Development of experimental microfluidic device and methodology for assessing microrheological properties of blood.

BACKGROUND AND OBJECTIVE: Microfluidics is a useful tool for investigating blood microrheology. The study aimed to present the development of a microfluidic device for assessing the microrheological properties of blood cells' suspensions and its application in patients with diabetes mellitus type 2 (T2DM). METHODS: A new microfluidic device was elaborated, connected to a system, including a microscope with a digital camera, a pump with a manometer and a computer with specially developed software. Blood cells' suspensions were investigated in a microchamber between two parallel optical slides within a 100µm distance. The motion of the blood cells in the microchamber was observed by the microscope and it was recorded and visualized by a digital camera. A method for evaluating the deformability of blood cells and a device for its implementation were used [1]. RESULTS: The pressure and flow rate ranges in the microfluidic device were specified by model suspensions of beta-ferroxy-hydroxide and red blood cells (RBC) suspensions. The pressure changes, realized by a pump (micropipette), connected to a manometer were established and the corresponding shear rates in the microfluidic device were determined. Data about the blood microrheological properties like RBC aggregation and deformability, leukocyte adhesion from a group of healthy volunteers and from patients with T2DM were obtained. CONCLUSIONS: The developed device and experimental system is a promising tool for the study of blood microrheology.

Hemorheological and microvascular disturbances in patients with type 2 diabetes mellitus.

BACKGROUND: In the blood vessels the impaired hemorheological parameters in patients with type 2 diabetes mellitus (T2DM) could lead to elevated flow resistance, increased forces at the endothelial wall and to microvascular disturbances. OBJECTIVE: The aim of the study is to investigate the hemorheological variables and the changes of the skin blood flow responses to cold stress in T2DM patients. METHODS: The basic hemorheological parameters: hematocrit (Ht), fibrinogen (Fib), whole blood viscosity (WBV) and plasma viscosity (PV) were examined in 20 patients with T2DM and a control group of 10 healthy age and sex matched controls. The mechanisms of vascular tone regulation were investigated using the wavelet analysis of the skin temperature oscillations (WAST). The degrees of the microvascular tone changes were determined during a cold test in the endothelial (0.02-0.0095 Hz), neurogenic (0.05- 0.02 Hz) and myogenic (0.05- 0.14 Hz) frequency ranges. RESULTS: Significant increase of Fib and WBV in the patients in comparison to controls was found. The mean values of the amplitudes of the skin temperature (ST) pulsations decreased significantly during the cold stress only in the endothelial frequency range for the diabetic patients. CONCLUSIONS: The results of our study reveal parallel impairment of the blood rheological parameters and the cutaneous microcirculation in T2DM patients.

Methods in hemorheology and their clinical applications1.

The lecture was presented during the Fåhraeus award ceremony for 2016 at the University of Lisbon. It summarizes the main results and some of the more important hemorheological contributions achieved in the Laboratory of Biodynamics and Biorheology of the Institute of Mechanics to BAS and in collaboration with other laboratories of the research group, involved in many studies explaining hemorheological disturbances in various clinical conditions. An original method for the study of microstructural changes in the biological fluids by measuring the electrical conductivity simultaneously with the the rheological properties of red blood cells (RBC) in the whole blood and red blood cell suspensions in a viscometric flow was suggested. The influence of the disturbed hemorheological parameters on the common carotid artery and cerebral blood flow was studied. Analysis of blood flow in the common carotid artery bifurcation with stenosis was done. This lecture does not claim to be a comprehensive review, and many important studies were not cited. The author would like to acknowledge the valuable collaboration of all those cited in the reference list.

Hemorheology and vascular reactivity in patients with diabetes mellitus type 2.

The study aimed to investigate the hemorheological parameters in patients with diabetes mellitus type 2 and to estimate their relationship with the cerebral and cutaneous blood flow and their responses to postural changes. The basic hemorheological constituents: hematocrit (Ht), fibrinogen (Fib), whole blood (WBV) and plasma viscosity (PV) were examined in 20 patients with diabetes mellitus type 2 and in 10 healthy age and sex matched controls. Blood flow velocity in the middle cerebral artery (MCA) was measured by transcranial Doppler monitoring at rest and during 5-min head-up tilt. Also laser Doppler-recorded tiptoe skin blood flow was investigated and venoarteriolar reflex perfusion responses to postural impact was monitored. Significant increase of Fib and WBV at shear rates of 0.0237 s(-1) to 128.5 s(-1) in the patients in comparison to controls was found. The postural challenge caused decrease of the cerebral blood flow velocity and increase of the resistance index (RI) in the diabetic patients. The initial mean skin perfusion values of the tiptoes and the venoarteriolar constriction response indices were significantly higher in the diabetes group. In the patients with diabetes mellitus type 2 the increased blood viscosity values were associated with impaired cerebrovascular and peripheral vascular responces.

