Red Cell Aggregation Research Articles

Effect of fibrinogen on leukocyte margination and adhesion in postcapillary venules.

To quantitatively evaluate the role of fibrinogen (Fb) as a determinant of leukocyte (WBC) margination in postcapillary venules in light of its ability to induce red blood cell (RBC) aggregation with reductions in shear rate (gamma) and increase adhesiveness of WBCs to endothelium (EC). Red cell aggregation (RCA), WBC margination (flux at the EC), rolling velocity, and adhesion to the EC were measured in rat mesenteric postcapillary venules upon reducing gamma, prior to and following systemic infusion of Fb. Proximal occlusion of feeding microvessels with a blunted probe facilitated reductions in gamma from 600 to 50 s(-1). An index of aggregation (G) was derived from light-scattering properties of RBCs, where G was proportional to the number of RBCs per aggregate. WBC margination was measured as the percentage of total luminal WBC flux that rolled on the EC, F*(WBC). For normal levels of Fb (0.07 g%), reductions in gamma resulted in a 4-fold rise in F*(WBC) and no change in G as gamma was reduced to 50 s(-1). Infusion of Fb to achieve a plasma concentration to 0.7 g% caused a modest 20% increase in G and a 2.5-fold increase in F*(WBC) at gamma = 50 s(-1). WBC-EC adhesion appeared to increase significantly, but much less than with infusion of high molecular weight dextran (Dx). With Dx, G increased 3-fold, with reductions in gamma, but F*(WBC) increased only half the amount incurred with Fb at low shear. The greater margination in the presence of Fb results from RBC rouleaux that promote radial migration of WBCs. In contrast, clumps of RBCs resulting from high molecular weight Dx entrain WBCs within plasma gaps along the vessel centerline. In the presence of Fb, margination of WBCs increases dramatically at low shear due to rouleaux formation, which enhances radial migration of WBCs. This effect is much greater than with Dx because disruption of the much weaker Fb induced rouleaux precludes reductions in H(MICRO), whereas clumping aggregates induced by Dx form plasma gaps. Thus, modest levels of RCA caused by increased Fb may greatly enhance margination and with an enhancement of adhesiveness synergistically promote firm WBC-EC adhesion in the low flow state.

Fibrinolytic and biochemical factors in patients with venous insufficiency

Long-term metabolic changes in vein tissue of the limbs and feet have been reported in the course of chronic venous insufficiency (CVI). Low fibrinolytic potential was first demonstrated using fibrin-embedded vein rings (Todd's biopsy), then in blood via increased euglobulin lysis time. This defect is especially pronounced when blood is taken from foot veins of patients with grades 5 and 6 of CVI or skin lesions. Haemorrheological disorders such as increased blood viscosity and red cell aggregation, related mainly to high fibrinogen levels, have also been shown in patients with CVI. Such disorders, particularly observed in blood from foot veins, play an important role in the interaction between the blood cell components and the vessel wall. More recently, the margination and adhesion of white cells to the endothelium and the subsequent activation of white cells have been assumed. Adhesion molecules such as ICAM-1, VCAM-1 and P-selectin have been identified surrounding skin ulceration. There is also evidence that markers of neutrophil degranulation reach high levels in blood taken from foot veins after exposure to experimental venous hypertension. White cell activation results in elevated plasma levels of L-selectin, increased production of oxygen free radicals, pseudopods, CD11b integrin, platelet-monocyte aggregates and abnormal lymphocyte L-selectin. Increased nitric oxide, presumably due to monocyte activation, as well as increased urine haemosiderin related to skin microvessel leakage have also been reported. VCAM-1 and metalloproteinase MMP9 activity have been stated as sensitive markers in relation to endothelium activation and white cell trapping, which follows hypoxia and/or alterations in wall shear stress, mainly in skin venulae and in the region of valves.

