WO1996029864A1

WO1996029864A1 - Rejuvenating outdated red cells

Info

Publication number: WO1996029864A1
Application number: PCT/US1996/003999
Authority: WO
Inventors: Harold T. Meryman
Original assignee: Organ, Inc.; American National Red Cross
Priority date: 1995-03-24
Filing date: 1996-03-22
Publication date: 1996-10-03
Also published as: AU5429396A

Abstract

A method for rejuvenating stored red blood cells in a red cell storage solution includes adding a biologically compatible solution to a red cell solution in a sterile closed system without breaking the sterile closed system and mixing a resultant red cell solution.

Description

REJUVENATING OUTDATED RED CELLS

BACKGROUND OF THE INVENTION This invention relates to a method for rejuvenating stored red cells. Red cells stored by refrigeration have a limited shelf-life depending on the solutions in which they are stored. Cells collected in CPDA1 (citrate, phosphate, dextrose, adenine) can be stored for 5 weeks without further treatment with or without removal of the plasma. More recent procedures involve the separation of red cells from the plasma and subsequent dilution of the cells with 100ml of an additive solution. The three currently- licensed solutions are Adsol, Nutricel and Sagman. Cells stored with these additive solutions have a six-week shelf-life.

Shelf-life is determined, in the United States at least, by measurements of the proportion of cells circulating in the recipient 24-hrs after transfusion. The FDA has unofficially established 75% as the minimum for a licensed product. The quantity of free hemoglobin that is transfused can also limit shelf-life. Although no official maximum has been established, there is general agreement that hemolysis should not exceed 1%.

During storage, human red blood cells undergo morphological and biochemical changes, including decreases in the cellular level of adenosine triphosphate (ATP) and

2,3-diphosphoglycerate (2,3-DPG), changes in cellular morphology, and progressive hemolysis. The concentration of ATP, after a brief initial rise, progressively declines to between 30 and 40% of its initial level after six weeks of storage. The fluidity of the cell membrane of red cells, which is essential for the passage of red cells through the narrow channels in the spleen and liver, is loosely correlated with the level of ATP. The primary function of red cells in the circulation is to deliver oxygen to the tissues. A unique characteristic of hemoglobin is that it can unload much of its oxygen even though the partial pressure of oxygen in the tissues may be relatively high. A compound called 2,3-diphosphoglycerate (2,3-DPG) is essential to this process and, in its absence, oxygen is not efficiently delivered to the tissues. During refrigerated storage as currently practiced, the level of 2,3-DPG falls rapidly after about three or four days of storage and approaches zero by about ten days.

In addition, morphological changes occur during storage, ultimately leading to the development of spicules on stored red cells (echinocytosis) . These spicules can bud off as vesicles, radically changing the surface-to- volume ratio of the cells and their ability to deform on passing through narrow channels. Such cells will be filtered out of the circulation by the spleen and liver following transfusion.

As stated above, to be acceptable for transfusion at least 75% of the red cells that are transfused must be capable of remaining in circulation twenty-four hours following the transfusion. The concentration of ATP and the morphology of red cells serve as indicators of the suitability of stored cells for transfusion.

In order to prolong the shelf-life of transfusable red blood cells, it is necessary to store the cells or treat them in some manner that prevents a rapid decline in ATP and, if possible, 2,3-DPG. Solutions that prolong the shelf-life of red cells are known (see, e.g. , Meryman, U.S. Patent No. 4,585,735, and Meryman, U.S. Patent No. 5,250,303, both of which are herein incorporated in their entirety by reference) . Typically such solutions contain citrate, phosphate, glucose and adenine and occasionally other ingredients that function to prolong shelf-life by maintaining the level of ATP in the cells. Glycolytic activity is enhanced in red blood cells if the intracellular pH (hereinafter H,) measured at 4°C is about 7.4.

