Clinical Hemorheology and Microcirculation

The red cell electrophoretic mobility test by Field and Joyce was used as an adjunct in the diagnosis of 820 clinically acceptable cases of multiple sclerosis. In 90% of the cases the mobility of the red cells was consistent with MS (87% in early and 93% in later cases). In 5% of 109 normal subjects the mobility was consistent with MS. In 4 of 8 cases of Amyotrophic Lateral Sclerosis, in 1 of 7 cerebral infarctions, and in 1 irradiated tumor of 3 brain tumors, the mobility was consistent with MS. The related sensitivity of MRI and red cell electrophoretic mobility to MS was compared. The relative sensitivity of the MRI and Mobility tests for MS was compared.

We observed significant cell swelling in the Ringer's and tris based suspension media which can interfere with filtration studies of red cell deformability. Mean corpuscular volume (MCV) in Ringer's solution increased from 95.6±2.5 fl to 109.2±2.5 fl (N=5,P<0.001) and from 95.4 fl±3.4 fl to 109.9±2.5 fl (N=5,P<0.005) in tris-saline within 2 hours of preparation of the cell suspension. These alterations in cell volume were sufficient to cause significant increases in filtration time when using 3.0 um polycarbonate filters. At 30 mm Hg pressure gradient, Ringer's suspensions showed a three-fold increase in filtration time from 11.48±1.6 sec to 32.71±6.8 sec (N=5,P<0.002), and in tris-saline, there was a six-fold increase in filtration time from 11.22±0.58 sec to 72.0±14.0 sec (N=5,P<0.001). At 45 mm Hg, in Ringer's suspensions, filtration time increased from 9.03±1.40 sec to 19.48±1.31 sec (N=5,P<0.001). These increases occured independent of hemolysis. Spontaneous cell swelling was prevented by using suspensions prepared with magnesium chloride and phosphate buffered saline. These experiments reveal that spurious changes in cell volume occur during filtration experiments using Ringer's and tris based suspension media and can confound the interpretation of deformability studies.

The aim was to define hemorheological changes (and their kinetics) in a non-exercising group of subjects after exposure to moderately high altitude of 2700m. 12 men were transported to this height without marked physical exercise and remained there for 6 days. During this time there was a significant increase in blood and plasma viscosity as well as fibrinogen. Hematocrit, blood cell counts and ESR did not change significantly. All alterations were reversible upon return to normal height. These findings suggest that a viscidation of blood occurs as a result of exposure even to moderate height.

Changes in the circulatory behaviour of white cells may contribute to the progression of tissue ischaemia. To investigate whether the therapeutic effects of pentoxifylline might result from an inhibition of white cell activation and rheological changes, we measured the filterability of cell suspensions derived from patients with critical leg ischaemia, before and after infusion of 600mg of this agent. Activation was also assessed, using morphological criteria and by measuring plasma levels of granulocyte elastase. It was found that the flow rate of mononuclear cells through 8 μm pore filters was increased after treatment, but no significant changes were found for granulocytes or unfractionated white cells. Nor were there any changes in the number of cells judged to be active or in plasma elastase. However, it was noticeable that those patients with worse granulocyte flow or higher levels of activation did tend to show improvement after treatment. Thus there is some evidence of an in vivo affect of pentoxifylline on white cell behaviour, possibly made evident in this study by its ability to reduce the cell response to the process of sample preparation.

Blood cell deformability, most often measured as filterability through a polycarbonate filter, is a major determinant of the rheological properties of whole blood. In this study the influence of the suspension medium on the filterability of red cells was studied. We found a considerable variation in filterability between suspensions of red cells in media of different kinds. Thus the cells were suspended in phosphate buffered saline (PBS), pH 7.4, alone, in PBS with glucose 0.9 g/l, in PBS with bovine serum albumin (BSA) 2.5, 5.0 and 50 g/l, in PBS with BSA 50 g/l, fibrinogen 0.02 g/l and cholesterol 0.025 g/l, PBS with human plasma 19:1 (i.e. 1 ml plasma added to 19 ml of PBS) and in plasma alone. Photographs of cells in different suspension media were taken in the microscope. The filterability of red cells in PBS was greatly increased by addition of BSA and also the reproducibility was increased, especially at low concentrations of BSA. Red cells suspended in PBS alone, gave visually grossly distorted appearance rut the addition of BSA or human plasma, normalized the morphology of the cells. With human plasma, however, difficulties in reproducing the results were found. It is concluded that the choice of buffer medium is a critical point in blood cell filtration studies. The present study indicates that phosphate buffered saline with low concentration of bovine serum albumin might be a suitable choice.