Effects of aggregation of red cells and linear velocity gradients on the correlation-based method for quantitative IVUS blood flow at 20 mhz

Recent computer simulations suggest that the presence of aggregates of red blood cells (RBCs), at random angles and lengths, does not affect the measurements of blood flow transverse to the ultrasound (US) beam direction using a correlation-based method and an intravascular (IV) US array catheter. However, in case of aggregates of RBCs aligned with the flow, measurements of simulated blood velocity are affected. Blood velocity gradients were also shown not to influence the correlation-based method for blood velocity estimation. The objective of this study was to quantify the influence of aggregates of RBCs and blood velocity gradients on the correlation-based method during in vitro experiments. For this purpose, measurements were performed on washed RBCs (no aggregation), normal human blood, and two types of diseased blood in which a lower or a higher level of aggregation was present. The decorrelation pattern of a circular US transducer as a function of transverse blood flow was studied using a Couette system. Changing the shear rate of the Couette system modified the aggregation level of RBCs and the velocity gradient. With the exception of the results at low shear rates and abnormally high aggregation levels, agreements were found between the autoconvolution of the acoustical beam (reference curve) and the radiofrequency (RF) decorrelation patterns. For the high shear rate present in coronary arteries, the correlation-based method for blood flow estimation should not be influenced by these phenomena. (E-mail: a.vandersteen@erasmusmc.nl)

L'hémorhéologie permet-elle de comprendre le « paradoxe de l'hématocrite » lors de l’entraînement et du surentraînement ?

Purpose. – Doping with EPO increases both hematocrit and performance. On the opposite, training reduces hematocrit due to plasma volume extension and overtraining both increases hematocrit and decreases performance. This apparent paradox seems to be explained by a new concept proposed interpreting the interactions between viscosity factors and blood flow. Facts. – Viscosity factors exert a non linear “all or none” effect that can be described according to the “ percolation theory”. Blood viscosity has no hemodynamic relevance by its own, but viscosity factors (hematocrit, red cell deformability and aggregability, plasma viscosity) can result according to flow conditions in self-potentiating phenomena of quick transition between a state of high fluidity and a state of red cell aggregate clumping. In muscle, this model predicts that: (a) during exercise the only viscosity factor that could influence flow is plasma viscosity; (b) at rest all viscosity factors (including high hematocrit) may induce a self-potentiating “viscidation” process. Conclusion. – According to these concepts, supraphysiologic hematocrit values are probably unable to disturb muscular perfusion at exercise, but are likely to promote self-potentiated stasis at rest and thus to increase the risk of thrombosis.

Le « syndrome des jambes lourdes » chez les athlètes surentraînés est-il le reflet de désordres hémorhéologiques ?

Introduction. – The aim of this work is to evaluate if the feeling of heavy legs in athletes is correlated with overtraining syndrome-related hemorheological disturbances. Results. – The subjects who quoted the item: “I have the feeling of heavy legs” had higher plasma viscosity and higher red cell aggregation parameters. The overtraining score was correlated positively with plasma viscosity, and aggregability parameters. The feeling of heavy legs was correlated with plasma viscosity and aggregability. Conclusion. – These findings suggest that the feeling of heavy legs in overtrained athletes is related to overtraining syndrome-related hemorheologic disturbances, namely mild plasma hyperviscosity and mild erythrocyte hyperaggregability.

C-reactive protein: associations with haematological variables, cardiovascular risk factors and prevalent cardiovascular disease.

C-reactive protein (CRP) has been proposed as a risk factor for cardiovascular disease; however, this association is confounded by mutual relationships with both classical and haematological cardiovascular risk factors. We, therefore, measured CRP with a high-sensitivity assay in stored plasma samples from 414 men and 515 women in the north Glasgow MONICA (MONItoring trends in CArdiovascular diseases) survey, to study its correlation with haematological variables, classical risk factors and prevalent cardiovascular disease. CRP correlated with age, oral contraceptive use, menopause and most classical cardiovascular risk factors (except blood pressure). CRP also correlated with plasma levels of the pro-inflammatory cytokine interleukin 6, and haematocrit, viscosity, red cell aggregation, white cell count, and coagulation factors [fibrinogen, factor (F) VII in women, FVIII, FIX] and inhibitors (antithrombin and protein C in women; protein S) but not coagulation activation markers. CRP was significantly associated with prevalent cardiovascular disease in both men (P = 0.03) and women (P = 0.009), however, the association became non-significant after adjustment for firstly classical risk factors, then fibrinogen. We conclude that correlations with classical and haematological risk factors account for a substantial component of the association of CRP with prevalent cardiovascular disease, but there is evidence of a residual, independent effect among women.

