WO2011095964A1

WO2011095964A1 - A method and apparatus for separating a liquid component from a liquid medium

Info

Publication number: WO2011095964A1
Application number: PCT/IE2011/000008
Authority: WO
Inventors: Margaret Mary Goggin; Andrew Dale Neal; David John Crimmins
Original assignee: Fuil Technologies Limited
Priority date: 2010-02-03
Filing date: 2011-02-02
Publication date: 2011-08-11

Abstract

Apparatus (1) for harvesting buffy coat from animal blood which has been centrifuged in a supply container (11). A valving system is operable in four states. Initially the valves (20) are operated in an initial state with the supply container (11) communicating with the first container (2), and blood plasma (11) is transferred to the first container (2). The valves (20) are then operated into a first state with the supply container (11) connected to the syringe (4), and the buffy coat is urged from the supply container (11) to the syringe (4). The apparatus (1) including the syringe (4) is centrifuged for stratifying residual blood plasma in the syringe (4). The valves (20) are then operated into a second state connecting the syringe (4) to the first container (2), and the blood plasma is transferred from the syringe (4) to the first container (2). The valves (20) are then operated into a third state and the buffy coat is transferred from the syringe (4) to the second container (3).

Description

"A method and apparatus for separating a liquid component from a liquid medium"

The present invention relates to apparatus for separating a specific component in liquid form from a liquid medium, and in particular, though not limited to apparatus for separating a buffy coat from blood. The invention also relates to a method for separating a specific component in liquid form from a liquid medium, and in particular, though not limited to a method for separating a buffy coat from blood.

It has been found that treating injuries, inflammations and other ailments in animals can be significantly enhanced by stem cell therapy whereby stem cell material is applied to the affected site of the animal. Ideally the stem cell therapy should be autologous stem cell therapy, whereby the stem cells are harvested from the animal being treated, as opposed to allogeneic stem cell therapy whereby the stem cells are harvested from another animal. Additionally, it is important that the stem cells are of the type which readily convert to the tissue or organ being treated. It has been found that the most adaptable stem cells are those which are harvested from the blood of the placenta and the umbilical cord attached to an animal at birth. Stem cell material is harvested from the blood of the placenta and the umbilical cord by centrifuging the blood in order to separate out the stem cell material from the rest of the blood. The stem cells reside within an area populated by white blood cells (leukocytes) and after centrifuging, the layer in which the stem cells reside is commonly referred to as a buffy coat. The buffy coat is a thin, greyish white layer which forms between the platelets and plasma at the top, and packed red cells of a haematocrite at the bottom after centrifuging.

In general, after harvesting of the buffy coat, the buffy coat is preserved by freezing for subsequent treatment of the animal from which the placenta and umbilical cord were separated at birth. Since the stem cells of the buffy coat may not be required for many years, it is essential that the buffy coat be harvested and stored in such a manner that it does not deteriorate. Accordingly, it is essential that the buffy coat be separated from the blood with minimum opportunity for contamination.

Unfortunately, many methods for harvesting buffy coat from stem cells can lead to contamination of the buffy coat material. Additionally, it is important that when harvesting the buffy coat, the harvested material should comprise buffy coat material only, and the presence of any other blood components in the harvested buffy coat should be minimised, and preferably, should be negligible. Unfortunately, it is difficult, if not impossible, with apparatus and methods known heretofore to minimise the amount and quantity of other components of blood which are harvested along with the buffy coat.

There is therefore a need for a method and apparatus which minimises the amount and quantity of other components, other than buffy coat in the harvested buffy coat after harvesting, and preferably, which reduces the amount and quantity of such other components in the harvested buffy coat to negligible amounts and quantities. There is also a need for a method and apparatus which minimises the risk of contamination of buffy coat material during harvesting thereof.

The present invention is directed towards providing such a method and apparatus.

