Cell Transit Analyzer Research Articles

Using Air Liquid Interface for studying Mechanical Deformation of Cells in Microfluidic Channels

Biological cells are known to respond to changes in their environment by changing their mechanical response. For several diseases (e.g. diabetes, spherocytosis) changes in mechanical properties of cells have been corelated with the progression of the disease. Changes in cells mechanical properties has also been corelated with their development and age. With the advent of advanced microfluidic characterisation techniques, the mechanical response of a large number of cells can be measured at the single cell level. These studies help in investigating fundamental questions in cell biology. These analysers are called microfluidic cell transit analysers. In this work we introduce a novel cell-transit analyser where an air-liquid interface is used as a mechanical element to induce deformability in cells. This technique enables both static and dynamic deformability measurements in live cells ex vivo. The devices were fabricated using PDMS microchannels bonded to glass substrates. The air-water interface is formed using manually controlled syringes. A syringe pump was used to flow the cell containing samples. In this work, stiffer air-water interface (surface tension 72 mN/m) to deform more compliant Red Blood Cells (effective 20 mN/m) in micro-channels. As the interface stiffnesses are of similar order, the air-water interface also deforms. In the static mode, measurement of the air-water interface deformation allows us to measure the Laplace pressure drop and hence estimate the stiffness of the Red Blood Cells. Using our technique, we estimated the stiffness of a single RBC was measured to be 18±2 mN/m which is in good agreement with previous reports. In the dynamic mode the RBC containing buffer was flowed from one side and air pressure was applied at the two perpendicular channels to create stable air-liquid interfaces with desired constriction widths. This allows us to perform high throughput measurements (>100 cells/s) of deformability index (DI) and transit time for different air-liquid interface constriction widths. The adjustments in constriction width can be made in real time and can be tuned to widths as small as 2µm by controlling the air-pressure. The technique also eliminates the cell-substrate interactions due to the use of a virtual wall. In order to validate the ability of the technique to differentiate cell population based on mechanical response, DI and transit time for cells passing through the air-water interface constriction were measured for healthy and 0.01% Glutaraldehyde (GA) treated RBCs. The DI of the GA-RBCs was found to be smaller than healthy RBCs thereby confirming the effectiveness of the technique in deformability cytometry. Further, the mechanical response of cells to different constriction widths was measured. We found that for differentiating Red Blood Cells of different stiffness using DI and transit time, constriction widths < 4µm are optimum.

Measurement of erythrocyte deformability by cell transit analyser

Erythrocyte deformability (filterability) as measured by the Cell Transit Analyser was studied in the presence and absence of contaminating leucocytes and platelets. The effects of two anticoagulants (lithium heparin and K 2 EDTA), no anticoagulant (defibrinated blood) and two buffers (HEPES and phosphate) were also evaluated

Anti-inflammatory effects of selected drugs on activated neonatal and adult neutrophils

Aim. In view of the central role of granulocytic neutrophils in the context of inflammatory reactions, the present study focuses on anti-inflammatory effects of drugs on activated neutrophils in neonates and adults. Methods. Sixteen blood samples of neonates and adults were investigated in a prospective study. Loss of deformability, morphological changes, and increases in neutrophil elastase were determined as measures of neutrophil activation due to incubation with the pro-inflammatory cytokine interleukin-8. For inhibition experiments, the blood samples were also incubated with the phosphodiesterase inhibitors milrinone and piclamilast, the protease inhibitor urinastatin, ketamine, protein C concentrate, and the nitric oxide donor FK 409. Changes in deformability were investigated with a cell transit analyzer, morphological changes by microscopic observation, and the extent of neutrophil elastase release with an enzyme immunoassay. Results. The drugs milrinone, piclamilast, urinastatin, ketamine, protein C concentrate and FK 409 showed deactivating effects on activated neutrophils in recommended clinical doses. They improved deformability as well as reduced pseudopod formation and the release of neutrophil elastase. The effects on neutrophils did not differ between neonates and adults despite their functional differences. Conclusion. We conclude that these drugs may reduce the inflammatory response and improve microcirculation in neonates and adults during inflammation.

