Optimización de unidades formadoras de colonias y detección del virus de papiloma humano en sangre de cordón umbilical / Colony-forming units optimization and human papillomavirus detection in umbilical cord blood

Resumen Introducción: Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos. Objetivo: Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU. Métodos: Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming. Resultados: La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p < 0.0001); así como la de SCU comparada con la de segmento (p < 0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+. Conclusiones: Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Abstract Introduction: Analysis of several markers is required for adequate quality control in umbilical cord blood units (UCBU) when are used for therapeutic purposes. Objective: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. Methods: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the SSP technique. Results: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY11 and GP5/GP6+. Conclusions: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.

Neutropenia aloimune neonatal em gêmeas idênticas / Neonatal alloimmune neutropenia in identical twins

A neutropenia aloimune neonatal (NAN) é uma patologia causada pelo antagonismo imunológico, como a doença hemolítica do recém-nascido ou a trombocitopenia aloimune neonatal, mas relacionada aos neutrófilos, em vez de glóbulos vermelhos ou plaquetas. Descreveremos um caso clínico de duas gêmeas idênticas nascidas a termo, com Apgar de 8 e 9, sendo que após algumas horas do nascimento apresentaram febre. Um exame de sangue revelou neutropenia grave que resultou em sepse. O diagnóstico da NAN foi realizado clinicamente e por testes de histocompatibilidade. A prova cruzada por citometria de fluxo foi positiva, usando soro da mãe e suspensões celulares (granulócitos e linfócitos) das gêmeas e do pai. Este teste não fornece informações sobre para qual sistema genético os anticorpos foram positivos, se contra os antígenos específicos de neutrófilos humanos (HNA) ou contra os antígenos leucocitários humanos (HLA). Para o esclarecimento, realizamos o teste de aglutinação de granulócitos (GAT) com um painel de doadores fidelizados e com antígenos HNA1-5 conhecidos, utilizando o soro materno como reagente. Foi também realizada a pesquisa de anticorpos anti-HLA e anti-HNA no soro materno. Os genótipos HLA e HNA foram identificados, permitindo conhecer as especificidades dos anticorpos maternos contra os antígenos dos neutrófilos do marido e das filhas. O diagnóstico de NAN não é realizado na maioria dos hospitais de nosso país e do exterior, devido à dificuldade de execução dos testes de histocompatibilidade, no entanto a prova cruzada por citometria de fluxo pode facilmente ser implantada nos laboratórios clínicos, sendo que está descrita detalhadamente nesse caso clínico.

Neonatal alloimmune neutropenia (NAN) is a disease caused by immunological antagonism, such as hemolytic disease of the newborn or neonatal alloimmune thrombocytopenia, but related to neutrophils rather than to red blood cells or platelets. We will describe a clinical case of two identical twins born with Apgar 8 and 9 that started with fever few hours after delivery. A blood test revealed severe neutropenia, which was followed by sepsis. The diagnosis of NAN was done clinically and by histocompatibility testing. Flow cytometry crossmatch was positive, using mother serum and cell suspensions (granulocytes and lymphocytes) from the twin girls and from the father. This test did not provide information about the genetic system for which the antibodies are positive, if against human neutrophil antigens (HNA) or human leucocyte antigens (HLA). To clear this, the granulocyte agglutination test (GAT) was performed with a panel of control donors with known HNA1-5 antigens, using the maternal serum as a reagent. We did also a Luminex screening assay for detection of anti-HLA and anti-HNA antibodies in the mother serum. The HLA and HNA genotypes were identified, which allowed to define specificities in mother's antibodies against the neutrophil surface antigens from her husband and from the twins. The diagnosis of NAN diagnose is not done in most hospitals worldwide, mainly by the difficulty in executing the histocompatibility test. However, the crossmatch by flow cytometry could be easily done in clinical laboratories following the method described in this article.

Optimization of HLA-B*27 ALLELE Genotyping by PCR-SSP

OBJECTIVES: HLA-B27 is strongly associated with ankylosing spondylitis (AS) and its presence helps to confirm AS diagnosis. Due to the high HLA polymorphism and the differentiated contribution of alleles and molecules encoded by them, HLA-B*27 allele identification is relevant in the clinical follow-up, diagnosis, and treatment of this spondyloarthropathy. Inexpensive genotyping techniques with high specificity and sensitivity are of great interest in histocompatibility laboratories. This work aimed to optimize HLA-B*27 genotyping by Polymerase Chain Reaction Sequence-specific Primer (PCR-SSP), which is an accessible and inexpensive technique. METHODS: The PCR-SSP was standardized using 26 HLA-B*27 positive and 3 HLA-B*27 negative samples previously defined by Polymerase Chain Reaction Sequence-specific Oligonucleotide Probes (PCR-SSOP) (medium resolution, One Lambda®) and primers described by Duangchanchot et al. (2009). For validating the technique, 397 samples were genotyped using PCR-SSP as well as PCR-SSOP. RESULTS: The PCR-SSP technique was standardized for identifying the alleles HLA-B*27:02, HLA-B*27:CAFRW (05/13/16/17/28/37/38/39/42), HLA-B*27:CAFRZ (08/26/40), HLA-B*27:09 and HLA-B*27:12, which were found in 90 positive samples (22.67%). There was 100% agreement between the two techniques for heterozygous samples; however, two homozygous samples could not be detected by PCR-SSP. CONCLUSION: The HLA-B*27 genotyping using PCR-SSP, an easy-to-use, specific, and affordable technique, was optimized for heterozygous samples. This technique may contribute to AS diagnosis.

