Caracterización de los anticuerpos anti-HLA en posibles receptores cubanos de trasplante cardiaco / Characterization of anti-HLA antibodies in Cuban possible receptors of heart transplant

Introducción: En la supervivencia del corazón trasplantado son de importancia el empleo de los anticuerpos contra el sistema principal de histocompatibilidad (anticuerpos anti-HLA). Hace seis años se introdujo en Cuba el porcentaje de anticuerpos anti-HLA frente a panel (PRA) por método de ensayo de inmunoabsorción ligado a enzima (ELISA) como parte de las pruebas de compatibilidad pretrasplante de los receptores de trasplante cardiaco. Objetivo: Caracterizar los anticuerpos anti-HLA en pacientes receptores cubanos de trasplante cardiaco. Métodos: Entre septiembre de 2013 y abril de 2017 se les realizó el PRA por ELISA a 38 muestras de pacientes recibidas en el laboratorio de histocompatibilidad del Instituto de Hematología e Inmunología. Se utilizó la comparación de proporciones para el análisis estadístico. Resultados: El 47,4 por ciento de los pacientes estudiados presentó anticuerpos anti-HLA, fueron los más frecuentes los de clase I. La proporción de pacientes con PRA del 0 por ciento fue mayor en PRA clase II que en I (p: 0,0027). Mientras que fue mayor la proporción de pacientes con PRA clase I entre el 20 y el 75 por ciento (p: 0,0046). El 77,8 por ciento de los pacientes tuvo un PRA clase I mayor al 10 por ciento y en el PRA clase II alcanzó el 80 por ciento. Conclusiones: El porcentaje de anticuerpos anti-HLA frente a panel por método de ensayo de inmunoabsorción ligado a enzima permitió una mejor caracterización de los anticuerpos anti-HLA, lo que contribuyó a mejorar la compatibilidad en este tipo de paciente(AU)

Introduction: In survival after heart transplantation, the use of antibodies against the main histocompatibility system (anti-HLA antibodies) is important. Six years ago, the percentage of anti-HLA antibodies against panel (PRA) by enzyme-linked immunosorbent assay (ELISA) method was introduced in Cuba as part of the pre-transplant compatibility tests of heart transplant recipients. Objective: To characterize anti-HLA antibodies in Cuban heart transplant recipients. Methods: Between September 2013 and April 2017, PRA by ELISA was performed on 38 patient samples received in the histocompatibility laboratory of the Institute of Hematology and Immunology. Comparison of proportions was used for statistical analysis. Results: 47.4 percent of the study patients presented anti-HLA antibodies; those in class were the most frequent. The proportion of patients with PRA of 0 percent was higher in PRA class II than in class I (p=0.0027). The proportion of patients with PRA class I was greater, accounting for 20-75 percent (p=0.0046). 77.8 percent of the patients had a class I PRA greater than 10 percent, while in class II PRA it reached 80 percent. Conclusions: The percentage of anti-HLA antibodies versus a panel of enzyme linked immunosorbent assay method allowed better characterization of anti-HLA antibodies, which contributed to improving compatibility in this type of patient(AU)

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

Comparative effects of different extracts of Kansui Radix stir-baked with vinegar on function of expelling water retention with drastic purgative in cancerous ascites model rats / 中草药

