Microfluidic-Based Detection of AML-Specific Biomarkers Using the Example of Promyelocyte Leukemia

A microfluidic assay for the detection of promyelocytic leukemia (PML)-retinoic acid receptor alpha (RAR alpha) fusion protein was developed. This microfluidic-based system can be used for rapid personalized differential diagnosis of acute promyelocyte leukemia (APL) with the aim of early initiation of individualized therapy. The fusion protein PML-RAR alpha occurs in 95% of acute promyelocytic leukemia cases and is considered as diagnostically relevant. The fusion protein is formed as a result of translocation t(15,17) and is detected in the laboratory by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). Diagnostic methods require many laboratory steps with specialized staff. The developed microfluidic assay includes a sandwich enzyme-linked immunosorbent assay (ELISA) system for PML-RAR alpha on surface of magnetic microparticles in a microfluidic chip. A rapid detection of PML-RAR alpha in cell lysates is achieved in less than one hour. A biotinylated PML-antibody on the surface of magnetic streptavidin coated microparticles is used as capture antibody. The bound translocation product is detected by a RAR alpha antibody conjugated with horseradish peroxidase and the substrate QuantaRed. The analysis is performed in microfluidic channels which involves automated liquid processing with stringent washing and short incubation times. The results of the developed assay show that cell lysates of PML-RAR alpha-positive cells (NB-4) can be clearly distinguished from PML-RAR alpha-negative cells (HL-60, MV4-11).