Frequência dos alelos do sistema antígeno leucocitário humano em doadores e pacientes pré-transplante de medula óssea / Human leukocyte antigen alleles frequency in donors and pre-transplant patients from bone marrow

Introdução: O estudo da frequência dos alelos detectados nos doadores e pacientes previamente selecionados para o transplante de medula óssea permite estimar as reais chances de um paciente em lista de espera encontrar um doador com antígeno leucocitário humano (Human leucocite antigen; HLA) idêntico não relacionado, além de facilitar e direcionar o planejamento do crescimento do Registro Nacional deDoadores de Medula Óssea. Objetivo: Descrever e analisar afrequência dos alelos do sistema HLA de classe I (HLA-A, -B e -C) e classe II (HLA-DRB1 e -DQB1) de doadores e pacientespré-transplante de medula óssea, do Hospital de Câncer deBarretos. Material e Métodos: Um total de 98 amostras dedoadores e 106 amostras de pacientes foi selecionado comtipificações em alta resolução, no período de outubro de 2014a outubro de 2015. As amostras foram tipificadas para os lociHLA-A, -B, -C, -DR e -DQ. Resultados: O predomínio daraça branca reflete a composição étnica do Brasil. As doençasde base mais comuns que levaram o paciente ao transplanteforam a leucemia aguda linfóide (34%) e mieloide (29,2%).Os grupos alélicos mais frequentes nos registros foramA*02, A*24, A*03, A*01, B*35, B*44, C*07, DQB1*03,DQB1*05, DQB1*06, DRB1*01 e DRB1*13. Conclusão: Osresultados encontrados reforçam a importância de conhecero perfil demográfico e imunogenético das regiões do Brasil,contribuindo desta forma na redução do tempo de espera porum doador histocompatível

Introduction: The study of allele frequencies detected in donors and patients previously selected for bone marrow transplantation allows us to estimate the real chances of a patient in the waiting list to find an Human leucocite antigen (HLA) identical unrelated donor. This also facilitates and drives the growth planning of the Brazilian Registry of planning Bone Marrow Transplantation (REDOME). Objective: Describe and analyze the frequency of HLA class I alleles (HLA-A*, -B* and ­C*) and class II alleles, genotypes, and haplotypes(HLA-DRB1* and -DQB1*) from donors and bone marrowpre-transplant patients. Material and Methods: A total of 98donor samples and 106 patient samples were selected withhigh resolution typing, from October 2014 to October 2015.Samples were typed for HLA-A, -B, -C, -DR and -DQ loci.Results: The predominance of the white race reflects theethnic composition of Brazil. The most common underlyingdiseases that led to transplantation patients were acutelymphoid leukemia (34%) and myeloid (29.2%). The mostfrequent allelic groups were A*02, A*24, A*03, A*01, B*35,B*44, C*07, DQB1*03, DQB1*05, DQB1*06, DRB1*01 andDRB1*13. Conclusion: The results reinforce the importanceof understanding the demographic and immunogenic profilefrom Brazilian Regions. This can contribute to the reduction ofwaiting time for a histocompatible donor.

Uso de everolimo de novo em receptores de transplante renal com doador vivo HLA idêntico / De novo everolimus for recipients of kidney transplants from HLA identical donors

Resumo Introdução: Receptores de rim de doadores vivos HLA-idêntico apresentam menor risco para rejeição aguda e maior sobrevida do enxerto, quando comparado a outros tipos de transplante. Um regime imunossupressor sem inibidor de calcineurina (ICN) pode melhorar ainda mais esses resultados, através da redução de eventos cardiovasculares, metabólicos e tóxicos secundários a esse fármaco. Objetivo: Avaliar eficácia e segurança do novo tratamento imunossupressor com suspensão planejada do ICN. Métodos: Estudo prospectivo, aberto, braço único de tratamento em único centro para avaliar resultados do transplante renal HLA-idêntico em pacientes que recebem everolimo (EVR), tacrolimo (TAC) e corticoide, seguido da descontinuação do TAC 30 dias pós-transplante. Após análise interina de eficácia, a descontinuação do TAC foi postergada para o terceiro mês pós-transplante, através de emenda ao protocolo. Resultados: Trinta e nove pacientes foram incluídos. Apesar de as médias das concentrações de TAC e EVR terem respeitado os intervalos propostos, cinco pacientes tiveram rejeição aguda comprovada por biópsia e um paciente apresentou um episódio de glomerulite com depósitos de C4D. Esse resultado demandou o fim das inclusões. A proporção de pacientes com proteinúria > 0.5g/L não atingiu mais que 22% dos pacientes em nenhuma visita. Os eventos adversos mais frequentes foram relacionados ao uso de EVR: úlceras orais, dislipidemia e edema periférico. Conclusão: O regime proposto não foi eficaz para essa população, principalmente pela alta incidência de rejeição aguda. O perfil de segurança mostrou que a exposição prolongada a altas concentrações sanguíneas de EVR aumenta a incidência dos eventos adversos relacionados ao fármaco.

