RESUMO
Crossmatching either by complement-dependent cytotoxicity (CDC) and/or by flow cytometry (FCXM) are routinely used for assessing anti-HLA donor antibodies before kidney transplantation. FCXM has demonstrated greater sensitivity and many transplant centers have opted for its use without the concomitant CDC assay. The objective of this study was to evaluate the accuracy of the median channel shift (MCS) in the FCXM in predicting the CDC assay results. A total of 1516â¯T cell FCXM and 1408 B cell FCXM were studied in deceased donors lymphocytes between January/2016 and March/2017. The high detection rate of CDC+ results by FCXM+ resulted in 87% (FCXM-T) and 90% (FCXM-B) sensitivity, and 98% negative predictive value, for both. The low specificity of FCXM B (43%) is atributed to cases of CDC-/FCXMB+. FCXM T and B were able to detect 53% and 76% of cases with donor specific antibodies of classes I and II with intensity of fluorescence ≥5001. The MCS differentiated CDC+ (Md, P25 and P75) results: MCS-T 390 (245-469) and MCS-B 282 (180-350). Through ROC curve analysis (AUC), the MCS showed satisfactory performance in detecting CDC+: MCS-T 0.909 (0.886-0.933) and MCS-B 0.775 (0.724-0.826). Considering the accuracy and sensitivity evaluation, the MCS-T 245 and MCS-B 282 cutoffs showed a better prediction of CDC+. This study showed that it is possible to calibrate MCS based on CDC+ with accuracy >90%, however, that leads to a risk in terms of non-detection of low-titer anti-HLA antibodies.
Assuntos
Linfócitos B/imunologia , Tipagem e Reações Cruzadas Sanguíneas/métodos , Citometria de Fluxo/métodos , Transplante de Rim , Linfócitos T/imunologia , Homólogo 5 da Proteína Cromobox , Proteínas do Sistema Complemento/metabolismo , Citotoxicidade Imunológica , Antígenos HLA/imunologia , Humanos , Isoanticorpos/sangue , Valor Preditivo dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Transplantados , Listas de EsperaRESUMO
Preformed anti-human leukocyte antigen (HLA) antibodies may be present in the blood of kidney transplant candidates. The production of these antibodies may occur in the post-transplant period, with the possible development of donor-specific antibodies (DSA). Luminex-based tests, such as the single antigen (SA) assay and the Luminex crossmatch (Xm-DSA) assay are the most commonly used tools to detect anti-HLA antibodies, due to their high sensitivity and specificity. This cross-sectional study aimed to compare the findings of two methods for the detection of DSAs after kidney transplant: SA and Xm-DSA. A total of 122 patients who underwent deceased donor kidney transplant at Hospital de Clínicas de Porto Alegre were included. The SA assay detected anti-class I HLA DSAs in 17 patients (13.9%) and anti-class II HLA DSAs in 22 patients (19.6%), whereas the Xm-DSA detected DSAs in 18 patients (14.8%) both against class I and class II antigens. There was agreement between the two methods for class I (kappaâ¯=â¯0.66, pâ¯=â¯0.001) and class II (kappaâ¯=â¯0.54, pâ¯=â¯0.025) antigens. The incidence of DSAs as obtained by the SA assay was 15.57%, and the most prevalent DSAs were those against HLA-DR antigens. Patient survival at 3â¯years was 92%. The two techniques assessed in this study provide important information on the presence of DSAs and may help in the post-transplant patient monitoring and in immunosuppressive strategy.
Assuntos
Rejeição de Enxerto/diagnóstico , Teste de Histocompatibilidade/métodos , Isoanticorpos/sangue , Nefropatias/terapia , Transplante de Rim , Adulto , Estudos Transversais , Feminino , Rejeição de Enxerto/mortalidade , Antígenos HLA/imunologia , Humanos , Isoantígenos/imunologia , Nefropatias/mortalidade , Masculino , Pessoa de Meia-Idade , Monitorização Fisiológica , Testes Sorológicos , Análise de Sobrevida , Doadores de Tecidos , Transplante HomólogoRESUMO
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.
