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

cell-free DNA를 이용한 유리체 망막 림프종 진단

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.

저자 스포트라이트: 유리체액에서 Cell-Free DNA 추출을 사용한 VRL 진단 발전 

유리체 망막 림프종의 진단 및 모니터링을 위한 유리체 및 수성 체액 검체의 Cell-Free DNA 추출

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  

안액에서 Cell-Free DNA 추출 및 분석

먼저, 눈액의 cell-free supernate infraction 1밀리리터당 70마이크로리터의 컨디셔닝 버퍼를 추가합니다. 클리어링 비드를 소용돌이치게 하여 완전히 섞습니다. 다음으로, 샘플에 비드를 추가한 다음 샘플과 비드 혼합물을 소용돌이칩니다.이 혼합물을 실온에서 3000G에서 15분 동안 원심분리합니다. 펠릿을 빼내지 않고 상층을 추출한 다음 동일한 양의 소변 펠릿 소화 완충액을 펠릿에 추가합니다. 용액을 위아래로 피펫팅하여 펠릿을 다시 부유시킵니다.이제 단백질 분해 효소 K를 재현탁 펠릿 혼합물에 첨가합니다. 혼합물을 섭씨 55도에서 30분 동안 배양한 다음 동일한 부피의 게놈 용해 완충액을 분해 혼합물에 추가합니다. 용액을 완전히 와류시켜 잘 섞습니다.이제 시중에서 판매되는 스핀 컬럼을 새 수집 튜브에 넣습니다. 스핀 컬럼에 200마이크로리터의 소변 DNA 준비 완충액을 추가합니다. 16, 000 G에 실내 온도에 1 분 동안 분리하고 flowthrough를 폐기하십시오.이제 피펫 700 마이크로리터의 소변 DNA를 컬럼과 원심분리기로 세척하여 버퍼를 원심분리합니다. 플로우스루를 폐기한 후 스핀 컬럼을 DNase, RNase-free 마이크로 원심분리기 튜브로 옮깁니다. 마지막으로, 적절한 부피의 DNA 용출 완충액을 컬럼 매트릭스에 직접 추가하고 실온에서 3-5분 동안 그대로 두었다가 다시 원심분리하여 cell-free DNA를 얻습니다.안구액의 세포 성분과 세포가 없는 성분의 DNA 수율은 비슷했습니다. 유리체 망막 림프종 관련 돌연변이의 세포 및 무세포 성분에 대한 PCR 분석을 수행했습니다. 더 높은 돌연변이율은 cell-free 성분에 존재했으며, 이는 낮아진 주기 역치에 의해 입증되었습니다.

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 ...

연구

암 연구학

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

DNA 추출을 위한 유리체 및 수용액의 세포 및 무세포 분획 분리

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

눈액의 세포 성분에서 DNA의 추출 및 분석