Characterization of Extracellular Vesicles Derived from Red Blood Cells Using Vibrational Spectroscopy and Imaging

Richard A. Dluhy,¹ Amareshwari Konutham¹, Shaiju S Nazeer¹, 
Joo-Yeun Oh², Amit Gaggar², and Rakesh P. Patel², 

¹Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294
²Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294

Degradative changes that occur during storage of red blood cells (RBCs) include hemolysis, followed by formation of microvesicles and exosomes. There are currently no direct methods for determining the extent of in-bag hemolysis, or its degradation products. We used Raman spectroscopy to assess hemolysis in RBCs. Raman spectra indicated increased concentrations of oxyhemoglobin (oxyHb) and methemoglobin (metHb), and decreased membrane fluidity with storage age. Changes in oxyHb and metHb were associated with the intraerythrocytic and extracellular fractions, respectively. Changes in Raman bands reflective of oxyHb, metHb, and RBC membranes correlated with hemolysis; the most statistically significant change was an increased intensity of metHb and decreased membrane fluidity. 

We also studied extracellular vesicles (EVs) released from RBCs. EVs, both microparticles (MPs) and exosomes (Exos), are produced by a variety of cell types in response to diverse stimuli and are potential diagnostic indicators of disease. We used Raman and infrared (IR) to differentiate MPs and Exos produced from RBCs. These are the first studies of EVs isolated from RBCs.  We have been able to identify differences in oxyHb and metHb profiles in MPs and Exos from their Raman spectra. Multivariate statistical methods have been used to characterize and classify MP and Exo spectra with 100% accuracy. These methods also provide a means for variable selection to identify the spectral markers most responsible for the differentiation of MP and Exo spectra. Vibrational imaging using Raman and IR identified the differential effects of MPs and Exos on pulmonary epithelial cells.  These studies identify Raman and IR microscopy as a valuable noninvasive diagnostic modality for the study EV’s in relation to overall age-induced hemolysis and toxicity in RBCs.