According to the invention there is provided apparatus for separating a specific component in liquid form from a liquid medium comprising the specific component and at least one other liquid component of a density different to the density of the specific component, the apparatus comprising a first container, a second container and a third container, a communicating means communicating the first, second and third containers to each other and to an inlet port, the inlet port being adapted for coupling to a supply container comprising the liquid medium, and a valving means located in the communicating means, the valving means being selectively operable in respective ones of a first state communicating the inlet port with the third container, a second state communicating the third container with the first container, and a third state communicating the third container with the second container, so that when the valving means is in the first state, the specific component is urgeable from the supply container through the inlet port to the third container, when the valving means is in the second state any liquid component of one of a lower density and a higher density than the density of the specific component in the third container, which may have been transferred from the supply container with the specific component, is urgeable from the third container to the first container, and when the valving means is in the third state the specific component is urgeable from the third container to the second container. In one embodiment of the invention the valving means is selectively operable in an initial state communicating the inlet port with the first container, so that when the valving means is in the initial state, a liquid component of one of a lower density and a higher density than the density of the specific component of the liquid medium is urgeable from the supply container through the inlet port to the first container.

In another embodiment of the invention the apparatus is adapted for placing in a centrifuge so that the liquid in the third container is centrifugable in the centrifuge.

In another embodiment of the invention a first connector element couples the first and second containers to a second connector element, and the second connector element couples the first connector element to the inlet port and to the third container.

In a further embodiment of the invention the valving means is adapted to selectively communicate the first connector element with the first container, the second container and the second connector element.

Preferably, the valving means is adapted to selectively communicate the second connector element with the third container, the inlet port and the first connector element.

Advantageously, the valving means is adapted to communicate the second connector element with the inlet port and with the first connector element, and to communicate the first connector element with the first container when the valving means is operated in the initial state. Ideally, the valving means is adapted to isolate the second connector element from the third container and to isolate the first connector element from the second container when the valving means is operated in the initial state. In one embodiment of the invention the valving means is adapted to communicate the second connector element with the inlet port and with the third container when the valving means is operated in the first state.

Preferably, the valving means is adapted to isolate the second connector element from the first connector element when the valving means is operated in the first state. In another embodiment of the invention the valving means is adapted to

communicate the second connector element with the third container and with the first connector element, and to communicate the first connector element with the first container when the valving means is operated in the second state. Preferably, the valving means is adapted to isolate the second connector element from the inlet port and to isolate the first connector element from the second container when the valving means is operated in the second state.

Advantageously, the valving means is adapted to communicate the second connector element with the third container and with the first connector element, and to communicate the first connector element with the second container when the valving means is operated in the third state.

Ideally, the valving means is adapted to isolate the second connector element from the inlet port and to isolate the first connector element from the first container when the valving means is operated in the third state.

In one embodiment of the invention the third container comprises an inlet/outlet port. In another embodiment of the invention the third container comprises a syringe. Preferably, the syringe comprises an elongated barrel terminating at one end in the inlet/outlet port, and a plunger urgeable through the barrel for urging liquid alternately into and out of the barrel through the inlet/outlet port. Advantageously, the barrel of the syringe is of a translucent material. Ideally, the barrel of the syringe is of a transparent material.

In an alternative embodiment of the invention the third container is adapted to facilitate urging of the liquid therefrom by a peristaltic action on the third container. Preferably, the third container comprises a bag formed of a flexible film material for facilitating the peristaltic action. Advantageously, the film material of the bag of the third container comprises a translucent material. Ideally, the film material of the bag of the third container comprises a transparent material.

Preferably, the third container is of varying transverse cross-sectional area adjacent the inlet/outlet port. Advantageously, the transverse cross-sectional area of the third container reduces progressively towards the inlet/outlet port. In another embodiment of the invention the second container comprises a film material. Preferably, the film material of the second container comprises fluorinated ethylene-propylene _.(FEP).

In one embodiment of the invention the communicating means comprises a system of tubes.

