Electrophoretic and aggregation behavior of bovine, horse and human red blood cells in plasma and in polymer solutions

The electrophoretic mobility of native and glutaraldehyde-fixed bovine, human, and horse red blood cells (RBC) was investigated as a function of ionic strength (5-150 mM) and concentration of 464 kDa dextran (2 and 3 g/dl); RBC aggregation in autologous plasma and in dextran solutions was also measured. In agreement with previous observations, human and horse RBC form stable rouleaux whereas bovine RBC do not aggregate in either plasma or in dextran 464 kDa solutions. Electrophoretic measurements showed a species-dependent adsorption and depletion of dextran that can be theoretically evaluated. Adsorption of polymer is not a prerequisite for RBC aggregation (bovine RBC show the highest amount of adsorbed dextran yet do not aggregate). Aggregate formation thus occurs as long as the Gibbs free energy difference, given by the osmotic pressure difference between the bulk phase and the polymer-depleted region between two RBC, is larger than the steric and electrostatic repulsive energy contributed by the macromolecules present on the RBC surface. With increasing bulk-phase polymer concentration the depletion layer thickness decreases and the amount of adsorbed macromolecules increases, thereby resulting in an increase of the repulsive component of the interaction energy and decreased aggregation. We thus view electrophoretic measurements of RBC in various media as an important tool for understanding polymer behavior near the red cell surface and hence the mechanisms involved in RBC aggregation.     

Aggregation behavior and electrophoretic mobility of red blood cells in various mammalian species

Differences of red blood cell (RBC) aggregation among various mammalian species has been previously reported for whole blood, for RBC in autologous plasma, and for washed RBC re-suspended in polymer solutions. The latter observation implies the role of cellular factors, yet comparative studies of such factors are relatively limited. The present study thus investigated RBC aggregation and RBC electrophoretic mobility (EPM) for guinea pigs, rabbits, rats, humans and horses; RBC were re-suspended in isotonic 500 kDa dextran solutions for the EPM and aggregation measurements, with aggregation studies also done in autologous plasma. Salient results included: (1) species-specific RBC aggregation in both plasma and dextran (horse > human > rat > rabbit approximately = guinea pig) with a significant correlation between aggregation in the two media; (2) similar EPM values in PBS for rat, human and horse, a lower value for guinea pig, and a markedly reduced EPM for rabbit RBC; (3) EPM values in dextran with a rank order identical to that for cells in PBS; (4) relative EPM results indicating formation of a polymer-poor, low viscosity depletion layer at the RBC surface (greatest depletion for horse RBC). EPM-aggregation correlations were evident and generally consistent with the Depletion Model for aggregation, yet did not fully explain differences between species; additional studies at various ionic strengths and with various dextran fractions thus seem warranted.     

Inhibition of polymer-induced red blood cell aggregation by poloxamer 188

Previous reports have suggested that non-ionic poloxamer surfactants of appropriate molecular mass and composition can reduce red blood cell (RBC) aggregation in whole blood and in RBC-plasma suspensions. We have thus evaluated this phenomenon for RBC aggregated by several water-soluble polymers, using poloxamer 188 (P188), a non-ionic, tri-block molecule (total molecular mass of 8.40 kDa, 80% polyoxyethylene). Human RBC were washed, then re-suspended in isotonic solutions of dextran 70 (70.3 kDa), dextran 500 (476 kDa), PVP (360 kDa) or P-L-GLU (61.2 kDa); density-separated RBC were also studied. RBC aggregation was quantitated via a computerized Myrenne Aggregometer (extent, strength) and by the Microscopic Aggregation Index (MAI) method. Over the range of 0.5 to 5 mg/ml, poloxamer 188 inhibited both the extent and strength of aggregation in a dose-dependent manner, with the magnitude of the decrease related to polymer type (e.g., at 5 mg/ml, 62% decrease for dextran 70 vs. 14% decrease for P-L-GLU); MAI results with dextran 70 also showed a dose-dependent decrease. Poloxamer 188 at 5 mg/ml was more effective with younger, less-dense cells. Based upon the depletion model for polymer-induced aggregation, these findings suggest that poloxamer 188 acts by penetrating the depletion layer near the glycocalyx, thereby reducing the osmotic gradient between the intercellular gap and the suspending medium. Regardless of the specific mechanism(s) of action, poloxamers appear to offer interesting approaches for future basic science and clinical studies, and thus the possibility for greater insight into RBC aggregation.     