Abstract Introduction: Kidney transplant recipients from HLA-identical living donor have lower risk of acute rejection and greater graft survival compared to other types of kidney transplantation. Immunosuppressive regimens without calcineurin inhibitors (CNI) can further improve these results by reducing cardiovascular, metabolic and toxic events related to this drug class. Objective: This study aimed to evaluate efficacy and safety of a new immunosuppressive regimen with planned suspension of CNI. Methods: This was a prospective, single center and single treatment arm study to evaluate HLA-identical kidney transplant recipients receiving everolimus (EVR), tacrolimus (TAC) and corticosteroids, followed by TAC discontinuation 30 days after transplantation. TAC discontinuation was later postponed to the third month after an interim efficacy analysis. Results: Thirty-nine patients were included. Although mean TAC and EVR blood concentrations have remained within the proposed therapeutic ranges, five patients had biopsy-proven acute rejection and one patient had an episode of C4D-positive glomerulitis. This result led to the end of the inclusions. Interestingly, the proportion of patients with proteinuria greater than 0.5 g/L has not reached more than 22% of patients in any visit. Adverse events related to EVR use were the most incident in this population: oral ulcers, dyslipidemia and peripheral edema. Conclusion: The proposed scheme was not effective for this population, particularly due to a high incidence of acute rejection. Safety profile showed that prolonged exposure to a high concentration of blood EVR increases the incidence of adverse events related to this drug.

Enfermedad celíaca: revisión / Celiac disease: a review

La Enfermedad Celiaca (EC) tiene una prevalencia cercana al 1% de la población general y se considera que hay un número importante de pacientes asintomáticos no diagnosticados. Su presentación clínica es variable comprendiendo el clásico síndrome de malabsorción, formas menores y la EC silente. El diagnóstico serológico tiene una elevada sensibilidad y especificidad y siempre debe confirmarse con biopsia. El diagnóstico en pacientes en dieta libre de gluten incluye test de tipificación de HLA y prueba de dieta con gluten con estudio serológico e histológico posterior. El pilar del tratamiento es la dieta libre de gluten, que debe ser supervisada por un nutriólogo con experiencia. La monitorización de la terapia debe realizarse con serología. La EC mal controlada puede determinar complicaciones como linfoma y adenocarcinoma de intestino delgado. En el futuro es probable que nuevas terapias farmacológicas sean de utilidad en el manejo de la EC.

Celiac disease has a prevalence near to 1% of general population and there is an important amount of asymptomatic people not yet diagnosed. Clinical presentation includes the classical malabsorption syndrome, minor and silent celiac disease. Serologic diagnosis has an elevated sensitivity and specificity, and must be confirmed by biopsy. Diagnosis in those on gluten free diet includes HLA type and gluten challenge with posterior serologic and histologic evaluation. The core of the treatment is the gluten free diet that must be supervised by an expert nutritionist. Monitoring is with serology. Poor disease control can determine complications such as lymphoma and small bowel adenocarcinoma. In the future, it is likely that new pharmacologic therapies will be available for the management of celiac disease.

Frecuencia de genes HLA en pacientes con insuficiencia renal crónica procedentes del occidente y centro de Cuba / Frequency of HLA genes in patients with chronic kidney insufficiency, from western and central Cuba

Introducción: el trasplante es la terapia que permite la mayor sobrevida a los pacientes con insuficiencia renal crónica. Para prevenir el rechazo del órgano, en primer lugar es necesario un estudio de la compatibilidad de los antígenos leucocitarios humanos (HLA) del paciente y de los posibles donantes. En Cuba solo se había realizado la tipificación HLA por métodos serológicos, pero en la actualidad se emplean técnicas moleculares. Objetivo: caracterizar el polimorfismo de los alelos HLA A, B, DR y DQ por métodos moleculares en pacientes cubanos en espera de trasplante renal. Métodos: se estudiaron 410 pacientes con insuficiencia renal crónica de las regiones occidental y central del país a los que se les realizó tipificación molecular de los loci mencionados. Los resultados se expresaron según la nueva nomenclatura y fueron registrados en una base de datos confeccionada al efecto. Se compararon las frecuencias alélicas de la población blanca y no blanca y se determinó el porcentaje de frecuencia de los haplotipos para los alelos clase I y II. Resultados: los alelos A*11, A*30, A*74, B*42, B*51 y B*53 fueron más frecuentes en la población blanca mientras que los alelos B*58 y DRB1* 15 predominaron en los no blancos. Las frecuencias haplotípicas más encontradas en la clase I en la población blanca fueron A*02 B*51, A*02 B*44, A*02 B*35; y en la no blanca, A*01B*08, A*02B*51, A*02B*44. Para los alelos de la clase II, en la población blanca fueron DQB1*03, DRB1*04, DQB1*06, DRB1*13, DRB1*05, DRB1*01; y en los no blancos, DQB1*03, DRB1*04, DQB1*06, DRB1*13, DQB1*05, DRB1*01. Conclusiones: la caracterización de los pacientes con insuficiencia renal crónica con respecto a su tipificación HLA permitirá trazar estrategias futuras relacionadas con la donación y el trasplante en todo el país(AU)

