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Updated: May 14, 2026
Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma
Published on: January 12, 2024
Multicolor flowcytometric immunophenotyping is a valuable tool for detection of intraocular lymphoma
Tom Missotten1, Dennis Tielemans, Jacolien E Bromberg
1Uveitis and Medical Retina Department, The Rotterdam Eye Hospital, Rotterdam, The Netherlands. t.missotten@oogziekenhuis.nl
Insights
Multicolor flowcytometric analysis accurately diagnoses intraocular lymphoma (IOL) by identifying clonal B-cells in ocular fluid. This method offers high sensitivity and specificity, aiding in distinguishing IOL from other conditions.
Area of Science:
- Ophthalmology
- Hematology
- Oncology
Background:
- Intraocular lymphoma (IOL) is a rare malignancy often misdiagnosed as uveitis.
- Current diagnostic methods like cytology and molecular analysis (IGH, cytokine) lack consistent validation.
- Accurate and validated diagnostic tools are crucial for timely IOL treatment.
Purpose of the Study:
- To evaluate the diagnostic accuracy of multicolor flowcytometric immunophenotyping for intraocular lymphoma.
- To assess the method's sensitivity and specificity in a cohort of patients with suspected IOL.
- To compare flowcytometry with existing diagnostic techniques for IOL.
Main Methods:
- Vitreous fluid analysis using multicolor flowcytometry with specific antibodies (CD45, CD3, CD19, CD20, anti-SmIgκ, anti-SmIgλ).
- Identification of a clonal B-cell population with Igκ/Igλ disequilibrium as a diagnostic criterion for non-Hodgkin lymphoma (NHL).
- Longitudinal follow-up of 51 patients for a minimum of 2 years to validate diagnostic accuracy.
Main Results:
- Flowcytometric analysis confirmed IOL in 14 of 51 patients (11 primary, 3 metastatic).
- The test demonstrated 82.4% sensitivity and 100% specificity for IOL diagnosis.
- In 3 patients diagnosed with central nervous system NHL later, the test initially failed to detect a clonal B-cell population.
Conclusions:
- Multicolor flowcytometry is a highly specific and sensitive method for diagnosing intraocular lymphoma.
- This technique provides a reliable alternative to existing methods, with results comparable to IGH gene rearrangement testing.
- Readily available in hematology labs, flowcytometry can significantly aid in the clinical diagnosis of IOL.
Objective:
Intraocular lymphoma (IOL) is a rare condition and frequently difficult to distinguish from uveitis or other uveitis-masquerading syndromes. The diagnosis is confirmed by cytologic examination of ocular fluid specimens and more recently by molecular-immunoglobulin heavy chain (IGH) translocation or cytokine analysis. However, some of these more recent methods have not been validated by follow-up studies.
Design:
Evaluation of a diagnostic test.
Participants:
In a cohort of 51 consecutive patients with a clinical suspicion of IOL, vitreous analysis was performed via multicolor flowcytometric immunophenotyping.
Methods:
Multicolor flowcytometric immunophenotyping was performed with CD45, CD3, CD19, CD20, anti-SmIgκ, and anti-SmIgλ antibodies. The presence of a clear B-cell population showing a disequilibrium of Igκ versus Igλ expression was used to confirm the diagnosis of non-Hodgkin lymphoma (NHL). Patients were followed for a minimum of 2 years (mean, 5.9 ± 2.0 years) to validate the accuracy of the method.
Main Outcome Measures:
The presence or absence of IOL during follow-up.
Results:
In 14 of 51 patients, a clinical diagnosis of IOL was confirmed using flowcytometric analysis. Of these 14 patients, 11 had primary IOL and 3 had metastasized secondary lymphomas. In 3 of 51 patients who were diagnosed with (central nervous system) NHL during follow-up, the test failed to confirm the presence of a clonal B-cell population. In 18 of the 34 other patients, an infectious or well-defined immunologic disorder was established during follow-up. The remaining 16 patients, with a minimal follow-up of 2 years, were diagnosed with idiopathic uveitis.
Conclusions:
Multicolor flowcytometric analysis had 82.4% sensitivity and 100% specificity in patients with suspected IOL. This is comparable to the reported vitreous interleukin (IL)-6/IL-10 testing sensitivity of 0.8 and sensitivity of 0.65 to 0.95 by immunoglobulin heavy chain (IGH) gene arrangement testing in clinical cohorts. Because flowcytometric tests are readily performed in hematologic laboratories, this can be regarded as a useful method for confirming the clinical diagnosis of IOL.
Financial Disclosure(S):
The author(s) have no proprietary or commercial interest in any materials discussed in this article.
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