Clinical Hemorheology and Microcirculation

The interpretation of the role of the hemorheological alterations in peripheral obliterative arterial diseases is still of considerable interest. After the first identification of the existence of a hyperviscosity condition in this disease, successive research has gradually tried to explain its meaning. First hyperviscosity was considered as associated to the disease, then some researchers have attributed it a decisive and essential role and have seen it capable, by itself, of reducing the blood supply to the tissue, while others have considered it just a subordinate expression of the disease. A last hypothesis tries to combine these two possibilities and considers the hyperviscosity of vascular diseases first as dependent on the tissue ischemia and then as responsible for the activation of a vicious circle which further contributes to reduce the peripheral blood flow. In such interpretation the therapeutical attempts to treat the peripheral vascular diseases by means of the improvement of blood fluidity should assume a positive role.

Research from many disciplines is coming together to give a more composite picture of the multitude of factors involved in cardiovascular and haematocrit control. In this review the term stress polycythaemia syndrome is used in its broadest sense, where all exogenous and endogenous stresses are considered. The contraction of plasma volume seen in association with the various stimuli is the end result of a final common pathway of neuroendocrine activation via the autonomic nervous system. In most circumstances the development of the stress polycythaemia syndrome is an appropriate response to a specific stimulus. However, there are haemorheological consequences which may not always be advantageous and in the presence of vascular disease may predispose the subject to the possibility of arterial occlusion. Management of the stress polycythaemia syndromes depends on identification and removal of the stimuli and correction of the haemorheological disturbances.

The role of blood rheology in retinal disorders is reviewed. Since retinal vessels can be observed directly, and capillary perfusion observed at fluorescein angiography, retinal studies have been of value in demonstrating the circulatory effects of overtly pathological blood rheology in haematological disorders, i.e. polycythaemias, paraproteinaemias, leukaemias and sickle cell disorders. Moreover, the susceptibility of the retinal circulation to arterial and venous occlusion, diabetic angiopathy, and increased intra-ocular pressure has revealed the potential of covertly pathological blood rheology to further impair blood flow and tissue function. In the compromised retinal circulation where shear forces are reduced, relatively small increases in haematocrit, plasma viscosity, red cell aggregation and cell rigidity may be sufficient to reduce capillary perfusion and hence promote visual complications. Conversely, rheological therapy may be of value in these blinding disorders.

Homozygous sickle cell (HbSS) disease is a clinical disorder characterized by abnormal hemorheology and a variety of pathophysiologic conditions; its underlying basis is a defect in the hemoglobin molecule which leads to polymer formation at low levels of oxygen tension. Micropipette analyses of oxygenated HbSS RBC indicate increased membrane elasticity and viscosity versus normal REC, especially for irreversibly sickled cells (ISC). HbSS RBC from a given donor are heterogeneous in their mechanical properties and large donor-to-donor variations are common. The abnormal rheological properties of HbSS blood become accentuated when oxygen tension is reduced, with micropipette data indicating an exponential increase of effective membrane rigidity and the time constant for deformation as oxygen tension is lowered. However, for HbSS REC which remain discoidal and for all normal RBC, no effects of oxygen tension are observed. Presently, correlations between rheologic measurements and clinical severity are less than satisfactory, perhaps due to the hemorheological tests employed; suggestions for alternate rheologic techniques and a brief review of therapeutic approaches are presented.

Red cell “flexibility”, generally accepted as a pivotal determinant of blood flow in the microvessels, represents the micromechanical “behavior” of a complex structure consisting in a viscoelastic membrane shell and a cytosolic fluid. It is impossible to describe its overall “material properties” in the sense of physical “constants”; only its behavior can be tested in fluid dynamic conditions kept as constant and well controlled as possible. Under such conditions, an overt abnormal flow behavior can easily be diagnosed. Earlier claims of “covertly abnormal red cell flow behavior” in diabetes mellitus, which were based on measurements of “more rigid” than normal cells have recently been contradicted, the earlier results being attributed to technological or to contamination errors (by the presence of white cells). New data obtained by techniques inherently independent of leukocyte artefacts are reviewed: various types of the “single erythrocyte rigidometer” (SER), polymicroviscometry (PMV) of packed red cells, MyNiPore microsieve conductometry (MMC) and whole blood viscometry. All data reviewed clearly confirm the presence, in certain diabetic patients, of microrheologically abnormal erythrocytes. The reason for the failure of conventional techniques to detect these abnormalities is discussed.

Patients with diabetes mellitus develop hyperproteinaemia with a consequential increase in erythrocyte aggregation and blood viscosity. Evidence for a loss of erythrocyte deformability is more controversial, however, with steady-state diabetics showing largely normal rheology when studied by micropipette, ektacytometric, or filtration techniques. Decompensated diabetics with hyperosmolar plasma or a very high intraerythrocytic sorbitol concentration may, however, have impaired erythrocyte deformability. Membrane studies of erythrocytes from diabetics have shown insulin-dependent abnormalities of lipid fluidity, cholesterol/phospholipid ratio, and Na⁺/K⁺ ATPase activity but most of the rheological methods used in clinical studies are not yet sufficiently sensitive or specific to assess the rheological consequences of these membrane changes.

