Document Type

Conference Proceeding

Publication Date

4-2026

Abstract

Background: Timely, precise diagnosis of Retinopathy of Prematurity (ROP) is key to implementing appropriate treatment, as distinctions between mild-ROP and severe-ROP are the primary factors in its characterization. Deconvolution analysis of bulk RNA sequencing is an indirect method to identify static and dynamic molecular and physiological associations.

Objective: The objective of this this study was to evaluate if deconvolution analysis can be used to identify cellular biomarkers of ROP.

Design/Methods: This is a prospective study (IRB#086113MP2E) with a convenience sample size of 8 neonates with no ROP, 6 neonates with mild to moderate ROP (M-ROP), and 7 neonates with severe ROP (S-ROP) that necessitated laser surgery born at less than 26 weeks gestation. Peripheral blood Mononuclear cells were used for total RNA isolation. The isolated RNA was submitted to the Genome Sciences Core at Wayne State
University for RNA sequencing (RNA-seq). The barcoded libraries were multiplexed at equimolar
concentrations and sequenced (75 bp reads; >5M reads per sample) on the NovaSeq 6000. Data was
demultiplexed using Illumina's CASAVA 1.8.2 software.
In order to identify blood cell proportions in the bulk RNASeq data, a deconvolution analysis was performed using xCell2 1.3.0 R package (https://bioconductor.org/packages/devel/bioc/html/xCell2.html). The pretrained Immune Compendium reference was used to deconvolute the data and identify relative proportions of 40 different blood cell types. To identify significantly different cell populations across the three groups, a pairwise Students t-test was performed and adjusted using an FDR of 0.05.

Results: The demographic characteristics were not statistically significantly different (Table1). The pairwise comparison results are presented in Table 2 and graphically in Figure 1. Amongst 40 cell types evaluated only Neutrophils were statistically significantly higher in newborns Severe ROP as compared to controls. There was no statistically significant difference when data was compared for mild/moderate ROP versus the control group. There was no difference in expression of hematopoietic and endothelial cell types when compared using pan cancer single cell RNA deconvolution analysis (data not shown).

Conclusion(s): This is a unique preliminary data with interesting results. Neutrophils were upregulated in extremely premature newborns at that subsequently developed severe ROP while there were no differences in hemopoietic and endothelial cell types. A prospective single cell RNA analysis is warranted to further identify mechanisms associated with development of severe ROP.

Comments

97th Pediatric Academic Societies (PAS) Meeting, April 24-27, 2026, Boston, MA

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