Method and apparatus for storing and preparing cryopreserved blood

A bag for containing blood or blood components comprising a closed container made of material which is permeable to a cryoprotective agent, such as glycerol, which may be added to the blood or blood components directly through the permeable walls of the blood bag. The cryoprotective agent protects the blood or blood components when the blood is frozen for storage and the cryoprotective agent can thereafter be removed through the permeable walls of the container without contaminating the blood therein. An apparatus and process is also disclosed for the cryoprotective agent to be added or removed from the blood bag using electric current, hydrostatic pressure, centrifugal force or osmosis.

FIELD AND BACKGROUND OF THE INVENTION 
The present invention relates in general to the preservation of blood and, 
in particular, to a new and useful method and apparatus for storing and 
preparing cryopreserved blood particularly red blood cells. 
The supply, storage and use of blood or components thereof poses various 
and long standing problems in the field of medicine. While large 
quantities of blood or its components are always in demand, the 
requirements are sporadic and unpredictable so that large quantities must 
be stored and maintained at various locations such as blood banks and 
large hospitals. While such quantities must always be maintained and held 
ready, a standing problem exists particularly with the storage of red 
blood cells, in that the red blood cells and other blood components are 
highly perishable. One very important solution in preserving blood has 
been the development of techniques in freezing red blood cells or whole 
blood to preserve them for long periods of time. To prevent the 
destruction of the red blood cells while they are being frozen, it has 
been found necessary to add a cryopreserving agent to the blood. When the 
blood is to be used, after it has been thawed, this cryopreserving agent 
must be washed out of the blood. The usual agent used is glycerol at a 
concentration of between about 40 to 50%. With the use of such high 
amounts of glycerol, the rate of freezing or thawing has not been found to 
be critical and storage of the blood or red blood cells is at between 
-60.degree. C. to -80.degree. C. Storage can be in liquid nitrogen, liquid 
nitrogen vapor or achieved by mechanical refrigeration devices or dry ice. 
The blood has been found to be stable even with rises in the storage 
temperature so that the so preserved blood can be transported in dry ice 
to areas of consumption. 
While the blood stored in high concentrations of glycerol is relatively 
insensitive to temperature changes, the concentration of glycerol itself 
has posed a problem in that it must be somehow removed before the blood is 
used. In one example, it has been found necessary to use no less than 
seven dilution and sedimentation cycles over a period of hours in order to 
remove the glycerol from the blood or red blood cells which have been 
frozen in 40% glycerol. This process known as deglycerolization, was 
improved somewhat by the introduction of an agglomeration process by which 
the red blood cells could be sedimented without the use of a centrifuge 
device. 
An improvement in the above method of freezing blood or red blood cells 
came with the introduction of a technique using relatively low 
concentrations of glycerol. It was found that the concentration of 
glycerol could be reduced to approximately 20% which permitted 
deglycerolization in three or four sedimentation cycles in a clinical 
centrifuge with the process time reduced to one or two hours. The 
reduction in glycerol concentration, however increased the sensitivity of 
the blood to temperature changes so that the blood preserved by this 
method required storage and transportation in liquid nitrogen or liquid 
nitrogen vapor. It was found that even transient rises in the storage 
temperature led to extreme hemolysis of the red blood cells. 
Some of the problems outlined above in removing the cryoprotective agent 
from the blood have been solved by the introduction and use of cell 
washing devices which are capable of automatically processing large 
quantities of blood. The cost of such units is high, however, and is only 
economical when large quantities of blood are available to be processed. 
