Atomic force microscopy (AFM) offers complementary imaging modes that can provide morphological and structural details of red blood cells (RBCs), and characterize interactions between specific biomolecules and RBC surface antigen. This review describes the applications of AFM in determining RBC health by the observation of cell morphology, elasticity and surface roughness. Measurement of interaction forces between plasma proteins and antibodies against RBC surface antigen using the AFM also brought new information to the immunohaematology field. With constant improvisation of the AFM in resolution and imaging time, the reaction of RBC to changes in the physico-chemistry of its environment and the presence of RBC surface antigen specific-biomolecules is achievable.

Erythrocytes (red blood cells, RBCs), the most common type of blood cells in humans are well known for their ability in transporting oxygen to the whole body through hemoglobin. Alterations in their membrane skeletal proteins modify shape and mechanical properties resulting in several diseases. Atomic force microscopy (AFM), a new emerging technique allows non-invasive imaging of cell, its membrane and characterization of surface roughness at micrometer/nanometer resolution with minimal sample preparation. AFM imaging provides direct measurement of single cell morphology, its alteration and quantitative data on surface properties. Hence, AFM studies of human RBCs have picked up pace in the last decade. The aim of this paper is to review the various applications of AFM for characterization of human RBCs topology. AFM has been used for studying surface characteristics like nanostructure of membranes, cytoskeleton, microstructure, fluidity, vascular endothelium, etc., of human RBCs. Various modes of AFM imaging has been used to measure surface properties like stiffness, roughness, and elasticity. Topological alterations of erythrocytes in response to different pathological conditions have also been investigated by AFM. Thus, AFM-based studies and application of image processing techniques can effectively provide detailed insights about the morphology and membrane properties of human erythrocytes at nanoscale.

Thrombelastography (TEG) is a viscoelastic hemostatic assay. We have observed that end-stage renal disease (ESRD) and trauma-induced coagulopathy (TIC) produce distinctive TEG tracings. We hypothesized that rigorously definable TEG patterns could discriminate between healthy controls and patients with ESRD and TIC.

TEG was performed on blood from ESRD patients (n = 54) and blood from trauma patients requiring a massive blood transfusion (n = 16). Plots of independent TEG parameters were analyzed for patterns coupled to disease state, compared with controls. Decision trees for taxonomic classification were then built using the "R-Project" statistical software.

Minimally overlapping clusters of TEG results were observed for the three patient groups when coordinate pairs of maximum amplitude (MA) and TEG-activated clotting time (ACT) were plotted on orthogonal axes. Based on these groupings, a taxonomical classification tree was constructed using MA and TEG ACT. Branch points were set at an ACT of 103 s, and these branches subdivided for MA at 60.8 mm for the high ACT branch and 72.6 mm for the low ACT branch, providing a correct classification rate of 93.4%.

ESRD and TIC demonstrate distinct TEG patterns. The coagulopathy of ESRD is typified by a prolonged enzymatic phase of clot formation, with normal-to-elevated final clot strength. Conversely, TIC is typified by prolonged clot formation and weakened clot strength. Our taxonomic categorization constitutes a rigorous system for the algorithmic interpretation of TEG based on cluster analysis. This will form the basis for clinical decision support software for viscoelastic hemostatic assays.

In hemodialysis (HD) patients, anemia relates to three main factors: insufficient production of erythropoietin; impaired management of iron; and decreased lifespan of red blood cells (RBCs). The third factor can relate to structural deterioration of RBCs due to extrinsic (extracorporeal circuit; biochemical activation and/or mechanical stress during dialysis) and intrinsic (uremic milieu; biochemical interference of the RBC membrane constituents with toxins) mechanisms. Herein, we evaluate information accessed with advanced imaging techniques at the cellular level.

Atomic force and scanning electron microscopes were employed to survey intact RBCs (iRBCs) of seven HD patients in comparison to seven healthy donors. The extrinsic factor was investigated by contrasting pre- and post-HD samples. The intrinsic environment was investigated by comparing the microscopy data with the clinical ones.

