U.S. patent number 3,874,384 [Application Number 05/345,961] was granted by the patent office on 1975-04-01 for improved blood storage unit and method of storing blood.
This patent grant is currently assigned to American Hospital Supply Corporation. Invention is credited to Jon M. Brake, Fred H. Deindoerfer.
United States Patent |
3,874,384 |
Deindoerfer , et
al. |
April 1, 1975 |
Improved blood storage unit and method of storing blood
Abstract
Blood storage units comprising containers with aqueous
preservative solutions therein are improved by incorporating
dihydroxyacetone (DHA) together with L-ascorbate (vitamin C) in the
preservative solutions. The invention also relates to a method of
storing human blood wherein viable red cells are stored in contact
with both DHA and L-ascorbate. The preservative solution of the
blood storage unit may also contain an anticoagulant such as
citrate, a sugar energy source such as dextrose, and an ATP
maintaining agent such as adenine. Where the blood storage unit is
to be heat sterilized, as preferred, a blood bag providing a
separate compartment for part of the preservative agents prevents
deterioration of the preservative agents, the DHA and sugar energy
source being heat sterilized separately for later admixture with
the ascorbate, citrate anticoagulant and adenine. Separate pH
control can also thereby be provided for the heat
sterilization.
Inventors: |
Deindoerfer; Fred H.
(Northridge, CA), Brake; Jon M. (Burbank, CA) |
Assignee: |
American Hospital Supply
Corporation (Evanston, IL)
|
Family
ID: |
26890255 |
Appl.
No.: |
05/345,961 |
Filed: |
March 29, 1973 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
194652 |
Nov 1, 1971 |
3847378 |
|
|
|
194689 |
Nov 1, 1971 |
3795581 |
|
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|
Current U.S.
Class: |
604/408; 424/529;
435/2 |
Current CPC
Class: |
A61J
1/10 (20130101); A01N 1/0226 (20130101); A01N
1/0263 (20130101); A61J 1/12 (20130101) |
Current International
Class: |
A61J
1/00 (20060101); A61J 1/14 (20060101); A61J
001/00 (); A61K 017/00 (); C12K 009/00 () |
Field of
Search: |
;195/1.8 ;424/101 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Rosen; Sam
Parent Case Text
CROSS-REFERENCES
This application is a continuation-in-part of our copending
application Ser. Nos. 194,652 and 194,689, both filed Nov. 1, 1971,
now U.S. Pat. Nos. 3,847,378 and 3,795,581, respectively.
Claims
We claim:
1. A blood storage unit comprising a container for receiving and
storing a predetermined volume of blood and preservative solution
admixable with the blood stored in said container, said
preservative solution being sterile and providing a sugar energy
source and an anti-coagulant for preserving said blood, said
preservative solution also providing for cooperative admixture with
said stored blood an amount of dihydroxyacetone (DHA) equal to 5 to
100 millimoles (mM) per liter of said predetermined blood volume
together with an amount of L-ascorbate equal to 0.5 to 20 mM per
liter of said predetermined blood volume.
2. The blood storage unit of claim 1 wherein said DHA is present in
an amount of from 10 to 30 mM of DHA per liter of said
predetermined blood volume.
3. The blood storage unit of claim 1 wherein said preservative
solution also provides adenine in an amount equal to from 0.1 to
1.0 mM per liter of said predetermined blood volume.
4. The blood storage unit of claim 1 wherein said preservative
solution provides from 1 to 10 mM of said L-ascorbate per liter of
said predetermined blood volume.
5. The method of maintaining the 2,3-diphosphoglycerate (2,3-DPG)
content of viable red cells of whole human blood, comprising
incorporating in said whole blood from 5 to 100 millimoles (mM) of
dihydroxyacetone (DHA) per liter of said blood together with 0.5 to
20 mM of L-ascorbate per liter of said blood, and holding said
blood with said red cells in contact with said DHA and L-ascorbate
for sufficient time to maintain their 2,3-DPG content at a level
resulting from the synergistic action of said DHA and said
L-ascorbate.
6. The method of claim 5 wherein said DHA and said L-ascorbate are
incorporated in said blood in amounts of from 15 to 45 mM DHA and
1.5 to 15 mM ascorbate per liter of blood.
7. The method of maintaining 2,3-diphosphoglycerate (2,3-DPG)
content of the red cells of whole human blood under storage
conditions, comprising adding to said whole blood from 5 to 100
millimoles (mM) of dihydroxyacetone (DHA) and from 0.5 to 20 mM of
L-ascorbate per liter of blood, and storing said DHA and ascorbate
containing blood without freezing at a temperature below 10.degree.
C.
8. The method of claim 7 wherein from 10 to 30 mM of said DHA and
from 1 to 10 mM of said L-ascorbate are added to said blood
immediately after the collection thereof.
