Vox Sang. 52: 182-185 (1987) 0 1987 S. Karger AG, Basel 0042-9007/87/0523-0182 $2.75/0

The Use of Nystatin to Restore the Flow Properties of Time-Expired Stored Erythrocytes

Susan Turnera, A. R. Williamsa, J. M. H. Reesb Departments of aMedical Biophysics and bPharmacology, University of Manchester, UK

Abstract. Human erythrocytes stored for more than 21 days in citrate phosphate dextrose with adenine (CPDA-1) exhibited a marked reduction in volume following incubation for 24 h at 37 "C in fresh autologous plasma. This incubation apparently mimics the effects of reinfusion of the stored cells in vivo. These shrunken stored cells have a decreased filterability as measured by their increased transit times through a 5-pm diameter Nuclepore filter. The polyene antifungal agent nystatin was used to reinflate these shrunken cells with different concentrations of potas- sium ions. A concentration of 90 Mpotassium chloride was found to reinflate the shrunken stored cells so that their mean cell volume, mean corpuscular haemoglobin concentration and average densities were the same as those of fresh cells. This reinflation also restored the filterability of the shrunken stored cells so as to be similar to that of fresh cells.

Introduction

In a previous study [Turner et al., 19871, the effects of storage in citrate phosphate dextrose with adenine (CPDA-1) on red blood cells were studied for a period of up to 42 days. Measurements of cell volume, mean cor- puscular haemoglobin concentration (MCHC), cell densi- ty and flow through polycarbonate membrane filters were made at weekly intervals. Cells taken directly from stor- age showed very little change in any of the parameters measured over the 42-day period. However, stored cells which had been incubated in autologous fresh plasma for 24 h at 37 "C before the measurements were made (i.e. a procedure which apparently mimics reinfusion) did show significant changes after a storage period of approximate- ly 21 days. Cells stored for periods greater than 21 days were found to be shrunken, have an elevated MCHC, have a greater percentage of dense cells within the popu- lation and a significantly decreased ability to flow through

' Some of this work was presented to the British Pharmaceutical Conference and was abstracted in J. Pharm. Pharmac. 37: 106

a Nuclepore polycarbonate membrane having a pore diameter of 5 km.

These changes were probably due to a progressive loss of intracellular ions (mainly K+) [Wallas, 19791 and there- fore water, without a corresponding loss of haemoglobin. This resulted in an increase in MCHC as the cells aged during storage in vitro [Clark et al., 19781. The increased MCHC in turn resulted in an increased erythrocyte inter- nal viscosity and cell density, and increased the resistance of cells to flow under positive pressure through polycar- bonate membranes. This increased resistance to flow in vivo would increase the transit time for cells through the splenic channels [Hams and Kellermeyer, 19701 and increase the probability of their sequestration by macro- phages [Weiss and Tavassoli, 19701.

An attempt to prevent shrinkage of red cells during 24 h of incubation has been made by pretreatment with the polyene antifungal agent nystatin. Nystatin has pre- viously been used to alter intracellular sodium and potas- sium concentrations, by producing pores in the mem- brane large enough to allow the passage of monovalent cations into the cells [Cass and Dalmark, 19731. The effects of nystatin on cell membrane permeability are

(1985). reversible as long as there is thorough washing of the cells

Reversal of the Blood Storage 'Lesion' 183

to remove the nystatin after treatment [Clark et al., 19781.

In the following study, measurements of cell volume, cell density, MCHC and flow rates through polycarbonate membranes were made for nystatin-treated cells and com- pared with those obtained for freshly taken cells and unin- flated cells which had been stored in CPDA-1 for 2 1 days.

Materials and Methods

Experienced clinicians withdrew blood samples from an antecu- bital vein of healthy adult volunteers. The blood was immediately anticoagulated with CPDA-I in a ratio of 450 ml blood to 63 ml CPDA-1, divided into aliquots and stored in glass Macartney bottles at +4 "C. Blood samples were removed after 21 days of storage, centrifuged for 5 min at 2000g in a Centaur 2 MSE centrifuge, and the plasma and buffy coat were removed. The packed cells were then washed twice using a sterile saline solution (0.9% wlv) and mixed thoroughly using a Vortex mixer.

