Red cells slide as they form doublets and deform in rouleaux.

Mammalian erythrocyte aggregation increases when the levels of plasma proteins favoring aggregation rise. Red cell aggregate formation has been attributed to noncovalent bonding of adjacent erythrocyte plasma membranes by these proteins and similar macromolecules. The proposed membrane to membrane noncovalent bonds would keep each membrane from sliding during aggregate formation because the bonds responsible for maintaining cell-cell contact would need to be disrupted. Because past studies of doublet formation suggested that the membranes might slide during contact expansion, we embedded latex particles in the membranes of individual human red cells and recorded doublet formation on video tape. The cells were suspended in a buffer that contained polyvinylpyrrolidone at a concentration sufficient to cause a moderately elevated sedimentation rate. The latex particles remained stable in position relative to each host cell during doublet formation, indicating that sliding was involved. We also stretched individual rouleaux using a glass rod and observed that latex particles attached to red cells whose contact area was reduced by the motion maintained their position during their return to a normal shape. These studies show that erythrocyte aggregate formation is accomplished by membrane sliding and that aggregate shape change and disruption during blood flow commonly involve sliding. The sliding motion argues that the attraction between red cell membranes generated by an array of elongated macromolecules involves a delocalized rather than a noncovalently coordinated adhesion.

Developing red cell flow orientation shown by changes in blood reflectivity.

The rate of onset of erythrocyte flow orientation at normal concentration has not previously been established. Reflectivity of blood and resuspended non-aggregated red cells at normal (41%) and elevated (60%) hematocrit has been used to examine this process. Video recordings were made before, during, and after shearing by bob motion in a cylinder-in-cylinder viscometer at shear rates ranging from 4 to 100 inverse seconds. Unaggregated erythrocytes in PBS, already more reflective than blood before shearing, became even more reflective during shearing even at the lowest shear rate studied. The time required for the increased reflectivity to stabilize was observed to be inversely proportional to shear rate for both blood and resuspended red cells. Reflection became constant after a total shear strain (bob shear rate x time) from 4 to 10 at all shear rates studied. Onset of increased reflection expressed in total shear strain units (an index of overall bob movement) was independent of shear rate in the absence of aggregation. When red cells were studied in native plasma, reflectivity increased as much as 30% during shearing, but always remained below unaggregated red cell reflectivity for the same hematocrit and shear rate. Greater reflectivity of unaggregated red cells persisted after cessation of shearing, while blood's reflectivity dropped progressively over several seconds to its pre-shearing value. The geometry chosen for study and insensitivity to light source composition indicate that specular reflection by red cells near the cup's inner surface is responsible for the increased light return during flow. Maximal rate of rise in reflectivity at all shear rates studied was observed to coincide with blood's previously reported transient shear stress overshoot and to correspond with an overall bob motion that would rotate suspended particles approximately 45 degrees. The close relation of both reflectivity increase and shear stress overshoot to such modest overall bob movement indicates that an efficient flow-mediated rotation of either individual or aggregated erythrocytes from initially random positions toward an orientation in the shear plane characterizes flow onset.

Rapidly recovered transient flow resistance: a newly discovered property of blood.

Although blood flows in a pulsatile fashion, little consideration has been given in past studies to its instantaneous resistance to motion when onset and cessation of flow occur abruptly. Hemorheological studies have documented three kinds of blood flow properties. 1) Shear thinning is a fall in viscolity as shear rate rises. 2) Viscoelasticity is a transient shear stress variation due to elastic deformation of erythrocytes. Dilatancy is a viscoelasticity-modifying property attributed to high shear rate erythrocyte rigidity; viscoelasticity is prominent only at low shear rate. 3) Thixotropy is an initial extra flow resistance linked to developing orientation and disaggregation of erythrocytes. Thixotropy returns fully to blood over a period longer than 1 min. Measurements utilizing a fast response Couette viscometer have revealed an extra 10% transient flow resistance after a flow cessation shorter than that between heart beats. The rapidly recovered transient flow resistance has a temporal pattern similar to thixotropy. Its peak and duration are directly related to total shear strain (shear rate x time) over the 8-30 s-1 shear rate range studied. Transient behavior was essentially identical in analyses carried out using three different viscometer gaps. Numerical simulation to test the effect of the newly observed transient behavior on sudden onset tube flow shows that the developing pattern of pulsatile arterial flow can be affected by its presence.