9. The method of claim 7 in which said blood is stored for a period
of from 3 to 6 weeks.
10. A preservative solution for addition to stored blood,
comprising a sterile aqueous solution of dihydroxyacetone (DHA) and
L-ascorbate, said solution containing from 0.5 to 20 mM of said
L-ascorbate per each 5 to 100 mM of said DHA.
11. A preservative solution for addition to substantially 0.5
liters of whole blood, comprising a sterile aqueous solution
containing from 2.5 to 50 mM dihydroxyacetone together with 0.25 to
10 mM of L-ascrobate.
12. A heat-sterilized blood storage unit, comprising a container
for receiving and storing a predetermined volume of blood, a first
sterile aqueous preservative solution in said container, means
providing a separate compartment, a second sterile aqueous
preservative solution in said compartment, means permitting said
second solution to be introduced into said container for admixture
with said first solution and with said predetermined volume of
blood, said first and second solutions together providing a sugar
energy source, an anti-coagulant, an amount of dihydroxyacetone
(DHA) equal to 5 to 100 millimoles (mM) per liter of said
predetermined blood volume, and an amount of L-ascorbate equal to
0.5 to 20 mM per liter of said predetermined blood volume, said
L-ascorbate being contained only in one of said first and second
solutions prior to said admixture thereof, and said sugar energy
source and said DHA being contained only in the other of said first
and second solutions.
13. The blood storage unit of claim 12 wherein said DHA is present
in an amount of from 10 to 30 mM of DHA per liter of said
predetermined blood volume.
14. The blood storage unit of claim 12 wherein one of said
preservative solutions also contains adenine in an amount equal to
from 0.1 to 1.0 mM per liter of said predetermined blood
volume.
15. The improved blood storage unit of claim 13 wherein said one
preservative solution contains from 1 to 10 mM of said L-ascorbate
per liter of said predetermined blood volume.
Description
DRAWINGS
The accompanying drawing, comprising FIGS. 1 to 7, illustrates one
form of a blood collection and storage unit for use in practicing
the present invention where the preservative solutions are heat
sterilized. The construction and method of use of such blood
storage units will be further described in Example IV.
BACKGROUND AND SUMMARY
The state of the art with respect to biochemical knowledge of the
chemical makeup and functioning of red cells (erthyrocytes) is
summarized in two recent publications: Red Cell Metabolism and
Function, edited by George J. Brewer, Plenum Press, 1970; and Red
Cell Metabolism, Ernest Beutler, Grune and Stratton, 1971. The
principal energy source for red cells is glucose (or equivalent
sugar) which is metabolized by the cells through complex
biochemical pathways involving enzymatic reactions. The principal
pathway, often referred to as the Embden-Meyerhoff pathway,
involves the anaerobic breakdown of glucose to pyruvic or lactic
acid. An additional pathway is referred to as the direct oxidative
shunt or hexose monophosphate shunt.
In the Embden-Meyerhoff pathway, the compound
1,3-diphosphoglycerate is produced from
D-glyceraldehyde-3-phosphate. The 1,3-diphosphoglycerate is
converted by interaction with ADP (adenosine diphosphate) to ATP
(adenosine triphosphate) and 3, phosphoglycerate, the reaction
being catalyzed by phosphoglycerate kinase. An alternate by-path
also leads to 3-phosphoglycerate, by way of 2,3-diphosphoglycerate
(hereinafter referred to as 2,3-DPG or more concisely as DPG) -- an
important regulator of the oxygen affinity of hemoglobin. The
complexing of 2,3-DPG with hemoglobin decreases the affinity of
oxygen (O.sub.2) to hemoglobin in a manner essential to the release
of oxygen to the body tissues.
In human blood, the normal level of 2,3-DPG is within the range
from 12 to 18 micromoles 2,3-DPG per gram of hemoglobin. Beutler
gives a more precise figure: 15.36 .+-. 1.98 micromoles 2,3-DPG/g.
hemoglobin (Red Cell Metabolism supra., p. 99). In the body, under
usual conditions, sufficient 2,3-DPG is produced by the red cells
in the metabolism of glucose by the Embden-Meyerhoff pathway to
provide the required amount for proper oxygen-hemoglobin-tissue
transfer. For reasons that are not understood, however, the 2,3-DPG
content of red cells in stored blood decreases to subnormal levels
interfering with oxygen released by the cells, even though blood is
stored under refrigerator conditions (1.degree.-6.degree. C.) in
admixture with an anticoagulant solution containing dextrose (or
equivalent sugar) as the principal energy source for the red cells.
Therefore, although the red cells remain viable, contain sufficient
ATP, and provide a satisfactory survival rate (70 percent or more
after 24 hours), the subnormal 2,3-DPG content of the red cells may
actually cause a decrease in the oxygen delivered to the tissues
for several hours after the transfusion, and as long as 24 hours
may be required for the transfused red cells to be restored to
normal 2,3-DPG levels for efficent delivery of oxygen to the
tissues. (Dawson, "The Hemoglobin Function of Blood Stored at
4.degree. C.," pp. 305-317, in Red Cell Metabolism and Function,
supra).
