ABSTRACT
The manuscript presents the potential of surface-enhanced Raman spectroscopy (SERS) and tip-enhanced Raman spectroscopy (TERS) for label-free characterization of extracellular microvesicles (EVs) and their isolated membranes derived from red blood cells (RBCs) at the nanoscale and at the single-molecule level, providing detection of a few individual amino acids, protein and lipid membrane compartments. The study shows future directions for research, such as investigating the use of the mentioned techniques for the detection and diagnosis of diseases. We demonstrate that SERS and TERS are powerful techniques for identifying the biochemical composition of EVs and their membranes, allowing the detection of small molecules, lipids, and proteins. Furthermore, extracellular vesicles released from red blood cells (REVs) can be broadly classified into exosomes, microvesicles, and apoptotic bodies, based on their size and biogenesis pathways. Our study specifically focuses on microvesicles that range from 100 to 1000 nanometres in diameter, as presented in AFM images. Using SERS and TERS spectra obtained for REVs and their membranes, we were able to characterize the chemical and structural properties of microvesicle membranes with high sensitivity and specificity. This information may help better distinguish and categorize different types of EVs, leading to a better understanding of their functions and potential biomedical applications.
Subject(s)
Extracellular Vesicles , Spectrum Analysis, Raman , Spectrum Analysis, Raman/methods , Erythrocyte Membrane , Nanotechnology/methods , Proteins/chemistryABSTRACT
This work presents a semi-quantitative spectroscopic approach, including FTIR-ATR and Raman spectroscopies, for the biochemical analysis of red blood cells (RBCs) supported by the biochemical, morphological and rheological reference techniques. This multi-modal approach provided the description of the RBC alterations at the molecular level in a model of accelerated aging induced by administration of D-galactose (D-gal), in comparison to natural aging. Such an approach allowed to conclude that most age-related biochemical RBC membrane changes (a decrease in lipid unsaturation and the level of phospholipids, or an increase in acyl chain shortening) as well as alterations in the morphological parameters and RBC deformability are well reflected in the D-gal model of accelerated aging. Similarly, as in natural aging, a decrease in LDL level in blood plasma and no changes in the fraction of glucose, creatinine, total cholesterol, HDL, iron, or triglycerides were observed during the course of accelerated aging. Contrary to natural aging, the D-gal model led to an increase in cholesterol esters and the fraction of total esterified lipids in RBC membranes, and evoked significant changes in the secondary structure of the membrane proteins. Moreover, a significant decrease in the phosphorous level of blood plasma was specific for the D-gal model. On the other hand, natural aging induced stronger changes in the secondary structures of the proteins of the RBCs' interior. This work proves that research on the aging mechanism, especially in circulation-related diseases, should employ the D-gal model with caution. Nonetheless, the D-gal model enables to imitate age-related rheological alterations in RBCs, although they are partially derived from different changes observed in the RBC membrane at the molecular level.
Subject(s)
Aging, Premature/chemically induced , Aging/blood , Disease Models, Animal , Erythrocyte Membrane/chemistry , Galactose/toxicity , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis, Raman , Aging, Premature/blood , Animals , Cytosol/chemistry , Erythrocyte Aging/drug effects , Erythrocyte Deformability/drug effects , Erythrocyte Indices/drug effects , Erythrocyte Membrane/drug effects , Free Radicals/toxicity , Galactose/pharmacology , Hemorheology/drug effects , Male , Mice , Mice, Inbred C57BL , Phosphorus/blood , Research DesignABSTRACT
A new type of aggregate, formed in human red blood cells (RBCs) in response to glutaraldehyde treatment, was discovered and analyzed with the classical and advanced biomolecular imaging techniques. Advanced Heinz body-like aggregates (AHBA) formed in a single human RBC are characterized by a higher level of hemoglobin (Hb) degradation compared to typical Heinz bodies, which consist of hemichromes. The complete destruction of the porphyrin structure of Hb and the aggregation of the degraded proteins in the presence of Fe3+ ions are observed. The presence of such aggregated, highly degraded proteins inside RBCs, without cell membrane destruction, has been never reported before. For the first time the spatial differentiation of two kinds of protein mixtures inside a single RBC, with different phenylalanine (Phe) conformations, is visualized. The non-resonant Raman spectra of altered RBCs with AHBA are characterized by the presence of a strong band located at 1037 cm-1, which confirms that glutaraldehyde interacts strongly with Phe. The shape-shifting of RBCs from a biconcave disk to a spherical structure and sinking of AHBA to the bottom of the cell are observed. Results reveal that the presence of AHBA should be considered when fixing RBCs and indicate the analytical potential of Raman spectroscopy, atomic force microscopy and scanning near-field optical microscopy in AHBA detection and analysis.
