Method of washing blood cells and container assembly thereof

Thawed glycerolized red blood cells are washed in a system (1) of closed collapsible containers of flexible material which are positioned concentrically in a centrifuge rotor. The blood cells are held in an annular primary container (2) into which wash liquid is centrifugally fed from a c entral container (3) and from which supernatant is expressed into a central waste container (4) while the primary container is being compressed as a result of centrifugal action on an elastic body (24) in the rotor. A container assembly (1) for use in carrying out the washing comprises an annular collapsible primary container (2), a collapsible circular closed wash liquid container (3), a collapsible circular, closed waste container (4), and valve controlled conduits for passing liquid from the wash liquid container into the primary container and from the primary container into the waste container. The wash liquid container (3) and the waste container (4) are positioned one on top of the other in the circular area surrounded by the primary container (2).

TECHNICAL FIELD 
This invention relates to a method of discontinuous washing of blood cells 
and a container assembly for use in washing discrete quantities or batches 
of blood cells in a centrifuge. 
BACKGROUND OF THE INVENTION 
Washing of blood cells is required e.g. when frozen and glycerolized red 
blood cells are to be reconstituted for transfusion to a recipient. After 
thawing, the blood cells are liberated from glycerol and other undesired 
components by repeated washing steps using a wash solution. Blood cells 
which have been processed by techniques other than glycerolization and 
freezing so as to be capable of long-term storage likewise have to be 
washed free of additives before they can be transfused to a recipient. 
U.S. Pat. No. 3,326,458, U.S. Pat. No. 3,679,128, U.S. Pat. No. 3,737,096 
and U.S. Pat. No. 3,858,796 disclose examples of methods for batch washing 
of blood cells and of centrifuges and container assemblies for use in 
carrying out such washing methods. 
More particularly, U.S. Pat. No. 3,326,458 discloses batch washing of 
glycerolized red blood cells in a system of closed collapsible containers 
of flexible material which are positioned concentrically in a centrifuge 
rotor. An annular processing or primary container holds the cells to be 
washed and communicates through collapsible conduits with other 
containers, including a circular, centrally positioned wash liquid 
container and an annular waste container which is positioned radially 
outwardly of the primary container. Pinch valves are provided to control 
the flow between the primary container, on the one hand, and the wash 
liquid container and the waste container, on the other hand. 
When a batch of thawed glycerolized red blood cells held in the primary 
container is to be reconstituted, the centrifuge rotor is spun at 
appropriate speed until the red blood cells have sedimented in the 
radially outer portion of the primary container. While the rotor is 
spinning, the valve controlling the flow from the primary container into 
the waste container is opened to allow the glycerol supernatant to flow 
into the waste container. To this end, a predetermined volume of 
compressing liquid is centrifugally actuated to cause compression of the 
primary container so that an equal volume of supernatant is expressed from 
it. 
Following closing of the just-mentioned valve, the valve controlling the 
flow from the wash liquid container into the primary container is opened 
to allow wash liquid to flow under action of the centrifugal field into 
the primary container, thereby expanding it and displacing the compressing 
liquid against action of the centrifugal field. The wash liquid mixes with 
the pack or concentrate of red blood cells and is then centrifugally 
separated from the cells to form a supernatant which is subsequently 
expressed into the waste container in the manner described above with 
reference to the glycerol supernatant. 
The steps of admitting a predetermined volume of wash liquid into the 
primary container and subsequently expressing it into the waste container 
together with liberated contaminating substances are repeated until the 
red blood cells are clinically acceptable. 
SUMMARY OF THE INVENTION 
An object of the invention is to provide an improved method of batch 
washing of blood cells in a centrifuge using a system of closed 
collapsible concentric containers of flexible material and utilizing the 
centrifugal field to effect the transfer of wash liquid and supernatant 
between a primary container holding the cells, on the one hand, and wash 
liquid and waste containers, on the other hand. 
Another object of the invention is to provide an improved container 
assembly for use in washing blood cells in a centrifuge. 
In view of the foregoing and other objects, the invention provides a method 
and a container assembly as defined in the claims. 
As will be explained in greater detail below, the wash liquid is 
transferred radially outwardly from the centrally positioned wash liquid 
container to the annular primary container and then, in the form of a 
supernatant, radially inwardly, against the direction of the centrifugal 
field, from the primary container to the waste container which is likewise 
positioned centrally, the transfer being effected in both directions with 
the aid of the centrifugal field. 
To this end, an elastic body (a body of solid material which changes its 
shape and size under action of opposing forces but recovers its original 
shape when the forces are removed) is used to apply to the primary 
container a centrifugally produced force which tends to compress the 
primary container and which prevails over the head of pressure of the 
liquid in the waste container when radially inward transfer is to be 
effected but is overcome by the head of pressure of the liquid in the wash 
liquid container when radially outward transfer is to be effected. In 
order that this feature of the compressing force may be achieved, the 
centrifuge is operated at different rotational speeds in different steps 
of the washing procedure, namely, a higher speed when radially inward 
transfer is to be effected and a lower speed when radially outward 
transfer is to be effected. 