Hemorheology and heart rate variability in patients with diabetes mellitus type 2.

Our study aimed to investigate the relationship between hemorheological parameters and heart rate variability (HRV) in patients with diabetes mellitus type 2. Hemorheological variables, including hematocrit (Ht), fibrinogen (Fib), whole blood (WBV) and plasma viscosity (PV) at shear rates of 0.0237 s(-1) to 128.5 s(-1) were examined in 20 patients with diabetes mellitus type 2 and in 10 control subjects. They all underwent non-invasive short-term monitoring of heart rate at rest and after passive head-up tilt. Measurement of the R-R intervals and calculation of the time domain parameters and the power spectral data were performed by our softwear, using fast Fourier transformation. Significant increase of Fib and WBV in the patients in comparison to controls was found within the range of shear rates 0.0237 s(-1) to 128.5 s(-1). In the diabetic patients parallel decrease of the total power (TP), the low frequency spectral power (LF) and of the mean RR and mild increase of the low frequency-high frequency ratio (LF/HF) at rest were established. This tendency was kept after the passive tilt. In patients with diabetes mellitus type 2 the increased blood viscosity was associated with reduced HRV.

Common carotid artery hemodynamic factors in patients with cerebral infarctions.

The aim of the study was to investigate the changes of the common carotid local hemodynamic factors like wall shear stress and tensile forces in 16 patients with chronic unilateral cerebral infarctions (CUCI), 58 patients with risk factors (RF) for cerebrovascular disease (CVD) and 25 healthy control subjects. The blood flow velocities (BFV), the internal diameters (D) and the vessel wall intima-media thickness (IMT) in the common carotid arteries (CCA) were recorded with color duplex sonography. Systolic (SBP) and diastolic (DBP) blood pressure were measured and mean blood pressure (MBP) was calculated by the formula of Wiggers. Whole blood viscosity (WBV) at the shear rate of 94.5 s-1 was measured on the day of the Doppler ultrasound examination with a rotational viscometer Contraves Low Shear 30. Wall shear stress (WSS), the circumferential wall tension (T) and the tensile stress tau were calculated. The main RF in the patients' groups were hypertension and hyperlipidemia. The SBP, WBV and IMT were significantly increased in the patients with UCI and RF for CVD in comparison to controls. Lower systolic WSS and tau and higher T were established in the patients with UCI. The IMT correlated with WSS and tau. The study confirms the complex influence of the changes in WBV and blood pressure for the development of carotid atherosclerosis.

Influence of hemorheological parameters and mean blood pressure on carotid blood flow asymmetry in patients with chronic unilateral cerebral infarctions.

The study aimed to follow the influence of some hemorheological parameters and mean blood pressure (MBP) on the carotid blood flow asymmetry in 20 patients with chronic unilateral cerebral infarctions (UCI) as compared to 30 patients with transient ischemic attacks (TIAs), 17 patients with risk factors (RF) for cerebrovascular disease (CVD) and 11 healthy controls. Hemorheological variables: hematocrit (HT), plasma (PV) and whole blood viscosity (WBV), serum lipids and echocardiographic parameters were investigated. Blood pressure (systolic, diastolic and mean) was registered and color duplex sonography of the carotid arteries was performed. The lumen diameter, the intima media thickness (IMT) and the blood flow velocity (systolic, mean and diastolic) in the carotid arteries were examined and the velocity asymmetry ratio (AR) was calculated. Significant increase of HT in UCI, of mean blood pressure (MBP) in the patients with RF and TIAs and of IMT and velocity parameters in all patients was established. The correlation analysis revealed parallel influence of HT, MBP, cardiac function, IMT, lumen diameter and some of the modifiable RF on the carotid AR in patients with chronic UCI affecting mainly the collateral circulation. The complex investigation of these factors would enable us to individualize the therapeutic approach in the patients with cerebral ischemic events.

Platelet morphological, functional and rheological properties attributable to addictions.

Hemorheological abnormalities such as elevated whole blood viscosity, plasma viscosity, erythrocyte deformability and platelet aggregation, hematocrit and fibrinogen levels, are frequently examined as diagnostic tool and prognostic relevance in socially important hemorheological disorders. Distinct biological - morphological and functional platelet alterations, have been described in different addictions (heroin-, cocaine-, nicotine-, alcohol-, etc.). Chronic addictions could cause biochemical and conformational changes in platelets and their membranes, thus modulating platelet receptor expression, morphology (anisocytosis, giant platelets) and activation (alpha-granule release), platelet aggregation and hemorheological properties. Some of these alterations in chronic addicts - documented at cellular- and molecular level, could be easily used as a precise diagnostic tool with regard to thromboembolic complications and microcirulation injuries attributable to addictions. The present review focuses on some changes in platelet morphological, functional and rheological properties induced by chronic opiate/opioid abuse. Hypothesis is accumulated that free fatty acids (FFAs) and especially oleic acid (OA) could cause positive molecular and conformational changes in platelets of addicts with hemorheological disorders.