The problem of administering stored blood deficient in 2,3-DPG is
rendered more acute under many clinical conditions, such as
patients in septic shock, patients receiving large volumes of
stored blood, and infants, particularly premature infants, with
infection or the respiratory disease syndrome, since the 2,3-DPG
levels of the patient's blood may already be depressed, and further
depression may occur on administration of the low 2,3-DPG level
blood.
Since the recognition of the function of 2,3-DPG as an oxygen
release regulator for hemoglobin, and the recognition that
depressed levels of 2,3DPG can occur in the body and under in vitro
storage of blood, there has been a widespread search for chemical
additives or other means of maintaining, or even increasing, the
2,3-DPG content of red cells. It has been found that frozen blood
stored under very cold conditions (viz. -85.degree. C.) can be
stored for many months without significant change in 2,3-DPG
levels. However, because of the added expense in freezing blood and
storing it in the frozen conditions, the use of frozen blood has
not become a commercial blood storage practice. The almost
universal procedure in the United States at the present time is to
combine the freshly collected blood with an anticoagulant solution
containing dextrose, such as a citrate-dextrose solution or a
citrate-phosphate-dextrose solution, and then to store the blood
under refrigeration at a substantially constant temperature within
the range from 1.degree. to 6.degree. C. Following this procedure,
blood bank storage is approved up to 21 days, and if the blood is
not administered by that time, it usually must be discarded.
In our copending applications, Ser. Nos. 194,652 and 194,689, cited
above, we have disclosed a blood storage unit and method of blood
storage wherein dihydroxyacetone (DHA) is incorporated in the
preservative solution and maintained in contact with the red cells
of the blood during storage for the purpose of maintaining and/or
increasing the 2,3-DPG content of the red cells. Subsequent to our
discovery of the effect of DHA on DPG levels of red cells, DR.
Ernest Beutler, City of Hope Medical Center, Duarte, Calif., has
found that the mechanism of action of the DHA involves triokinase
enzyme activity, a type of enzyme activity which had not previously
been known to exist in red cells. By the postulated mechanism,
dihydroxyacetone is converted to dihydroxyacetone phosphate by the
mediation of triokinase enzyme activity, and the dihydroxyacetone
phosphate enters the main metabolic pathway of the red cells.
Dr. Ernest Beutler has also reported that L-ascorbic acid (vitamin
C) has a positive effect on the maintenance of DPG levels in stored
blood, but that the mechanism of action of L-ascorbate is unknown:
Transfusion Congress, American Association of Blood Banks, XXV
Annual Meeting, Aug. 27-Sept. 2, 1972; and Western Society of
Clinical Research, Meeting Feb. 3-5, 1972, Carmel, Calif. As
reported by Dr. Beutler, red cells stored with L-ascorbate use
significantly less dextrose than controls, and the intracellular pH
is significantly higher. Further, during storage of the red cells
with ascorbate, less lactate and more pyruvate is formed from the
sugar energy source. It therefore appears that the mechanism of
action of ascorbate in maintaining DPG levels, although not fully
understood, is quite different from the mechanism of action of DHA.
Our discovery, which forms an important part of the present
invention, was therefore unexpected; namely, that DHA and
L-ascorbic acid (vitamin C) can function synergistically in
maintaining and/or increasing DPG levels of red cells in stored
blood, especially where the blood is stored for 2 or 3 weeks or
longer, such as storage periods of from 3 to 6 weeks.
DETAILED DESCRIPTION
In practicing the present invention, approved types of blood
collection and preservation containers are preferred. Either glass
or plastic containers can be utilized, providing they meet the
U.S.P. requirements. (See U.S. Pharmacopeia XVIII, pages 887 and
923.) The containers will be sized for receiving and storing a
predetermined volume of blood, such as 1,000 ml., 500 ml., etc.
Typically, the containers will have an internal volume adapted for
receiving 500 ml. (1/2 1.) of blood together with 70 to 125 ml. of
anticoagulant solution. In other words, the containers can have an
internal volume of around 570 to 625 ml. The containers will also
be equipped with means for introducing the fresh blood as it is
collected, and for delivery of the blood in transfusion. Such
transfusion and infusion assemblies used with the blood collection
and storage units should meet U.S.P. requirements. (See U.S.
Pharmacopeia XVIII, p. 887).
As in the established practice, the anticoagulant solution for
admixture with blood collected in the containers should contain an
anticoagulant substance to prevent coagulation of the blood,
preferably, also, a sugar energy source for the red cells in
addition to the DHA. The preferred anticoagulant is "citrate ions"
which may be supplied by sodium citrate, or mixtures of citric acid
and sodium citrate. The quantities to be employed can be the same
as in present practice (see U.S. pharmacopeia XVIII, pages
47-49).
