Prolonged cold storage of red blood cells by oxygen removal and additive usage

Prolonged cold storage of red blood cells by oxygen removal and additive usage. A cost-effective, 4.degree. C. storage procedure that preserves red cell quality and prolongs post-transfusion in vivo survival is described. The improved in vivo survival and the preservation of adenosine triphosphate levels, along with reduction in hemolysis and membrane vesicle production of red blood cells stored at 4.degree. C. for prolonged periods of time, is achieved by reducing the oxygen level therein at the time of storage; in particular, by flushing the cells with an inert gas, and storing them in an aqueous solution which includes adenine, dextrose, mannitol, citrate ion, and dihydrogen phosphate ion, but no sodium chloride, in an oxygen-permeable container which is located in an oxygen-free environment containing oxygen-scavenging materials.

BACKGROUND OF THE INVENTION 
The current blood supply is considerably smaller than the need therefor. 
Stored blood is considered unusable after about 6 weeks of steady 
deterioration in storage as determined by the inability of such cells to 
survive in the circulation after transfusion, which in part is caused by 
hemoglobin oxidation and degradation and adenosine triphosphate (ATP) 
depletion. Moreover, the risks involved in receiving blood from 
nonautologous donors remain significant. In order to address current 
needs, blood storage techniques must be simple, inexpensive and long term. 
Red blood cells (RBCs) survive for about 4 months under conditions of 
turbulent flow in the body without protein synthesis. Oxygen (O.sub.2) is 
essential for the conversion of hemoglobin (Hb) to met-Hb, the breakdown 
of which produces toxic products such as hemichrome, hemin and free 
Fe.sup.3+. Together with O.sub.2, these products catalyze the formation of 
hydroxyl radicals (OH.circle-solid.), and both OH.circle-solid. and the 
met-Hb breakdown products damage the red cell lipid membrane, the membrane 
skeleton, and the cell contents. As will be discussed hereinbelow, current 
approaches to red cell preservation do not address the hemoglobin 
breakdown damage pathway. 
Refrigeration reversibly disables the enzymes essential for met-Hb 
reduction in vivo, increases the solubility of damaging O.sub.2 (almost by 
a factor of two) in the environment of the red blood cells, and permits 
the level of ATP to decrease by diminishing the glycolytic rate (at 
4.degree. C. the rate is about 1% of that found at 37.degree. C.). 
Reduction of red cell ATP concentration results in echinocyte (an unstable 
form of red blood cells) formation, increased rates of membrane 
vesiculation, loss of red cell surface area, and accelerated sequestration 
by splenic macrophages. Vesiculation continues throughout the cold storage 
period, is exacerbated by echinocyte formation, and decreases red blood 
cell survival by decreasing red blood cell membrane area. 
The effects of elevation and preservation of ATP levels in blood storage 
situations has been studied. For example, in "Studies In Red Blood Cell 
Preservation-7. In Vivo and in Vitro Studies With A Modified 
Phosphate-Ammonium Additive Solution," by Greenwalt et al., Vox Sang 65, 
87-94 (1993), the authors determined that the experimental additive 
solution (EAS-2) containing in mM: 20 NH.sub.4 Cl, 30 Na.sub.2 HPO.sub.4, 
2 adenine, 110 dextrose, 55 mannitol, pH 7.15, is useful in extending the 
storage shelf-life of human RBCs from the current standard of 6 weeks to 
an improved standard of 8-9 weeks. Packed RBCs are suitable for 
transfusion following the removal of the supernatant with a single washing 
step. Greenwalt et al. also conclude that factors other than ATP 
concentration appear to play an increasingly important role in determining 
RBC viability after 50 days of storage. They cite the results of L. Wood 
and E. Beutler in "The Viability Of Human Blood Stored In Phosphate 
Adenine Media," Transfusion 7, 401-408 (1967), and find in their own 
experiments that the relationship between ATP concentration and 24h RBC 
survival measurements appears to become less clear after about 8 weeks of 
storage. E. Beutler and C. West restate that the relationship between red 
cell ATP concentration and viability is a weak one after prolonged periods 
of storage in "Storage Of Red Cell Concentrates In CPD-A2 For 42 and 49 
Days," J. Lab. Clin. Med. 102, 53-62 (1983). 
