Centrifugation-induced release of ATP from red blood cells

Abstract

Centrifugation is the primary preparation step for isolating red blood cells (RBCs) from whole blood, including for use in studies focused on transduction of adenosine triphosphate (ATP), an important vasodilatory signaling molecule. Despite the wide use of centrifugation, little work has focused on how the centrifugation itself affects release of ATP from RBCs prior to subsequent experimentation. Here we report that both the centrifugation force and duration have a pronounced impact on the concentration of ATP present in the packed RBCs following centrifugation. Multiple subsequent centrifugations yield extracellular ATP concentrations comparable to the amount released during the initial centrifugation, suggesting this effect is cumulative. Pairwise measurements of hemoglobin and ATP suggest the presence of ATP is primarily due to an increase in centrifugation-induced hemolysis. These results indicate that common centrifugation parameters, within the ranges explored here, can release ATP in quantities comparable to the low end of the range of values measured in typical ATP transduction experiments, potentially complicating experimental interpretation of those results.

Blood apheresis technologies – a critical review on challenges towards efficient blood separation and treatment

“…53 The adverse effects to cells as associated with the spinning duration and centrifugal force was also explored and correlated to adenosine triphosphate (ATP) (organic compound that drives many metabolic processes in cells) release with haemolysis. 35 An increase in the concentration of ATP was observed (from 1.4-4.7 nM) as the centrifugation time increased from 1 to 5 min (Fig. 1c) while varying the applied centrifugal force from 900-16 000 Â g. 35 The magnitude of ATP in the packed RBCs obtained using the parameters explored suggests that care must be taken when interpreting ATP transduction data in experiments that utilize centrifugation.…”

“…The main challenge of using centrifugation-based apheresis is the occurrence of haemolysis, or rupture of blood cells, caused by shear stress introduced during spinning. 35 The extent of haemolysis is dependent on centrifugal operating conditions such as operating mode to be used, centrifugal force, spinning duration and number of centrifugal cycles and will be discussed in this section.…”

Temporomandibular joint biosupplementation using platelet concentrates: a narrative review

“…Careful evaluation of the state of RBCs post processing is paramount in selecting the ideal biologic. Several laboratory investigations have confirmed that centrifugal forces in the range of 500-1,500 g for 10 minutes impart no hemolytic damage to blood samples; however repeat centrifugation cycles within such a g-force range could raise levels of PFH within the supernatant (79).…”

Red Blood Cell Deformability, Vasoactive Mediators, and Adhesion

“…Supraphysiologic levels of mechanical force, resulting either from poorly controlled deformation as the primary stimulus, or from inadvertent mechanical force of gases used to bubble RBC solutions to induce hypoxia, will unquestionably lead to cell rupture and the release of cellular contents (RBC lysis) including ATP. Importantly, the centrifugation used to separate the plasma or supernatant from treated RBCs can itself be sufficient to produce hemolysis, with liberation of ATP (Mancuso et al, 2018). We recommend gentle centrifugation for supernatant separation.…”