Subject(s)
Cytoskeleton/metabolism , Heinz Bodies/pathology , Glutaral/toxicity , Heinz Bodies/ultrastructure , Heme/metabolism , Hemoglobins/metabolism , Humans , Male , Protein Aggregates/physiologyABSTRACT
Lungs, due to their high oxygen availability and vascularization, are an ideal environment for cancer cell migration, metastasis and tumour formation. These processes are directly connected with extracellular matrix (ECM) remodelling, resulting from cancer cell infiltration and preparation of the environment suitable for tumour growth. Herein, we compare the potential of fast, label-free and non-destructive methods of Fourier-transform infrared spectroscopy (FT-IR) in standard and high definition (HD) modes with nonlinear coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF) and a fluorescence lifetime imaging (FLIM) technique for lung metastasis detection. We show their potential in the detection of lung macrometastasis, in which we already observed the ECM remodelling. The CARS image revealed a dense cell fraction typical of ECM remodeling and reduction of the TPEF signal together with an increase of fluorescence lifetime predominantly due to NAD(P)H suggesting metabolic changes in the metastatic foci. FT-IR spectroscopy allowed not only for macrometastasis detection but also their stage definition based mainly on the analysis of proteins, RNA and glycogen fractions. The multimodal approach additionally suggested partial enzymatic degradation of elastin in ECM and collagen remodelling.
Subject(s)
Breast Neoplasms , Lung Neoplasms , Animals , Breast Neoplasms/diagnostic imaging , Humans , Lung Neoplasms/diagnostic imaging , Mice , Photons , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis, RamanABSTRACT
New technologies to diagnose malaria at high sensitivity and specificity are urgently needed in the developing world where the disease continues to pose a huge burden on society. Infrared and Raman spectroscopy-based diagnostic methods have a number of advantages compared with other diagnostic tests currently on the market. These include high sensitivity and specificity for detecting low levels of parasitemia along with ease of use and portability. Here, we review the application of vibrational spectroscopic techniques for monitoring and detecting malaria infection. We discuss the role of vibrational (infrared and Raman) spectroscopy in understanding the processes of parasite biology and its application to the study of interactions with antimalarial drugs. The distinct molecular phenotype that characterizes malaria infection and the high sensitivity enabling detection of low parasite densities provides a genuine opportunity for vibrational spectroscopy to become a front-line tool in the elimination of this deadly disease and provide molecular insights into the chemistry of this unique organism.
Subject(s)
Malaria/diagnosis , Spectroscopy, Fourier Transform Infrared/methods , Spectrum Analysis, Raman/methods , Animals , Erythrocytes/microbiology , Erythrocytes/pathology , Heme/analysis , Hemeproteins/analysis , Humans , Plasmodium/growth & development , Spectroscopy, Fourier Transform Infrared/instrumentation , Spectrum Analysis, Raman/instrumentation , VibrationABSTRACT
Based on the multimodal characterization of human red blood cells (RBCs), the link between the storage-related sequence of the nanoscale changes in RBC membranes in the relation to their biochemical profile as well as mechanical and functional properties was presented. On the background of the accumulation of RBCs waste products, programmed cell death and impaired rheological properties, progressive alterations in the RBC membranes including changes in their height and diameter as well as the in situ characterization of RBC-derived microparticles (RMPs) on the RBCs surface were presented. The advantage of atomic force microscopy (AFM) in RMPs visualization, even at the very early stage of vesiculation, was shown based on the results revealed by other reference techniques. The nanoscale characterization of RMPs was correlated with a decrease in cholesterol and triglycerides levels in the RBC membranes, proving the link between the lipids leakage from RBCs and the process of vesiculation.
