Erythrocyte swelling and membrane hole formation in hypotonic media as studied by conductometry.

Hypoosmotic swelling of erythrocytes and the formation of membrane holes were studied by measuring the dc conductance (G). In accordance with the theoretical predictions, these processes are manifested by a decrease in G followed by its increase. Thus, unlike the conventional osmotic fragility test, the proposed methodological approach allows investigations of both the kinetics of swelling and the erythrocyte fragility. It is shown that the initial rate of swelling and the equilibrium size of the cells are affected by the tonicity of a hypotonic solution and the membrane rheological properties. Because the rupture of biological membranes is a stochastic process, a time-dependent increase in the conductance follows an integral distribution function of the membrane lifetime. The main conclusion which stems from reported results is that information about rheological properties of red blood cell (RBC) membranes and the resistivity of RBCs to a certain osmotic shock may be extracted from conductance signals.

A novel approach for assessments of erythrocyte sedimentation rate.

INTRODUCTION: Previous studies have shown that the dispersed phase of sedimenting blood undergoes dramatic structural changes: Discrete red blood cell (RBC) aggregates formed shortly after a settling tube is filled with blood are combined into a continuous network followed by its collapse via the formation of plasma channels, and finally, the collapsed network is dispersed into individual fragments. Based on this scheme of structural transformation, a novel approach for assessments of erythrocyte sedimentation is suggested. METHODS: Information about erythrocyte sedimentation is extracted from time records of the blood conductivity measured after a dispersion of RBC network into individual fragments. RESULTS: It was found that the sedimentation velocity of RBC network fragments correlates positively with the intensity of attractive intercellular interactions, whereas no effect of hematocrit (Hct) was observed. CONCLUSION: Thus, unlike Westergren erythrocyte sedimentation rate, sedimentation data obtained by the proposed method do not require correction for Hct.

The mechanism of erythrocyte sedimentation. Part 2: The global collapse of settling erythrocyte network.

Results reported in the companion paper showed that erythrocytes in quiescent blood are combined into a network followed by the formation of plasma channels within it. This study is focused on structural changes in the settling dispersed phase subsequent to the channeling and the effect of the structural organization on the sedimentation rate. It is suggested that the initial, slow stage of erythrocyte sedimentation is mainly controlled by the gravitational compactness of the collapsed network. The lifetime of RBC network and hence the duration of the slow regime of erythrocyte sedimentation decrease with an increase in the intercellular pair potential and with a decrease in Hct. The gravitational compactness of the collapsed network causes its rupture into individual fragments. The catastrophic collapse of the network transforms erythrocyte sedimentation from slow to fast regime. The size of RBC network fragment is insignificantly affected by Hct and is mainly determined by the intensity of intercellular attractive interactions. When cells were suspended in the weak aggregating medium, the Stokes radius of fragments does not differ measurably from that of individual RBCs. The proposed mechanism provides a reasonable explanation of the effects of RBC aggregation, Hct and the initial height of the blood column on the delayed erythrocyte sedimentation.

The mechanism of erythrocyte sedimentation. Part 1: Channeling in sedimenting blood.

Despite extensive efforts to elucidate the mechanism of erythrocyte sedimentation, the understanding of this mechanism still remains obscure. In attempt to clarify this issue, we studied the effect of hematocrit (Hct) on the complex admittance of quiescent blood measured at different axial positions of the 2 mm x 2 mm cross-section chambers. It was found that after the aggregation process is completed, the admittance reveals delayed changes caused by the formation of cell-free zones within the settling dispersed phase. The delay time (tau(d)) correlates positively with Hct and the distance between the axial position where measurements were performed and the bottom and is unaffected by the gravitational load. These findings and literature reports for colloidal gels suggest that erythrocytes in aggregating media form a network followed by the formation of plasma channels within it. The cell-free zones form initially near the bottom and then propagate toward the top until they reach the plasma/blood interface. These channels increase the permeability of a network and, as a result, accelerate the sedimentation velocity. The energy of the flow field in channels is sufficiently strong to erode their walls. The upward movement of network fragments in channels is manifested by erratic fluctuations of the conductivity. The main conclusion, which may be drawn from the results of this study, is that the phase separation of blood is associated with the formation of plasma channels within the sedimenting dispersed phase.

A novel technique for quantification of erythrocyte aggregation abnormalities in pathophysiological situations.