The invention will be described in greater detail below with reference to 
the accompanying drawings, in which:

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In FIGS. 1 and 2 reference numeral 1 generally designates a container 
assembly which comprises an annular primary container 2 and two circular 
secondary containers, a wash liquid container 3 and a waste container 4, 
positioned one on top of the other in the circular space enclosed by the 
primary container 1. The three containers are formed of flexible plastic 
sheet material. A flexible conduit 5 has one end thereof connected with 
the interior of the primary container 2 and is used for feeding liquid 
into the primary container and for discharging liquid therefrom. The other 
end of the conduit 5 is provided with a sterile connector 6. 
A collapsible flexible conduit 7 provides a flow path between the interiors 
of the primary container 2 and the wash liquid container 3. At the 
location where the conduit 7 is attached to the primary container 2 a 
one-way valve 8 is provided which comprises a flap of thin flexible sheet 
material attached to the inner side of the top wall of the primary 
container 2 so as to overlie the opening of the conduit 7. One end of the 
flap is free to move relative to the container wall to permit flow of 
liquid from the wash liquid container into the primary container and 
prevent flow in the opposite direction. 
The wash liquid container 3 is also provided with a flexible conduit 9 
which is used for feeding wash liquid into the container. After a 
predetermined amount of wash liquid has been introduced, the conduit is 
sealed. 
A collapsible flexible conduit 10 provides a flow path between the radially 
inner portion of the interior of the primary container 2 and the interior 
of the waste container 4. At the location where the conduit 10 is attached 
to the waste container a one-way valve 11 similar to the above-mentioned 
valve 8 is provided on the inner side of the top wall of the container to 
permit flow of liquid from the primary container into the waste container 
but prevent flow in the opposite direction. 
The container assembly 1 is made of plastic sheets, e.g. of polyvinyl or 
polyethylene, which are permanently joined by heat sealing. Suitably, the 
container assembly is formed of three circular concentric sheets A, B and 
C placed one over the other, the intermediate sheet B having a smaller 
diameter corresponding to the inner diameter of the annular primary 
container 2 and the top and bottom sheets A and C having a diameter 
corresponding to the outer diameter of the primary container. The three 
sheets are joined by heat sealing at an annular outer seam 12 and an 
annular inner seam 13 to form the annular primary container 2 and the two 
circular central containers 3 and 4 which have a common wall formed by the 
intermediate sheet B. 
In order that all of the flexible conduits may be positioned on the top 
side of the container assembly so as to be readily accessible from above, 
the top and intermediate sheets A and B are joined by heat sealing also 
over an area where the conduit 10 and the one-way valve 11 are attached to 
the waste container 4. 
FIG. 3 diagrammatically shows a centrifuge rotor adapted for use with the 
container assembly 1 of FIGS. 1 and 2 in carrying out blood cell washing 
in accordance with the invention. A similar centrifuge rotor is described 
in greater detail in WO 87/06857. 
An annular separation compartment extends about the centrifuge head along 
its periphery. The central compartment communicates with the separation 
compartment through a slot-like connecting zone. A centrifuge cone is 
driven by a program controlled motor, and fits in a hub of the centrifuge 
head. An upper ring is permanently clamped to a bowl-shaped lower portion 
of the head. An elastic diaphragm is clamped between the bowl and the 
upper ring. A transparent cover is held onto the centrifuge head by a snap 
ring. 
The centrifuge rotor has an annular outer compartment 17 adapted to receive 
and enclose the primary container 2 of the container assembly 1 and a 
circular central compartment 18 adapted to receive the wash liquid and 
waste containers 3, 4. A central opening 20 is provided in the cover 19 of 
the rotor. 
When the container assembly 1 has been positioned in the rotor compartments 
17, 18 and the rotor cover 19 has been positioned over the container 
assembly, the conduit 5 is pulled up through the cover opening 20 so as to 
be accessible from above the rotor. The loops formed by the conduits 7 and 
10 are also pulled up through the cover opening 20 and positioned in 
centrifugally actuated pinch valves 21 and 22, respectively, on the rotor 
cover. To this end, a sealing member (not shown) through which the 
conduits extend may be pulled upwardly into the cover opening 20 to seal 
off the rotor compartments. Thereupon the rotor compartments may be placed 
under overpressure or negative pressure by way of a passage 23. 
