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

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

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  

Extraktion und Analyse von DNA aus der zellulären Komponente des Augenhumors

Um eine DNA-Extraktion durchzuführen, wird zunächst eine geeignete Menge Zelllyselösung in das aus dem Augenhumor gewonnene Zellpellet gegeben. Pipettieren Sie die Lösung auf und ab, um sie gründlich zu mischen. Sobald die Zellen vollständig lysiert sind, fügen Sie eine Proteinpräzipitatlösung hinzu, die ein Drittel des Gesamtvolumens ausmacht.Nun 20 bis 30 Sekunden lang kräftig mischen. Dann drei Minuten bei 3000g bei Raumtemperatur schleudern. In der Zwischenzeit 300 Mikroliter Isopropanol in ein sauberes Mikrozentrifugenröhrchen überführen.Sobald die Zentrifugation abgeschlossen ist, pipettieren Sie den Überstand vorsichtig in das Röhrchen mit Isopropanol. Als nächstes gibst du 1,5 Mikroliter Glykogen in das Röhrchen und drehst das Röhrchen um, um es gründlich zu mischen. Stellen Sie das Röhrchen bei minus 20 Grad Celsius für eine Stunde oder über Nacht in einen Gefrierschrank, um die DNA-Ausfällung zu erleichtern.Nach dem Einfrieren zentrifugieren Sie das Röhrchen fünf Minuten lang bei 3000 g bei Raumtemperatur. Pipettieren Sie dann das Isopropanol heraus. Um das Pellet zu waschen, fügen Sie 0,5 Milliliter 70%iges Ethanol hinzu.Drehen Sie den Schlauch vorsichtig um, um eine gleichmäßige Wäsche zu gewährleisten. Dann das Röhrchen eine Minute lang bei 3000g bei Raumtemperatur schleudern. Entsorgen Sie den Überstand aus dem Zentrifugenröhrchen.Drehen Sie nun das Röhrchen auf saubere Gaze, um die Lösung abzutropfen und das Pellet zu trocknen. Nach einem kurzen Schleudern in der Mikrozentrifuge wird der verbleibende Ethanoltröpfchen mit einer feinen Einwegpipettenspitze pipettiert. Als nächstes fügen Sie dem getrockneten DNA-Pellet 45 Mikroliter DNA-Hydratationslösung hinzu.Legen Sie dann den Schlauch in einen Heizblock, der auf 50 Grad Celsius eingestellt ist. Die DNA-Ausbeute sowohl der zellulären als auch der zellfreien Bestandteile der Augenflüssigkeit war ähnlich. Es wurde eine PCR-Analyse der zellulären und zellfreien Komponenten einer vitreoretinalen Lymphom-assoziierten Mutation durchgeführt.In der zellfreien Komponente war eine höhere Mutationsrate vorhanden, was sich in einer erniedrigten Zyklusschwelle zeigte.

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 ...

Forschung

Krebsforschung

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

Cell-Free DNA Extraction and Analysis from Eye Humor

Zellfreie DNA-Extraktion und -Analyse aus Augenhumor