Repeatability of Gene Expression Analysis in Circulating Cell Populations of Subjects with Neovascular Age-Related Macular Degeneration (nvAMD) Undergoing anti-VEGF Treatment

Abstract

Purpose: The etiology of refractoriness to anti-VEGF injections in patients with nvAMD is unknown. However, expression of the pro-angiogenic chemokine Bv8 in CD11b-positive cells has been linked to anti-VEGF refractoriness in animal models (Shojaei et al Nature 2007). Here we investigate the repeatability in measuring Bv8 gene expression in CD11b-positive circulating cells in successive blood draws from nvAMD subjects undergoing anti-VEGF treatment.

Methods: Two blood draws were obtained from 49 subjects with active nvAMD 1-9 months apart while patients were receiving standard of care anti-VEGF injections. Peripheral blood mononuclear cells (PBMCs) were isolated and CD11b-positive cells purified using magnetic bead sorting. RNA was isolated from the sorted cells. Quantitative gene expression was analyzed using Taqman qPCR. Relative Quantification (RQ) values were determined by normalizing to the subject with the lowest gene expression in the 1st blood draw. RQ values determined from the 1st blood draw are referred to as RQ₁, and from the 2nd blood draw as RQ₂.

Results: The average of the RQ₁ values was 6.23 ± 4.58 (range 1 - 19.861). The average of the RQ₂ values was 5.95 ± 3.85 (range 0.948 - 16.96). C_T values from triplicate reactions for each sample varied by less than 1%. Bland-Altman analysis was used to inspect the repeatability of the RQ measurements between the blood draws. The mean RQ₂-RQ₁ value was 0.05, indicating no systematic bias towards either measurement. The Coefficient of Repeatability (CR) value for subjects in the lower three quartiles of Bv8 expression (RQ_avg < 7.5) was 2.727. The CR value for subjects in the upper quartile of Bv8 expression (RQ_avg > 7.5) was 9.876. Alternatively, average RQ values were determined for each subject, and the percentage of variation from the mean calculated. The average percentage variation from the mean for this group of subjects was 14.43 ± 11.64%. Variations under 25% of the mean were found in 90% of the subjects.

Conclusions: The variability of gene expression was within acceptable limits for repeatability. The low variability in the triplicate assay results suggests that the overall variability is found within the subjects and not due to technical factors and supports the use of qPCR in analyzing gene expression in circulating cells of patients with nvAMD.