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Purpose: To investigate which risk factor affects the development dnDSA.Methods: 315 patients (pts) with available antibodies study and follow upgreater then 6 months (who un derwent KTx from January 2005 to December2017) are object of our study. Mean recipients age was 52 years (range 24-71), mean donors age was 50 years (range 15-81). Mean follow up post KTxwas 59 months (range 10-60).Post transplant sera were studied at months 3 and then yearly, (or by clinicalneeds) to access the presence of dnDSA. Screening was obtained with class Iand II Flow PRA test, in positive sera class I and II Luminex single antigenbeads was performed.Patients were divided in groups depending of immunosuppressive protocol:tacrolimus and mycophenolic acid (MPA) (n = 153), cyclosporine (CsA) andMPA (n = 37), CsA and Proliferation Signal Inhibitor (PSI) (sirolimus oreverolimus) (n = 99), PSI and MPA (n = 18), other (n = 8).Development of dnDSA was correlated with known risk factors : pretransplant class I and II PRA, donors age, recipients age, immunosuppressiveprotocols.Lastly 10 years graft survival and renal function (assessed by creatinine)was compared for pts with and without dnDSA.Results: In our study 59 patients (19%) developed dnDSA after a mean timeof 32 months (range 1-136) since KTx.Immunosuppressive protocols showed similar incidence of dnDSA:tacrolimus+MPA = 26 pts (17%), CsA+MPA = 10 pts (27%), CsA+PSI = 17pts (17.2%), PSI+MPA = 6 pts (33%). (p=ns).Ten years graft survival was adversely influenced by dnDSA (78.0% vs.90.7%) (p = 0.017).Median last serum creatinine level was higher in patients with dnDSApositivity (2.0 vs. 1.5 mg/dl) (p = 0.045).Among all the variables examinated, dnDSA is positively influenced only byrecipients age: patients under 50 years of age more prone to develop dnDSA(p = 0.013).Conclusion: In our experience the only variable influencing development ofdnDSA is the recipient age minor of 50 years. more...

Clinical impact of preformed HLA donor specific antibodies(DSA) ranges from an acceptable risk to an absolute contra-indication for kidney transplantation, according to the technique used to detect them (CDC vs. flow cytometry/solid phase). The new FlowDSA-XM technique combinessensitivity of the classical FCXM with specificity of the sin-gle antigen bead assay, allowing a better correlationbetween crossmatch result and patients' HLA class I and IIsensitization detected by solid phase assay. We performed acomparative analysis between FCXM and FlowDSA-XMresults by testing 46 sera (9 PRA=0%, 14 HLA class I posi-tive, seven HLA class II positive and 16 HLA class I&IIpositive) with five different lymphocytes population (typedfor all HLA class I&II loci by high resolution PCR-SSP/SSO techniques).The standard FCXM technique alloweddetection of IgG-antibodies specific for T and B lympho-cytes. The FlowDSA-XM consisted of an incubation oflymphocytes and serum was performed. Then, three differ-ent Capture Beads (HLA I A, B, C beads, HLA IIa DQbeads and HLA IIb DR, DQ, DP beads) were added. Lastly,a Lysis/Stain buffer was used to solubilize the HLA mole-cules and to label the antibodies bound to them. FlowDSA-XM and FCXM showed strong concordance in the ninePRA negative sera and in the 14 anti-HLA class I positivesera; FlowDSA-XM was partially discordant in one ofseven anti-HLA class II positive sera because of FCXM Bcells showed a very weak positivity while FlowDSA-XMHLA IIb showed a result near cut-off. Moreover, theFlowDSA-XM was more sensitive than the FCXM showinghigher values of positivity, especially in the presence ofanti-HLA class I DSA and gave "specific" results discrimi-nating between HLA class I and/or II DSA. On the contrarythe FCXM gave B positive results for the presence of bothHLA class I and class II DSA. In conclusion the FlowDSA-XM, allowing a better interpretation of the crossmatchresult, could represent the crossmatch of the newmillennium. more...