Objective: To study the effects of different extracts of Kansui Radix stir-baked with vinegar (KRV) on the function of expelling water retention with drastic purgative in cancerous ascites model rats. Methods: The cancerous ascites model rats were respectively ig administered with KRV powder, ethanol extract, aqueous extract, and ehanol and aqueous extract of KRV (340 mg/kg) for 7 d. The amounts of urine and ascites, the levels of urinary sodium, potassium, chloride ion, and pH value, and the contents of PRA, Ang II, and ALD in serum were investigated. UPLC-QTOF MS technology was used to explore the components differences in various extracts of KRV. Results: Compared with the control group, the amount of urine in model group was significantly reduced (P<0.05), the ascites generated, and the urinary sodium, potassium, chloride ion, pH value, and the contents of PRA, Ang II, and ALD in serum were all significantly increased (P<0.01). Compared with the model groups, the treatment groups showed decreasing trend in ascites; The amounts of urine in positive groups, powder groups, ethanol and aqueous extract groups showed a significant increase (P<0.05); The level of urinary sodium of water extraction groups showed significant decrease (P<0.05); The levels of urinary sodium, potassium, chloride ion, pH value, and the contents of PRA, Ang II, and ALD in serum of positive groups, powder groups, ethanol extract groups, and ethanol and aqueous extract groups all showed a significant decrease (P<0.05, 0.01). Diterpenes were inspected in the alcohol extract and alcohol and aqueous extract, fewer in the aqueous extract. Conclusion: Powder groups and ethanol and aqueous extract groups of KRV have remarkable effect on expelling water retention with drastic purgative, and there is no significant difference between the two groups, which could provide the basis for clinical medication of KRV that is made into the pill and powder. Diterpenes in KRV may be the active components on the function of expelling water retention with drastic purgative.

Alteration of level of panel reactive antibodies, TGF-βin serum and expression rate of nTreg cells in peripheral blood in women with unexplained recurrent spontaneous abortion after immunotherapy / 中国免疫学杂志

Objective: To investigate the effects of immunotherapy on PRA, TGF-β and CD4+CD25+Foxp3+ nTreg cells in women with unexplained recurrent spontaneous abortion. Methods:We detect the level of PRA,TGF-βin serum and the expression of nTreg cells in peripheral blood of pregnant, non pregnant and natural fertility women by enzyme immunoassay and flow cytometry. Results:After immunotherapy,the median level of PRA and TGF-βin non pregnant group was lower than that in pregnant group(27. 5%,107. 7 pg/L vs 68. 75%,189. 9 pg/L,P<0. 01). Compared to pregnant group,the median expression rate of nTreg cells were lower in non pregnant group(5. 05% vs 8. 05%,P<0. 01). Conclusion:The PRA,TGF-βand nTreg cells are critical factors for pregnancy in RSA patients. It is helpful to identify and judge the effect of immunotherapy by detect the level of panel reactive antibodies,TGF-β in serum and the expression rate of nTreg cells in peripheral blood.

Expression and clinical significances of progesterone receptor isoforms A and B in ovarian se-rous cystadenocarcinoma / 临床与实验病理学杂志

Purpose To explore the effects of progesterone receptor isoforms A and B ( PR-A,PR-B) on carcinogenesis and progres-sion of ovarian serous cystadenocarcinoma ( OSC) . Methods The expressions of PR-A and PR-B in 52 cases of OSC, 22 cases of o-varian borderline serous cystadenoma ( OBSC) , 22 cases of umbrella of normal fallopian ( UNF) were detected by immunohistochmical Elivision technique. Results The expression of PR-A in OSCs, OBSCs and UNFs were 94. 5%, 94. 5%, and 68. 38%, respective-ly, with there were statistical significance among three groups (P<0. 05). The expression of PR-B in OSCs, OBSCs and UNFs were 100%, 77. 27%, and 40. 38%, respectively, with there were statistical significance among three groups (P<0. 05). The difference of PR-A/PR-B ratio in OSCs, OBSCs and UNFs was statistical significance ( P<0. 05 ) . The expressions of PR-A and PR-B in OSCs were lower, there were statistical significance between the clinical stageⅠ+Ⅱ andⅢ+Ⅳ (P<0. 05), and histological gradeⅠandⅡ. The difference of PR-A/PR-B ratio between the histological gradeⅠandⅡin OSC was statistical significance. There was statistical significance of PR-B between OSCs with lymph metastasis and without lymph metastasis (P<0. 05). Expression of PR-A and PR-B in OSC was positive correlation (P<0. 05). Conclusion With the carcinogenesis and progression of OSC, the expressions of PR-A and PR-B gradually declined, and the downregulation of PR-B is more obvious, may be an important biological sign of malignant transfor-mation in ovarian tissue. The increasing ratio of PR-A to PR-B in OSC may indicate poor differentiation. The relatively higher expres-sion of PR-B may inhibit the lymph metastasis in OSC.