The sugar energy source for the red cells is preferably dextrose.
However, it is known that other sugars are equivalent to dextrose
for this purpose, including fructose, mannose, and galactose. The
amount of dextrose or equivalent sugar employed can be the same as
in present practice (see U.S. Pharmacopeia XVIII, pages 47-48).
More specifically, from about 1.7 to 1.9 grams dextrose based on
dextrose monohydrate can be utilized per 500 mililiters of
blood.
In practicing the present invention, the blood collection and
preservation unit should contain at least 5 and preferably at least
10, millimoles (mM) DHA per liter of blood. Consequently, when the
unit is designed to collect 500 ml. of blood, at least 2.5 and
preferably 5 mM of DHA will be incorporated in the aqueous
anticoagulant solution. While there does not appear to be any
critical upper limit on the content of DHA, there appears to be no
reason to exceed 100 mM DHA per liter of blood. When the container
is designed for 500 ml. of blood, therefore, it will not be
necessary to incorporate more than 50 mM of DHA in the
anti-coagulant solution. Where the DHA is being utilized for
2,3-DPG maintenance, and a sugar energy source is provided, as in
present practice, it will usually not be necessary to employ more
than 30 mM of DHA per liter of blood, or 15 mM per 500 ml. of
blood.
Since red cells occupy approximately one-third the volume of whole
blood, it will be appreciated that the red cells in storage will be
in contact with an aqueous solution containing from 7.5 to 150 mM
of DHA per liter of solution, or preferably 15 to 45 mM DHA per
liter of solution. Preferably, the DHA is incorporated in the blood
immediately after its collection.
For cooperation with the DHA in maintaining and/or increasing the
DPG content of the red cells of the blood, the present invention
utilizes L-ascorbic acid (vitamin C) as a cooperating additive. The
L-ascorbic acid may be incorporated in the preservative solution in
its free acid form, or as a water-soluble, blood-compatible,
non-toxic ascorbate salt. For example, the sodium salt of
L-ascorbic acid can advantageously be used to obtain the same
effect as adding ascorbate as free L-ascorbic acid. As used
subsequently herein, therefore, the term "L-ascorbate" or,
"ascorbate" is intended to refer to and include the L-ascorbate
moiety both as free acid and in salt form, either form being
biologically equivalent for the purposes of the present
invention.
On the basis of L-ascorbate content, the preservative solution for
admixture with the stored blood should provide 0.5 to 20 mM of
L-ascorbate per liter of blood. For example, where the preservative
solution is for admixture with substantially 0.5 liters of whole
blood, from 0.25 to 10 mM L-ascorbate should be used in combination
with 2.5 to 50 mM DHA. Preferably, from 1 to 10 mM of L-ascorbate
per liter of blood is employed. For example, when the preservative
solution is to be added to substantially 0.5 liters of blood, it
can advantageously contain from 0.25 to 10 mM L-ascorbate together
with 2.5 to 50 mM DHA. The red cells will therefore be stored in
contact with an aqueous solution containing from 0.75 to 30 mM
L-ascorbate per liter of solution, or preferably from 1.5 to 15 mM
L-ascorbate per liter of solution.
The DHA-ascorbate preservative solution also preferably contains
adenine. For example, from 0.1 to 1.0 mM adenine can be
incorporated in the preservative solution per liter of
predetermined blood volume. In other words, where the preservative
solution is for admixture with substantially 0.5 liters of blood,
the amount of adenine can range from 0.05 to 0.5 mM.
For refrigeration storage, as described above, the conjoint action
of the DHA-ascorbate combination of the present invention in
maintaining DPG levels is accentuated as the length of the storage
period increases. After storage of about 2 to 3 weeks, the
synergistic cooperation of the L-ascorbate and the DHA becomes the
predominant effect. With the DHA-ascorbate combination of the
present invention, blood may be stored while maintaining acceptable
DPG levels for periods of time over 3 weeks and up to 5 to 6 weeks.
Data demonstrating the remarkable synergism of DHA and L-ascorbate
during the extended storage of blood is presented below in Example
I. Where adenine is incoporated in the preservative solution, as
preferred, the ATP (adenosine triphosphate) content of the red
cells can also be maintained at a satisfactory level during such
extended storage periods.
The DHA-ascorbate combination of this invention can be utilized at
preservative pH's from neutrality (approximately pH 7.0) down to
acid pH's as low as 5.0. PH's on the acid side may be advantageous.
For example, an admixture of the preservative solution with the
blood, a pH in the range of 5.3 to 5.9, such as a pH of
substantially 5.6, is particularly advantageous.
