of Luminex Single-Antigen Beads Is a Reliable Predictor of Acute Rejection in Living-Relative Kidney Recipients

BackgroundLuminex single-antigen assay is a promising technique for determination of donor-specific antibodies (DSAs) in renal transplantation. MethodsSerum samples from living-relative renal recipients before and after renal transplantation were examined with the use of Luminex single-antigen assay. The impact of de novo DSAs on the early clinical outcomes of renal allograft was analyzed. ResultsOf the 61 patients included, 15 patients (24.6%) presented de novo DSA (4 class I, 4 class II, and 7 both classes) after transplantation, and the average (median) cumulative strength of DSA was 1,283 (range, 0–10,802) mean fluorescence index (MFI) for class I versus 1,324 (range, 0–14,985) MFI for class II (P > .05). Twelve (19.7%) of the 61 patients experienced a clinical/subclinical acute rejection (AR) episode within the 1st 2 years after transplantation. A clinical/subclinical AR episode was diagnosed in 40% of DSA(+) patients and 13.0% of DSA(−) patients (P < .05). DSA(+) patients who developed an AR episode had a higher mean cumulative MFI value (8,118.3 ± 5,287.4; range, 1,785–14,985) than patients who did not develop an AR episode (3,283.7 ± 2,601.0; range, 786–8,113; P < .05). Serum creatinine levels in the DSA(+) group were significantly higher than in the DSA(−) group at 12 and 24 months after transplantation. The graft survival rates at 2 years in the DSA(+) and DSA(−) groups were not different (86.7% vs 91.3%; P > .05). ConclusionsPatients with de novo DSAs at high strength may suffer a high risk of developing an AR episode. Therefore, careful monitoring of de novo DSAs with the use of Luminex single-antigen beads may help in early intervention for AR in renal allografts and to minimize renal damage caused by the antibodies.

Analysis of preformed donor-specific anti-HLA antibodies characteristics for prediction of antibody-mediated rejection in kidney transplantation

BackgroundThe relevance of preformed donor specific antibodies (DSA) detected by Luminex assays, with a negative complement-dependent cytotoxicity (CDC) crossmatch, remains unsettled in kidney transplantation (KT). We aimed to analyze the impact of preformed DSA characteristics on kidney graft outcomes. MethodsIn 462 patients that received a kidney graft in our unit, between 2007 and 2012, pre-transplant sera were analyzed by Luminex screening assay to determine the presence of anti-human leukocyte antigen (HLA) antibodies and single-antigen bead assay [positive if mean fluorescence intensity (MFI)≥1000] to assign anti-HLA specificities. ResultsAnti-HLA antibodies were present in 95 patients (20.6%), but only 40 (8.7%) had DSA. Antibody-mediated rejection (AMR) at 1-year was higher in patients with DSA (35.0%) than in those without them (0.9%) (P<0.001). Only DSA with a MFI of >3000 were significantly associated with AMR occurrence. Receiver operator curves revealed that a MFI of >4900 in the highest DSA bead had a high sensitivity (85.7%) and that the sum of all DSA beads MFI>11,000 had a high specificity (92.3%) for AMR prediction. Anti-thymocyte globulin versus basiliximab induction was more frequent in DSA+ AMR− (65.4%) versus DSA+ AMR+ (34.6%) patients (P=0.072). Five-year censored graft survival was lower in DSA+ than in DSA− patients (respectively, 84.8% versus 94.9%, P=0.006), although survival was only reduced in DSA+ AMR+ (68.8%) versus DSA+ AMR− (96.0%) patients (P=0.038). ConclusionsPreformed DSA is associated with kidney graft loss, in relation with AMR occurrence. DSA strength may be used to improve immunological risk stratification of sensitized patients and their clinical management.