U.S. Pat. No. 4,585,735, for "Prolonged Storage Of Red Blood Cells," which 
issued to Harold T. Meryman et al. on Apr. 29, 1986, discloses a hypotonic 
suspension medium and a method for prolonged storage of red blood cells at 
about 4.degree. C. The preferred hypotonic suspending solution contained 
110 mM glucose, 55 mM mannitol, 7.9 mM potassium citrate, 25.8 mM 
potassium phosphate, 14.7 mM potassium dihydrogen phosphate, 2 mM adenine, 
and 50 mM NH.sub.4 Cl. It was compared with an isotonic solution 
containing 110 mM glucose, 55 mM mannitol, 58.6 mM potassium citrate, 25.8 
mM potassium phosphate, 14.7 mM potassium dihydrogen phosphate, and 2 mM 
adenine in a demonstration of 24 h in vivo survival for red cells stored 
in the two solutions for periods ranging up to 125 days. From FIG. 2 of 
the '735 patent, it may be observed that the hypotonic solution provides 
significantly better protection for the red blood cells. Prior to 
transfusion, the cells were sedimented by centrifugation and resuspended 
in a transfusable solution. 
In "Effects Of Oxygen On Red Cells During Liquid Storage at +4.degree. C.," 
by Hogman et al., Vox Sang 51, 27-34 (1986), the authors disclose that the 
red cell content of ATP is slightly better maintained after 2-3 weeks when 
blood bags prepared by the standard procedures were stored in an 
oxygen-free atmosphere. Venous blood was refrigerated and deprived of 
additional oxygen during storage, by placing the moderately 
oxygen-permeable storage bags (standard polyvinyl chloride (PVC) bags were 
employed and not the highly permeable bags currently available) in a 
nitrogen environment and thereby gradually reducing the level of oxygen 
saturation. The reduction in oxygen concentration occurs slowly during 
storage at 4.degree. C., and is far from complete, starting at .about.60% 
and reaching .about.30% hemoglobin saturation at 5 weeks. No conclusion 
could be drawn concerning the effects of this procedure on the overall 
quality of stored cells. These authors did not address or significantly 
reduce the oxygen-dependent damage to hemoglobin and the oxygen-mediated 
damage caused by hemoglobin breakdown products. 
U.S. Pat. No. 5,624,794 for "Method Using Oxygen Removal For Extending The 
Useful Shelf-Life Of Refrigerated Red Blood Cells," which issued to Mark 
W. Bitensky et al. on Apr. 29, 1997, discloses a method, using oxygen 
removal at the time of storage, for preserving adenosine triphosphate 
levels and reducing hemolysis and membrane vesicle production in red blood 
cells stored at 4.degree. C. for prolonged periods of time. To achieve a 
low oxygen concentration, the red blood cells are flushed with an inert 
gas and stored in an oxygen gettering environment. 
Accordingly, it is an object of the present invention to provide a 
procedure for blood storage which takes advantage of the positive effects 
of oxygen removal and of the use of additive solutions for addressing the 
problems of hemoglobin degradation, red blood cell lysis (hemolysis) and 
ATP depletion in a manner consistent with the practice of autologous 
transfusion and enhanced heterologous transfusion logistics, and which 
achieves significant prolongation of the time during which refrigerated 
storage of red blood cells is not detrimental to their subsequent use. 
Another object of the present invention is to provide a procedure for 
prolonged blood storage while minimizing the complexity of the procedures 
required for preparing transfusible samples. 
Yet another object of the present invention is to provide a procedure for 
prolonged blood storage in which use of additive solutions is synergistic 
with anaerobic storage. 
Additional objects, advantages and novel features of the invention will be 
set forth in part in the description which follows, and in part will 
become apparent to those skilled in the art upon examination of the 
following or may be learned by practice of the invention. The objects and 
advantages of the invention may be realized and attained by means of the 
instrumentalities and combinations particularly pointed out in the 
appended claims. 