Competitive role between fibrinogen and albumin on thixotropy of red cell suspensions

Plasmatic proteins, namely fibrinogen and globulins, play a major role in red blood cell (RBC) aggregation which is accountable for the three-dimensional structure of blood. Consequently, blood rheological properties linked to this structure must be modified when the protein plasma content changes. This paper gives results and related comments on thixotropic properties of RBC suspensions (0.45 hematocrit) in isotonic solutions containing various amount of fibrinogen to which albumin is added. Thixotropic behavior of these RBC suspensions is studied with a low inertia coaxial cylinders viscometer at a shear rate step of Y = 1 s-1. Rheograms are interpreted in term of thixotropy coefficient. The main conclusion is that albumin improves RBC disaggregability of whole blood, resulting probably from a competitive effect between fibrinogen and albumin in the RBC aggregation process.     

Action of hydroxyethyl starch on the flow properties of human erythrocyte suspensions

Hydroxyethyl starch (HES) has often been used as a plasma expander, but questions still remain concerning the mechanisms by which it produces changes in the rheological properties of blood and erythrocyte (RBC) suspensions under various flow conditions. The present investigation has shown that the dynamic viscosity of HES (232,000 and 565,000 daltons) solutions rises in a nonlinear fashion with increasing HES concentration, and for a given concentration of HES exhibits Newtonian behavior at shear rates between 0.15 to 124 sec-1. At low (less than 0.9 sec-1) shear rates the apparent viscosity of a 40% RBC suspension increases with lower concentrations of HES because of RBC aggregation. At higher concentrations of HES, increases in suspension viscosity are due to an increase in the viscosity of the continuous phase since the RBC are largely disaggregated. At high (greater than 36 sec-1) shear rates the relative viscosity (eta/eta O) of RBC suspensions slowly decreases with increasing HES concentration. At low shear rates eta/eta O increases and then decreases with increasing HES concentration. Evidence of the concentration-dependent effects of HES on RBC aggregation is provided not only by the viscometric analysis but also from measurements of erythrocyte sedimentation rate (ESR) and the zeta sedimentation ratio (ZSR). HES is a more potent aggregating agent in phosphate buffered saline (PBS) than it is in plasma. Polymer size has only a slight effect on the extent of RBC aggregation produced, but does have a significant effect on the concentration of polymer at which maximum aggregation occurs. The viscosity-corrected electrophoretic mobility of RBC in HES rises monotonically with the concentration of HES in the suspending medium. Decreases in the extent of RBC aggregation with increasing polymer concentrations probably result from an increase in the electrostatic repulsive forces between the cells.     

Alteration of Blood Flow in a Venular Network by Infusion of Dextran 500: Evaluation with a Laser Speckle Contrast Imaging System

This study examined the effect of dextran-induced RBC aggregation on the venular flow in microvasculature. We utilized the laser speckle contrast imaging (LSCI) as a wide-field imaging technique to visualize the flow distribution in venules influenced by abnormally elevated levels of RBC aggregation at a network-scale level, which was unprecedented in previous studies. RBC aggregation in rats was induced by infusing Dextran 500. To elucidate the impact of RBC aggregation on microvascular perfusion, blood flow in the venular network of a rat cremaster muscle was analyzed with a stepwise reduction of the arterial pressure (100 → 30 mmHg). The LSCI analysis revealed a substantial decrease in the functional vascular density after the infusion of dextran. The relative decrease in flow velocity after dextran infusion was notably pronounced at low arterial pressures. Whole blood viscosity measurements implied that the reduction in venular flow with dextran infusion could be due to the elevation of medium viscosity in high shear conditions (> 45 s^-1). In contrast, further augmentation to the flow reduction at low arterial pressures could be attributed to the formation of RBC aggregates (< 45 s^-1). This study confirmed that RBC aggregation could play a dominant role in modulating microvascular perfusion, particularly in the venular networks.     

Tangent simple systems method applied to a precise study of viscoelastic behaviour of human blood

The tangent simple systems (TSS) method, proposed in (1), is applied in order to study the viscoelastic behaviour of human blood in transient flow for a rectangular low shear rate step. The tangent simple systems which were used are Maxwell liquids. These systems allow one to obtain plots of variations of instantaneous values of viscosity coefficient mu, elasticity modulus G and retardation time tau = mu/G of the studied blood samples, as a function of flow duration. Variations of both parameters mu and G versus time are represented by two exponential functions which involve three couples of parameters (mu o, mu infinity), (Go, G infinity) and (tau mu, tau G). These parameters can be considered as the characteristics of each blood sample. Another representation of the results, called the dual rheogram, is also indicated. The dual rheogram enables one to follow the evolution of the blood structure. Several examples of application of the TSS method to normal blood sample and to suspensions of artificially modified red blood cells (RBC) are given.     