Subject(s)
Erythrocytes/metabolism , Microscopy, Atomic Force/methods , Cell-Derived Microparticles/metabolism , Erythrocyte Count , Flow Cytometry , HumansABSTRACT
This work focused on a detailed assessment of lung tissue affected by metastasis of breast cancer. We used large-area chemical scanning implemented in Fourier transform infrared (FTIR) spectroscopic imaging supported with classical histological and morphological characterization. For the first time, we differentiated and defined biochemical changes due to metastasis observed in the lung parenchyma, atelectasis, fibrous, and muscle cells, as well as bronchi ciliate cells, in a qualitative and semi-quantitative manner based on spectral features. The results suggested that systematic extracellular matrix remodeling with the progress of the metastasis process evoked a decrease in the fraction of the total protein in atelectasis, fibrous, and muscle cells, as well as an increase of fibrillar proteins in the parenchyma. We also detected alterations in the secondary conformations of proteins in parenchyma and atelectasis and changes in the level of hydroxyproline residues and carbohydrate moieties in the parenchyma. The results indicate the usability of FTIR spectroscopy as a tool for the detection of extracellular matrix remodeling, thereby enabling the prediction of pre-metastatic niche formation.
Subject(s)
Breast Neoplasms/pathology , Extracellular Matrix , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Spectroscopy, Fourier Transform Infrared , Animals , Disease Models, Animal , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Female , Humans , Immunohistochemistry , MiceABSTRACT
This work presents the potential of vibrational spectroscopy, Vis and NIR Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR) in reflection and transmission modes, and nano-FTIR microscopy to study the biochemical alterations in membranes of isolated and intact red blood cells (RBCs). The main goal was to propose the best spectroscopic method which enabled following biochemical alterations in the RBC membranes and then to translate this spectroscopic signature of degradation to in situ analysis of RBCs. Two models corresponding to two distinct cases of RBC membrane conditions were employed, and they were derived from healthy and young mice and mature mice with advanced atherosclerosis. It was shown that each technique provided essential information about biochemical alterations of the isolated membranes as well as membranes in the intact RBCs, which can be used in the development of a rapid and in situ analytical technology. Finally, we proposed that the combination of macro- and nanoprobing implemented in IR spectroscopy provided a wide chemical characterization of the RBC membranes, including alterations in lipid and protein fractions. This study also examined the effect of the sample preparation to determine destructive factors influencing a spectroscopic analysis of isolated membranes and intact RBCs derived from healthy and disease-affected mice.
Subject(s)
Erythrocyte Membrane/chemistry , Nanotechnology/methods , Spectroscopy, Fourier Transform Infrared/methods , Spectrum Analysis, Raman/methods , Animals , Cholesterol/chemistry , Esterification , Male , Mice , Mice, Inbred C57BL , Phospholipids/chemistryABSTRACT
The work presents the complementary approach to characterize the formation of various Hb species inside isolated human RBCs exposed to NO, with a focus on the formed Hb-NO adducts. This work presents a complementary approach based on Resonance Raman Spectroscopy (RRS) supported by Blood Gas Analysis, Electron Paramagnetic Resonance Spectroscopy, UV-Vis Absorption Spectroscopy and Mössbauer Spectroscopy to characterize the formation of various Hb species, with a focus on the Hb-NO adducts formed inside isolated human RBCs exposed to NO, under the experimental conditions of low and high levels of oxygen Hb saturation. In the present work, we induced Hb-NO adducts using PAPA-NONOate, a NO-donor with known chemistry and kinetics of NO release, and confirmed the formation of Hb-NO adducts in RBCs incubated with Human Aortic Endothelial Cells (HAECs) stimulated to produce NO. Our results provide a new insight into the formation of Hb-NO adducts after the exposure of RBCs with high oxyHb content to exogenous NO with special attention to the formation of LSHbIIINO in addition to LSHbIINO and metHb (HS/LSHbIIIH2O). We also point out that reliable characterization of Hb-NO adducts requires complementary techniques. Among them, RRS, as a label-free and non-destructive tool, appears to be an important discrimination technique in the studies of Hb-NO adducts inside intact RBCs.