Additional problems in removing the cryopreserving agent from frozen blood 
or red blood cells is the possible introduction of contaminants since, at 
some point in the known processes, the blood is brought into direct 
contact with washing solutions and the like which must be supplied from 
some source external to the container in which the blood was collected and 
stored. Another problem in using frozen blood is the relatively long time 
period which is required to render the blood usable, that is 
deglycerolized. Especially when the blood is used in small institutions 
such as hospitals located at remote areas, an emergency situation 
requiring unexpected large quantities of blood cannot be treated 
effectively since the time lag required in processing the frozen blood 
would be prohibitive. Another problem existing where emergencies are 
anticipated and large quantities of blood are prepared is that the frozen 
blood once thawed and deglycerolized has a life of only about 24 hours. If 
for some reason the emergency fails to materialize, the blood must either 
be used elsewhere within the 24 hours or else it is outdated and no longer 
useful. It must also be noted that the blood must be matched to potential 
donors so that this reduces further the number of potential recipients 
which can use the thawed blood. This loss of otherwise useful blood is 
particularly onerous in view of the difficulty in obtaining and 
maintaining sufficient blood supplies throughout the United States. The 
discovery and use of a technique of thawing and deglycerolizing frozen 
bood or red blood cells in a relatively short period of time would be 
particularly useful not only in permitting the emergency use of frozen 
blood but would also enhance the inventory control of blood since frozen 
blood can be stockpiled in periods of high availability and then 
selectively used in periods of shortage. 
SUMMARY OF THE INVENTION 
The present invention is drawn to a process and apparatus for adding or 
removing the cryoprotective agent from blood preserved by freezing and 
particularly frozen red blood cells. 
A blood bag is used which has walls made of a material which is permeable 
to glycerol. Once the bag is sterilized and supplied with blood from a 
donor, the steps of glycerolizing, freezing and deglycerolizing the blood 
may be performed without any direct contact with the interior of the bag 
so that a sterile condition can always be maintained, and no conditions 
are provied to endanger the sterile condition. This may substantially 
extend the post-thaw useful life of the blood or red blood cells since the 
known post-thaw life of 24 hours is established due to the danger of 
contamination from bacterial or virol growth after this period. 
The invention is also drawn to an apparatus for processing the blood or red 
blood cells stored in the glycerol permeable bag and to a process of 
glycerolizing and deglycerolizing blood or red blood cells so stored. It 
has been found that glycerolization and deglycerolization through the 
permeable wall of the blood bag can be achieved either by electoosmotic 
transfer of the glycerol into or out of the bag or by applying a 
hydrostatic pressure which drives fluid through the bag carrying glycerol 
into or out of the bag, or by using a centrifugal force to drive fluid 
through the bag thereby carrying glycerol into or out of the bag, or an 
osmotic gradient which would drive fluid through the bag and carry 
glycerol into or out of the bag. The characteristics of the material used 
for the blood bag walls, in addition to their being highly permeable to 
glycerol, are that the material be resistant to fracture in its frozen 
state, it be flexible, sterilizable, preferably transparent for the visual 
inspection of the blood or blood cell condition and that it have minimal 
leaching of plasticizers. These criteria are met by several materials for 
example the polycarbonate membrane developed for the NIH used in 
hemodialysis and manufactured by American Membrane, Inc. Membranes can now 
be manufactured both highly permeable and sufficiently strong that the 
membrane material itself could be used to form the blood bag. For example, 
a prototype blood bag made from polycarbonate membrane and filled with 
blood (300 ml) was dropped 4 ft. to the floor several times without 
breaking the bag. However, it may also be found useful to seal the 
polycarbonate membrane directly to an electroosmosis membrane. 
Electroosmosis membranes are generally about 0.5 mm thick with a nylon 
backing so that they can withstand severe trauma. With either blood bag, 
the blood or red blood cells should only come in contact with a non-ionic 
membrane with a blood compatibility similar to the polycarbonate or 
Cuprophane membranes used for example during hemodialysis. 
Polycarbonate membranes of the described type are freezable to -80.degree. 
C. in 4 moles of glycerol without cracking. The bags are also highly 
permeable to glycerol and have high tear resistance and can also be 
heat-sealed for the convenient formation of the blood bags. The blood bags 
may be formed, for example, with one or two ports to permit the connection 
of an infusion set. Integrated tubing may also be provided to permit 
collection of blood from a donor and for the transfer of bood to satellite 
packs and the like. This permits a system of collection, freezing with 
glycerolization, storage, deglycerolization and readministration using a 
single bag system. 