The NO-donor FK409 improves mechanical properties of activated neonatal PMN

Activated polymorphonuclear neutrophils (PMN) play an important role in the microcirculation. Nitric oxide (NO) reduces the sequestration of PMN in the narrow vessels of various organs and, therefore, may reduce organ injury during inflammation. Since PMN of term neonates show various functional differences compared to PMN in adults (decreased chemotaxis, decreased intracellular killing, decreased adhesion), we studied the influence of the semi-synthetical NO-donor FK-409 (4-Ethyl-2-hydroxyimino-5-nitro-3-hexenamide) on the deformability of IL-8 activated PMN in term neonates and adults. A cell transit analyzer (CTA) was used to study transit times of individual PMN through 8 μm filter pores, neutrophil elastase concentrations were determined by enzyme-immunoessay and activation of PMN was classified by mircroscopic evaluation. The transit times of PMN activated by IL-8 in adults were 9.3 ± 2.9 s, in term neonates 10.7 ± 3.3 s. FK-409 improved the transit time of activated PMN in adults (5.4 ± 1.6 s) and in term neonates (5.6 ± 1.1 s). Despite of the functional differences of PMN in term neonates and adults, the improvement of the transit times by FK-409 was not different between the two groups. The NO donor decreased the neutrophil elastase concentrations and the morphological signs of activation in neonates and adults. We conclude that the NO-donor FK-409 improves the microcirculation by increasing the deformability of IL-8 activated PMN. NO may reduce in neonates tissue damage by reduced PMN sequestration due to decreased PMN rigidity.

Effects of phosphodiesterase (III/IV)-inhibitors and cytokines on mechanical properties of neutrophilic granulocytes in neonates and adults

Sequestration of activated PMN and enrichment in tissues play a key role in tissue damage during septicaemia and after ischemia/reperfusion. Since polymorphonuclear neutrophilic granulocytes (PMN) of term neonates show various functional differences compared to PMN in adults (decreased chemotaxis, decreased intracellular killing, decreased adhesion) we studied the influence of interleukin 8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) on the reduction of deformability of PMN in neonates and adults. The following phosphodiesterase (PDE)-inhibitors were applied to ameliorate the reduction in deformability when the PMN were stimulated with fMLP or IL-8: Enoximone, Milrinone (PDE-III-inhibitors), Pentoxifylline (PTX) and Piclamilast (PDE-IV-inhibitors). The micropipette technique and the cell transit analyzer (CTA) were used and compared. Aspiration times into micropipettes with an internal diameter of 5 microm, transit times through 8 microm filter pores and neutrophil elastase concentrations were determined. Despite of the functional differences of PMN in neonates compared to adults the significant decrease of deformability of PMN activated with cytokines compared to passive PMN was not different in both groups. The neutrophil elastase concentrations reflect the activation of the PMN: highest concentrations during activation, decreased concentrations due to PDE-inhibitors, and PMN in a passive state. The neutrophil elastase concentrations were not different from PMN of neonates and adults. These PDE-inhibitors significantly increased the deformability of activated PMN but significant differences between the deformability of PMN in neonates and adults were not found. Despite the functional differences of PMN in neonates PDE-III/IV-inhibitors lead to similar improvement of mechanical properties of activated PMN in neonates and adults. These drugs may ameliorate impaired microcirculation also in neonates during inflammation.

Hemorheologic Abnormalities Associated with HIV Infection: In Vivo Assessment of Retinal Microvascular Blood Flow