Subject(s)
Endothelial Cells/chemistry , Erythrocytes/chemistry , Hemoglobins/chemistry , Nitric Oxide/chemistry , Aorta/cytology , Cells, Cultured , Electron Spin Resonance Spectroscopy , Endothelium, Vascular/cytology , Humans , Kinetics , Spectrum Analysis, RamanABSTRACT
Growing interest in the role of endothelium under physiological and pathological conditions has led to an increasing demand for its representative in vitro models especially suitable for drug tests and medical diagnostics. There are several endothelial cell lines commercially available whose biochemistry, and hence response to various stimuli, can be different. Recently, two vibrational techniques, Raman and Fourier-transform infrared microscopy, have been found to be potent tools for studying the biochemical composition of a single cell in an easy, rapid and label-free way. However, depending on the applied technique, the results may exhibit some divergence due to different selection rules as well as distinct experimental conditions. This paper presents the methodology of examination and characterization of three popular human endothelial cell lines: HAoEC (primary cells), HMEC-1 and EA.hy926 (immortalized cells). Based on high lateral resolution Raman imaging together with standard and high magnification Fourier-transform infrared measurements, the differences in spectral information and the distribution of biomolecules are presented and discussed.
Subject(s)
Cell Line/cytology , Endothelial Cells/cytology , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis, Raman , Humans , VibrationABSTRACT
The combination of FT-IR and Raman spectroscopies allowed the biochemical profiling of lungs in the early stage of pulmonary metastasis in the murine model of breast cancer. Histological staining was used as a reference. Raman spectroscopy was especially useful in the detection and semi-quantitative analysis of the vitamin A content in lung lipofibroblasts, whereas the IR technique provided semi-quantitative information on the contents of nucleic acids, carbohydrates including glycogen, and lipids as well as changes in the secondary structures of tissue proteins. Our spectroscopic results suggest that the early phase of metastasis in the lung is characterized by a decrease in the endogenous retinoid content in combination with a decrease in the content of glycogen and lipids.
Subject(s)
Breast Neoplasms/pathology , Lung Neoplasms/diagnosis , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis, Raman , Animals , Cell Line, Tumor , Glycogen/analysis , Lipids/analysis , Lung Neoplasms/secondary , Mice , Mice, Inbred BALB C , Retinoids/analysisABSTRACT
Confocal Raman mapping and FT-IR imaging combined with chemometric analysis was used to study the alterations in murine brain tissue induced by the development of atherosclerosis. FT-IR imaging allowed us to obtain lower spatial resolution data (â¼5.5 µm) from large, representative cross-sectional brain areas, while Raman mapping provided a more detailed insight into chosen regions of interest with high spatial resolution (â¼0.4 µm). A comparison of white (WM) and grey matter (GM) from control (C57BL/6J) and ApoE/LDLR(-/-) mice with advanced atherosclerosis revealed disease-induced changes in both: GM and WM. The alterations included an increased lipid to protein ratio and higher total content of cholesterol.
