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Updated: Sep 18, 2025
Author Spotlight: Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor
Published on: January 12, 2024
Optimal utilization of paucicellular vitreous sample for diagnosis of primary vitreoretinal lymphoma
Armughan S Khan1, Anam Khan1, Zarrin Hossein-Zadeh1
1Department of Pathology and Laboratory Medicine, North Shore University Hospital and Long Island Jewish Medical Center, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell Health, Greenvale, New York, USA.
Insights
Diagnosing primary vitreoretinal lymphoma (PVRL) is challenging due to low cellularity. Combining whole slide imaging (WSI) with MYD88 mutation analysis and targeted immunohistochemistry (IHC) improves diagnostic accuracy for this rare cancer.
Area of Science:
- Ophthalmology
- Hematology
- Oncology
- Pathology
Background:
- Primary vitreoretinal lymphoma (PVRL) is a rare, aggressive intraocular non-Hodgkin lymphoma, often diffuse large B-cell lymphoma.
- Vitreous cytology, the diagnostic gold standard, faces limitations with poor sample preservation and low cellularity.
- MYD88 mutation analysis shows promise for improved sensitivity in challenging samples.
Purpose of the Study:
- To evaluate the diagnostic utility of whole slide imaging (WSI), immunohistochemistry (IHC), and MYD88 mutation analysis in primary vitreoretinal lymphoma (PVRL).
- To assess the effectiveness of these ancillary tests in paucicellular or poorly preserved vitreous samples.
Main Methods:
- A 5-year retrospective study analyzed vitreous samples from 3 tertiary centers.
- Samples were reviewed by cytopathologists and hematopathologists, classified as negative, atypical, or positive.
- Whole slide imaging (WSI) quantified atypical lymphocytes, with ancillary tests including flow cytometry, IHC, and MYD88 mutation analysis.
Main Results:
- Of 226 samples, 6 were positive for PVRL.
- WSI improved the quantification of atypical lymphocytes.
- MYD88 analysis was conclusive in 4/5 cases, IHC in 7/8, and flow cytometry in 2/8.
Conclusions:
- Cytology remains essential, but WSI, targeted IHC, and MYD88 analysis enhance diagnostic precision for PVRL, especially in paucicellular samples.
- Flow cytometry is best reserved for high-cellularity cases with strong clinical suspicion.
Introduction:
Primary vitreoretinal lymphoma (PVRL) is a rare, aggressive, and intraocular non-Hodgkin lymphoma, typically manifesting as diffuse large B-cell lymphoma (95%). Vitreous fluid cytology is the gold standard for diagnosis; however, its utility is limited by poor preservation and low cellularity. Recent studies indicate that myeloid differentation primary response protein 88 (MYD88) mutation analysis is more sensitive and accurate on low-cellularity or poorly preserved samples. The incidence of PVRL has reportedly tripled with an annual average of ∼50 cases in the United States. Delayed diagnosis can lead to mortality within 2 years, underscoring the need for improved diagnostic methods.
Materials And Methods:
We conducted a 5-year retrospective study of vitreous samples from 3 tertiary centers. A cytopathologist and a hematopathologist reviewed the samples and classified them as "negative," "atypical," or "positive." Whole slide imaging (WSI) was incorporated to quantify atypical lymphocytes using an arbitrary cutoff (≥25% considered positive; <25% considered atypical) and to document necrosis and apoptosis. Ancillary tests included flow cytometry, immunohistochemistry (IHC), and MYD88 mutation analysis.
Results:
Of the 226 samples, 214 were diagnosed as negative, 6 as atypical, and 6 as positive. WSI enhanced the diagnosis by precisely quantifying atypical lymphocytes. Flow cytometry was conclusive in 2 of 8 cases, IHC in 7 of 8, and MYD88 analysis in 4 of 5 cases.
Conclusions:
While cytology remains the gold standard, a combination of WSI, targeted IHC, and MYD88 analysis enhances diagnostic precision in paucicellular samples. Flow cytometry should be reserved for cases with high cellularity and strong clinical suspicion.