Cyclic and radial variation of the Doppler power from porcine whole blood.

The Doppler power from porcine blood was observed in a mock flow loop to have cyclic and radial variation during a pulsatile cycle. It was found to decrease with shear rate under steady flow, except near the center of the tube at which other mechanisms such as the effects of radial distribution on the rouleaux might be involved. Under pulsatile flow, the timing of the peak of the Doppler power measured at the center of the tube became closer to the peak systole from 20 to 60 beats/minute (BPM), and the power and velocity peaks coincided at 60 BPM. The overall radial variation of the Doppler power during a whole pulsatile cycle was prominent due to the increase of shear rate from the center to 4.5 mm radial position within a tube of 6.35 mm radius. The cyclic variation of the Doppler power varied with the radial position, being relatively large at the center, reaching a minimum at an intermediate radial position, and increasing again near the wall. The peak of the Doppler power occurred at early systole near the tube wall and lagged the flow closer to the center. The "black hole" phenomenon was observed only over portions of the flow cycle. All these complex variations of the Doppler power across the tube over a cycle are thought to be the result of red cell aggregation, which can be affected by shear rate and acceleration.

Modeling the frequency dependence (5-120 MHz) of ultrasound backscattering by red cell aggregates in shear flow at a normal hematocrit.

The frequency dependence of the ultrasound signal backscattered by blood in shear flow was studied using a simulation model. The ultrasound backscattered signal was computed with a linear model that considers the characteristics of the ultrasound system and tissue acoustic properties. The tissue scattering properties were related to the position and shape of the red blood cells (RBCs). A 2D microrheological model simulated the RBC dynamics in a Couette shear flow system. This iterative model, described earlier [Biophys. J. 82, 1696-1710 (2002)], integrates the hydrodynamic effect of the flow, as well as adhesive and repulsive forces between RBCs. RBC aggregation was simulated at 40% hematocrit and shear rates of 0.05-2 s(-1). The RBC aggregate sizes ranged, on average, from 3.3 RBCs at 2 s(-1) to 33.5 cells at 0.05 s(-1). The ultrasound backscattered power was studied at frequencies between 5-120 MHz and insonification angles between 0-180 degrees. At frequencies below approximately 30 MHz, the ultrasound backscattered power increased as the shear rate was decreased and the size of the aggregates was raised. A totally different scattering behavior was noted above 30 MHz. Typical spectral slopes of the backscattered power (log-log scale) between 5-25 MHz equaled 3.8, whereas slopes down to 0.6 were measured at 0.05 s(-1), between 40-60 MHz. The ultrasound backscattered power was shown to be angle dependent at low frequencies (5-25 MHz). The anisotropy persisted at high frequencies (>25 MHz) for small aggregates (at 2 s(-1)). In conclusion, this study sheds some light on the blood backscattering behavior with an emphasis on the non-Rayleigh regime. Additional experimental studies may be necessary to validate the simulation results, and to fully understand the relation between the ultrasound backscattered power, level of RBC aggregation, shear rate, frequency, and insonification angle.

Rheological abnormalities and thromboembolic complications in heart disease: spontaneous echo contrast and red cell aggregation.

