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

Summary

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.

Abstract

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.

Introduction

Vitreoretinal lymphoma (VRL) is an aggressive lymphoma associated with primary central nervous system diffuse large B-cell lymphoma^1,2,3. VRL is typically fatal due to its involvement in the central nervous system^1,2. Although rare^1,4, VRL often presents with symptoms similar to posterior uveitis and other vitreoretinal diseases^4,5. Consequently, patients exhibiting uveitis symptoms require a diagnosis....

Protocol

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 col.......

Discussion

Vitreoretinal lymphoma (VRL) is an aggressive large B-cell lymphoma^1,2,3 whose symptoms can mimic other vitreoretinal diseases^4,5. 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.......

Materials

Name	Company	Catalog Number	Comments
2-Propanol (Isopropanol)	Fischer	A415-500
DNA Clean & Concentrator-10	Zymo Research	D4011
DNA Clean & Concentrator-5	Zymo Research	D4003
Gentra Puregene Cell Lysis Solution	Qiagen	158906
Gentra Puregene DNA Hydration Solution	Qiagen	158916
Gentra Puregene Protein Precipitation Solution	Qiagen	158912
Phosphate Buffered Saline (PBS)	Sigma	P-4417
Quick-DNA Urine Kit	Zymo Research	D3061	Conditioning buffer; also includes clearing beads, Proteinase K and spin columns
Ultrapure Glycogen, 20 µg/µL (20 mg/mL)	Thermo Fisher/Invitrogen	10814010