SUMMARY OF THE INVENTION 
To achieve the foregoing and other objects, and in accordance with the 
purposes of the present invention, as embodied and broadly described 
herein, the method for storing red blood cells hereof includes the steps 
of: mixing a sample of whole blood containing the red blood cells to be 
stored with an anticoagulant solution, forming thereby a first suspension 
of red blood cells, concentrating the red blood cells from the liquid 
portion (plasma) of the first suspension, forming thereby a mass of packed 
red blood cells, mixing the packed red blood cells so produced with an 
additive solution which includes an aqueous solution of adenine, dextrose, 
mannitol, citrate ion, and dihydrogen phosphate ion, with no sodium 
chloride, forming thereby a second suspension of red blood cells, reducing 
the oxygen level of the red blood cells in the second suspension of red 
blood cells to less than 10% of the level of oxygen therein when obtained 
by flushing the red blood cells with an inert gas, and storing the red 
blood cells in the second suspension of red blood cells at 4.degree. C. 
Preferably, no further exposure of the cooled red blood cells to oxygen is 
permitted. 
Preferably also, the level of oxygen in the stored red blood cells is 
reduced during storage. 
It is preferred that the amount of additive solution be sufficient to 
achieve a final hematocrit of between 30% and 60%. 
It is also preferred that the pH of the additive solution be adjusted to 
approximately 7.1. 
In another aspect of the present invention, and in accordance with its 
objects and purposes, the method for storing red blood cells hereof 
includes the steps of: forming a mass of packed red blood cells, mixing 
the packed red blood cells with an additive solution which includes an 
aqueous solution of adenine, dextrose, mannitol, citrate ion, and 
dihydrogen phosphate ion, with no sodium chloride, forming thereby a 
suspension of red blood cells, reducing the level of oxygen of the red 
blood cells in the suspension of red blood cells to less than 10% of the 
level of oxygen therein when obtained, and storing the red blood cells in 
the suspension of red blood cells at 4.degree. C. 
Preferably, no further exposure of the cooled red blood cells to oxygen is 
permitted. 
Preferably also, the level of oxygen in the stored red blood cells is 
reduced during storage. 
It is preferred that the amount of additive solution be sufficient to 
achieve a final hematocrit of between 30% and 60%. 
It is also preferred that the pH of the additive solution be adjusted to 
approximately 7.1. 
Benefits and advantages of the present invention include the preservation 
of ATP levels and the reduction of hemolysis and accumulation of membrane 
vesicles in the refrigerated RBCs, as a consequence of creating an 
environment (O.sub.2 removal) that prevents hemoglobin degradation, with 
the result that useful refrigerated storage periods may be prolonged.

DETAILED DESCRIPTION 
Briefly, the present invention includes improvement of the in vivo survival 
characteristics of transfused red blood cells (RBCs) that have been stored 
at 4.degree. C. for prolonged periods of time by removing oxygen therefrom 
at the time of storage, adding a preservation solution, OFAS1, which 
consists essentially of an aqueous solution of adenine, dextrose, 
mannitol, citrate ion, and dihydrogen phosphate ion in place of 
conventional storage solutions, and preventing any further exposure of the 
stored RBCs to oxygen. The in vitro diagnostics of hemolysis, vesicle 
production and ATP levels, when taken together, provide a useful 
indication of in vivo survival. Additionally, evidence for the synergism 
of anaerobic storage and the use of OFAS1 was obtained from these in vitro 
diagnostic measurements. The beneficial effects of oxygen removal during 
refrigerated storage of red blood cells in OFAS1 additive solution were 
further investigated by in vivo recovery measurements in humans. These 
measurements completely support the in vitro conclusions. The OFAS1 
additive solution contains ingredients that are found in the FDA approved 
solutions AS-1 (2 mM of adenine, 122 mM of dextrose, 42 mM of mannitol, 
and 154 mM of sodium chloride as currently formulated), and AS-3 (2.2 mM 
of adenine, 61 mM of dextrose, 70 mM of sodium chloride, 20 mM of sodium 
citrate, 2 mM of citric acid, and 20 mM of sodium hydrogen phosphate, as 
currently formulated), and does not incorporate any new ingredients. The 
OFAS1 solution does not contain sodium chloride, however. The pH of the 
OFAS1 solution was adjusted to approximately 7.1 with the addition of 
sodium hydroxide. Clearly, other bases can be used to accomplish this 
purpose. The solution was then sterilized by filtration through 0.2 .mu.m 
pore filters, since it was believed that deterioration would occur if the 
additive solution was sterilized according to the generally used procedure 
of autoclaving. 