In ten POAD patients, 800 mg. of Defibrotide (polideoxynucleotide extracted from mammalian lung, with antitrombotic and fibrinolytic activity) were infused i.v. Blood and plasma viscosity, haematocrit, blood filterability and fibrinogen concentration were controlled, in basal conditions and after one hour from the end of infusion. Haemodynamic parameters: rest flow, peak flow, time to peak flow, half time and total time of reactive hyperemia by means of strain gauge pletismography, were controlled at lower limbs before infusion and after 1 h., 2 h., 6 h., from the end of infusion. The present investigation showed an improvement of rheological parameters and a decrease of total time and half time of reactive hyperemia. These data demonstrate a rheological activity of Defibrotide as well as the fibrinolytic one.

Twenty patients suffering from peripheral obliterative arterial disease underwent metabolic and haemorheological study. The femoral arteriovenous differences in lactate and oxygen extraction varied with the severity of the ischemic process, as assessed clinically. In patients with claudication lactate release and oxygen extraction were comparable with those in the control group while were significantly increased in Fontaine’s III and IV stage patients. Moreover this latter group showed the most relevant changes in whole blood viscosity and filterability. Such findings bring a further contribution to the correlation between the regional circulatory imbalance and changes in haemorheological parameters.

Red cell concentration is the main determinant of bulk blood viscosity. the evaluation of other determinants of blood rheology may require elimination of differences due to hematocrit alone. A simple and efficient procedure is described for calculating blood viscosity at a standard hematocrit, based on plasma viscosity, native sample hematocrit and whole blood viscosity at native hematocrit. The method considers individual variations of the viscosity – hematocrit relationship, as well as its dependence on shear rate.

The investigation of erythrocyte deformability changes during cell ageing might give one insight on circulatory behaviour of young and old red blood cells.

Erythrocytes of healthy subjects of both sexes had been separated into 3 fractions of increasing age on a Percoll discontinous density gradient and their different age has been tested by pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PD) activities.

Each fraction, free of leucocytes, suspended to 10% haematocrit, was filtered through Nuclepore filters 5 micron diameter; erythrocytes lying on filters were observed by Scanning Electron microscopy. Erythrocyte deformability has shown small decrease from middle aged cells to old and youngest ones.

The existence of changes in blood viscosity and filterability in some diseases supported several methods to wash red cells, hypothesizing that these modifications could be due mainly to red cells. In order to investigate these changes, washed red cells were studied in several diseases. Washed red cells were suspended to 10% haematocrit and filtered through Nucleopore filters 5 micron diameter; this study was meda in healthy subjects and in patients suffering from peripheral obliterative arterial disease, stroke, myocardial infarction and cancers; the same parameter was studied also in arterial and venous blood. The analysis of these data did not show very significant differences between the healthy donors and each group of patients studied.

On the basis of these results we could suppose that the various diseases really did not induce any change of the erythrocyte deformability and that the changes we see in whole blood filterability depends on other blood cells or components.

On the contrary we can hypothesize that the washing technique by itself provokes a damage of the erythrocyte membrane; this damage may be so great as to inable us to detect a significant difference among the groups studied.

Chronic occlusive arterial disease has been shown to be accompanied by blood hyperviscosity which is associated with increased plasma viscosity, red cell aggregation and red cell rigidity. There exists a linear correlation between fibrinogen and plasma viscosity and the flow behaviour of red cells meshed to each other by fibrinogen. Therapeutical decrease of the fibrinogen level aims at improving blood fluidity and increasing tissue perfusion by raising blood flow velocity in conjunction with an increase in red cell oxygen transport rate. Pharmacological approaches to realize this therapeutical principal are: fibrinolysis (streptokinase, urokinase, in myocardial infarction); defibrinogenation (ancrod, defibrase, in peripheral vascular disease); anabolics (stanazol in Raynaud’s-Syndrome), lipid lowering drugs (clofibrate analogous in peripheral vascular disease), hemorheologically active drugs (pentoxifylline). Confirmed effects in respect of improvement of blood fluidity and tissue perfusion via fibrinogen reduction are at present only available for defibrinogenation by ancrod and by pentoxifylline.

25 patients with breast cancer after primary surgery and 10 patients with metastatic disease were investigated regarding hemorheological parameters such as erythrocyte aggregation, plasma viscosity, hematocrit and fibrinogen during chemotherapy with methotrexate, 5-fluorouracil and cyclophosphamide. Erythrocyte aggregation increased significantly during the first course of chemotherapy and remained elevated thereafter. There were no significant changes regarding the other parameters. The influence of tumor volume was much more important leading to significant differences between the two groups regarding erythrocyte aggregation, plasma viscosity and fibrinogen, while hematocrit and total protein concentration were not different.