Red blood cell (RBC) aggregation in blood samples taken from healthy volunteers and from multiple myeloma (MM), iron deficiency (IDA) and beta-minor thalassemia (T) patients was studied by a novel method based on electrical properties of colloidal systems. It was found that RBC aggregation changes in the following order: MM > IDA > control > or = T. Comparison of aggregation data obtained by this and other techniques shows that the sensitivity of the proposed technique to detect abnormal changes in RBC aggregation is substantially higher. For example, the mean values of relative aggregation indices measured for MM by this method and that based on the phenomenon of light scattering are 13.0 and 4.2, respectively. The high sensitivity of this technique allows investigations of the effect of moderate aggregating agents (i.e., IgG) on RBC aggregation. It is assumed that the higher sensitivity of the proposed technique to abnormal changes in RBC aggregation may be helpful both in basic studies to improve the understanding of the reason(s) for these abnormal changes, and in clinical investigations for earlier diagnostics.

The mechanism of the dextran-induced red blood cell aggregation.

In order to clarify the mechanism of dextran-induced aggregation, the effect of the ionic strength (I) on the minimal shear stress (tau(c)) required to rupture RBC doublets was studied for suspensions with the external media containing 76 and 298 kDa dextrans. At low and high ionic strengths, tau(c) increases with increasing I, whereas at intermediate I values, tau(c) versus I dependencies reveal a plateau step. The non-monotonous shape of these curves disagrees with the depletion model of RBC aggregation and is consistent with the predictions of the bridging mechanism. Literature reports point out that elastic behavior of dextran molecules in low and high I regions is fairly typical of Hookean springs and hence predict an increase in tau(c) with increasing I. A plateau step is accounted for by the enthalpic component of the dextran elasticity due to the shear-induced chair-boat transition of the dextran's glucopyranose rings. A longer plateau step for suspensions with a higher molecular weight dextran is explained by a larger contribution of the enthalpic component to the dextran elasticity. Thus, the results reported in this study provide evidence that RBC aggregation is caused by the formation of dextran bridges between the cells.

Increased red cell aggregation is correlated with HbA1C and lipid levels in type 1 but not type 2 diabetes.

The present study was designed to study RBC aggregability in type 1 and type 2 DM by a new method based on the dielectric properties of disperse systems. This dielectric method has a significantly higher sensitivity to detect enhanced RBC aggregation in DM than other methods. Aggregability is increased in type 1 DM and even more markedly in type 2 diabetic patients. The enhanced RBC aggregation in type 1 diabetes was significantly correlated with the levels of HbA(1C), cholesterol and triglycerides. However, no correlation between metabolic control and RBC aggregability was found in type 2 DM. The in vitro addition of non-toxic, low molecular weight dextran improves the high RBC aggregation in diabetes type 2. In the future, low molecular weight dextran may be used in DM patients clinically to lower the risk for vascular complications, after the problem of filtration is solved.

The role of erythrocyte aggregation in the abnormal hemorheology of multiple myeloma patients.

OBJECTIVES: The aim of this study is to clarify whether increased aggregation of red blood cells (RBCs) of multiple myeloma (MM) patients is caused by changes in plasma chemical composition or is associated with alterations in RBC properties and in addition, to suggest an approach to revert the enhanced aggregation in MM toward normal. MATERIALS AND METHODS: 40 blood samples of MM patients and suspensions of control RBCs in MM plasma were examined. In addition, RBC aggregation in MM blood was studied in the presence of dextrans with mean molecular weights of 9.6 and 40 kDa (D9.6 and D40). A method based on electrical and dielectric properties of cellular suspensions was used to study RBC aggregation. In this method, a lower aggregation index demonstrates a higher aggregability. RESULTS: The mean values of aggregation index for whole blood of healthy individuals, control cells in MM plasma and MM blood sample are 19.0, 7.2 and 3.2%, respectively. The kinetics of RBC aggregation slow down with the decrease in the fraction of MM plasma. No correlation was found between RBC aggregation and the immunoglobulin plasma level. Addition of D9.6 to MM blood reverts the enhanced aggregation toward normal. DISCUSSION: The findings that RBC aggregation changes in the following order: MM blood > normal RBCs in MM plasma > control blood sample, suggest that surface-active plasma molecules play a role in enhanced aggregation in MM. The surface concentration of these molecules and hence RBC aggregability is reduced in the presence of dextrans due to their competitive adsorption onto RBC membrane. Because the end-to-end distance of D40 is quite comparable with the Debye length, the effect of this particular dextran on RBC aggregation is negligible.

Conductometric study of shear-dependent processes in red cell suspensions. I. Effect of red blood cell aggregate morphology on blood conductance.

