RESUMO
Abstract Flow cytometry (FCM) was implemented in 2008 at the Pontificia Universidad Javeriana and later at the Hospital Universitario San Ignacio to examine special samples of patients with hematological malignancies and solid tumors other than bone marrow and peripheral blood for diagnosis and monitoring. This study describes the main findings of special sample evaluation over a six-year period. In all, 1070 samples of body fluids from patients with benign and malignant diseases were examined by FCM. These samples were stabilized with TransFixTM and stained with six-color immunophenotyping panels. Samples included cerebrospinal fluid, bronchoalveolar lavage, pleural fluid, pericardial fluid and ascite fluid from patients with acute and chronic leukemia, myelodysplastic syndromes, lymphomas, myeloma, autoimmune diseases, immunodeficiencies and solid tumors, among others. Flow cytometry provided important information for the classification and detection of minimal numbers of tumor cells in leukemia and lymphoma cases. This work represents the first national report describing FCM implementation in special samples for diagnosis and clinical monitoring of patients with malignant and benign pathologies.
Resumen La citometría de flujo fue implementada en 2008 en la Pontificia Universidad Javeriana y posteriormente en el Hospital San Ignacio con el fin de examinar, para diagnóstico y monitoreo, muestras especiales de pacientes con malignidades hematológicas y tumores sólidos, distintos de los de médula ósea y sangre periférica. Este estudio describe los principales hallazgos de la evaluación de estas muestras especiales en un periodo de seis años. En total, se examinaron por citometría de flujo 1070 muestras de fluidos corporales de pacientes con enfermedades malignas y benignas. Estas muestras se estabilizaron con TransFix™ y teñidas con paneles inmunofenotípicos de seis colores. Las muestras incluyeron líquido cefalorraquídeo, lavado broncoalveolar, fluido pleural, fluido pericárdico y fluido ascítico, provenientes de pacientes con leucemia aguda y crónica, síndromes mielodisplásicos, linfomas, mieloma, enfermedades autoinmunes, inmunodeficiencias y tumores sólidos, entre otras enfermedades. La citometría de flujo proporcionó información importante sobre la clasificación y detección de números mínimos de células tumorales en casos de leucemia y linfoma. Este trabajo representa el primer reporte nacional que describe la implementación de citometría de flujo en muestras especiales para diagnóstico y monitoreo clínico de pacientes con patologías malignas y benignas.
Resumo A citometria de fluxo foi implementada em 2008 na Pontificia Universidad Javeriana e posteriormente no Hospital San Ignacio com a finalidade de examinar amostras especiais de pacientes com malignidades hematológicas e tumores sólidos diferentes aos de medula óssea e sangue periférico para diagnóstico e monitoramento. Este estudo descreve as principais descobertas a partir da avaliação de amostras especiais em um período de 6 anos. Se examinaram por citometria de fluxo um total de 1.070 amostras de fluídos corporais de pacientes doenças malignas e benignas. Estas amostras se estabilizaram com TransFix™ e coradas com painéis imunofenotípicos de seis cores. As amostras incluíram fluído cérebro-espinhal, lavado broncoalveolar, fluído pleural, fluído pericárdico e fluído ascético, provenientes de pacientes com leucemias aguda e crónica, síndromes mielodisplásicos, linfomas, mielomas, doenças autoimunes, imunodeficiência, e tumores sólidos, entre outras doenças. A citometria de fluxo proporcionou informações importantes sobre a classificação e detecção de um número mínimo de células tumorais nos casos de leucemia e linfomas. Este trabalho representa o primeiro relato nacional que descreve a implementação de citometria de fluxo em amostras especiais para diagnóstico e monitoramento clínico de pacientes com patologias malignas e benignas.
Assuntos
Humanos , Líquidos Corporais , Colômbia , Citometria de FluxoRESUMO
BACKGROUND: TcTLE is a nonamer peptide from Trypanosoma cruzi KMP-11 protein that is conserved among different parasite strains and that is presented by different HLA-A molecules from the A2 supertype. Because peptides presented by several major histocompatibility complex (MHC) supertypes are potential targets for immunotherapy, the aim of this study was to determine whether MHC molecules other than the A2 supertype present the TcTLE peptide. METHODOLOGY/PRINCIPAL FINDINGS: From 36 HLA-A2-negative chagasic patients, the HLA-A genotypes of twenty-eight patients with CD8+ T cells that recognized the TcTLE peptide using tetramer (twenty) or functional (eight) assays, were determined. SSP-PCR was used to identify the A locus and the allelic variants. Flow cytometry was used to analyze the frequency of TcTLE-specific CD8+ T cells, and their functional activity (IFN-γ, TNFα, IL-2, perforin, granzyme and CD107a/b production) was induced by exposure to the TcTLE peptide. All patients tested had TcTLE-specific CD8+ T cells with frequencies ranging from 0.07-0.37%. Interestingly, seven of the twenty-eight patients had HLA-A homozygous alleles: A*24 (5 patients), A*23 (1 patient) and A*01 (1 patient), which belong to the A24 and A1 supertypes. In the remaining 21 patients with HLA-A heterozygous alleles, the most prominent alleles were A24 and A68. The most common allele sub-type was A*2402 (sixteen patients), which belongs to the A24 supertype, followed by A*6802 (six patients) from the A2 supertype. Additionally, the A*3002/A*3201 alleles from the A1 supertype were detected in one patient. All patients presented CD8+ T cells producing at least one cytokine after TcTLE peptide stimulation. CONCLUSION/SIGNIFICANCE: These results show that TcTLE is a promiscuous peptide that is presented by the A24 and A1 supertypes, in addition to the A2 supertype, suggesting its potential as a target for immunotherapy.