0 1987 S.Karger AG, Basel 0042-9007/87/0523-0186 $ 2 . 7 9 0 Vox Sang. 52: 186-190 (1987)

The Effects of Reinflation on the Survival of Time-Expired Stored Rabbit Erythrocytes in vivo

Susan T u r n e r a , A. R. W i l l i a m s a , J.M. H. Reesb Departments of aMedical Biophysics and bPharmacology, University of Manchester, UK

Abstract. Rabbit blood cells stored for prolonged periods in citrate phosphate dextrose with adenine (CPDA- 1) at +4 "C show the same decreased volume, increased cell density and decreased filterability as human cells stored under similar conditions. As with human erythrocytes, the stored rabbit cells had to be incubated with autologous fresh plasma for 24 h at 37 'C before these changes could be observed (this incubation process apparently mimics the effects of reinfusion). These shrunken stored rabbit cells could also be reinflated using nystatin, so that their mean cell volume, mean corpuscular haemoglobin concentration, average cell densities and filterabilities were restored to normal values. This in vitro reinflation of the stored cells significantly prolonged their in vivo survival rate as determined by radiochromium labelling studies. This reinflation technique may therefore have an application in the prolongation of the useful storage life of human blood.

Introduction

It has been shown that prolonged storage of human erythrocytes in vitro in citrate phosphate dextrose with adenine (CPDA-1) results in cell shrinkage which in turn causes an increased mean corpuscular haemoglobin con- centration (MCHC), an increase in the percentage of dense cells within the population and a decrease in the ability of cells to flow through polycarbonate membranes [Turner et al., 1987al. These effects may be reversed in vitro using nystatin and an incubation medium contain- ing 27 Wsucrose, 140 W s o d i u m chloride and 90 mM potassium chloride. Time-expired stored cells treated with nystatin show cell volumes, MCHC, the percentage of dense cells within the population and flow character- istics through polycarbonate membranes which are simi- lar to those measured for freshly taken cells.

The effects of storage of rabbit erythrocytes in CPDA- 1 in vitro has also been investigated, and the same effects were found as with human blood. Stored rabbit erythro- cytes were found to be shrunken, have high MCHCs, have a high percentage of dense cells within the population and

I Some of this work was presented to the British Pharmaceutical Conference and was abstracted in J. Pharm. Pharmac. 37: 106 (1 985).

a decreased ability to flow through polycarbonate mem- branes. These changes were again only observed when the stored cells were incubated at 37 "C in fresh autologous plasma. Similarly, this storage lesion in rabbit cells was reversed using nystatin and a medium containing 27 mM sucrose, 22 mM sodium chloride and 140 mM potassium chloride.

In vivo survival of nystatin-treated stored rabbit eryth- rocytes has been measured using sodium chromate ( Cr)- labelled cells and compared with that for freshly taken cells. This enabled us to assess whether the apparent 'rejuvenation' seen in vitro was confirmed with an increase in mean cell lifespan in vivo.

Methods

The mean cell lifespan of three different groups of cells was estimated using 51Cr-labelled erythrocytes. The groups of cells stud- ied were freshly taken cells, cells stored in CPDA-1 for 21 days and cells stored in CPDA-1 for 21 days treated with nystatin and 140mM potassium chloride as previously described [Turner et al., 1987bl. For the nystatin group, cells were incubated at 21 'C. The high concentration of K+ was necessary to alter the intracellular concen- tration. Before labelling, each sample was centrifuged for 5 min at 2,000 gin 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% w/v) and mixed thoroughly using a

Increased Survival of Reinflated Stored Erythrocytes 187

Vortex mixer. Sterile saline (0.9% w/v) was then added to give haematocrits of approximately 45%. The cells were washed free from nystatin prior to reinfusion.

A 2-ml blood sample was taken from each group and mixed with 50 pCi 51Cr (Amersham Radiochemicals) for a period of 30 rnin at 21 'C. The sodium chromate solution provided hexavalent chrom- ium ions which readily penetrated the erythrocyte. The chromate ions attach themselves to the haemoglobin molecules and undergo reduction to the trivalent state [Gray and Sterling, 19501. Chromium is maintained in the trivalent state within the erythrocyte until the cell undergoes breakdown by the spleen, when the chromium is released to the plasma and is excreted by the kidneys. Chromium does not penetrate the erythrocytes in the trivalent state and is therefore not available to label additional cells [Ebaugh et al., 19531.