The role of abnormal rheological changes in the pathogenesis of thromboembolism has received much attention in recent years, especially in the field of cardiology. Such changes are sometimes seen in an echocardiogram as a smokelike haze known as spontaneous echo contrast (SEC). The presence and severity of SEC correlate with dilated cardiac chambers and the incidence of thromboembolic complications. It is caused by increased red cell aggregation and increased fibrinogen levels, both of which are known risk factors for thrombosis. Although not used clinically, measurements of red cell aggregation can be made in research settings. This can provide findings that give insight into factors causing increased red cell aggregation. A small series of patients with angina pectoris was studied with the Myrenne aggregometer for red cell aggregation. The results, which show correlation between the plasma fibrinogen and triglyceride levels, are presented. As yet, there are only a few therapeutic guidelines for the correction of abnormally high fibrinogen levels in patients at risk.

Reduced acute phase response to differentiate between viral and bacterial infections in children.

The objective of this study was to document the reduced acute phase response that appears in children with viral as opposed to bacterial infections. The white blood cell count (WBCC), the erythrocyte sedimentation rate (ESR), and leukocyte and erythrocyte adhesiveness/aggregation were determined in 36 children with acute bacterial infection, 29 children with viral infection, and 19 controls. A significant reduced WBCC, ESR, and leukocyte and erythrocyte adhesiveness/aggregation was noted in the children with acute viral infection as opposed to those with bacterial infection: 10,800 +/- 3600 and 20,000 +/- 10,000 cells/cm2, 29 +/- 21 and 53 +/- 35 mm Hg, 23 +/- 9 and 41 +/- 15%, and 3.4 +/- 5.1 and 9.8 +/- 13.6 microns, respectively. The results indicate that a reduced acute phase response can be observed in children with an acute viral infection. This can have diagnostic implications and pathophysiological consequences in terms of less flow impairment in the microcirculation due to less red and white blood cell aggregation.

Variation of the ‘‘Black Hole’’ phenomenon with speed and stroke rate

The Black Hole (BH) phenomenon, a central hypoechoic hole surrounded by a hyperechoic zone, has been observed and investigated only under steady flow. The phenomenon was recently observed under pulsatile flow, but its variation during a cycle has never been reported yet. In order to better understand the BH phenomenon and its variation, a GE LOGIQ 700 Expert system was used to collect B mode images from porcine blood in a rigid tube (ID: 9.5 mm). The BH became weaker or disappeared during the systole in terms of contrast and size. The variation of the BH over a cycle was investigated with the peak speed levels (10–25 cm/s), stroke rates (20, 40, 60 beats/min), mean speed levels (10, 20 cm/s), and hematocrits (12%–46%). Under steady flow, the BH phenomenon started to appear at an asymmetrical radial position at very low speeds when speed was increased. The BH shifted to the center of the tube at higher speeds and finally disappeared at ∼100 cm/s. However, the asymmetrical position at lower speeds was not observed when speed was decreased. The variation in the BH is presumably due to the red cell aggregation, which is affected by the shear rate and acceleration during a pulsatile cycle.

Effects of moderate weight loss on anginal symptoms and indices of coagulation and fibrinolysis in overweight patients with angina pectoris.

To evaluate the effects of moderate weight loss, in overweight patients with angina, on plasma coagulation, fibrinolytic indicies and pain frequency. Single-stranded 12-week dietary intervention, an individualised eating plan with quantitative advice delivered by a dietitian. Target weight loss of 0.5 kg per week. Outpatient research clinic. Fifty-four volunteers with angina pectoris were recruited. Five subjects withdrew, so 27 males, 22 females, mean body mass index (BMI) 29.3 (s.d. 4.3) kg/m(2) and age 60.3 (s.d. 6.5) y completed the intervention. Body weight and frequency of anginal pain. Plasma fibrinogen, red cell aggregation (RCA), viscosity, factor VII activity, plasminogen activator inhibitor (PAI) activity, tissue plasminogen activator antigen (t-PA), plasma cholesterol, triglyceride and insulin. After the 12-week dietary intervention period, mean body weight fell by 3.5 (s.d. 2.6) kg or 4.3% (P=0.0001), range -11.7 to +1.7 kg. Mean angina frequency fell by 1.8 (s.d. 3.6) from 3.2 to 1.4 episodes/week (P=0.009) and plasma cholesterol by 0.4 (s.d. 0.7) from 6.3 to 5.9 mmol/l (P=0.0001). HDL cholesterol and triglyceride were unchanged. Of the coagulation and fibrinolytic factors, factor VII activity and RCA were significantly reduced by 5 (s.d. 20), IU/dl (P=0.04) and 1.3 (s.d. 1.3) arbitrary units (P=0.014), respectively. A conventional dietetic intervention, resulting in 4% weight loss, offers the potential to reduce atherosclerotic and thrombotic risk, and to reduce pain frequency, in angina patients. Given the importance of this result in a public health context, these results indicate that this may be a fruitful area for future nutrition research.

