Name: Extraction and Analysis of DNA from the Cellular Component of the Eye Humor
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extraction and analysis of dna from the cellular component of the eye humor

Diagnóstico de linfoma vitreorretiniano usando DNA livre de células

Vitreoretinal lymphoma (VRL) is an aggressive lymphoma associated with primary central nervous system diffuse large B-cell lymphoma1,2,3. VRL is typically fatal due to its involvement in the central nervous system1,2. Although rare1,4, VRL often presents with symptoms similar to posterior uveitis and other vitreoretinal diseases4,5. Consequently, patients exhibiting uveitis symptoms require a diagnosis to either confirm or rule out VRL.
Recently, consensus criteria for diagnosing VRL were published, which involve a combination of clinical examination and laboratory findings6. Specimens commonly used to diagnose VRL include vitreous humor and, more recently, aqueous humor7. Vitreous humor is obtained through a surgical procedure called pars plana vitrectomy, which allows access to the posterior segment of the eye8.
In the presented protocol, both aqueous humor and vitreous humor specimens were collected for cellular and cfDNA extraction. After anesthetizing the patients and placing trocars approximately 4 mm from the corneal limbus, an aqueous humor sample of approximately 100-200 &#181;L was obtained using a 1 mL tuberculin syringe at the corneal limbus. For pseudophakic patients, undiluted vitreous was obtained by introducing sterile air into the infusion, enabling the collection of a larger amount of undiluted vitreous (up to 3.5 mL). In phakic patients, approximately 500 to 1000 &#181;L of undiluted vitreous was removed before turning on an infusion of balanced salt solution. In some cases, secondarily diluted vitreous (500 to 2,000 &#181;L) was collected by switching the infusion to fluid and placing the vitrector within the vitreous skirt to obtain this sample. The most dilute vitreous fraction was collected by preserving the cassette bag (Supplemental Figure 1) at the end of the surgery. Once this bag reached the pathology department, dilute vitreous was obtained from draining fluid out of this bag into conical tubes for subsequent DNA extraction.
Cytopathology of vitreous fluid is often considered the gold standard9. However, several studies have demonstrated limited sensitivity due to processing and minimal cellularity10,11,12. Flow cytometry can aid in identifying clonal B-cells but can also be limited by low cellularity and the fragility of large lymphoma cells13,14,15. Both cytopathology and flow cytometry requires intact whole cells. Many of these cells are destroyed during collection, storage, and processing. When molecular testing is performed using DNA extracted from intact cells (cellular DNA), it suffers from this same limitation. In addition, dividing the limited vitreous specimen for all of these tests reduces the amount of material available for each test.
Cell-free DNA (cfDNA) represents another source of DNA that does not require intact cells. cfDNA from vitreous specimens has been used for the detection of VRL16,17 as well as uveal melanoma18. In this protocol, cellular and cell-free DNA are extracted from vitreous and aqueous fluid to detect VRL.

Autor em destaque: Avançando no diagnóstico de VRL usando extração de DNA livre de células do humor vítreo 

Extração de DNA livre de células de espécimes de humor vítreo e aquoso para diagnóstico e monitoramento de linfoma vitreorretiniano

With this research, we are attempting to use molecular testing of vitreous humor collected from the eye by vitrectomy to diagnose vitreoretinal lymphoma or VRL, which is an aggressive disease whose symptoms overlap those of common inflammatory ocular conditions. Cytopathology flow cytometry and molecular testing are current methods used to diagnose VRL. Published sensitivities of non molecular methods are 44 to 90%for cytopathology and 50 to 60%for flow cytometry, cytopathology, flow cytometry and molecular testing with cellular DNA requires intact neoplastic cells.That poses a problem in vitreous humor specimens because the volume and cellularity of these specimens is low and cells can be degraded by apoptosis as well as specimen collection, processing and storage. We found that cell-free DNA extracted from vitreous fluid specimens provides another source of DNA from molecular testing that does not require intact cells, and that also shows evidence of VRL and can be used to diagnose this lymphoma. Cell-free DNA extraction offers molecular testing without intact neoplastic cells.This allows maximizing the utility of vitreous specimens as intact cells can be reserved for cytopathology and flow cytometry while utilizing the remaining supernatant fluid that would otherwise be discarded for molecular testing.

Watch this Scientific Journal Video about Extraction and Analysis of DNA from the Cellular Component of the Eye Humor at JoVE.com

Extraction and Analysis of DNA from the Cellular Component of the Eye Humor  

Extração e Análise de DNA do Componente Celular do Humor Ocular

Para realizar a extração de DNA, primeiro adicione uma quantidade apropriada de solução de lise celular ao pellet celular obtido do humor ocular. Pipetar a solução para cima e para baixo para misturá-la completamente. Uma vez que as células estão totalmente lisadas, adicione uma solução de precipitado de proteína para compor um terço do volume total.Agora, vórtice e misture vigorosamente por 20 a 30 segundos. Em seguida, gire por três minutos a 3000g em temperatura ambiente. Enquanto isso, transfira 300 microlitros de isopropanol para um tubo de microcentrífuga limpo.Uma vez concluída a centrifugação, pipetar cuidadosamente o sobrenadante para o tubo contendo isopropanol. Em seguida, adicione 1,5 microlitros de glicogênio ao tubo e inverta o tubo para misturar completamente. Coloque o tubo a menos 20 graus Celsius em um freezer por uma hora ou durante a noite para facilitar a precipitação de DNA.Após o congelamento, centrifugar o tubo em cinco minutos a 3000g à temperatura ambiente. Em seguida, pipetar o isopropanol. Para lavar o pellet, adicione 0,5 mililitros de etanol 70%.Inverta o tubo suavemente para garantir uma lavagem uniforme. Em seguida, gire o tubo por um minuto a 3000g à temperatura ambiente. Descarte o sobrenadante do tubo da centrífuga.Agora, inverta o tubo em gaze limpa para drenar a solução e secar o pellet. Após um giro rápido na microcentrífuga, use uma ponta de pipeta descartável de ponta fina para pipetar a gota de etanol restante. Em seguida, adicione 45 microlitros de solução de hidratação de DNA ao pellet de DNA seco.Em seguida, coloque o tubo em um bloco de aquecimento regulado para 50 graus Celsius. Os rendimentos de DNA de ambos os componentes celulares e livres de células dos fluidos oculares foram semelhantes. Foi realizada análise por PCR dos componentes celulares e livres de células de uma mutação associada ao linfoma vitreorretiniano.Uma maior taxa de mutação estava presente no componente livre de células, como demonstrado por um limiar de ciclo reduzido.

Research

JoVE Journal

Cancer Research

Extraction and Analysis of DNA from the Cellular Component of the Eye Humor

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To ...