The method used for equilibrium dialysis of monovalent ions was adapted from the protocols used by Cass and Dalmark [ 19731 and Cavieres and Ellory [ 19741. Cells were suspended to approximately 5% haematocrit in solutions containing 27 mM sucrose, 140 mM sodium chloride and potassium chloride in a range of concentrations between 50 and 500 mM. These high concentrations were needed to increase cell volume. Nystatin was added to a final concentration of 30 pg/ml using a stock solution of 5 mg nystatin in 1 ml anhydrous methanol. Methanol (rather than ethanol) was used because nystatin is poorly soluble in ethanol. The final concentration of methanol was 6 pVml. In control experiments lacking nystatin, methanol (like saline) was without effect. Cells were allowed to equilibrate for 30 min at 21 'C, as nystatin is more effective at lower temperatures [Finkelstein and Cass, 19681. The nystatin was washed out using four changes of the same equilibration medium but without the nystatin, to restore the cell's normal permeability bamer to sodium and potassium. The cells were then washed twice with a sterile saline solution (0.9Oh wlv) and then incubated in autologous fresh plasma for 24 h at 37 'C. Cells used for control measurements were treated in the same manner except that methanol alone was added to the equilibration media during the incubation.

A Coulter counter model ZB in conjunction with a size distribu- tion analyser P128 (Coulter Electronics Limited) was used to mea- sure cell volumes. The percentage of dense cells within the cell populations was measured by high-speed centrifugation (at approxi- mately 12,000 g) in the presence of a mixture of phthalic acid esters (density= 1.098 g/ml) as previously described [Turner et al., 19871. Measurement of mean cell volume (MCV) and MCHC were made using standard blood bank methods by means of the Coulter S analyser (Coulter Electronics Limited). Flow characteristics were determined using a Nuclepore filtration method, utilising filters with a 5-pm pore diameter, as previously described [Turner et al., 19871.

The effects of treatment of red blood cells with nystatin on cell fragility were assessed using absorption measurements. Absorbances of washing solutions were made using a Shimadzu recording spectro- photometer UV-240 P/N 204-5800, Schimadzu Corporation) at a h of 540 nm.

Results

Incubation of cells with nystatin and potassium chlo- ride concentrations ranging from 1 x lo-' to 5 x 10-l M resulted in an increase in cell volume of approximately 40 pm3. However, incubation of the cells in the absence of nystatin had little effect on cell volume, showing that nystatin was necessary to bring about these changes in volume. The increase in cell volume could be demon- strated on freshly taken cells and on cells which had been stored in CPDA- 1 for 2 1 days at +4 "C, as long as the nystatin was present in the original incubation me- dium.

The effects of increasing the concentration of potas- sium chloride in the equilibration medium on cells which had been stored in CPDA-1 for 21 days are shown in figure 1.

It appeared that the concentration of potassium chlo- ride required in the equilibration medium to produce an MCV similar to that found for freshly taken cells (i.e. approximately 90 pm3) was 1 x 10-l M. Equilibration media used in subsequent experiments had potassium chloride concentrations within a range of 50-1 30 mM.

The percentage of cells more dense than a mixture of phthalic acid esters (density= 1.098 g/ml) could also be altered using the nystatin reinflation method. Figure 2 shows the effects of altering the concentration of potas- sium chloride in the equilibration medium over a range of 50-130 mM on the percentage of dense cells. In the absence of nystatin, the cells retained the same density over the entire range of potassium chloride used. For cells which had been stored for 21 days in CPDA-1 (and then incubated in fresh plasma for 24 h), this ranged between 30 and 40% of the cell population having a density greater than that of the ester mixture, and approximately 5% of the population for freshly taken cells (which had been incubated in a similar manner). In the presence of nystatin, the percentage of dense cells was reduced from 32.14 k 5.97 with 50 mM potassium chloride to 1.22 k 0.21 with 130 &potassium chloride in the equil- ibration medium. From these results it was estimated that approximately 90 mM potassium chloride produced a percentage of dense cells within the stored cell population similar to that found for freshly taken cells.

Coulter S measurements were therefore made on cells treated with nystatin and 90 &potassium chloride, and they were compared with those for methanol-treated fresh and 21-day stored cells (all samples being incubated for 24 h at 37 "C in autologous fresh plasma before being measured). These results are presented in table I.

184 Turner/Williams/Rees

Fig. 1. Effects of increasing the concentration of KCI on the mean red cell volume of human blood stored for 2 1 days in CPDA- 1 at 4 'C and then treated with nystatin (a) or methanoysaline (0). Mean of 6 determinations ? SEM. 'pt0.05.

Fig. 2. Changes in density of human red blood cells following treatment with KCI of fresh cells (m) and cells stored for 2 1 days in CPDA-I and then treated with nystatin (A) or methanoVsaline (0).

The phthalate-ester mixture had a density of 1.098 g/ml. Mean of 6 determinations 2 SEM. 'p<0.05.

Table I. MCV and MCHC of fresh human cells, in stored cells and in stored cells treated with nystatin. Mean k SEM of 6 determi- nations.

Fresh cells Stored cells

untreated Nystatin-treated

MCV, fl 90.83k 1.89 86.67k0.49 90.67+ 1.74

MCHC, g/dl 30.20k0.94 37.38k0.28 31.73k 1.03

Fig. 3. Flow ratios of fresh red blood cells (o), stored cells treated with methanol (A) and storedcells treated with nystatin (a). Mean of 6 determinations kSEM. 'p<0.05.

