Timothy Daniels, MLS(ASCP), MB, QLS, and Helmut Weigelin, MLS(ASCP) were instrumental in establishing this extraction method within our laboratory.

The present protocol follows the human care guidelines and with approval of the institutional review board (IRB) of the University of Michigan. A waiver of informed consent was obtained for this from IRB. There are no relevant inclusion or exclusion criteria for the patients involved.
1. Separation of cellular and cell-free components
NOTE: Three types of samples can be received for VRL diagnostic testing: vitreous (undilute; fluid from vitrectomy collected prior to beginning infusion), diluted vitreous within a cassette bag (Figure 1), and aqueous humor (fluid from the anterior chamber of the eye).
<ol>
	<li>For undiluted vitreous fluid, which is very viscous, dilute with 3-5 mL PBS to facilitate pipetting.</li>
	<li>For dilute vitreous within the cassette bag (Supplemental Figure 1), drain fluid into 50 mL conical tubes (one for every 45 mL of fluid).</li>
	<li>Aqueous humor is very low in volume (&#60;200 &#181;L). To ensure maximum recovery of cells, transfer the sample to a 1.5 mL tube, rinse the original 500 &#181;L microcentrifuge tube the sample was collected in with an equal volume of PBS, and add to the 1.5 mL tube.</li>
	<li>Centrifuge vitreous or aqueous fluid at 3,000 x g for 15 min at room temperature.</li>
	<li>Carefully remove the supernatant using a pipette, being careful not to disturb the pelleted cells.</li>
</ol>
2. DNA extraction from the cellular component
<ol>
	<li>Add an appropriate amount of cell lysis solution (300 &#181;L for small pellet, 1 mL or more for larger pellet) (see Table of Materials). Mix by pipetting up and down.</li>
	<li>When completely lysed, add 1/3 total volume of the protein precipitate solution (100 &#181;L for 300 &#181;L lysate) (see Table of Materials).</li>
	<li>Vortex vigorously for 20-30 s. Spin for 3 min at 3,000 x g at room temperature.</li>
	<li>Pipette 300 &#181;L of isopropanol into a clean microcentrifuge tube. Carefully pipette the supernatant from the previous step to the isopropanol tube.</li>
	<li>Add 1.5 &#181;L of 20 mg/mL glycogen (for 300 &#181;L lysis) (see Table of Materials).</li>
	<li>Mix well by inversion, then place the sample in a -20 &#176;C freezer for 1 h to overnight to aid in DNA precipitation.</li>
	<li>Centrifuge the 1.5 mL tube for 5 min at 3,000 x g at room temperature. Pipette off the isopropanol.</li>
	<li>Add 0.5 mL of 70% ethanol and invert the tube to wash the pellet.</li>
	<li>Spin for 1 min at 3,000 x g at room temperature and decant the ethanol. Repeat the ethanol wash once.</li>
	<li>Invert the tube on clean gauze to drain the solution and dry the pellet or decant. Quickly spin the DNA in a microcentrifuge and pipette the remaining drop of ethanol with a fine-tip disposable pipet tip (once no residual ethanol is left in the tube, the DNA should be dry and ready to hydrate in 5-10 min).</li>
	<li>Add 45 &#181;L of DNA hydration solution (see Table of Materials) to the dried DNA pellet.</li>
	<li>Place the tube in a heating block at 50 &#176;C and allow it to solubilize for 1-2 h. Pipet the DNA up and down to speed up the hydration process. 
	&#8203;NOTE: Add more DNA hydration solution if the sample appears too viscous.</li>
</ol>
3. Cell-free DNA extraction
<ol>
	<li>The amount of supernatant from step 1.5 is variable. Add 70 &#181;L of conditioning buffer (see Table of Materials) for every milliliter of the supernatant.</li>
	<li>Mix clearing beads well by vortexing. Add 10 &#181;L of clearing beads if processing &#60;14 mL of cell-free supernatant, 20 &#181;L if processing 14-40 mL.</li>
	<li>Mix sample/bead mixture well by vortexing. Centrifuge at 3,000 x g for 15 min at room temperature.</li>
	<li>Without disturbing the pellet, pipette out the supernatant leaving behind 100 &#181;L if 10 &#181;L of clearing beads are used, and 200 &#181;L if 20 &#181;L of clearing beads are used.</li>
	<li>Add an equal volume of Urine Pellet Digestion buffer (see Table of Materials) to the pellet and resuspend the pellet well by vortexing or pipetting.</li>
	<li>Add Proteinase K (5% (v/v)) to the resuspended pellet (e.g., add 10 &#181;L of Proteinase K to 200 &#181;L mixture) and mix well by gentle vortexing.</li>
	<li>Incubate the pellet mixture at 55 &#176;C for 30 min.</li>
	<li>Add one volume of Genomic lysis buffer (see Table of Materials) to the digestion mixture (e.g., 210 &#181;L Genomic lysis buffer to 210 &#181;L of digestion mixture) and mix by vortexing.</li>
	<li>Transfer the commercially available spin column (see Table of Materials) to a new collection tube.</li>
	<li>Add 200 &#181;L of Urine DNA Prep buffer to the spin column, centrifuge at &#8805;16,000 x g for 1 min at room temperature, and discard flow-through.</li>
	<li>Add 700 &#181;L of Urine DNA wash buffer to the column, centrifuge at &#8805;16,000 x g for 1 min at room temperature and discard flow-through.</li>
	<li>Repeat the step with 200 &#181;L Urine DNA wash buffer.</li>
	<li>Transfer the spin column to a DNase/RNase-free microcentrifuge tube.</li>
	<li>Add the appropriate volume of DNA elution buffer (based on the molecular tests to be performed) directly onto the column matrix and allow it to stand for 3-5 min at room temperature. 
	NOTE: 30 &#181;L of elution buffer was used in the example cases. More elution buffer is used if additional testing is required.</li>
	<li>Centrifuge at &#8805;16,000 x g at room temperature for 1 min to obtain the cell-free DNA.</li>
</ol>