After 30 min of incubation, 50 mg of ascorbic acid was added as a sterile solution to reduce any unbound chromate to the trivalent state. 10 ml ofa sterile saline solution (0.9% w/v) was then used to wash the labelled cells and the cells centrifuged at 2,000 g for 5 min. The supernatant liquid was removed and its gamma activity counted to assess radiolabelling efficiency. The washing procedure was repeated, then the labelled cells made up to the original 2-ml volume for injection, using a sterile saline solution (0.9% w/v). A strict aseptic technique was employed to maintain sterility throughout the label- ling procedure.

0.2 ml/kg of labelled cells were injected into each animal via a marginal ear vein. After 10 min, a blood sample was taken into a heparinized syringe from a vein other than that used for injection. Further samples were taken after 24 h and three samples between days 2 and 7. Thereafter, at least two samples per week were taken until 21 days after the injection. Samples were immediately haemo- lysed by addition of water to the counting tubes and stored at +4 "C until the end of the collection period. A sample of 51Cr-labelled cells which was not injected into the animal was also stored and counted, so that the amount of activity injected could be calculated.

On the final day of the study, the radioactivity of each sample was counted using a Packard Auto-Gamma 800C counting system (Pack- ard Instruments), and a background activity count measured using a sampling tube containing water only. The results were obtained as counts per minute per millilitre of red blood cells and were corrected for background counts. The data were plotted in accordance with the directions given in the International Committee report on Radioiso- tope Red Cell Studies, and the mean cell life-span for each group of cells was calculated [ICSH, 19801.

The distribution of 51 Cr-labelled cells within the population of red cells was also determined using a centrifugation method in vitro. In each case, 1 ml of labelled cells was taken and added to 9 ml freshly taken autologous blood. The cells were incubated for 24 h at 37 'C, then spun at 2,000 g for 5 min so that the plasma and buffy coat could be removed. The packed red cells were well mixed using a Vortex mixer and transferred into centrifuge tubes (Bilbate) which were sealed at one end using Cristaseal (Hawksley and Sons Ltd.). The tubes were then spun at an average centrifugal force of 12,000 gfor 30 min in an MSE high-speed ultracentrifuge at +4 'C. The tubes of packed cells were cut into equal lengths and the radioactivity of the fractions counted for gamma activity as before.

The mean cell volume of each group of cells was determined using a Coulter counter and also the percentage of cells with a density greater than that of a mixture of phthalic acid esters (1.106 g/ml) was measured as previously described [Turner et al., 1987al.

Fig. 1. Mean cell life-span, MCV and percentage of dense cells (see text) for freshly taken cells (open columns), 2 I-day stored cells (shaded columns) and 21-day stored cells treated with nystatin and KCI ( 140 mM, stippled columns). n = 6 f SEM.

Results

Mean cell life-span, mean cell volume (MCV) and the percentage of dense cells within the population are shown for each group of cells in figure 1, and their statistical analyses are summarised in table I.

51Cr-labelled fresh erythrocytes were found to have a mean cell life-span of approximately 29 days. The mean cell life-span of cells stored in CPDA- 1 was almost halved after 2 1 days of storage to approximately 16 days. This decrease in mean cell life-span was accompanied by a reduction in cell volume and a dramatic increase in the

188 Turner/Williams/Rees

Table I. Significance levels derived from figure 1 (Student’s t test).

Sample Mean cell MCV Percentage of life-span pm3 dense cells days

Fresh vs. stored pt0.01 p t0 .05 ~ ( 0 . 0 1

Fresh vs. nystatin NS NS NS

Stored vs. nystatin p<O.Ol NS ptO.O1

NS =Not significant; indicates p>0.05.

Table 11. The efficiency of labelling with W r of fresh cells, cells stored for 21 days and 21-day stored cells treated with nystatin and KC1(140mM), n-6rtSEM.