Cell-cell affinity of senescent human erythrocytes

During their 120-day life span, human red blood cells (RBC) undergo several physicochemical changes, including an increased tendency to aggregate in plasma or polymer solutions. This study was designed to examine potential associations between age-related differences in RBC mobility, aggregation, and membrane glycocalyx properties for cells suspended in buffer and in 3 g/dl solutions of 70.3 kDa dextran. A recent model for depletion-mediated RBC aggregation was employed to calculate the changes of glycocalyx properties that were consistent with experimental electrophoretic mobility (EPM) and aggregation data. Young and old cells were obtained by density separation, after which aggregation and EPM were determined versus ionic strength; old cells exhibited a two- to threefold greater aggregation in dextran. EPM of old cells was identical to young cells in polymer-free media yet was 4% greater in dextran. The greater EPM for old RBC indicates a larger polymer depletion layer, which could be explained either by a 10-15% decrease of their glycocalyx thickness or a similar percentage decrease of polymer penetration into their glycocalyx. The larger depletion layer leads to markedly elevated cell-cell affinities for old cells, with the computed affinity increases consistent with enhanced old RBC aggregation. These results provide a rational explanation for the aggregation and EPM behavior of old RBC, and raise the possibility of depletion-mediated interactions contributing to senescent cell removal from the circulation.     

Dielectric approach to the investigation of erythrocyte aggregation: I. Experimental basis of the method

A method based on dielectric properties of dispersed systems was developed to investigate red blood cell (RBC) aggregation in blood and RBC suspensions. Measurements of capacitance and resistance were made in a rectangular channel at low (0.2 MHz) and high (14 MHz) frequencies relative to the mid-point of the beta-dispersion range. Compared to capacitance, minimal post-shearing changes of resistance were observed; capacitance changes at 0.2 MHz were two orders of magnitude larger than those at 14 MHz and hence subsequent measurements were carried out at the lower frequency. It is shown that post-shearing changes in the capacitance are affected by the recovery of RBC shape and relaxation processes at the electrode-suspension interface. However, the dominant factor contributing to time-dependent changes in the capacitance is the dynamic process of RBC aggregation. It is experimentally shown that the time record of the capacitance at 0.2 MHz quantitatively reflects the aggregation process in RBC-plasma suspensions with hematocrit up to 0.56 (v/v) and in suspensions of RBCs in artificial aggregating media. It is concluded that a dielectric approach to the study of RBC aggregation in whole blood offers great potential for basic studies and for diagnostic use.     

Conductometric study of shear-dependent processes in red cell suspensions. II. Transient cross-stream hematocrit distribution

A novel experimental approach based on electrical properties of red blood cell (RBC) suspensions was applied to study the effects of the size and morphology of RBC aggregates on the transient cross-stream hematocrit distribution in suspensions flowing through a square cross-section flow channel. The information about the effective size of RBC aggregates and their morphology is extracted from the capacitance (C) and conductance (G) recorded during RBC aggregation, whereas a slower process of particle migration is manifested by delayed long-term changes in the conductance. Migration-induced changes in the conductance measured at low shear rates (< or =3.1 s(-1)) for suspensions of RBCs in a strongly aggregating medium reveal an increase to a maximum followed by a decrease to the stationary level. The ascending branch of G(t) curves reflects the aggregate migration in the direction of decreasing shear rate. A further RBC aggregation in the region of lower shear stresses leads to the formation of RBC networks and results in the transformation of the rheological behavior of suspensions from the thinning to the thickening. It is suggested that the descending branches of the G(t) curves recorded at low shear rates reflect an adjustment of the Hct distribution to a new state caused by a partial dispersion of RBC networks. For suspensions of non-aggregating RBCs it is found that depending on whether the shear rate is higher or lower compared with the prior value, individual RBCs migrate either toward the centerline of the flow or in the opposite direction.     

