A simple way to detect disease‐associated cellular molecular alterations from mixed‐cell blood samples

&NA; Blood is a promising surrogate for solid tissue to investigate disease‐associated molecular biomarkers. However, proportion changes of the constituent cells in the often‐used peripheral whole blood (PWB) or peripheral blood mononuclear cell (PBMC) samples may influence the detection of cell‐specific alterations under disease states. We propose a simple method, Ref‐REO, to detect molecular alterations in leukocytes using the mixed‐cell blood samples. The method is based on the predetermined within‐sample relative expression orderings (REOs) of genes in purified leukocytes of healthy people. Both the simulated and real mixed‐cell blood gene expression profiles were used to evaluate the method. Approximately 99% of the differentially expressed genes (DEGs) detected by Ref‐REO in the simulated mixed‐cell data are owing to the transcriptional alterations in leukocytes rather than the proportion changes of leukocytes. For the real mixed‐cell data, the DEGs detected by Ref‐REO in the PBMCs expression data for systemic lupus erythematosus (SLE) patients overlap significantly with the DEGs detected in the expression data of SLE CD4 + T cells and B cells and they are mainly enriched with mRNA editing and interferon‐associated genes. The detected DEGs in the PWB data for lung carcinoma patients are significantly enriched with coagulation‐associated functional categories that are closely associated with cancer progression. In conclusion, the proposed method is capable of detecting the disease‐associated leukocyte‐specific molecular alterations, using mixed‐cell blood samples, which provides simple, transferable and easy‐to‐use candidates for disease biomarkers.