Biosensing of Haemorheological Properties Using Microblood Flow Manipulation and Quantification.

The biomechanical properties of blood have been used to detect haematological diseases and disorders. The simultaneous measurement of multiple haemorheological properties has been considered an important aspect for separating the individual contributions of red blood cells (RBCs) and plasma. In this study, three haemorheological properties (viscosity, time constant, and RBC aggregation) were obtained by analysing blood flow, which was set to a square-wave profile (steady and transient flow). Based on a simplified differential equation derived using a discrete circuit model, the time constant for viscoelasticity was obtained by solving the governing equation rather than using the curve-fitting technique. The time constant (λ) varies linearly with respect to the interface in the coflowing channel (ß). Two parameters (i.e., average value: <λ>, linear slope: dλdß) were newly suggested to effectively represent linearly varying time constant. <λ> exhibited more consistent results than dλdß. To detect variations in the haematocrit in blood, we observed that the blood viscosity (i.e., steady flow) is better than the time constant (i.e., transient flow). The blood viscosity and time constant exhibited significant differences for the hardened RBCs. The present method was then successfully employed to detect continuously varying haematocrit resulting from RBC sedimentation in a driving syringe. The present method can consistently detect variations in blood in terms of the three haemorheological properties.

Red Blood Cell Morphodynamics in Patients with Polycythemia Vera and Stroke.

Polycythemia vera (PV) is a Ph-negative myeloproliferative neoplasm (MPN) which is characterized by erythrocytosis and a high incidence of thrombotic complications, including stroke. The study aimed to evaluate red blood cell (RBC) morphodynamic properties in PV patients and their possible association with stroke. We enrolled 48 patients with PV in this cross-sectional study, 13 of which have a history of ischemic stroke. The control group consisted of 90 healthy subjects. RBC deformability and aggregation analysis were performed using a laser-assisted optical rotational red cell analyzer. The following parameters were calculated: aggregation amplitude (Amp), RBC rouleaux formation time constant (Tf), time of formation of three-dimensional aggregates (Ts), aggregation index (AI), rate of complete disaggregation (y-dis), and the maximal elongation of RBC (EImax). Statistical analysis was performed with the R programming language. There were significant differences in RBCs morphodynamics features between patients with PV and the control group. Lower EImax (0.47 (0.44; 0.51) vs. 0.51 (0.47; 0.54), p < 0.001) and γ-dis (100 (100; 140) vs. 140 (106; 188) s-1, p < 0.001) along with higher amplitude (10.1 (8.6; 12.2) vs. 7.7 (6.6; 9.2), p < 0.001) was seen in patients with PV compared with control. A statistically significant difference between PV patients with and without stroke in aggregation amplitude was found (p = 0.03). A logistic regression model for stroke was built based on RBC morphodynamics which performed reasonably well (p = 0.01). RBC alterations may be associated with overt cerebrovascular disease in PV, suggesting a possible link between erythrocyte morphodynamics and increased risk of stroke.

The impact of circulation in a heart-lung machine on function and survival characteristics of red blood cells.

Extracorporeal circulation is accompanied by changes in red blood cell morphology and structural integrity that affect cell function and survival, and thereby may contribute to the various side effects of heart-lung machine-assisted surgery. Our main objectives were to determine the effect of circulation of red blood cells in a stand-alone extracorporeal circuit on several parameters that are known to be affected by, as well as contribute to red blood cell aging. As a source of RBCs, we employed blood bank storage units of different ages. In order to assess the relevance of our in vitro observations for the characterization of extracorporal circulation technology, we compared these changes in those of patients undergoing extracorporeal circulation-assisted cardiac surgery. Our results show that circulation in a heart-lung machine is accompanied by changes in red blood cell volume, an increase in osmotic fragility, changes in deformability and aggregation behavior, and alterations in the exposure of phosphatidylserine and in microvesicle generation. RBCs from 1-week-old concentrates showed the highest similarities with the in vivo situation. These changes in key characteristics of the red blood cell aging process likely increase the susceptibility of red blood cells to the various mechanical, osmotic, and immunological stress conditions encountered during and after surgery in the patient's circulation, and thereby contribute to the side effects of surgery. Thus, aging-related parameters in red blood cell structure and function provide a foundation for the validation and improvement of extracorporeal circulation technology.

