ABSTRACT For patients with end stage renal disease, kidney transplant offers significant survival and quality-of-life advantages compared with dialysis. But for patients seeking transplant who are highly sensitized, waiting times have traditionally been long and options limited. We present the case of a 34-year-old hypersensitized female who underwent renal retransplantation. Histocompatibility tests revealed a calculated panel-reactive antibody of 99.53% with multiple antibodies against class I and II human leucocyte antigens and an eplet analysis was performed. The donor’s potential unacceptable antigens were re-defined and the calculated panel-reactive antibody decreased to 88.38%. After one month the patient received a deceased-donor kidney transplant. Complement dependent cytotoxicity crossmatch was negative; virtual crossmatch and flow cytometry crossmatch with historical serum were positive. High-dose intravenous immunoglobulin and rituximab were added to the thymoglobulin-based induction immunosuppression. Three donor-specific antibodies were detected and plasmapheresis was performed. Renal allograft biopsy revealed no manifestations of rejection. Repeated testing observed a decrease in donor-specific antibodies median fluorescence intensity values. Four months post-transplant, the patient remained with normal graft function without proteinuria. She is receiving a standard maintenance immunosuppression regime with prednisolone, mycophenolate mofetil and tacrolimus. The careful discussion among the transplantation center and histocompatibility laboratory in association with intense immunosuppression and close laboratory monitoring allowed a successful human leukocyte antigen-incompatible deceased donor kidney transplantation in the most critical phase for the occurrence of humoral rejection. It is noteworthy that the new histocompatibility and immunogenetics methodologies provide a more affirmative and comprehensive assessment of mismatch acceptability.

Alloantibodies against donor human leukocyte antigens (HLA), termed as donor-specific antibodies (DSA), are one of the most important factors for both early and late kidney allograft dysfunction. In the past, these antibodies were mainly detected through cell-based crossmatch tests. Recently, new techniques such as solid phase immunoassays (SPI) have revealed these antibodies in patient sera with a high degree of detail, previously unimaginable. They have allowed us to accurately determine recipients’ allosensitization status, improve pre-transplant risk assessment with a potential donor and post-transplant alloimmune monitoring. However, the high sensitivity of these new assays has also created areas of uncertainty about their clinical impact. In the pre-transplant setting, the presence of preformed DSA has been associated with an increased risk of antibody-mediated rejection (AMR) and subsequent allograft loss. Nevertheless, several studies have shown that not all DSA are deleterious. Hence, understanding the clinical correlations of DSA characteristics, namely strength, HLA class, complement-fixing ability or IgG subclasses, is paramount for an adequate stratification of the immunological risk at transplant. Furthermore, given that the number of allosensitized patients on waiting lists is increasing, the added information from these new SPI is essential to improve their chance of being transplanted with an admissible immunological risk. After transplantation, the appearance of de novo DSA (dnDSA) has also been associated with a deleterious effect on kidney allograft survival. Moreover, it has been acknowledged that a majority of late allograft failures are caused by alloantibody-driven injury. The current challenges, in this setting, are determining cost-effective DSA screening protocols and understanding which patients could benefit from specific interventions. Furthermore, although therapeutic strategies to control antibody-induced damage remain limited, the longitudinal surveillance of dnDSA emergence and the clinical correlations of their characteristics will play a crucial role in the improvement of late kidney allograft survival.

Abstract Patients who are candidates for a second kidney transplant (SKT) frequently have a higher level of panel reactive antibodies (PRA). We assessed the allosensitisation change after a first graft failure (GF), its predictors and impact on retransplantat outcomes. We retrospectively selected 140 adult patients who received a SKT. Recipient and donor characteristics were analyzed. We defined the delta PRA (dPRA) as the difference between peak PRA before the SKT and first one (cohort median value = +10%). Logistic regression analysis was used to determine risk factors for dPRA ≥ 10% and acute rejection (AR) in the SKT. Univariable and multivariable Cox analysis was applied to assess independent predictors of second GF. Risk factors for dPRA ≥ 10% at SKT were AR (OR = 2.57; P = 0.022), first graft survival <1 year (OR = 2.47; P = 0.030) and ABDR HLA mismatch (OR = 1.38 per each mismatch; P = 0.038). AR in the SKT was associated with dPRA ≥ 10% (OR = 2.79; P = 0.047). Induction with a lymphocyte-depleting agent had a protective effect (OR = 0.23; P = 0.010). SKT survival was lower (P = 0.008) in patients with a dPRA ≥ 10% (75.6%, 60.5% in dPRA ≥ 10%; 88.6%, 88.6% in dPRA < 10% patients at 5 and 10 years, post-transplant respectively). Multivariable Cox regression showed that dPRA ≥ 10% (HR = 2.38, P = 0.042), delayed graft function (HR = 2.82, P = 0.006) and AR (HR = 3.30, P = 0.001) in the SKT were independent predictors of retransplant failure. This study shows that an increased allosensitisation at retransplant was associated with the degree of HLA mismatch and led to poorer outcomes. De-emphasis of HLA matching in current allocation policies may be undesirable, particularly in patients with a higher chance of needing a SKT.