Blood rheology in deep venous thrombosis—relation to persistent and transient risk factors

Introduction: The risk of recurrence in patients with symptomatic deep venous thrombosis (DVT) is higher in patients with persistent risk factors than in patients with transient risk factors. The purpose of this prospective study was to investigate the course of rheological variables in patients with DVT up to 1 year after the acute event in relation to risk factors. Patients and methods: In 37 patients with proven DVT plasma fibrinogen, plasma viscosity, red cell aggregation, whole blood viscosity, hematocrit and platelet aggregation were studied in the acute phase, at 6 weeks and at 12 months. Results: In the acute phase, patients had higher fibrinogen (medians and ranges; 450 [270–611] vs. 247 [170–340] mg/dl, p<0.01), plasma viscosity (1.67 [1.48–1.96] vs. 1.60 [1.50–1.70] mPa s, p<0.01), red cell aggregation (9.76 [5.87–12.66] vs. 5.66 [3.67–8.46] arbitrary units; p<0.01) and whole blood viscosity (5.78 [5.61–5.87] vs. 5.59 [5.27–5.9] mPa s, p<0.01), but lower hematocrit (40 [32–46] vs. 45 [38–50]%, p<0.01) and platelet aggregation (by epinephrine: 41 [13–85] vs. 79 [29–91]%, ( p<0.01) than controls. During the 1-year follow-up, fibrinogen, plasma viscosity, red cell aggregation and whole blood viscosity constantly decreased, whereas hematocrit and platelet aggregation increased in the total of patients (all p<0.01). Subgroup analysis according to risk factors showed that at 12 months patients with persistent risk factors ( N=21) had higher plasma fibrinogen (357 [235–450] vs. 247 [214–335] mg/dl, p<0.01), plasma viscosity (1.65 [1.50–1.80] vs. 1.59 [1.42–1.77] mPa s, p<0.05) and red cell aggregation (7.82 [6.0–11.3] vs. 6.3 [5.2–7.1] arb. units, p<0.01) than patients with transient risk factors ( N=16). Compared with controls, these variables were increased in patients with persistent risk factors (all p<0.01), but not in patients with transient risk factors (all n.s.). Conclusion: In patients with persistent risk factors rheological changes are still present 1 year after acute DVT, whereas in patients with transient risk factors blood rheology returns to normal. Further studies are needed to clarify whether blood rheology might be helpful to identify patients at high risk of recurrence.

Detection of a Thrombolysis-Related Reduction in Red Blood Cell Adhesiveness/Aggregation Using a Simple Slide Test

Increased red cell aggregability might have unfavorable rheological effects in the microcirculation. It has been suggested that thrombolysis-related hypofibrinogenemia might be associated with a reduced erythrocyte adhesiveness/aggregation. We followed the reduction in erythrocyte adhesiveness/aggregation using a simple slide test and image analysis that measures the spaces that are formed between the aggregated erythrocytes. A significant (p = 0.01) reduction in the degree of erythrocyte adhesiveness/aggregation was noted in patients with acute myocardial infarction who received thrombolysis as opposed to individuals with Braunwald Class IIIB unstable angina who had normal fibrinogen concentrations. No change was found in the Westergren erythrocyte sedimentation rate, which is an indirect method to detect changes in aggregability of red blood cells. The present study shows the superiority of using a direct measurement of red blood cell adhesiveness/aggregation. This extremely rapid, cheap and almost bedside methodology to detect changes in erythrocyte adhesiveness/aggregation might be useful to detect changes of hemorheological relevance following thrombolysis.