High-intensity interval training irrespective of its intensity improves markers of blood fluidity in hypertensive patients.

Aim: The present study examined and compared the effects of two different HIIT (High-intensity interval training) protocols on markers of blood fluidity in hypertensive patients.Methods: Thirty hypertensive (stage 1, systolic BP >140 and diastolic BP>90 mmHg) patients (age, 47.96 ± 3.20 yrs), were randomly allocated to short-duration HIIT (SDHIIT, n = 10), long-duration HIIT (LDHIIT, n = 10), and control (n = 10) groups. After 2 weeks of continuous mild training, patients in SDHIIT group performed 8 weeks of HIIT included 27 min HIIT that encompassed 27 repetitions of 30 s activity at 80%-100% of VO2peak interspersed by 30 s passive/active (10%-20% of VO2peak) recovery, while, patients in LDHIIT group performed 8 weeks of HIIT (32 min per session) included 4 repetitions of 4 min activity at 75%-90% of VO2peak interspersed by 4 min passive/active (15%-30% of VO2peak) recovery. Two blood samples were taken before and after training and were analyzed for hemorheological variables.Results: Significant (P < .05) reductions in systolic blood pressure (SBP), blood and plasma viscosity, fibrinogen concentration and red blood cell (RBC) aggregation (8-12%) were found following two training protocols (P < .05), though, the differences between adaptations were not statistically significant (P > .05). In addition, HIIT protocols increased RBC deformability significantly (P < .05), with no significant differences being observed between two protocols.Conclusion: It is concluded that HIIT training reduces SBP and markers of blood fluidity in patients with stage 1 hypertension irrespective of the HIIT intensity and duration. Therefore, this type of exercise training could be prescribed for improving the blood fluidity markers in hypertensive patients.

Ultrasound Standing Wave-Based Cell-to-liquid Separation for Measuring Viscosity and Aggregation of Blood Sample.

When quantifying mechanical properties of blood samples flowing in closed fluidic circuits, blood samples are collected at specific intervals. Centrifugal separation is considered as a required procedure for preparing blood samples. However, the use of centrifuge is associated with several issues, including the potential for red blood cell (RBC) lysis, clotting activation, and RBC adhesions in the tube. In this study, an ultrasonic transducer is employed to separate RBCs or diluent from blood sample. The ultrasonic radiation force is much smaller than the centrifugal force acting in centrifuge, it can avoid critical issues occurring under centrifuge. Then, the RBC aggregation and blood viscosity of the blood sample are obtained using the microfluidic technique. According to the numerical results, ultrasonic transducers exhibited a maximum quality factor at an excitation frequency of 2.1 MHz. Periodic pattern of acoustic pressure fields were visualized experimentally as a column mode. The half wavelength obtained was as 0.5 λ = 0.378 ± 0.07 mm. The experimental results agreed with the analytical estimation sufficiently. An acoustic power of 2 W was selected carefully for separating RBCs or diluent from various blood samples (i.e., Hct = 20% ~ 50%; diluent: plasma, 1x phosphate-buffered saline (PBS), and dextran solution). The present method was employed to separate fixed blood samples which tended to stack inside the tube while using the centrifuge. Fixed RBCs were collected easily with an ultrasonic transducer. After various fixed blood samples with different base solutions (i.e., glutaraldehyde solution, 1x PBS, and dextran solution) were prepared using the present method, RBC aggregation and the viscosity of the blood sample are successfully obtained. In the near future, the present method will be integrated into ex vivo or in vitro fluidic circuit for measuring multiple mechanical properties of blood samples for a certain longer period.

Quantitative Measurement and Evaluation of Red Blood Cell Aggregation in Normal Blood Based on a Modified Hanai Equation.