Introduction: transplantation is the therapy allowing the highest possible survival in patients with chronic kidney insufficiency. To prevent rejection of the organ, first of all it is necessary to make a compatibility test of human leukocyte antigens (HLA) from the patient and the possible donors. In Cuba, only serological HLA typing had been made but at present, molecular techniques are being applied. Aim: characterization of polimorfirsm of alleles HLA A, B, DR y DQ by molecular techniques in Cuban patients awaiting renal transplantation. Methods: four hundred and ten patients with chronic kidney insufficiency from Western and Central Cuba were studied by molecular typing of the above mentioned loci. Results were expressed by the new nomenclature and were registered In a data base prepared for that purpose. Allele frequencies of white and no white population were compared and percentage of haplotype frequencies for alleles class I and II were determined. Results: alleles A*11, A*30, A*74, B*42, B*51and B*53 were more frequent in white population while B*58 y DRB1*, 15 were mostly found in no whites. Haplotypic frequencies most found in class I in white population were A*02 B*51, A*02 B*44, A*02 B*35; and in no whites, A*01B*08, A*02B*51, A*02B*44. For class II alleles, DQB1*03, DRB1*04, DQB1*06, DRB1*13, DRB1*05, DRB1*01 were the most found in white population; and in no whites, DQB1*03, DRB1*04, DQB1*06, DRB1*13, DQB1*05, DRB1*01. Conclusions: characterization of patients with chronic kidney insufficiency in respect to HLA typing will allow future strategies related to kidney donation and transplantation in the whole country(AU)

Discrepancies in HLA typing: use of different methods to confirm doubtful or inconclusive results / Discrepâncias na tipagem HLA: diferentes métodos para confirmar resultados duvidosos ou inconclusivos

The major histocompatibility complex (MHC) is a set of genes found on the short arm of chromosome 6. MHC molecules in human beings are known as human leukocyte antigens (HLA). HLA polymorphism can be determined by serological and molecular typing methods, which may yield discordant results. The present analysis performed HLA typing of samples with discordant results by PCR-SSP and PCR-SSO, so that typing discrepancies could be clarified. The cross-sectional study analyzed 33 samples from individuals included in an HLA-disease association study. Discrepant alleles were observed in 6 of 33 samples. Discordant samples were retyped using One Lambda Micro SSP™, Dynal RELI™ SSO and Luminex™ SSO assays for HLA class I (HLA-A, HLA-B) and class II (HLA-DRB1) molecules. The three methods produced concordant results after HLA retyping. Human error occurred in interpreting the initial results, which led to discrepancies in the results obtained. The participation of experienced professionals and the availability of at least two different methods to confirm doubtful or inconclusive results are mandatory for effective HLA typing.

O complexo principal de histocompatibilidade (MHC) é um conjunto de genes encontrados no braço curto do cromossomo 6. Em humanos, as moléculas de MHC são conhecidas como antígenos leucocitários humanos (HLA). Polimorfismo HLA pode ser determinado por métodos de tipagem sorológica e molecular que são susceptíveis de produzir resultados discordantes. Este estudo teve como objetivo realizar a tipagem HLA de amostras com resultados discordantes por PCR-SSP e-SSO e para esclarecer discrepâncias de digitação. Este estudo transversal analisou 33 amostras de indivíduos incluídos em um estudo de associação HLA-doença. Alelos discrepantes foram observados em seis das 33 amostras. Amostras discordantes foram retyped usando One Lambda Micro SSP™, Dynal RELI™ SSO Luminex e ensaios ™ SSO para HLA de classe I (HLA-A, HLA-B) e classe II (HLA-DRB1) moléculas. Todos os três métodos apresentaram resultados concordantes após HLA redigitação. Houve erro humano na interpretação dos resultados iniciais o que levou a uma discrepância entre os resultados obtidos. Concluiu-se que a participação de profissionais experientes e com a disponibilidade de pelo menos dois métodos diferentes para confirmar os resultados duvidosos ou inconclusivos são essenciais para a tipagem de HLA eficaz.

Challenges with sensitized recipients in pediatric heart transplantation

The sensitization of patients to human leukocyte antigens prior to heart transplantation is increasingly being recognized as an important challenge both before and after the transplant, and the effects of sensitization on clinical outcomes are just beginning to be understood. Many patients are listed with the requirement of a negative prospective or virtual crossmatch prior to accepting a donor organ. This strategy has been associated with both longer waitlist times and higher waitlist mortality. An alternative approach is to transplant across a potentially positive crossmatch while utilizing strategies to decrease the significance of the human leukocyte antigen antibodies. This review will examine the challenges and the impact of sensitization on pediatric patients prior to and following heart transplantation.