The conductance and capacitance of flowing and quiescent red blood cell (RBC) suspensions were measured at a frequency of 0.2 MHz. The results demonstrate that the time-dependent changes in the conductance recorded during the aggregation process differ in nature for suspensions of short linear rouleaux, branched aggregates and RBC networks. It is shown that the conductance of RBC suspensions measured during the aggregation and disaggregation processes follows the morphological transformations of the RBC aggregates. Thus, this method enables characterization of the morphology of RBC aggregates formed in whole blood and in suspensions with physiological hematocrits both under flow conditions and in stasis. These results in combination with previous ones suggest that this technique can be used for studies of dynamic RBC aggregation and probably for diagnostic use.

Conductometric study of shear-dependent processes in red cell suspensions. II. Transient cross-stream hematocrit distribution.

A novel experimental approach based on electrical properties of red blood cell (RBC) suspensions was applied to study the effects of the size and morphology of RBC aggregates on the transient cross-stream hematocrit distribution in suspensions flowing through a square cross-section flow channel. The information about the effective size of RBC aggregates and their morphology is extracted from the capacitance (C) and conductance (G) recorded during RBC aggregation, whereas a slower process of particle migration is manifested by delayed long-term changes in the conductance. Migration-induced changes in the conductance measured at low shear rates (< or =3.1 s(-1)) for suspensions of RBCs in a strongly aggregating medium reveal an increase to a maximum followed by a decrease to the stationary level. The ascending branch of G(t) curves reflects the aggregate migration in the direction of decreasing shear rate. A further RBC aggregation in the region of lower shear stresses leads to the formation of RBC networks and results in the transformation of the rheological behavior of suspensions from the thinning to the thickening. It is suggested that the descending branches of the G(t) curves recorded at low shear rates reflect an adjustment of the Hct distribution to a new state caused by a partial dispersion of RBC networks. For suspensions of non-aggregating RBCs it is found that depending on whether the shear rate is higher or lower compared with the prior value, individual RBCs migrate either toward the centerline of the flow or in the opposite direction.

The reduction of a nitroxide spin label as a probe of human blood antioxidant properties.

The kinetics of reduction of the radical R*, 5-dimethylaminonaphthalene-1-sulfonyl-4-amino-2,2,6,6-tetramethyl-1-piperidine-oxyl by blood and its components were studied using the EPR technique. The results demonstrate that R* is adsorbed to the outer surface of the membrane and does not penetrate into the erythrocytes. A series of control experiments in PBS demonstrate that ascorbate is the only natural reducing agent that reacts with R*. The observed first order rate of disappearance of the nitroxide radical k, is: k(blood) > k(eryth) > k(plasma) and k(blood) approximately = k(eryth) + k(plasma). The results demonstrate that: a. The erythrocytes catalyze the reduction of R* by ascorbate. b. The rate of reduction of the radical is high though it does not penetrate the cells. c. In human erythrocytes there is an efficient electron transfer route through the cell membrane. d. The study points out that R* is a suitable spin label for measuring the reduction kinetics and antioxidant capacity in blood as expressed by reduction by ascorbate.

EPR analysis of radicals generated in ultrasound-assisted lipoplasty simulated environment.

The generation of various radicals by application of continuous wave (CW) high-intensity ultrasound energy (HIUE) to an aqueous biologic medium containing spin traps, under conditions simulating ultrasound-assisted lipoplasty (UAL), was demonstrated by EPR spectroscopy. The addition of water- soluble antioxidants, ascorbic acid and glutathione to the wetting solution substantially reduces the levels of hydroxyl radicals in the sonicated medium. These findings provide direct evidence for the generation of cavitation in the simulated intercellular environment, corroborating previous data, and pointing out that generation of transient cavitation in clinical UAL and other therapeutic and surgical applications of ultrasound is possible. The findings indicate that the effect of transient cavitation in aqueous biologic media may be similar to the effects of ionizing radiation, and raise the question of the long-term biosafety of the use of CW HIUE in UAL. The introduction of biocompatible water-soluble antioxidants to the sonicated medium may be utilized to suppress accumulation of radicals and reduce their possible adverse effects.

Thiopurine methyltransferase activity in the Jewish population of Israel.