The aggregation of red blood cells (RBCs) in normal blood (non-coagulation) has been quantitatively measured by blood pulsatile flow based on multiple-frequency electrical impedance spectroscopy. The relaxation frequencies fc under static and flowing conditions of blood pulsatile flow are utilized to evaluate the RBC aggregation quantitatively with the consideration of blood flow factors (RBC orientation, deformation, thickness of electrical double layer (EDL)). Both porcine blood and bovine blood are investigated in experiments, for the reason that porcine blood easily forms RBC aggregates, while bovine blood does not. The results show that the relaxation frequencies fc of porcine blood and bovine blood present opposite performance, which indicates that the proposed relaxation frequency fc is efficient to measure RBCs aggregation. Furthermore, the modified Hanai equation is proposed to quantitatively calculate the influence of RBCs aggregation on relaxation frequency fc. The study confirms the feasibility of a high speed, on-line RBC aggregation sensing method in extracorporeal circulation systems.

MONITORING OF HEMORHEOLOGICAL PARAMETERS IN PATIENTS WITH ATRIAL FIBRILLATION (INITIAL DATA).

The goals of this project were to determine how hemodynamics and hemorheological change in patients diagnosed with different forms of atrial fibrillation; also, how relievers of changes in fibrinogen monitoring in patients with permanent, persistent, paroxysmal forms of arrhythmias. There was examined 30 patients (the average age of patients 65). Patients and control groups have been tested the following studies: index of erythrocytes aggregability, deformability, plasma viscosity to evaluate the blood rheological parameter, also -Fibrinogen to determine of coagulation condition. The Index of the Erythrocytes aggregation was done with the system of textural analyzes. These new innovative methods "Georgian Technique" is created by Georgian scientists and they are famous in the world as direct, numeral and exact. The index of the deformation of the erythrocytes was done with filtration method. Plasma viscosity was measured in the capillary viscosimeter in 370 C. According to the obtained data patients with atrial fibrillation have the same conditions of rheological and coagulation systems, despite of the forms of the atrial fibrillation. And it is different compared to the control group. In the statistical processing of the total row of fibrinogen, the patients were divided into 3 categories. As it turned out fibrinogen and Index of erythrocytes aggregation are in a linear relationship. The quantity of fibrinogen and of erythrocytes aggregation increase simultaneously and the greater the sequential number of the category is, the changes are more pronounced. However, the change/variability of each biological parameter, as shown from our data, is not uniform and linear. The obtained results clearly illustrate the existence of two parallel mechanisms in the body. These are on the one hand the systems of coagulation, anti-coagulation and fibtinollysis. These processes are in a state of the weighted condition, and they are characterized by dynamic equilibrium. On the other hand, the rheology system, which involves the combination of blood flow, blood velocity, vascular stiffness / elasticity, is characterized by one direction going on, with the adequacy and non-existing antipodal mechanisms. The hemorheological system does not have a physiologically balanced opposing anti-rheological system. All of this generate that the hemorheological status is very important in the development and formation of some disease. The arrangement of a rheologycal system is taking preventive character. On the one hand, the rheologycal system is a consolidation of diagnosis and evaluation of the mechanisms. Also, it is the treatment target. Normalization of them is very important in the therapeutic standpoint of the individual.

Hemorheological dysfunction in cardiac syndrome X.

BACKGROUND: Cardiac syndrome X (CSX) is often described as angina or angina-like chest pain with a normal coronary arteriogram, yet the underlying pathophysiological mechanisms have not been fully elucidated. The aim of the current study was to determine alterations in blood rheology (erythrocyte aggregation and deformability, plasma viscosity - PV) in patients with CSX. METHODS: The study comprised 26 CSX patients (55.77 ± 12.33 years) and 37 age- and sex-matched (56.32 ± 11.98 years) healthy controls. Erythrocyte aggregation and deformability were measured by an ektacytometer and PV with a rotational viscometer. RESULTS: Erythrocyte deformability measured at 1.69 and 3.00 Pa was lower in the CSX patients compared to the controls (p = .0001 and .017, respectively). Erythrocyte aggregation index (AI) (72.758 ± 7.65 vs. 66.483 ± 6.63, p = .002) and PV measured at a shear rate of 375 s-1 (1.932 ± 0.225 vs. 1.725 ± 0.331, p = .019) were significantly higher in patients with CSX. When AI, RDW and erythrocyte deformability measured at 1.69 Pa were evaluated together, it was observed that the increase in AI and RDW augments the risk of having CSX (OR: 1.2 and 2.65, respectively), while the rise in deformability decreases this risk (OR = 0.02). CONCLUSIONS: Hemorheological impairments are associated with CSX.