Issues in solid-organ transplantation in children: translational research from bench to bedside

In this review, we identify important challenges facing physicians responsible for renal and cardiac transplantation in children based on a review of the contemporary medical literature. Regarding pediatric renal transplantation, we discuss the challenge of antibody-mediated rejection, focusing on both acute and chronic antibody-mediated rejection. We review new diagnostic approaches to antibody-mediated rejection, such as panel-reactive antibodies, donor-specific cross-matching, antibody assays, risk assessment and diagnosis of antibody-mediated rejection, the pathology of antibody-mediated rejection, the issue of ABO incompatibility in renal transplantation, new therapies for antibody-mediated rejection, inhibiting of residual antibodies, the suppression or depletion of B-cells, genetic approaches to treating acute antibody-mediated rejection, and identifying future translational research directions in kidney transplantation in children. Regarding pediatric cardiac transplantation, we discuss the mechanisms of cardiac transplant rejection, including the role of endomyocardial biopsy in detecting graft rejection and the role of biomarkers in detecting cardiac graft rejection, including biomarkers of inflammation, cardiomyocyte injury, or stress. We review cardiac allograft vasculopathy. We also address the role of genetic analyses, including genome-wide association studies, gene expression profiling using entities such as AlloMap®, and adenosine triphosphate release as a measure of immune function using the Cylex® ImmuKnow™ cell function assay. Finally, we identify future translational research directions in heart transplantation in children.

Frecuencias alélicas, genotípicas y haplotípicas del sistema HLA clase I y II en donantes de una población del suroccidente colombiano / Allele, genotype and haplotype frequencies of HLA system class I and II in donors in a population in southwestern Colombia

Introducción: la determinación de los alelos que se expresan con mayor frecuencia en una población permite conocer la probabilidad que tiene una persona de encontrar un donante no relacionado compatible en esa población y sirve de referencia para estudios de asociación del HLA con diferentes enfermedades y aplicaciones en estudios antropogenéticos. Objetivo general: determinar las frecuencias alélicas HLA-A*, B* y DRB1* en una población del suroccidente colombiano. Diseño: estudio descriptivo retrospectivo. Material y métodos: estudio descriptivo y retrospectivo en el cual se incluyeron 1230 donantes (vivos y fallecidos) evaluados en el protocolo para trasplante renal y/o de médula ósea de la Fundación Valle del Lili. La tipificación se hizo por métodos moleculares y el análisis estadístico de las frecuencias alélicas, genotípicas y haplotípicas se realizó con los programa genéticos GENEPOP y Arlequín. Resultados: se identificaron 20, 29 y 13 alelos para los locus HLA-A*, HLA-B* y HLA-DRB1* respectivamente y los alelos más frecuentes fueron A*02 (25%), B*35 (17,7%) y DRB1*04 (23%). Los haplotipos más frecuentes para dos loci fueron A*24B*35(8,9%), B*35DRB*04 (8,1%) y A*24DRB*04 (10,9%) y para tres loci A*24B*35DRB1*04 (5.4%) y A*24B*40DRB1*04 (3,2%). Conclusión: los alelos que se expresan con mayor frecuencia en la población del suroccidente colombiano están representados por los alelos más comunes en la población colombiana. (Acta Med Colomb 2013; 38: 16-21).

Introduction: the determination of the alleles expressed more often in a population allows to know the probability of a person to find a matched unrelated donor in that population and serves as reference for the HLA association studies with different diseases and applications in anthropogenetic studies. Objective: to determine the allelic frequencies HLA-A*, B* and DRB1* in the Colombia southwestern population. Design: retrospective descriptive study. Methods: in this study we included 1230 donors (living and deceased) evaluated witin our renal transplantation or bone marrow transplant protocol following at the Fundación Valle del Lili in Cali, Colombia. The typing was made by molecular methods and the statistical analysis of allele, genotype and haplotype frequencies was performed with the genetic programs GENEPOP and Arlequin. Results: we identified 20, 29 and 13 alleles for the HLA-A*, HLA-B* and HLA-DRB1* locus respectively and the most frequent alleles were A* 02 (25%), B* 35 (17.7% ) and DRB1* 04 (23%). The haplotypes for the two more frequent loci were A* 24B* 35 (8.9%), B* 35DRB* 04 (8.1%) and A* 24DRB* 04 (10.9%) and for three loci A* 24B * 35DRB1* 04 (5.4%) and A* 24B* 40DRB1* 04 (3.2%). Conclusion: the alleles expressed more frequently in the Colombia southwestern population are represented by the most common alleles in the colombian population. (Acta Med Colomb 2013; 38: 16-21).