Assuntos
Recém-Nascido , Gêmeos Monozigóticos , Trombocitopenia Neonatal Aloimune , Antígenos HLA , Pais , Testes de Aglutinação , Teste de Histocompatibilidade , Linfócitos , Células , Aglutinação , Parto , Diagnóstico , Citometria de Fluxo , Testes Hematológicos , Histocompatibilidade , NeutropeniaRESUMO
Mutações no gene STAT1 (signal transducer and activator of transcription 1) têm sido identificadas como responsáveis pela maioria dos casos sindrômicos da candidíase mucocutânea crônica com herança autossômica dominante (AD). Nesse artigo, descrevemos uma menina de 7 anos que apresentou candidíase da mucosa oral e unhas, além de infecção disseminada da pele e couro cabeludo por Microspora gipseum. Recentemente, a paciente foi diagnosticada e tratada de meningite por Cryptococcus neoformans. Na família não existem outros casos de candidíase. A avaliação imunológica incluiu a detecção de subpopulações de linfócitos (CD3, CD4, CD8, CD20 e células NK), assim como a dosagem de IgG, IgA, IgM e IgE, subclasses de IgG e autoanticorpos. Excluindo-se discreta diminuição de CD3, CD4, CD8, NK e leve aumento de IgG1, os demais exames estiveram dentro da normalidade. O sequenciamento do exoma detectou uma rara mutação em heterozigose no exon 14 do domínio de ligação do DNA (DNA-binding domain) do gene STAT1, ocasionando um provável ganho de função (GOF) responsável pela doença (Gly384Asp). Essa variação foi também identificada pelo sequenciamento de Sanger, não estando reportada nos bancos de dados públicos e apresentando elevado potencial de dano (índice CADD=32). Será interessante contarmos com informações clínicas e estudos com outros pacientes para conhecermos mais essa mutação patológica. Além da apresentação do caso, discutiremos as formas de tratamento existentes.
STAT1 (signal transducer and activator of transcription 1) gene mutations have been identified as responsible for most syndromic cases of chronic mucocutaneous candidiasis with autosomal dominant (AD) inheritance. In this article, we described a 7-year-old girl who presented with candidiasis of the oral mucosa and nails, as well as disseminated infection of the skin and scalp caused by Microsporum gypseum. Recently, the patient was diagnosed and treated for Cryptococcus neoformans meningitis. There are no other cases of candidiasis in the family. The immunological evaluation consisted of detection of subpopulations of lymphocytes (CD3, CD4, CD8, CD20, and NK cells), as well as measurement of IgG, IgA, IgM, and IgE, IgG subclasses, and autoantibodies. Excluding a slight decrease in CD3, CD4, CD8, NK and a minimal increase in IgG1, the others were within normal limits. Exome sequencing detected a rare heterozygous variation in exon 14 of the DNA-binding domain of the STAT1 gene, causing a probable gain of function (GOF) responsible for the disease (Gly384Asp). This variation was also identified by Sanger sequencing, but it was not reported in public databases and had a high potential for damage (Combined Annotation-Dependent Depletion [CADD] score = 32). Having clinical information and conducting studies of other patients will be helpful to learn more about this pathological mutation. In addition to the presentation of the case, we will discuss the existing forms of treatment.
Assuntos
Humanos , Feminino , Criança , Candidíase Mucocutânea Crônica , Cryptococcus neoformans , Fator de Transcrição STAT1 , Pacientes , Autoanticorpos , Terapêutica , Imunoglobulina A , Imunoglobulina E , Imunoglobulina G , Imunoglobulina M , Linfócitos , Antígenos CD4 , Éxons , Antígenos CD8 , Exoma , Meningite , MicrosporumRESUMO
OBJECTIVES: To evaluate B-cell expression patterns and association with function and survival in dysfunctional kidney allografts. MATERIALS AND METHODS: There were 110 kidney transplant recipients included who had for-cause biopsies. Demographic and transplant data were collected. Immunostaining for B cells, plasma cells, and C4d was performed by the immunoperoxidase technique in paraffin-embedded samples. Circulating antihuman leukocyte antigen donor-specific antibodies were detected in a single-antigen assay at biopsy. The main outcomes were kidney graft survival and function. The patients were evaluated in 3 groups according to the Banff classification: no rejection (40 patients), T-cell-mediated rejection (50 patients), and antibody-mediated rejection (20 patients). RESULTS: The CD138-positive plasma cell-rich infiltrates predominated in antibody-mediated rejection and were associated with stronger reactivity against panel antibodies (r = 0.41; P ≤ .001) and positive donor-specific antibodies (r = 0.32; P ≤ .006). The CD20-positive lymphocytes were associated with T-cell-mediated rejection, increased human leukocyte antigen mismatch, and frequency of retransplant. The CD138-positive cell infiltrates also were significantly greater in patients who had late than early rejection. There was no correlation between cellular CD20 and CD138 expression, and neither CD20 nor CD138 predicted worse graft function or survival. Other markers of antibody-mediated rejection such as C4d and donor-specific antibodies were associated with worse graft function and survival at 4 years after transplant. In multivariate analysis, C4d was the only risk factor associated with graft loss. CONCLUSIONS: After kidney transplant, CD20-positive B-cell infiltrates were associated with T-cell-mediated rejection, and CD138-positive plasma cells were associated with antibody-mediated rejection. Graft loss was associated with the presence of C4d.
