Characterization of freeze-dried oxidized human red blood cells for pre-transfusion testing by synchrotron FTIR microspectroscopy live-cell analysis

Thulya Chakkumpulakkal Puthan Veettil, Diana Alves, Jitraporn Vongsvivut, Rosemary L. Sparrow, Bayden R. Wood, Gil Garnier

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Oxidative treatment of human red blood cells (RBCs) prior to freeze-drying appears to stabilize the RBCs to withstand dried storage at room temperature. To better understand the effects of oxidation and freeze-drying/rehydration on RBC lipids and proteins, single-cell measurements were performed by synchrotron-based Fourier transform infrared (FTIR) microspectroscopy ‘live-cell’ (unfixed) analysis. Lipid and protein spectral data of tert-butyl hydroperoxide (TBHP)-oxidized RBCs (oxRBCs), FDoxRBCs and control (untreated) RBCs were compared using principal component analysis (PCA) and band integration ratios. The oxRBCs and FDoxRBCs samples had similar spectral profiles that were clearly different to control RBCs. Spectral changes in the CH stretching region of oxRBCs and FDoxRBCs indicated the presence of increased saturated and shorter-chain lipids, consistent with lipid peroxidation and stiffening of the RBC membrane compared to control RBCs. The PCA loadings plot for the fingerprint region of control RBCs corresponding to the α-helical structure of hemoglobin, shows that oxRBCs and FDoxRBCs have conformational changes in the protein secondary structure to β-pleated sheets and β-turns. Finally, the freeze-drying process did not appear to compound or induce additional changes. In this context, FDoxRBCs could become a stable source of reagent RBCs for pre-transfusion blood serology testing. The synchrotron FTIR microspectroscopic live-cell protocol provides a powerful analytical tool to characterize and contrast the effects of different treatments on RBC chemical composition at the single cell level.

Original languageEnglish
Pages (from-to)1595-1602
Number of pages8
Issue number7
Early online date7 Mar 2023
Publication statusPublished - 7 Mar 2023

Bibliographical note

Funding Information:
This study was performed on the IRM beamline at Australia Synchrotron (Victoria, Australia), ANSTO. The use of IRM was funded as part of the Projects at the Australian Synchrotron (Project ID: M17147) and has been made available to users of the IRM Beamline. TCPV is a recipient of an Australian Institute of Nuclear Science and Engineering (AINSE) postgraduate research award (PGRA) 2021. We thank Dr Keith Bambery and Callum Gassner for their valuable suggestions on resonant mie scattering correction algorithm. This work was funded in part by the Australian Research Council (ARC) and Haemokinesis Ltd (Melbourne, Australia) through a linkage program, grant, LP160100544.

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy
  • Electrochemistry


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