Subject(s)
Atherosclerosis/diagnostic imaging , Brain/diagnostic imaging , Spectroscopy, Fourier Transform Infrared , Spectrum Analysis, Raman , Animals , Mice , Mice, Inbred C57BL , Mice, Knockout, ApoEABSTRACT
The widespread and cost-effective use of transflection substrates in Fourier transform infrared (FTIR) imaging of clinical samples is affected by the presence of artefacts including the electric field standing wave (EFSW) and contributions from light dispersion. For IR-based diagnostics, the manifestation of undesirable artifacts can distort the spectra and lead to erroneous diagnosis. Nevertheless, there is no clear consensus in the literature about the degree of influence of these effects. The aim of this work is to contribute to this discussion by comparing transflection and transmission images of the same tissue. For this purpose two adjacent sections of the same tissue (lymphoma sample) were fixed onto a CaF2 window and a transflective slide for FTIR imaging. The samples in this case had a central area where based on morphology it was presumed the fixative did not penetrate to the same extent hence providing a comparable region for the two different substrates with a distinct physical/chemical difference. Transmission and transflection spectra from adjacent hyperspectral tissue images were combined in an extended dataset. Surprisingly, unsupervised hierarchical cluster analysis clustered together transflection and transmission spectra, being classified according to differences in tissue fixation instead of the geometry employed for the image acquisition. A more detailed examination of spectra from the peripheral zone of the tissue indicated that the main differences between the transflection and transmission spectra were: (1) a small shift of the amide I, (2) a larger "noise" component in the transflection spectra requiring more averaging to obtain representative spectra of tissue types, and (3) the phosphate bands were generally higher in absorbance in the transflection measurements compared to the transmission ones. The amide I shift and the larger spectral variance was consistent with results obtained in previous studies where the EFWS was present. The findings indicate that artifacts resulting from transflection measurements were small but consistent across the tissue, and therefore the use of transflection measurements could be employed for disease diagnosis. Accordingly, we recommend a straightforward multivariate comparison of images from transmission and transflection measurements in a combined data matrix obtained from adjacent sections of the tissue as a useful preliminary study for establishing the impact of the EFWS on the samples, before considering the routine use of transflection substrates for any new tissue studied.
Subject(s)
Optical Imaging/methods , Spectroscopy, Fourier Transform Infrared , Tissue Fixation , Artifacts , Lymphoma/pathologyABSTRACT
In general, the first overtone modes produce weak bands that appear at approximately twice the wavenumber value of the fundamental transitions in vibrational spectra. Here, we report the existence of a series of enhanced non-fundamental bands in resonance Raman (RR) spectra recorded for hemoglobin (Hb) inside the highly concentrated heme environment of the red blood cell (RBC) by exciting with a 514.5 nm laser line. Such bands are most intense when detecting parallel-polarized light. The enhancement is explained through excitonic theory invoking a type C scattering mechanism and bands have been assigned to overtone and combination bands based on symmetry arguments and polarization measurements. By using malaria diagnosis as an example, we demonstrate that combining the non-fundamental and fundamental regions of the RR spectrum improves the sensitivity and diagnostic capability of the technique. The discovery will have considerable implications for the ongoing development of Raman spectroscopy for blood disease diagnoses and monitoring heme perturbation in response to environmental stimuli.
Subject(s)
Erythrocytes/chemistry , Erythrocytes/parasitology , Hemoglobins/analysis , Malaria, Falciparum/diagnosis , Spectrum Analysis, Raman/methods , Heme/analysis , Humans , Lasers , Plasmodium falciparum/isolation & purificationABSTRACT
Raman microspectroscopic imaging has been utilized for the investigation of pathological changes in the liver induced by diabetes and atherosclerosis.
Subject(s)
Atherosclerosis/pathology , Biomarkers/analysis , Diabetes Mellitus, Experimental/pathology , Lipids/analysis , Liver/pathology , Spectrum Analysis, Raman/methods , Animals , Apolipoproteins E/physiology , Atherosclerosis/metabolism , Diabetes Mellitus, Experimental/metabolism , Heme/metabolism , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, LDL/physiology , Vitamin A/metabolismABSTRACT
In the present study, we characterized the secondary structure alterations of intact red blood cells (RBCs) cytosol with special attention to the sex-related alterations in 8- and 24-week-old female and male ApoE/LDLR-/- mice, compared to age-matched female and male C57BL/6J control animals. Results were obtained with previously established methodology based on Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Additionally, we evaluated 2,3-DPG levels in the RBCs and showed its potential link to the hemoglobin (Hb) secondary structure alterations. Considering Hb structure alterations probed by FTIR-ATR, the ratio of turns to α-helices in 8-week-old ApoE/LDLR-/- mice suggested more pronounced secondary structure alterations within the RBCs than in the age-matched control. Sex-related differences were observed solely in 24-week-old male ApoE/LDLR-/- mice, which showed statistically significant increase in the secondary structure alterations compared to 24-week-old female ApoE/LDLR-/- mice. Similar to the secondary structure alterations, no sex-related differences were observed in the levels of 2,3-DPG in RBCs, except for 24-week-old male ApoE/LDLR-/- mice, which showed significantly higher levels compared to the age-matched female ApoE/LDLR-/- mice. Considering the age-related alterations, we observed significant increases in the intracellular 2,3-DPG of RBCs with animals' age in all studied groups, except for female ApoE/LDLR-/- mice, where a significant difference was not reported. This suggests the clear correlation between secondary structure of Hb alterations and 2,3-DPG levels for male and female murine RBC and proves a higher resistance of older female RBCs to the secondary structure changes with progression of atherosclerosis. Moreover, it may be concluded that higher 2,3-DPG levels in RBCs occurred in response to the secondary structure alterations of Hb in ApoE/LDLR-/- mice.