Application of modern comprehensive three-dimensional teaching method in clinical practice of respiratory medicine / 中华医学教育探索杂志

Objective To evaluate the effect of modern comprehensive three-dimensional teaching method in clinical practice of respiratory medicine. Method The problem based teaching method PBL (problem-based learning), the standardized patient-based practice SPE/Xs (standardized patient experiences/exam) and Mini clinical examination practice Mini-CEX (Mini clinical examination exercise) method and other methods were organically combined and used in respiratory medicine clini-cal practice, and it was named“modern comprehensive three-dimensional teaching mode”. 60 students were randomly divided into the modern comprehensive three-dimensional teaching method group (30 students) and the traditional teaching method (SBL) (30 students), using the modern com-prehensive three-dimensional teaching method and the SBL breathing internal medicine practice teaching respec-tively. After the teaching, the methods of performance testing and questionnalre survey were used to evaluate the teaching effect of two groups of students. SPSS software was used to compare t test. Results The test results of modern three-dimensional integrated teaching method group was (86.91±4.77), higher than that of the control group (79.00±7.51) points (t=12.4, P=0.026 0). In the subjective questions (such as discourse analysis problem and case), modern three-dimensional integrated teaching group (47.63± 2.14) points is obvi-ously superior to the traditional group (36.00±6.71) points (t=21.6, P=0.001 9). According to the results of the survey, the modern comprehensive teaching method can improve students' subjective initiative and enthusiasm for learning, improve students' ability of comprehensive analysis, promote the formation of clinical thinking, and help students master the basic knowledge and basic skills of respiratory diseases. Conclusions In the respiratory medicine teaching, implementation of modern comprehensive three-dimensional teaching method is helpful to improving the teaching effect.

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

Doença hemolítica: a atuação do enfermeiro enquanto cuidador e orientador / Hemolytic sisease: the role of the nurse as caregiver and advisor

Objetivo: adquirir um expressivo conteúdo referente à doença hemolítica e abordar a atuação do enfermeiro enquanto cuidador e orientador. Método: para a elaboração deste artigo, foi realizada revisão de literatura, a partir das bases de dados Bireme, Scielo, Pubmed, Lilacs, SciElo. Considerações finais: foi possível concluir que o enfermeiro deve possuir conhecimento significativo sobre a doença hemolítica, para que possa atuar de forma efetiva enquanto cuidador e orientador, a fim de proporcionar um bom tratamento e prevenir agravos.

Objective: to acquire a expressive content related to hemolytic disease and to broach the nurse’s role while carer. Method: to prepare this article, we reviewed the literature, from databases Bireme, SciELO, PubMed, Lilacs, SciELO. Conclusion: it was concluded that the nurse must possess significant knowledge about the disease hemolytic, so you can work effectively as a carer and advisor in order to provide a good treat and prevent diseases.

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rondonia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.

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.

Effect of desensitization treatment for highly sensitized uremic patients before kidney transplantation / 中华器官移植杂志