Hemorheologic studies using clinical filtration pressure techniques have suggested that changes in erythrocyte (RBC) deformability occur during left ventricular assist system (LVAS) support. In order to assess whether changes suggested by clinical filtration pressure were associated with alterations in the actual material properties of the membrane complex, micropipette aspiration experiments were performed during NOVACOR LVAS support ranging between 28 and 126 days in four patients. The micropipette aspiration method provides a direct measurement of the surface shear elastic modulus μ of the RBC membrane complex. The modulus consistently fell within the range reported for normals, and no apparent trend with time was evident. In one instance, however, an abnormal spike in μ, which paralleled clinical filtration data, coincided with symptoms of a transient neurologic event. For the LVAS patients studied, clinical abnormalities in hemorheology as indicated by filtration were not attributed to changes in μ.

There is in vivo evidence that dextrans have antiplatelet effect. We have performed a series of experiments to elucidate a possible hemorheological action through which dextrans might alter platelet function. A perfusion system allowing morphometric evaluation of platelet interaction with subendothelial structures was used for this purpose. Perfusion experiments were performed in the presence of dextran 40 or 70 (10% v:v) or with blood reconstituted with dextran-incubated red blood cells. Experiments in which blood or red blood cells were treated with the same proportion of 4% human serum albumin (HSA) were used as a control. Changes in the mechanical properties of red blood cells were morphologically assessed with a micropipette aspiration technique. None of the dextrans used modified the mechanical properties of red blood cells. However, both dextran 40 (D40) and dextran 70 (D70) increased the intensity of the interaction of platelets with subendothelial structures when added to whole blood. Our experiments suggest that when added in vitro dextrans do not alter the hemorheological mechanisms of platelet function.

The effects of idebenone (6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone) on hemorheologic variables were evaluated in 7 geriatric patients with cerebral infarction, 3 men and 4 women (ages 61–83, mean age 70.7 years). Idebenone was given orally at a dose of 30–60mg three times daily after meals for 12 weeks. The hemorheologic variables were determined twice prior to the administration and at 8 and 12 weeks after the administration. Hemorheologic measurements included erythrocyte deformability was determined according to the method based on the examination of passing erythrocytes through a filter containing pores smaller than the undeformed cells. Erythrocyte aggregation was measured by the method based on the increase of the light transmission through whole blood that occurs when individual cells aggregate into rouleaux or rouleaux-rouleaux complexes. Erythrocyte deformability was increased and erythrocyte aggregation measured at a high shear rate and a low shear rate, plasma viscosity and collagen-induced platelet aggregation were also decreased after the administration. Thus the improvement of hemorheologic variables in cerebral infarction may prevent from disturbing blood flow and may contribute to prevention of formation and progression of thrombosis and atherosclerosis in geriatric patients with cerebral infarction.

It has been demonstrated that iron and iron compounds facilitate hydroxyl radical generation from activated oxygen species. Free haemoglobin (Hb), like other iron-rich substances, might also mediate hydroxyl radical production and induce lipid peroxidation and cell damage.

On the basis of the hypothesis that Hb may influence circulating blood cells, namely red cells, studies have been carried out on the effect of Hb on rheological properties of whole blood.

Hb is capable of significantly influencing whole blood filterability and, to a lesser extent, whole blood viscosity. The addition of Fe²⁺ has a potentiating effect; superoxide dismutase and thiourea which are respectively selective inhibitors of superoxide anion and hydroxyl radicals are able to antagonize the Hb-induced rheological impairment.

From these observations, the hypothesis may suggest that Hb negatively influences some rheological properties of whole blood by producing oxygen free radicals. Hb released in vivo at the site of arterial stenosis and of atherosclerotic plaque rupture may induce a rheological impairment and, so doing, play a role in microcirculatory derangement.