Particle migration induced by rheological mechanisms in a flowing suspension is studied. Most of this paper is devoted to calculate the migration force due to aggregation of the particles which form the bulk suspension. The last section deals with the effect of particles deformability on the margination phenomena. The mechanism of white blood cell margination and the following steps leading to emigration can be fully understood owing to our theoretical model. Theory is compared to experimental data obtained by P. GAEHTGENS and his group in blood perfused glass capillaries in which white blood cells adhesion does not occur.

Deteriorated blood rheology has been demonstrated in obstetric diseases with decreased placental perfusion. Many of the patients investigated in these studies suffered from conditions associated with hemorheological abnormalities, such as diabetes or pre-eclampsy. Therefore it is difficult to establish the pathogenetic role of blood rheology in fetal growth retardation. In the present study placental perfusion and whole blood viscosity were measured in 26 pregnant women with intrauterine fetal growth retardation, without pre-eclampsy or other disease of the mother. These patients were compared to 20 controls with normal, uncomplicated pregnancy. Significantly higher whole blood viscosity accompanied with deteriorated placental perfusion was found in the patient’s group. Results suggest a pathogenetic role of hyperviscostiy in cases of intrauterine growth retardation without pre-eclampsy.

The technique of television microscopy has been applied to study human nailfold capillary blood flow in a variety of conditions that result in changes in blood rheological behaviour. The most striking changes were observed in leukaemia: in the presence of high total leucocyte counts stationary capillaries were frequently observed. A highly significant improvement in capillary flow velocity followed cytoreduction therapy. In contrast capillary flow velocity was normal at rest in patients with polycythaemia and was not altered following isovolaemic haemodilution. In patients with Waldenström’s macroglobulinaemia capillary flow tended to be lower than age matched control subjects but few stationary capillaries were observed. Plasmapheresis in two subjects resulted in an improvement in capillary flow. These results emphasize the need to take into account the autoregulatory capacity of the microvasculature when extrapolating from the viscometer to the in vivo state.

A new method of estimation of mechanical properties of erythrocytes is presented, based on the measurement of red cell susceptibility to centrifugal packing. Blood is spun in a microhematocrit centrifuge run at a voltage lowered to provide a rotation speed corresponding to 200 × g and apparent hematocrit is measured after 15-min centrifugation (Ha₁₅). Then the centrifuge is run at a standard speed and the final hematocrit is read (Hk). The difference between these two values, denoted as Erythrocyte Packing Difference (EPD) is a measure of red cell susceptibility to centrifugal packing. EPD is independent of hematocrit in a broad range of hematocrit values. A survey of a population of 489 healthy donors points to a significant sex difference in EPD which amounted to 10% ± 2% for males and 8.5% ± 2.5% for females. A decreased EPD was found in diabetes. EPD changes under conditions affecting red cell deformability. This simple and easily standarizable method of estimation of mechanical properties of unmanipulated erythrocytes is recommended for routine use.

Hemoconcentration was induced by administration of oral Furosemide in a group of healthy athletes. Before and after this treatment a variety of hemorheological variables was measured. Simultaneously the time needed to reach the anaerobic threshold during a standardized ergometer test was recorded. The results show that the changes in blood rheology correlate with the changes in threshold – the more hemoconcentration, the more prolongation of the time to reach the threshold. This suggests that athletes may benefit in terms of performance by hemoconcentration. The findings support the hypothesis that the optimal hematocrit for oxygen transport rate is higher than normal when average shear stress in the circulation is high such as in athletes. Conversely it may be lower than normal when shear stress is low like distal to a stenosis in occlusive arterial diseases.

The effect of pentoxifylline (PTX) on red blood cell-endothelial cell adherence was studied for sickle and normal red blood cells (RBC). Human umbilical vein endothelial cells grown to confluence on glass slides formed the base of a parallel plate flow chamber into which RBC suspensions were perfused at a constant flow rate, producing a wall shear stress of 1 dyne/cm². RBC were incubated with PTX at 37°C for one hour at concentrations ranging from 0.1 to 10.0 mmol/L. Adhesion was monitored using video microscopy. The number of adherent RBC was determined at 10-minute intervals during a wash-out period which followed a 10-minute perfusion of red cells. When compared with unincubated control RBC, the adherence of sickle RBC incubated with 0.1, 1.0 and 10.0 mmol/L of pentoxifylline was reduced to 65%, 53% and 39% of control values, respectively. There was no significant difference between the adherence of drug incubated and untreated normal RBC. These results indicate that pentoxifylline significantly affects the adhesion of sickle RBC to cultured human endothelial cells, but has no effect on the adherence of normal RBC.

The electrophoretic mobility of erythrocytes from patients with primary Raynaud’s phenomenon was measured in near physiological strength saline using laser Doppler microelectrophoresis and has been shown not to be significantly different from that of matched normal controls. It was found that washing the erythrocytes caused a significant, but small, increase in mobility of up to 3.5%. Two anticoagulation procedures, defibrination and heparinization, were compared and found not to affect measured mobility.