Simulation of Ultrasound Backscattering by Red Cell Aggregates:Effect of Shear Rate and Anisotropy

Tissue characterization using ultrasound (US) scattering allows extraction of relevant cellular biophysical information noninvasively. Characterization of the level of red blood cell (RBC) aggregation is one of the proposed application. In the current paper, it is hypothesized that the microstructure of the RBCs is a main determinant of the US backscattered power. A simulation model was developed to study the effect of various RBC configurations on the backscattered power. It is an iterative dynamical model that considers the effect of the adhesive and repulsive forces between RBCs, and the effect of the flow. The method is shown to be efficient to model polydispersity in size, shape, and orientation of the aggregates due to the flow, and to relate these variations to the US backscattering properties. Three levels of aggregability at shear rates varying between 0.05 and 10 s −1 were modeled at 40% hematocrit. The simulated backscattered power increased with a decrease in the shear rate or an increase in the RBC aggregability. Angular dependence of the backscattered power was observed. It is the first attempt to model the US power backscattered by RBC aggregates polydisperse in size and shape due to the shearing of the flow.

Effects of direct adsorption of lipoproteins apheresis on lipoproteins, low-density lipoprotein subtypes, and hemorheology in hypercholesterolemic patients with coronary artery disease.

Direct adsorption of lipoproteins (DALI) apheresis has been shown to reduce effectively low-density lipoprotein (LDL) cholesterol and lipoprotein (a) concentrations. However, the effects on nontraditional risk indicators such as hemorheology and LDL subtypes have not been investigated so far. Five patients (2 women, 3 men, age 53 +/- 8 years) with coronary artery disease and severe LDL hypercholesterolemia regularly treated with other LDL apheresis devices entered the study and were then treated with DALI for the first time. Hemorheological and lipoprotein parameters were measured before and immediately after the initial DALI apheresis as well as before the fourth DALI apheresis. Compared to baseline (before the first DALI apheresis), the following parameters were significantly improved (p < 0.05) after the first DALI apheresis: LDL cholesterol (69 +/- 28 versus 208 +/- 82 mg/dl) and cholesterol in each LDL subfraction as well as plasma viscosity (1.23 +/- 0.04 versus 1.37 +/- 0.06 mPa), C-reactive protein, native blood viscosity, red cell aggregation, and red cell deformability. When parameters before the fourth DALI apheresis were compared to baseline, LDL cholesterol was still lower, and red cell deformability was still improved while cholesterol in each subfraction showed a statistical trend to lower concentrations (0.08 < p < 0.14). In conclusion, DALI apheresis not only reduces LDL cholesterol but also induced a significant reduction of cholesterol in all LDL subfractions and improved various hemorheological parameters.

A point process approach to assess the frequency dependence of ultrasound backscattering by aggregating red blood cells.

To study the shear-thinning rheological behavior of blood, an acoustical measurement of the erythrocyte aggregation level can be obtained by analyzing the frequency dependence of ultrasonic backscattering from blood. However, the relation that exists among the variables describing the aggregation level and the backscattering coefficient needs to be better clarified. To achieve this purpose, a three-dimensional random model, the Neyman-Scott point process, is proposed to simulate red cell clustering in aggregative conditions at a low hematocrit (H<5%). The frequency dependence of the backscattering coefficient of blood, in non-Rayleigh conditions, is analytically derived from the model, as a function of the size distribution of the aggregates and of their mass fractal dimension. Quantitative predictions of the backscatter increase due to red cell aggregation are given. The parametric model of backscatter enables two descriptive indices of red cell aggregation to be extracted from experimental data, the packing factor W and the size factor delta. Previously published backscatter measurements from porcine whole blood at 4.5% hematocrit, in the frequency range of 3.5 MHz-12.5 MHz, are used to study the shear-rate dependence of these two indices.