In solid organ transplantation the HLA-A, -B, -DR, -DQ donor-recipient matching results are insufficient for highly sensitized patient. In fact, solid phase single antigen assays, used to define HLA antibodies in transplant candidates, show the presence of antibodies specific for all HLA molecules. Thus, an extended HLA typing of deceased donor is necessary to improve selection of the most suitable transplant candidate. Until July 2016, in our laboratory all donors were typed for HLA-A,-B,-C,-DR,-DQ loci by PCR-SSP technique; when a sensitized patient was selected, the donor typing was prospectively enlarged to pertinent HLA molecules. To simplify this procedure and improves organ allocation we introduced the new RT PCR-SSP technique, based on an innovative chemistry (Linkage Bioscience Inc.), it enables us to provide intermediate resolution typing of 11 HLA loci, in less than 90 minutes. Allele-specific amplification combined with SYBR Green and real-time PCR instruments are used to detect amplification products and to collect dissociation data for automatic interpretation by SureTyper software. Since August 2016, 76 potential deceased donors were typed by this technique. No allelic ambiguities were evidenced but rather high resolution typing were obtained in several cases: 11 HLA-A alleles, 55 HLA-B alleles, 48 HLA-C alleles, 41 HLA-DRB1 alleles, 37 HLA-DRB345 alleles, 20 HLA-DQA1 alleles, 30 HLA-DQB1 alleles, one HLA-DPA1 allele, and 50 HLA-DPB1 alleles. Moreover, the extended HLA typing obtained by RT PCR-SSP avoided additional HLA-C,-DRB1, -DQA1 and-DPB1 typing in 12 cases (15,8%) and allowed to define donor molecules against which 10 patients (13,2%) showed preformed high fluorescence intensity antibodies (MFI >5000). In conclusion, RT PCR-SSP is less hands-on and, considering the number of typed HLA loci, cheaper than traditional PCR-SSP techniques. The extended donor HLA typing gives useful information for a more precise pre-transplant virtual crossmatch and for a better donor-recipient selection to improve clinical transplant outcome. more...

Recent studies associated non-HLA antibodies speci?c forAngiotensin II Type-1 Receptor (AT1R) with antibody-mediated rejection (AMR) of kidney allograft. This study analyzed thepresence of AT1R antibodies in kidney transplanted patientsshowing or not showing HLA donor-speci?c antibodies (DSA)production and the correlation of these antibodies with graftoutcome. Sera from 63 kidney transplanted patients were analyzedfor anti-AT1R using a new ELISA assay. Luminex Single Antigentechnique (LSA) was used to analyze de novo DSA production.The capability of detected DSA to ?x C1q was tested by a modi?edLSA assay. Analyzing graft outcome, 50 patients suffered graftfailure (GF)/graft dysfunction (GD) for AMR. Sixteen (26%)patients were DSA-negative/anti-AT1R-negative and did notshow AMR. Twenty-seven (43%) patients were DSA-positive/anti-AT1R-negative; all but one suffered GF/GD and hadC1q-positive DSA. Sixteen (26%) patients were DSA-positive/anti-AT1R-positive; all suffered GF/GD and had C1q-positiveDSA. The remaining 4 (6%) patients were DSA negative/anti-AT1R-positive and suffered GD. The production of DSA onlyand DSA plus anti-AT1R showed signi?cant associations with kid-ney GF/GD (P < 0.0001). At 40 months after antibody appearance,graft survival was 72% for DSA-negative/anti-AT1R-negativepatients, 4% for DSA-positive/anti-AT1R-negative, but allDSA-positive/anti-AT1R-positive patients lost the graft within12 months from antibody appearance. Our study con?rmsthe crucial role of DSA production in kidney transplantationand underlines the synergic effects of anti-AT1R develop-ment on graft outcome. Assessment of anti-AT1R status alongwith de novo DSA development should help in the diagno-sis of AMR and can give the opportunity for speci?c clinicalintervention. more...

