The relevance of cell size on ultrasound-induced hemolysis in mouse and human blood in vitro.

This paper describes a further test of the hypothesis that cell size is an important physical parameter in ultrasound (US)-induced hemolysis, that is, the larger the cell the greater the potential for sonolysis by a cavitational mechanism. Mouse (M) and human (Hu) erythrocytes in vitro were used; their mean corpuscular volumes were 49.0 and 89.5 fL, respectively. At a US exposure in vitro in the presence of Albunex that yielded an average of 36.8% hemolysis for M blood, the Hu blood yielded an average of 54.0% hemolysis. The data supported the hypothesis. This paper also briefly discusses the difficulty of extrapolating sonolytic in vitro results to those derived in vivo.

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: medium tonicity.

Whole human anticoagulated blood in vitro underwent controlled plasma replacement with either isotonic (0.9%) or hypotonic (0.5%) saline to 1. restore the blood to its original volume (which resulted in different hematocrits) or 2. bring the blood to a singular hematocrit (40%). The hypotonic cell MCVs were, on average, considerably larger than their isotonic counterpart by a ratio of 1.4:1. The blood samples were then subjected to two tests, one of mechanical fragility, the other to ultrasound (US)-induced hemolysis. The US exposure metrics were: 1.0-MHz center frequency, 200-micros pulse duration, 20-ms interpulse interval, exposure durations of 10 to 30 s in the presence of Albunex, as a control on blood gas nucleation, and exposure vessel rotation at 200 rpm. In all instances, the hypotonic blood displayed higher levels of hemolysis than the corresponding isotonic treatment. The highest ratio of US-induced hemolysis for the hypotonic:isotonic regimens was 2.2. In some instances, the ratio was somewhat less but appeared to be related to differences in whole blood viscosities among the regimens or other factors. The data supported the a priori hypothesis that hypotonicity will result in an increased tension on the cell membrane and render it more susceptible to shear-induced hemolysis, including exposure to US under conditions known to foster the occurrence of inertial cavitation. There was no temperature increase during the insonations of the blood.

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 3. Antioxidant (Trolox) inclusion.

This project tested the hypothesis that human erythrocytes pretreated with Trolox (a water-soluble analog of vitamin E) would be more susceptible to ultrasound (US)-induced hemolysis by a cavitational mechanism because of an increased fragility of the erythrocyte membrane over that without Trolox supplementation. Samples of whole human blood from apparently healthy donors (hematocrit approximately 40%) in vitro were supplemented or not supplemented with Trolox at various concentrations, ranging from 1.8 to 0.0018 mg/mL plasma. Mechanical fragility tests indicated the Trolox-treated blood in vitro exhibited greater hemolysis than untreated blood in vitro (p < 0.001). US exposures at comparable acoustic amplitude, pulse length and duty factor in the presence of the US contrast agent Albunex yielded differing results; at 1 MHz, the Trolox-supplemented blood had significantly greater hemolysis in vitro than non-Trolox-supplemented blood; at 3 MHz, there was a substantial reduction in hemolysis relative to that obtained at 1 MHz, and no statistically significant difference between the Trolox-supplemented and -unsupplemented blood. There was also essentially no support for an alternative hypothesis that the Trolox was functioning primarily as a pro-oxidant. These collective experimental results support the hypothesis and suggest duality in the functionality of membranous antioxidant inclusions or associations; they may foster protection against oxidative damage, yet render the cell less capable of withstanding mechanical stress.

Comparative hemolytic effectiveness of 1 MHz ultrasound on human and rabbit blood in vitro.

This project continued testing of the general working hypothesis that cell size is a physical determinant in extent of ultrasound (US)-induced hemolysis, the larger the cell the greater the lysis. For this project, the specific hypothesis tested was that human erythrocytes, being larger than rabbit erythrocytes, would be the more sensitive to sonolysis induced by inertial cavitation in the presence of Albunex, a US contrast agent. The rationale behind this hypothesis was 1. an earlier-published analytic construct indicating an inverse relation between particle size and the shear force required for deformation, and 2. a number of independent demonstrations that, among sized populations of erythrocytes, an inverse relation exists between erythrocyte volume and mechanically-induced shear forces in the cell-bathing medium; namely, the larger the cell, the less shear force required to rupture the cell's membrane. The present data support the hypothesis; over six independent trials, the mean corpuscular volumes of human (H) and rabbit (R) erythrocytes were 89.5 and 64.1 microm(3), respectively, H > R (p << 0.001), and the ratio of US-induced hemolysis in H to R blood in vitro was 1.12:1.0 (p < 0.004).

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 2. Medium dissolved gas (pO2) content.

The data collected in this project supported the a priori hypothesis that the concentration of dissolved oxygen in whole human blood in vitro affected the extent of ultrasound (US)-induced hemolysis under conditions conducive to the occurrence of inertial cavitation. Aliquots of whole human blood in vitro with a relatively high O(2) level had statistically significantly more 1-MHz US-induced hemolysis than aliquots with a relatively low O(2) level in the presence of controlled gas nucleation (Albunex or ALX, supplementation), with US-induced hemolytic yields being substantially less at 2.2- and 3.5-MHz exposures or in the absence of ALX-supplementation at otherwise comparable acoustic pressures, pulse lengths and duty factors. Passive cavitation detection (pcd) measures indicated a linear relationship for hemolysis up to about 70% and pcd values (R(2) = 0.99).

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 1. HIV macrocytosis (cell size).

This paper reports the results of a further test of the hypothesis that the extent of ultrasound (US)-induced cell lysis in the presence of a US contrast agent to enhance cavitational effects is a function of cell size. The present data support the hypothesis. Human adult erythrocytes in vitro derived from patients with HIV (n = 15) and apparently healthy individuals (n = 15) were compared for US-induced hemolysis in vitro. The anticoagulated whole blood from patients with HIV and macrocytic erythrocytes had significantly greater (p <0.0001) mean corpuscular volume (MCV) and a significantly greater (p <0.03) extent of US-induced hemolysis in vitro relative to blood from apparently normal, healthy individuals. As a control to determine if disease state (i.e., HIV infection per se) might be a contributing factor in US-induced hemolysis in vitro, the blood from patients with HIV and apparently normal MCVs (n = 15) was also tested against an additional population of apparently normal, healthy individuals (n = 15); there were no statistically significant differences in MCVs or US-induced hemolysis between the two groups (p >> 0.05). There were also no statistically significant differences in viscosities or hematocrits of the whole blood or plasma in vitro from HIV-macrocytic or apparently healthy individuals but, for all blood types, a pooled correlation existed between hematocrit and whole blood viscosity.