OR13: HIGH TITER ANTIBODY STRENGTH CANNOT RELIABLY BE DESCRIBED WITHOUT DILUTION

Background Luminex Single Antigen Bead (SAB) assay is commonly used to detect circulating donor specific antibody (DSA) in serum of post-transplant patients. DSA Mean Florescent Index (MFI) may be used to impute strength and titer of DSA, and to follow treatment response over time. It is not accurate to impute the strength of high titer antibody based on MFI without testing serum dilutions. Methods Serum samples were heat inactivated (HI) for 30 min at 56° to remove complement inhibition. Serum dilutions were done in phosphate buffered saline. The starting dilution was 1:50 and increased as needed. HLA antibody testing was done by the Luminex Single Antigen Bead (SAB) assay. Results Dilution of serum can have a range of effects on the MFI of the antibody (see figure ). Decreasing MFIs with dilution are the expected result of an accurate MFI measurement within the reportable range of the assay. When there is an excess of antibody relative to the amount of binding sites on the beads, this is not the case. The MFI may maintain a constant value, or it may actually increase as the serum is diluted, indicating a prozone or “hook” effect not attributable to complement. Some patient sera were diluted as much as 1:200 or 1:2,000 in order to obtain mfi values that could be used to follow trends in DSA with treatment and over time (see table). Conclusion To accurately assess the strength of a high titer antibody, serum should be diluted to concentrations that result in consistently decreasing MFIs. Extremely strong antibodies may saturate the binding sites on the beads resulting in MFI values at the upper limits of the assay without truly binding and measuring all the antibody present. Hook effects in HI sera may indicate interfering factors besides complement.

Virtual Crossmatch in Kidney Transplantation

BackgroundThe Luminex Single-Antigen Beads (LSA) assay allows an accurate detection and characterization of preexisting donor-specific antibodies (DSA) in kidney transplant candidates. But the ability of LSA to detect quite low levels of antibodies makes it hard to correctly predict crossmatch results in donor selection. In this study we retrospectively analyzed the accuracy of our virtual crossmatch (v-XM) protocol, which was used for selection of potential kidney transplant recipients, in predicting the results of actual crossmatch (a-XM) in cadaver-donor renal transplantation. We also investigated correlation between negative a-XM results and strength/specificity of preformed DSA. MethodsThe correlation between negative v-XMs and a-XMs performed in 2007–2012 at the Regional Transplant Center of the Lazio Region, Italy, was analyzed. In carrying out v-XM, the donor HLA molecules against which patients showed LSA-detected DSA with normalized mean fluorescence intensity (MFI) ≥5,000 were considered to be “unacceptable DSA,” and LSA-DSA showing MFI <5,000 were defined as “acceptable DSA.” All cadaver donors had been typed for HLA-A, -B, -DR, and -DQB molecules by sequence-specific primer methods. On the basis of a negative v-XM, we performed 507 a-XMs between serum samples from 256 renal transplant candidates and T/B lymphocytes from 302 cadaver donors with the use of both complement-dependent cytotoxicity (CDC) and flow cytometry (FC) methods. ResultsThe v-XM negative results showed good correlation with both CDC and FC a-XMs (97% and 90%, respectively). The sensitivity of v-XM was 100%; this high value was related to the lack of false-negative DSA results. The limited specificity with both techniques (CDC-XM, 74%; FC-XM, 79%) was due to the presence of “acceptable” and/or anti-DQA/DPB DSA in some patient sera used to perform the a-XMs. During the study period, 171 (67%) of the 256 sensitized patients received a kidney transplant: 30% of these had “acceptable DSA” and/or anti-DQA/DPB DSA. No antibody-mediated rejection due to preformed HLA-DSA was observed. ConclusionsOur v-XM protocol showed high sensitivity in predicting donor-recipient immunologic compatibility. The results of this study also demonstrated the importance of evaluating DSA strength for implementing v-XM results in the selection of kidney transplant recipients. Moreover, the finding of anti-DQA/DPB DSA, especially in serum samples that gave positive results with the use of both CDC and FC a-XMs, highlights the importance of defining all of the donor HLA molecules to perform an accurate v-XM.

Donor Specific Antibody (DSA) Formation in Tacrolimus-Based, Mycophenolate vs mTOR Immunosuppression Regimens.