Observed differences in dextran and polyvinylpyrrolidone as rouleaux-inducing agents

The effectiveness of dextran fractions (Dx-500, Dx-100, Dx-70) and polyvinylpyrrolidone (PVP-360, PVP-40) in inducing aggregation of red blood cells (RBC) was studied in a nonflowing environment. The Dx fractions, at low concentrations, induced aggregation of human RBC but failed to do so at high concentrations (concentrations greater than 70 g/L). The effect was different on RBC from animal species (cat and rabbit); aggregation increased steadily with the Dx concentration and there was no critical concentration beyond which Dx failed to induce aggregation. The PVP was found to be very effective, at all concentrations, in inducing aggregation of RBC from both human and the animal species. These results have a twofold significance (1) they suggest that Dx and PVP, both neutral polymers, interact differently with the human RBC membrane; and (2) the association of Dx with the human RBC membrane is different from that with cat and rabbit RBC membranes.     

Conductometric study of shear-dependent processes in red cell suspensions. I. Effect of red blood cell aggregate morphology on blood conductance

The conductance and capacitance of flowing and quiescent red blood cell (RBC) suspensions were measured at a frequency of 0.2 MHz. The results demonstrate that the time-dependent changes in the conductance recorded during the aggregation process differ in nature for suspensions of short linear rouleaux, branched aggregates and RBC networks. It is shown that the conductance of RBC suspensions measured during the aggregation and disaggregation processes follows the morphological transformations of the RBC aggregates. Thus, this method enables characterization of the morphology of RBC aggregates formed in whole blood and in suspensions with physiological hematocrits both under flow conditions and in stasis. These results in combination with previous ones suggest that this technique can be used for studies of dynamic RBC aggregation and probably for diagnostic use.     

Anionic contribution for fibrous maturation of protofibrillar assemblies of the human tau repeat domain in a fluoroalcohol solution

Tau protein forms fibrous aggregates in the brain of patients with Alzheimer's disease. This type of aggregation in vitro is promoted efficiently by polyanions and anionic micelles. Here, we report another cosolvent system that induces the fibrous aggregation of human tau four-repeat domain (tau4RD). The protein aggregation was primarily achieved by a nonanionic agent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), while the ionic condition was modified by inorganic salts. The aggregation analysis by three spectroscopic methods revealed a two-phase kinetics of the aggregation of tau4RD in the presence of HFIP at approximately 4-6%. Large increases in the light-scattering, the thioflavin-binding, and the secondary structure content of tau4RD have progressed within a few minutes at 37 degrees C, which was followed by another slower aggregation phase. Electron microscopic analysis demonstrated that the amorphous granules are formed in the faster step, which acquired a fibrous shape in the slower step in the solution containing NaCl. In the absence of the salt, however, the fibrous maturation was inhibited. Examination of various salt species in place of NaCl demonstrated that binding of anions to the precursor aggregates was essential for the fibrous maturation. On the basis of the results, we proposed an aggregation scheme of tau in which the formation of a thioflavin-binding intermediate occurred ahead of its fibrous maturation. The anionic environment was suggested to play a crucial role in the fibrous maturation and, therefore, could be an in vivo determinant of the morphology of the aggregates of tau.     

Effects of dextran molecular weight on red blood cell aggregation

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biologic and biophysical interest, yet the mechanistic details governing the process are still being explored. Although a depletion model with osmotic attractive forces due to polymer depletion near the RBC surface has been proposed for aggregation by the neutral polyglucose dextran, its applicability at high molecular mass has not been established. In this study, RBC aggregation was measured over a wide range of dextran molecular mass (70 kDa to 28 MDa) at concentrations ≤2 g/dL. Our results indicate that aggregation does not monotonically increase with polymer size; instead, it demonstrates an optimum dextran molecular mass around 200-500 kDa. We used a model for depletion-mediated RBC aggregation to calculate the expected depletion energies. This model was found to be consistent with the experimental results and thus provides new insight into polymer-RBC interactions.     

Size determination of red blood cell aggregates induced by dextran using ultrasound backscattering phenomenon.

