Kinetics of increased deformability of deoxygenated sickle cells upon oxygenation

Abstract

We have examined the kinetics of changes in the deformability of deoxygenated sickle red blood cells when they are exposed to oxygen (O(2)) or carbon monoxide. A flow-channel laser diffraction technique, similar to ektacytometry, was used to assess sickle cell deformability after mixing deoxygenated cells with buffer that was partially or fully saturated with either O(2) or carbon monoxide. We found that the deformability of deoxygenated sickle cells did not regain its optimal value for several seconds after mixing. Among density-fractionated cells, the deformability of the densest fraction was poor and didn't change as a function of O(2) pressure. The deformability of cells from the light and middle fraction increased when exposed to O(2) but only reached maximum deformability when equilibrated with supraphysiological O(2) concentrations. Cells from the middle and lightest fraction took several seconds to regain maximum deformability. These data imply that persistence of sickle cell hemoglobin polymers during circulation in vivo is likely, due to slow and incomplete polymer melting, contributing to the pathophysiology of sickle cell disease.

FIGURE 1

The experimental setup. The syringe pump pushed two syringes containing deoxygenated sickle erythrocytes in one and oxygenated or carbon monoxide (CO)-saturated buffer in the other to reach a final flow rate of 20 mL/min through the mixer into the flow channel. The mixer, composed of a homemade mixer followed by a 4.5 cm section of an in-line mixer, was used to achieve complete mixing of the two solutions. The time it took from the mixing to the observation point was varied by changing the length of the tubing (d) between the in-line mixer and the flow channel. Longer tubing gave longer times for the deoxygenated erythrocytes to regain their optimal deformability. The deformability was probed by measuring the diffraction pattern from a laser projected onto a screen.

FIGURE 2

Diffraction patterns of sickle and normal erythrocytes: fully deoxygenated, fully oxygenated, and fully CO-saturated. The calculated deformability coefficients (DCs) for the sickle cells are 1.28 for 100% deoxygenated, 1.64 for fully oxygenated, and 1.73 for fully CO-saturated. The calculated DCs for the normal cells are 3.60 for 100% deoxygenated, 3.62 for 100% oxygenated, and 3.60 for fully CO-saturated.

FIGURE 3

Diffraction patterns of sickle and normal erythrocytes of deoxygenated sickle erythrocytes mixed with oxygen (O₂, 0.70 mM) at various times after mixing. The calculated DCs for the sickle cells are 1.38 for 0.7 s, 1.58 for 2.4 s, and 1.64 for 5.3 s. The calculated DCs for the normal cells are 3.62 for 0.7 s, 3.58 for 2.4 s, and 3.59 for 5.3 s.

FIGURE 4

The DC of deoxygenated sickle erythrocytes mixed with oxygenated or CO-saturated buffer plotted against time after mixing. (A) Deoxygenated sickle erythrocytes were mixed with 0.56 mM oxygenated buffer to achieve a final O₂ concentration of 0.28 mM. The difference between the trials was due to different blood source, the freshness of the erythrocytes, and the storage time after deoxygenation. The error bars on trial 6 represent the standard deviation from two measurements on the same blood sample taken ∼30 min apart. (B) Deoxygenated sickle erythrocytes were mixed with 50% CO-saturated buffer to reach a final concentration of dissolved CO of 25% (250 μM). The error bars on trial 6 represent the standard deviation from two measurements on the same blood sample taken ∼30 min apart. (C) Deoxygenated sickle erythrocytes were mixed with 1.4 mM oxygenated (fully saturated) buffer to achieve a final O₂ concentration of 0.70 mM. (D) Deoxygenated sickle erythrocytes were mixed with 100% CO-saturated buffer to reach a final concentration of dissolved CO of 50% or 0.5 mM.

FIGURE 5

Normalized average DC of deoxygenated density-fractionated sickle erythrocytes mixed with partially O₂-saturated buffer plotted against time after mixing. Measurements were taken on five different blood samples and the normalized average deformability, was calculated as described in the Materials and Methods section. The deformability at each time after mixing was divided by the maximum deformability for that particular blood sample fraction obtained by equilibration with 100% O₂. This ratio is then multiplied by the average maximum deformability of all the blood samples for the particular fraction (light, medium, or dense). The average of these normalized deformabilities from five different blood samples are plotted with ±SD. Light, medium, and dense indicate the increase in density of the three density fractions of sickle erythrocytes. Each fraction of erythrocytes was deoxygenated and then mixed with 0.56 mM oxygenated buffer to achieve a final O₂ concentration of 0.28 mM.

FIGURE 6

Average DC of deoxygenated density-fractionated sickle erythrocytes 6.5 s after mixing with partially O₂-saturated buffer compared with that of fully oxygenated erythrocytes for the same density fractions. The average and standard deviations were calculated from the original un-normalized data for five different blood samples. p-values were derived from a paired t-test.

FIGURE 7

Average temperature effect on the DC of deoxygenated sickle erythrocytes mixed with partially O₂-saturated buffer plotted against time after mixing. The un-normalized average DC and standard deviations were calculated from three paired measurements, at 20°C and 37°C, and each measurement was conducted using a different blood sample. For the DC measurements at 37°C, the blood samples were incubated at 37°C for 30 min before use. The deformability of the samples after equilibration with 100% O₂-saturated buffer was the same at both temperatures; for example, on one blood sample, we measured DC = 2.16 at 37°C and 2.22 at 20°C. The average DC for samples equilibrated with 100% O₂ at 37°C DC was 2.09 ± 0.14. Further measurements at 37°C at 6.5 s after mixing gave a value of 1.74 ± 0.05, confirming that the deformability of the cells was still changing at 5.3 s where the DC was 1.71 ± 0.21 after mixing at this temperature.