Name: Extraction and Analysis of DNA from the Cellular Component of the Eye Humor
Uploaded: 2024-01-12T12:55:00
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extraction and analysis of dna from the cellular component of the eye humor

Diagnóstico del linfoma vitreorretiniano mediante ADN libre 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 destacado: Avance en el diagnóstico de VRL mediante la extracción de ADN libre de células del humor vítreo 

Extracción de ADN libre de células de muestras de humor vítreo y acuoso para el diagnóstico y seguimiento del 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  

Extracción y Análisis de ADN del Componente Celular del Humor Ocular

Para realizar la extracción de ADN, primero agregue una cantidad adecuada de solución de lisis celular al gránulo celular obtenido del humor ocular. Pipetea la solución hacia arriba y hacia abajo para mezclarla bien. Una vez que las células estén completamente lisadas, agregue una solución de precipitado de proteína para constituir un tercio del volumen total.Ahora, haga vórtice y mezcle vigorosamente durante 20 a 30 segundos. Luego gíralo durante tres minutos a 3000 g a temperatura ambiente. Mientras tanto, transfiera 300 microlitros de isopropanol a un tubo de microcentrífuga limpio.Una vez completada la centrifugación, pipetee cuidadosamente el sobrenadante en el tubo que contiene isopropanol. A continuación, agregue 1,5 microlitros de glucógeno al tubo e invierta el tubo para mezclar bien. Coloque el tubo a -20 grados centígrados en un congelador durante una hora o toda la noche para facilitar la precipitación del ADN.Después de congelar, centrifugar el tubo a los cinco minutos a 3000 g a temperatura ambiente. A continuación, pipetea el isopropanol. Para lavar el pellet, agregue 0,5 mililitros de etanol al 70%.Invierta el tubo suavemente para asegurar un lavado uniforme. A continuación, gire el tubo durante un minuto a 3000 g a temperatura ambiente. Deseche el sobrenadante del tubo de la centrífuga.Ahora, invierta el tubo sobre una gasa limpia para drenar la solución y secar el gránulo. Después de un centrifugado rápido en la microcentrífuga, utilice una punta de pipeta desechable de punta fina para pipetear la gota de etanol restante. A continuación, agregue 45 microlitros de solución de hidratación de ADN al gránulo de ADN seco.Luego coloque el tubo en un bloque calefactor ajustado a 50 grados centígrados. Los rendimientos de ADN de los componentes celulares y libres de células de los fluidos oculares fueron similares. Se realizó un análisis por PCR de los componentes celulares y celulares libres de una mutación asociada al linfoma vitreorretiniano.Se presentó una mayor tasa de mutación en el componente libre de células, como lo demuestra un umbral de ciclo más bajo.

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 ...

Investigación

Investigación sobre el cáncer

Separation of Cellular and Cell-Free Fractions of the Vitreous and Aqueous Humor for DNA Extraction

Separación de fracciones celulares y libres de células del humor vítreo y acuoso para la extracción de ADN

Cell-Free DNA Extraction and Analysis from Eye Humor

Extracción y análisis de ADN libre de células a partir del humor ocular