Single-center ex-vivo study using blood samples from healthy donors processed through a 25-gauge vitrectomy system. The study compares four combinations of operational parameters (vacuum pressure and cut rate) to evaluate their impact on cellular recovery and morphological preservation. Pre- and post-procedure complete blood counts and cytology smears will be analyzed to identify the settings that minimize cell loss and mechanical damage, with the ultimate goal of optimizing diagnostic yield in vitreoretinal lymphoma (VRL) vitrectomy.

Primary vitreoretinal lymphoma (VRL) is a rare but aggressive intraocular malignancy, typically classified as a subtype of primary central nervous system lymphoma. It predominantly involves the retina and vitreous and is frequently associated with central nervous system disease. Clinical presentation often mimics chronic uveitis, leading to diagnostic delays and suboptimal management.

Definitive diagnosis of VRL requires cytopathologic confirmation through diagnostic vitrectomy, which provides vitreous samples for cytology, immunohistochemistry, flow cytometry, and molecular analyses (e.g., MYD88 mutation testing, IL-10/IL-6 ratio). However, despite the central role of vitreous biopsy, its diagnostic sensitivity remains limited to approximately 70% (Iuliano L et al. Int Ophthalmol 43, 2841-2849 (2023)), primarily due to the mechanical fragility and scarcity of malignant lymphoid cells.

During vitrectomy, the vitreous is fragmented and aspirated through a high-speed cutting probe (vitrector), which applies both mechanical cutting and vacuum forces. These operational parameters, while optimized for surgical efficiency and visualization, may inadvertently damage cells by rupturing membranes or disrupting nuclei, thereby reducing the yield and integrity of cellular material available for analysis.

Given the critical importance of maximizing diagnostic sensitivity in a disease with high morbidity and mortality, refining vitrectomy parameters to minimize cellular damage could represent a major advancement in the diagnostic workflow of VRL.

The present study aims to systematically evaluate the effect of different combinations of vacuum pressure and cut rate on cellular recovery and morphological preservation in a controlled ex-vivo vitreous model. Approximately 100 healthy donors will be enrolled and stratified into four experimental groups, each undergoing simulated diagnostic vitrectomy procedures under distinct parameter settings:

• Group A: Low vacuum / Low cut rate
• Group B: Low vacuum / High cut rate
• Group C: High vacuum / Low cut rate
• Group D: High vacuum / High cut rate This design enables direct comparison of operational conditions to identify settings that optimize cell preservation and recovery. The ultimate goal is to inform best-practice recommendations for diagnostic vitrectomy in suspected VRL, potentially improving cytologic yield and overall diagnostic accuracy.

Inclusion Criteria:

• Provision of written informed consent authorizing the anonymized research use of residual blood samples.
• Age 18-65 years at the time of donation.
• Eligibility for routine whole blood donation, according to the institutional and national standards of the Blood Transfusion Center.
• Good general health, as per standard donor screening procedures.

Exclusion Criteria:

No additional exclusion criteria are defined beyond standard blood donation regulations. Exclusion from the study will therefore coincide with ineligibility for blood donation according to institutional protocols (e.g., due to infection, hematologic disease, recent surgery, or medication use that affects blood composition).

Approximately 100 healthy adult volunteers meeting standard blood donation eligibility criteria at the Blood Transfusion Center of IRCCS Ospedale San Raffaele will be included.

Only residual anonymized blood samples obtained from routine donor activity will be used for this study. No additional collection, manipulation, or intervention will be performed on donors for research purposes.

Given that the objective of the study is to assess the variation (Δ) in cellular composition before and after ex-vivo processing, rather than the absolute baseline characteristics of individual samples, no disease-specific inclusion or exclusion criteria are required beyond those routinely applied for standard blood donation eligibility.