Timothy Daniels，MLS（ASCP），MB，QLS，和Helmut Weigelin，MLS（ASCP）在我们的实验室内建立这种提取方法方面发挥了重要作用。

本协议遵循人类护理指南，并经密歇根大学机构审查委员会 （IRB） 批准。为此，IRB获得了知情同意的豁免。对于所涉及的患者，没有相关的纳入或排除标准。
1. 细胞和无细胞成分的分离
注意：可以接收三种类型的样本进行 VRL 诊断测试：玻璃体（未稀释;在开始输注前收集的玻璃体切除术液体）、盒袋内稀释的玻璃体（图 1）和房水（来自眼睛前房的液体）。
<ol><li>对于非常粘稠的未稀释玻璃体液，用 3-5 mL PBS 稀释以方便移液。</li>
	<li>对于盒袋内的稀玻璃体（补充图1），将液体排入50 mL锥形管中（每45 mL液体一个）。</li>
	<li>房水的体积非常低 （<200 μL）。为确保细胞的最大回收率，将样品转移到 1.5 mL 管中，用等体积的 PBS 冲洗收集样品的原始 500 μL 微量离心管，然后加入 1.5 mL 管中。</li>
	<li>在室温下以3,000× g 离心玻璃体或水溶液15分钟。</li>
	<li>使用移液管小心地除去上清液，注意不要干扰沉淀细胞。</li>
</ol>2. 从细胞成分中提取 DNA
<ol><li>加入适量的细胞裂解液（小沉淀为300μL，大沉淀为1mL或更多）（参见 材料表）。通过上下移液混合。</li>
	<li>完全裂解时，加入总体积1/3的蛋白质沉淀溶液（300μL裂解物为100μL）（参见 材料表）。</li>
	<li>剧烈涡旋20-30秒。在室温下以3,000× g 旋转3分钟。</li>
	<li>将 300 μL 异丙醇移液到干净的微量离心管中。小心地将上清液从上一步移液到异丙醇管中。</li>
	<li>加入 1.5 μL 20 mg/mL 糖原（用于 300 μL 裂解）（参见 材料表）。</li>
	<li>倒置充分混合，然后将样品置于-20°C冰箱中1小时至过夜，以帮助DNA沉淀。</li>
	<li>在室温下以 3,000 x g 离心 1.5 mL 管 5 分钟。移液器取出异丙醇。</li>
	<li>加入 0.5 mL 70% 乙醇并倒置试管以洗涤沉淀。</li>
	<li>在室温下以3,000× g 旋转1分钟，并倒出乙醇。重复乙醇洗涤一次。</li>
	<li>将管子倒置在干净的纱布上以排出溶液并干燥沉淀或倾析剂。在微量离心机中快速旋转DNA，并用细头一次性移液器吸头移液剩余的乙醇滴（一旦试管中没有残留乙醇，DNA应干燥并准备好在5-10分钟内水合）。</li>
	<li>向干燥的DNA沉淀中加入45μLDNA水合溶液（参见 材料表）。</li>
	<li>将管置于50°C的加热块中，使其溶解1-2小时。上下移液 DNA 以加快水合作用过程。 注意：如果样品看起来太粘稠，请添加更多的 DNA 水合溶液。</li>
</ol>3. 游离DNA提取
<ol><li>步骤1.5的上清液量是可变的。每毫升上清液加入 70 μL 调节缓冲液（参见 材料表）。</li>
	<li>通过涡旋充分混合透明珠。如果处理 14 mL 无细胞上清液，则加入 10 μL 透明珠<14 mL，如果处理 14-40 mL，则加入 20 μL。</li>
	<li>通过涡旋充分混合样品/微珠混合物。在室温下以3,000× g 离心15分钟。</li>
	<li>在不干扰沉淀的情况下，如果使用 10 μL 透明珠，则移液出留下 100 μL 的上清液，如果使用 20 μL 透明珠，则移出 200 μL。</li>
	<li>向沉淀中加入等体积的尿沉淀消化缓冲液（参见 材料表），并通过涡旋或移液将沉淀重悬。</li>
	<li>将蛋白酶 K （5% （v/v）） 加入重悬沉淀中（例如，向 200 μL 混合物中加入 10 μL 蛋白酶 K），并通过轻轻涡旋充分混合。</li>
	<li>将沉淀混合物在55°C孵育30分钟。</li>
	<li>向酶解混合物中加入一体积的基因组裂解缓冲液（参见 材料表）（例如，210μL基因组裂解缓冲液与210μL酶解混合物中）并通过涡旋混合。</li>
	<li>将市售的离心柱（参见 材料表）转移到新的收集管中。</li>
	<li>向离心柱中加入 200 μL 尿液 DNA 制备缓冲液，在室温下以 ≥16,000 x g 离心 1 分钟，弃去流出物。</li>
	<li>向色谱柱中加入 700 μL 尿液 DNA 洗涤缓冲液，在室温下以 ≥16,000 x g 离心 1 分钟，弃去流出。</li>
	<li>用 200 μL 尿液 DNA 洗涤缓冲液重复该步骤。</li>
	<li>将离心柱转移到无 DNase/RNase 的微量离心管中。</li>
	<li>将适当体积的DNA洗脱缓冲液（基于要进行的分子测试）直接添加到柱基质上，并在室温下静置3-5分钟。 注：示例病例中使用了 30 μL 洗脱缓冲液。如果需要额外的测试，则使用更多的洗脱缓冲液。</li>
	<li>在室温下以≥16,000× g 离心1分钟以获得游离DNA。</li>
</ol>