Blood's critical Taylor number and its flow behavior at supercritical Taylor numbers.

When the inner cylinder of a fluid-filled Couette viscometer is rotated rapidly, a vortical flow pattern develops when a dimensionless value referred to as the critical Taylor number (Tc) is reached. We have determined its magnitude in our viscometer for three Newtonian fluids and for blood at 37 degrees C, using the inflection point of torque/RPM vs. RPM (sudden rise in apparent viscosity). Its position was identified by least squares line fitting. Because blood was studied, the viscosity used in Tc calculation was the apparent bob shear stress/shear rate ratio at the inflection marking vortical flow onset. For glycerol-water mixtures Tc was 41.8 +/- 0.3 (N = 11), for propylene glycol 42.0 +/- 0.2 (N = 14), for silicone oil 41.8 +/- 0.2 (N = 11). For healthy blood Tc was 40.7 +/- 0.9 (N = 140). This evidence against blood's increased resistance to flow instability was accompanied by a slower rate of rise in torque both above and below Tc compared to the three Newtonian fluids. Newtonian fluids and blood both developed wavy vortical flow at a rotation rate moderately higher than Tc. Blood resisted this unstable flow behavior more than the Newtonian fluids but it also experienced a slower rate of rise in torque with increasing rotation rate above the critical Taylor number. Shear-thinning is the simplest explanation for blood's mildly altered Taylor vortex behavior; blood's resistance to flow instability is otherwise not found to be sufficient to affect its flow stability in man.

An instrument to evaluate the time dependent flow properties of blood at moderate shear rates.

The rheology of blood is characterized by shear thinning, viscoelasticity, and thixotropy. Its rheological evaluation is usually accomplished using a torque balance technique during rotational viscometry. Because a stable torque balance does not develop instantly, studies of thixotropy and viscoelasticity of blood have usually been carried out only at low shear rate where their development is slow enough to be monitored accurately. The torque balance technique may be converted from static to dynamic by incorporating the rate of change of sensing system angular momentum. We have modified our Couette viscometer, adding a computer-controlled stepping motor and a second digital voltmeter. The latter is used to determine the angular position of the sensing system every 25 or 50 msec. The new approach allows rapid observation of the development and disappearance of shear stress, enabling us to examine the transient behavior of blood at moderate shear rate (1 to 100 inverse seconds). The transient flow behavior of blood at moderate shear rate is most easily compared directly with the behavior of Newtonian fluids. We present information about the response of our system using a torque balance observation rate of 20 per second. Blood's viscoelasticity is observed to fall substantially as shear rate rises, while its thixotropic transient excess stress rises steadily with increasing shear rate.

Deformational strain energy and erythrocyte shape.

Analyses of erythrocyte deformation commonly assume the discocytic shape to be unstressed and hence to be without strain energy. This assumption is based on micropipet experiments and a theoretical disparity between observed and requisite curvature rigidity. The polynomial description of Evans and Fung and strain energy expressions introduced by Zarda et al. have been used to explore the dependence of erythrocyte strain energy on unstressed surface curvature and elastic moduli. As unstressed membrane curvature is varied from flatness to curvatures greater than that of the sphered erythrocyte, the strain energy is found to fall to a value about two-thirds that of the sphered cell. This value occurs for a cell with a thickness, surface area and volume comparable to those of observed erythrocytes assuming a curvature elastic modulus of 1.5 X 10(-11) ergs and an extensional elastic modulus of 6.6 X 10(-3) ergs cm-1. The erythrocyte so characterized has a slightly smaller diameter than that of the measured erythrocyte as does a cell similar in thickness to the expanded cell studied by Evans and Fung. A cup-shaped erythrocyte with similar surface area and volume has a slightly greater strain energy than the standard discocyte when its unstressed curvature is that of a sphered cell. Its strain energy falls below that of the discocyte as its unstressed curvature approaches flatness. Curvature strain energy was found to be concentrated in the dimple of the erythrocyte, the effect being more striking when unstressed curvature was greater. Extensional strain energy, lower in density, was concentrated at the equator. The basis for current emphasis on the unstressed discocyte is reviewed. While no clear conclusion can be reached, the alternative model presented here is attractive in explaining cup-shaped erythrocyte formation but requires a rather high resistance to curvature deformation. Until conclusive evidence is developed both the unstressed and the present discocyte models should be evaluated in examining erythrocyte deformation and adhesive behavior.