Sample Labelling efficiency, 96

Fresh cells

Stored cells

Stored cells treated with nystatin

17.53 rt0.98

16.74k 1.97

14.40rt 1.88

percentage of dense cells within the population of red cells.

After reinflation of 2 l-day stored erythrocytes using nystatin and 140 mM potassium chloride, the mean cell life-span was increased to 23 days, and this was found to be not significantly different to that measured for freshly taken cells (p>0.05). The reinflation of stored cells using nystatin was also accompanied by a dramatic decrease in the percentage of the cells more dense than a mixture of phthalic acid esters (1.106 g/ml).

The efficiency of labelling with 51Cr was found to be the same for each group of cells as is shown in table 11, even though aged erythrocytes have been shown to take up 51Cr at slower rates than young erythrocytes [Usami et al., 19711. The figures as a group are lower than those for human red cells. No obvious reason for this presents itself.

The distribution of SICr-labelled cells within autolo- gous red cell populations in vitro is shown in figure 2. It was found that SICr-labelled fresh cells were evenly dis- tributed throughout the packed red cell population,

Fig. 2. The proportional distribution of 51Cr in red cell popula- tions in haematocrit tubes for fresh cells, stored cells, stored cells treated with nystatin and stored cells treated with methanol. The top layer of the tube is designated 5 , the bottom layer 1.

whereas cells stored for 2 1 days in CPDA-1 were not. The 2 1 -day stored radiolabelled cells were distributed so that there was a greater percentage of the cells at the bottom of the haematocrit tubes than at the top of the tubes. A significant difference in the percentage of radiolabelled cells was measured between the bottom and top fractions for 21-day stored cells (pt0.01), whereas this was found to be not significantly different (p>0.05) for freshly taken radiolabelled cells.

This uneven distribution of cells found after 2 1 days of storage in CPDA-1 could be altered using nystatin and 140 mM potassium chloride, to give similar distributions to those found for freshly taken cells. Treatment of 2 1 -day stored cells with methanol and 140 mM potassium chlo- ride (i.e. in the absence of nystatin) had little effect on the distribution of labelled cells within the erythrocyte pop- ulation. Student’s t test was used to compare the activity of labelled cells counted within the fraction at the bottom of the packed red cell population (fraction 1). This showed that there was a significant difference in the percentage of dense cells within a packed red cell popu-

Increased Survival of Reinflated Stored Erythrocytes 189

lation between freshly taken cells and 2 1-day stored cells (p>0.05). It was also shown that incubation of stored cells with nystatin and 140 mM potassium chloride could re- verse this effect, whereas incubation with methanol and 140 mM potassium chloride could not (p>0.05).

Discussion

High-speed centrifugation has had many applications in the separation of red blood cells into fractions accord- ing to their density. For any normal sample of erythro- cytes, the oldest and densest cells migrate to the bottom of the tube, and the youngest and least dense cells are dis- placed to the top of the tube forming a continuous density gradient [Ganzioni et al., 19711. In these experiments it was found that 51Cr-labelled freshly taken erythrocytes were evenly distributed throughout a normal red cell population as determined using high-speed centrifuga- tion. After 21 days of storage in CPDA-l, however, the labelled cells were found to be positioned towards the bottom of the packed cell column after being mixed with fresh blood cells. This indicated that the 21-day stored cells had a greater percentage of dense cells than the normal red cell population, i.e. they were associated with the senescent erythrocytes.

Adaptation of a method by Cass and Dalmark [ 19731 to alter cellular sodium and potassium concentrations using nystatin enabled us to reduce the high percentage of dense cells within the red cell population after 2 1 days of storage in CPDA- 1. Stored cells treated with nystatin and 140 mM potassium chloride showed an even distribution throughout the red cell population after being mixed with fresh blood cells, whereas methanol-treated cells employ- ing the same incubation media but in the absence of nystatin did not. The ability of nystatin to induce mem- brane permeability to monovalent cations allowed cells stored in CPDA-1 for 2 1 days to be reinflated [Clark et al., 19781. This produced a decrease in MCHC and a corre- sponding decrease in cell density, so that density distri- butions similar to those for freshly taken cells were mea- sured.