使用游离 DNA 诊断玻璃体视网膜淋巴瘤

<ol>
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E., Damato, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+intraocular+lymphomas.">Understanding intraocular lymphomas.</a> Clinical &amp; Experimental Ophthalmology. 36 (6), 564-578 (2008).</li><li>Cassoux, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+and+central+nervous+system+lymphoma:+clinical+features+and+diagnosis.">Ocular and central nervous system lymphoma: clinical features and diagnosis.</a> Ocular Immunology and Inflammation. 8 (4), 243-250 (2000).</li><li>Read, R. W., Zamir, E., Rao, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+masquerade+syndrome.">Neoplastic masquerade syndrome.</a> Survey Ophthalmology. 47, 81-124 (2002).</li><li>Carbonell, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consensus+recommendations+for+the+diagnosis+of+vitreoretinal+lymphoma.">Consensus recommendations for the diagnosis of vitreoretinal lymphoma.</a> Ocular Immunology and Inflammation. 23 (3), 507-520 (2021).</li><li>Demirci, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+humor-derived+MYD88+L265P+mutation+analysis+in+vitreoretinal+lymphoma:+a+potential+less+invasive+method+for+diagnosis+and+treatment+response+assessment.">Aqueous humor-derived MYD88 L265P mutation analysis in vitreoretinal lymphoma: a potential less invasive method for diagnosis and treatment response assessment.</a> Ophthalmology Retina. 7 (2), 189-195 (2023).</li><li>Machemer, R., Buettner, H., Norton, E. W., Parel, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitrectomy:+a+pars+plana+approach.">Vitrectomy: a pars plana approach.</a> Transactions - American Academy of Ophthalmology and Otolaryngology. 75 (4), 813-820 (1971).</li><li>Vogel, M. H., Font, R. L., Zimmerman, L. E., Levine, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reticulum+cell+sarcoma+of+the+retina+and+uvea.+Report+of+sex+cases+and+review+of+the+literature.">Reticulum cell sarcoma of the retina and uvea. Report of sex cases and review of the literature.</a> American Journal of Ophthalmology. 66 (2), 205-215 (1968).</li><li>Kimura, K., Usui, Y., Goto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Japanese+intraocular+lymphoma+study+group.+clinical+features+and+diagnostic+significance+of+the+intraocular+fluid+of+217+patients+with+intraocular+lymphoma.">Japanese intraocular lymphoma study group. clinical features and diagnostic significance of the intraocular fluid of 217 patients with intraocular lymphoma.</a> Japanese Journal of Ophthalmology. 56 (4), 383-389 (2012).</li><li>Davis, J. L., Miller, D. M., Ruiz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+testing+of+vitrectomy+specimens.">Diagnostic testing of vitrectomy specimens.</a> American Journal of Ophthalmology. 140 (5), 822-829 (2005).</li><li>Char, D. H., Ljung, B. M., Miller, T., Phillips, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+intraocular+lymphoma+(ocular+reticulum+cell+sarcoma)+diagnosis+and+management.">Primary intraocular lymphoma (ocular reticulum cell sarcoma) diagnosis and management.</a> Ophthalmology. 95 (5), 625-630 (1988).</li><li>Tanaka, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+accurate+diagnosis+of+vitreoretinal+lymphoma+using+a+combination+of+diagnostic+test+results:+a+prospective+observational+study.">More accurate diagnosis of vitreoretinal lymphoma using a combination of diagnostic test results: a prospective observational study.</a> Ocular Immunology and Inflammation. 30 (6), 1354-1360 (2022).</li><li>Missotten, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+flow+cytometric+immunophenotyping+is+a+valuable+tool+for+detection+of+intraocular+lymphoma.">Multicolor flow cytometric immunophenotyping is a valuable tool for detection of intraocular lymphoma.</a> Ophthalmology. 120 (5), 991-996 (2013).</li><li>Bertram, H. C., Check, I. J., Milano, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotyping+large+B-cell+lymphomas.+Flow+cytometric+pitfalls+and+pathologic+correlation.">Immunophenotyping large B-cell lymphomas. Flow cytometric pitfalls and pathologic correlation.</a> American Journal of Clinical Pathology. 116 (2), 191-203 (2001).</li><li>Bonzheim, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+hallmarks+of+primary+and+secondary+vitreoretinal+lymphoma.">The molecular hallmarks of primary and secondary vitreoretinal lymphoma.</a> Blood Advances. 6 (5), 1598-1607 (2022).</li><li>Shi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+relevance+of+the+high+prevalence+of+MYD88+L265P+mutated+vitreoretinal+lymphoma+identified+by+droplet+digital+polymerase+chain+reaction.">Clinical relevance of the high prevalence of MYD88 L265P mutated vitreoretinal lymphoma identified by droplet digital polymerase chain reaction.</a> Ocular Immunology and Inflammation. 29 (3), 448-455 (2021).</li><li>Bustamante, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+DNA+tracking+through+driver+mutations+as+a+liquid+biopsy-based+marker+for+uveal+melanoma.">Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based marker for uveal melanoma.</a> Journal of Experimental and Clinical Cancer Research. 40 (1), 196 (2021).</li></ol>

