Washing in hypotonic saline reduces the fraction of irreversibly-damaged cells in stored blood: a proof-of-concept study

Abstract

Background: During hypothermic storage, a substantial fraction of red blood cells (RBCs) transforms from flexible discocytes to rigid sphero-echinocytes and spherocytes. Infusion of these irreversibly-damaged cells into the recipient during transfusion serves no therapeutic purpose and may contribute to adverse outcomes in some patients. In this proof-of-concept study we describe the use of hypotonic washing for selective removal of the irreversibly-damaged cells from stored blood.

Materials and methods: Stored RBCs were mixed with saline of various concentrations to identify optimal concentration for inducing osmotic swelling and selective bursting of spherical cells (sphero-echinocytes, spherocytes), while minimising indiscriminate lysis of other RBCs. Effectiveness of optimal treatment was assessed by measuring morphology, rheological properties, and surface phosphatidylserine (PS) exposure for cells from several RBCs units (n=5, CPD>AS-1, leucoreduced, 6 weeks storage duration) washed in hypotonic vs isotonic saline.

Results: Washing in mildly hypotonic saline (0.585 g/dL, osmolality: 221.7±2.3 mmol/kg) reduced the fraction of spherical cells 3-fold from 9.5±3.4% to 3.2±2.8%, while cutting PS exposure in half from 1.48±0.86% to 0.59±0.29%. Isotonic washing had no effect on PS exposure or the fraction of spherical cells. Both isotonic and hypotonic washing increased the fraction of well-preserved cells (discocytes, echinocytes 1) substantially, and improved the ability of stored RBCs to perfuse an artificial microvascular network by approximately 25%, as compared with the initial sample.

Discussion: This study demonstrated that washing in hypotonic saline could selectively remove a significant fraction of the spherical and PS-exposing cells from stored blood, while significantly improving the rheological properties of remaining well-preserved RBCs. Further studies are needed to access the potential effect from hypotonic washing on transfusion outcomes.

Figure 1

Typical morphological appearance of (a) fresh red blood cells (RBCs) containing primarily discocytes (D), and (b) RBCs that have been stored hypothermically for 42 days showing cells in various stages of morphological deterioration, including echinocyte 1 (E1), echinocyte 2 (E2), echinocyte 3 (E3), sphero-echinocyte (SE), spherocyte (S) and stomatocyte (ST). Scale bar: 10 μm.

Figure 2

Effect of hypotonic treatment on fresh red blood cells (RBCs) of different morphologies. Individual RBCs were trapped in circular microwells of 20 μm diameter and 5 μm depth. The osmolality of the suspending medium was adjusted to 225 mmol/kg at t=20 sec, and back to 290 mmol/kg at t=250 sec. There was an approximately 120 sec delay for the osmolality within the microwells to equilibrate with that of the medium flowing over them through the main channel of the device. Scale bar: 10 μm.

Figure 3

Optimisation of the hypotonic treatment. (a) Dependence of percentage of haemolysis on duration of treatment with 0.225, 0.45, 0.495, 0.585, and 0.9 g/dL saline. (b) Dependence percentage of haemolysis on concentration of saline (range 0.225–0.9 g/dL) for different treatment durations (t=0.5, 2, and 30 min). (c) Dependence of red blood cell RBC morphology on the concentration of saline for t=30 min treatment duration. (d) Dependence of surface phosphatidylserine (PS) exposure on concentration of saline for t=30 min treatment duration. All values shown as mean ± standard deviation (n=3) for all studies.