Aim Preformed HLA cytotoxic antibodies, specific for mismatched HLA molecules of the potential donor, represent an absolute contraindication in kidney transplantation. New techniques, like the Luminex-Single Antigen Beads assay, are very sensitive allowing detection of HLA donor specific antibodies (DSAs) at low mean fluorescence intensity (MFI) values. However, few evidence on the clinical relevance of such low "strength" DSAs have been reported. Methods Graft outcome (follow-up 34.2 ± 19.8 months) of 99 pre-sensitized patients (%FlowPRA class I = 41 ± 33; %FlowPRA class II = 30 ± 36), transplanted between May 2007 and June 2014 on the basis of both CDC-XM and FC-XM negative results, was analyzed. Five patients were excluded from the study because of primary non-function of the graft (3 patients) or death for non-immunologic causes with non-functional graft (2 patients). Forty-seven (47%) patients did not have pre-formed HLA DSAs; the remaining 52 (53%) patients had "acceptable" DSAs (MFI ? 5000) specific for HLA-A/B/C/DR/DQB molecules or anti-DPB/DQA DSAs with high MFI values (>5000). HLA class I DSAs were present in 28 patients; five of these showed DSAs against more than one donor HLA molecules. HLA class II DSAs were present in 19 patients; three of these showed anti-DPB/DQA DSAs with MFI > 5000. The remaining five patients had both HLA class I and class II DSAs; one of these had high level of anti-DP DSA. Results Analyzing graft outcome of the remaining 94 transplanted patients with functioning graft, we did not evidenced any significant difference between DSA positive patients and DSA negative patients (rejection: 10.4% vs. 6.5%, P = ns; graft loss: 12.5% vs. 10.9%, P = ns). In particular, among the 48 DSA positive patients, three (6.2%) had humoral rejection without graft loss; two (4.2%) had cellular rejection, one of these lost the graft. Six DSA positive patients suffered graft failure that was never due to antibody-mediated rejection related to pre-formed DSAs. Conclusion The results of this study show that pre-formed DSAs with low mean fluorescence intensity values do not represent a contraindication in kidney transplantation. An accurate evaluation of the "strength" of Luminex-detected DSA allows transplanting patients with clinically "irrelevant" HLA antibodies. more...

The development of donor-specific HLA antibodies (DSA) represents the major risk factor of graft failure in kidney transplantation. However, some patients show persistent presence of circulating DSA without occurrence of graft loss. Solid phase assays, such as Luminex Single Antigen (LSA) beads, are highly sensitive in detecting DSA but not very predictive of transplant outcome because of detection of complement-fixing and less clinically relevant no complement-fixing antibodies. Using the novel LSA-C1q assay, we investigate the clinical relevance of de novo DSA in relation to their capability to fix complement. In serum samples from 53 kidney transplanted patients who had developed IgG-LSA DSA after transplantation, we measured complement­fixing ability of detected DSA by Class I and Class II LSA-C1q assay. As for transplant outcome, 32 (60%) patients suffered graft failure and 21 (40%) had good graft function during the follow up period. As for complement-fixing capability of DSA, 35 patients showed production of C1q-positive DSA; the remaining 18 produced C1q-negative DSA. C1q-positive DSA were present in all but three patient who had graft failure; only six of the 21 patients showing C1q-negative DSA suffered graft failure (91% vs. 29% respectively, P?¬0.0001). As for HLA specificity of DSA, C1q-positive DQA antibodies were only present in three patients showing good graft function. On the contrary, DQB-DSA were present in 21 patients showing C1q-positive HLA antibodies; all but one of these patients suffered graft loss. In conclusion our data suggest that C1q-LSA assay shows the capability to identify the subset of IgG-LSA DSA strongly associated to graft loss in kidney transplantation. Moreover the ability of C1q assay in distinguishing less harmful no complement-fixing DSA from clinically relevant C1q-fixing DSA represent a non-invasive tool for identifying patients who need specific immunosuppressive-therapy strategy to prolong graft survival. more...

In kidney transplantation, current HLA class II matching strategies consider DR antigens only. Both a and b chains of HLA-DQ and -DP heterodimers are polymorphic and can elicit a humoral immune response. Preformed HLA class II donor-speci.c antibodies (DSA) have a deleterious impact on graft outcome and post-transplant development of anti-class II DSA is strongly associated with acute and chronic rejection occurrence. This study was done to investigate the incidence of anti-DQ and -DP antibod­ies in 56 kidney transplant (KTx) recipients who developed HLA class II DSA and 145 HLA class II-positive re-transplant candidates. Antibody characteriza­tion was performed by using Single antigen bead assay (microbeads coated with 29 DQA1/DQB1, 24 DPA1/DPB1 heterodimers besides DRB1/3/4/5, molecules). In the KTx group of patients, incidence of anti-DQ/DP DSA was signi.cantly higher than anti-DR DSA (76% vs. 20%, P < 0.0001); in 3 patients we only found anti-DQA1 DSA. During the follow up period, graft failure occurred in 14 of the 38 (36.8%) patients who only developed anti-DQ and/or -DP DSA. Analyzing speci.city of HLA class II antibodies detected in re-transplant can­didates, 131 of the 145 (90%) patients showed production of anti-DQ and/or anti-DP antibodies. Nineteen percent of these patients had only anti-DQ a/b chain antibodies, 5% had only anti-DP a/b chain antibodies and 7% had both anti-DQ and anti-DP antibodies. It is important to underline that 87% of anti- Histocompatibility DQA1/DQB1 positive patients and 98% of anti-DPA1/DPB1 positive patients developed wide antibody patterns due to the recognition of a "public epitope" of the mismatched donor molecule/s. These data demonstrate the great immunogenicity of mismatched DQA1/DQB1 and DPA1/DPB1 molecules of a kidney transplant and underline the need to consider all the HLA class II molecules in matching strategies especially for re-transplant candidates. more...