Different methods are commonly used to study the red blood cell aggregation phenomenon. The major interest of the ultrasonic method presently discussed is to assess the mean size of red blood cell (RBC) aggregates by measuring ultrasonic intensity backscattered by blood. Applying Rayleigh theory of sound to blood medium, one can show that the scattered ultrasonic intensity is proportional to the 6th power of the size of the RBC aggregates. The ultrasonic method is used to evaluate the mean size of RBC aggregates induced by dextrans. RBCs are suspended at various hematocrits H, in solution of dextrans of different molecular weights M and at different weight concentrations Cw. Results are presented by using the ultrasonic backscattering coefficient chi which is a relevant quantity in a scattering experiment. For suspensions of RBCs aggregated with dextran of molecular weight 70,000 dalton (dextran 70) at concentration Cw = 40 g/l, variations of chi as a function of H are similar to those obtained for normal blood. At a fixed hematocrit, variation of chi versus Cw for dextran 70 exhibits a maximum at 40 g/l. In the case of RBCs suspended at hematocrit 20% and aggregated with dextrans of molecular weight M, 70,000 less than or equal to M less than or equal to 2,000,000, the variations of chi versus molar concentration Cm are similar to those of the microscopic aggregation index defined by Chien (1). Finally, a statistical model of the blood structure previously described (2) is applied to evaluate the mean size of the aggregates. According to this model, the mean size of aggregates is independent of hematocrit for H less than or equal to 40% and independent of the molecular weight of dextran for M greater than or equal to 150,000 dalton.     

Red blood cell aggregation quantitated via Myrenne aggregometer and yield shear stress

Although the study of red blood cell (RBC) aggregation continues to be of basic science and clinical interest, aggregation standards for calibration do not exist, and most aggregation studies report data in terms of arbitrary units: quantitative comparisons between studies are thus essentially precluded. However, use of low shear viscometry plus the Casson equation provides a yield shear stress that has defined units and is known to reflect RBC aggregation. Employing human RBC-plasma suspensions exhibiting a wide range of aggregation, the present study examined relations between yield shear stress values and aggregation indices obtained using the Myrenne aggregometer: the latter approach uses a light-transmission technique and provides an "M" index at stasis and an "M1" at very low shear. Our results for normal controls and for angina patients without coronary artery disease indicate highly significant correlations (p<0.001) between the yield stress and both M and M1. Thus, within the range of aggregation studied, these findings lend support to the rheological validity of the Myrenne approach; extension of our findings to intensely aggregating RBC suspensions may require additional validation studies.     

Aggregation of human RBC in binary dextran-PEG polymer mixtures

The present study was prompted by prior reports suggesting that small polymers can affect RBC aggregation induced by large macromolecules. Human RBC were washed and re-suspended in isotonic buffer solutions containing 72.5 kDa dextran (DEX 70, 2 g/dl) or 35.0 kDa poly(ethylene glycol) (PEG 35, 0.35 g/dl), then tested for aggregation in these solutions with and without various concentrations of smaller dextrans (10.5 and 18.1 kDa) or PEGs (3.35, 7.5 and 10.0 kDa). RBC aggregation was measured at stasis and at low shear using a photometric cone-plate system (Myrenne Aggregometer) and RBC electrophoretic mobility (EPM) in the various polymer solutions via an automated system (E4, HaSoTec GmbH). Our results indicate: (1) a heterogeneous effect with greater reduction of aggregation for small PEGs added to DEX 70 or for small dextrans added to PEG 35 than for small polymers of the same species; (2) for cells in DEX 70, aggregation decreased with increasing molecular mass and concentration of the small dextrans or PEGs; (3) for cells in PEG 35, small dextrans decreased aggregation with increasing molecular mass and concentration, whereas small PEGs had minimal effects with a minor influence of concentration and an inverse association between molecular mass and inhibition of aggregation. RBC EPM results indicated the expected polymer depletion for cells in DEX 70 or PEG 35, and that small PEGs yielded greater EPM values than small dextrans for cells in PEG 35 whereas the opposite was true for cells in DEX 70. Interpretation of our results in terms of the depletion model for RBC aggregations appears appropriate, and our findings are consistent with the assumption that inhibition of aggregation occurs because of an increase of small molecules in the depletion region. Our results thus suggest the merit of further studies of red blood cell aggregation in binary polymer systems.     

Modulation of endothelial nitric oxide synthase expression by red blood cell aggregation

