Monitoreo de anticuerpos anti-HLA en pacientes con insuficiencia renal crónica en lista de espera para trasplante / Anti-hla antibodies monitoring in patients with chronic re- nal failure on waiting list for renal transplant in Paraguay

INTRODUCCIÓN: El estudio de anticuerpos anti-HLA en el suero del paciente en lista de espera para trasplante renal es fundamental para optimizar la elección de un donante así como el esquema de inmunosupresión de inducción y mantenimiento acorde al riesgo inmunológico. Estos anticuerpos pueden Encontrarse de manera preexistente al trasplante como resultado de exposición del individuo a transfusiones sanguíneas, embarazos y trasplantes previos. El objetivo del estudio fue determinar la incidencia de inmunización frente a antígenos de HLA, los factores asociados y su impacto en pacientes en espera de un trasplante renal. MATERIAL Y MÉTODOS: En este estudio, observacional retrospectivo de corte trasversal, fueron incluidos 254 pacientes en lista de espera para trasplante renal que acudieron al Laboratorio Central de Salud Pública en el período comprendido entre julio de 2013 y julio de 2015. RESULTADOS: De los 254 pacientes estudiados, 30% presentaron anticuerpos anti-HLA. El evento sensibilizante más significativo fue la exposición a un trasplante previo, presentando anticuerpos anti-HLA el 84% de los candidatos a retrasplante (p<0,05). En segundo lugar se encontraron las mujeres multíparas, presentando un PRA (Panel Reactivo de Anticuerpos) positivo el 69% de ellas (p<0,05). Por último, el 24% de los pacientes poli-transfundidos presentaron anticuerpos anti HLA (p<0,05). CONCLUSIONES: En el trascurso de los dos años de estudio, 51 pacientes fueron trasplantados, de los cuales un solo paciente presentaba anticuerpos anti-HLA antes del trasplante. Esto indica claramente que la inmunización frente a antígenos de HLA representa una barrera para el acceso al trasplante

INTRODUCTION: Anti-HLA antibodies determination in the serum of patients on a waiting list for renal transplant is essential to optimize donor selection as well as for the induction and maintenance immunosuppression scheme, according to immunological risk. These antibodies could be present before transplantation as a result of being exposed to blood transfusions, pregnancies and previous transplants. The objective of the study was to determine immunization against HLA antigens, associated factors and their impact on the waiting list for a renal transplant. METHODS: In this observational retrospective cross sectional study, 254 patients on the waiting list for renal transplant were included. These patients attended the Public Health central laboratory between July 2013 and July 2015. RESULTS: 30% of the 254 studied patients presented anti-HLA antibodies. The most significant sensitizing event was the exposure to a previous transplant (p=<0.05). Multiparous women were in second place, 69% of them presenting positive PRA (panel reactive antibodies) (p=<0.05). Finally 24% of poly transfused patients presented anti-HLA antibodies (p=<0.05). CONCLUSIONS: During the 2 year of the study, 51 patients were transplanted, presenting only one of them anti-HLA antibodies before transplantation. This results clearly indicate that the immunization against HLA represents a barrier for transplantation access

Application of Calculated Panel Reactive Antibody Using HLA Frequencies in Koreans

BACKGROUND: Introduction of the Luminex panel reactive antibody (PRA)-single antigen (SA) assay has increased the detection rates of unacceptable antigens in sensitized patients; the calculated PRA (CPRA) level represents the percentage of actual organ donors that express 1 or more of these unacceptable antigens. We developed a CPRA calculator based on the HLA frequencies in Koreans to measure sensitization levels in Korean patients. METHODS: To develop the calculator, we obtained the HLA-A, HLA-B, and HLA-DR phenotypes of 1,622 Koreans, and compared these with previously reported frequencies in Koreans. Sera from patients awaiting kidney transplantation were tested for HLA antibodies by Luminex PRA-screen, PRA-identification (ID), and PRA-SA assays. The measured %PRA from the PRA-screen (N=55) and PRA-ID (N=71) were compared to the %CPRA for the unacceptable antigens obtained from PRA-SA. RESULTS: Phenotype frequencies used for the CPRA calculator agreed with previously reported data. The concordance rates among the 3 PRA methods for the detection of class I and class II antibodies were 76.1-81.8% (kappa, 0.519-0.636) and 72.7-83.6% (0.463-0.650), respectively. For the detection of broadly sensitized sera (>50% or >80%), the concordance rates were over 80%. In sera with 80-100% CPRA, 91.7% and 94.4% of the samples had concordant results (80-100% PRA) in the PRA-screen and PRA-ID assay, respectively. CONCLUSIONS: Although further clinical studies are required to confirm the benefits of CPRA values, adoption of CPRA analysis based on HLA frequencies in Koreans may be useful for sensitization measurements and organ-allocation algorithms.