To evaluate retinal microvascular blood flow in human immunodeficiency virus (HIV)-infected individuals using scanning laser Doppler flowmetry (SLDF) and to seek correlations between flow and various laboratory measures that may predict alterations in flow. The Heidelberg Retina Flowmeter and SLDF software were used to acquire in vivo retinal blood flow data from 24 HIV-infected individuals and 16 HIV-negative control subjects. In each subject, separate scans were performed in each of six retinal regions: nasal parapapillary retina; macula; and the superior, nasal, inferior, and temporal periphery. Erythrocyte aggregation (assessed in vitro by a fully automatic erythrocyte aggregometer and by zeta sedimentation ratio [ZSR, a hematocrit-independent sedimentation rate]), serum fibrinogen level, plasma viscosity, and leukocyte rigidity (assessed in vitro with a cell transit analyzer) were compared with flow in selected regions. Flow was significantly higher in the periphery (superior, nasal, inferior, temporal) than in the posterior retina (nasal parapapillary retina, macula). Flow was highest in the temporal periphery for both HIV-infected subjects and control subjects. Flow in the posterior retina was significantly lower in HIV-infected subjects than in control subjects (P < 0.0001). Among HIV-infected individuals, flow in the macula correlated negatively with ZSR (r = -0.397, P = 0.0547) and leukocyte rigidity (r = -0.505, P = 0.0119). Microvascular blood flow in the posterior retina is reduced in HIV-infected individuals. Both increased erythrocyte aggregation and increased leukocyte rigidity contribute to this hemorheologic abnormality.

Use of Cell Transit Analyser pulse height to study the deformation of erythrocytes in microchannels

Information on microvascular rheology can be used to develop mathematical models of network hemodynamics in various contexts: tumor-induced angiogenesis, reaction to ischemia and collateralization, hypertension, … The rheological method which probably most closely simulates red blood cell (RBC) flow behavior in the microcirculation is the determination of individual erythrocyte transit time and shape changes during flow through cylindrical micropores. However, these filtration experiments remain difficult to interpret in terms of cell flow properties, because the shape of the cell inside the pore is unknown. This paper uses the cell transit analyser (CTA) electrical pulse (especially the pulse height) to determine the relationship between the cell velocity and deformed shape in the pore, depending on the flow strength, suspending medium viscosity and cell membrane elasticity. As predicted by published numerical simulations for the flow of deformable particles through narrow channels, increasing pressure leads to increased deformation, but for a given pressure, increased viscosity leads to a slight increase in deformation. The sensitivity of filtration experiments to cell mechanical properties is improved when using low driving pressures and narrow and sufficiently long pores.

In vitro effects of high glucose concentrations on membrane protein oxidation, G-actin and deformability of human erythrocytes.

The object of this study was to examine the effect of elevated in vitro glucose concentrations on protein modification and functional changes in human erythrocytes. Groups were exposed to 5-45 mM glucose concentrations. The time effect of any changes was also evaluated. In erythrocyte ghosts, protein glycation and oxidation were evaluated using spectrophotometric methods. G-actin was measured by a DNase I inhibition assay in cell lysates. Erythrocyte deformability was assessed using a cell transit analyser. At 24 h, a significant protein oxidation (at 25 and 45 mM glucose; p < 0.05), and G-actin increase was observed for all concentrations (p < 0.05). At 48 h, a significant increase in glycation (25 and 45 mM glucose; p < 0.05), protein oxidation (p < 0.05), and G-actin (p < 0.05) was observed in all groups. A significant positive correlation was observed between glucose /protein oxidation, glucose/G-actin and protein oxidation/G-actin at 24 and 48 h. Our findings show that the oxidative effect of glucose on erythrocytes depends on concentration and incubation time. We also present the first evidence of increased G-actin in human erythrocytes exposed to high glucose concentrations as a diabetes model.

Increased polymorphonuclear leucocyte rigidity in HIV infected individuals

AIMIndividuals with human immunodeficiency virus (HIV) infection were evaluated for evidence of abnormal polymorphonuclear leucocyte (PMN) rigidity, which can alter capillary blood flow.METHODSThe transit time of individual PMN through 8...

Increased rigidity and priming of polymorphonuclear leukocytes in sepsis.