Differentiating between cold agglutinins and rouleaux: a case series of seven patients.

We present a case series of seven patients with suspected cold agglutinin antibodies, discovered after initiation of bypass. Laboratory analysis of blood samples intraoperatively determined the cause of the aggregation to be rouleaux formation in three of the patients and cold agglutinins in the other four.

Optical aggregometry of red blood cells associated with the blood-clotting reaction in extracorporeal circulation support.

The aggregability of red blood cell (RBCs) is associated with the contribution of plasma proteins, such as fibrinogen and lipoproteids, to blood-clotting. Hence, we hypothesized that RBC aggregability reflects the blood-clotting reaction. A noninvasive optical monitoring method to measure RBC aggregability for the assessment of blood-clotting stage during mechanical circulatory support was developed. An in vitro thrombogenic test was conducted with a rotary blood pump using heparinized fresh porcine blood. Near-infrared laser light at a wavelength of 785 nm was guided by an optical fiber. The fibers for detecting incident, forward-, and backward-scattered light were fixed on the circuit tubing with an inner diameter of 1/4 inch. Because there is substantial RBC aggregation at low shear flow rates, a pulsatile flow was generated by controlling the pump rotational speed. The flow rate was changed from 0 to 8.5 L/min at a period of 40 s. The intensities of forward- and backward-scattered light changed dramatically when the flow stopped. The aggregability was evaluated by the increase ratio of the transmitted light intensity from the flow stopping in the low-flow condition. The experiment started when the anticoagulation was stopped by the addition of protamine into the circulating blood. Reduction in RBC aggregability was associated with a decrease in the amount of fibrinogen and the number of platelets. Continuous, noninvasive monitoring of thrombosis risk is possible using optical measurements combining pulsatile flow control of a rotary blood pump. RBC aggregometry is a potential label-free method for evaluating blood-clotting risk.

Enhanced RBC Aggregation in Type 2 Diabetes Patients.

BACKGROUND: This study aimed to investigate the relationship between HbA1c and RBC aggregation in type 2 diabetes mellitus (T2DM) patients by analysis of data from routine clinical tests and from in vitro experiments. METHODS: A total of 2,111 inpatients with type 2 diabetes were selected and among them, 364 patients (Group A) had limited influence of plasma proteins on erythrocyte sedimentation rate (ESR) and was compared with the rest of the 1,747 inpatients (Group B). ESR, HbA1c , WBC, CRP, Fbg, and HCT were measured in all samples. Sixty samples were also collected from T2DM patients and used for in vitro ESR studies. Spearman's correlation coefficients were employed to reflect the correlation between ESR and other parameters. Mann-Whitney U test was used to compare the study parameters. RESULTS: The test results for Group A were lower than Group B with respect to ESR, age, HCT, HbA1c , CRP, WBC, and Fbg. Only the difference in HbA1c , CRP, and Fbg values had statistical significance (P < 0.05). In addition, HbA1c correlated better with ESR for Group A (R = 0.622) than Group B (R = 0.563), whereas CRP and Fbg were contrary to this. In the in vitro studies, the HbA1c values were classified into the subgroups of 6.5-8.0%, 8.1-10%, and >10%. The corresponding ESR values were 28 ± 5.1 mm/h, 33 ± 2.7 mm/h, and 40 ± 4.1 mm/h, respectively. CONCLUSION: ESR results of T2DM patients were elevated that was mainly caused by Fbg levels, and in addition HbA1c in part contributed to RBC aggregation.

Relative permittivity measurement during the thrombus formation process using the dielectric relaxation method for various hematocrit values.

The relative permittivity ε' and the dielectric loss ε″ for various hematocrit values H for static bovine blood condition have been measured using the dielectric relaxation method to detect thrombosis in real time. The suitable measurement frequency f m ranged within 60 kHz to 1 MHz, and the relaxation frequency of red blood cells (RBCs) f rc was observed to be 2 MHz. In the f m, the temporal change of normalized ε' exhibited a minimum (called as bottom point). The bottom point was observed to be exponentially shortened as H increased. This characteristic of the ε'* minimum is discussed from three viewpoints: during fibrin formation, direct thrombus formation, and rouleaux formation processes. ε'* during the fibrin formation process decreased over time, irrespective of f. However, ε'* in f m during the direct thrombus formation process and during the aggregation formation process increased immediately and rapidly over time. Therefore, the ε'* bottom point in f m might be the indication of micrometer-scale thrombus formation by RBC aggregation due to fibrin formation.

