Comparative analysis of macroscopic and microscopic optical absorbance in hemagglutination assay

We report a comparative study of macroscopic and microscopic optical absorbance in hemagglutination (HA) assay. Red blood cells (RBCs) exhibit unique optical absorbance properties with characteristic peaks including Soret, Qv, and Qo. In addition, RBCs absorb light and appear as dark contrast in bright-field microscopy images, indicating an increase in local optical density (OD). By systematic analysis of macroscopic and microscopic OD measurements and UV–Visible (UV–Vis) spectroscopy, we developed a phenomenological model of RBC agglutination and non-agglutination. The antigen–antibody reaction in RBC agglutination behaves as a catastrophic event such that networking of RBC clumps is initiated at a critical RBC concentration. We analyzed the dependence of OD on RBC concentration. At the critical RBC concentration, OD values are dropped or saturated for RBC agglutination, on the other hand, ODs keep increasing as the increase of RBC concentration for RBC non-agglutination. By the analysis of UV–Vis spectroscopy for HA assay, we provide an optimal wavelength range as 480-520 nm, away from RBC characteristic absorption peaks. For further validation, we demonstrated the OD-based HA assay for the detection of H1N1 influenza A virus. Our investigation provides insights into how to utilize the physical properties of RBCs for novel HA assay platforms.