Frecuencia génica de antígenos menores de histocompatibilidad en la población chilena y estimación de sus efectos inmunológicos en el trasplante alogénico de progenitores hematopoyéticos / Frequency of minor histocompatibility antigens among Chilean blood donors

Background: Minor histocompatibility antigens (mHAgs) play a critical role in the immune responses associated with allogeneic stem cell transplantation, such as graft versus host disease (GVHD) and graft-versus-tumor (GVT). Aim: To determine the gene frequencies of the mHAgs HA-1, HA-2 and HA-8 in Chilean Blood Bank donors. Material and Methods: Blood from 192 blood donors was analyzed. The presence of haplotype HLA-A*02 was determined by flow cytometry. The frequency of mHAgs was determined by allele specific polymerase chain reaction in genomic DNA. Results: Sixty one participants were carriers of the haplotype HLA-A*02. The relative allele frequency HA-1H was 45%, HA-Ir 55%, HA-2V 80.6%, HA-2M 19.4%, HA-8R 49.8% and HA-8P was 50.2%. Based on mHAgs disparity between HA-1, HA-2 or HA-8, the probability to generate a GVT response in HLA-A*02 individuals was 40%. Conclusions: The mHAgs frequency in Chilean population is under Hardy-Weinberg equilibrium and they are similar to those of other ethnic populations in the world.

Suero de conejo con actividad de complemento para tipificación del complejo mayor de histocompatibilidad humano / Rabbit serum with complement activity to typify human major histocompatibility complex

En el ensayo de microlinfocitotoxicidad, el suero de conejo como fuente de complemento desempeña un rol esencial en la clasificación del complejo mayor de histocompatibilidad humano, tanto para la tipificación antigénica como en el cross-match linfocitario de la pareja donante-receptor antes de realizar los trasplantes. En este trabajo, se obtuvieron 3 lotes experimentales de dicho hemoderivado con óptimos indicadores de rendimiento, actividad biológica y estabilidad. El exanguíneo de los animales se realizó por la técnica de yugulación. El suero fue separado por centrifugación en condiciones ambientales y de temperatura controladas. Posteriormente, el producto se sometió a filtración esterilizante y se tomaron muestras para los ensayos de calidad. El estudio mostró que el producto conserva la actividad biológica al menos durante 18 meses y que los valores de citotoxicidad se encuentran dentro de los límites de aceptación para su empleo en los ensayos de histocompatibilidad pretrasplante

Microlinfocitotoxicity assay is one of the serological methods used to classify human major histocompatibility complex. In this technique the rabbit´s serum as complement source, plays an essential role in antigens determination and in lymphocytes cross match assay as necessary stage before the selection of the best donor-receiver pair to realize the organ transplant. Three experimental lots of normal rabbit serum were developed with excellent indicators of efficiency, biological activity and stability. Rabbit's blood was obtained through jugulating technique and serum was separated by centrifugation in controlled environment and temperature´s conditions. The sterilization process was performed by filtration and the samples were taken for quality´s control assays. Stability studies showed that the product kept the biological activity at least during 18 months after it was processed and cytotoxicity's values were adequate for its employment in histocompatibility pre-transplant assays

Importance of killer immunoglobulin-like receptors in allogeneic hematopoietic stem cell transplantation

Hematopoietic stem cell transplantation is the treatment of choice for many hematologic diseases, such as multiple myeloma, bone marrow aplasia and leukemia. Human leukocyte antigen (HLA) compatibility is an important tool to prevent post-transplant complications such as graft rejection and graft-versus-host disease, but the high rates of relapse limit the survival of transplant patients. Natural Killer cells, a type of lymphocyte that is a key element in the defense against tumor cells, cells infected with viruses and intracellular microbes, have different receptors on their surfaces that regulate their cytotoxicity. Killer immunoglobulin-like receptors are the most important, interacting consistently with human leukocyte antigen class I molecules present in other cells and thus controlling the activation of natural killer cells. Several studies have shown that certain combinations of killer immunoglobulin-like receptors and human leukocyte antigens (in both donors and recipients) can affect the chances of survival of transplant patients, particularly in relation to the graft-versusleukemia effect, which may be associated to decreased relapse rates in certain groups. This review aims to shed light on the mechanisms and effects of killer immunoglobulin-like receptors - human leukocyte antigen associations and their implications following hematopoietic stem cell transplantation, and to critically analyze the results obtained by the studies presented herein.

Analysis of HLA-A, HLA-B, HLA-DRB1 allelic, genotypic, and haplotypic frequencies in Colombian population / Análisis de frecuencias alélicas, genotípicas y haplotípicas HLA -A, HLA-B, HLA-DRB1 en población colombiana

Introduction: The high polymorphism of the HLA system allows its typification to be used as valuable tool in establishing association to various illnesses, immune and genetic profiles; it also provides a guide to identifying compatibility among donors and receptors of organs transplants. Objective: To establish HLA-A, HLA-B, and HLA.DRB1 allele, genotype and haplotype frequencies among patients treated at Clinica Colsanitas SA. Methods: 561 patients coming from different regions in Colombia, who were attended in 8 centers of the clinical laboratory of the Clinica Colsanitas in different cities of the country from January 2004 to August 2008, were included in this study. All were HLA-A,-B, and -DRB1 typified via SSP PCR. Allele, genotype and haplotype frequencies were estimated with STATA Software Version 9.0 and the GENEPOP genetic analysis package. Results: 19, 28, and 15 different alleles were identified for loci HLA-A,-B and -DRB1, respectively. Alleles found most frequently were A*24 (26.2%), A*02 (26%), B*35(22.7%), and DRB1*04 (24%). The most frequent genotypes were A*02,24 (14.2%), B*07,35 (5.5%), DRB1*01,04, and DRB1*04,04 (6.9%); while most the frequent haplotypes were HLA A*24, B*35 (9.2%), A*24, DRB1*04 (8.1%); B*35, DRB1*04 (7.8%), A*2 DRB1*04 (7.4%). Conclusion: The results obtained provide a useful reference framework for the population studied, allowing compatibility probability calculations to be performed for organ transplants.