Subject(s)
Apolipoproteins E , Erythrocytes , 2,3-Diphosphoglycerate , Animals , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Spectroscopy, Fourier Transform InfraredABSTRACT
Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) with the use of a slide-on germanium accessory followed by chemometric analysis allowed for providing meaningful information about the biochemical composition of a single endothelial cell. In this work, the methodology of the ATR-FTIR measurements of dried cells and dried cells immersed in water solution is presented. The contact of the cell and Ge crystal was set up manually and monitored through the integration of the amide I band. Additionally, the cell imaging in transreflection mode was tested, but the spectral differences between sub-cellular structures were not prominent in the registered spectra. It has been shown that the ATR-FTIR method gives better results due to the increased spatial resolution and S/R ratio as well as small contribution of the optical artifacts.
Subject(s)
Endothelial Cells/cytology , Spectroscopy, Fourier Transform Infrared/methods , Cell Line , Endothelial Cells/chemistry , Germanium , HumansABSTRACT
Label-free molecular imaging is a promising utility to study tissues in terms of the identification of their compartments as well as chemical features and alterations induced by disease. The aim of this work was to assess if higher magnification of optics in the Fourier transform infrared (FT-IR) microscope coupled with the focal plane detector resulted in better resolution of lung structures and if the histopathological features correlated with clustering of spectral images. FT-IR spectroscopic imaging was performed on paraffinized lung tissue sections from mice with optics providing a total magnification of 61× and 36×. Then, IR images were subjected to unsupervised cluster analysis and, subsequently, cluster maps were compared with hematoxylin and eosin staining of the same tissue section. Based on these results, we observed minute features such as cellular compartments in single alveoli and bronchiole, blood cells and megakaryocytes in a vessel as well as atelectasis of the lung. In the case of the latter, differences in composition were also noted between the tissue from the non-cancerous and cancerous specimen. This study demonstrated the ability of high-definition FT-IR imaging to evaluate the chemical features of well-resolved lung structures that could complement the histological examination widely used in animal models of disease.
Subject(s)
Neoplasms , Animals , Disease Models, Animal , Fourier Analysis , Lung/diagnostic imaging , Mice , Spectroscopy, Fourier Transform Infrared/methodsABSTRACT
In this study for the first time, we investigated the correlation between sex-specific differences in adenosine triphosphate (ATP) levels in red blood cells (RBCs) and their mechanical, biochemical, and morphological alterations during the progression of atherosclerosis in ApoE/LDLR double-deficient (ApoE/LDLR-/-) mice. Our results indicate that both sex and age affect alterations in RBCs of both ApoE/LDLR-/- and C57BL/6J mice. When compared with male RBCs, female RBCs were characterized by lower basal ATP and mean corpuscular hemoglobin concentration (MCHC), higher hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), deformability, and phosphatidylserine (PS) exposure levels, regardless of age in both, ApoE/LDLR-/- and C57BL/6J mice. ApoE/LDLR-/- mice compared with age-matched controls showed lower basal ATP levels regardless of age and sex. Intracellular ATP level of RBCs was decreased solely in senescent female C57BL/6J mice, while it was elevated in males. Basal extracellular ATP levels were 400 times lower than corresponding intracellular level. In conclusion, basal ATP levels, RBC morphology, deformability, PS exposure levels alterations are sex-dependent in mice. Changes in basal ATP levels were correlated with PS exposure and trends of changes in MCV. Trends of changes of the most RBC parameters were similar in both sexes of ApoE/LDLR-/- mice compared with age-matched controls; however, their kinetics and levels vary greatly between different stages of disease progression.