Resumen Los pacientes candidatos a un segundo trasplante de riñón (STR) tienen a menudo un nivel superior de panel reactivo de anticuerpos (PRA). Evaluamos el cambio de alosensibilización después de un primer fracaso del injerto, sus predictores y repercusión en los resultados del retrasplante. Elegimos retrospectivamente a 140 pacientes adultos que recibieron un STR. Analizamos las características de los receptores y de los donantes. Definimos el delta PRA (dPRA) como la diferencia entre el pico de PRA antes del STR y el primero (cohorte mediana = +10%). Se aplicó análisis de regresión logística para determinar los factores de riesgo para dPRA ≥ 10% y rechazo agudo (RA) en STR y análisis univariado y multivariado de Cox para evaluar predictores independientes de fallo del retrasplante. Los factores de riesgo para dPRA ≥ 10% fueron: RA (OR = 2,57; p = 0,022), supervivencia del primero injerto menor a un año (OR = 2,47; p = 0,030) e incompatibilidad HLA ABDR (OR = 1,38 por cada mismatch; p = 0,038). El RA en el STR fue asociado con dPRA ≥ 10% (OR = 2,79; p = 0,047). La inducción con un agente depletor de linfocitos tuvo un efecto protector (OR = 0,23; p = 0,010). La supervivencia del STR fue menor en pacientes con dPRA ≥ 10% (75,6; 60,5% en dPRA ≥ 1 0%; y 88,6; 88,6% en dPRA < 10%; a los 5 y 10 años). La regresión multivariada de Cox mostró que dPRA ≥ 10% (HR = 2,38; p = 0,042), función retardada de injerto (HR = 2,82; p = 0.006) y RA (HR = 3,30; p = 0,001) en STR fueron factores predictores independientes de fracaso en el retrasplante. Este estudio muestra que un incremento en la alosensibilización de retrasplante se ha asociado con el grado de incompatibilidad HLA y puede conducir a resultados más pobres en STR. La falta de énfasis en la compatibilidad de HLA en las políticas actuales de asignación puede no ser deseable, especialmente en pacientes con una mayor probabilidad de necesitar un STR.

Introduction: Kidney transplant improves survival even in highly-sensitized(HS) patients. To overcome their disadvantage in accessing transplantation, those with high Complement Dependent Cytotoxic PRA (CDC-PRA) receive additional points during allocation. Whether this strategy reaches all HS patients and how long they wait for a transplant is largely undetermined. Methods: Patients on our unit’s active wait-list for kidney transplantation in the year 2014 were analyzed. CDC-PRA and calculated PRA (cPRA) were recorded. To obtain cPRA, antibodies in the last serum available specific for HLA-A,-B or -DR with an intensity > 1000 MFI were considered. Results: The cPRA values in the population (N=551) were 0% (N=312), 1-79% (N=118) and ≥ 80% (22%; N=121). Among these groups, the proportion of women (29.5, 55.9 and 61.2%, P<0.001), prior sensitizing events (43.3, 80.5 and 96.7%, P<0.001) and time on dialysis (median of 3.9, 4.1 and 6.0 years, P<0.001) increased with cPRA, respectively. In most of those with a cPRA ≥ 80%, the CDC-PRA raised no suspicion of HS status (median 0%, P25-75 0-8%) and only 35 (28.9%) or 12 patients (9.9%) had a CDC-PRA in the peak serum higher than 50 or 80%, respectively (cut-offs needed to obtain additional points during allocation). HS patients by cPRA corresponded to 71% vs 15% of patients waiting for ≥ or <8 years, respectively (P<0.001). Even after exclusion of patients with a CDC-PRA above 50%, this disproportionate representation remained (58% versus 13%, P<0.001). Conclusion: HS patients as measured by cPRA remained longer on the wait-list, both in the primary analysis and when excluding those with a CDC-PRA> 50%. Moreover, only 30% of HS by cPRA patients received the extra points designed to improve their transplantability. We consider that both CDC-PRA and cPRA should be taken into account when defining HS status

