Nova abordagem técnica no estudo citogenético da leucemia linfocítica crónica-B
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2018-12-17
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A leucemia linfocítica crónica B (LLC-B) é a forma de leucemia mais prevalente no hemisfério Ocidental, afetando sobretudo indivíduos adultos, com idade superior a 65 anos. É uma doença clonal linfoproliferativa, caracterizada pela proliferação anormal e acumulação de linfócitos B neoplásicos nos tecidos sanguíneos, órgãos linfáticos e medula óssea. A análise citogenética desempenha um papel muito importante no estudo desta patologia, uma vez que a deteção de alterações cromossómicas específicas tem implicações prognósticas e terapêuticas. Para a análise citogenética destas amostras é necessário a realização de culturas com estimuladores de linfócitos B. O estimulador normalmente utilizado para a obtenção de metafases é o 1,2-Otetradecanoilforbol-13-acetato (TPA), que deteta alterações cromossómicas em 40 a 50% dos casos por citogenética convencional. Recentemente, novos estimuladores, como o oligodesoxinucleótido GpG-DSP30 (DSP30) e a Interleucina (IL-2) foram aplicados às culturas celulares com o objetivo de melhorar o número de metafases e a taxa de deteção de anomalias cromossómicas. O desenvolvimento de novas técnicas de citogenética molecular, nomeadamente a técnica hibridação in situ por fluorescência (FISH), vieram prestar um grande contributo para o estudo da LLC-B, permitindo a deteção de anomalias em 80% dos casos. O objetivo desta dissertação consiste na implementação do protocolo mais eficaz na estimulação das células com LLC-B. Para atingir os objetivos, foram testados vários protocolos, onde se aplicaram dois estimulares (DSP30 e IL-2), com diferentes concentrações, isoladamente e em combinação. Foi ainda aplicado, em simultâneo, o estimulador convencional (TPA). A combinação dos três estimuladores (TPA/DSP30/IL-2), foi também testada. Para cada protocolo foi realizada a citogenética convencional e a FISH. As culturas estimuladas com os dois ou os três estimuladores em simultâneo (DSP30/IL-2 (2 µM; 0,2 µg/mL) ou com TPA/DSP30/IL-2 (20 ng/mL; 2 µM; 0,2 µg/mL)) permitiram a obtenção de um maior número de metafases e de metafases com melhor qualidade (cromossomas mais alongados e com melhor resolução de bandas), quando comparados com as culturas estimuladas só com TPA (20 ng/mL). Verificou-se ainda que a aplicação destes estimuladores aumentou a taxa de deteção de alterações cromossómicas, nomeadamente, de novos rearranjos e de cariótipos complexos. A técnica FISH, foi útil na deteção de deleções submicroscópicas, não detetadas pela citogenética convencional. Em suma, o protocolo para o estudo de doentes com LLC-B deve incluir uma das seguintes combinações mitogénicas: DSP30/IL-2 (2 µM; 0,2 µg/mL) ou TPA/DSP30/IL-2 (20 ng/mL; 2 µM; 0,2 µg/mL). O estudo citogenético desta patologia deve abranger a citogenética convencional e a FISH, uma vez que estas técnicas desempenham um papel complementar no diagnóstico e prognóstico de doentes com LLC-B.