Where the preservative solutions are sterilized by heat
(autoclaving), as preferred, it has been discovered that the
decomposition of the DHA and the ascorbate can be minimized by
dividing the preservative solution into two separate solutions for
purposes of sterilization, the solutions being recombinable for
admixture with the blood within the blood collection container.
Specifically it has been discovered that ascorbate when heat
sterilized tends to be decomposed by DHA and also by dextrose.
Consequently, it is preferred to provide the blood storage unit
with a separate compartment containing an aqueous solution of DHA
and dextrose, the blood bag, or other compartment, containing an
aqueous solution of the ascorbate. The DHA-dextrose aqueous
solution component has been found to be most stable when heat
sterilized at a pH within the range from 3.8 to 4.2, such as a pH
of substantially 4.0. This pH is therefore preferred. The ascorbate
containing solution component can advantageously have a pH of 5.3
to 5.9, such as a pH of substantially 5.6. This component can also
contain the citrate anticoagulant and the adenine, all of these
ingredients being substantially stable under heat sterilization in
admixture with each other under the stated pH. Alternatively,
however, all ingredients of the preservative solution can be
combined, and the aqueous solution can be sterilized by passing it
through a sterilization filter before being filled into the blood
storage container. This procedure, however, is more difficult and
expensive than heat sterilization.
Various aspects of the present invention are further illustrated by
the specific examples set out below:
EXAMPLE I
This example describes actual laboratory experiments and reports
the data obtained, which demonstrate the synergistic effect of
dihydroxyacetone (DHA) and L-ascorbic acid (vitamin C) on
2,3-diphosphoglycerate (DPG) in stored blood. In three separate
experiments, blood from a single donor was divided into four
portions. One was stored with CPD-adenine, one with
CPD-adenine-ascorbate, one with CPD-adenine-DHA, and one with
CPD-adenine-ascorbate-DHA. The concentrations of the components
were as follows: CPD-adenine, CPD (citrate-phosphate-dextrose) per
U.S.P. XVIII, pg. 48-49, and adenine, 0.5 mM per liter of blood;
L-ascorbic acid (L-ascorbate), 100 mg. per each 100 ml. blood; and
dihydroxyacetone (DHA), 20 mM per liter blood. The pH of the
preservative solution was 5.6.
Samples were stored in 100 ml. plastic blood bags at 4.degree. C.
and sampled at intervals. DPG was determined by the enzymatic
method of Prins and Loos, as described in "Red Cell Metabolism and
Function," ed. G. J. Brewer, pp. 227-288 (Plenum Press, 1970).
In Experiment No. 1, blood was drawn into heparin (2115 U.S.P.
units/500 ml. blood). Forty ml. of blood were transferred to the
sterile 100 ml. plastic bags containing 6 ml. of CPD-adenine. In
Experiments 2 and 3, blood was drawn in CPD-adenine (70 ml.
anticoagulant/500 ml. of blood). Aliquots of the blood were then
transferred aseptically to sterile 100 ml. plastic bags.
A 10% solution of DHA was prepared and sterilized by autoclaving.
It was added to selected bags in a ratio of 0.5 ml. per 500 ml of
blood. A solution of L-ascorbate was prepared by dissolving 5 grams
of L-ascorbic acid in 100 ml. of water and adjusting to pH 5.5 with
1 N sodium hydroxide. It was sterilized by filtering through a 0.22
micron sterilizing filter, and was added to selected bags in a
ratio of 1 ml. per 50 ml. of blood.
The results of these experiments are shown in Table A. After 3
weeks of storage, the synergism of DHA and ascorbate on DPG levels
is revealed. For this purpose, synergism can be measured when the
DPG level of the DHA plus ascorbate sample exceeds the sum of the
DPG level of ascorbate alone plus DHA alone. At 3 weeks, such
synergism was measured in one of three experiments. At 4 weeks and
5 weeks, the synergism was measured in 2 out of 3 experiments. At 6
weeks, synergism was measured in all 3 experiments.
In Table B, the same data are recalculated as difference values,
sample minus control. This isolates the effect on DPG due to the
additive from the effect due to the CPD-adenine preservative. The
synergism is even more clearly evidenced in these results; namely
synergistic action is disclosed in 2 out of 3 experiments at 3
weeks, and in 3 out of 3 experiments at 4, 5,and 6 weeks. It is
therefore apparent that synergistic cooperation of DHA and
ascorbate in maintaining DPG levels in stored blood provides a
means for greatly improving the quality of the blood.
TABLE A
__________________________________________________________________________
Effect of Ascorbate, DHA and the Combination of Ascorbate/DHA on
DPG Levels of Blood Stored in CPD-Adenine
__________________________________________________________________________
Storage Time DPG (% of initial) Additive (weeks) Exp. No. 1 Exp.
