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

Background To 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. Methods The 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 <12%) and high monocyte specificity. They were used as the denominator and together with another monocyte informative target gene as the numerator, the value of this new DIRECT LS-TA can be determined which is a kind of ratio-based biomarker (RBB). The clinical utility was the differentiation of patients with bacterial infection from control subjects in a discovery dataset and 4 other replication datasets. Methods to convert DIRECT LS-TA results to multiple of control median (MoM) provided approximations to delta-delta CT values of relative quantification commonly used in qPCR or digital PCR assays. Results Over 50 monocyte-informative genes were identified, including key immune response genes like IFI44L , IL1B , VNN1 and NFKBIZ . DIRECT LS-TA results showed excellent correlation with the gold standard results, gene expression in isolated monocyte expression (R 2 =0.55-0.97). Then, DIRECT LS-TA of these 50 target genes were evaluated to identify the best RBB to detect the host response to bacterial infection. Monocyte DIRECT LS-TA VNN1 RBB showed consistent upregulation across five independent datasets (median fold change 2.7-fold, p<10 -8 ) with strong diagnostic performance (AUC=0.84-0.99). The expected corresponding delta-delta CT value is more than 1.4 cycle which can be reliably measured by qPCR. Additional monocyte-informative genes including NLRC4 , CYP1B1 , PFKFB3 , LILRA5 , NFKBIA , and NFKBIZ also demonstrated significant diagnostic ability (AUC>0.8). Conclusions The 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