Assuntos
Linfócitos B/imunologia , Rejeição de Enxerto/imunologia , Transplante de Rim/efeitos adversos , Rim/imunologia , Linfócitos T/imunologia , Doença Aguda , Adolescente , Adulto , Antígenos CD20/análise , Autoanticorpos/sangue , Linfócitos B/metabolismo , Biomarcadores/análise , Biópsia , Distribuição de Qui-Quadrado , Complemento C4b/análise , Feminino , Rejeição de Enxerto/sangue , Rejeição de Enxerto/diagnóstico , Sobrevivência de Enxerto , Antígenos HLA/imunologia , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Rim/metabolismo , Rim/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Fragmentos de Peptídeos/análise , Plasmócitos/imunologia , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Fatores de Risco , Sindecana-1/análise , Linfócitos T/metabolismo , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Introdução: A reação em cadeia da polimerase (PCR) foi teste de grande impacto no diagnóstico das meningites e encefalites linfocíticas durante a última década. Esse método foi extensivamente usado no diagnóstico das infecções do sistema nervoso central (SNC), devido a sua habilidade em detectar amostras mínimas de DNA-alvo no líquido cefalorraquiano. Objetivo: O objetivo deste estudo foi identificar a prevalência dos patógenos oportunistas responsáveis por causar problemas neurológicos em pacientes infectados com o vírus da imunodeficiência humana (HIV) e avaliar sua associação com os achados clínicos, laboratoriais e da tomografia computadorizada cerebral (TCC). Pacientes e métodos: Um estudo transversal foi realizado em 203 amostras de líquido cefalorraquiano (LCR) de pacientes do sul do Brasil infectados com HIV e com aparente encefalite e meningite linfocíticas. As amostras foram analisadas para os seguintes agentes pelo método da reação em cadeia da polimerase "nested" ou dupla (N-PCR): citomegalovírus, vírus do Epstein-Barr, vírus do herpes simplex tipos 1 e 2, vírus da varicella zoster, vírus do herpes humano tipo 6, vírus JC, Toxoplasma gondii e micobactérias. Resultado: Pelo menos um patógeno foi encontrado em 77 (38%) dos indivíduos. O Epstein-Barr foi o mais prevalente, com 40 casos (19,7%), seguido pelo citomegalovívus, com 12 casos (15%) e pelo vírus JC, em 9 casos (4,4%). Um N-PCR positivo mostrou associação com aumento de proteínas e de celularidade (P=0,001), meningismo (P=0,017) e tomografia computadorizada anormal (P=0,006). Conclusão: O painel de PCR empregado foi efetivo na identificação de infecções neurológicas severas em pacientes HIV positivos (AU)
Introduction: Polymerase chain reaction (PCR) has had great impact on the diagnosis of lymphocytic meningitis and encephalitis over the last decade. It has been extensively used in the diagnosis of central nervous system (CNS) infections for its ability to detect small amounts of target DNA in the cerebrospinal fluid (CSF). Objective: The aim of this study was to identify the prevalence of opportunistic pathogens responsible for neurological disorders in patients infected with human immunodeficiency virus (HIV) and to evaluate its association with clinical, laboratory and cerebral computed tomography (CCT) findings. Patients and methods: A cross-sectional study was performed on 203 cerebrospinal fluids (CSF) from HIV-infected patients from Southern Brazil, with apparent lymphocytic meningitis and encephalitis. CSF samples were analyzed with probes for cytomegalovirus, Epstein-Barr virus, herpes simplex virus types 1 and 2, varicella zoster virus, human herpes virus type 6, JC virus, Toxoplasma gondii and mycobacterium in nested polymerase chain reaction (N-PCR). Results: At least one pathogen was found in 77 (38.0%) individuals. Epstein-Barr virus was the most prevalent with 40 cases (19.7%), followed by cytomegalovirus with 12 cases (5.9%) and JC virus with 9 cases (4.4%). Positive NPCR showed association with high spinal fluid protein and cell count (P=0.001), meningism (P=0.017) and abnormal CCT (P=0.006). Conclusion: The PCR panel used was effective in screening several neurological infections in HIV positive patients (AU)