Since the membranes used to form the bags are also usually highly permeable 
to water, to improve handling characteristics, it has been found 
advantageous to place the membrane bag into an additional plastic 
container which is not permeable to water. The additional plastic bag 
would, of course, be removed during deglycerolization. 
Examples of the use of electric fields on blood samples concerning 
electrodialysis of blood can be found in the following articles: 
Bier, M., Brucker, G. C. and Roy, H. E., "Blood Electrolysis", Trans. Am. 
Soc. Artif. Int. Organs, 13:227 (1967); 
Smith, A. L., Berkowitz, H. D. and Bluemle, L. W., "Electrodialysis of 
Blood: Evaluation of a High Capacity Unit", Trans. Am. Soc. Artif. Int. 
Organs 10:273 (1964); 
Adachi, R., "The Studies on the Jikei Electrodialyzer", Report VII, Acta 
Aurol., 7:274 (1961); 
Berkowitz, H. and Bluemle, L. W., "Electrodialysis of Blood: Some Problems 
and Approaches to Their Solution", Trans. Am. Soc. Artif. Int. Organs, 
9:97 (1963). 
From these studies, it has been found that the electrodialysis of blood 
does not cause direct damage to the corpuscular elements of the blood such 
as the red blood cells. The reason for this appears to be the high 
electrical resistance of the cell walls of these elements which prevents 
the passage of electrical current therethrough. There has also been found 
to be no loss of biological activity of the proteins exposed to the 
electrical current as demonstrated in the article "Preparative 
Electrophoresis Without Supporting Media", Bier, M., Electrophoresis, 
Academic Press Inc., New York (1959). 
Accordingly, an object of the present invention is to provide a bag for 
containing blood or blood components and in particular red blood cells 
comprising a closed container having at least one port for the entry and 
exit of blood and at least one wall portion which is permeable to a 
cryoprotective agent whereby the cryoprotective agent can be removed or 
added to the blood through said at least one wall portion without removing 
the blood from the container or breaking sterility. 
Another object of the present invention is to provide a process for 
removing or adding a crypoprotective agent to blood or blood components 
which is to be or has been frozen, providing a container for the blood and 
cryoprotective agent mixture having at least one wall which is permeable 
to the cryoprotective agent, and exposing the container to an electric 
field for causing the active movement of the cryoprotective agent through 
the wall portion and out of or into the container and blood mixture. 
A still further object of the present invention is to provide a process for 
adding or removing a cryoprotective agent from blood or blood components 
which is to be or has been frozen in a mixture with the cryoprotective 
agent comprising, providing a container for the blood and cryoprotective 
agent mixture having at least one wall portion which is permeable to the 
cryoprotective agent, and providing a hydrostatic pressure difference 
across the permeable wall for forcing the cryoprotective agent through the 
wall portion and out of, or into said container. 
A still further object of the present invention is to provide a process for 
adding or removing a cryoprotective agent from blood or blood components 
which is to be or has been frozen in a mixture with the cryoprotective 
agent comprising, providing a container for the blood and cryoprotective 
agent mixture having at least one wall portion which is permeable to the 
cryoprotective agent, and providing a centrifugal force to at least one 
side of the container for forcing the cryoprotective agent through the 
wall portion and into or out of said container. 
A still further object of the present invention is to provide a process for 
adding or removing a cryoprotective agent from blood or blood components 
which is to be or has been frozen in a mixture with the cryoprotective 
agent mixture having at least one wall portion which is permeable to the 
cryoprotective agent, and providing osmotic pressure to at least one side 
of the container for forcing the cryoprotective agent through the wall 
portion and into or out of said container. 
