Direct single cell-type gene expression analysis in Whole blood: Novel ratio-based gene expression biomarkers using 2 novel monocyte reference genes (PSAP and CTSS) for monitoring bacterial infection

Abstract

BackgroundTo determine single cell-type specific gene expression in peripheral blood (PB) requires either prior labour-intense cell sorting or expensive single-cell RNA sequencing. We developed and validated a novel ratio-based biomarker (RBB) called Direct Leukocyte Subpopulation-Transcript Abundance (DIRECT LS-TA) assay that allows quantification of monocyte-specific gene expression directly from peripheral blood samples without cell sorting. MethodsThe DIRECT LS-TA method leverages known cell-type proportions and differential gene expression profiles among leukocyte subpopulations (e.g. monocytes, lymphocytes and granulocytes) to identify monocyte-informative genes. We shortlisted genes that had 2.5-fold higher expression in isolated monocytes compared to PB samples, indicating >50% transcript of genes in PB are contributed by monocytes alone. Public gene expression datasets were used to generate a list of monocyte informative genes with which DIRECT LS-TA assay is applicable in PB samples. PSAP and CTSS were identified as the monocyte informative reference genes, based on low biological variation (CV 0.8). ConclusionsThe DIRECT LS-TA method (as a simple RBB) provides reliable quantification of monocyte-specific gene expression directly from whole blood samples without cell separation. The robust performance in bacterial infection diagnosis demonstrates its potential clinical utility for rapid infection differentiation and informed antibiotic stewardship. DIRECT LS-TA will emerge as a new kind of in vitro diagnostics (IVD) which can convey single cell-type gene expression information from PB samples. The new kind of IVD and uniqueness of the information, together with the ease of implementation will make it very useful in clinics