Bolus infusion of human leukocytes (Polys and/or Monos) during continuous, constant pressure filtration of 5 μm pore (Nuclepore^®) filters was performed in order to calculate cell passage time distributions. The ratio cell to pore number was kept small (about 0.4) to prevent clogging of the filters and to simplify the analyses of the flow recordings that were made continuously with an ultrasonic transit time flow probe.

In a first step the flow curves were simply inverted to show the changes in resistance during and after cell filtration. The resistance change in the filters due to the passage of the leukocyte bolus was similar, both in shape and magnitude, to that seen earlier in isolated organs perfused with a leukocyte bolus. Thus, there was an initial peak in the resistance followed by a slow decline towards a plateau value 5–20% above the baseline. In addition, the results showed that permanent plugging of the filters was inversely related to the driving pressure (25, 50 or 100 mm H₂O), which also correlates well with the results from organ perfusion experiments. The percentage of cells that became permanently trapped in the pores differed between Polys and Monos only at the lowest pressure (35% Monos and 25% Polys).

The dynamics of the bolus' approach to and contact with the filter was investigated using glutaraldehyde fixated (rigid) Polys, and then used to de-convolute the flow recordings from the normal cells in order to obtain their passage time distributions. In these analyses, it appeared that the normal leukocytes could be divided into a number of sub-populations with passage times ranging from 0 to infinity. When pressure was changed there was a shift of cells between these sub-populations with the sub-population with infinite passage time being greatest at the lowest pressure. This finding indicates that the importance of the viscous element of the leukocytes, which has been regarded to be the most significant determinator of leukocyte rheology, must be re-evaluated. The present results suggest that cell properties such as adhesiveness and elasticity influence leukocyte rheology to a greater extent than has hitherto been recognized.

Micropipettes with internal diameters of 1 to 5 μm are frequently used for manipulation of erythrocytes and leukocytes. A modified method for the production of narrow micropipettes with flat tips is described. A stainless steel rod with a cylindrical bore hole is filled with low-melting point solder glass, which is rapidly melted by a high electrical current. The tip of a micropipette of desired diameter is moved into the softened glass, the electrical current is interrupted and the glass is chilled. When the pipette is removed it usually breaks off with a flat tip.

A set of experimental data was previously obtained from blood of healthy controls and insulin-treated diabetics clinically divided into three different groups: diabetics without retinopathy, diabetics with minimal retinopathy and diabetics with severe retinopathy (1). Results of the multivariate analysis of both biological and rheological data suggest the presence in diabetic plasma of two kinds of fibrinogen in contrast with normal plasma.

Hemorheological profile of a group of menopausal female subjects have been studied. Parameters such as whole blood viscosity, plasma viscosity, hematocrit, red cell rigidity and biochemical values such as plasma fibrinogen, proteins are evaluated. Females in premenopausal phase are taken as controls. Menopausal women showed significantly high whole blood viscosity, plasma viscosity and red cell rigidity. There was no significant rise in hematocrit and plasma fibrinogen. The results suggest that the menopausal phase plays important role in altering the rheology of blood. This change in rheology can be a potential risk factor in the development of ischaemic heart diseases.

Glycated erythrocyte membrane proteins and some metabolic parameters (plasma glucose profile, glycosuria, glycated haemoglobin, glycated serum proteins, cholesterol, triglycerides) were evaluated in 35 subjects, that is, 20 insulin dependent diabetics and 15 controls, matched for sex and age. Whole blood and washed erythrocyte filtrability, whole blood, - washed erythrocyte - and plasma viscosity were evaluated in all subjects. Glycated erythrocyte membrane proteins were significantly higher in diabetics with respect to controls (10.6±0.4 vs 4.3±0.2 nmlHMF/mg prot;p<0.001) and correlated with all the metabolic parameters considered. Significantly lower values of whole blood and washed erythrocyte filtrability (2.0±0.13 vs 2.4±0.13 ml/min,p<0.01; and 5.85±0.11 vs 6.58±0.18 ml/min,p<0.001) and significantly higher values of washed erythrocyte viscosity were found in diabetics with respect to controls (2.20±0.05 vs 2.03±0.04 cP,p<0.02). Glycated erythrocyte membrane proteins were inversely related to washed erythrocyte filtrability in diabetic patients. A demonstrable effect on erythrocyte filtrability is caused by the degree of glycation on erythrocyte membrane proteins of non diabetic subjects incubated with glucose. A very close relationship was found between the two alterations (r=0.82,p<0.001). These results suggest that the glycation of erythrocyte membrane proteins could be an important factor determining some haemorheological alterations in diabetics.