The iRBC membranes of the enrolled HD patients were overpopulated with orifice-like (high incidence; typical size within 100-1,000 nm) and crevice-like (low incidence; typical size within 500-4,000 nm) defects that exhibited a statistically significant (P < 0.05) relative increase (+55% and +350%, respectively) in respect to healthy donors. The relative variation of the orifice and crevice indices (mean population of orifices and crevices per top membrane surface) between pre- and post-HD was not statistically significant (-3.3% and +4.5%, respectively). The orifice index correlates with the concentrations of urea, calcium, and phosphorus, but not, however, with that of creatinine.

Extracorporeal circulation is not detrimental to the structural integrity of RBC membranes. Uremic milieu is a candidate cause of RBC membrane deterioration, which possibly worsens anemia.

Together with impaired production of erythropoietin and iron deficiency, the decreased lifespan of red blood cells (RBCs) is a main factor contributing to the chronic anaemia observed in haemodialysis (HD) patients. Atomic force microscopy is employed in this work to thoroughly survey the membrane of intact RBCs (iRBCs) of HD patients in comparison to those of healthy donors, aiming to obtain direct information on the structural status of RBCs that can be related to their decreased lifespan. We observed that the iRBC membrane of the HD patients is overpopulated with extended circular defects, termed 'orifices', that have typical dimension ranging between 0.2 and 1.0 μm. The 'orifice' index-that is, the mean population of 'orifices' per top membrane surface-exhibits a pronounced relative increase of order 54 ± 12% for the HD patients as compared to healthy donors. Interestingly, for the HD patients, the 'orifice' index, which relates to the structural status of the RBC membrane, correlates strongly with urea concentration, which is a basic index of the uraemic milieu. Thus, these results indicate that the uraemic milieu downgrades the structural status of the RBC membrane, possibly triggering biochemical processes that result in their premature elimination from the circulation. This process could decrease the lifespan of RBCs, as observed in HD patients.

We have developed what we believe is an efficient method to determine the electric parameters (the specific membrane capacitance C(m) and the cytoplasm conductivity kappa(i)) of cells from their dielectric dispersion. First, a limited number of dispersion curves are numerically calculated for a three-dimensional cell model by changing C(m) and kappa(i), and their amplitudes Deltaepsilon and relaxation times tau are determined by assuming a Cole-Cole function. Second, regression formulas are obtained from the values of Deltaepsilon and tau and then used for the determination of C(m) and kappa(i) from the experimental Deltaepsilon and tau. This method was applied to the dielectric dispersion measured for rabbit erythrocytes (discocytes and echinocytes) and human erythrocytes (normocytes), and provided reasonable C(m) and kappa(i) of the erythrocytes and excellent agreement between the theoretical and experimental dispersion curves.

We performed a systematic study of the sensitivity of dielectric spectroscopy to erythrocyte morphology. Namely, rabbit erythrocytes of four different shapes were prepared by precisely controlling the pH of the suspending medium, and their complex permittivities over the frequency range from 0.1 to 110 MHz were measured and analyzed. Their quantitative analysis shows that the characteristic frequency and the broadening parameter of the dielectric relaxation of interfacial polarization are highly specific to the erythrocyte shape, while they are insensitive to the cell volume fraction. Therefore, these two dielectric parameters can be used to differentiate erythrocytes of different shapes, if dielectric spectroscopy is applied to flow-cytometric inspection of single blood cells. In addition, we revealed the applicability and limitations of the analytical theory of interfacial polarization to explain the experimental permittivities of non-spherical erythrocytes.