Oxygen removal, and the effects of the OFAS1 additive solutions were 
investigated with red blood cells stored in standard polyvinyl chloride 
(PVC) blood bags with di-(2-ethylhexyl) phthalate (DEHP) plasticizer. 
Oxygen was removed from warm RBCs by flushing the blood bags with argon 
between 6 and 10 times, which reduced the oxygen level of the RBCs to 
below 10% of the level of oxygen when obtained (each transfer bag 
containing the red cells was filled with purified Ar and shaken gently for 
approximately 10 min. before expelling the gas). For red blood cells 
prepared for in vitro diagnostics a unit of blood was typically stored in 
AS-1/AS-3 additive solution in a standard storage bag for between 2 and 5 
days after collection at a blood bank. Each unit of blood was then 
subdivided into about 120 mL aliquots, placed in DEHP plasticized PVC 
transfer bags with 150 mL capacity, and stored at 4.degree. C. in a 
light-shielded blood bank refrigerator. No measurements were performed 
with red blood cells which were not shielded from the light; however, it 
is believed by the present inventors that fluorescent light does not cause 
significant red blood cell deterioration. Samples were withdrawn as needed 
via a sterile septum sampling port. Rapid cooling after rapid purging is 
essential to prevent lactic acid buildup in the RBCs. Moreover, it should 
be mentioned that the oxygen can also be removed after the RBCs are 
cooled. However, since the RBCs are unprotected from the effects of 
oxidation once cooled, and since oxygen removal is more rapid at 
37.degree. C. or 21.degree. C. when compared with 4.degree. C., the 
preferred procedure is to cool them after oxygen removal. As reported by 
Hogman et al., supra, conventional PVC blood storage bags are permeable to 
O.sub.2. It takes about 4 weeks of conventional storage for a unit of 
packed red blood cells to become fully oxygenated. In order to evaluate 
the long-term effects of replacing the storage gas, transfer bags were 
stored in an anaerobic chamber filled with an inert gas such as argon. 
Blood bag gas exchange was further enhanced by 2-3 cycles of exposing the 
anaerobic chamber to partial vacuum followed by filling with the chosen 
inert gas. In addition, about 10% (v/v) of hydrogen gas was added to the 
argon storage gas along with a palladium catalyst in the anaerobic chamber 
that houses the stored blood to continuously remove traces of O.sub.2 
emerging from the blood bags. 
For "control" samples, cells were stored in the 150 ml transfer packs 
without further treatment. For both aerobic and anaerobic storage in 
OFAS1, cells were centrifuged at 2,000x g in the transfer pack, and the 
supernatant was removed and replaced with an appropriate amount of the 
additive solution to achieve a final hematocrit (Hct) of about 40. 
Membrane vesicle production was quantified by measuring the protein 
content of isolated vesicle fractions. The ATP concentration was measured 
with a commercial diagnostic kit. All data are given as the average value 
obtained from 4-6 units of blood. 
In vivo tests were conducted using a cohort of 10 subjects divided into two 
groups. A unit of blood was first collected into CP2D anticoagulant 
solution. Subsequently, platelets and plasma were removed and OFAS1 added. 
Oxygen was then removed as described hereinabove. Whole blood units were 
stored undisturbed in the OFAS1 additive solution under both anaerobic and 
aerobic conditions for 8 and 9 weeks. The 24 h post-transfusion recovery 
was determined using the well-known Tc-99m/Cr-51 double labeling protocol. 
The results of the 24 h in vivo recovery experiments are shown in the 
Table. 
TABLE 
______________________________________ 
ANAEROBIC AEROBIC 
SUBJECT Cr-51/Tc-99 m 
Cr-51 Cr-51/Tc-99 m 
Cr-51 
______________________________________ 
9 wk A 73.4 80.3 62.3 68.3 
9 wk B 71.6 80.0 47.7 60.3 
9 wk C 67.4 72.6 
9 wk D 73.4 80.9 56.5 63.5 
8 wk E 71.4 79.1 60.2 68.4 
8 wk F 70.7 77.1 69.7 76.6 
8 wk G 74.1 83.7 77.8 
8 wk H 68.6 81.5 67.1 70.0 
8 wk I 68.2 74.8 
9 wk average 
71.5 78.5 55.5 64.0 
9 wk std. Dev. 
