Determination of erythrocyte deformability and its correlation to cellular ATP release using microbore tubing with diameters that approximate resistance vessels in vivo

Abstract

A novel method is described for measuring the deformability of red blood cells (RBCs) in tubing whose diameters approximate forces encountered in vivo. Here, RBCs from rabbits are loaded into a 50 cm section of 75 µm id microbore tubing and connected to a syringe pump. This section of tubing is then connected to a 15 cm section of 25 µm id tubing. As buffer is pumped through the flow system, the RBCs are evacuated from both sections of tubing. However, the inability of the RBCs to move freely through the 25 µm id section of tubing results in a buildup of cells at the inlet of this portion of tubing. The continued force output by the syringe pump results in a deformation of the RBCs until all of the cells are eventually evacuated from the flow system. It was found that a measurement of the time required to reach half of the maximum pressure (½P_max) may be used as an indicator of the RBC deformability. For a given sample, a simple buffer results in less time to reach ½P_max (6.9 ± 0.2 s) than deformable RBCs (21.6 ± 0.8 s). To verify that the increased amount of time to reach ½P_max is indeed due to the RBCs, various hematocrits of an RBC sample were investigated and, as expected, it was found that a 12% RBC hematocrit had a higher ½P_max value (26.0 s ± 2.2 s) when compared to a 7% hematocrit (19.1 ± 0.3 s). In addition, RBCs chemically stiffened with glutaraldehyde were shown to be 25% less deformable than normal RBCs. Finally, a study was performed to examine the relationship between RBC deformability and ATP release and it was found that ATP release increased as a function of RBC deformability. This method greatly simplifies deformability measurements, employing only a syringe pump and microbore tubing, and may lead to a more complete understanding of the physiological significance of erythrocyte deformability.