Laser Tweezers Raman Spectroscopy As a Novel Red Blood Cell Functional Assay for Sickle Cell Disease

Abstract 4847

The number of individuals with sickle cell disease (SCD) in the U.S. is estimated to be over 70,000, and the average life span of a SCD patient is 39 years. Despite the high incidence of SCD in the U.S. and its significant negative effects on patient health, there are currently only limited treatment options for this disease. Also, although a single mutation in the globin gene is known to cause SCD, there is significant clinical diversity. As technology advances, many studies, including genome-wide analyses and proteomics, have been conducted to understand the disease mechanism and find better treatments. However, none has yet led to the development of novel therapies. We need a practical approach to study red blood cells (RBCs) to increase our knowledge of this heterogeneous disease.

Laser tweezers Raman spectroscopy (LTRS) is a label-free, laser-based method for the biochemical analysis of single living cells. Based on the phenomenon of inelastic light scattering of photons by molecular bonds, Raman spectroscopy can provide detailed information, in the form of a Raman spectrum, about the molecular structure and conformations inside single living cells. Moreover, the integration of laser tweezers enables 1) simple, convenient analysis of single suspension cells such as RBCs and 2) a method to impart a mechanical force on a cell (via the optical forces) and simultaneously monitor the biochemical response of that cell to the applied force. Previous studies using optical tweezers have shown different RBC elasticity between samples from SCD patients treated with hydroxyurea and those that were not treated with hydroxyurea. Raman spectroscopy has been applied to both normal and pathological RBCs in previous studies, but the results were limited to the identification of characteristic signatures. Our integration of the two methods, Raman spectroscopy and laser tweezers, is a new biophotonic approach for studying the intricate relationship between the biochemical and mechanical properties of a single cell that was not previously possible and is not easily carried out with existing methods. As such, we believe that this recent emerging single cell-based technology can be developed and used as an RBC functional assay.

We have previously shown that LTRS can detect a force dependent oxygenation state transition in individual RBCs trapped at different laser powers, presumably due to the optical forces stretching the cell and inducing changes in the hemoglobin-hemoglobin and hemoglobin-membrane interactions that change the oxygen content of the cell. We have also shown characteristic Raman signatures indicating different oxygen content in different RBC samples at specific applied forces. Signatures were different among RBCs from cord, normal adult, and SCD patient blood, suggesting laser tweezers Raman spectroscopy can be used to measure the different force dependent oxygen content in a single RBC. We are currently validating our system to determine whether Raman spectroscopy can be used to identify and distinguish gene-corrected RBCs from normal and sickle RBCs. Our preliminary results showed distinct Raman fingerprints associated with the membrane structure of RBCs. Spectral fingerprints were observed to be different between normal and sickle RBCs, and gene-corrected RBCs were observed to have Raman profiles similar to those of normal RBCs. We speculate that the signature overlap between normal and gene-corrected samples is due to partial gene correction. These results are encouraging and strongly suggest that Raman spectroscopy can not only identify molecular structures but also be used as a functional assay.