During or before surgery, 100 μL of AH was taken from all the patients after an ACP, for IL-10 measurement and microbiologic analysis, depending on clinical suspicion. If the ACP was performed before vitrectomy it was done under topical anesthesia with a 30-gauge needle in the operating theater with a surgical microscope. If it was performed during the surgery or vitrectomy, the patient was under general anesthesia. All vitreous specimens were obtained through a standard three-port pars plana vitrectomy. First, 0.5 mL of AH was obtained after ACP and then 1 mL of pure vitreous was obtained by a syringe attached to the vitrectomy cutter handpiece via a stopcock. The specimen was immediately delivered to the cytopathology laboratory. A minimum of 100 μL of the undiluted vitreous and 100 μL of AH were submitted for IL-10 measurement. Specimens were stored at −70°C, and cytokine assays were performed on all specimens, but in lots of 20 freshly thawed samples every 15 days. IL-10 levels were determined by using a standard quantitative sandwich enzyme immunoassay technique (Quantikine; R&D Systems, Abingdon, UK). This procedure has been reported recently.
15 The rest of the undiluted vitreous was prepared for cytologic analysis. The remaining pure vitreous was cytocentrifuged at 600 rpm for 8 minutes, to obtain one or two slides. A core vitrectomy was performed after infusion, and the diluted vitreous was collected in the vitrector cassette and delivered to the laboratory. Cell culture medium (10% fetal calf serum) was added to improve cell viability. The sample was then centrifuged at 1000 rpm for 8 minutes at 4°C. The pellet was resuspended in 2 mL of balanced saline and cytocentrifuged in the same conditions as was used for fresh vitreous. Ten slides were prepared with both undiluted and diluted vitreous. Two slides were stained with May-Grunwald Giemsa. The remaining slides were air dried and fixed by immersion in acetone for 7 minutes. Immunocytochemical staining was performed according to the alkaline phosphatase anti-alkaline phosphatase (APAAP) method (EnVision System-Alkaline Phosphatase; Dako, Trappes, France), with monoclonal antibodies against pan-T, T-helper, and suppressor/cytotoxic antigens (CD3, CD4, CD8), B-cell antigen CD20, and κ or λ light chain.
Diluted vitreous samples were also prepared for PCR analyses. Detection of clonal Ig and TCR gene rearrangements were performed according to previously published protocols.
16 In ambiguous cases, PCR was performed after microdissection of atypical cells at the National Eye Institute (NEI, Bethesda, MD).
17
Samples of diluted vitreous were also delivered to microbiology laboratories for culture and PCR, to screen for infectious microorganisms according to clinical suspicion.