Analysis of Panel Reactive Antibody in Patients Awaiting Renal Transplantation / 대한이식학회지

BACKGROUND: We performed panel reactive antibody (PRA) tests in renal transplantation candidates registered to the Korean Network for Organ Sharing (KONOS) and analyzed the results of PRA tests in relation to the results of HLA crossmatch (XM) tests and transplantation history. METHODS: From 833 patients awaiting cadaveric renal transplantation in the KONOS registry, 122 (98 patients) XM (NIH or AHG)-positive and 147 (147 patients) XM-negative serum samples were selected for PRA test. Enzyme linked immunosorbent assay (ELISA)-PRA screening test was performed and HLA antibody specificities were identified by NIH and AHG PRA methods. RESULTS: PRA positive rate was significantly higher in XM-positive group compared with XM-negative group (ELISA-PRA, 70.5% vs. 21.8%; AHG-PRA [PRA > or =10%], 73.9% vs. 9.6%). Donor specific antibodies were defined in 52.5% (64/122), whereas false positive XM results were suspected in 20.5% (25/122) of the XM-positive samples. Patients with transplantation histories showed significantly higher positive rates for ELISA-PRA (78.7% vs. 30.8%) and AHG-XM tests (78.2% vs. 29.3%). Highly sensitized patients (AHG-PRA > or =80%) showed significantly higher cumulative waiting rate (88.9% vs. 60.2% at 4 years) and longer waiting time (3.8 vs. 3.6 years) (Kaplan Meier method, P=0.037). PRA positive rate in the total renal transplantation candidates in the KONOS registry was estimated to be 33.9% for ELISA-PRA and 21.7% for AHG-PRA (PRA > or =10%), and the proportion of highly sensitized (PRA > or =80%) patients was estimated to be 5.4%. CONCLUSIONS: Pre-transplantation PRA as a routine test is needed in cadaveric renal transplantation for effective and fair allocation of organs in Korea.

Comparison of Anti-HLA Detecting Methods; Cytotoxicity, Flow Cytometric Crossmatch, Multiple Antigen-ELISA, Single Antigen-ELISA / 대한이식학회지

PURPOSE: Identification of antibody specificity is difficult using a multiple antigen PRA (MA-PRA) assay. The purpose of this study was to determine the clinical impact of single antigen PRA (SA-PRA) ELISA assay on the transplant outcome and to analyze the clinical significance of SA-PRA compared with CDC-AHG, flow cytometric crossmatch (FCXM) and MA-PRA. METHODS: A total of 151 kidney transplanted patients were tested for the presence of HLA antibodies in the pre- and posttransplant period. The HLA specificities were classified as donor-specific antibodies (DSA) including donor private antigen specific (DS-HLA) or donor public antigen specific (DS-cross reactive group (CREG)), and nondonor specific HLA antibodies. RESULTS: Of the 151 recipients, 28 patients experienced acute rejection episodes (ARE). The pretransplant CDC-AHG, FCXM and MA-PRA tests were positive in 2, 8 and 18 patients, respectively and the concordance between FCXM and MA-PRA was 89.4% (135/151). Of the 47 sera which were tested with both MA-PRA and SA-PRA, 4 sera were SA-PRA positive and MA-PRA negative. The HLA specificities which were not determined with MA-PRA were detected with SA-PRA test. The patients with DSA showed higher incidence of ARE (7/12, 58% vs. 21/139, 15%; P<0.001) and lower glomerular filtration rate (GFR) at 6 posttransplant months (54.9+/-10.2 vs. 66.2+/-19.3; P=0.023) than the patients without DSA. The patients with ARE had higher incidence of posttransplant DS-HLA (6 (21%) vs. 0 (0%); P<0.001), DS- CREG (7 (25%) vs. 0 (0%); P<0.001), de novo HLA antibody (6 (21%) vs. 0 (0%); P<0.001) than the patients without ARE. CONCLUSION: This study suggests that analysis of HLA specificities using the SA-PRA may be useful as a supportive crossmatch test or as a monitoring test after transplantation for early detection of patients at risk of poor clinical outcome.