Another object of the present invention is to provide an apparatus for 
removing, or adding a cryoprotective agent to blood which has been or is 
to be frozen in a mixture with the cryoprotective agent comprising, a 
housing having a blood mixture chamber adapted for containing the mixture 
of blood and cryoprotective agent in the blood bag and a wash solution 
chamber, a partition in said housing separating said blood mixture chamber 
from said wash solution chamber having at least one wall portion which is 
permeable to the cryoprotective agent but not to at least one component of 
the blood, wash solution supply and discharge means connected to said wash 
solution chamber for renewing a supply of wash solution therein, and 
cryoprotective agent transport means connected across said wall portion 
for forcing the cryoprotective agent therethrough and out of or into said 
blood mixture chamber. 
A still other object of the present invention is to provide an apparatus 
for removing or adding a cryoprotective agent from blood wherein the 
transport means for forcing the cryoprotective agent through the wall 
portion comprises an electrode positioned on either side of said wall 
portion and electric field means connected to said electrodes for 
establishing an electric field therebetween and across said wall portion 
to force the cryoprotective agent thereacross by electro-osmosis. 
A still further object of the present invention is to provide an apparatus 
for adding a cryoprotective agent, or removing the same from blood or 
blood components wherein the cryoprotective agent transport means 
comprises means for establishing hydrostatic pressure on one side of said 
wall portion for forcing the cryoprotective agent into or out of the blood 
bag. 
A still further object of the present invention is to provide an apparatus 
for a cryoprotective agent to be added or removed from blood or blood 
components wherein the cryoprotective agent transport means comprises 
means for establishing a centrifugal force on one side of said wall 
portion for forcing the cryoprotective agent into or out of the blood bag. 
A still further object of the present invention is to provide a blood bag 
design and apparatus therefor to remove a cryoprotective agent from the 
blood or add the agent to the blood which is simple in design, rugged in 
construction and economical to manufacture. 
The various features of novelty which characterize the invention are 
pointed out with particularity in the claims annexed to and forming a part 
of this disclosure. For a better understanding of the invention, its 
operating advantages and specific objects attained by its uses, reference 
is made to the accompanying drawings and descriptive matter in which 
preferred embodiments of the invention are illustrated.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Referring now to the figures in particular, the invention embodied therein, 
FIG. 1 comprises a blood bag generally designated 10 for containing whole 
blood or components thereof, particularly red blood cells. In accordance 
with the invention, the blood or red blood cells are mixed with a 
cryoprotective agent which is usually glycerol solution. The entire bag is 
then quick-frozen using, for example, liquid nitrogen or mechanical 
freezing units which is a known technique for preserving and storing the 
blood or blood components for future use. In accordance with the 
invention, the blood bag includes at least one wall portion which is 
permeable to the cryoprotective agent, in this case, glycerol. While only 
a portion of the bag 10 need be permeable, it is preferable to have the 
entire bag wall 11 which comes into contact with the blood made of the 
glycerol permeable material. An example of material which is permeable to 
glycerol while being capable of retaining red blood cells and excluding 
contaminants such as bacteria and the like is a polycarbonate membrane for 
hemodialysis manufactured by American Membrane, Inc. While the wall 11 
could itself form the bag, it is advantageous to seal the permeable wall 
11 to a reinforcing membrane 12 which may include nylon threads extending 
therethrough. An example of material which can be used for this 
reinforcing wall is a suplonated polystyrene electro-osmosis membrane. 
Since the membranes 11 and 12 are also permeable to water, an outer 
plastic container or bag 14 may be provided into which the blood bag 
comprising walls 11 and 12 may be placed for storage and handling. When 
the cryoprotective agent is to be removed from bag 10 in accordance with 
the invention, however, this outer impermeable bag 14 is removed. The 
reinforcing wall and outer bag 12 and 14 are shown in section but where 
such layers are used, it is understood that they totally encompass the 
inner permeable wall 11. 
