Proteomic Comparison of Standard Undiluted Vitreous Sampling and Large Volume Vitreous Biopsy Obtained by Perfluorocarbon–Perfused Pars Plana Vitrectomy (PCPV)

Abstract

Purpose: : Evaluation of vitreous samples for bioactivity or rare constituents is commonly hindered by small volumes of undiluted vitreous obtainable by standard pars plana vitrectomy (PPV). Use of PCPV has the potential for acquisition of large vitreous volumes undiluted by aqueous infusion solutions. This study examines whether vitreous obtained through PCPV has the same proteomic and bioactivity characteristics as standard sample acquisition.

Methods: : Samples were obtained from 3 patients undergoing PPV for proliferative diabetic retinopathy. Undiluted vitreous was collected by standard approach prior to beginning perfusion. Perflourocarbon liquid (PFC) was then infused and a second sample obtained encompassing vitreous displaced by the PFC. Samples were immediately placed on ice and stored at –80^oC. Proteomic analysis was performed on 50 µl samples using 12% SDS–PAGE gel electrophoresis, trypsin digest, liquid chromatography–tandem mass spectroscopy and the Sequest algorithm. The number of unique peptides for each protein was identified. Immunoblotting was performed using primary antibodies against angiotensinogen (AGT) and pigment–epithelium derived factor (PEDF).

Results: : The volume of vitreous obtained using PCPV was 2– to 5–fold greater than the standard approach with mean recoveries of 373±97µl and 1173±586µl, respectively (p=0.04). There were a total of 136 proteins identified, with 100 and 117 identified in the PCPV and standard samples, respectively. Identical shared proteins constituted 60%. Molecules known to increase in PDR (PEDF, AGT, and DKK3) were present in similar total peptide numbers regardless of collection technique. Western blot analysis of standard and PCPV techniques confirmed that PEDF (mean 1.08±0.17 vs 0.92± 0.15) and AGT (mean, 1.11±0.37 vs 0.89±0.36) were present in all samples from all patients with no statistically significant difference attributable to biopsy technique. Bioactivity equivalency studies are ongoing.

Conclusions: : PCPV can yield larger vitreous volumes than standard collection techniques while retaining similar (but not identical) protein content and concentration. Comparisons of vitreous bioactivity will be presented. If proven comparable, this collection technique may facilitate research studies that require analysis of vitreous fluid.

Keywords: clinical (human) or epidemiologic studies: systems/equipment/techniques • vitreous • diabetic retinopathy