Development of a Web-based Program for the Identification of Human Leukocyte Antigen Antibody Specificities / 대한진단검사의학회지

BACKGROUND: Panel reactive antibody (PRA) test is used to determine whether a patient awaiting transplantation is previously sensitized. Tail analysis algorithm is widely used to identify antibody specificities, but it is very difficult to perform manually. METHODS: To develop a web-based program, PHP (5.1.2), Apache (2.0.55), and MySQL (5.0.22) were used. Tail analysis algorithm was applied to identify specificities, which analyzed statistically 2 x 2 tables representing reactivities to broad antigens, splits and cross reactive groups (CREG). Exploiting two CREG classifications of Rodey (R) and Takemoto (T), antibody specificities were identified by 3 methods (ABC, R-ABC, T-ABC) simultaneously. Performance of the system was evaluated using 159 samples that showed > or =6 PRA% by a lymphocytotoxicity assay. RESULTS: A web-based system that can identify HLA antibody specificities was implemented on www.koreanhla.com. Among 159 samples tested, antibody specificities were identified in 151 (95.0 %), but not in 8 samples with PRA >97%. Among the 151 samples, 110 showed broad or split specificities and 41 CREG specificities. CONCLUSIONS: We developed a web-based computer program for the identification of HLA antibody specificities. Accessible to everyone on the internet, this program should be of help in sharing PRA results among laboratories.

Detection of Donor Specific Anti-HLA Antibodies Using Antibody Monitoring System / 대한이식학회지

PURPOSE: The antibody monitoring system (AMS, GTI Inc.) is a solid phase ELISA crossmatch test for the detection of IgG antibody to the donor-specific solubilized HLA class I and class II antigens. The objective of this study was to compare the results of AMS assay with donor specific anti-HLA IgG antibodies (DS-HLA Abs), as determined by ELISA-PRA and flowcytometric crossmatch test (FCXM). METHODS: A total of 132 sera were tested for the presence of DS-HLA Abs by ELISA-EIA, FCXM and AMS assay. RESULTS: DS-HLA Abs were determined in 41 serum samples by an ELISA-PRA panel and FCXM. There was a significant degree of concordance (84.8%) between the results from the FCXM and AMS (P<0.001). The sensitivity, specificity, the positive predictive value and the negative predictive value of AMS assay to detect DS-HLA Abs was 90.2%, 93.4%, 86.0%, 95.5%, respectively. The AMS is a simple, objective test and it has several advantages over the cell-based crossmatch test such as elimination of non-HLA antibody reactivity, elimination of the non-donor specific antibody reactivity, no need for viable cells, and the donor's HLA antigens can be prepared in advance. CONCLUSION: This study suggests that AMS may be useful as a supportive crossmatch test or as a monitoring test after transplantation for detecting class I and/or class II DS-HLA Abs.

Pruebas de Histocompatibilidad en el Programa de Trasplantes / Histocompatibility tasting provides data to evaluate the immunological risk for transplantation

The importance of the role of the histocompatibility laboratory in solid organ transplantation is to perform HLA typing and determine the degree of HLA matching between recipient/donor. It is a useful tool to increase graft survival and decrease chronic rejection. HLA matching has a positive effect on kidney transplants and it has variable impact on other organ transplants. The crossmatch procedure is the most important test in a solid organ transplantation to evaluate the presence of recipient antibodies to antigens expressed on donor white cells. This test decreases the risk of hyperacute humoral rejection or early graft loss. Positive crossmatch is a contraindication for transplantation because it represents the existence of IgG recipient antibodies that will reath againts donor antigens. Antibody evaluation is important in donor-recipient selection and the responsability of the histocompatibility laboratory is to identify clinically relevant anti-donor HLA antibodies. This detection is useful to determine the degree of humoral alloimmunization, expressed as a percent panel reactive antibody (96PRA). This test also provides information about the antibody specificity and can be used for evaluate a patient's immune status providing a significant correlation in selecting donors.