The blood bag 10 is also advantageously provided with a port fitting 16 
which is adapted for receiving blood from a donor in known fashion, for 
receiving the cryoprotective agent and, when the blood is to be dispensed, 
for supplying the blood to a recipient. A second port fitting 18 may be 
provided which may include a plurality of sectional chambers 20 which may 
be filled with blood from bag 10 and, in the frozen state, removed one at 
a time for typing purposes. The blood contained in bag 10 can therefore be 
typed for certain factors without invading the body of bag 10 and also 
without requiring the blood to be thawed. 
With reference to FIG. 2, the principles of electroosmosis as used in 
accordance with the invention will be described. Electroosmosis is the 
process by which a solution can be transferred through convection as a 
result of an electric current flow. Ionic polymer membranes notably have 
high electro-osmotic activity. The electric current applied across such a 
membrane travels primarily by counter ions. Molecular collisions between 
these counter ions and the solution, drive the solvent in the direction of 
the electric current. If the solvent is bound directly to these counter 
ions, this will increase the total solvent transfer through the membrane. 
Therefore, if any non-electrolytes are present and permeable to the 
membrane, they will be transferred through convection with the solvent. 
Electrolytes which are present, however, will not be transferred as a 
result of so-called Donnan exclusion from the membrane. Electroosmosis 
thus leads to an efficient and active separation of non-electrolytes from 
electrolytes. 
Turning to FIG. 2, a two-component chamber 22 is shown having a wash 
solution compartment 24 and a blood mixture compartment 26 divided by a 
permeable membrane 28. Electrodes 30 and 32 are provided in the respective 
compartments and energized with a programmable current supply. It has been 
found that the use of alternating current rather than direct current is 
preferable since direct current leads to concentration polarization at the 
solution-membrane interface, that is the region of the respective 
compartments immediately adjacent the membrane 28. 
The use of alternating current at selected frequencies removes the 
polarization concentration each time the current reverses its direction. 
Alternating current alos permits the use of reversible electrodes since 
depletion during one cycle of the current is replenished during the 
following cycle. The use of reversible electrodes eliminates pH changes 
and the evolution of gas. While the embodiment of the invention shown in 
FIG. 2 can be used for deglycerolizing blood in chamber 26, a problem of 
dehydration arises since the flow of water out of the blood in chamber 26 
is not compensated. An electrolyte or wash solution supply means 34 is 
connected through a line 27 to the wash solution or electrolyte chamber 24 
and continually drained through a drain 36. Glycerol and water forced 
through membrane 28 is thus continually washed from the chamber 24. 
Chamber 26 containing the blood mixture is analogous to an electrolyte 
plus non-electrolyte mixture chamber. The blood fluid can be maintained by 
a feed solution supply 23. For glycerolizing the blood, supply 34 can 
provide the agent rather than wash it away. 
Details of the electro-osmosis process when applied generally to the 
transfer of non-electrolytes is disclosed in an article entitled "Rapid 
Filtration of Non-Electrolytes by Electro-osmosis", A. Zelman, D. Walsh, 
H. Wayt and D. Gisser (1978). 
Turning to FIG. 3, the problem of dehydration of the blood or blood 
components is solved by providing a three-compartment chamber 50 having 
two compartments 40 and 41 for containing the wash or electrolyte solution 
each supplied with electrolyte or wash solution from an apparatus similar 
to wash solution supply means 34 through lines 42 and 43. The solutions 
are continually drained through drain lines 44 and 45. First and second 
electrodes 47 and 49, respectively, are provided in the compartments 40 
and 41 and can be supplied with electricity through their terminals by a 
programmable current supply. Between compartments 40 and 41 is a central 
blood mixture compartment 52 which is defined between first and second 
membranes 54 amd 56, respectively. 