It has been proposed that abnormal mechanical properties may contribute to capillary retention of polymorphonuclear leukocytes (PMN) in sepsis, leading to the development of organ dysfunction. The present study was designed to determine whether PMN rigidity is increased in severe sepsis, and whether changes in the rheologic behavior of PMN correlate with the clinical course in sepsis. Eighteen adults with severe sepsis were studied over a period of 14 d; 11 survived and seven died. PMN deformation behavior was investigated via micropore filtration, using the cell transit analyzer. On Day 0, PMN rigidity was 2.5-fold greater for sepsis patients than for five normal controls (p < 0.001). PMN rigidity progressively improved over the 14 d study period for patients who recovered, but not for those who died; clinical indicators correlated with PMN rigidity. Patient PMN also exhibited a 5-fold greater increase in rigidity in response to formyl-methionylleucylphenylalanine (fMLP) than did control PMN. Both the increased rigidity and enhanced response to fMLP could be simulated in vitro by incubation of normal PMN with tumor necrosis factor-alpha (TNF-alpha). We conclude that circulating PMN are more rigid in severe sepsis, and are "primed" for an augmented response to chemotactic stimuli. These findings support the hypothesis that cytokine-mediated increases of PMN rigidity may lead to sequestration of these cells in capillaries and to the consequent impairment of microvascular perfusion in sepsis.

Effects of dietary supplementation of saturated fatty acids and of n-6 or n-3 polyunsaturated fatty acids on plasma and red blood cell membrane phospholipids and deformability in weanling guinea pigs.

The fatty acid composition of plasma cholesteryl esters, plasma phospholipids, red blood cell (RBC) membrane phosphatidylcholine (corresponding to the outer membrane leaflet), and phosphatidylethanolamine (corresponding to the inner membrane leaflet) was investigated in weanling guinea pigs fed with diets of cacao (saturated fatty acids), sunflower oil [n-6 polyunsaturated fatty acids (PUFA)] or fish oil (n-3 PUFA) for 20 wk. RBC deformation was measured by means of a cell-transit analyzer (filtration) and a cone-plate rheoscope. The contents of saturated fatty acids in plasma phospholipids and RBC membrane leaflets were similar in all three groups. Diets with sunflower oil resulted in a high content of linoleic acid in plasma cholesteryl esters and in the outer leaflet of RBC membranes. Fatty acids of fish oil were mainly incorporated in plasma phospholipids and in the inner leaflet of RBC membranes. The arachidonic acid content was high in all groups in the plasma phospholipids and in the inner leaflet. The n-6 and n-3 PUFA were mainly incorporated in the inner leaflet. In all groups the polyunsaturated/saturated fatty acid ratio and the total PUFA content were similar in the inner RBC membrane. The RBC filtration times and the RBC deformation indices were not affected by the dietary treatment.

A standardized parameter for the distribution of individual Red Blood Cell deformability with the Cell Transit Analyzer

The purpose of this study was to identify a parameter allowing the standardization of the Cell Transit Analyzer (CTA) in order to study the individual deformability of each explored Red Blood Cell (RBC). Using theoretical arguments based on the principle of the CTA, we calculated the thickness "x" of the crown of fluid surrounding each RBC during its entry phase into the micropore. A mathematical equation (x = 62.5/magnitude of dU) was established between the difference of potential (dU, mV) that occurs during this phase and the corresponding thickness ("x", micron) of the crown. By exploring fresh control RBCs of healthy subjects and assuming that the rigid RBCs proportion in a fresh blood sample of healthy subject is less than 3.5%, we performed a thresholding of "x" to distinguish rigid RBC from deformable ones. That thresholding was necessary to stamp the variability of counts linked to polycarbonate filters (PF) used to carry out measures. According to the PF, the value of the threshold "Tx" provided by the thresholding ranged between 0.222 and 0.246 micron. Using the values of "Tx", we counted approximately 10-25% rigid RBCs in the pathological samples of four patients with sickle cells SS disease and of one diabetic patient with splenomegaly. We observed in addition that the percentages of rigid RBCs counted after thresholding are identical from a filter to another one with an absolute error less than 2% in fresh RBCs (normal or pathological) samples. We concluded that the method of standardization by thresholding presented here could be used in clinic routine to count the rigid RBCs percentage contained in a given sample.

Flow resistance of individual neutrophils in coronary artery disease: decreased pore transit times in acute myocardial infarction.

OBJECTIVE: To investigate single neutrophil flow resistance in coronary artery disease, including myocardial infarction before initiation of reperfusion therapy. DESIGN: Neutrophil flow resistance was measured in 93 subjects in five...