The investigation of interspecies diversity of erythrocyte aggregation properties by two different photometric methods in four animal species.

Among the haemorheological parameters, red blood cell (RBC) aggregation shows the largest interspecies diversity, and often controversial data can be found in the literature, besides the methodology-dependent issues. In this present investigation, we compared four experimental/laboratory animal species' RBC aggregation by two different photometric methods for better revealing the differences. Blood samples (K3-EDTA, 1.5 mg/ml) were taken from female animals: 16 inbred mice (Mus musculus, cardiac puncture), 15 outbred rats (Rattus norvegicus, caudal caval vein puncture), 15 beagle dogs (Canis canis, cephalic vein) and 23 juvenile pigs (Sus scrofa domesticus, medial saphenous vein). Haematological parameters (microcell counter) and RBC aggregation (light transmission and syllectometry-laser backscatter methods) were determined within 2 h after sampling. Describing the first 5-10 s of the aggregation process, additional parameters were calculated out of the syllectometric raw data. Standardized difference was calculated to determine the sensitivity of the two devices. Parameters describing the extent and magnitude of red blood cell aggregation showed the lowest values in the rat and the highest in the pig and canine blood. In turn, parameters describing the kinetics of aggregation showed the lowest values in the mouse and the highest in the rat. The standardized difference values for the laser backscattering method were 2-4 times larger vs. the light transmission one. The magnitude of the differences was not consequent in the aggregation parameters. These comparative results show that the laser backscattering method can detect the RBC aggregation differences between the investigated species more sensitively than the light transmission method.

Global Tests in Evaluation of the Function of Pro-and Anticoagulant Systems: Present and Future.

Comparative analysis of the effectiveness of global tests for evaluation of the blood coagulation systems demonstrated the possibility of obtaining express data on hemostasis stages I-II and III when working with native substrate (whole blood). Changes in viscous characteristics of the whole blood recorded ex vivo during hemocoagulation allowed us to propose the concept of permanency of fibrinogenesis as the obligatory in vivo process determining the hemostatic potential, an integral characteristic of the hemocoagulation cycle providing sufficient blood fluidity and limiting extravasation of blood components under conditions of permeability disturbances and damage to the vascular wall.

Effect of uneven red cell influx on formation of cell-free layer in small venules.

This study examined how the uneven influx of red blood cells (RBCs) from feeding vessels influences formation of cell-free layer (CFL) in the downstream vessel of a venular bifurcation. Spatio-temporal variations of the CFL width along the downstream vessel (19-41-µm inner diameter, D) were determined at 0.5D intervals from 0.5D to 3.0D away from the bifurcation. Upstream flow conditions were quantified by the ratio of volume flow rates (Q*=Q(High)/Q(Low)) between high flow (Q(High)) and low flow feeding (Q(Low)) vessels. The RBC aggregation level in the rats was adjusted to be at healthy human levels by infusing Dextran 500. Our results suggested that the CFL formation process could be seen only from 2.0D away from the bifurcating point. The mean CFL width at the wall adjacent to the feeding vessel with a higher flow rate was consistently greater than that at the opposite wall, leading to an asymmetric CFL formation in the vessel. A positive relation (P<0.05) between the asymmetry of the CFL width and the volume flow rate ratio (Q*) was found. Our numerical prediction showed that flow resistance in the venular network could be significantly increased by the asymmetric formation of CFL downstream and this effect might become more pronounced under pathological flow conditions such as hyper-aggregating and/or low shear conditions.

Which sub-compartments of fat mass and fat-free mass are related to blood viscosity factors?