Introdução: O efeito deletério dos anticorpos para antigénios MICA (major histocompatibility class I chainrelated A) na incidência de rejeição aguda e sobrevida do enxerto ainda não está consensualmente estabelecido. Metódos: Estudo retrospetivo de 554 transplantados renais. A análise comparativa entre doentes positivos e negativos para anticorpos anti-MICA pré-formados foi realizada para avaliar eventos sensibilizadores. A incidência de rejeição aguda no primeiro ano pós transplante renal e a sobrevida do enxerto renal foram determinadas consoante o resultado da citometria de fluxo pré-transplante para anticorpos anti-MICA e/ou anti- HLA (anti-human leukocyte antigen). Aplicou-se um modelo de análise multivariada para identificação de preditores independentes para rejeição aguda. Resultados: Foram identificados 41 doentes (7.4%) com anticorpos anti-MICA pré formados. A sensibilização para HLA, as transfusões sanguíneas e gestações prévias foram mais frequentes nos doentes MICA + mas apenas a presença de anticorpos anti-HLA classe I apresentou uma associação independente (OR 2.67, p = 0.02). Comparativamente ao grupo MICA-/HLA-, o grupo MICA-/HLA+ apresentou menor sobrevida livre de rejeição ao 1º ano (78.6% vs. 89.3%, p < 0.01), maioritariamente ocorrida nos primeiros seis meses, enquanto que nenhuma diferença foi encontrada com o grupo MICA+/HLA- (88.9% vs. 89.3%, p = ns). Apenas o status MICA-/HLA+ teve impacto independente na incidência de rejeição (OR 2.09, p = 0.03), ao contrário do status MICA+/HLA- (OR 1.08, p = ns). O grupo MICA-/HLA+ apresentou menor sobrevida do enxerto censurada para a morte aos 4 anos (85.8% vs. 95.3%, p = 0.03), não se verificando diferenças no grupo MICA+/HLA- (95.1% vs. 95.3%, p = ns). Conclusão: Os nossos resultados não suportam um efeito deletério dos anticorpos pré-formados para MICA, independente da sensibilização HLA, na incidência de rejeição aguda no 1º ano pós transplante e na sobrevida do enxerto

Background: Evidence supporting deleterious effect of preformed major histocompatibility class I chain-related A (MICA) antibodies in rejection incidence and graft survival is still unclear. Methods: Retrospective analysis of 554 kidney transplanted patients. Comparison between positive or negative for MICA antibodies patients was performed to characterize sensitizing triggers. Further classification according to pre-transplant flow cytometry-recorded anti-MICA and/or anti-human leukocyte antigen (HLA) antibodies was made to determine first year rejection incidence and graft survival. Multivariate analysis was applied to determine predictors for acute rejection. Results: Pre-formed anti-MICA antibodies were detected in 41 patients (7.4%). HLA sensitization, blood transfusions and pregnancies were frequently found in anti-MICA+ patients but only pre-formed anti-HLA class I antibodies showed independent association (OR 2.67, p = 0.02). Comparing to MICA-/HLA-, MICA-/HLA+ group presented significantly lower first year rejection-free survival (78.6% vs. 89.3%, p < 0.01), mostly occurred in the first six months, while no difference was found in MICA+/HLA- (88.9% vs. 89.3%, p = ns). MICA-/HLA+ showed independent impact in rejection (OR 2.09, p = 0.03), while no evidence was found in MICA+/HLA- (OR 1.08, p = ns). At 4 years, MICA-/HLA+ group presented lower graft survival (85.8% vs. 95.3%, p = 0.03). Again, no difference was found in MICA+/HLA- group (95.1% vs. 95.3%, p = ns). Conclusion: Our results do not support HLA-independent deleterious pathogenic role of pre-formed MICA antibodies on first year rejection incidence and graft survival