Changes in neutrophil rheology after acute ischemia and reperfusion in the rat hindlimb

Neutrophils may contribute to the pathogenesis of ischemia-reperfusion injury by impairing microcirculatory flow and causing tissue damage. In this study neutrophil deformability and activation were studied after acute ischemia and reperfusion of the rat hindlimb. A cell transit analyzer with 8 μm pore filters was used to measure the flow resistance of individual neutrophils isolated from arterial blood drawn 30 seconds and 5 minutes after reperfusion, after 2 hours of femoral or iliac artery occlusion, in anesthetized rats. Femoral occlusion followed by 30 seconds of reperfusion resulted in a 16% rise in mean pore transit time, and iliac occlusion caused a 31% increase. Neutrophil activation (percentage of cells with nonspherical shape) rose from 6% to 23% (femoral occlusion) or from 7% to 29% (iliac occlusion). By 5 minutes of reperfusion, transit times, but not shape change, had almost returned to preischemic levels. Sham-occluded rats had no significant change in neutrophil behavior. Plasma isolated from rats after 30 seconds of reperfusion after 2 hours of iliac occlusion caused a 27% increase in mean transit time when incubated with nonautologous neutrophils, while activation rose from 32% to 55%. Thus plasma factor(s) released after ischemia and reperfusion of rat hindlimb caused transient rheologic alteration of circulating neutrophils, which may contribute to local or disseminated vascular occlusion and tissue injury.

Decreased deformability of polymorphonuclear leukocytes in diabetic cats.

Polymorphonuclear leukocytes (PMN) have been found to be less deformable in humans with non-insulin-dependent diabetes. It has therefore been hypothesized that white blood cells (WBC) may affect the development of diabetic microangiopathy. This study was designed to determine whether PMN or small and large lymphocytes were less deformable in a large animal model of diabetes-chronically hyperglycemic pancreatectomized cats. Venous blood was withdrawn from 13 normal and 14 diabetic cats. The diabetic cats had been kept in poor metabolic control since their pancreatectomy (7-113 months before this study). Blood cell counts, hemoglobin concentration, hematocrit, erythrocyte volume, and WBC differential counts were obtained from the blood samples. Purified mononuclear WBC and PMN fractions were obtained by separating the blood on a discontinuous Percoll gradient. The deformability of each cell fraction was determined using a Cell Transit Analyzer (ABX, Montpellier, France) that measures the transit time of cells through 7.5-microns pores. By varying the sampling rate of the CTA and the pressure difference across the filter, the subpopulations of the mononuclear fractions (small and large lymphocytes) could be identified and each subpopulation analyzed separately. Median transit times for the PMN and small lymphocytes were significantly greater for the diabetic cats, but no difference was found in the large lymphocyte fractions. These results are in accordance with the finding that PMN are less deformable in humans with diabetes. We also showed that the small lymphocytes from diabetic cats have prolonged transit times. The results suggest that PMN may contribute to the development of microangiopathies like diabetic retinopathy. Diabetic cats may prove useful for testing potential therapies to improve WBC deformability.

Decreased erythrocyte deformability in glycogen storage disease

Liver glycogen storagen diseases (GSG) are disorders associated with severe dyslipidaemia which can induce cell membrane alterations and possibly reduced cell deformability. Since decreased erythrocyte deformability is known to disturb blood flow in capillaries and may promote ischaemic diseases, this study was designed to investigate erythrocyte deformability using a new filtration system, the Cell Transit Analyser (CTA), and to examine lipid compounds in the blood of 23 patients affected with GSD, aged from 1 to 20 years and 18 controls aged from 1 to 17 years. The patients showed a mixed hyperlipidaemia with predominent hypertriglyceridaemia and an increase in erythrocytes mean transit times (TT) due to the presence of more rigid erythrocytes subpopulations when compared to controls. Thus the erythrocyte rigidity, in addition to the lipid abnormalities must be taken into account for long-term evolution of GSD patients. Moreover this cellular alteration may contribute to shortened erythrocyte survival.