Procedures and solutions have been devised that permit some of the declines in ATP and 2,3-DPG and the morphological changes associated with long-term storage to be reversed and thereby rejuvenate the red blood cells. Known rejuvenating solutions, however, are not suitable for transfusion; they must be removed prior to transfusing the cells. In particular, it is possible to restore normal or even supra-normal levels of 2,3-DPG by incubating the red cells at 37°C for one to two hours in a solution containing glucose, phosphate, inosine and pyruvate. Such a solution has been licensed for this purpose. However, the inosine and pyruvate are not acceptable for transfusion. Therefore, the cells must be washed prior to transfusion.

Currently used apparatus for washing utilizes rotating seals to introduce and remove wash solutions. This is considered a violation of the closed system. Current regulations require that cells washed in such apparatus be stored for no more than twenty-four hours because of the potential for bacterial contamination. In addition, conventional wash solutions, such as glucose- saline solutions, are not suitable for red cell storage beyond twenty-four hours. Although it is also possible to freeze the rejuvenated cells once they have been washed, these red cells still must be used within twenty-four hours of thawing.

In certain circumstances it is desirable to extend the shelf-life of refrigerated red blood cells beyond the current forty-two days. For example, autologous units drawn for use in elective surgery may expire before the surgery can be performed. In addition, storing blood for several months before use would permit retesting the donor for evidence of AIDS or hepatitis infection and help to avoid the transfusion of infected blood. Other than by freezing, which is labor intensive and expensive, no such capability is known to exist.

Because of the critical need for transfusable red blood cells, it is of great importance not only to develop methods that maintain high intracellular levels of both ATP and 2,3-DPG, good morphology and low hemolysis after washing, but also to develop methods for the routine collection and resuspension of unwashed red cells with better storage characteristics than are achieved by current procedures.

SUMMARY OF THE INVENTION The invention provides a method for rejuvenating red blood cells designed not only to restore 2,3-DPG but also to improve twenty-four hour survival of the cells and to permit a prolonged period of post-rejuvenation storage.

This invention provides a method for rejuvenating red cells stored in a red cell storage solution comprising adding a biologically compatible solution to the red cell storage solution without breaking the sterile closed- system of the red cell storage solution; and mixing the resultant solution. The method may further comprise separating the red cells from a supernatant by sedimentation and removal of an amount of the supernatant such that the volume of the final red cell storage solution is preferably substantially equivalent to the volume of the original red cell storage solution. In a particularly preferred embodiment, this invention provides a method for rejuvenating stored red cells comprising: (a) attaching a storage bag comprising stored red cells to a sterile transfer pack containing a biologically compatible storage solution using a sterile connection device without breaking said sterile closed system; (b) introducing said biologically compatible storage solution into said storage bag; (c) mixing a resultant solution; (d) separating the red cells from a supernatant by sedimentation; (e) transferring at least a portion of the supernatant to the sterile transfer pack; (f) sealing the sterile connection device; and (g) detaching the sterile transfer pack from the storage bag so that the storage bag remains sealed.

BRIEF DESCRIPTION OF THE DRAWING Figure l depicts the morphological index of red cells after a rejuvenation procedure of this invention after six weeks of storage. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All publications mentioned herein are incorporated by reference.

As used herein, "stored red blood cells" or "stored red cells" refers to red blood cells that have been stored, for example under conventional methods of refrigeration known to those of ordinary skill in the art, for at least one week and include red cells that have been stored for more than six weeks.

As used herein, "rejuvenated stored red blood cells" or "rejuvenated stored red cells" refers to red blood cells that can be further stored under refrigerated conditions with low hemolysis and with cell morphological index and levels of ATP and 2,3-DPG that are greater than the levels of morphological index, ATP and 2,3-DPG in cells that have been stored under refrigerated conditions for the same amount of time without being rejuvenated.

As used herein, a "red cell storage solution" is any solution for storing red blood cells. Red cell storage solutions include, but are not limited to those known in the art such as those listed in Tables 1 and 2, infra.