According to one method of the invention, the two membranes 54 and 56 may 
be of the same ionic character so that dehydration of the blood within 
compartment 52 is avoided. FIG. 3 shows the use of a pair of cation 
exchange membranes 54 and 56. When the current supplied to the electrodes 
is such to make electrode 47 an anode and electrode 49 a cathode, the 
electric current flows from electrode 47 through the two cationic 
membranes 54 and 56 and to the electrode 49. Concurrently with this 
current flow, a total volume flux or flow of equal magnitude is 
established across member 54 from compartment 40 to compartment 52 and 
across membrane 56 from compartment 52 to compartment 41. Since the 
glycerol will follow with the current, glycerol or any other 
cryoprotective agent in the blood contained in compartment 52 will move 
from that compartment into the solution wash compartment 41 whereas water 
alone will flow from compartment 40 into compartment 52. 
When the current is reversed, making electrode 47 a cathode and electrode 
49 an anode, current will flow to the left as seen in FIG. 3 across the 
membranes 54 and 56 and again glycerol will leave compartment 52 and enter 
compartment 40 while an equal volume of water will enter compartment 52 
through membrane 56. 
According to another object of the invention, the electrodes 47 and 49 may 
be excluded or not energized and the function replaced by hydrostatic 
pressure, centrifugal force or osmotic pressure means to force fluid 
through the blood bag. Either of these means alone can be relied on to 
force water from compartment 41 to the blood mixture compartment 52 and, 
to maintaining equilibrium, to force glycerol from compartment 52 to 
compartment 40. Simultaneously with this application of pressure, 
compartment 40 is continually washed of glycerol through the lines 42 and 
44. 
Turning to FIG. 4, an alternate method of removing glycerol or other 
cryoprotective agent from the cryopreserved blood is shown. In this 
embodiment of the invention, a three-component chamber 60 is provided 
which has electrolyte or wash solution compartment 70 and 71 which are 
supplied with wash solutiions through lines 72 and 73 which solution is 
continuously drained through drain lines 74, 75. A pair of electrodes 77, 
78 are provided in these compartments and connected to a programmable 
current supply for supplying a current across membranes 84 and 86 which 
define therebetween a blood mixture compartment 82. In accordance with 
this embodiment of the invention, the membrane 84 is a cation exchange 
membrane and the membrane 86 is an anion exchange membrane. The wash or 
electrolyte solutions supplied to compartments 70 and 71 contain, for 
example, NaCl. When electric current passes from left to right as shown in 
FIG. 4 so that electrode 77 acts as anode and electrode 78 acts as 
cathode, Na ions being positive will pass into compartment 82 from the 
left-hand compartment 70 and C1 ions being negative will pass from the 
right-hand compartment 71 into the central compartment 82. This will cause 
a forced change in the concentration gradient between the red blood cell 
and its external solution. The cell will shrink forcing glycerol out of 
the red blood cell into the solution. 
When the current is reversed making electrode 77 the cathode and electrode 
78 the anode, the negative C1 ions will leave compartment 82 through the 
anode membrane 86 and positive Na ions will leave compartment 82 through 
the cathode membrane 84. During this half cycle, water and glycerol will 
be removed from the extra cellular fluid of the blood in compartment 82. 
The lowering of centration in the fluid surrounding the red blood cell 
causes the cells to swell. 
If the second cycle above described is permitted to proceed until the 
removal of salt from the water causes the red blood cells to swell, and 
the next cycle adds sufficient salt to compartment 82 to cause the cells 
to shrink, pumping of the glycerol from the red blood cells will occur. By 
ending the cycle with the same volume as originally contained in chamber 
82, one is assured of a proper electrolyte balance. 
Since the frequency of the alternating current as well as the magnitude 
thereof can be easily adjusted through a programmable current supply, the 
rate of glycerol addition or removal can be completely automated in any of 
the methods illustrated in FIGS. 3 or 4. 
It should be noted that glycerol should not be removed too quickly in the 
initial stages of deglycerolization since a danger exists that the red 
blood cells will swell and hemolyze. Thus, during the beginning stages of 
deglycerolization using the inventive method and apparatus, the current is 
supplied at low levels and gradually increases as the glycerol 
concentration of the cells decreases. Frequencies of between 0.001 to 0.1 
Hz are useful in practicing the invention and the magnitude of current 
should be variable in time between about 1-75 ma/cm.sup.2. 
