Clinical Hemorheology and Microcirculation

The weak aggregation of red cells, referred to as rouleaux formation, has been explained by the non-specific adsorption of plasma proteins to these cells in a bridging configuration. However, no unequivocal measurement of sufficient protein adsorption to cause aggregation has been reported. Aggregation induced by a depletion mechanism which requires no protein adsorption is also possible. No definitive information is currently available which proves either model but we find little support for the bridging hypothesis in either our own work or in that of others.

This paper deals with the effect of albumin and immunoglobulins on erythrocyte aggregation parameters. The study was carried out with an automatic system based on the light reflectometry technique which allows to determine kinetic parameters and dissociation shear rates. The plasma substitution by physiological solution or albumin showed the role of albumin on dissociation shear rates. Albumin properties were altered by heating at 56–60°C. In presence of immunoglobulins, the role of albumin appeared to be complex, depending on the Alb/Glo ratio.

The present study was designed to extend our prior observations of RBC aggregation (Clinical Hemorheology 7, 93–108, 1987) in order to examine the electrokinetic behavior of age-separated RBC in various media, aggregation behavior of rat and bovine RBC in plasma, dextran and PVP, and donor-specific differences in human RBC aggregation. Our results indicate: 1) age-separated human RBC exhibit identical mobilities in PBS or in albumin, whereas older RBC have significantly (p<0.02) greater mobilities than younger cells in plasma or serum; 2) extracellular calcium reduces equally the mobility of young and old human RBC and increases equally the aggregation of both cell populations; 3) markedly reduced aggregation for unfractionated rat RBC in dextran T70, greater aggregation for old rat RBC in T70 yet a more negative mobility for these old cells, and no aggregation for bovine RBC in plasma, dextran (T70, T500, T2000) or PVP-360. Of particular interest were donor-specific differences for thrice-washed human RBC suspended in dextran T70; 1) cells exhibit a > two-fold range of aggregation between donors unrelated to ABO blood group or cell volume; 2) significant (p<0.05) correlations were observed between RBC aggregation behavior in autologous plasma and T70, thus suggesting possible in vivo conditioning of the cells. In overview, our current results further emphasize the importance of erythrocyte factors in RBC aggregation and suggest that when evaluating situations associated with abnormal aggregation, possible contributions of cellular as well as suspending media factors need to be considered.

A biological evaluation of the erythroaggregameter commercialized by SEFAM (Nancy, France) is reported. The main characteristics of the apparatus are: Couette-type viscometer, laser light source (wavelength near infra-red), analysis of back-scattered light Several quantitative parameters are calculated: aggregation times, partial and total dissociation thresholds. Reproductibility and variance are satisfactory. The method is very dependent on the experimental conditions such as: delay between sampling to measurement, temperature, nature of anticoagulant. We confirm the importance of both hematocrit and fibrinogen. There is a sex related difference of aggregation parameters which is not dependent on hematocrit. At last, we have found that erythroaggregameter was able to detect RBC damages induced by either moderate heating or surface charge alterations.

The process of red blood cell (RBC) aggregation is always associated with its counterpart: phase separation. The possibility of predicting the occurrence of a specific hemodynamic effect produced by these two processes in the living vasculature is frequently limited by insufficient information on the extremely complex set of local flow conditions.

The aggregation phenomenon is an important hemorheological factor which influences blood flow in human capillary bed. Aggregation can be evaluated by a number of methods but none of them can be applied simply to in vivo measurements. In contrast, use of ultrasound waves could allow measurements of mechanical and structural properties of red blood cell (RBC) suspensions as well in vitro as in vivo. To characterize more precisely and quantitatively the aggregation phenomenon, an ultrasonic method based on quantitative echography is used. The aim of this paper is to present effects of some parameters (hematocrit, dextran concentration. RBC electrical charge, shear stress) on RBC aggregation studied by ultrasonic echography. Sickle cell aggregation is also considered.

Linking between blood viscosity and erythrocyte aggregation is interpreted as resulting of structural changes of red blood cell aggregates and their clusters, in the absence of blood flow. Limits of applying such an interpretation is discussed with the help of few examples.

Because blood is a non-Newtonian fluid, its energy dissipation during flow is more complex than that of Newtonian fluids. We have shown that, when flow is reinitiated after a pause comparable to physiologic diastole, that a substantial extra transient shear stress develops. This briefly increased resistance is related in shape and duration to blood thixotropy. Both are linked through their dependence on total shear strain (shear rate × time) and its associated rapidly increasing orthogonal reflectivity to the development of cell orientation as flow is initiated and restored. Studies of blood from diabetic patients suggest that a greater predisposition to aggregation of red blood cells mediated by fibrinogen and other plasma proteins influences the magnitude of this transient resistance. The timing and magnitude of blood’s transient resistance are such that, combined with blood’s shear-thinning, it burdens cardiac systole in the large arteries by an increase as great as ten percent and with a brief local peak excess of more than a third.

RBCs aggregation plays a very important role at the microcirculatory level. In order to have a better understanding of the different parameters involved, we first presented results obtained in glass capillaries. The existence of a plasmatic layer and of a yield shear stress and the effect on the viscosity of RBCs suspensions has been proved. We then after presented results obtained on rats when the RBCs aggregation level was modified. The increase of the hydraulic resistance, important modifications of the systemic blood pressure, of the cardiac frequency and of the electrocardiogram were observed. Effect of troxerutine were studied.