The influence of mTOR inhibition upon DSA formation and later clinical events is controversial, with small reports suggesting a higher rate of DSA with mTOR but experimental data suggesting potential inhibitory signaling pathways. Prior clinical studies did not include regimens with tacrolimus (TAC) and/or prednisone (P) with adequate controls. We compared de novo DSA formation (DSA+) rates between two common immunosuppression regimens (TAC/MPA/P vs. lower exposure TAC/mTOR/P) and clinical outcomes Methods: 66 kidney and SPK recipients on TAC (lo)/mTOR/P (36 with everolimus, 30 with sirolimus) from hospital discharge were compared in a case-control study to 132 patients on TAC/MPA/P, matched for age, gender, race, and type/timing of transplant from 2007-2012. DSA assessment was performed prospectively at time of transplant and at 1, 6, 12, and 24 months post-transplant using single antigen beads. We compared de novo DSA formation ( ≥500 MFI), proteinuria, GFR and acute rejection (AR) between the two groups. Results: With a mean follow up of 796 days, TAC (lo)/mTOR had a higher proportion DSA+ at all time points but did not meet statistical significance. By one year, DSA+ rates were 35% with TAC (lo)/mTOR vs 28% with TAC/MPA (OR 1.37; 95% CI 0.69-2.75; p= 0.37) There was no significant difference between the two arms when comparing clinical outcomes, including GFR at 12 months, proteinuria at 12 months and AR at 12 months.Table: No Caption available.When comparing Sirolimus vs Everolimus, there was no significant difference in DSA formation or clinical outcomes. Neither the type of DSA (class I vs class II) nor strength of DSA (MFI) were significantly different among groups. Comparing those that formed DSA vs those that did not, GFR was not different at 1 year but proteinuria was significantly higher in DSA+ vs DSA- (0.35 vs 0.17g/d, p=0.03). Conclusion:In kidney and SPK transplant patients on TAC/prednisone regimens, mTOR inhibition was associated with similar rates of DSA formation compared to MPA. Our study did not demonstrate a difference in GFR, proteinuria or acute rejection at one year between the two arms. mTOR therapy is neither protective nor permissive in DSA formation or downstream clinical surrogates of antibody-mediated injury. DISCLOSURES:Wiseman, A.: Other, Novartis, consultant, Tolera, consultant, Astellas, consultant.

Predictive Value of the Strength and Class of Pre Transplant Donor Specific Antibodies (DSA) On Clinical Outcomes in Renal Allograft Recipients.

Background: The strength of pre-transplant donor-specific HLA antibodies (DSA) has been questioned as a quantifiable tool in the prediction of future immunologic injury. Desensitization is often considered based upon DSA MFI in the setting of a negative flow cytometry crossmatch (FCXM). The type of DSA has also been purported to be predictive of post-transplant outcomes. However, attempts to identify pre-transplant DSA characteristics that correlate with graft outcomes have yielded inconsistent results.[/bold] Methods: 547 consecutive recipients of a first kidney or kidney/pancreas transplant with a negative pre-transplant FCXM between September 2007 and August 2012 at our center were included in the analysis. All patients underwent cell-based FCXM and SAB analysis (LABScreen beads, One Lambda Inc.) on current and historic sera prior to transplantation. Results: Of 547 patients with a negative FCXM included in the analysis, 196 had DSA (DSA+, MFI ≥500) detected at any point prior to transplant by SAB analysis. One year acute rejection (AR) and three year graft survival were similar in DSA+ vs. DSA- patients. AR was more likely in patients with DSA MFI ≥1000 at any time prior to transplant (20% vs. 9% and 11% for MFI < 1000 and DSA-, p=0.039), but did not correlate with DSA class. With a median follow-up period of 2.3 years, graft function and proteinuria were similar in all groups irrespective of DSA strength or class. Three year graft survival was similar with DSA strength ≥ 1000 MFI, ≥ 3000 MFI (p=0.74), and with respect to class.Figure: No Caption available.Conclusion: Pre-transplant DSA with a negative FCXM is not associated with poor graft outcomes, and further characterization of DSA by strength and class does not reveal a correlation with long term graft survival. While pre-transplant DSA MFI ≥ 1000 warrants careful monitoring for AR, neither antibody strength nor class should represent a barrier to transplant in the setting of negative FCXM.

Role of Non-Donor Specific HLA Antibodies (NDSA) in Kidney Transplant Rejection.