An annular elastic body 24, e.g. a rubber body, is positioned in the rotor 
and centered on the rotor axis L. The elastic body 24 forms the bottom 
wall of the annular outer rotor compartment 17 and is elastically 
deformable under action of the centrifugal field to reduce the volume of 
this rotor compartment and thereby to compress the collapsible primary 
container received therein. The deformation and resulting compressing 
action of the elastic body may be amplified or modified by means of 
radially movable weight segments 25 arranged in a ring about the inner 
periphery of the elastic body. 
A programmed-controlled motor (not shown) rotates the centrifuge rotor at 
selected speeds. 
When a batch of red blood cells is to be washed, e.g. following thawing and 
in preparation for use of the blood cells for transfusion, the container 
assembly 1 is positioned in the rotor compartments as explained above. A 
predetermined volume of wash liquid, e.g. a solution containing 0.9 
percent of NaCl and 0.2 percent of glucose, has previously been introduced 
in the wash liquid container 3 and the conduit 9 has then been sealed by 
means of a heat sealing tool. 
Moreover, the conduit 7 has been provided with a closure device, e.g. a 
pinch clamp, which can readily be removed when desired, or an internal 
flow barrier, such as shown at 16, which can be broken by bending the 
conduit. The connector 6 of the conduit 5 is made accessible from above 
the rotor and the conduits 7 and 10 are inserted in the normally closed 
pinch clamps 21 and 22, respectively. Thereupon, the closure device of the 
conduit 7 is removed or the flow barrier 16 is broken. 
FIGS. 4a to 4j diagrammatically illustrate the processing sequence 
following the insertion of the container assembly 1 in the centrifuge 
rotor. 
As an initial step (FIG. 4a) a batch of red blood cells, e.g. red blood 
cells which have previously been glycerolized and stored in frozen state 
and then thawed in preparation for reuse, is fed into the primary 
container 2 through the conduit 5. In this step the centrifugally actuated 
valves 21 and 22 are held in closed condition. Thereupon, the conduit 5 is 
sealed. 
In a second step (FIG. 4b) the centrifuge rotor is spun at a predetermined 
first speed sufficient to cause the valve 21 to open but insufficient for 
the valve 22 to open. Although the valve 21 is opened, the conduit 7 is 
still blocked to flow from the primary container 2 because the one-way 
valve 8 is closed. As a result of the rotor spinning, the red blood cells 
are sedimented in the circumferential outer portion of the primary 
container 2 and a supernatant fraction (glycerol and other substances 
having a density less than that of the red blood cells) is formed in the 
circumferential inner portion. 
The third step (FIG. 4c) comprises accelerating the rotor to a 
predetermined second, higher speed sufficient to cause the centrifugally 
actuated valve 22 to open. This speed is also sufficient to cause the 
elastic body 24 to deform under action of the centrifugal field and exert 
a pressure on the primary container 2 and thereby compress it so that the 
supernatant fraction is expressed radially inwardly through the conduit 10 
into the waste container 4. 
In the fourth step (FIG. 4d) the rotor is decelerated sufficiently to cause 
the valve 22 to close. The speed at which the valve 22 closes is 
sufficiently low to allow the elastic body 24 to retract so that the 
primary container 22 can expand, but still sufficiently high to keep the 
valve 21 open. As a consequence, wash liquid will pass through the conduit 
7 into the primary container 2 until this container has expanded to the 
limit set by the walls of the outer rotor compartment 17. 
In the fifth step (FIG. 4e) the centrifuge rotor is braked rapidly so that 
the valve 21 is also closed and the cells become suspended in the wash 
liquid that has been transferred into the primary container 2. Following 
the rapid deceleration caused by the braking, the rotor is oscillated 
about the axis of rotation L to bring about an intensive agitation of the 
cells in the wash liquid. 
In the sixth step (FIG. 4f), the rotor is again accelerated to the first 
speed so that the cells are again sedimented in the circumferential outer 
portion while a supernatant fraction consisting mainly of wash liquid and 
liberated contaminants is formed in the circumferential inner portion. 
This step is more or less identical with the second step. 
Then the third and following steps are repeated (FIGS. 4g to 4j) as many 
times, normally 3 or 4 times, as are required to make the cells clinically 
acceptable, e.g. for transfusion to a patient. 
The last quantity of wash liquid transferred into the primary container is 
left therein to serve as a suspending or carrier liquid for the blood 
cells, and finally the contents of the primary container are transferred 
to a standard transfusion bag through the conduit 5. 
As is readily appreciated, the flow pattern and container configuration 
according to the invention makes it possible to utilize substantially the 
full diameter of the centrifuge rotor for the separation, because there is 
no need for a container positioned radially outwardly of the container 
holding the cells. Moreover there is no need for solid transverse walls 
separating adjacent containers in the centrifuge rotor; such walls would 
hamper the loading of the container assembly into the centrifuge rotor and 
the removal of the container assembly from the rotor. 