The effects of enhanced red blood cell (RBC) aggregation on nitric oxide (NO)-dependent vascular control mechanisms have been investigated in a rat exchange transfusion model. RBC aggregation for cells in native plasma was increased via a novel method using RBCs covalently coated with a 13-kDa poloxamer copolymer (Pluronic F-98); control experiments used RBCs coated with a nonaggregating 8.4-kDa poloxamer (Pluronic F-68). Rats exchange transfused with aggregating RBC suspensions demonstrated significantly enhanced RBC aggregation throughout the 5-day follow-up period, with mean arterial blood pressure increasing gradually over this period. Arterial segments ( approximately 300 microm in diameter) were isolated from gracilis muscle on the fifth day and mounted between two glass micropipettes in a special chamber equipped with pressure servo-control system. Dose-dependent dilation by ACh and flow-mediated dilation of arterial segments pressurized to 30 mmHg and preconstricted to 45-55% of the original diameter by phenylephrine were significantly blunted in rats with enhanced RBC aggregation. Both responses were totally abolished by nonspecific NO synthase (NOS) inhibitor (Nomega-nitro-l-arginine methyl ester) treatment of arterial segments, indicating that the responses were NO related. Additionally, expression of endothelial NOS protein was found to be decreased in muscle samples obtained from rats exchanged with aggregating cell suspensions. These results imply that enhanced RBC aggregation results in suppressed expression of NO synthesizing mechanisms, thereby leading to altered vasomotor tonus; the mechanisms involved most likely relate to decreased wall shear stresses due to decreased blood flow and/or increased axial accumulation of RBCs.     

Physical and chemical effects of red cells in the shear-induced aggregation of human platelets.

Both chemical and physical effects of red cells have been implicated in the spontaneous aggregation of platelets in sheared whole blood (WB). To determine whether the chemical effect is due to ADP leaking from the red cells, a previously described technique for measuring the concentration and size of single platelets and aggregates was used to study the shear-induced aggregation of platelets in WB flowing through 1.19-mm-diameter polyethylene tubing in the presence and absence of the ADP scavenger enzyme system phosphocreatine-creatine phosphokinase (CP-CPK). Significant spontaneous aggregation was observed at mean tube shear rates, (G) = 41.9 and 335 s-1 (42% and 13% decrease in single platelets after a mean transit time (t) = 43 s, compared to 89 and 95% decrease with 0.2 microM ADP). The addition of CP-CPK, either at the time of, or 30 min before each run, completely abolished aggregation. In the presence of 0.2 microM ADP, CP-CPK caused a reversal of aggregation at (t) = 17 s after 30% of single cells had aggregated. To determine whether red cells exert a physical effect by increasing the time of interaction of two colliding platelets (thereby increasing the proportion of collisions resulting in the formation of aggregates), an optically transparent suspension of 40% reconstituted red cell ghosts in serum containing 2.5-micron-diameter latex spheres (3 x 10(5)/microliters) flowing through 100-microns-diameter tubes was used as a model of platelets in blood, and the results were compared with those obtained in a control suspension of latex spheres in serum alone. Two-body collisions between microspheres in the interior of the flowing ghost cell or serum suspensions at shear rates from 5 to 90 s-1 were recorded on cine film. The films were subsequently analyzed, and the measured doublet lifetime, tau meas, was compared with that predicted by theory in the absence of interactions with other particles, tau theor. The mean (tau meas/tau theor) for doublets in ghost cell suspensions was 1.614 +/- 1.795 (SD; n = 320), compared to a value of 1.001 +/- 0.312 (n = 90) for doublets in serum. Whereas 11% of doublets in ghost cell suspensions had lifetimes from 2.5 to 5 times greater than predicted, in serum, no doublets had lifetimes greater than 1.91 times that predicted. There was no statistically significant correlation between tau meas/tau theor and shear rate, but the values of tau meas/tau theor for low-angle collisions in ghost cell suspensions were significantly greater than for high-angle collisions.     

Cellular determinants of low-shear blood viscosity

Low-shear viscometry is one of the methods commonly used to estimate the degree of red blood cell (RBC) aggregation in various bloods and RBC suspensions. However, it has been previously shown that alterations in RBC morphology and mechanical behavior can affect the low-shear apparent viscosity of RBC suspensions; RBC aggregation is also sensitive to these cellular factors. This study used heat treatment (48°C, 5 min), glutaraldehyde (0.005–0.02%) and hydrogen peroxide (1 mM) to modify cell geometry and deformability. Red blood cell aggregation was assessed via a Myrenne Aggregometer (“M” and “Ml” indexes), RBC suspension viscosity was measured using a Contraves LS-30 viscometer, and RBC shape response to fluid shear stresses (i.e., deformability) was determined by ektacytometry (LORCA system). Our results indicate that low-shear apparent viscosity and related indexes may not always reflect changes of RBC aggregation if cellular properties are altered: for situations where RBC aggregation has been only moderately affected, cellular mechanical factors may be the major determinant of low-shear viscosity. These findings thus imply that in situations which may be associated alterations of RBC geometry and/or deformability, low-shear viscometry should not be the sole measurement technique used to assess RBC aggregation.