Influence of red cell concentration on filtration of blood cell suspensions.

Pressure-time curves obtained by passing suspensions of blood cells in Ringer solution through a 5 microns polycarbonate filter at constant flow (1.6 ml/min) were evaluated for their ability to reflect the deformability of the erythrocytes. The initial pressure reading (Pi) obtained in a quasi-steady state during the first 1-2 sec of pumping was found to be reproducible for hematocrit values between 10 and 30 percent. This Pi value was normalized by the pressure generated by the cell-free suspending medium (PO) at the same flow rate. The ratio Pi/PO was found to be linearly proportional to hematocrit up to 30 percent but independent of leukocyte concentration up to 12,000/mm3. Later portions of the curve did vary with leukocyte count. By using the equations developed from theoretical modeling of cells passing through a filter, the experimentally determined relation of Pi/PO to hematocrit, and the known geometry of the filter pores, we were able to calculate parameters reflecting the deformability of red cells. These include beta, the ratio of resistance in a pore containing a red cell to that in a pore containing only the suspending medium, and alpha, the proportion of pores filled by erythrocytes in transit. The application of theoretical analysis to experimental data has provided quantitative insights into the behavior of red cells during filtration tests in normal and disease states.

Folding of red blood cells in capillaries and narrow pores.

The geometric features of red blood cells in narrow channels in vivo and in vitro were studied by electron microscopy. In rabbit myocardial capillaries about half of the red cells were folded. In polycarbonate filters with pore diameters of 2.2-4.5 microns approximately one third of the trapped red blood cells were folded. The frequency of folding did not depend on the applied pressure, which ranged from 0.1 to 8.0 cm H2O. The folding of the red blood cells in filter pores was used to estimate the bending stiffness of the membrane. An analysis based on the large deformation theory of bending of an elastic sheet was developed. Using pressures of 0.2 and 1.0 cm H2O, the bending stiffness of human red cell membranes was estimated to be approximately 2.4 - 11.6 x 10(-12) dyn-cm, which is in good agreement with other methods. A limiting radius of curvature of about 85 nm was found at higher pressures.

The dynamics of shear disaggregation of red blood cells in a flow channel.

Red blood cell (RBC) rouleaux were formed in a flow channel in the presence of 2 g/dl dextran (molecular weight 76,000). The partial separation of RBC rouleau doublets adhering to the floor of the flow channel in response to small oscillatory shear stresses was observed experimentally. Theoretical analyses on displacement and drag force were performed to determine whether the motion of the cell involves membrane rotation (i.e., rolling) or sliding. From the experimental data and the results of theoretical analyses, it is concluded that, under the conditions of the experiments, the RBCs in a doublet separate from each other by rolling, rather than sliding of the sheared cell.

Evaluation of a filter aspiration technique to determine membrane deformability.

A filter aspiration technique for the measurement of red cell membrane deformability described by Brailsford et al. (Blood Cells 1977;3:25) was evaluated. Pore diameters of 0.6 and 1.0 micron were used. Portions of red cells were aspirated with various hydrostatic pressures between 1 and 9 mm H2O. After fixation the filter was removed and the length and radius of the pressure-induced tongues measured by scanning electron microscopy. One to several tongues per cell were observed. The number of tongues had no influence on the measurements at pressures less than or equal to 7 mm H2O. The elastic modulus of the red cell membrane was calculated; it yielded 2.9 (+/- 0.4) X 10(-3) dynes/cm. Data obtained with filter aspiration were compared with the results obtained concurrently with the micropipette technique. A good correlation was found for normal red cells (r = 0.74, P less than 0.001). Stomatocytes produced by chlorpromazine in vitro had an increased elastic modulus with both methods. For echinocytes produced by salicylate or adenosine triphosphate depletion, the elastic modulus as determined by filter aspiration was normal, but the modulus as measured by micropipette aspiration was increased. Possible reasons for the difference are discussed. We conclude that the filter aspiration is a valid technique to measure elastic properties of red cell membranes, except when there are surface irregularities, such as those in the echinocytes.

A model of microvascular oxygen transport in sickle cell disease.

The model of local control of oxygen delivery in the microvasculature developed by H. J. Granger and A. P. Shepherd (1973, Microvasc. Res. 5, 49-72) was extended to describe microcirculatory blood flow in sickle cell disease. Two major characteristics of sickle cell blood were incorporated into the model: an abnormal blood viscosity which is dependent on the degree of hemoglobin oxygen saturation and hematocrit, and a reduced affinity of hemoglobin (Hb) for oxygen. Sickle cell blood viscosity as a function of oxygen saturation and hematocrit was modeled empirically based upon existing data. Alterations in HbO2 affinity were studied in the model by introducing P50 as an independent variable. The altered oxygen supply/demand relationship in sickle cell disease was simulated following an increase in tissue metabolic demand and a decrease in arteriolar blood flow. The results were analyzed to evaluate the roles of the various rheological characteristics of sickle cell blood in affecting microcirculatory dynamics and tissue oxygen delivery. It was demonstrated that, within the hematocrit range of 20 to 45%, the elevation of P50 from 27 to 38 mm Hg in sickle cell blood is adequate to compensate for the diminished O2 content, despite an elevated blood viscosity, and maintain near normal tissue pO2.

Pleiotropic drug resistance in cystic fibrosis fibroblasts: increased resistance to cyclic AMP.

In previous studies, cystic fibrosis (CF) fibroblasts were demonstrated to be resistant to the cytotoxic effects of ouabain, dexamethasone, and the sex hormones, dihydrotestosterone, 17beta-estradiol, and progesterone. We now show that CF fibroblasts also exhibit greatly increased resistance to the cytotoxic effects of exogenous dibutyryl cyclic AMP (cAMP), as well as to isoproterenol and theophylline, drugs which are known to increase endogenous levels of cAMP. CF cells were also shown to have normal amounts of (3H)cAMP binding to protein kinase as well as normal amounts of cAMP-stimulated protein kinase activity. Phosphodiesterase in CF cells was also found to be stimulated by cAMP to the same degree as in normal cells. These findings suggest that there is no detectable protein kinase deficiency in CF cells. cf cells thus appear to be unlike some cAMP-resistant mutants described by others which are defective in protein kinase activity and cAMP regulation of phosphodiesterase levels. The cross-resistance of CF fibroblasts to ouabain, steroid hormones, and cAMP may provide a unique opportunity to study the biochemical events involved in the metabolism of these drugs as well as the basic biochemical defect in a common human genetic disease.