Detachment of adherent human PMN from cultured endothelial cells (human umbilical vein, HUVEC) and from immobilized albumin (bovine serum albumin, BSA) was investigated as a function of shear stress. Non-activated PMN did not adhere to either of the substrates (<2%). 20 min after activation with PMA (phorbol myristate acetate, 10 nM), 48 ± 5% (mean ± SEM, n=15) of the total number of PMN added had adhered to albumin, and 18 ± 4% (n=9) to HUVEC. The dishes with pre-adhered PMN were subjected to shear stress in the range occurring in venules in vivo, using a rotating plate-and-cone (cone angle 2.5°) apparatus made of transparent plastic to allow observation of the cells during the assay. On HUVEC, the majority of adhered PMN (66 ± 3%, n=9) was removed by a shear stress of 0.12 Pa (1.2 dyn/cm²), while the same shear stress removed only 15 ± 5% (n=9) of the PMN adhered to BSA. Further increase of shear stress to 2.3 Pa (23 dyn/cm²) removed 39 ± 4% (n=14) of the PMN adhered to BSA. Using spherical and semispherical approximations for the shape of the adhered PMN, the adhesive forces between PMA-activated PMN and HUVEC were calculated to be on the order of 0.05 nN per PMN, while most PMN on BSA adhered with a force of more than 4 nN per PMN. The force needed to detach PMN from HUVEC is more than one order of magnitude lower than value reported for PMN adhesion to venular endothelium in vivo. The present results suggest that the endothelium may playa regulatory role in PMN adhesion and limit the attachment force to allow emigration.

The aim of the study is to define the kinetics of hemorheological changes during and after a standardized bicycle ergometry lasting for 3 hours. Hematocrit, plasma and blood viscosity, red cell aggregation and deformability are measured in 15 volunteers hourly during exercise and followed for 21 hours thereafter. During exercise there is a marked viscidation of the blood which is caused by hemoconcentration and partial loss of red cell deformability. Subsequently blood cell rheology factors normalize. Three hours after exercise there is a marked fall of hematocrit and plasma viscosity. Native blood viscosity declines significantly below baseline values 5 hours after discontinuation of exercise and is still reduced 24 hours after the start of the experiment. The results suggest that the well known exercise-induced hemoconcentration is reversed after discontinuation of exercise when a marked “autohemodilution” takes place.

Rheological measurements were made on the blood of 22 chronic cigarette smokers (8 males, 14 females) 2 weeks before and 2 weeks after complete abstention from smoking. Significant reductions were observed in blood viscosity at both high and low shear rates and in plasma viscosity. These changes could be related to alterations in packed cell volume and plasma fibrinogen concentration, both of which fell significantly over the same period. The male and female groups responded similarly. These data indicate that a significant improvement occurs in rheologically-related cardiovascular risk factors in cigarette smokers within two weeks of abstaining from tobacco.

In order to define normal reference limits of a power law fitting method for evaluating whole blood viscosity, 110 healthy subjects were studied. We found that whole blood viscosity and blood viscosity standardized for hematocrit and plasma viscosity influence were significantly different in adult males, adult females and subjects over 60 years old (p<0.0001). In our opinion, it was also possible to distinguish blood viscosity differences into red blood cells aggregability and deformability components.