No. 2 Exp. No. 3 Average
__________________________________________________________________________
None 3 12 12 22 15.3 Ascorbate 3 18 66 71 51.7 DHA 3 67 71 95 77.7
DHA + Ascorbate 3 137* 109 150 132* None 4 10 15 14 13 Ascorbate 4
14 71 60 48.3 DHA 4 41 26 25 30.7 DHA + Ascorbate 4 119* 86 125*
110* None 5 13 8 15 12 Ascorbate 5 25 39 56 40 DHA 5 28 10 20 19.3
DHA + Ascorbate 5 125* 48 115* 96* None 6 13 11 19 14.3 Ascorbate 6
32 13 50 31.7 DHA 6 5 8 20 11 DHA + Ascorbate 6 88* 36* 84* 69.3*
__________________________________________________________________________
*Sum of DPG value for ascorbate and DHA alone is less than DPG
value for ascorbate and DHA together.
TABLE B
__________________________________________________________________________
Effect of Ascorbate, DHA and the Combination of Ascorbate /DHA on
Differential DPG Levels of Blood Stored in CPD-Adenine
__________________________________________________________________________
Storage Time DPG (% of initial)** Additive (weeks) Exp. No. 1 Exp.
No. 2 Exp. No. 3 Average
__________________________________________________________________________
Ascorbate 3 6 54 59 36.4 DHA 3 55 59 73 62.4 DHA + Ascorbate 3 125*
77 138* 116.7* Ascorbate 4 4 56 46 35.3 DHA 4 31 11 11 17.7 DHA +
Ascorbate 4 109* 71* 111* 97 Ascorbate 5 12 31 41 28 DHA 5 15 2 5
7.3 DHA + Ascorbate 5 112* 40* 100* 84* Ascorbate 6 19 2 31 17.3
DHA 6 0 0 1 0.3 DHA + Ascorbate 6 65* 25* 25* 51.7*
__________________________________________________________________________
*Sum of DPG values of ascorbate and DHA separately is less than DPG
value of the combination of the two. **Calculated as the difference
in DPG between the sample and the control CPD-adenine.
EXAMPLE II
In one embodiment, the invention may be practiced as follows:
To prepare a CPD-adenine-ascorbate-dihydroxyacetone system,
dissolve the following chemicals in 800 ml of water for injection
U.S.P. and add water to make one liter of solution: sodium citrate
dihydrate 30.8 grams (g), dextrose (anhydrous) 22.2 g,
dihydroxyacetone 14.7 g, adenine 0.55 g, L-ascorbic acid 8.1 g, and
sodium biphosphate monohydrate 2.22 g. Sterilize by filtration
through a 0.22 micron sterilizing filter. Using aseptic technique
fill 70 milliliters (ml) into sterile blood bags of volume capacity
for collection of 500 ml of blood. Pack the prepared units in metal
cans under nitrogen until needed for blood collection and storage
use.
Where the blood in admixture with the DHA and ascorbate is stored
for periods beyond 1 week, as preferred, it is desirable to invert
the storage containers at least at the end of each week of storage.
In one preferred procedure, the storage containers are inverted
daily, or at least 5 days per week. Such inversion serves to
provide a mild agitation of the contents of the blood bag, thereby
maintaining the red cells in more uniform contact with the solution
of DHA and ascorbate. This will help to assure that the combined
effects of the DHA and the ascorbate are maximized.
EXAMPLE III
A preferred mode of practicing the invention is illustrated as
follows:
A special design of blood storage unit is used, such as the blood
bag unit of the attached drawing, which will be subsequently
described in detail in Example IV. In general, the unit consists of
a 500 ml. blood bag with a 15 ml. pilot tube attached. The solution
for the blood bag (Solution A) is prepared by dissolving the
following in 800 ml. of water and adding water to make one liter
final volume: sodium citrate dihydrate 38.3 g., adenine 0.68 g.,
ascorbic acid 11.1 g., and sodium biphosphate monohydrate 2.76
g.
With the pilot tube clamped off, 56.4 ml. of this solution is
filled through a donor tube into the blood bag. Then cyclohexane is
applied to the end of the donor tube, and it is inserted into a
needle adaptor with attached needle. This seals the needle to the
tubing.
The solution for the pilot tube (Solution B) is prepared by
dissolving the following in 800 ml. of water and bringing to a
final volume of one liter: dextrose (anhydrous) 105.6 g. and
dihydroxyacetone (66.9 g.). The pH is adjusted to 4 by adding 1 N
sodium hydroxide. The separate compartment provided by the pilot
tube is connected at its inner end to the blood bag, by a
releasably clamped tubing. Then 15.4 ml. of this solution is added
to the pilot tube, through the short filling tubing connected to
the outer end of the pilot tube. This filling is then heat
sealed.