Background: To avoid Calcineurin Inhibitor (CI) nephrotoxicity, a CI-freeprotocol was introduced in our centre, as immunosuppressive therapy (IT) indual kidney transplantation (DKT) from marginal donors.Methods: Donor kidneys were histologically evaluated according to Remuzziscore (J Am Soc Nephrol 1999). Fifteen DKT were performed since February2009 with the following IT: basiliximab, steroids, mycophenolate sodium(1440 mg/die), sirolimus (n = 12) or everolimus (n = 3). Mean age of recipientswas 66 years (range 61-71) whereas mean donors age was 68.5 years (range55-78). All patients were analyzed for donor specific HLA-antibodies (HLA-DSA) by solid phase Luminex Single Antigen bead assay, at 1 and 6 monthsfrom KT and then yearly.Results: Median follow up was 35.7 months (range 1-73). Cumulativeincidence of acute rejection (AR) was 33.3% (5/15). ARs were biopsy proven,within 90 postoperative days and responsive to steroids treatment. The mediancreatinine at last follow-up was 1.5 mg/dl (range 0.8-2.8). Steroids werestopped in 7 recipients due to new onset diabetes. No deaths or graft lossesoccurred throughout the follow-up. No patient developed HLA-DSA throughoutthe follow-up.Discussion/Conclusions: AR episodes occurred when recipients wereunder-immunosuppressed for IT adverse events: sirolimus/everolimus bloodlevel under 5 ng/ml (n = 3), early steroid withdrawal (n = 1), or during acutetubular necrosis (n = 1). In our experience CI free IT provides good results inDKT, with excellent long term graft function. Special care should be taken insuch aged recipients with several comorbidity factors. Dual KT is oftenperformed with low HLA compatibility and adequate IT should be constantlyachieved. Hence we recommend sirolimus/everolimus trough levels on the highside of their therapeutic range in the first 6 months post KT. more...

Background: The once-daily (QD) formulation of tacrolimus (Tac) showedsimilar efficacy than twice-daily (BD) Tac and seems to improve adherence inkidney transplant (KT) recipients. The aim of our study was to define theefficacy and the safety of "de novo" QD Tac administration in KT recipients whoshowed pre KT high panel reactive antibodies (PRA).Methods: Twentyeight patients with a median PRA of 88.3%(range 64-100)who underwent KT were enrolled in the study. Patients were transplanted with anegative crossmatch performed using both cytotoxic and flow-cytom etricmethods. All patients received following immunosuppress ion: basiliximab,steroids, QD-Tac based at 0.2 mg/kg to reach a trough levels of 7-10 ng/mL;27 patients received mycophenolate and one everolimus also. All patients wereanalyzed for donor specific HLA-antibodies (HLA-DSA) by solid phase LuminexSingle Antigen bead assay, at 1 and 6 months from KT and then yearly.Results: Median follow-up was 26.4 months (range 2-57). The 3 yearspatients and graft survival were 100%. The median creatinine at last follow-upwas 1.5 mg/dl (range 0.9-3.1); no patients showed proteinuria. One patientexperienced acute rejection (category 4 IIA Banff '07) at POD 9; after steroidsbolus he was switched to BD Tac because the difficulties to achieve atherapeutic Tac level. No patient developed HLA-DSA throughout the follow up.Conclusion: QD formulation of Tac showed excellent graft and patientsurvival also in highly sensitized patients. A careful care is mandatory in earlyKT to achieve a therapeutic trough Tac levels. more...