Introducción: El alto polimorfismo del sistema HLA, hace que su tipificación sea una herramienta de gran valor al establecer asociación con diferentes enfermedades, patrones inmunológicos, antropogenéticos, así como para establecer probabilidades de encontrar donantes compatibles con receptores de diferentes tipos de trasplante de órganos. Objetivo: Establecer las frecuencias alélicas, genotípicas y haplotípicas en pacientes atendidos en la Clínica Colsanitas SA. Metodología: Se incluyeron un total de 561 pacientes atendidos en el Laboratorio Clínico de La Clínica Colsanitas SA, en 8 sedes en diferentes ciudades del Colombia, durante el período comprendido entre enero de 2004 a agosto de 2008. Se realizó tipificación de HLA -A,-B,-DRB1 por PCR SSP. Las frecuencias alélicas, genotípicas y haplotípicas fueron estimadas mediante el paquete estadístico Stata y el paquete de análisis genético Genepop. Resultados: Fue posible la identificación de 19, 28 y 15 alelos de los loci HLA A-B-DRB1 respectivamente, de los cuales los más frecuentes fueron A*24 (26.2%), A*02 (26%), B*35 (22.7%), DRB1*04 (24%). Los genotipos más frecuentes encontrados fueron A*02,24 (14.2%), B*07,35 (5.5%), DRB1*01,04 y DRB1*04,04 (6.9%). Los haplotipos más frecuentes fueron: HLA A*24, B*35 (9.2%), A*24, DRB1*04 (8.1%); B* 35, DRB1*04 (7.8%), A*2 DRB1*04 (7.4%). Conclusión: Los resultados obtenidos permiten tener referencia para aplicaciones en la población estudiada, así como para establecer probabilidades de compatibilidad en la creciente área de trasplante de órganos.

Does minor histocompatibility antigen HA-1 disparity affect the occurrence of graft-versus-host disease in tunisian recipients of hematopoietic stem cells?

INTRODUCTION: Minor histocompatibility antigen HA-1 (MiHAg-HA-1) disparity between a patient and his or her human leukocyte antigen (HLA) genoidentical donor has been widely associated with an increased risk of graft-versus-host disease following allogeneic hematopoietic stem cell transplantation. OBJECTIVE: To examine the effect of HA-1 disparity on the incidence of both acute and chronic graft-versus-host disease in Tunisian recipients of hematopoietic stem cells. METHODS: A total of 60 patients and their 60 respective sibling hematopoietic stem cell donors were enrolled in this study. All patients prophylactically received cyclosporine A and/or methotrexate for graft-versus-host disease. An HA-1 genotyping assay was performed with the SSP-PCR method, and HLA-A*0201- and/or HLA-A*0206-positive samples were identified using the Luminex HLA typing method. RESULTS: The Luminex HLA typing assay showed that 54 patients were positive for either the HLA-A*0201 or HLA-A*0206 alleles. Among these cases, six pairs were mismatched for MiHAg-HA-1. Both acute and chronic graft-versus-host disease occurred in four mismatched patients (Fisher's p-values were 0.044 and 0.170, respectively). A univariate logistic regression model analysis showed that only acute graft-versus-host disease may be affected by recipient MiHAg-HA-1 disparity (p: 0.041, OR: 6.727), while chronic graft-versus-host disease correlates with both age and recipient/donor sex mismatch (p: 0.014, OR: 8.556 and p: 0.033, OR: 8.664, respectively). CONCLUSION: Our findings support previously reported data suggesting a significant association between HA-1 disparity and the risk of acute graft-versus-host disease following hematopoietic stem cell transplantation.

Análise do polimorfismo do gene HLA-A na infecção pelo vírus dengue e as inter-relações na resistência/susceptibilidade à febre hemorráica da dengue / Analysis of polymorphism of the HLA-A gene in dengue infection and interrelations in resistance / susceptibility to dengue fever hemorráica

As formas graves da dengue, a febre hemorrágica da dengue (FHD) e a síndrome de choque da dengue (SCD) são as expressões de um mecanismo patogênico complexo no qual interagem o vírus, o ambiente e o hospedeiro, e principalmente ressalta-se a resposta imunológica deste. A influência da composição genética do hospedeiro na susceptibilidade ou resistência a doenças tem sido estabelecida em diversos estudos. O sistema HLA é bem conhecido pela sua importância na rejeição de transplantes e na regulação da resposta imune, protegendo o organismo contra agentes infecciosos. O numeroso polimorfismo dos genes HLA que expressam as moléculas de CPH de classe I e II, pode acarretar diferenças na natureza e magnitude da resposta imunológica. Estas diferenças são geradas pela apresentação de antígenos do vírus dengue pela molécula de classe I aos linfócitos T CD8...