Compaction stasis, which occurs in certain diseases and conditions, is generally considered to be without increased capillary or vascular permeability. In inflammation, however, compaction stasis occurs together with increased capillary permeability for which the term ‘Cohnheim compaction phenomenon’ was introduced. Our studies of whole human blood with hematocrits from 60 to 96 per cent have been made, since reversible compaction stasis occurs with high concentrations of erythrocytes. Measurements of blood with these high hematocrits in whole human blood, employing the cone and plate geometry of the Weissenberg Rheogoniometer, showed that in steady shear normal stress was present an that at very low shear rates plug flow was indicated. Experiments in oscillatory flow showed and elastic modulus. A brief survey of studies related to compaction stasis and the Cohnheim compaction phenomenon is given. The survey is also concerned with acute, sterile inflammation, in which the erythrocytes are aggregated or reversibly clumped. A differentiation is made between reversible and irreversible compaction stasis. In reversible compaction stasis, the erythrocytes are aggregated or reversibly clumped and thus can desaggragate. Irreversible compaction stasis, in which the erythrocytes are irreversibly clumped or agglutinated, may be associated with infections. The affected vessel walls and erythrocyte agglutinates may then contain microorganisms and/or their products. We found experimental evidence that in conditions of reversible compaction stasis, in which increased capillary permeability was not suspected, such as in polycythemia, shock and mountain sickness, increased focal capillary permeability can occur, indicating the Cohnheim compaction phenomenon.

We studied hematocrit, blood and plasma viscosity as well as the erythrocyte aggregation index in a group of 55 healthy newborns and in 16 mothers without risk factors for neonatal diseases. 39 healthy adults were considered as control group. We found that the newborns showed a significantly lower erythrocyte aggregation index compared to that of the control group, whereas their hematocrit was significantly higher. The healthy newborns also showed very low plasma viscosity and plasma fibrinogen level compared to those of the adults. The peculiar hemorheologic pattern of the healthy newborn, although rather different from that of the healthy adult, may be considered as “near normal”.

Viscoelasticity of whole blood and plasma viscosity have been measured with an oscillating capillary rheometer routinely in patients with arteriosclerotic risk factors. Significantly higher values of the hemorheologic parameters are found in such patients. No correlation to other risk factors (hypertension, diabetes, hyperlipemia, smoking) could be found. Long term treatment of the patients with hemorheologically active drugs can induce a decrease of the pathologically increased viscoelastic parameters which become significant after three months of treatment. The response of the patients to different drugs varies individually, therefore, each patient and each drug should be checked during the treatment continuously.

We evaluated the red cell membrane dynamic property and the mean erythrocyte aggregation (MEA) in vascular atherosclerotic disease (VAD). Our study includes two group of subjects with VAD. In the first group the ghosts were marked with DPH and the membrane microviscosity was calculated from the polarization degree. In the second group the red cell membrane fluidity was evaluated on intact red cells using the fluorescent label pyrene and calculating the Iex/Im ratio; in this group the subjects were subdivided according to the mono (MVAD) or polivascular (PVAD) lesions. From the obtained data the different trend of the membrane microviscosity and of the membrane fluidity is evident; more homogeneous is the behaviour of the MEA that in VAD is significantly increased. No significant correlations are evident between and MEA and between Iex/Im and MEA. Interesting the findings regarding the relationships between the rheological determinants and some individual phospholipids.

In this study, hematocrit, plasma viscosity, fibrinogen, albumin, red cell aggregation and disaggregation threshold were measured on 20 control subjects and on 438 diabetic patients with and without vascular complications, with good and poor metabolic control. The rheological disturbances were characterized by a significant increase of red cell aggregation tendency at low shear rate, of red cell disaggregation thresholds, and by a significant increase of fibrinogen level and decrease of albumin level. The rheological disturbances observed may confirm the role of rheological phenomena in the genesis of diabetic microangiopathy.

Erythrocyte adhesion to endothelial cells was measured using human umbilical vein endothelial cells in culture and a radiometric method. Erythrocyte adhesion was found to be increased in a group of twelve diabetics as compared to normal. The increase in adhesion was statistically significant (p<0.001) and was modified by the addition of Troxerutine in vitro. Troxerutine at a concentration of 10⁻⁶M impaired the abnormal erythrocyte adhesion observed in diabetics but from these results we cannot extrapolate the in vivo effect of the drug.

Blood rheological abnormalities play an important role in venous pathology as does varicose vein disease. In this study the essential microrheological parameters (hematocrit, plasma viscosity, fibrinogen, erythrocyte aggregation and deformability) were tested on 20 control subjects and 20 patients having varicose veins in the legs. The measurements were taken on samples collected at an antecubital vein, on leg veins before and after 30 minutes of stasis. Those patients having varicose veins of the lower limbs showed aggravated rheological disturbances after stasis. In the veins, where the blood flow rate is the lowest, the erythrocyte aggregation appeared as the fundamental parameter associated with the alteration of red cell deformability and with the increase of plasma viscosity.

Rheological factors appear to be of a great importance in Venous Insufficiency. They are related to several biological disorders, as haemoconcentration and imbalance of plasma proteins, and are markedly increased in varicose and/or altered veins. Erythrocyte hyperaggregation is thought to be the most interesting physiopathological disturbance, related, in such patients, to alteration of thyxotropic properties of the blood, as well as structural defects of low flow forming red blood cells aggregates. Troxerutine, a potent drug on vessel wall, exhibits in patients a direct action on red cell aggregation, particularly marked at high doses. The beneficial effects of troxerutine should therefore be exerted via actions on vessel wall and red blood cells.

Erythrocyte aggregation was studied in patients with dysglobulinemia. By using a light reflectometry technique, it was demonstrated an hyperaggregation state in all patients. Following plasma exchange, it was demonstrated an improvement of aggregation. However, the extent of aggregation parameter variations was not identical in all cases, suggesting that patients may react differently to plasma exchange.