Kidney transplant course is often complicated with development of Donor Specific Antibody (DSA) which have been associated with antibody-mediated rejection (ABMR) and poor allograft survival. However the role of Non-Donor Specific Antibody (NDSA) in occurrence of graft rejection and loss remains controversial. We studied patients who received a kidney transplant at Harper University Hospital from 2000-2010 and grouped them based on development of antibodies. The sera of patients were routinely screened for DSA and NDSA at 6 month to one year of transplant via ELISA or Luminex assay and only those with MFI >2500 were considered positive. The patients were evaluated for occurrence of biopsy proven acute rejection which was classified as cellular or ABMR per Banff criteria. Graft function was determined by most recent blood creatinine levels as of 12-31-2012 and graft loss was defined as need to return to dialysis or re-transplant and was censored for death. Of the 200 patients included in the study, 63 (31.5%) had NDSA only, 10 (5%) produced DSA only, 25 (12.5%) produced both and 102 (51%) non-producers at an average of 7.9 months post-transplant (range: 1-18 months). The baseline recipient characteristics were comparable except that recipients producing NDSA only or both were more likely to have had a previous transplant and therefore had higher peak PRA than non-producers and DSA only, which is suggestive of preformed NDSA due to prior sensitization. Those who produced both were more likely to be on maintenance steroid as compared to non-producing, NDSA only, and DSA only (12% vs. 32.35%, 22.22%, and 30% respectively; p<0.05). The fraction of patients with greater strength of DSA and NDSA i.e. MFI >10K was higher in the combined group than those in NDSA only (80% vs. 51.28%; p=NS) and DSA only (66.7% vs. 0%; p<0.05). There was an increased incidence of ABMR in those who produced both as compared to non-producers, NDSA only, and DSA only (20% vs. 3.92%, 1.59%, 0% respectively; p<0.01), while there was no difference in cellular rejection rates among the groups (p=NS). While the serum creatinine at last follow-up was not different between the groups, the graft loss was higher in those that produced both than others. In conclusion, NDSA alone especially at MFI <10K are not associated with increased risk of ABMR or graft loss. It is unclear if higher levels of NDSA are detrimental, especially in presence of DSA.

Pretransplant Donor-Specific Anti-HLA Antibodies and Their Characteristics Are Associated With Poorer Kidney Graft Outcomes in Kidney and Kidney-Pancreas Recipients.

The role of donor-specific antibodies (DSA) and their characteristics in kidney transplantation remains not fully understood. We aimed to analyze in a cohort of kidney and kidney-pancreas transplant recipients the effect of pretransplant DSA in kidney graft adverse outcomes: acute humoral rejection (AHR) and graft failure (GF). A cohort of 434 patients (pts) that received a kidney graft (67 pts also receiving a pancreas graft) between 2008-12 after a negative CDC crossmatch (XM) were included. Presence of anti-human leukocyte antigen (HLA) antibodies was investigated in pretransplant sera using a Luminex® screening assay and anti-HLA specificities were assigned performing a Luminex® single antigen assay [positive: mean fluorescence intensity (MFI) was ≥1000 (1K)]. In DSA+ pts, ROC curves were constructed to assess the ability of MFI values (DSAmax: highest DSA bead; DSAcum: detected DSA MFI sum) to predict AHR. Comparison between DSA defined groups:Table: No Caption available.1-year incidence of AHR by DSA class was 17%, 57% and 57%, in I (n=23), II (n=7) and I+II (n=7), respectively (logrank p=0.023); by DSA MFI was 6%, 46% and 67%, in 1k-3k (n=17), 3k-7k (n=11) and >7K (n=9), respectively (logrank p<0.001). Considering only DSA+ pts, comparisons were performed using χ2 or Mann-Whitney U test between AHR- (n=25) and AHR+ (n=12) groups. Differences were found in mean HLA mismatch (3.5 vs 4.3; p=0.008), median DSA number (1 vs 3; p=0.005), MFI DSAmax (2.5K vs 8K; p=0.001), DSAcum (3.5K vs 19.5K, p=0.001), DSAmax class II (12% vs 58%; p=0.006) and DSA class (AHR-: I 76%, II 12%, I+II 12%; p=0.02); number of kidney-pancreas (2 vs 2; p=0.6) and flow XM+ pts (2 vs 3; p=0.3) were similar. In ROC analysis, both MFI DSAmax (AUC 0.83; 3.75K had 92% sensitivity, 72% specificity) and DSAcum (AUC 0.85; 11.2K had 67% sensitivity, 92% specificity) were good predictors of AHR. Pretransplant DSA have a deleterious effect on kidney graft outcomes. Class, number and specially strength of DSA modulate that effect and should be considered in DSA+ pts management, such as the complementary behavior of MFI DSAmax and DSAcum as predictors of AHR.