作者没有什么可透露的。

玻璃体视网膜淋巴瘤 （VRL） 是一种侵袭性大 B 细胞淋巴瘤 1,2,3，其症状可以模仿其他玻璃体视网膜疾病 4,5。玻璃体和最近的房水分子检测已成为诊断或排除 VRL 的关键方法。然而，这些液体的体积非常小，并且通常具有低细胞性。这些液体中的许多细胞也可能在收集、储存和处理?...

玻璃体视网膜淋巴瘤 （VRL） 是一种与原发性中枢神经系统弥漫性大 B 细胞淋巴瘤相关的侵袭性淋巴瘤 1,2,3。VRL通常是致命的，因为它受累于中枢神经系统1,2。虽然罕见1,4，但 VRL 通常表现为类似于后葡萄膜炎和其他玻璃体视网膜疾病的症状 4,5。因此，出现葡萄膜炎症状的患者需要诊断以确认或排除 VRL。
最近，发布了诊断 VRL 的共识标准，其中包括临床检查和实验室检查结果的结合6。通常用于诊断 VRL 的标本包括玻璃体液和最近的房水7。玻璃体液是通过称为扁平玻璃体切除术的外科手术获得的，该手术允许进入眼睛的后段8.
在所提出的方案中，收集了房水和玻璃体液标本用于细胞和cfDNA提取。在麻醉患者并将套管针放置在距离角膜缘约 4 mm 处后，使用 1 mL 结核菌素注射器在角膜缘处获得约 100-200 μL 的房水样本。对于假晶状体患者，通过在输注中引入无菌空气来获得未稀释的玻璃体，从而能够收集大量未稀释的玻璃体（最多 3.5 mL）。在有晶状体患者中，在打开平衡盐溶液输注之前，取出约 500 至 1000 μL 未稀释的玻璃体。在某些情况下，通过将输注转换为液体并将玻璃体放置在玻璃体裙内以获得该样品来收集二次稀释的玻璃体（500 至 2,000 μL）。通过在手术结束时保留盒袋（补充图1）来收集最稀的玻璃体部分。一旦这个袋子到达病理科，通过将液体从这个袋子中排出到锥形管中，得到稀释的玻璃体，以便随后提取DNA。
玻璃体液的细胞病理学通常被认为是金标准9.然而，一些研究表明，由于加工和最小细胞性，灵敏度有限10,11,12。流式细胞术可以帮助识别克隆性 B 细胞，但也可能受到低细胞和大淋巴瘤细胞脆性的限制13,14,15。细胞病理学和流式细胞术都需要完整的全细胞。其中许多细胞在收集、储存和处理过程中被破坏。当使用从完整细胞（细胞 DNA）中提取的 DNA 进行分子测试时，它也受到同样的限制。此外，将有限的玻璃体标本分开进行所有这些测试可以减少每个测试的可用材料量。
游离 DNA （cfDNA） 是另一种不需要完整细胞的 DNA 来源。玻璃体标本的 cfDNA 已用于检测 VRL 16,17 以及葡萄膜黑色素瘤18。在该方案中，从玻璃体和水溶液中提取细胞和游离DNA以检测VRL。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Propanol (Isopropanol)</td>
			<td>Fischer</td>
			<td>A415-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Clean &#38; Concentrator-10</td>
			<td>Zymo Research</td>
			<td>D4011</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Clean &#38; Concentrator-5</td>
			<td>Zymo Research</td>
			<td>D4003</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentra Puregene Cell Lysis Solution</td>
			<td>Qiagen</td>
			<td>158906</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentra Puregene DNA Hydration Solution</td>
			<td>Qiagen</td>
			<td>158916</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentra Puregene Protein Precipitation Solution</td>
			<td>Qiagen</td>
			<td>158912</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma</td>
			<td>P-4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick-DNA Urine Kit</td>
			<td>Zymo Research</td>
			<td>D3061</td>
			<td>Conditioning buffer; also includes clearing beads, Proteinase K and spin columns</td>
		</tr>
		<tr>
			<td>Ultrapure Glycogen, 20 &#181;g/&#181;L (20 mg/mL)</td>
			<td>Thermo Fisher/Invitrogen</td>
			<td>10814010</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-free dna extraction of vitreous and aqueous humor specimens for diagnosis and monitoring of vitreoretinal lymphoma