2.8 3.9 7.4 4.0 
8 wk average 
70.6 79.2 65.7 73.2 
8 wk std. Dev. 
2.4 3.5 4.9 4.7 
______________________________________ 
Reference will now be made in detail to the preferred embodiments of the 
present invention, examples of which are illustrated in the accompanying 
drawings. Turning now to the FIGS., FIG. 1a shows the results derived from 
the Cr-51 and Tc-99m double-labeling procedures, while FIG. 1b shows 
results derived from Cr-51 data only. Red blood cells were stored in OFAS1 
with a Hct of about 40% (obtained using a hematocrit centrifuge), with 
oxygen removal (left-sloping hatching) and without oxygen removal 
(right-sloping hatching). Current practice in the blood storage industry 
is to store red blood cells with a hematocrit of about 60% (40% by volume 
of storage solution and 60% by volume of packed red cells). In the present 
invention, hematocrits of between 30% and 60% are expected to give 
acceptable storage characteristics; however, with a Hct of less than 40%, 
the volume of the storage bags begins to become large. 
The number, n, designates the number of subjects averaged. For one subject 
in the 8 week aerobic sample, a Tc-99m label was not available; therefore, 
only single labeling data were obtained for that individual. Due to a 
number of drop-outs, averages of fewer than 5 subjects were included in 
the results presented. The following conclusions may be derived from the 
Table and from FIGS. 1a and 1b: 
1. Oxygen removal enhances the 24 h recovery by 16% after 9 weeks of 
storage; 
2. As expected, the recovery rates for the single label are considerably 
higher than those for the double label procedure in all cases (by 7-9%); 
3. Virtually no change in the rate of 24 h recovery was seen between 8 and 
9 weeks of anaerobic storage, in contrast to an 10% drop in recovery for 
samples stored in the presence of oxygen; and 
4. The smaller cohorts (4 subjects instead of 5) in the anaerobic trial did 
not have increased subject-to-subject variability both at 8 and 9 weeks of 
storage. After conducting the above-described 24 h in vivo recovery 
studies, the red blood cell samples were stored for an additional 4-8 
weeks beyond the infusion date. Several in vitro diagnostic tests 
(including ATP levels, vesicle production and the extent of hemolysis) 
were carried out for these samples, the results of which are shown in 
FIGS. 2-4. Unlike for blood used solely for in vitro investigations, 
samples were taken weekly from the original storage bag, and ATP levels 
were determined beginning after 8 or 9 weeks of storage. All data obtained 
from blood prepared for the in vivo experiments were averaged, and the 
standard deviations are shown in the FIGS. 
FIG. 2a compares cellular ATP levels as a function of time for aerobically 
(open circles) and anaerobically stored (black circles) cells in OFAS1, 
FIG. 2b is a comparison of ATP levels between red blood cells stored 
anaerobically in OFAS1 (black circles) and "control" red cells stored 
aerobically in AS1/AS3 (Xs), and FIG. 2c is a comparison of ATP levels 
between cells stored aerobically in OFAS1 (open circles) with the 
"control" red cells stored aerobically in AS1/AS3 (Xs) as a function of 
time. The dotted lines indicate the 6 week storage point and the ATP level 
of conventionally stored red cells. For red cells anaerobically stored in 
OFAS1, comparable levels of ATP were reached after 13 weeks of storage. 
For the in vitro control experiments, each data point represents an 
average of 4-7 red blood cell samples aerobically stored in AS-1 and AS-3, 
as described hereinabove. These data were gathered from over 120 units of 
stored red cells over a two-year period. FIG. 2c is a comparison of ATP 
levels between conventional ("control") aerobic storage (Xs) and aerobic 
storage in OFAS1 (open circles). It is seen that red blood cells 
aerobically stored in OFAS1 show significant elevation of ATP levels when 
compared to cells aerobically stored in AS-1/AS-3 at 7 weeks. However, the 
decline of ATP levels over time was more rapid than for samples stored in 
OFAS1, and approached the levels of conventional additive solutions beyond 
10 weeks. 
FIG. 3 shows vesicle production for red cells on which in vivo measurements 
were made as a function of time. The amount of vesicle isolated from the 
cell suspensions was evaluated using standard protein assay techniques. 