Late antibody-mediated rejection in renal allografts: outcome after conventional and novel therapies.

Although several strategies for treating early antibody-mediated rejection (AMR) in kidney transplants have been investigated, evidence on treatment of late AMR manifesting after 6 months is sparse. In this single-center series, we present data on 23 consecutive patients treated for late AMR. Late AMR was diagnosed using Banff 2007 criteria along with presence of donor-specific antibodies (DSA) and acute rise in serum creatinine (SCr). Response to therapy was assessed by improvement in SCr, histologic improvement, and decline in DSA strength. Overall, 17% (4/23) had documented nonadherence while 69% (16/23) had physician-recommended reduction in immunosuppression before AMR. Eighteen patients (78%) were treated with plasmapheresis or low-dose IVIg+rituximab; 11 (49%) with refractory AMR also received one to three cycles of bortezomib. While there was an improvement (P=0.02) in mean SCr (2.4 mg/dL) at the end of therapy compared with SCr at the time of diagnosis (2.9 mg/dL), this improvement was not sustained at most recent follow-up. Eleven (48%) patients had no histologic resolution on follow-up biopsy. Lack of histologic response was associated with older patients (odds ratio [OR]=3.17; P=0.04), presence of cytotoxic DSA at time of diagnosis (OR=200; P=0.04), and severe chronic vasculopathy (cv≥2) on index biopsy (OR=50; P=0.06). A major setting in which late AMR occurred in our cohort was reduction or change in immunosuppression. Our data demonstrate an inadequate response of late AMR to current and novel (bortezomib) therapies. The benefits of therapy need to be counterweighed with potential adverse effects especially in older patients, large antibody loads, and chronic allograft vasculopathy.

3-P

In the recent years, many HLA laboratories predict crossmatch results based on the strength of Donor Specific Antibodies (DSA) measured by single antigen (SA) solid phase assays. This study evaluates the prediction of CDC crossmatch results to flow cytometry Median Channel Sshift (MCS) versus SA cumulative Median Fluorescent Intensity (MFI) of DSA. 93 T cell AHG-CDC and flow cytometry crossmatch (FCXM) were reviewed, results compared to DSA MFI (0-10000) (>10000). In the other hand, the same samples were compared to MCS (0-250)& (>250). Percentage frequency, chi-square and P-value were calculated to assess the association between mentioned variables. DSA MFI’s and MCS were grouped into two groups (0-10000), (>10000), (0-250) & (>250) respectively and are presented in this table.[ Table 1 ] From the above data there was a significant association in prediction of positive CDC crossmatch based on MCS (chi square of 67.95, P < .0001) compared to SA cumulative MFI (Chi square 11.38, P=0.0007. The positive predictive value of the CDC crossmatch based on MCS was higher than MFI with 88% and 57% respectively. Therefore SA cumulative MFI is not significant to consider as predictor for a positive CDC crossmatch rather than transplant risk factor.

Eosinophils Are Not Required for the Induction and Maintenance of an Alloantibody Response

Eosinophil-produced cytokines have been shown to participate in the maintenance of antigen-specific plasma cells (PC) in bone marrow (BM), suggesting that eosinophils are required in the development and/or maintenance of alloantibody responses posttransplant. To test this hypothesis, we sensitized eosinophil-deficient ΔdblGATA1 mice and wild-type (WT) control mice with allogeneic splenocytes or with allogeneic heart grafts and compared the kinetics and titers of serum donor-specific antibodies (DSA), as well as BM and spleen CD130 + B220 low PC populations between groups. Spleen cells from naïve ΔdblGATA1 BALB/c mice contained higher percentages of PC than WT without detectable differences in BM PCs. After sensitization with allogeneic splenocytes, BALB/c ΔdblGATA1 mice contained fewer BM PCs but more splenic PCs compared to controls. These differences were associated with modestly lower titers of serum DSA 4 and 12 weeks after sensitization but secondary immunizations induced similar increases in both groups. Moreover, the kinetics and strength of DSA did not differ in WT and ΔdblGATA1 BALB/c mice transplanted with B6 cardiac allografts, nor did they differ in transplanted ΔdblGATA1 and WT mice on a B6 background. Therefore, eosinophils are not required for alloantibody formation or maintenance in mice and are thus unlikely to be effective targets for antibody desensitization.