Objective To explore the feasibility and efficacy of desensitization protocol for highly sensitized renal transplant patients (HSP). Methods Thirty-five HSPs ( HLA class-Ⅰ panel reactive antibody ＞50 %), including 27 patients with a positive T and/or B cell cytotoxicity crossmatch (XM) and 8 patients with a negative T and B cell XM, received plasmapheresis plus intravenous immunoglobulin (PP-IVIG)treatment. Results The positive XM was rendered negative by PP-IVIG treatment in 25 of 27 (92.6 %)HSPs, and subsequent transplantation was performed. Two patients did not receive renal transplants due to persistent positive XM. In 25 patients receiving renal transplants, no hyperacute rejection occurred. There were 8 cases of acute rejection, including 5 cases of acute humoral rejection (AHR). All rejection episodes were reversed. During a follow-up period of 52 ± 26 months, the serum creatinine levels at 12th and 24th month were 112± 18 and 130 ± 38 mol/L respectively. The 1- and 3-year graft survival rate was 96. 0 %and 80. 0 % respectively. Conclusion The desensitization therapy by PP-IVIG is effective for HSP. High rate of AHR is the major defect of this protocol. The short-term graft survival rate after this protocol is acceptable but the long-term survival rate needs to be defined.

Application of FLOW-PRA detection in clinical organ transplantation / 中华器官移植杂志

Objective To apply flow cytometry-panel reactive antibody (FLOW-PRA) and compare the application of traditional (enzyme-linked imrnunosorbent assay) ELISA-PRA in clinical organ transplantarion,so as to evaluate the concordance,sensitivity,accuracy and practicability of FLOW-PRA.Methods PRA was detected in 212 serum samples from 185 patients awaiting organ transplantation using FLOW-PRA and ELISA-PRA.Results It took 1.5 h and 3 h for FLOW-PRA vs ELISA-PRA.Concordance correlation coefficient for the results of the two methods was 94% (class Ⅰ)and 89% (class Ⅱ),respectively.Of all sera,24.5% (in comparison to ELISA-PRA,P<0.005)were class I positive,18.4% (P<0.05) class Ⅱ positive by flow cytometry,and 17.9% and 14.6% by ELISA,respectively.The positive incidence in Flow group was higher than in ELISA group.Low titer of antibodies was detected positively only by flow eytometry,furthermore,the antigen specificity of PRA could only be discriminated by FLOW-PRA.Conclusion Flow cytometry is more sensitive and more accurate than ELISA in PRA detection.FLOW-PRA is easy to operate and time-effective,and suitable for clinical application.

Pure Retrograde Amnesia: Is This Real Organic or Psychogenic?

Cases showing memory disturbances with isolated retrograde memory without any definitive brain lesions are classified as dissociative amnesia. Improvements in imaging techniques have allowed the organic causes of this disease to be identified in several recent cases. Several studies have diagnosed cases of memory disorders as pure retrograde amnesia (PRA). The patient reported here had no psychiatric disorder and had normal MRI results, but showed hypometabolism in PET that met the criteria for PRA.

Comparative Evaluation of ELISA and Luminex Panel Reactive Antibody Assays for HLA Alloantibody Screening / 대한진단검사의학회지

BACKGROUND: For the detection of HLA antibodies, solid-phase tests using purified HLA antigens are increasingly used. In this study, we analyzed the panel reactive antibody (PRA) test results using ELISA and Luminex methods, and the results were compared with those of crossmatch test. METHODS: A total of 111 sera including 90 sera from kidney transplanted patients were tested. ELISA-PRA was performed using Lambda Antigen Tray Class I and II Mixed kits (One Lambda Inc., USA) and additional test was performed to identify HLA specificities. Luminex-PRA tests were performed using LABScreen Mixed kits (One Lambda Inc., USA) and LIFECODES LifeScreen Deluxe kits (Tepnel Co., USA). RESULTS: The positive rates of PRA were higher in Tepnel (P=0.006) and One Lambda Luminex (P<0.001) methods than ELISA, without significant difference between two Luminex methods (P=0.087). The overall concordance rate among the three PRA tests was 62.2% (69/111). The positive and negative predictive values of PRA tests for the flow cytometric crossmatch were 33.3-45.7% and 85.7-89.5%, respectively. Of the two Luminex methods, One Lambda showed higher positive rate than Tepnel for the detection of class I antibodies. The sensitivity of pretransplant PRA for the detection of posttransplant acute rejection episodes was higher in Luminex (P=0.007 for Tepnel, P=0.003 for One lambda) than ELISA method. CONCLUSIONS: Different methods used to detect HLA antibodies showed discrepant results. As the Luminex method was more sensitive than ELISA for the detection of HLA antibodies, it can be used as a routine test in the transplantation laboratory.