TABLE 1

Biologically Compatible Buffered Solutions Currently Used for Cell Storage Solution Concentration

CPDA-1 ADSOL NUTRICEL

Ingredient (mM) (mM^*) ( M)

NaCilrate 89.6 - 20.0

dextrose 161.0 111.0 55.5

NaH,P0₄ 16.1 - 20.0

Adenine 2.0 2.0 2.2

Manmtol 41.2 -

NaCl 154.0 70.1

Osmolality 323 342 244 (mOsm)

PH 5 7 5_ 5.8

CDPA-l and ADSOL are sold by Baxter Travenol and NUTRICEL is sold by Cutter. Osmolality is the effective osmolality comπbuted by the non-pencirating constituents. As used herein, a "biologically compatible storage solution" is a transfusable solution in which red cells can be stored under refrigerated conditions and the red cells that are contacted therewith retain viability. Contacting includes any process in which the cells are in some manner exposed to the solution and includes, but is not limited to, suspension of the cells in the solution. A biologically compatible storage solution has a pH and a salt concentration that are suitable for maintaining the integrity of the cell membrane and do not inhibit or destroy the biological and physiological reactions of the cells contacted therewith. Typically a biologically compatible storage solution has a pH between 6 and 9.5 and is isotonic or only moderately hypotonic or hypertonic. Biologically compatible storage solutions include, but are not limited to known solutions such as those listed in Table 2, infra. TABLE 2

Examples of Biologically Compatible Buffered Solutions That Can Effect an Increase in the Intracellular pH of Red Blood Cells

Concentration Grams Osmolality

Name Ingredient (mM) (%) (mOsm) PH

ARCS glucose 139 2.5 126 7.4

NaCilrate 33.3 0.98 (w/o glucose)

Na,HP0₄ 12.0 0.17

NaH,P0₄ H₂0 2.9 0.04

Ademne 2.0 0.028

ARC C glucose 177.0 3.19 121 7_5

NaCitrate 27.2 0.8 (w/o glucose)

Na,HP0₄ 12.0 0.17

NaH,P0₄ H-0 2.9 0.04

Ademne 2.0 0.028

ARC32 glucose 177.0 3.19 28.3 8.0

NaCitrate 89 2.63 (w/o glucose)

Na,HP0₄ 16 0.227

Ademne 2.0 0.028

ARC27 glucose 69 1.24 126 7.4

NaCitrate 33.3 0.98 (w/o glucose)

Na,HP0₄ 12.0 0.17

NaH₂P0₄ H₂0 2.9 0.04

Ademne 1.14 0.016

ARC30 glucose 50 0.9 136 7.5

NaCitrate 22.0 04 (w/o glucose)

Na,HP0₄ 10.6 0.15

NaH,P0₄ H,0 2.5 0.04

Ademne 0.01 0.00014

Manmiol 44 0.8

As used herein, "an effectively hypotonic solution" is one in which the combined osmolality of solutes that do not penetrate the red cell membranes is less than the combined osmolality of the extracellular red cell storage solution. Such solutions are known to one of ordinary skill in the art.

As used herein, "refrigerated conditions" refers to conditions for storing red blood cells under refrigeration. Refrigerated conditions include, but are not limited to, temperatures of 4±2°C.

It has been discovered that if outdated red cells are resuspended in a biologically compatible storage solution in accordance with this invention and subsequently stored under refrigerated conditions, there is not only a restoration of 2,3-DPG but there is also an improvement in red cell morphology which is indicative of the ability of red cells to survive in circulation. This form of rejuvenation will provide an extended opportunity to utilize red cells that would otherwise be outdated. The process also provides an alternative to the existing practice of freezing rejuvenated cells, which is relatively very expensive. Red cells rejuvenated using this method may be subsequently stored for additional time before being transfused. The red cells may be stored for more than 48 hours after the rejuvenation procedure and, preferably, they are stored for more than one week. The rejuvenated cells may be transfused after being stored for more than four weeks and may be used even after subsequent storage for six weeks.