La importancia del laboratorio de histocompatibilidad en los programas de trasplante de órganos sólidos es llevar a cabo la tipificación HLA para determinar el grado de compatibilidad que exhibe la pareja receptor/donador para el trasplante. Se tiene conocimiento que el grado de compatibilidad HLA representa un efecto positivo en el trasplante renal y en la disminución de los episodios de rechazo. Su impacto en la sobrevida de los injertos es variable en otros órganos. El procedimiento más importante para evaluar la presencia de anticuerpos preformados presentes en el suero del receptor en contra de los antígenos expresados en los linfocitos del donador es la prueba cruzada. Esta prueba permite disminuir el riesgo de un rechazo hiperagudo o la pérdida temprana del injerto. Una prueba cruzada positiva se considera como contraindicación para el trasplante por presencia de anticuerpos preformados detectables en el suero del receptor del tipo IgG en contra de los antígenos del donador en estudio. Parte de la responsabilidad del laboratorio de histocompatibilidad es la detección en el receptor de anticuerpos anti-HLA clínicamente relevantes dirigidos en contra de las especificidades antigénicas de su potencial donador. Esta evaluación es útil para conocer el grado de aloinmunización humoral del paciente (sensibilización) y se expresa como un porcentaje de reactividad de anticuerpos (%PRA). Esta prueba también permite conocer la especificidad del anticuerpo anti HLA presente, y así evaluar el estatus inmunológico del paciente y la selección del donador.

Analysis of Pretransplant ELISA-Panel Reactive Antibody in Kidney Transplant Patients / 대한이식학회지

BACKGROUND: HLA antibodies have been shown to be associated with graft loss of organ transplants in prior studies. This study was designed to analyze the results of ELISA- panel reactive antibody (ELISA-PRA) in kidney transplant patients and the impact of this test on the clinical outcome. METHODS: We have investigated ELISA-PRA results from 110 living donor renal transplant patients from Nov. 2001 to Apr. 2004. RESULTS: ELISA-PRA positivity was found in 22 (20%) patients and was higher in the female patients than male (P<0.05). Pretransplant transfusion, pregnancy or transplantation history was not significantly correlated with ELISA- PRA result. ELISA-PRA (+)patients had more rejection episodes of 41% (n=9) (P=0.0005) and graft failures of 18% (n=4) (P=0.028) than ELISA-PRA (-), which had 8% (n=7) and 3% (n=3), respectively. Patients group with a result of ELISA-PRA/flowcytometric crossmatch (FCXM) (+/ -) or (+ / +) had worse clinical outcome than ELISA- PRA/FCXM ( -/- ). ELISA-PRA/FCXM (+ /+ ) correlated with higher incidence of allograft rejection than ELIS- PRA/FCXM (+ /- ) or ( -/+ ). CONCLUSION: These results suggest that in conjunction with FCXM results, pretransplant ELISA-PRA test is useful predictor of clinical outcome in renal transplant recipients.

Clinical Implications of Pre-Transplantation Panel Reactive Antibody in Renal Transplantation in Koreans / 대한이식학회지

PURPOSE: Panel reactive antibody (PRA) is a screening test for HLA alloimmunization. The purpose of this study is to clarify the clinical implications of pre-transplantation PRA in renal transplantation in Koreans. METHODS: Study subjects included 99 renal transplant recipients whose HLA cross match tests were conducted between Jan. 1994 and Dec. 1995. Their sera were tested for PRA using 50 lymphocyte panels from Koreans by NIH and AHG methods. RESULTS: PRA was positive in 18 (18%) patients by NIH method, and in 19 (19%) patients by AHG method. NIH PRA positivity was higher in the female group (33% vs. 12% in males, P=0.01) while the age of AHG PRA (+) group was higher than the (-) group (37+/-16 vs. 28+/-13, P 45, donor age > 50 and AHG PRA (+) were associated with worse graft survival. In multivariate analysis, donor age > 50 along with AHG PRA (+), or donor age > 50 with recipient age > 45 were significant prognostic factors for graft survival. Recipient age >45, donor age > 50 and AHG PRA (+) were still prognostic of long-term graft fates in cadaveric transplants. CONCLUSION: AHG PRA correlates better with clinical data than NIH PRA and pre-transplant PRA is a significant prognostic factor for long-term graft fates in cadaveric renal recipients in Koreans.