In order to find a new suspending medium for Ektacytometric measurement, comparisons of measured rheological properties of erythrocyte suspension in different buffers were carried out. Two kinds of buffer were used, e.g. PBS and PVP. And four types of erythrocyte were measured by using both suspending media, namely: normal rabbit's erythrocytes (RBC), rabbit's RBC hardened by formalin with different concentrations, rabbit's RBC treated by trypsin and human erythrocyte sampled from patients. Microscopic and scanning electron microscopic photographs of erythrocytes rigidizied at constant shear rate were observed. Results showed that (1) for everyone of the these four kinds of erythrocytes, measured deformation index (D1)—shear rate ( $\dot{\gamma}$ ) curves in PBS are similar qualitatively to those measured in PVP. (2) Microscopic and scanning electron microscopic photographs showed that erythrocytes indeed deformed when the shear rate of flow field for PBS was as low as $\dot{\gamma}=20s^{-1}$ . It indicated that PBS could be used as suspending medium. On the contrary when we used PVP ( $\dot{\eta}=2$ mPas Fig. 1 [1]) as suspending medium at $\dot{\gamma}=800s^{-1}$ , no obvious deformation could be obtained by using Ektacytometer. It seems to mean that, as the suspending medium for Ektacytometric measurement, PBS would be better than PVP, especially in low shear stress area. This point of view is different from that of privous works [2] which do 1101 consider PBS as suspending medium of Ektacytometer.

The effect of subcutaneous treatment of turpentine on hemorheological parameters was studied ex vivo in rats. A dose-response study (0.2 to 5 ml/kg) was performed on a series of parameters: whole blood apparent viscosity, plasma viscosity, plasma fibrinogen content, total plasma protein, hematocrit, erythrocyte aggregation and filterability. Significant alterations of all hemorheological parameters were observed 24 hrs after the turpentine treatment except for erythrocyte filterability and hematocrit values. In most cases, these alterations were significant (p<0.01) for doses higher than 0.3 ml/kg, with a maximum effect usually observed for a dose ≥ 1 ml/kg. These hemorheological alterations were still present 48 hrs after turpentine treatment and in the same order of magnitude. Thus, the present study demonstrated for the first time that turpentine treatment induced significant alterations of hemorheological parameters in dose-dependent manner, probably via an increase in plasma protein components.

Whether erythrocyte deformability is reduced in patients with peripheral occlusive arterial disease (POAD) is discussed controversially. Within this study erythrocyte filterability through microfilters with different pore diameters (3, 5, and 8 μm) was investigated in patients with POAD and age-matched healthy volunteers under physiological and ischaemia-related boundary conditions (lactate acidosis, hypercapnia, hypoxia, hyperosmolarity). 70 subjects were assigned to two control and three POAD groups characterized by varying combinations of risk factors (smoking, diabetes mellitus). Red blood cells (RBC) were suspended in four different media (pH/ mosmol *l⁻¹ : 7.4/ 300, 7.4/400, 6.5/300, 6.5/400) containing a physiological bicarbonate buffer adequately gased with O₂/CO₂/N₂. Mean hematocrit-corrected relative erythrocyte transit times (RCTT) and concentrations of clogging particles (CP) were measured by means of the St. George's Filtrometer. In agreement with previous reports, filtration data varied with erythrocyte volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) used as estimates of surface area-to-volume ratio (SAVR) and inner viscosity (IV) of RBC, respectively. While RBC filtration rates were dominated by IV in 5 and 8 μm pores they were mainly limited by SAVR in 3 μm pores. For RCTT statistically significant differences between controls and POAD patients could not be found. CP correlated with leucocyte counts of the suspensions if filterability was not primarily limited by MCV. A quantitative agreement among these parameters, however, was only obtained in 8 μm pores and, therefore, interactions between both erythrocytes themselves and erythrocytes and leucocytes should have contributed to filter obstruction. For CP statistically significant differences were only found between controls and diabetic POAD patients in whom also higher leucocyte counts were registered. It is concluded that in clinical or pharmacological studies of RBC filterability the most important in-vitro boundary conditions (milieu, filter pore size) should be varied in order to account for the complex and partially antagonistical relationships between erythrocyte properties and pore geometry as well as blood cell interactions. Because the capillary morphology of ischaemic tissue areas is usually not known or may be subjected to dynamic alterations the meaning of the invitro filterability with respect to the in-vivo microcirculation is additionally insecured.