The size of body compartments is a determinant of several factors of blood viscosity. Red cell aggregation is proportional to fat mass while hematocrit is proportional to both fat-free mass and abdominal adiposity, but which parts of these body components are involved in this relationship is not known. Segmental bioelectrical impedance analysis (sBIA) provides a possibility to delineate the relationships more precisely between various subdivisions of the body and blood viscosity factors, going farther than preceding studies using non segmental BIA. In this study we investigated in 38 subjects undergoing a standardized breakfast test with mathematical modelling of glucose homeostasis and a segmental bioelectrical impedance analysis (sBIA) the relationships between the various compartments of the body and viscosity factors. Blood and plasma viscosity were measured with the Anton Paar rheometer and analyzed with Quemada's model. The parameters better correlated to hematocrit are fat free mass (r = 0.562) and its two components muscle mass (r = 0.516) and non-muscular fat-free mass (r = 0.452), and also trunk fat mass (r = 0.383) and waist-to hip ratio (r = 0.394). Red cell aggregation measurements were correlated with both truncal and appendicular fat mass (r ranging between 0.603 and 0.728). Weaker correlations of M and M1 are found with waist circumference and hip circumference. This study shows that the correlation between lean mass and hematocrit involves both muscle and non-muscle moieties of lean mass, and that both central and appendicular fat are determinants of red cell aggregation.

Two-phase model for prediction of cell-free layer width in blood flow.

This study aimed to develop a numerical model capable of predicting changes in the cell-free layer (CFL) width in narrow tubes with consideration of red blood cell aggregation effects. The model development integrates to empirical relations for relative viscosity (ratio of apparent viscosity to medium viscosity) and core viscosity measured on independent blood samples to create a continuum model that includes these two regions. The constitutive relations were derived from in vitro experiments performed with three different glass-capillary tubes (inner diameter=30, 50 and 100 µm) over a wide range of pseudoshear rates (5-300 s(-1)). The aggregation tendency of the blood samples was also varied by adding Dextran 500 kDa. Our model predicted that the CFL width was strongly modulated by the relative viscosity function. Aggregation increased the width of CFL, and this effect became more pronounced at low shear rates. The CFL widths predicted in the present study at high shear conditions were in agreement with those reported in previous studies. However, unlike previous multi-particle models, our model did not require a high computing cost, and it was capable of reproducing results for a thicker CFL width at low shear conditions, depending on aggregating tendency of the blood.

Numerical simulations of deformation and aggregation of red blood cells in shear flow.

This article reviews numerical simulations of red blood cells (RBCs) mainly using the lattice Boltzmann method (LBM), focusing on the 2-dimensional deformation and aggregation of the cells in simple shear flow. We outline the incorporation of the immersed boundary method into the LBM, in which the membrane forces are obtained from the membrane model. The RBCs are simulated as a single biconcave capsule and as a doublet of biconcave capsules. The transition from swinging to tumbling motions of the RBCs, as induced by reducing the shear rate or increasing the membrane bending stiffness, is discussed. Also discussed is the aggregation tendency of the doublet of RBCs, for which homogenous deformability maintained RBC aggregation, whereas an increased deformability difference resulted in RBC dissociation.

[Non-invasive methods for the assessment of platelet, leukocyte, erythrocyte aggregation and coagulation hemostasis].

A non-invasive method for the study of platelet aggregation and formation of leukocyte-erythrocyte-platelet aggregates as well as certain hemostatic parameters is proposed. The method is based on the speckle-analysis of coherent light scattering from the surface of erythrocytes moving in an artificially isolated vessel segment. It was shown that light scattering index significantly correlated with ADP-, adrenalin-, or collagen-induced platelet aggregation, with the formation of leukocyte-erythrocyte or platelet-erythrocyte aggregates, and with the levels of fibrinogen, soluble fibrin-monomer complexes and related parameters. It is concluded that the proposed method for the study of hemostatic system can be used to roughly evaluate intensity of intravascular blood coagulation and probability of thrombosis.

[Two-week magnetic deprivation does not alter rheologic parameters of rat's erythrocytes].

Purpose of the work was to explore likelihood of impairment of erythrocytes deformation and aggregation properties in a compensated magnetic field of the Earth. It was shown that complete magnetic deprivation does not alter rat's hemorheology substantially. Stress-related changes were found neither in blood nor adrenals of the animals.