玻璃体视网膜淋巴瘤 （VRL） 是一种侵袭性淋巴瘤，通常被归类为原发性中枢神经系统弥漫性大 B 细胞淋巴瘤。为了诊断VRL，需要收集玻璃体液和最近的房水等标本。对这些标本进行 VRL 的诊断测试包括细胞学、流式细胞术和分子检测。然而，细胞病理学和流式细胞术，以及使用细胞DNA的分子检测，都需要完整的全细胞。挑战在于玻璃体和房水通常具有低细胞性，并且许多细胞在收集、储存和处理过程中被破坏。此外，由于玻璃体液粘度高，玻璃体液和房水体积小，这些标本给分子检测带来了额外的困难。本研究提出了一种从玻璃体和水性标本中提取游离DNA的方法。这种方法补充了细胞DNA的提取，或允许这些标本的细胞成分用于其他诊断方法，包括细胞学和流式细胞术。

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.

这些提取方法在有限数量的病例上进行，以确保与玻璃体（4）和水（4）标本的细胞DNA相比，游离DNA具有足够的产量和扩增性。从这些样品中，这些液体的游离成分的DNA产量与细胞成分的DNA产量相似（表1）。还使用分子检测评估了这些样本的细胞和 cfDNA，例如 VRL 相关突变 MYD88 L265P。 MYD88 L265P的等位基因特异性实时PCR（图1）说明了在细胞DNA和cfDNA中...

Watch this Scientific Journal Video about Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor at JoVE.com

本文建立了一种从玻璃体和房水中提取游离 DNA 以进行分子研究以诊断玻璃体视网膜淋巴瘤的程序。该方法能够同时从样品的细胞成分中提取 DNA 或将其保留用于辅助测试。

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To ...

通过这项研究，我们试图使用玻璃体切除术从眼睛收集的玻璃体液的分子检测来诊断玻璃体视网膜淋巴瘤或VRL，这是一种侵袭性疾病，其症状与常见的炎症性眼部疾病重叠。细胞病理学、流式细胞术和分子检测是目前用于诊断 VRL 的方法。非分子方法的已发表灵敏度为44%至90%用于细胞病理学，50%至60%用于流式细胞术、细胞病理学、流式细胞术和分子检测，细胞DNA需要完整的肿瘤细胞。这给玻璃体标本带来了一个问题，因为这些标本的体积和细胞含量很低，细胞可以通过细胞凋亡以及标本收集、处理和储存而降解。我们发现，从玻璃体液标本中提取的游离 DNA 提供了另一种分子检测的 DNA 来源，它不需要完整的细胞，并且还显示了 VRL 的证据，可用于诊断这种淋巴瘤。游离 DNA 提取可在不检测完整肿瘤细胞的情况下进行分子检测。这样可以最大限度地发挥玻璃体标本的效用，因为完整的细胞可以保留用于细胞病理学和流式细胞术，同时利用剩余的上清液，否则这些上清液将被丢弃用于分子检测。

cell-free-dna-extraction-vitreous-aqueous-humor-specimens-for

Research

JoVE Journal

Cancer Research

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

1.2K 次观看.  University of Michigan, Ann Arbor. 通过这项研究，我们试图使用玻璃体切除术从眼睛收集的玻璃体液的分子检测来诊断玻璃体视网膜淋巴瘤或VRL，这是一种侵袭性疾病，其症状与常见的炎症性眼部疾病重叠。细胞病理学、流式细胞术和分子检测是目前用于诊断 VRL 的方法。非分子方法的已发表灵敏度为44%至90%用于细胞病理学，50%至60%用于流式细胞术、细胞病理学、流式细胞术和分子检测，细胞DNA需要完整的肿...

视频: 作者聚焦：使用玻璃体液中的游离 DNA 提取推进 VRL 诊断 

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.