Figure 1 shows the mean and standard deviation for red cell morphology and percent hemolysis for six units of red cells that are filtered into collection bags and stored unmixed for six weeks at 4°C using Nutricel additive, rejuvenated using a solution containing 69 mM Glucose, 30.6 mM NaCitrate, 13.2 mM Na₂HP0₄ and 2 mM Adenine, and stored for an additional six weeks at 4°C. To rejuvenate the cells, each unit is connected to a sterile transfer pack (Fenwal 4R2014) filled with approximately 370 ml of the solution; the solution is transferred into the collection bag until full; the bags are centrifuged at high speed for 8 minutes; the supernatant is transferred to the transfer pack until full; and the bags are disconnected.

Figure 1 demonstrates that when the rejuvenation procedure is applied after six weeks of storage, the morphology index of the red cells increases over the next week of refrigerated storage. Over the subsequent weeks, the morphology index slowly returns to where it was at six weeks. The values recorded during the twelve week period are indicative of clinical acceptability.

It has been discovered that stored red cells can be rejuvenated using a biologically compatible storage solution. Preferably, the biologically compatible solution comprises glucose, phosphate, adenine and citrate. Preferably, the solution is effectively hypotonic. Preferably, the solution has a higher pH than currently licensed red cell storage solutions; more preferably, the solution is selected to raise the intracellular pH of the red blood cells. In particular, the pH of the solution preferably should be at least 7.

The method provides for adding the solution to red blood cells stored in a red cell storage solution without breaking the sterile closed system of the red cell storage solution and mixing the resultant solution. The closed system is maintained so that the resulting solution can be stored under refrigerated conditions without risk of bacterial contamination or a need to discard the rejuvenated cells after only 24 hours to reduce that risk.

The solution may be added by attaching a storage bag comprising stored red cells to a sterile transfer pack containing the rejuvenating solution using a sterile connection device and introducing the solution into the storage bag through the sterile connection device. For example, Haemonetics model SCD 312 may be used. It is often desirable to maintain the volume of the red cell storage solution in order to transfuse the red cells into a patient. Therefore, it may be necessary to remove excess solution from the red cells. Excess solution may be removed after the red cells have been separated from a supernatant by sedimentation. The sedimentation may occur by, for example, centrifugation. In particular, the assembly of the two bags may be placed in a centrifuge in an orientation that causes the red cells to sediment to the bottom of the storage bag. After sedimentation has occurred, an amount of supernatant may be removed such that the volume of the final red cell solution is substantially equivalent to the volume of the original red cell storage solution.

If a storage bag attached to a sterile plastic transfer pack is used, as discussed above, the volume of the red cell storage solution may be maintained easily. In particular, the sterile connection device may be temporarily sealed after the biologically compatible solution is transferred into the storage bag so that the solution cannot flow back into the sterile plastic transfer pack. For example a removable clip can be attached to the sterile connection device. The sedimentation of red cells can occur naturally or, much more preferably, for example, by placing the storage bag in a centrifuge to separate the red cells from the supernatant. After the separation is complete, the seal in the sterile connection device can be removed to allow supernatant to be transferred into the sterile plastic pack by, for example, a plasma press.

Preferably when the sterile plastic transfer pack is attached to the storage bag, the pack is full of the biologically compatible solution. After the solution is centrifuged, the supernatant can be transferred to the sterile plastic transfer pack until it is again full, thereby maintaining a substantially equivalent volume in the storage bag to the volume present before the rejuvenation process. Alternatively, the sterile plastic transfer pack can be calibrated so that the amount of solution in the pack before and after the rejuvenating process can be measured and adjusted as desired.