Erythrocyte aggregation at non-steady flow conditions: a comparison of characteristics measured with electrorheology and image analysis.

In the present study electro-rheology (Contraves LS30 viscometer-based system) and optical shearing microscopy (Lincam CSS450 system and image analysis) techniques have been utilized in order to provide quantitative data on the behaviour of the microstructural properties of whole normal human blood at non-steady flow conditions. The objective of this work is to contribute towards a better understanding of red blood cell aggregation at flow conditions similar to that occurring in a circulatory system and to aid the interpretation and validation of electro-rheological data through a quantitative comparison with data acquired with optical shearing microscopy. Electro-rheology is a promising technique that has been used to provide bulk fluid properties, showing potential for basic research and diagnostic purposes, whereas optical shearing techniques offer a direct assessment of blood microstructure at a cellular level. However, little information exists in the literature regarding the relationships between electro-rheological measurements and blood microstructural characteristics. The results showed that the different non-steady flow conditions affect differently the dynamics of aggregation varying from a parabolic-decrease to an inverted S-shape curve with time. For a wide range of the non-steady flows results obtained with the two different techniques agree to a difference between 1.2 and 12%.

Time dependent variation of human blood conductivity as a method for an estimation of RBC aggregation.

Time variation of whole human blood conductivity and shear stresses were investigated at rectangular and trapezium-shaped Couette viscometric flow under electric field of 2 kHz. The kinetics of conductivity signals were recorded both under transient flow and after the complete stoppage of shearing at shear rates from 0.94 to 94.5 s(-1) and temperatures T=25 degrees C and 37 degrees C. Contraves Low Shear 30 rotational viscometer as a base unit and a concurrent measuring system, including a device, developed by the conductometric method with a software for measurement of conductivity of biological fluids (data acquisition system), previously described (IFMBE Proceedings Series, Vol. 11, 2005, pp. 4247-4252; Clin. Hemorheol. Microcirc. 35(1/2) (2006), 19-29), were used for the experiments. The obtained experimental relationships show that the human blood conductivity is time, shear rate, hematocrit and temperature dependent under transient flow. It is also dependent on the regime of the applied shear rates. Non-linear curve approximation of the growth and relaxation curves is done. The results show that valuable information could be received about the kinetics of "rouleaux formation" and the time dependences of the blood conductivity follow the structural transformations of RBC aggregates during the aggregation-disaggregation processes. The results suggest that this technique may be used to clarify the mechanism of dynamics of RBC aggregates.

Plasma lipids and blood viscosity in patients with cerebrovascular disease.

It is known that plasma lipids could increase the cerebrovascular risk through alteration of the hemorheological profile. The aim of the study was to evaluate the relationship between blood viscosity parameters and plasma lipids in patients with cerebrovascular disease (CVD). The study included 43 patients with transient ischemic attacks (TIAs), 53 patients with chronic unilateral cerebral infarctions (UCI) and 57 patients with risk factors (RF) for CVD. Whole blood viscosity (WBV) at different shear rates by Couette rotational viscometer Contraves Low Shear 30, plasma viscosity (PV) with capillary viscometer, hematocrit (Hct), fibrinogen (Fib), Cholesterol (Chol), triglycerides (Tg) and high-density lipoproteins (HDL) were examined in all patients and in a control group of 56 presumed healthy subjects. The hemorheological results showed increase of Hct, Fib, WBV and PV in the patients with TIAs and UCI; it was more pronounced in the UCI patients. Significant increase of Hct and WBV in the group with RF for CVD was also found. The elevation of Chol and Tg predominated in the patients with UCI and in the subjects with RF for CVD. These lipid variables correlated significantly with PV in the TIAs and RF for CVD groups. Conclusion is drawn about the significance of plasma Tg for decrease of blood fluidity and for impairment of the cerebral circulation in CVD.

Hemorheological parameters in correlation with the risk factors for carotid atherosclerosis.

