Effects of a single sickling event on the mechanical fragility of sickle cell trait erythrocytes

Abstract

Hemolysis contributes to the pathology associated with sickle cell disease. However, the mechanism of hemolysis or relative contribution of sickling due to hemoglobin (Hb) polymerization vs. oxidative damage remains unknown. Earlier studies aimed at deciphering the relative importance of these two mechanisms have been complicated by the fact that sickle red cells (SS) have already been affected by multiple rounds of sickling and oxidative damage before they are collected. In our study, we examine the mechanical fragility of sickle cell trait cells, which do not sickle in vivo, but can be made to do so in vitro. Thus, our novel approach explores the effects of sickle Hb polymerization on cells that have never been sickled before. We find that the mechanical fragility of these cells increases dramatically after a single sickling event, suggesting that a substantial amount of hemolysis in vivo probably occurs in polymer-containing cells.

Figure 1. Deformability of AA, AS and SS cells

A) Images of red cell deformability for oxy and deoxy AA (control), AS (sickle cell trait) and SS (sickle) cells at a flow rate of 5 ml/min (106.1 Pa) B) The deformability coefficient (DC) is plotted for various shear stress. Oxygenation did not affect AA cells (oxygenated: 2.1 ± 0.5; deoxygenated 1.9 ± 0.4). Under oxygenated conditions, the AS cells average DC was 1.8 ± 0.4 (n=3), whereas the oxy SS cells average DC was 1.6 ± 0.3 (n=3). These data are an average of several trials taken from three different blood samples from three separate donors. Under deoxygenated conditions, the DC remains constant for both AS and SS cells at different shear (1.2 ± 0.04 and 1.3 ± 0.05 for AS and SS deoxy, respectively). This indicates that both cell types are poorly deformable due to polymerization.

Figure 2. Measure of hemolysis in AA cells

AA (control) cells were rotated at 33 rpm in the presence of glass beads. The percentage hemolysis was measured and plotted with respect to the time the cells were exposed to shear. No significant difference in fragility in the presence or absence of oxygen is displayed, where the data is an average of numerous measurements (n=3). These data are an average of several trials taken from three different blood samples from three separate donors. The lysis after one hour of shear for these oxy AA cells was 5.2 ± 1.6 % for 1 hr, 11.7 ± 2.7 % for 2 h, and 18.5 ± 6.1 % for 3 h. When the cells were deoxygenated, % hemolysis = 11.1 ± 5.9 % for 1 h, 18.7 ± 15.7 % for 2 h, and 23.3 ± 19.5 % for 3 h. Upon reoxygenation via dialysis, % hemolysis = 13.2 ± 9.0 % for 1 h, 26.1 ± 19.6 % for 2 h, and 38.7 ± 26.9 % for 3 h. As a control, another set of cells was treated identically as the reoxygenated ones except without any deoxygenation. These are referred to as “dialyzed oxy”. For dialyzed oxy, % hemolysis = 3.5 ± 0.6 % for 1 h, 8.0 ± 1.6 % for 2 h, and 12.4 ± 2.0 % for 3 h. No significant differences were observed when comparing oxygenated to deoxygenated conditions or dialyzed oxygenated conditions to reoxygenated conditions (p > 0.05, using t-test and one-way ANOVA).

Figure 3. Mechanical fragility of SS cells

SS (sickle) cells were rotated against glass beads under both oxy and deoxy conditions. Percent Hemolysis was measured and plotted as a function of the time of the exposure to mechanical stress. Under oxygenated conditions, % hemolysis = 5.6 ± 1.3 % for 1 h, 9.9 ± 0.4 % for 2 h, and 16.8 ± 2.0 % for 3 h. In the absence of oxygen, hemolysis increased nearly three-fold in comparison to an oxy environment. For one hour, the % hemolysis = 36.2 ± 14.2 %. The lysis increased over time to 52.9 ± 23.8 % and 67.4 ± 41.8 % for 2 h and 3 h, respectively. Once the cells were reoxygenated, the hemolysis was significantly reduced to 11.5 ± 9.5 % for 1 h, 19.0 ± 12.5 % for 2 h, and 29.4 ± 11.5 % for 3 h. For dialyzed oxy, % hemolysis = 5.1 ± 2.4 % for 1 h, 10.5 ± 4.1 % for 2 h, and 14.5 % ± 5.1 % for 3 h (n=3). These data are an average of several trials taken from three different blood samples from three separate donors. *P < 0.05 for a t-test and one-way ANOVA, deoxy compared to oxy or reoxy compared to dialyzed oxy.

Figure 4. Mechanical Fragility of AS cells

AS (sickle trait) cells were subjected to mechanical stress similar to the SS (sickle) cells and hemolysis was measured. For oxy, % hemolysis = 10.9 ± 8.1 % for 1 h, 18.1 ± 12.2 % for 2 h, and 25.2 ± 15.1 % for 3 h. For deoxy, % hemolysis = 28.1 ± 10.2 % for 1 h, 47.2 ± 18.6 % for 2 h, and 84.4 ± 28.4 % for 3 h. (n=3). These data are an average of several trials taken from three different blood samples from three separate donors. When reoxygenated, the cells recovered to 4.9 ± 2.2 % for 1 h, 16.4 ± 7.2 % for 2 h, and 16.8 ± 3.1 % for 3 h. These values are comparable to the dialyzed oxy conditions (% hemolysis = 4.5 ± 1.9 % for 1 h, 7.4 ± 3.3 % for 2 h, and 12.4 ± 3.8 % for 3 h). *P < 0.05 for a t-test and one-way ANOVA, deoxy compared to oxy or reoxy compared to dialyzed oxy.