Changes in density were also measured using the posi- tion of a mixture of phthalic acid esters within a tube of packed red cells to indicate the percentage of dense cells within a cell population [Danon and Marikovsky, 19641. As seen previously, the percentage of cells more dense than the ester mixture increased with storage. This per- centage could be decreased to the normal values for fresh blood samples using nystatin treatment and 140 mM

potassium chloride. A corresponding decrease in cell vol- ume was seen on storage which was increased by reinfla- tion using nystatin and 140 mM potassium chloride.

As far as in vivo survival was concerned, the labelling of erythrocytes with 51Cr provided a simple method to determine red cell life-span [ICSH panel, 197 11. Reinfla- tion of 2 1-day stored erythrocytes with nystatin increased the mean cell life-span so as to be similar to that measured for freshly taken cells. The fact that the mean cell life-span was not quite as long for nystatin-treated stored cells as for freshly taken cells may indicate either that other factors (as well as elevated MCHC) are involved in the determination of red cell survival in vivo, or that our washing procedure did not remove all the nystatin or else somehow altered the cell surface.

The question arises as to the clinical application of this technique. Clearly, a chemical method which extends the viable storage life of blood would be of help to the trans- fusion services, especially at a time when these services in (e.g.) the UK are under some pressure.

The fact that a theoretical approach has led to such an extension suggests that the method could have practical application. However, we do not predict that this method can be used for prolonging the storage life of blood rou- tinely for two reasons, firstly the toxicity of nystatin, and secondly the amount of manipulation of the blood that the method requires and the possibility of contamina- tion.

Nystatin has considerable systemic toxicity. In prac- tice, our method should result in a preparation of blood which - though treated with nystatin - is free from the antifungal agent at the time of transfusion. Indeed on a theoretical basis the extension in life of cells treated with nystatin can only be achieved after the nystatin has been washed off. What does minimise the application of the method is that it requires too much manipulation of blood stocks for routine use. The method involves addi- tion of nystatin and ions and thorough and successive washings which would be impracticable for routine use.

Immediately we see an application of the technique for laboratory studies, or perhaps where blood of remarkably rare characteristics needs storage for an additional short period.

More importantly we suggest that since we can clearly prolong the useful storage life of blood from a theoretical approach, such a prolongation of blood storage life could theoretically be achieved using the same concept, but not by using nystatin, nor such a complex washing procedure. The problems envisaged would be technical and quite surmountable.

190 Turner/ WilliamdRees

References

Cass, A.; Dalmark, M.: Equilibrium dialysis of ions in nystatin- treated red cells. Nature new Biol. 244: 47-49 (1 973).

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).

Danon, D.; Marikovsky, Y.: Determination of density distribution of red cell population. J. Lab. clin. Med. 64: 668-674 (1964).

Ebaugh, F.G.; Emerson, C.P.; Ross, J.F.: The use of radioactive chromium-5 1 as an erythrocyte tagging agent for the determina- tion of red cell survival in vivo. J. clin. Invest. 32: 1260-1276 (1953).

Ganzioni, A.M.; Oakes, R.; Hillman, R. S.: Red cell aging in vivo. J. clin. Invest. 50: 1373-1378 (1971).

Gray, S. J.; Sterling, K.: The tagging of red cells and plasma proteins with radioactive chromium. J. clin. Invest. 29: 1604-1 6 13 ( I 950).

International Committee for Standardisation in Haematology: Rec- ommended methods for radioisotope red cell survival studies. Br. J. Haemat. 21: 241-250 (1971).

International Committee for Standardisation in Haematology: Rec- ommended method for radioisotope red cell survival studies. Br. J. Haemat. 45: 659-666 (1980).

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: 177-181 (1987a).

Turner, S.; Williams, A.R.; Rees, J.M.H.: The use of nystatin to restore the flow properties of time-expired stored erythrocytes. Vox Sang. 52: 182-185 (1987b).

Usami, S.; Chien, S.; Gregersen, M.I.: Viscometric behaviour of young and aged erythrocytes; in Hartert, Copley, Theoretical and clinical haemorheology, pp. 266-270 (Springer, Berlin 197 1).

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

J. M. H. Rees, PhD, Department of Pharmacology, University Medical School, GB-Manchester M13 9PT (UK)