The study aimed to follow the relationship between some hemorheological variables and the main risk factors (RF) for carotid atherosclerosis (CA). Carotid atherosclerosis was evaluated by color duplex sonography of the carotid arteries in 18 patients with RF for CA, 31 patients with transient ischemic attacks (TIAs), 21 patients with chronic unilateral cerebral infarctions (UCI) and 11 healthy subjects without RF for CA. The examined hemorheological variables were whole blood and plasma viscosity, hematocrit and fibrinogen. They were correlated with intima-media thickness (IMT) of the common carotid and the internal carotid arteries and with other main RF for CA: hypertension, diabetes mellitus, coronary heart disease, and hyperlipidemia. The hemorheological investigation showed an increase in blood and plasma viscosity at different shear rates and it was more expressed in the group with UCI. The neurosonographic investigation revealed an increase in the IMT and carotid artery stenoses in the patients' groups with CVD. These were also more frequent in the patients with UCI. Different correlations were established between the hemorheological parameters, the IMT of the carotid arteries and other RF for CA. In the group with UCI, the hematocrit and the whole blood viscosity correlated significantly with the IMT, arterial blood pressure and cholesterol values. These data confirm the influence of the hemorheological parameters on carotid blood vessel walls and on blood flow in patients with CVD.

Evaluation of the hemorheological and neurosonographic relationship in patients with cerebrovascular diseases.

During the last fifteen years some of our priority scientific topics of research were hemorheological and neurosonographic investigations in 229 patients with different forms of cerebrovascular diseases (CVD): 75 patients with asymptomatic CVD (ACVD), 65 patients with transient ischemic attacks (TIAs) and 89 patients with chronic unilateral cerebral infarctions (UCI). The findings were compared with 70 healthy persons. The main estimated hemorheological parameters were hematocrit (Hct), apparent whole blood viscosity (WBV), plasma viscosity (PV) and fibrinogen (Fib). They were correlated with the following sonographic parameters, obtained by extracranial and transcranial Doppler sonography: blood flow velocities (BFV) and peripheral resistance index of Pourcelot (RP) of the major arteries of the head and the basal cerebral arteries and vasomotor reactivity indices (VMRI) of the middle cerebral arteries (MCA). Among the hemorheological variables the correlations of Hct with the velocity sonographic parameters predominated in all groups. Significant positive correlations between Hct, WBV and the RP of the internal carotid artery and MCA were found in patients with CVD. In UCI the increase in Hct and Fib was associated with a decrease in BFV of the collateral circulation where aging and high mean blood pressure were additional risk factors for impairment of the cerebral hemodynamics. Plasma viscosity was found to correlate with cerebral VMRI of MCA in patients with UCI. The clinical impact of these findings and their relation to the therapeutic strategy in CVD are discussed.

Hemorheological and hemodynamic effects of high molecular weight polyethylene oxide solutions.

Hemorheological and hemodynamic effects of high molecular weight polyethylene oxide (PEO) solutions have been studied in vitro and in vivo at 30 experimental dogs. The rheological behaviour of the PEO solutions and of blood samples has been assessed by rotational viscometers at 20 degrees C, 25 degrees C and 37 degrees C. An addition of PEO solutions to the blood in vitro has modified its rheological behavior, depending on the shear rates, concentration and temperature. Saline with aqueous PEO solutions at the concentration of 500 ppm has been infused into the blood circulation of the experimental dogs to achieve the total concentration in blood of 20-30 ppm. The following parameters cardiac output, mean arterial blood pressure, central venous pressure, pulse frequency, blood volume flow in the femoral artery, total vascular resistance and blood viscosity before and after the infusion of PEO solutions have been studied. The main observed effect was a decrease of the hemodynamic resistance in the cardiovascular system up to 40% below the baseline after infusion of PEO solutions.

Rheological behavior and parameters of the in vitro model of lung surfactant systems: the role of the main phospholipid component.

The proposed in vitro model for studying the alveolar surface layer of the lungs enables one to investigate the surface intermolecular forces which influence the stability of the alveolus. The general role for the stability of the alveolus belongs to the phospholipids in the alveolar surfactant and predominantly to their main component dipalmitoylphosphatidylcholine (DPPC). The aim of the study was to investigate the rheological behavior of DPPC and exogenous surfactant preparations used in neonatal clinical practice. Data for the rheological behavior of the solutions of the commercially available surfactants, Infasurf, Exosurf and Survanta, as well as of DPPC (their main phospholipid component) at shear rates from 0.024 to 94.5 s(-1) under steady and transient flow conditions at 23 degrees C were obtained. Infasurf and Exosurf showed Newtonian rheological behavior, while Survanta revealed the shear-thinning behavior of a non-Newtonian pseudoplastic fluid. The rheological properties of aqueous solutions of DPPC containing 0.14 M NaCl at concentrations from 100 and 630 microg/ml of phospholipid (chosen from the dependence of the probability for bilayer film formation) were studied. Differences observed in the rheological properties of the exogenous surfactants were interpreted on the basis of their composition, the presence of other phospholipid components, certain additives and surfactant proteins, as well as the bulk structures formed from them. The relevance of the results for the delivery of exogenous surfactants and their spreading in replacement therapy is discussed.