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

Diagnose des vitreoretinalen Lymphoms mit zellfreier DNA

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 im Rampenlicht: Weiterentwicklung der VRL-Diagnose durch zellfreie DNA-Extraktion aus Glaskörper 

Zellfreie DNA-Extraktion von Glaskörper- und Kammerwasserproben zur Diagnose und Überwachung des vitreoretinalen Lymphoms

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  

Trennung von zellulären und zellfreien Fraktionen des Glaskörpers und Kammerwassers für die DNA-Extraktion

Verdünnen Sie zunächst viskose Glaskörperflüssigkeit mit drei bis fünf Millilitern PBS, um das Pipettieren zu erleichtern. Die verdünnte Glaskörperflüssigkeit, die Sie in einem Kassettenbeutel erhalten haben, wird in 50-Milliliter-Röhrchen abgelassen. Für Kammerwasser mit geringem Volumen überführen Sie die Probe in ein 1,5-Milliliter-Röhrchen und spülen Sie dann das ursprüngliche 500-Mikro-Mikrozentrifugenröhrchen mit einem gleichen Volumen PBS.Geben Sie die Spüllösung in das 1,5-Milliliter-Röhrchen. Zum Schluss zentrifugieren Sie die Glaskörperflüssigkeit bei 3000 g für 15 Minuten bei Raumtemperatur. Anschließend pipeptieren Sie den Überstand vorsichtig in ein neues Röhrchen, ohne das Zellpellet zu stören.

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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

196 Aufrufe. Verdünnen Sie zunächst viskose Glaskörperflüssigkeit mit drei bis fünf Millilitern PBS, um das Pipettieren zu erleichtern. Die verdünnte Glaskörperflüssigkeit, die Sie in einem Kassettenbeutel erhalten haben, wird in 50-Milliliter-Röhrchen abgelassen. Für Kammerwasser mit geringem Volumen überführen Sie die Probe in ein 1,5-Milliliter-Röhrchen und spülen Sie dann das ursprüngliche 500-Mikro-Mikrozentrifugenröhrchen mit einem gleichen Volumen PBS.Geben Sie die Spüllösu...

Serie: 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 ...