Doublet formation of diabetic erythrocytes as a model of impaired membrane viscous deformation.

Erythrocyte deformation involves both viscous dissipation in the cell interior and viscoelastic motion of the cell membrane. Reports that describe reduced filterability of diabetic erythrocytes, altered response to oscillatory motion in a capillary-sized pipet, and impaired packing during centrifugation indicate a disturbance of red cell rheology in diabetes. We have selected conditions that minimize the macromolecule-mediated energy of attraction between erythrocytes and studied erythrocyte motion during doublet formation. Under such conditions, doublet formation frequency is strikingly reduced in diabetes. For nondiabetic erythrocytes the formation rate is 0.73 doublets per minute, whereas for diabetic erythrocytes the rate is 0.23 doublets per minute. In addition, mean velocity of doublet formation was found to be decreased to half of normal in diabetes. Completeness of doublet formation, regularly diminished when cell size of the two component cells was similar, was the same for diabetic and nondiabetic erythrocytes. Observation of several features of doublet formation gave a picture of the mechanical process. The initial cell making contact with the glass microscope slide was observed to remain fixed in position. The late arriving cell's ability to form a doublet was seen to decrease rapidly, apparently because it came to adhere to the glass surface. The attractive force between the cells overcomes the force of gravity, but cell deformation resistance slows doublet formation by balancing the tendency for cell-cell contact area to increase. An integral equation combining strain energy and viscous dissipation was applied to the doublet formation process. Slowing of doublet formation in diabetes appears to be produced by a doubling of resistance to rate of change of curvature of diabetic erythrocytes.

Effect of anionic amphophiles on erythrocyte properties.

This preliminary study describes effects of two pharmacologic agents on erythrocyte behavior. Increased erythrocyte aggregation has been proposed as important in the pathogenesis of a number of disorders, but the exact mechanism by which it plays a role in disease production remains unclear. Several anionic amphophiles have been reported to benefit diabetic vascular disease and atherosclerosis. If anionic amphophiles enter the erythrocyte plasma membrane they can increase its negative charge, reducing the energy of attraction between red blood cells and diminishing erythrocyte aggregation. Erythrocytes were studied after suspension in phosphate-buffered saline containing dextran as an aggregation-promoting agent. A marginal reduction of the suspension's viscosity was found at low shear rate when 2,5- dihydroxybenzene sulfonate was added. Additionally, erythrocyte sedimentation rate was marginally influenced. Both dihydroxybenzene sulfonate and acetylsalicylate protected human erythrocytes from hemolysis at concentrations from 10(-3) to 10(-5) M. The removal of erythrocyte sialic acid using neuraminidase to reduce surface negative charge led to unequivocal interference with aggregation (MAI technique of CHIEN et al., J. Gen. Physiol., 1973) by both anionic amphophiles were studied. Dihydroxybenzene sulfonate and actylsalicylate reduced the aggregation propensity of sialic-free erythrocytes, suggesting that the effect on the low shear rate viscosity of sialic acid-containing erythrocytes, though modest, is real.

Impaired erythrocyte doublet formation in diabetes.

A new means by which to examine erythrocyte deformability, the rate at which erythrocytes in dilute suspension form doublets after settling to the surface of a microscope slide, has been developed and tested. Doublet formation consists of the evaluation and subsequent apposition of one cell over another, a process limited by the ability of each red cell membrane to bend. The cell-cell attraction that promotes doublet formation is controlled by adding an appropriate amount of dextran to the artificial suspending medium. Videotaping permits careful analysis of doublet formation rate and maintains a permanent record. When erythrocytes from 20 diabetic and 20 non-diabetic subjects were studied, doublet formation rates were found to be strikingly different. Cells from half of the diabetics studied formed less than three doublets in 20 min while the non-diabetic mean for the same period was 15 doublets. No overall correlation between doublet formation and fasting glucose could be found. No relation between doublet formation rate and type of diabetes, treatment, or microvascular complications was observed. Doublet formation rate is a simple and rapid means of detecting and studying reduced erythrocyte deformability in diabetes.