Clinical Hemorheology and Microcirculation

During storage at +4°C, red blood cells undergo biochemical and physicochemical modifications, which alter their rheological characteristics especially the deformability. Even so until now not precisely defined deformability is undoubtedly a function of whole cell elasticity and viscosity. In a previous study we have investigated changes of elasticity of whole RBCs during a 6 weeks storage by quasi‐static experiments using our Cell‐Elastometer method. Since the changes in deformability we observed with that experimental approach have not been significant we extended the hard/software capabilities of this instrument to enable dynamic measurements also. We applied this modified hard‐/software set‐up to examine again changes in viscoelasticity of erythrocytes from concentrates during a six weeks storage at a blood bank. The cells were resuspended in CPD‐SAG‐M and stored at +4°C. Quasi‐static and dynamic experiments were performed on stored erythrocytes and showed for both significant changes in elasticity and viscoelasticity from the fourth week on. So it can be stated that due to our experimental results decrease in deformability of RBCs during storage occurs after a four weeks period of relative stability. To get further insight in changes of underlying or related biochemical properties according experiments have been performed in parallel. Especially the decrease in ATP showed a nearly parallel time course with a significant decrease after the 4th week. All other parameters especially the 2,3 DPG level showed a nearly linear de‐ or increase with time which are in accordance with the results of the additionally performed elongation experiments. Our quasi‐static and dynamic deformability measurements have been proven to provide a simple and reliable tool to follow up erythrocyte senescence during storage where a pronounced change in mechanical properties may be used as an indicator for a change in bioviability. This has to be verified in further experiments.

Polycystic kidney disease (PKD) is associated with an increased incidence of hypertension and cardiovascular abnormalities. As hemorheology is an important hemodynamic determinant and may contribute to vasculopathies we measured whole blood viscosity and red blood cell (RBC) and plasma rheological factors in 38 patients with PKD and compared this data with similar measurements in age‐ and sex‐matched healthy controls. Renal function was assessed by plasma creatinine concentration. Analysis of the data showed that the PKD group had a significant reduction in mean hematocrit and an increase in mean plasma viscosity and mean plasma fibrinogen concentration. Intrinsic RBC rheology assessed by standardised viscosity measurements was impaired in patients with PKD compared to control subjects. The changes in plasma and RBC rheology did not however result in increased whole blood viscosity in the patients with PKD due to the reduction in hematocrit level. Correlation analyses demonstrated a significant relationship between increased plasma creatinine concentration and lower hematocrit, decreased whole blood viscosity and impaired RBC deformability but not with increased plasma viscosity or plasma fibrinogen concentration. This study shows that although PKD is associated with mild abnormalities in plasma rheology and intrinsic RBC rheology these changes are offset by a reduction in hematocrit. The changes in RBC rheological determinants in PKD appeared to be related to the degree of renal impairment.

In order to ascertain whether erythrocyte deformability (ED) is involved in chronic coronary syndromes, this rheological property was determined in 92 survivors of acute myocardial infarction (AMI) who had had the acute event 3 years ago and in 150 volunteers. From the 92 AMI survivors in 50 (43 males, 7 females aged 61±9 years) ED was determined with filtrometric techniques (Hanss Hemorheometre) and in 42 (32 males, 10 females aged 63±11 years) with laser diffractometric ones (Rheodyn SSD). The control group consisted of 66 and 84 volunteers whose ED was measured with the above mentioned devices respectively. Patients and controls were matched for age, sex, total cholesterol and triglyceride levels. With the Hanss Hemorheometre, the Rigidity Index (RI) was higher in patients than in controls (9.4±1.2 vs 8.7±1.5; p=0.01) although after adjusting for confounding variables (MCV and leukocyte count) in a logistic regression analysis the RI was no longer statistically significant. With the Rheodyn SSD the Erythrocyte Elongation Index (EEI) determined at 12, 30 and 60 Pa, did not show statistically significant differences between cases and controls at any of the shear stresses tested. Our results suggest that AMI survivors who had had the ischemic event 3 years ago do not show decrease RBC deformability with either of the two methodologies used. Red blood cell deformability does not appear to contribute to impaired microcirculatory blood flow in chronic coronary syndromes.