There is no restriction on the capacity of the two bags. Standard blood storage bags have a capacity of approximately 600 milliliters, while the average volume of the stored cells which they contain is generally approximately 360 milliliters. Preferably, the volume of the rejuvenation solution bag exceeds the additional volume available in the red cell storage bag, therefore allowing the storage bag to be filled. In fact, the subsequent storage of the red cells following rejuvenation will be improved if it is possible to introduce a larger volume of solution into the blood bag. This could also be achieved by repeating the process with a plurality of smaller bags of rejuvenation solution, but this approach is less preferred.

The foregoing embodiments are intended to illustrate and not limit the present invention. It will be apparent that various modifications can be made without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for rejuvenating stored red blood cells comprising: storing a first biologically compatible storage solution containing red blood cells under refrigerated conditions for at least one week; adding a second biologically compatible storage solution to said first biologically compatible storage solution to form a resultant red cell solution; and mixing the resultant red cell solution.

2. The method of claim 1, further comprising: separating the red blood cells in the resultant red cell solution from a supernatant by sedimentation; and removing an amount of the supernatant to form a final red cell solution.

3. The method of claim 2, wherein the red blood cells are separated from the supernatant by centrifugation.

4. The method of claim 2, wherein a volume of the final red cell solution is substantially equal to a volume of the first biologically compatible storage solution.

5. The method of claim 1, wherein the second biologically compatible storage solution is effectively hypotonic to the red blood cells.

6. The method of claim 1, wherein the second biologically compatible storage solution has a pH of at least 7.

7. The method of claim 1, wherein the second biologically compatible storage solution raises an intracellular pH of the red blood cells.

8. The method claim 1, wherein the second biologically compatible storage solution comprises glucose, phosphate, adenine and citrate.

9. The method of claim 1, wherein the resultant red cell solution is stored for more than 48 hours.

10. The method of claim 9, wherein the resultant red cell solution is stored for more than one week.

11. The method of claim 9, wherein the resultant red cell solution is stored for more than four weeks.

12. The method of claim 1, wherein a storage bag comprising the first biologically compatible storage solution is attached to a sterile transfer pack comprising the second biologically compatible storage solution by a sterile connection device and, in said adding step, the second biologically compatible storage solution is transferred into the storage bag.

13. A method for rejuvenating stored red blood cells in a sterile closed system, comprising:

(a) attached a storage bag comprising a red cell storage solution containing stored red blood cells to a sterile transfer pack filled with a biologically compatible storage solution by a sterile connection device to form a sterile closed system;

(b) introducing said biologically compatible storage solution into said storage bag to form a resultant solution;

(c) mixing the resultant solution;

(d) separating the red blood cells from a supernatant by sedimentation; (e) transferring a portion of the supernatant from the storage bag to the sterile transfer pack such that the sterile transfer pack is filled;

(f) sealing the sterile connection device; and (g) detaching the sterile transfer pack from the storage bag so that the storage bag remains sealed.

14. The method of claim 13, wherein, during step (d) , the sterile connection device is temporarily sealed.

15. The method of claim 13, wherein the red blood cells are separated from the supernatant by centrifuging the storage bag in a centrifuge in an orientation that causes sedimentation of the red blood cells to a bottom of the bag.

16. The method of claim 13, wherein, in step (e) , supernatant is transferred into the sterile transfer pack by a plasma press.

17. The method of claim 13, further comprising: (h) storing the storage bag under refrigerated conditions for at least 48 hours.

18. The method of claim 13, wherein the biologically compatible storage solution is effectively hypotonic to the red blood cells.

19. The method of claim 13, wherein the biologically compatible storage solution comprises glucose, phosphate, adenine and citrate.

20. A method for rejuvenating stored red blood cells in a sterile closed system, comprising: (a) attaching a storage bag comprising a first biologically compatible storage solution containing stored red blood cells to a sterile transfer pack containing a second biologically compatible storage solution to form a sterile closed system; (b) introducing said second biologically compatible storage solution into said storage bag to form a resultant solution; and

(c) mixing the resultant solution.