All data were averaged and the standard deviations are shown in FIG. 3. 
Anaerobic storage in OFAS1 is represented by the black circles, while 
aerobic storage in OFAS1 is represented by the open circles. 
FIG. 4 shows the hemolysis of red cells for which in vivo measurements were 
made as a function of time. The extent of hemolysis was determined 
according to standard procedures. All data obtained from blood prepared 
for the in vivo experiments were averaged, and the standard deviations are 
shown in the FIG. Hemolysis control data from aerobic storage in AS-1/AS-3 
("control") are also shown for comparison. The extent of hemolysis for the 
aerobic AS1/AS3 control (Xs) was found to be significantly higher than for 
the aerobically stored in vivo samples in OFAS1 (open circles) and the 
anaerobically stored cells in OFAS1 (black circles). It is currently 
believed by the present inventors that the observed increase in hemolysis 
rates of the control samples resulted from more frequent thorough mixing 
to which these samples were subjected during weekly sampling processes. By 
contrast, the in vivo samples were only inverted gently once per week, 
until the date of infusion. 
From the in vitro results, it may be concluded that: 
1. ATP levels are significantly higher with anaerobic storage than with 
aerobic storage in OFAS1 (FIG. 2a); 
2. The reduced levels of ATP found for aerobic controls (aerobic in vitro 
data at 6 weeks) are approached only after 90 days of storage in the 
absence of oxygen (FIG. 2b); 
3. Higher ATP levels were observed with aerobic samples that were stored in 
OFAS1 when compared to conventional AS-1 and AS-3 controls. These data 
indicate a modest benefit of OFAS1 in elevating ATP levels, even under 
aerobic storage. However, the higher ATP level did not directly enhance 
the 24 h recovery rate after 8 weeks of storage (FIG. 2c); 
4. Rates of vesicle production are similar at 8 and 9 weeks between the two 
forms of storage. However, vesicle production accelerates beyond 10 weeks 
in the aerobic samples (FIG. 3); and 
5. Rates of hemolysis are similar for aerobic and anaerobic samples for up 
to 11 weeks of refrigerated storage (FIG. 4). Control in vitro 
measurements show considerably higher hemolysis rates in aerobically 
stored samples. As mentioned, this may be partly due to the handling 
needed to thoroughly mix the bag for weekly sampling (that is, the extra 
handling for sampling between weeks 0 and 9, compared with the in vivo 
samples). Such handling did not appear to adversely impact the blood 
stored anaerobically (data not shown). It may be that the OFAS1 also 
contributes to a reduction in hemolysis rates, but no data is currently 
available concerning this point. 
FIG. 5 shows a comparison of the level of ATP as a function of time among 
red blood cells stored aerobically in conventional storage media (small 
circles), cells stored anaerobically in conventional storage media 
(diamonds), cells stored aerobically in OFAS1 (pluses), and cells stored 
anaerobically in OFAS1 (inverted open triangles), and shows the 
synergistic effect of anaerobic storage in OFAS1. None of the red blood 
cells used in FIG. 5 were used for in vivo measurements. If one defines 
"synergy" as being equal to .mu.mol of ATP per gram of hemoglobin for 
anaerobic storage of the red cells in OFAS1 divided by the sum of .mu.mol 
of ATP per gram of hemoglobin for cells aerobically stored in AS-1 
(control), plus the difference in .mu.mol of ATP per gram of hemoglobin 
between the control and that for cells aerobically stored in OFAS1, plus 
the difference in .mu.mol of ATP per gram of hemoglobin between the 
control and that for is cells anaerobically stored in AS-1 (the sum being 
plotted as upright hollow triangles), one observes the synergistic effect 
of aerobically storing the cells OFAS1 plotted as inverted black triangles 
in FIG. 5. 
The foregoing description of the invention has been presented for purposes 
of illustration and description and is not intended to be exhaustive or to 
limit the invention to the precise form disclosed, and obviously many 
modifications and variations are possible in light of the above teaching. 
The embodiments were chosen and described in order to best explain the 
principles of the invention and its practical application to thereby 
enable others skilled in the art to best utilize the invention in various 
embodiments and with various modifications as are suited to the particular 
use contemplated. It is intended that the scope of the invention be 
defined by the claims appended hereto.