Name: Cell-Free DNA Extraction and Analysis from Eye Humor
Uploaded: 2024-01-12T12:55:00
Description: Watch this Scientific Journal Video about Cell-Free DNA Extraction and Analysis from Eye Humor at JoVE.com

cell-free dna extraction and analysis from eye humor

使用游离 DNA 诊断玻璃体视网膜淋巴瘤

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.

作者聚焦：使用玻璃体液中的游离 DNA 提取推进 VRL 诊断 

玻璃体和房水标本的游离 DNA 提取用于玻璃体视网膜淋巴瘤的诊断和监测

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 Cell-Free DNA Extraction and Analysis from Eye Humor at JoVE.com

Cell-Free DNA Extraction and Analysis from Eye Humor  

从眼部幽默中提取和分析游离 DNA

首先，每毫升眼液的无细胞上层侵犯，加入70微升调节缓冲液。涡旋透明珠以充分混合。接下来，将微珠添加到样品中，然后涡旋样品和微珠混合物。在室温下将该混合物以 3000 g 离心 15 分钟。在不去除沉淀的情况下，提取上层，然后向沉淀中加入等体积的尿沉淀消化缓冲液。上下移液溶液以重新悬浮沉淀。现在将蛋白酶 K 添加到重新悬浮的颗粒混合物中。将混合物在 55 摄氏度下孵育 30 分钟，然后向消化混合物中加入等体积的基因组裂解缓冲液。彻底涡旋溶液以充分混合。现在，将市售的离心柱放入新的收集管中。向离心柱中加入 200 微升尿液 DNA 制备缓冲液。在室温下以16， 000 G离心一分钟，然后丢弃流出物。现在将 700 微升尿液 DNA 洗涤缓冲液移液到色谱柱中，然后如图所示离心。丢弃流出物后，将离心柱转移到无 RNase 的微量离心管中。最后，将适当体积的DNA洗脱缓冲液直接加入柱基质中，在室温下静置三到五分钟，然后再次离心以获得游离DNA。眼液中细胞成分和游离成分的DNA产量相似。对玻璃体视网膜淋巴瘤相关突变的细胞和游离成分进行PCR分析。无细胞组分中存在较高的突变率，如降低的循环阈值所示。

Research

JoVE Journal

Cancer Research

Cell-Free DNA Extraction and Analysis from 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 ...

科研

癌症研究

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

分离玻璃体和房水的细胞和游离部分以提取 DNA

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

从眼部液的细胞成分中提取和分析DNA