OBJECTIVE: 6-Mercaptopurine is used therapeutically for its immunosuppressant and cytotoxic properties. It is deactivated by thiopurine methyltransferase (TPMT), which shows genetic polymorphism in many populations. In North American populations, TMPT activity exhibits a trimodal activity pattern. In Oriental populations, TPMT shows almost a unimodal pattern of activity. The purpose of the present study was to assess the activity of TPMT in a Jewish male population sample in Israel. METHODS: The study was approved by the Israeli Ministry of Health. Blood samples of 2.5 ml were collected in heparinized tubes from 134 males. The red blood cell (RBC) fraction of each individual was washed and hemolyzed. TPMT activity in the RBC hemolysate was determined using a radioactive assay with tritiated S-adenosyl methionine as a methyl donor. RESULTS: The activity of TPMT ranged from 3.2 nmol/h/ml to 42.9 nmol/h/ml packed RBCs with mean and median activities of 18.6 nmol/h/ml and 17.9 nmol/h/ml packed RBCs, respectively. The distribution frequency of TPMT was very close to the unimodal by analysis of normal distribution. CONCLUSION: The pattern of distribution of TPMT in the Jewish population of Israel is closer to that of East Asian populations than European and North American populations. This observation may have relevance for the usage of 6-mercaptopurine and azathioprine as therapeutic agents in the Jewish population.

Possible oxidative stress involvement in congenital dyserythropoietic anemia type 1.

INTRODUCTION: Congenital dyserythropoietic anemia type 1 (CDA1) patients may suffer from iron overload, associated with oxidative damage. The aim of this study was to evaluate possible involvement of oxidative stress in the pathogenesis of CDA1. STUDY DESIGN: : Blood samples from 10 children diagnosed as CDA1 patients from five Bedouin families, were studied. In this study, activities of superoxide dismutase and catalase were evaluated as well as methemoglobin, plasma total thiols, plasma total antioxidant capacity and glycerol lysis time. RESULTS: Normal values were found for superoxide dismutase, methemoglobin, trolox equivalent antioxidant capacity and total plasma thiols in CDA1 patients. However average catalase levels were significantly reduced (P<0.001) and glycerol lysis test was significantly prolonged (P<0.001). Ferritin levels, which were slightly increased in all patients, positively correlated with catalase values (r = 0.74, P = 0.022). CONCLUSION: Oxidative stress has not been proven in CDA1 pediatric patients. Some indications of oxidative damage exist, but it may not be directly related to the mechanism of anemia.

The effect of low-molecular weight dextran on erythrocyte aggregation in normal and preeclamptic pregnancy.

Erythrocyte aggregation was determined by a novel method enabling the quantification of the aggregation process in whole blood. Blood samples of 47 healthy pregnant women and 39 preeclamptic patients were examined. Subjects within each group were matched for the gestational age. It was found that RBC aggregation increases with the gestational age in healthy pregnancy and further increases in preeclampsia. Addition of low-molecular weight dextran (MW = 9300) to blood samples of both healthy pregnant women and preeclamptic patients reduces RBC aggregation in a concentration-dependent manner. The obtained results indicate alterations in plasma composition as the primary factor for the increased RBC aggregation in both normal and pathological pregnancy. It is suggested that adsorption of low-molecular weight dextran on the RBC membrane reduces the surface concentration of plasma bridging molecules thereby reducing RBC aggregation toward normal.

Dielectric approach to investigation of erythrocyte aggregation. II. Kinetics of erythrocyte aggregation-disaggregation in quiescent and flowing blood.

A method based on dielectric properties of dispersed systems was applied to investigate the kinetics of RBC aggregation and the break-up of the aggregates. Experimentally, this method consists of measuring the capacitance at a frequency in the beginning of the beta-dispersion. Two experimental protocols were used to investigate the aggregation process. In the first case, blood samples were fully dispersed and then the flow was decreased or stopped to promote RBC aggregation. It was found that the initial phases of RBC aggregation are not affected by the shear rate. This finding indicates that RBC aggregation is a slow coagulation process. In the second case, RBCs aggregated under flow conditions at different shear rates and after the capacitance reached plateau levels, the flow was ceased. The steady-state capacitance of the quiescent blood and the kinetics of RBC aggregation after stoppage of shearing depend on the prior shear rate. To clarify the reasons for this effect, the kinetics of the disaggregation process was studied. In these experiments, time courses of the capacitance were recorded under different flow conditions and then a higher shear stress was applied to break up RBC aggregates. It was found that the kinetics of the disaggregation process depend on both the prior and current shear stresses. Results obtained in this study and their analysis show that the kinetics of RBC aggregation in stasis consists of two consecutive phases: At the onset, red blood cells interact face-to-face to form linear aggregates and then, after an accumulation of an appropriate concentration of these aggregates, branched rouleaux are formed via reactions of ends of the linear rouleaux with sides of other rouleaux (face-to-side interactions). Branching points are broken by low shear stresses whereas dispersion of the linear rouleaux requires significantly higher energy.