Turning to FIG. 5, additional apparatus in accordance with the invention 
are shown which comprise a five-compartment chamber generally designated 
90 made of two Plexiglass plates 96, 96 each having an electrode 94 
thereon separated by five rubber spacers 92. The electrode plates 94 used 
in an experiment conducted in accordance with the invention had dimensions 
of 21 cm by 10 cm and the assembly was bolted together to form the five 
compartments. Between the five spacers 92 were positioned two 
electrodialysis membranes 88, 88, and two electro-osmosis membranes 98, 98 
which, together defined two electrode wash compartments 110, two dialysate 
compartments 120 and one blood bag compartment 130 into which was 
positioned a blood bag 100 having a wall made of polycarbonate membrane. 
Wash solution in the form of a dialysate was supplied from wash solution 
system or means 80 through a line 97 to each of the dialysate and 
electrode wash compartments 110 and 120. This wash solution was 
continuously removed through a drain line 99. The electrodes were 
energized by programmable current source 81. For additional details 
concerning the electrodialysis technique, the types of dialysates used and 
the materials for forming the electrodialysis membranes see the article 
"Blood Electrodialysis" cited above. In compartments 110 and 120 isotonic 
NaAC (sodium acetate) was circulated while compartments 130 contained 
glycerolized blood in bag 100. Alternating electric current was then 
applied to the electrode 94. The frequencies used were 0.0017 Hz, 0.0033 
Hz, 0.0067 Hz and 0.033 Hz. Current densities were adjustable from 0 to 50 
ma/cm.sup.2. 
The central compartment solution containing blood had a glycerol 
concentration of 3.54 M to start. At the end of the experiment the blood 
was analyzed for glycerol using osmometry. The difference in the 
osmolality between the blood before and after deglycerolization gave the 
number of moles of glycerol removed from the blood. 
Glycerol transfer was found to vary both with frequency and current 
density. For all frequencies and current densities tested, the total 
glycerol transfer remained greater than that which would be caused by 
diffusion alone. 
The results are summarized in Tables I - III. 
Table I represents the data on the % deglycerolization as a function of 
time for 23.8 ma/cm.sup.2 and 0.0067 Hz AC. Each line in these tables 
represents a new blood sample, therefore there is biological variability 
in this data as well as experimental error. 
TABLE I 
______________________________________ 
time .DELTA.osm 
n.sub.t in 
.DELTA.n.sub.t 
% deglycercol- 
(min) mOsm (moles) (moles) ization 
______________________________________ 
0 0 .460 0.000 0.0 
5 958 .460 .144 31.3 
10 771 .496 .131 26.4 
15 1167 .460 .175 38.0 
______________________________________ 
.DELTA.osm represents change in osomolality of blood sample 
n.sub.t in represents total number of moles of glycerol initially 
.DELTA.n.sub.t represents change in the total number of moles of glycerol 
 
Table II represents the data on the % deglycerolization as a function of 
current density for different switching frequencies. 
TABLE II 
______________________________________ 
Current 
Freq density .DELTA.osm 
n.sub.t in 
.DELTA.n.sub.t 
% deglycer- -(Hz) (ma/cm.sup.2) (mOsm 
) (moles) (moles) olization 
______________________________________ 
0 86 1.240 .030 2.4 
14 1304 .425 .156 36.8 
.0017 
24 2564 .425 .188 44.2 
34 1620 .708 .324 45.8 
44 1340 .708 .268 44.9 
0 86 1.24 .030 2.4 
.033 24 381 1.77 .191 10.8 
48 172.5 2.00 .097 4.9 
______________________________________ 
Table III represents the data on the % deglycerolization as a function of 
frequency. 
