Name: Extraction and Analysis of DNA from the Cellular Component of the Eye Humor
Uploaded: 2024-01-12T12:55:00
Description: 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

無細胞DNAを用いた硝子体網膜リンパ腫の診断

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.

著者スポットライト:硝子体液からの無細胞DNA抽出を使用したVRL診断の進歩 

網膜硝子体リンパ腫の診断とモニタリングのための硝子体および房水検体の無細胞DNA抽出

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.

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Extraction and Analysis of DNA from the Cellular Component of the Eye Humor  

眼液の細胞成分からのDNAの抽出と分析

DNA抽出を行うには、まず、眼液から得られた細胞ペレットに適量の細胞溶解液を添加します。溶液を上下にピペットで移し、完全に混合します。細胞が完全に溶解したら、タンパク質沈殿液を加えて、総容量の3分の1を構成します。次に、20〜30秒間激しくボルテックスして混ぜます。その後、室温で3000gで3分間回転させます。その間、300マイクロリットルのイソプロパノールを清潔な微量遠心チューブに移します。遠心分離が完了したら、上清をイソプロパノールの入ったチューブに慎重にピペットで移します。次に、チューブに1.5マイクロリットルのグリコーゲンを加え、チューブをひっくり返して完全に混合します。チューブをマイナス20°Cの冷凍庫に1時間または一晩置いて、DNA沈殿を促進します。凍結後、室温で3000gで5分間遠心分離します。次に、イソプロパノールをピペットで取り出します。ペレットを洗浄するには、0.5ミリリットルの70%エタノールを加えます。チューブを静かに裏返して、均一に洗うようにします。その後、チューブを室温で3000gで1分間回転させます。遠心チューブから上清を廃棄します。次に、チューブをきれいなガーゼで裏返して溶液を排出し、ペレットを乾燥させます。微量遠心分離機で素早く回転させた後、先端の細い使い捨てピペットチップを使用して、残りのエタノール液滴をピペットで移動します。次に、乾燥したDNAペレットに45マイクロリットルのDNA水和溶液を加えます。次に、チューブを摂氏50度に設定された加熱ブロックに入れます。眼液の細胞成分と無細胞成分の両方のDNA収量は類似していました。網膜硝子体リンパ腫関連変異の細胞成分および無細胞成分のPCR解析を実施した。より高い突然変異率は、サイクル閾値の低下によって実証されたように、無細胞成分に存在しました。

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 ...