Diagnosis of Vitreoretinal Lymphoma Using Cell-Free DNA

<ol>
	<li>Chan, C. C., 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=Primary+vitreoretinal+lymphoma:+a+report+from+an+International+Primary+Central+Nervous+System+Lymphoma+Collaborative+Group+symposium.">Primary vitreoretinal lymphoma: a report from an International Primary Central Nervous System Lymphoma Collaborative Group symposium.</a> Oncologist. 16 (11), 1589-1599 (2011).</li><li>Sagoo, M. S., 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=Primary+intraocular+lymphomas.">Primary intraocular lymphomas.</a> Clinical &amp; Experimental Ophthalmology. 59 (5), 503-516 (2014).</li><li>Coupland, S. 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>

The authors have nothing to disclose.

Vitreoretinal lymphoma (VRL) is an aggressive large B-cell lymphoma1,2,3 whose symptoms can mimic other vitreoretinal diseases4,5. Molecular testing of vitreous and, more recently, aqueous humor has become a critical method for making the diagnosis of VRL or ruling it out. However, these fluids are very low in volume and often have low cellularity. Many of the cells with...

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.

<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

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.

Author Spotlight: Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor 

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

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.

These extraction methods were performed on a limited number of cases to ensure adequate yield and amplifiability of cell-free DNA in comparison with cellular DNA from the vitreous (four) and aqueous (four) specimens. From these samples, DNA yields from the cell-free component of these fluids are similar to that of the cellular component (Table 1). Cellular and cfDNA from these samples were also evaluated using molecular testing, such as for the VRL-associated mutation MYD88 L265P. An allele-spec...

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

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.

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

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1.1K Views.  University of Michigan, Ann Arbor. 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 cytometr...