Introdução: A susceptibilidade genética na ocorrência da sarcoidose é sugerida por alguns factores, nomeadamente pela observação de casos de agregação familiar e a associação da raça a diferentes tipos de incidência e gravidade da doença. Vários estudos têm evidenciado a associação da classe I e especialmente da classe II do sistema HLA com a susceptibilidade à sarcoidose. Objectivos: Estudo dos polimorfismos genéticos da classe I e II do sistema HLA e do TNF -á num grupo de doentes com sarcoidose, nomeadamente a sua influência na susceptibilidade, apresentação clínica e evolução da doença. Material e métodos: Foram incluídos 104 doentes com sarcoidose, tendo sido estudadas a apresentação clínica, funcional, radiológica e os resultados do LBA. Foram usados métodos de biologia molecular na genotipagem do HLA -A*, B*, C*, DRB1*, DQB1* e TNF -á. O ADN foi extraído do sangue periférico e foram usados os métodos PCR -SSP e PCR -reverse hibridization. As frequências alélicas foram comparadas com controlos da mesma região geográfica pelo teste ÷2, sendo usado o teste Kruskal -Wallis para variáveis contínuas. Resultados: Comparativamente com os controlos, os doentes incluídos apresentavam frequências aumentadas de: B*08 (10,6% vs 6,1%), OR=1,8, IC=[1,1;3,1], p=0,02; DRB1*12 (4,3% vs 1,7%), OR=2,63, IC=[1,1;6,1], p=0,03. Os doentes com eritema nodoso apresentaram aumento das frequências alélicas de DRB1*03 (28% vs 9,3%), RR=2,39, IC=[1,5;3,8], pc=0,01 e DQB1*02 (38% vs 18%), RR=2,1, IC=[1,3;3,3], pc=0,02. O alelo DQB1*03 está diminuído nos doentes que apresentam síndroma ventilatória obstrutiva, RR=0,53, IC=[0,3;0,9],pc=0,05. O alelo DRB1*15 encontra -se significativamente associado quer à síndroma ventilatória restritiva quer à diminuição da transferência alveolocapilar (21,1% vs 6,6%), RR=2,46, IC=[1,35;4,48], p=0,01 e (18,1% vs 3,8%), RR=1,87, pc=0,05, respectivamente. Por sua vez, o genótipo A/A (high) do TNF -á apresentou uma frequência aumentada (p=0,04) nos doentes com eritema nodoso. Conclusões: Os resultados obtidos adicionam evidência ao facto de, quer a classe I quer a classe II do sistema HLA influenciarem a susceptibilidade, o tipo de apresentação, o grau de gravidade e a evolução na sarcoidose. Por outro lado, o eritema nodoso parece relacionar -se com o genótipo de elevada produção de TNF -á, associação esta já anteriormente descrita.

Introduction: Several factors suggest a genetic predisposition to sarcoidosis, namely the recognition of race as a risk factor and the occurrence of familial clustering of cases. Several studies have reported an association of sarcoidosis and HLA class I and especially class II alleles in different populations. Aim: HLA class I, class II and TNF-á genotyping in a group of sarcoidosis patients and its relation with clinical presentation and outcome. Material and methods: A total of 104 sarcoidosis patients were included. Clinical presentation, functional, radiology, BAL findings and organ involvement were studied. HLA- A*, -B*, -C*, DRB1*, DQB1* and TNF-á were genotyped by molecular biology methods. DNA was extracted from peripheral blood and PCR-SSP and PCR-reverse hybridisation methods were used. Allele frequencies were compared with controls from the same region. The X2 test was used for discrete values and the Kruskal-Wallis test for continuous values. Results: When patients were compared with controls we noticed increased frequencies of B*08 (10.6% vs. 6.1%), O.R.=1.8, C.I.=[1.1;3.1], p=0.02; DRB1*12 (4.3% vs. 1.7%), O.R.=2.63, C.I.=[1.1;6.1], p=0.03. Patients with erythema nodosum have increased frequencies of the alleles DRB1*03 (28% vs. 9.3%), R.R.=2.39, C.I.=[1.5;3.8], pc=0.01 and DQB1*02 (38% vs. 18%), R.R.=2.1, C.I.=[1.3;3.3], pc=0.02. Allele DQB1*03 is decreased in patients with obstructive pattern R.R.=0.53, C.I.=[0.3;0.9], pc=0.05. Allele DRB1*15 is related to restrictive pattern and reduced diffusion capacity (21.1% vs. 6.6%), R.R.=2.46, C.I.=[1.35;4.48], p=0.01 and (18.1% vs. 3.8%), R.R.=1.87, pc= 0.05 respectively. The TNF-á A/A (high) genotype is significantly associated with erythema nodosum (p=0.04). Conclusions: These data add support to the genetic association of HLA class I and II with sarcoidosis in terms of susceptibility, type of presentation, severity and outcome. Moreover as previously described in other populations, the TNF-á A/A (high) genotype has a significant association with erythema nodosum.