Deformability of red blood cells from different species studied by resistive pulse shape analysis technique

The Cell Transit Analyzer (CTA) is now being used widely in clinical hemorheology. Most of the data obtained by CTA are limited to human blood, although the CTA has an important potential to be used in experimental studies on animal models. However, behavior of red blood cells (RBC) from various species might be different in CTA. Eight parameters reflecting different aspects of cell passage through pores with 5 μm diameter and 15 μm length were determined or human, guinea pig, dog, rabbit, rat, mouse and sheep RBC, together with instrument precision and biological variation. These parameters have a wide range when measured in different species and correlate with cell volume. Sensitivity of these parameters to the glutaraldehyde-induced alterations in RBC deformability was not same for different laboratory mammals. The main reason for this difference seems to be related to the cell size and thus sensitivity might be significantly limited if 5 μm pore-size filters are used to test the smaller RBC. The results of this study may help in designing experimental studies on laboratory mammals using the CTA.

The influence of suspending phase viscosity on the passage of red blood cells through capillary-size micropores

Much attention has been paid to the study of blood flow in long, narrow tubes. While the influence of tube diameter and driving pressure have been examined in detail, the influence of suspending phase viscosity has generally been assumed only to affect the blood viscosity in a linearly proportional manner, hence the practice of normalizing apparent blood viscosity values by the suspending phase viscosity to give a relative viscosity ( e.g., Pries et al., 1992). While this assumption is probably valid for long tubes, it apparently does not hold for blood flow in short tubes (and by extension also for flow in short or branching capillary segments in vivo) in which RBC deformation plays a more significant role. In this paper we present a series of experiments using the Cell Transit Analyzer (CTA) in which the influence of driving pressure and suspending phase viscosity on RBC passage through short, narrow tubes has been systematically evaluated. Over the range studied (1 to 10 cm water), the influence of driving pressure was found to be unremarkable, in that RBC velocity scaled directly and linearly with pressure. This finding is consistent with previous studies. However, a distinct intercept was observed in the linear relationship between RBC pore transit time and suspending phase viscosity, which presumably arises as a consequence of RBC deformation either at the pore entrance or within the pore. Two simple mathematical models for the suspending phase-viscosity/transit-time relationship were considered. The results show that making CTA measurements over a range of suspending medium viscosities is a simple and practical way to obtain additional information about RBC mechanical properties.

Activated granulocyte induced alterations in red blood cells and protection by antioxidant enzymes

Activated leukocytes generate oxygen free radicals and cause injury in a variety of surrounding cells. Red blood cells (RBC) are among the most susceptible cells to this injury. Deformability, lipid peroxidation and membrane proteins were investigated in RBC after incubation with activated granulocytes (2 hours, at 37 QC), obtained from guinea pigs with experimental sepsis. A second group of experiments was conducted with non-activated granulocytes isolated from blood of healthy guinea pigs. RBC transit time through S 11m pores measured by a cell transit analyzer was 3.2S±0.12 msec and 2.86±0.02 msec in the suspensions containing activated and non-activated granulocytes, respectively (p<O.OS). Addition of superoxide dismutase (SOD) (20 Ilg/ml) and catalase (40 Ilg/rnl) inhibited the effect of activated granulocytes on RBC deformability. Lipid peroxidation was also increased under the influence of activated granulocytes (31.S0±3.36 nmol/grHb versus 20.02±2.97 nmol/grHb); SOD failed to prevent this increment, while catalase was found to be effective. A widening of Band 1 (spectrin) was observed in SDS-PAGE electrophoresis of membrane proteins of RBC incubated with activated granulocytes, indicating spectrin-hemoglobin crosslinking. This alteration was prevented by both SOD and catalase, as in the case of transit time. The superoxide radical seems to be responsible for the membrane protein and mechanical alterations, while lipid peroxidation was induced by a different oxidative mechanism.

Sensitivity of the cell transit analyzer (CTA) to alterations of red blood cell deformability: Role of cell size-pore size ratio and sample preparation

Sensitivity of the cell transit analyzer (CTA) to alterations of red blood cell deformability: Role of cell size-pore size ratio and sample preparation