TABLE III 
______________________________________ 
Freq. .DELTA.osm 
n.sub.t in 
.DELTA.n.sub.t 
% deglycer- 
(Hz) (mOsm) (moles) (moles) 
olization 
______________________________________ 
0.0017 1564 .425 .188 44.2 
0.0033 993 .425 .119 28.0 
0.0067 959 .460 .125 27.1 
0.033 381 1.77 .191 10.18 
______________________________________ 
Since the wash solution from means 80 never comes into direct contact with 
the blood in blood bag 100, it need not be subject to stringent sterile 
conditions which substantially reduces the costs thereof so that it can 
economically be continually washed through the chambers 110 and 120. 
In FIG. 6, a plurality of devices similar to the arrangement shown in FIG. 
5 are assembled in series. The blood bags 100 are flanked by chambers 201, 
202, 203, and 204 which can be supplied with washing solution from washing 
solution system 208. A pair of electrodes 209 and 210 are disposed on 
opposite ends of the respective end chambers 201 and 204 and supplied with 
programmed current from a programmed current source 211. Only a single 
programmed current source 211 need be provided which forms the 
cryoprotective agent forcing means to treat the plurality of blood bags 
100 in series. It is understood that the means 211 can be replaced by 
hydrostatic pressure means or osmotic pressure means or the entire 
arrangement can be mounted to impart a centrifugal force on the 
arrangement to move the cryoprotective agent in accordance with the 
invention. 
FIG. 7 shows a simplified form of an apparatus used to practice the 
invention which comprises a carousel 300 for supporting one or more blood 
bags 100. Carousel 300 includes an inner chamber 301 and an outer chamber 
302. Carousel 300 is mounted for rotation on shaft 303 and rotated by 
motor means 304 in known fashion. Inner chamber 301 can be supplied with a 
washing solution or cryoprotective agent solution depending on whether the 
bags 100 are to be treated with the cryoprotective agent or cleansed of 
the cryoprotective agent. When motor means 304 rotates carousel 300, 
centrifugal force is produced forcing the fluid from chamber 301 into and 
through the bags 100 and into the outer chamber 302. 
Although frozen, deglycerolized red cells are now generally available in 
large urban hospitals and through regional blood centers, because of the 
high cost of the procedure, this product is generally used only for 
special applications. Frozen red cells are generally not available in 
rural communities, third world nations, or in small military installations 
because of the technical demands of the procedure. The present invention 
offers the possibility of a system requiring minimum technician 
involvement, fully automated processing equipment, a system in which the 
cells remain within a single container throughout the entire sequence of 
glycerolizing, freezing, deglycerolizing and administration, and a system 
in which the processing bag is never entered, thus permitting an extension 
of the restrictive 24-hour outdating period. 
The introduction of a method of storing cells at -20.degree. C. eliminates 
the need for expensive and specialized storage facilities. Only the cost 
and clumsiness of deglycerolization stands in the way of cost-effective 
use of frozen cells for other than special applications. The inventive 
method provides a rapid, safe, effective and economical deglycerolizing 
procedure. The electro-osmotic preparation of blood cells of the invention 
is especially suited for automation. Thus, less technical expertise will 
be required and there will be less chance for error. For these reasons, 
cryogenically preserved cells can be made available to rural regions. 
The number of units to be processed simultaneously is essentially without 
limit and it is unimportant how many patients receive these units. 
In emergency situations several units of blood may have to be processed 
simultaneously for a single individual or several units may have to be 
processed simultaneously for several individuals. Present methods of cell 
washing from a single bowl are laborious, slow and in principle hazardous 
due to the possibility of contamination. The electro-osmotic processing of 
frozen cells is intrinsically simple in these situations. The blood 
processor can be designed in stacks so that the current passes through 
each blood bag simultaneously. Each bag is self-contained, sterile and can 
be washed in series with other bags with no possibility of cross 
contamination. 
While specific embodiments of the invention have been shown and described 
in detail to illustrate the application of the principles of the 
invention, it will be understood that the invention may be embodied 
otherwise without departing from such principles.