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.

Identificación de micobacterias no tuberculosas: comparación de métodos bioquímicos y moleculares / Identification of non-tuberculosis mycobacteria: comparison between biochemical and molecular methods

Las infecciones causadas por micobacterias no tuberculosas (MNT) o atípicas constituyen en la actualidad un grave problema de salud, especialmente en pacientes inmunocomprometidos. Estas micobacterias presentan patrones de susceptibilidad a antibióticos particulares y distintos a M. tuberculosis, por lo que la administración del tratamiento adecuado requiere de un método rápido, sencillo y sensible de identificación. La técnica de PRA (Análisis de Restricción de Productos de PCR), basada en la digestión enzimática del producto de amplificación del gen hsp65, ha mostrado ser un método adecuado de identificación de micobacterias. En el presente trabajo se comparó la técnica de PRA con el estándar de identificación de micobacterias representado por las pruebas bioquímicas en 30 aislados provenientes del Laboratorio de Tuberculosis del Instituto de Biomedicina. La técnica de PRA permitió identificar 96% de las cepas analizadas, en comparación con 92.% de cepas identificadas por las técnicas bioquímicas. Los resultados obtenidos fueron idénticos en 18 de 22 cepas, correspondiendo al 82% de los resultados. Se concluye que el PRA es un método rápido, sencillo y económico que produce resultados concordantes con las técnicas tradicionales, con un menor grado de error. Basados en estos resultados se recomienda el uso del PRA en los laboratorios clínicos como método de identificación de rutina para micobacterias.

Infections caused by atypical mycobacteria at present constitute a serious health problem, especially in immunocompromised patients. These mycobacteria present particular susceptibility patterns, different from M. tuberculosis, due to which the administration of an adequate treatment requires a fast, simple and sensitive identification method. The PRA technique (PCR Restriction), based on the enzymatic digestion of the amplification product of the hsp65 gene has shown to be an adequate method for the identification of mycobacteria. In this study we compared the PRA technique with the standard mycobacterial identification method, represented by biochemical tests, in 30 isolates from the Tuberculosis Laboratory of the Instituto de Biomedicina. The PRA technique allowed the identification of 96% of the strains analyzed, as compared with 92% of strains identified through biochemical methods. The results obtained were identical in 18 of 22 strains, corresponding to 82% of the results. It is concluded that the PRA technique is a fast, simple and economical method that produces results in concord with traditional techniques, with a lesser degree of error. Based in these results, the use of PRA as routine identification technique for mycobacteria is recommended for clinical laboratories.

Allograft Immune Reaction of Kidney Transplantation Part 2. Immunosuppression and Methods to Assess Alloimmunity

For solid organ transplant, ABO blood type of donor and recipient should be compatible in principle. Recent improvement of immunosuppressant made HLA typing not so important while no-mismatch transplant still shows the longest graft survival. PRA(panel reactive antibody) test is to screen and identify recipients with HLA sensitization. When solid organ transplant is scheduled, cross-match test of donor cell and recipient serum should be performed and positive result of cross-match prohibits transplantation. Donor specific antibody (DSA) test can predict the severity of recipient immune reaction against donor organ. Today's mainstay of allograft immunosuppressant regimen is triple therapy of steroid, calcineurin inhibitor(cyclosporine, tacrolimus), azathioprine or mycophenolate mofetil(MMF). Antibody induction using Thymoglobulin or anti-IL-2 receptor antibody(basiliximab or daclizumab) is frequently practiced as well.

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.