separation of cellular and cell-free fractions of the vitreous and aqueous humor for dna extraction

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 Separation of Cellular and Cell-Free Fractions of the Vitreous and Aqueous Humor for DNA Extraction at JoVE.com

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

Separação das frações celular e livre de células do humor vítreo e aquoso para extração de DNA

Para começar, dilua o líquido vítreo viscoso com três a cinco mililitros de PBS para facilitar a pipetagem. Escorra o líquido vítreo diluído recebido dentro de um saco de em tubos de 50 mililitros. Para humor aquoso de baixo volume, transfira a amostra para um tubo de 1,5 mililitro e, em seguida, lave o tubo de microcentrífuga original de 500 microlitros com um volume igual de PBS.Adicione a solução de enxágue ao tubo de 1,5 mililitro. Por último, centrifugar o líquido vítreo a 3000 G durante 15 minutos à temperatura ambiente. Em seguida, pipete cuidadosamente o sobrenadante para um novo tubo sem perturbar a pelota celular.

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Research

JoVE Journal

Cancer Research

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

190 visualizações.  University of Michigan, Ann Arbor. Para começar, dilua o líquido vítreo viscoso com três a cinco mililitros de PBS para facilitar a pipetagem. Escorra o líquido vítreo diluído recebido dentro de um saco de em tubos de 50 mililitros. Para humor aquoso de baixo volume, transfira a amostra para um tubo de 1,5 mililitro e, em seguida, lave o tubo de microcentrífuga original de 500 microlitros com um volume igual de PBS.Adicione a solução de enxágue ao tubo de 1,5 mililitro. Por último, centrifugar o líquido ví...

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

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 diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.

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