Quantitative determinations of microcytic-hypochromic red blood cell population and glycerol permeability in iron-deficiency anemia and beta thalassemia minor.

The Hl/H2 Technicon automated cell analyzer measures, in addition to the usual red blood cell (RBC) parameters, subpopulations of microcytic (M) and hypochromic (H) red blood cells. The M/H ratio may be useful in the differential diagnosis of iron-deficiency anemia (IDA) and beta thalassemia minor (Thal). Thirty-three iron-deficient patients and 26 thalassemia patients were studied. The M/H ratio was found to be higher in thalassemia patients than in IDA patients. Using a cut-off point of 1.9 M/H ratio, the calculated discriminant efficiency was 88%. When glycerol lysis values were determined at 70 s as a cut-off point, the discriminant efficiency was slightly higher, at 91%. Thus, the combination of the M/H ratio and the glycerol lysis time (GLT) improves the discriminant efficiency and provides a good diagnostic tool to differentiate between the two microcytic-hypochromic anemias. The study suggests that the M/H ratio together with the GLT could serve as a useful screening tool, prior to the application of other more sophisticated methods.

Dielectric approach to the investigation of erythrocyte aggregation: I. Experimental basis of the method.

A method based on dielectric properties of dispersed systems was developed to investigate red blood cell (RBC) aggregation in blood and RBC suspensions. Measurements of capacitance and resistance were made in a rectangular channel at low (0.2 MHz) and high (14 MHz) frequencies relative to the mid-point of the beta-dispersion range. Compared to capacitance, minimal post-shearing changes of resistance were observed; capacitance changes at 0.2 MHz were two orders of magnitude larger than those at 14 MHz and hence subsequent measurements were carried out at the lower frequency. It is shown that post-shearing changes in the capacitance are affected by the recovery of RBC shape and relaxation processes at the electrode-suspension interface. However, the dominant factor contributing to time-dependent changes in the capacitance is the dynamic process of RBC aggregation. It is experimentally shown that the time record of the capacitance at 0.2 MHz quantitatively reflects the aggregation process in RBC-plasma suspensions with hematocrit up to 0.56 (v/v) and in suspensions of RBCs in artificial aggregating media. It is concluded that a dielectric approach to the study of RBC aggregation in whole blood offers great potential for basic studies and for diagnostic use.

Effect of vitamin K1 on glucose-6-phosphate dehydrogenase deficient neonatal erythrocytes in vitro.

AIM: To determine whether vitamin K1, which is routinely administered to neonates, could act as an exogenous oxidising agent and be partly responsible for haemolysis in glucose-6-phosphat-dehydrogenase (G-6-PD). METHODS: G-6-PD deficient (n = 7) and control (n = 10) umbilical cord blood red blood cells were incubated in vitro with a vitamin K1 preparation (Konakion). Two concentrations of Vitamin K1 were used, both higher than that of expected serum concentrations, following routine injection of 1 mg vitamin K1. Concentrations of reduced glutathione (GSH) and methaemoglobin, indicators of oxidative red blood cell damage, were determined before and after incubation, and the mean percentage change from baseline calculated. RESULTS: Values (mean (SD)) for GSH, at baseline, and after incubation with vitamin K1 at concentrations of 44 and 444 microM, respectively, and percentage change from baseline (mean (SD)) were 1.97 + 0.31 mumol/g haemoglobin, 1.89 +/- 0.44 mumol/g (-4.3 +/- 13.1%), and 1.69 +/- 0.41 mumol/g (-14.5 +/- 9.3%) for the G-6-PD deficient red blood cells, and 2.27 +/- 0.31 mumol/g haemoglobin, 2.09 +/- 0.56 mumol/g (-7.2 +/- 23.2%), and 2.12 +/- 0.38 mumol/g (-6.0 + 14.1%) for the control cells. For methaemoglobin (percentage of total haemoglobin), the corresponding values were 2.01 +/- 0.53%, 1.93 +/- 0.37% (-0.6 +/- 17.4%) and 2.06 +/- 0.43% (5.7 +/- 14.2%) for the G-6-PD deficient red blood cells, and 1.56 +/- 0.74%, 1.70 +/- 0.78% (12.7 +/- 21.9%), and 1.78 +/- 0.71% (20.6 +/- 26.8%) for the control red blood cells. None of the corresponding percentage changes from baseline was significantly different when G-6-PD deficient and control red blood cells were compared. CONCLUSIONS: These findings suggest that G-6-PD deficient red blood cells are not at increased risk of oxidative damage from vitamin K1.