Neste sentido, visando a contribuir para o entendimento da imunopatologia da dengue, descrevemos as características demográficas e clínicas dos pacientes e investigamos a associação alélica do gene HLA-Acom a evolução para a FHD. O HLA-Ade 136 indivíduos residentes no Rio de Janeiro provenientes das epidemias no período de 2001 a 2008 (67 classificados como FD, 42 como FHD e 27 negativos para a infecção por dengue) foi tipificado através da técnica de alta resolução SBT (Sequence-based Typing) e as distribuições alélicas comparadas entre dois grupos de pacientes (FD e FHD). A maior parte dos pacientes com FHD era do sexo feminino (54,8%),ao contrário do grupo FD cuja maioria era homens (53,7%)Pacientes de cor branca foram predominantes independentemente da forma clínica dadengue. As análises estatísticas confirmaram a associação do grupo alélico HLA-A*01 com a evolução para FHD (p=0,01; OR=2,7; IC 95% 1,2-6,2),enquanto o grupo alélico HLA-A*31, apesar de não apresentar significância estatística (p=0,11; OR=0,5; IC 95% 0,2-1,2), pela comparação das frequências, foi maior no grupo FD, sugerindo que o HLA-A*31 possivelmente ofereça proteção contra a FHD. Através desse estudo foi possível comprovar que fatores genéticos têm um papel importante na susceptibilidade e na resistência para as formas clínicas da dengue. No entanto, novos estudos clínicos com maior número de pacientes são necessários para confirmar esses dados...

Desensibilización y trasplante renal: plasmaféresis-IVIG a dosis estándar en pacientes con alto riesgo inmunológico / Desensitization and renal transplant: plasmapheresis/IVIG standard dose in patients with high immunological risk

Introducción: Los pacientes con alto riesgo inmunológico siguen siendo relegados a la cada vez más larga lista de espera de un donador inmunológicamente compatible. El objetivo de esta comunicación es informar la experiencia de un centro de trasplantes en la desensibilización de pacientes con alto riesgo inmunológico. Material y métodos: Estudio descriptivo y retrospectivo de todos los pacientes sometidos a trasplante renal de noviembre de 1999 a enero de 2008, en quienes se llevó a cabo desensibilización pretrasplante renal. Resultados: Ocho pacientes presentaron aloinmunización (pruebas cruzadas positivas o panel reactivo de anticuerpos alto, PRA > 30 %). La desensibilización se realizó mediante sesiones de plasmaféresis con recambio de 1.5 volúmenes plasmáticos, y posterior a cada una se administró una dosis estándar de inmunoglubulina intravenosa (IVIG 5 g/dosis). La inmunosupresión se inició en la primera sesión de plasmaféresis con base en un inhibidor de calcineurinas (tacrolimus); en seis pacientes se añadió mofetil micofenolato y en dos, sirolimus. En siete se obtuvieron pruebas cruzadas negativas con el donador previo al trasplante; en el octavo no se efectuaron. En dos se administró anticuerpos humanizados contra CD25 (20 mg/dosis de basiliximab). Todos los pacientes han mantenido función estable del injerto. Conclusiones: De acuerdo con nuestra experiencia, la sobrevida del injerto renal en pacientes con alto riesgo inmunológico posterior a un adecuado protocolo de desensibilización y estrecha vigilancia postrasplante es similar a la observada en pacientes no sensibilizados, al menos durante el primer año del trasplante.

BACKGROUND: Patients with high immunological risk have been relegated to the growing waiting list for an immunologically compatible donor. Our objective was to report the experience of a transplant center in desensitization of patients with high immunological risk. METHODS: We carried out a descriptive and retrospective study. Included were all the renal transplant patients from November 1999 to January 2008 in which we used plasmapheresis and standard dose of intravenous immunoglobulin (IVIG) as desensitization. RESULTS: Eight patients had history of alloimmunity (positive crossmatch or high panel-reactive antibodies (PRA >30%). Desensitization was accomplished with plasmapheresis and exchange of 1.5 plasma volume. Subsequent to each session we administered a standard dose of IVIG (5 g/dose). Immunosuppression began equal to the first plasmapheresis with calcineurin inhibitor (tacrolimus) plus six patients with mycophenolate mofetil and two patients with sirolimus. In seven cases, negative crossmatches were obtained before the transplantation, except in the eighth case in whom it was not done. Two patients received human antibodies against CD25 (basiliximab, 20 mg/dose). During their evolution, all patients maintained stable graft function. CONCLUSIONS: According to our experience, renal graft outcome in patients with high immunological risk after an adequate desensitization protocol is similar to that observed in nonsensitized patients, at least during the first year of transplantation.