Evaluation of In-house Lymphocyte Panel of 72 Wells for the Identification of HLA Antibody Specificity / 대한임상병리학회지

BACKGROUND: To detect anti-human leukocyte antigen(HLA) class I alloantibodies in patients awaiting solid organ transplantation, panel reactive antibody(PRA) test using complement-dependent lymphocytotoxicity(CDC) has been used. The enough size of lymphocyte panel in PRA test enables the identification of HLA antibody specificities. So we made lymphocyte panel of 72 wells to evaluate the usefulness comparing with 36 wells screening panel. METHODS: A total of 55 sera(positive 20, negative 25, quality control materials provided by "International Cell Exchange" program of UCLA Tissue Typing Laboratory 10), which had been tested for PRA using 36 wells screening panel, were re-tested using newly produced 72 wells lymphocyte panel. RESULTS: The results of the 25 negative sera were same except one serum, which might be due to non-specific reaction. The %PRA values of the 20 positive sera using 36 wells screening panel were distributed into 1-10%(n=4), 10-50%(n=9), 50-80%(n=5), and 80-100%(n=2). Using lymphocyte panel of 72 wells, %PRA values of 20 positive sera showed no difference(p=0.61) from that of 36 wells and we could not identify the specificity of HLA antibodies for the 10 sera, which previously had not been identified with 36 wells screening panel. But we additionally or newly identified the specificity of HLA antibodies in 4 positive sera and 2 quality control materials. CONCLUSION: Identification of HLA antibodies was not much improved using a PRA test with 72 lymphocyte panel and therefore 36 lymphocyte panel is considered to be enough to screen the HLA antibodies. However the increase of the size of lymphocyte panel is expected to resolve the difficulty, caused by linkage disequilibrium, for the identification of HLA antibody specificity.

Comparison of NIH-CDC and AHG-CDC methods for the Detection of Panel Reactive Antibodies / 대한임상병리학회지

BACKGROUND: Panel reactive antibody (PRA) test is used for anti-HLA antibody screening and characterization in patients awaiting organ transplantation. Complement-dependent cytotoxicity (CDC) is the most widely used standard procedure and addition of antihuman globulin (AHG) reagent to the basic (NIH) CDC method increases the sensitivity of detection of HLA antibodies. We compared NIH-CDC and AHG-CDC methods for the detection of HLA class I panel reactive antibodies. METHODS: A total of 314 sera from 253 patients were analysed for the detection of HLA class I antibodies by NIH-CDC and AHG-CDC methods using a panel of 50 lymphocytes. PRA% and reaction strength (mean score) were calculated and antibody specificities were identified with r value calculated for antibody specificity. RESULTS: A total of 46 (15%) out of 314 sera were PRA-positive (PRA%> or =10%) by either NIH-CDC (33 sera) or AHG-CDC (43 sera). Concordance of PRA test results between these two methods was 96% (301/314). AHG-CDC was more sensitive in the detection of HLA antibodies compared with NIH-CDC, showing significantly higher PRA% (44% vs 29%, P=0.0001) and reaction strength (mean score 7.3 vs 6.1, P=0.0015) for PRA-positive samples. Among 46 PRA-positive sera, HLA antibody specificities were identified in 21 samples (46%) by NIH-CDC and in 32 samples (70%) by AHG-CDC. AHG-CDC methods frequently detected a wider range of antibody specificities compared with NIH-CDC and provided a more accurate assessment of the antibody specificities. Follow up PRA tests were useful providing information on change of alloimmunization status and antibody specificities in prospective organ transplantation patients. CONCLUSIONS: Compared with NIH-CDC, AHG-CDC method is more sensitive in the detection of panel reactive antibodies and provides a more accurate assessment of the HLA antibody specificities.