The bag unit can be used as follows: After opening the can, the bag
is removed and the clamp between the pilot tube and the bag is
opened. The pilot tube is squeezed, forcing Solution B into the
main bag. The clamp on the pilot line is closed, and the bag is
agitated to mix Solutions A and B thoroughly. The needle protector
is removed, and a venipuncture was made by the usual technique in a
human volunterr. After 500 ml. (530 g.) of blood is collected, the
clamp on the donor line is closed. The bag is stored on its side in
a 4.degree. C. refrigerator, and agitated to resuspend the red
cells in the plasma, as described in Example II.
EXAMPLE IV
In the accompanying drawings, there is shown a blood storage unit
which is adapted for the practice of the present invention. The
figures of this drawing are related as follows:
FIG. 1 is an elevational view of a complete blood storage unit
ready for the collection of blood;
FIG. 2 is a perspective view of one of the two clamps of the unit
of FIG. 1;
FIG. 3 is an exploded elevational view of the needle adaptor and
needle cover of the unit of FIG. 1;
FIG. 4 is a detailed view showing the clamped portion of one of the
tubes of FIG. 1;
FIG. 5 illustrates the appearance of the clamped portion of the
tube of FIG. 4 immediately after the removal of the clamp;
FIG. 6 illustrates the appearance of the clamped portion of the
tube of FIG. 4 after the clamp has been removed and the tube opened
for the flow of liquid; and
FIG. 7 is a sectional view taken on line 7--7 of FIG. 5 showing the
tube in collapsed condition as it would appear when clamped or
before opening the tube for liquid flow.
As referred to in Example III, the blood storage unit includes a
standard flexible plastic blood bag 10 having a blood storage
compartment 11 therein, and a pilot tube 12 providing a separate
smaller liquid storage compartment 13 therein. As indicated on FIG.
1, compartment 11 contains Solution A while compartment 13 contains
Solution B. It will be understood that these solutions may be
prepared and incorporated in these compartments as described in
Example III.
Although the constructional details of the blood bag unit of FIG. 1
are conventional and well known in the blood collection and storage
art, they will be briefly described in order that the use of the
blood storage unit for the purpose of the present invention may be
clearly understood. Blood bag 10 which may be formed by a heat
sealing procedure from a suitable plastic sheet material such as
polyvinylchloride is provided with an inlet 14 connected to an
inlet tube 15. As illustrated, tube 15, which may be longer than
illustrated if desired, connects to a Y-connector 16. From the
Y-connector there extends a blood collection tube 17 having a
needle assembly 18 at the outer end thereof and a line clamp 19
thereon adjacent a slidable sleeve 20. As shown more clearly in
FIG. 3, the needle assembly 18 includes a hub 19, a needle 20 and a
needle cover or protector 21. From connector 16 there also extends
a tube 22 which connects to the inner end of the enlarged pilot
tube 12 and with the compartment 13 therein. On tube 22, there is
also provided a line clamp 19 and an adjacent sleeve 20. It will be
understood that the tube 17 and 22 may be longer than shown if
desired. At the other end of the pilot tube 20, compartment 13
connects to a short filling tube 23.
As indicated in Example III, Solution A will be filled into
compartment 11 through tube 17 before the needle assembly 18 is
attached to the outer end thereof, the clamp 19 on line 17 being
open during this filling operation, while the clamp 19 on line 22
is closed. Following the filling of Solution A through tube 17,
clamp 19 can be moved to closed position and needle assembly 18
attached. As shown more clearly in FIG. 2, clamp 19 provides an
enlarged opening 19a through which the tubing can extend without
being clamped, and this opening communicates with the restricted
slot 19b within which the tubing is clamped to a temporarily sealed
condition.
Also, as indicated in Example III, Solution B is introduced into
the separate compartment 13 through the filler tube 23 with the
clamp 19 on line 22 in closed position. After the filling
operation, the filler tube 23 may be heat sealed as indicated at
24. During heat sterilization which may be carried out as described
in Example III, the clamps 19 on lines 17 and 22 may remain closed.
For collection of blood, the protector 21 will be removed from the
needle 20, clamp 19 opened and the tube held in oepn condition by
means of sleeve 20. The blood from the donor will then be
transferred through lines 17 and 15 to the compartment 11. After
the blood has been collected, clamp 19 on line 17 may again be
moved to closed position. Either prior to the collection of the
blood or subsequent thereto, Solution B may be mixed with Solution
A and with the blood in compartment 11 by opening clamp 19 and
moving sleeve 20 to hold tube 22 in open condition. Since the pilot
tube 20 is formed of a flexible plastic material, it can be
squeezed to provide a pump action forcing Solution B through tubes
22 and 15 into compartment 11. Tube 12 may also be elevated to
assist this transfer by gravity flow. After the transfer of
Solution B to compartment 11, the clamp 19 on line 22 may again be
moved to closed position. Where it is desired to make the unit more
compact for storage of the collected blood, and after the blood and
Solution B are both in compartment 11, the tube 15 may be heat
sealed, as indicated at 25 and then clipped off, as indicated at
26.