A novel approach to the study of RBCs based on the collection of three-dimensional high-resolution AFM images and on the measure of the surface roughness of their plasma membrane is presented. The dependence of the roughness from several parameters of the imaging was investigated and a general rule for a trustful analysis and comparison has been suggested. The roughness of RBCs is a morphology-related parameter which has been shown to be characteristic of the single cells composing a sample, but independent of the overall geometric shape (discocyte or spherocyte) of the erythrocytes, thus providing extra-information with respect to a conventional morphology study. The use of the average roughness value as a label of a whole sample was tested on different kinds of samples. Analyzed data revealed that the quantitative roughness value does not change after treatment of RBCs with various commonly used fixation and staining methods while a drastic decrease occurs when studying cells with membrane-skeletal alteration both naturally occurring or artificially induced by chemical treatments. The present method provides a quantitative and powerful tool for a novel approach to the study of erythrocytes structure through an ultrastructural morphological analysis with the potential to give information, in a non-invasive way, on the RBCs function.

We present a new tool for the estimation of the age of bloodstains, which could probably be used during forensic casework. For this, we used atomic force microscopy (AFM) for high-resolution imaging of erythrocytes in a blood sample and the detection of elasticity changes on a nanometer scale. For the analytic procedure we applied a fresh blood spot on a glass slide and started the AFM detection after drying of the blood drop. In a first step, an overview image was generated showing the presence of several red blood cells, which could easily be detected due to their typical "doughnut-like" appearance. The consecutively morphological investigations in a timeframe of 4 weeks could not show any alterations. Secondly, AFM was used to test the elasticity by recording force-distance curves. The measurements were performed immediately after drying, 1.5 h, 30 h and 31 days. The conditions were kept constant at room temperature (20 degrees C) and a humidity of 30%. The obtained elasticity parameters were plotted against a timeline and repeated several times. The elasticity pattern showed a decrease over time, which are most probably influenced by the alteration of the blood spot during the drying and coagulation process. The preliminary data demonstrates the capacity of this method to use it for development of calibration curves, which can be used for estimation of bloodstain ages during forensic investigations.

Estimation of the time of death is one of the most important problems for forensic medicine and law. Physical and chemical postmortem changes are evaluated together while estimating the time of death. The pattern analysis of antemortem and postmortem bloodstains is one of the important parameters for forensic science, and cellular changes of blood cells can be useful for the quantitative assessment of the time of death. In this study, by successively investigating erythrocytes exposed in air on mica for 5 days using tapping mode atomic force microscopy (TM-AFM), we observed deformation of whole cell and membrane surface of unfixed erythrocytes with time lapse. We found that the time-dependent cellular changes occurred after exposure of erythrocytes in air for several days. At 0.5 days of exposure, fissures and cell shrinkage were observed. At 2.5 days of exposure, the emergence of nanometer-scale protuberances were observed and these protuberances increased in number with increasing time. The changes of cell shape and cell membrane surface ultrastructure can be used to estimate the time of death. Futhermore, smear-induced abnormal erythrocytes and immunostained erythrocytes were observed here. The need for more precise research is indicated, such as the correlation of membrane changes to intervals of less than 0.5 day of air exposure, and use of various substrates in addition to mica, including glass, metals, fabrics, among others, on which the bloodstains appear in crime scenes. The results of this research demonstrate the efficacy of AFM as a potentially powerful analytical tool in forensic science.

For humans the sizes and shapes of their red blood cells are important indicators of well being. In this study, the feasibility of using the atomic force microscope (AFM) to provide the sizes and shapes of red blood cells has been investigated. An immobilisation procedure has been developed that enabled red blood cells to be reliably imaged by contact AFM in air. The shapes of the red blood cells were readily apparent in the AFM images. Various cell quantification parameters were investigated, including thickness, width, surface area and volume. Excellent correlation was found between the AFM-derived immobilised mean cell volume (IMCV) parameter and the mean cell volume (MCV) parameter used in current haematological practice. The correlation between MCV and IMCV values has validated the immobilisation procedure by demonstrating that the significant cell shrinkage that occurs during immobilisation and drying does not introduce quantification artifacts. Reliable IMCV values were obtained by quantifying 100 red blood cells and this typically required 3-5 AFM images of 100 microm x 100 microm area. This work has demonstrated that the AFM can provide in a single test the red blood cell size and shape data needed in the assessment of human health.