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

Diagnosi del linfoma vitreoretinico mediante DNA libero da cellule

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.

Autore in primo piano: Avanzamento della diagnosi di VRL mediante l'estrazione di DNA libero da cellule dall'umor vitreo 

Estrazione di DNA senza cellule di campioni di vitreo e umor acqueo per la diagnosi e il monitoraggio del linfoma vitreoretinico

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  

Estrazione e analisi del DNA dalla componente cellulare dell'umore oculare

Per eseguire l'estrazione del DNA, aggiungere prima una quantità appropriata di soluzione di lisi cellulare al pellet cellulare ottenuto dall'umore oculare. Pipettare la soluzione su e giù per mescolarla accuratamente. Una volta che le cellule sono completamente lisate, aggiungere una soluzione di precipitato proteico per costituire un terzo del volume totale.Ora, vorticare e mescolare energicamente per 20-30 secondi. Quindi centrifugarlo per tre minuti a 3000 g a temperatura ambiente. Nel frattempo, trasferire 300 microlitri di isopropanolo in una provetta per microcentrifuga pulita.Una volta completata la centrifugazione, pipettare con cura il surnatante nella provetta contenente isopropanolo. Quindi, aggiungi 1,5 microlitri di glicogeno al tubo e capovolgi il tubo per mescolare accuratamente. Metti la provetta a meno 20 gradi Celsius in un congelatore per un'ora o per tutta la notte per facilitare la precipitazione del DNA.Dopo il congelamento, centrifugare la provetta per cinque minuti a 3000 g a temperatura ambiente. Quindi, pipettare l'isopropanolo. Per lavare il pellet, aggiungere 0,5 millilitri di etanolo al 70%.Capovolgere delicatamente il tubo per garantire un lavaggio uniforme. Quindi centrifugare il tubo per un minuto a 3000 g a temperatura ambiente. Eliminare il surnatante dalla provetta da centrifuga.Ora, capovolgere il tubo su una garza pulita per drenare la soluzione e asciugare il pellet. Dopo un rapido giro nella microcentrifuga, utilizzare un puntale per pipetta monouso a punta fine per pipettare la gocciolina di etanolo rimanente. Successivamente, aggiungere 45 microlitri di soluzione di idratazione del DNA al pellet di DNA essiccato.Quindi posizionare il tubo in un blocco riscaldante impostato a 50 gradi Celsius. I rendimenti del DNA dei componenti cellulari e privi di cellule dei fluidi oculari erano simili. È stata eseguita l'analisi PCR dei componenti cellulari e privi di cellule di una mutazione associata al linfoma vitreoretinico.Un tasso di mutazione più elevato era presente nella componente priva di cellule, come dimostrato da una soglia di ciclo abbassata.

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 ...

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Ricerca sul cancro

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

Separazione delle frazioni cellulari e libere dell'umor vitreo e acqueo per l'estrazione del DNA

Cell-Free DNA Extraction and Analysis from Eye Humor

Estrazione e analisi del DNA senza cellule dall'umore oculare