The procedure for manipulating the clamp 19 and the sleeve 20 in
relation to a tube T, such as the tubes 17 or 22 of FIG. 1, is
illustrated in FIGS. 4 to 7. In FIG. 4, clamp 19 is shown in its
raised or clamping position, the tube T being squeezed to a
temporarily sealed condition by its engagement in the slot 19b. to
open the tube, clamp 19 is moved in relation to tube T so that the
tube extends through the larger opening 19a, and is then moved away
from the previously clamped portion by sliding it down the tube. As
shown in FIGS. 5 and 7, the clamped portion 27 of the tube T tends
to remain sealed after removal of the clamp 19. It can be opened by
squeezing it between a thumb and forefinger. After opening, the
sleeve 20 is pushed over the previously clamped portion of the tube
to hold the tube in open condition. This position is illustrated by
FIG. 6. Since such use and manipulation of such clamps and sleeves
are well known in the blood collection and administration art, it
is not believed to be necessary to further describe them
herein.
Conveniently, all of the components of the blood collection and
storage units of FIG. 1 can be formed of suitable plastic
materials. For example, bag 10, pilot tube 12, tubes 15, 17, 22 and
23 and Y connector 16 may be formed of polyvinyl chloride, slide
clamps 19 of nylon or other relatively rigid thermoplastic, and hub
19 and protector 21 of polyvinyl chloride or other suitable
thermoplastic. Needle 21 is preferably formed of stainless steel of
a standard needle size, such as a 16 gauge needle.
It will be understood, as shown, that bag 10 is provided with the
standard hanging loops and perforations, for example, as indicated
at 28 and 29. The top of the bag is also provided with a pair of
tubular connector outlets 30 having their outer ends closed by
tear-off caps 31. For administration of the blood to a patient, one
of the caps 31 can be removed, and a blood administration set
connected to one of the tubes 30.
It will be apparent to those skilled in the art that the blood
collection unit of FIG. 1 can be modified in various ways while
still being usable for the practice of the present invention. For
example, the pilot tube 12 may be replaced by a small separate bag,
or bag 10 can be manufactured with two compartments, and means
provided for opening a seal between the two compartments to mix
Solutions A and B after completion of the heat sterilization.
EXAMPLE V
This example describes laboratory experiments demonstrating that
DHA and ascorbate can be added to blood after one week of storage,
resulting in the maintenance of high DPG levels for 6 weeks. Five
hundred ml. of human blood were collected in a blood bag containing
70 ml. of CPD-adenine (composition given in Example I). Four 35-ml.
aliquots of the blood were transferred to sterile 100 ml. blood
bags, one bag serving as a control and the others being used in
other experiments. The bags were stored at 4.degree. C. for 1 week,
and then DHA (20 mM/l.) and L-ascorbate (5.7 mM/l.) were added to
one bag as follows: a sterile injection site (a spike with a rubber
septum attached) was placed in one of the ports of the blood bag.
Then using a sterile syringe, the following solutions were injected
into the large blood bag: 3.8 ml. of a 2 molar solution of DHA,
sterilized by autoclaving at 250.degree. F. for 10 minutes, and 7.6
ml. of a 5 percent solution of L-ascorbic acid adjusted to pH 5.6
with sodium hydroxide and sterilized by filtration through a 0.22
micron sterile filter. All bags were mixed daily except
weekends.
The results of DPG assays of the blood are shown in Table C. The
control showed a rapid fall in DPG levels after the first week,
while the blood supplemented with DHA and ascorbate at 1 week of
storage maintained normal or higher than normal DPG levels for 6
weeks.
TABLE C ______________________________________ DPG Levels in Blood
Collected in CPD-Adenine With and Without Addition of DHA/Ascorbate
After One Week of Storage at 4.degree. C. DPG (mM/g Hb) Storage
Time CPD-ad CPD-ad+DHA+ascorbate (weeks)
______________________________________ 0 12.9 12.9 1 12.8 12.6 2
4.8 13.1 3 2.9 14.2 4 1.8 15.4 5 1.9 15.6 6 2.4 13.5
______________________________________
EXAMPLE VI
For practicing the method described in Example V, the blood can be
collected in any standard blood storage bag or container, and at
the time of collection, mixed with a standard anti-coagulant
containing citrate ions and a sugar energy source such as dextrose.
For example, the CPD anti-coagulant described in Example I can be
employed, and, if desired, adenine may also be included, as
described in Example I. The container should be provided with means
for subsequently introducing an aqueous solution of
dihydroxyacetone and L-ascorbate. For example, a solution for
addition to 0.5 liters of blood can be prepared by dissolving 0.90
grams of DHA and 0.44 grams of L-ascorbic acid in 25 ml. of water,
and then subjecting the solution to sterile filtration.
* * * * *