Chronic lymphocytic leukemia B (B-CLL) is the most prevalent form of leukemia in the Western Hemisphere, primarily affecting adult individuals over the age of 65 years. It is a lymphoproliferative clonal disease characterized by abnormal proliferation and accumulation of neoplastic B lymphocytes in blood tissues, lymphatic organs and bone marrow. Cytogenetic analysis plays a very important role in the study of this pathology, since the detection of specific chromosomal alterations has prognostic and therapeutic implications. For the cytogenetic analysis of these samples it is necessary to perform cultures with B-lymphocyte stimulators. The stimulator commonly used to obtain metaphases is 12-0-tetradecanoyl-phorbol-13-acetate (TPA), which detects chromosomal changes in 40 to 50% of cases by conventional cytogenetics. Recently, new stimulators, such as CpG-oligonucleotide DSP30 (DSP30) and Interleukin (IL-2) were applied to cell cultures with the aim of improve number of metaphases and a rate of detection of chromosomal abnormalities. The development of new molecular cytogenetic techniques, namely fluorescent in situ hybridization (FISH), have made a great contribution to the study of B-CLL, allowing the detection of anomalies in 80% of the cases. The purpose of this dissertation is to implement the most efficient protocol for the stimulation of cells with B-CLL. In order to achieve the objectives, several protocols were tested, in which two stimulators (DSP30 and IL-2) were applied, with different concentrations, alone and in combination. The conventional stimulator (TPA) was also applied simultaneously. The combination of the three stimulators (TPA/DSP30/IL-2) was also tested. For each protocol, were performed conventional cytogenetics and FISH. The cultures stimulated with the two or three stimulators simultaneously (2 μM, 0,2 μg/mL) or TPA/DSP30/IL-2 (20 ng/mL, 2 μM, 0,2 μg/mL) allowed to obtain a greater number of metaphases and with better quality (more elongated chromosomes and better band resolution) compared to TPA stimulated cultures (20 ng/mL). It was also verified that the application of these stimulators increased the rate of detection of chromosomal alterations, namely, of new rearrangements and complex karyotypes. The FISH technique was useful in the detection of submicroscopic deletions not detected by conventional cytogenetics. Summarizing, the protocol for the study of patients with B-CLL should include one of the following mitogenic combinations: DSP30/IL-2 (2 μM; 0,2 μg/mL) or TPA/DSP30/IL-2 (20 ng/mL; 2 μM; 0,2 μg/mL). The cytogenetic study of this pathology should cover conventional cytogenetics and FISH, since these techniques play a complementary role in the diagnosis and prognosis of patients with B-CLL.
Chronic lymphocytic leukemia B (B-CLL) is the most prevalent form of leukemia in the Western Hemisphere, primarily affecting adult individuals over the age of 65 years. It is a lymphoproliferative clonal disease characterized by abnormal proliferation and accumulation of neoplastic B lymphocytes in blood tissues, lymphatic organs and bone marrow. Cytogenetic analysis plays a very important role in the study of this pathology, since the detection of specific chromosomal alterations has prognostic and therapeutic implications. For the cytogenetic analysis of these samples it is necessary to perform cultures with B-lymphocyte stimulators. The stimulator commonly used to obtain metaphases is 12-0-tetradecanoyl-phorbol-13-acetate (TPA), which detects chromosomal changes in 40 to 50% of cases by conventional cytogenetics. Recently, new stimulators, such as CpG-oligonucleotide DSP30 (DSP30) and Interleukin (IL-2) were applied to cell cultures with the aim of improve number of metaphases and a rate of detection of chromosomal abnormalities. The development of new molecular cytogenetic techniques, namely fluorescent in situ hybridization (FISH), have made a great contribution to the study of B-CLL, allowing the detection of anomalies in 80% of the cases. The purpose of this dissertation is to implement the most efficient protocol for the stimulation of cells with B-CLL. In order to achieve the objectives, several protocols were tested, in which two stimulators (DSP30 and IL-2) were applied, with different concentrations, alone and in combination. The conventional stimulator (TPA) was also applied simultaneously. The combination of the three stimulators (TPA/DSP30/IL-2) was also tested. For each protocol, were performed conventional cytogenetics and FISH. The cultures stimulated with the two or three stimulators simultaneously (2 μM, 0,2 μg/mL) or TPA/DSP30/IL-2 (20 ng/mL, 2 μM, 0,2 μg/mL) allowed to obtain a greater number of metaphases and with better quality (more elongated chromosomes and better band resolution) compared to TPA stimulated cultures (20 ng/mL). It was also verified that the application of these stimulators increased the rate of detection of chromosomal alterations, namely, of new rearrangements and complex karyotypes. The FISH technique was useful in the detection of submicroscopic deletions not detected by conventional cytogenetics. Summarizing, the protocol for the study of patients with B-CLL should include one of the following mitogenic combinations: DSP30/IL-2 (2 μM; 0,2 μg/mL) or TPA/DSP30/IL-2 (20 ng/mL; 2 μM; 0,2 μg/mL). The cytogenetic study of this pathology should cover conventional cytogenetics and FISH, since these techniques play a complementary role in the diagnosis and prognosis of patients with B-CLL.
Descrição
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde apresentada à Universidade de Trás-os-Montes e Alto Douro
Palavras-chave
LLC-B , TPA , DSP30 , IL-2 , alterações cromossómicas