Purpose: Spontaneous blood echogenicity in vein ultrasound images may be a marker for an increased erythrocyte aggregability, but a reliable quantitative evaluation method is a prerequisite for its use in clinical studies. We compared a simple scoring system of blood echogenicity intensity and pattern, with automatic image analysis.

Material and methods: 157 femoral and popliteal vein digitized ultrasound sequences were reviewed by two independent observers who chose an image, delimited an area of interest (ROI), and graded blood echogenicity intensity and pattern, using a four class score. Each observer reviewed the images selected by the other, without and with ROI. The computer calculated first and second order parameters describing echo intensity and spatial organization.

Results: Inter‐observer reproducibility of subjective assessment was poor (Kappa<0.5), whereas the automatically calculated ROI average gray level intensity relatively to the whole image (τ₁) effectively separated all grades of intensity. No parameter effectively separated patterns.

Conclusion: τ₁ is a simple parameter for the in vivo evaluation of blood echogenicity intensity. It should be evaluated in standardized conditions for clinical hemorheology studies in correlation with in vitro erythrocyte aggregation measurements.

The effects of oxygen radical generating system of H₂O₂ on neutrophil deformability, lipid peroxidation, protein degradation, chemotaxis, random migration and activation were studied in the presence and absence of selected anti‐oxidants such as vitamin E and carbon monoxide. Hydrogen peroxide caused a significant loss of neutrophil deformability (measured as an index of filtration), loss of chemotaxis (using chemoattractant) and loss of random migration (spontaneous migration) under agarose gel. Hydrogen peroxide also caused a significant increase in lipid peroxidation (measured as malonyldialdehye) and protein degradation (measured as free alanine). Hydrogen peroxide also caused activation of neutrophils (measured by nitroblue tetrazolium dye test). Pre‐incubation of neutrophils with vitamin E prevented significantly the increase in lipid peroxidation without affecting the loss of neutrophil deformability. However, pre‐incubation with carbon monoxide prevented significantly the increase in protein degradation and the loss of neutrophil deformability. The results indicate the importance of oxidatively damaged cell proteins in compromising the rheologic behaviour and migration of neutrophils, particularly under oxidative stress conditions.

The effect of long‐term supplementation of vitamin C on leukocyte adhesion to the cerebral endothelium was investigated in streptozotocin (STZ)‐induced diabetic rats. Diabetes was induced in male Wistar Furth rats by intravenous injection of STZ. The vitamin C, ascorbic acid, was supplemented with drinking water (1 g/l). The rats were divided into control and diabetic groups without or with supplementation of vitamin C. The cerebral microcirculation was directly observed through a cranial window after different periods (12, 24 and 36 weeks) of vitamin C supplementation, using fluorescence videomicroscopy. Leukocyte adhesion to the venular endothelium was examined by labeling leukocytes with rhodamin 6G.

The number density of adherent leukocytes in STZ‐diabetic rats was increased singnificantly, compared with control rats. This increase in leukocyte adhesion was prevented by the long‐term supplemented vitamin C. It was suggested that the antioxidant effect of vitamin C might be responsible for the prevention of leukocyte adhesion in diabetes mellitus.

The effect of long‐term supplementation of vitamin C on leukocyte adhesion to the cerebral endothelium was investigated in streptozotocin (STZ)‐induced diabetic rats. Diabetes was induced in male Wistar Furth rats by intravenous injection of STZ. The vitamin C, ascorbic acid, was supplemented with drinking water (1 g/l). The rats were divided into control and diabetic groups without or with supplementation of vitamin C. The cerebral microcirculation was directly observed through a cranial window after different periods (12, 24 and 36 weeks) of vitamin C supplementation, using fluorescence videomicroscopy. Leukocyte adhesion to the venular endothelium was examined by labeling leukocytes with rhodamin 6G.

The number density of adherent leukocytes in STZ‐diabetic rats was increased singnificantly, compared with control rats. This increase in leukocyte adhesion was prevented by the long‐term supplemented vitamin C. It was suggested that the antioxidant effect of vitamin C might be responsible for the prevention of leukocyte adhesion in diabetes mellitus.