Atorvastatin improves blood rheology in patients with familial hypercholesterolemia (FH) on long-term LDL apheresis treatment

To determine the effect of atorvastatin on blood rheology in patients with familial hypercholesterolemia (FH) on regular LDL apheresis, we prospectively studied the rheological variables fibrinogen, plasma viscosity, red cell aggregation, whole blood viscosity, hematocrit and platelet aggregation in 12 patients (two homozygous, ten heterozygous) before and during treatment with atorvastatin. Baseline values of red cell aggregation and whole blood viscosity were increased in FH patients on regular LDL apheresis compared with healthy controls ( P<0.05), whereas fibrinogen, plasma viscosity and hematocrit were similar in the two groups. Treatment with atorvastatin reduced red cell aggregation ( P<0.01), whole blood viscosity ( P<0.01), plasma viscosity ( P<0.01) and platelet aggregation ( P<0.05), but caused a slight increase in plasma fibrinogen (by 5%; P<0.01). Our findings suggest that atorvastatin improves blood rheology in patients with FH on regular LDL-apheresis. This improvement in blood flow properties may contribute to the well-known beneficial effects of atorvastatin on cardiovascular risk in patients with severe hyperlipidemia and atherosclerotic vascular disease.

In vivo measurements of ultrasonic backscattering in blood.

Ultrasonic backscattering in blood including its dependence on the hematocrit, plasma proteins, shear rate, and flow disturbance, has been studied extensively theoretically and experimentally in vitro. However, much of the result has never been validated in vivo. To do so, backscattering measurements were made on pigs using a 10-MHz non-focused intravascular transducer in direct contact with blood. The probe was placed in either the abdominal aorta or the inferior vena cava. The backscattering coefficient (BSC) of blood flowing in these vessels as well as downstream from a stenosis was measured using an approach that was originally developed for measurements with focused transducers. With this approach, 6% porcine red cell saline suspensions prepared immediately after each in vivo measurement were used as the reference medium. Result from seven pigs at hematocrits ranging from 29 to 36% (31.9 +/- 2.5%) demonstrated that BSC of blood in the vena cava, (4.62 +/- 2.06) x 10(-5) cm-sr-1, is consistently higher than that in the aorta, (2.65 +/- 1.22) x 10(-5) cm-sr-1. The difference has been attributed to the lower shear rate and the formation of red cell aggregation in venous blood. These in vivo results are in agreement with those obtained in vitro. In response to stenoses created by ligating the aorta, backscattering of the blood measured downstream from the stenosis showed that the closer the site of measurement relative to the stenosis, the higher the backscatter, presumably resulting from the higher degree of flow disturbance. In vitro backscattering results on porcine whole blood were also acquired at 20 MHz with a Diasonics intravascular scanner.

Characteristics of leucocyte adhesion directly observed in flowing whole blood in vitro.

Use of whole blood in adhesion assays allows analysis of the rheological and haematological factors that may influence adhesion, and avoids the need for isolation procedures that may modify the properties of leucocytes. We have adapted an in vitro flow model to allow videomicroscopy of leucocytes fluorescently labelled with rhodamine 6G (20 microg/ml) in anticoagulated whole blood. Blood was perfused at a range of wall shear rates (35-280/s) through a vertical glass capillary with a rectangular cross-section (microslide) that had been coated with P-selectin (10 microg/ml). Nearly all adherent cells were rolling in blood that had been anticoagulated with buffered citrate, but 40-50% became immobilized when heparin or thrombin inhibitor (PPACK) were used. The efficiency of leucocyte adhesion decreased steadily during 1-4 h of blood storage. Smaller fluorescent cells (lymphocytes) adhered less efficiently than larger cells (granulocytes) and rolled faster. Adhesion decreased monotonically with increasing wall shear rate or stress, but the velocity of rolling varied little. Among healthy volunteer donors, adhesion correlated with blood leucocyte count, but did not vary significantly with natural variation in haematocrit, blood viscosity or red cell aggregation. In conclusion, we have characterized adhesion of leucocytes in flowing whole blood, identified key experimental variables and demonstrated that physical environmental factors can markedly influence adhesive behaviour.