The investigation of the physical properties of peripheral blood mononuclear cells (PBMC) is of great relevance, as they play a key role in regulating human body health. Here we report the possibility to characterize human PBMC in their physiological conditions in a microfluidic-based measurement system. A viscoelastic polymer solution is adopted for 3D alignment of individual cells inflow. An optical signature (OS) acquisition of each flowing cell is performed using a wide angle light scattering apparatus. Besides, a quantitative phase imaging (QPI) holographic system is employed with the aim (i) to check the position in flow of individual cells using a holographic 3D cell tracking method; and (ii) to estimate their 3D morphometric features, such as their refractive index (RI). Results obtained by combining OS and QPI have been compared with literature values, showing good agreement. The results confirm the possibility to obtain sub-micrometric details of physical cell properties in microfluidic flow, avoiding chemical staining or fluorescent labelling.