FIG. 5 shows a container assembly 1 which is generally similar to that 
shown in FIGS. 1 and 2 except in that it comprises additional bag-like 
containers connected with the conduit 5. This modified container asembly 
is suitable for use in the washing of blood that has been treated 
according to the high-glycerol technique and accordingly contains about 40 
percent by weight of glycerol. In FIG. 5 reference numerals 1 to 16 
designate elements already described with reference to FIGS. 1 and 2. 
Connected to the conduit 5 are an additional wash liquid container 26 
provided with a rupturable closure 27, an empty transfusion container 28 
which has a rupturable closure 29 and a connector for a container S 
holding stored glycerolized red blood cells. The container 26 holds 
hypertonic (12 percent) saline. 
Except as described below, the container assembly 1 of FIG. 5 is used 
substantially in the same manner as the container assembly shown in FIGS. 
1 and 2. 
After the blood cell container S has been connected to the conduit 5 and 
the blood cells have been transferred with the glycerol into the primary 
container 2, the connection is closed by means of a heat sealing tool. The 
glycerolized blood cells are centrifuged with the containers 26 and 28 
positioned on top of the wash liquid container 3 in the central rotor 
compartment 18, and the glycerol supernatant is transferred into the waste 
container 4. Thereupon the centrifuge is stopped, the closure 27 is 
broken, and wash liquid held in the additional wash liquid container 26 is 
transferred into the primary container. This transfer may be effected e.g. 
under action of negative pressure in the centrifuge rotor. When the 
container 26 is emptied its connection with the conduit 5 is cut and heat 
sealed. At the same time the temporary closure device 16 of the conduit 7 
is opened. 
The blood cells suspended in the hypertonic wash liquid are then 
centrifuged and washed in the manner described above with reference to 
FIG. 4 using the wash liquid held in the wash liquid container 3. When the 
washing procedure is completed, the blood cells are suspended in the last 
quantity of wash liquid and transferred into the transfusion container 28 
after its closure 29 has been ruptured. It is also possible to replace the 
transfusion container 28 with a transfusion kit as shown in FIG. 6. 
FIG. 6 shows a blood processing kit which can conveniently be used to (1) 
separate whole blood into cells and plasma, (2) treat the cells with a 
liquid preservative, and (3) wash the thus preserved cells when they are 
to be reused. 
In FIG. 6 reference numerals 1 to 16 designate elements which have already 
been described with reference to FIGS. 1 and 2. 
Connected to the primary container 2 is a supply conduit 30 through which 
whole blood may be fed from a blood donor into the primary container. A 
branch conduit 31 is connected at one end to the conduit 10 and at the 
other end to an initially empty plasma container 32 and to a container 33 
holding a liquid preservative for blood cells, e.g. according to Meryman 
et al, Transfusion, Nov.-Dec. 1986, Vol. 26, pp. 500-505. 
A rupturable closure 34 of the conduit 31 may be opened manually by bending 
the conduit. 
A discharge conduit 36 connected to the primary container 2 includes a 
sterile coupling 37 for connection to a transfusion kit or it may be 
connected to such a kit in the production process. In the latter case the 
sterile coupling 37 is replaced with a rupturable closure. Alternatively, 
a transfusion container may be connected. 
In use of the processing kit of FIG. 6, the kit is positioned in the 
centrifuge rotor with the containers 32 and 33 placed in the central rotor 
compartment 18 on top of the wash liquid container 3. The conduit 30 is 
made accessible from above the rotor through the rotor cover opening 20 
and loops formed by the conduits 7 and 10 are inserted in the pinch valves 
21 and 22, respectively. 
Whole blood is withdrawn from a blood donor and fed through the conduit 30 
into the primary container 2 which has previously been charged with a 
suitable amount of anticoagulant, such as CPD (citrate-phosphate-dextrose) 
solution. The conduit 30 is then cut and sealed. 
The rotor is spun at a first speed such that blood cells and plasma are 
separated before the rotor is accelerated to a second speed to cause the 
centrifugally actuated valve 22 to open and to cause the elastic body 24 
to express the plasma through the conduits 10, 31 into the plasma 
container 32. 
Then the plasma container 32 is cut free by means of a heat sealing tool, 
the conduit 10 is removed from the valve 22, the closure 35 is opened, and 
the liquid preservative is transferred to the blood cells in the primary 
container 2. This transfer may be assisted by a negative pressure within 
the rotor and the rotor may be oscillated about its axis of rotation to 
agitate the cells in the liquid preservative. Thereupon, the conduit 31 is 
cut and the preserved blood is ready for storage. 
While the above-described steps are carried out, the conduits 7 and 10 are 
blocked by the temporary closures 16 and 35. 
When the preserved blood is to be reused, the processing kit, now 
comprising only the containers 2, 3, 4, is again positioned in the rotor, 
the closures 16 and 35 are opened, and washing is carried out as described 
with reference to FIG. 4.