Cells were found to be smaller after 2 1 days of storage followed by incubation in fresh plasma. They could be reinflated using nystatin in the presence of 90 mM potas- sium chloride to give the same volumes as freshly taken cells. This cell shrinkage caused an increase in MCHC for stored cells, but this was lowered by reinflating the cells to give values similar to those found for freshly taken cells.

Flow ratios for nystatin-treated stored cells, methanol- treated stored cells and freshly taken cells are shown in figure 3. The 21-day stored cells treated with nystatin and 90mM potassium chloride show flow ratios similar to those found for freshly taken cells. There were no signif- icant differences in flow ratios at any haematocrit between freshly taken cells and nystatin-treated stored cells (p>0.05). Stored cells treated with methanol alone within the incubation medium showed flow ratios lower than those found for nystatin-treated cells at all haema- tocrits. The flow ratios were significantly lower for metha- nol-treated stored cells at haematocrits of 15 and 20% (p<0.05) and 25% (pt0.01). Flow ratios could not be measured for haematocrits greater than 2 5% because attempted filtration resulted in filter blockage so that straight line plots could not be obtained.

Treatment of 2 1-day stored cells with nystatin and 90 mM potassium chloride reduced the resistance of the cells to flow and increased the flow ratio at any given haematocrit. The fact that treatment with methanol and the incubation medium had no effect on the flow ratio of stored cells indicated that nystatin was necessary to bring about the changes in flow rates through the Nuclepore filters. There was negligible evidence of lysis.

Reversal of the Blood Storage ‘Lesion’ 185

Discussion

The use of nystatin as a tool to alter internal ionic composition, and hence volume, of erythrocytes has been reported many times [Cass and Dalmark, 19731. Our experiments confirmed the ability of nystatin to alter cell volume using an incubation medium consisting of potas- sium chloride, sodium chloride and sucrose. An increase in the potassium concentration within the incubation medium produced an increase in the final volume of the treated cells. The concentration of potassium chloride required in the incubation medium to increase the vol- ume of shrunken 2 1-day stored erythrocytes to be similar to that found for freshly taken cells was estimated to be about 100 mM. Changes in red cell volume induced by incubation with nystatin and the incubation media also reduced the percentage of cells with a density greater than 1.098 g/ml within the treated cell population. As the cell volume increased, the cell density decreased, with a potas- sium chloride concentration of 90 mM being required to produce approximately 5% dense cells within the popula- tion (equivalent to that found for freshly taken cells).

A concentration of 90 mM potassium chloride plus nystatin was used to reinflate 21-day stored shrunken erythrocytes. Stored cells treated with this concentration of potassium chloride had cell volumes and MCHC val- ues which were the same as those measured for freshly taken cells (as measured using the Coulter S analyser) and showed flow characteristics similar to those measured for freshly taken cells using a polycarbonate membrane siev- ing method.

The apparent ‘rejuvenation’ of 2 1 -day stored erythro- cytes using nystatin may provide a method to increase the

usable life-span of CPDA- 1 -stored blood, but the reinfla- tion method must first be studied using an animal model in vivo to confirm these in vitro findings.

References

Cass, A.; Dalmark, M.: Equilibrium dialysis of ions in nystatin- treated red cells. Nature new Biol. 244: 47-49 (1973).

Cavieres, J.D.; Ellory, J.C.: Thallium and the sodium pump in human red cells. J. Physiol., Lond. 242: 243-266 (1974).

Clark, M.R.; Mohandas, N.; Caggiano, V.; Shohet, S.B.: Effects of abnormal cation transport on deformability of desiccytes. J. supramol. Struct. 8: 521-532 (1978).

Finkelstein, A.; Cass, A.: Permeability and electrical properties of thin lipid membranes. J. gen. Physiol. 52: 1 4 5 ~ 1 7 2 s (1968).

Harris, J. W.; Kellermeyer, R. W.: The red cell, pp. 28 1-626 (Harvard University Press, Cambridge 1970).

Turner, S.; Williams, A. R.; Rees, J. M. H.: The role of mean corpus- cular haemoglobin concentration in limiting the storage life of human blood. Vox Sang. 52: 186-190 (1987).

Wallas, C. H.: Sodium and potassium changes in blood bank stored human erythrocytes. Transfusion 19: 2 10-2 15 ( 1 979).

Weiss, L.; Tavassoli, M.: Anatomical hazards to the passage of erythrocytes through the spleen. Semin. Hematol. 7: 372-380 ( I 970).

Received: May 28, 1985 Accepted: September 5 , 1986

J.M.H. Rees, PhD, Department of Pharmacology, University Medical School, GB-Manchester MI3 9PT (UK)