In another embodiment of the invention the valving means comprises a plurality of isolating valves. Preferably, the valving means comprises an isolating valve located between the first and second connector elements and respective isolating valves located between the first connector element and the first and second containers, respectively, and between the second connector element and the inlet port and the third container, respectively.

In another embodiment of the invention the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the third container prior to urging the specific component therefrom is of a lower density than the density of the specific component. Preferably, the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container prior to urging the specific component therefrom is of a lower density than the density of the specific component.

Advantageously, some of a liquid component of one of a lower density and a higher density than the density of the specific component of the liquid medium is urged from the supply container with the specific component to the third container. Ideally, the quantity of the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is up to 10% by volume of the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid. In one embodiment of the invention the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is the liquid component of the lower density than the density of the specific component.

In another embodiment of the invention the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is the liquid component of the higher density than the density of the specific component.

Preferably, some of the liquid components of both the lower and higher densities than the density of the specific component of the liquid medium are urged from the supply container with the specific component to the third container.

In a further embodiment of the invention the apparatus is adapted for separating a buffy coat from blood. Preferably, the apparatus is adapted for separating a buffy coat from blood of an animal. Advantageously, the buffy coat is rich in stem cells. Preferably, the component of the blood of the one of the lower density and the higher density than the density of the buffy coat which is urged from the third container prior to urging of the buffy coat therefrom is blood plasma. Advantageously, the component of the blood of the one of the lower density and the higher density than the density of the buffy coat which is urged from the supply container prior to urging the buffy coat therefrom is blood plasma.

In one embodiment of the invention the component of the blood of the lower density than the density of the buffy coat which is urged from the supply container with the buffy coat to the third container is blood plasma.

In another embodiment of the invention the component of the blood of the higher density than the density of the buffy coat which is urged from the supply container with the buffy coat to the third container is red blood cells.

The invention also provides a method for separating a specific component in liquid form from a liquid medium comprising at least one other liquid component of a density different to the density of the specific component, the method comprising providing first, second and third containers, providing a communicating means communicating the first, second and third containers with each other and with an inlet port whereby the inlet port is adapted for coupling to a supply container comprising the liquid medium, and providing a valving means in the communicating means, the valving means being selectively operable in respective ones of a first state communicating the inlet port with the third container, a second state

communicating the third container with the first container, and a third state communicating the third container with the second container, the method further comprising coupling a supply container containing the liquid medium with the inlet port, operating the valving means in the first state, urging the specific component from the supply container through the inlet port to the third container, operating the valving means in the second state and urging any liquid component of one of a lower density and a higher density than the density of the specific component in the third container, which may have been transferred from the supply container with the specific component, from the third container to the first container, and operating the valving means in the third state and urging the specific component from the third container to the second container. In one embodiment of the invention the valving means is selectively operable in an initial state, and the valving means is operated in the initial state and a liquid component of one of a lower density and a higher density than the density of the specific component of the liquid medium is urged from the supply container through the inlet port to the first container prior to urging the specific component from the supply container to the third container.

In another embodiment of the invention the third container is subjected to centrifuging after the specific component has been urged into the third container from the supply container. Preferably, the third container is centrifuged so that the one of the liquid component of one of a lower density and a higher density than the density of the specific component in the third container is located adjacent an inlet/outlet port of the third container. Advantageously, the third container is subjected to centrifuging when the third container is connected to the first and second containers and to the inlet port. Ideally, the third container is subjected to centrifuging when the third container is connected to the first and second containers and to the supply container through the inlet port.

In another embodiment of the invention the supply container is subjected to centrifuging prior to urging the specific component from the supply container to the third container.

In a further embodiment of the invention the supply container is subjected to centrifuging prior to urging the one of the liquid component of one of a lower density and a higher density than the density of the specific component of the liquid medium from the supply container to the first container.

Preferably, the supply container is subjected to the centrifuging when connected to the inlet port. Advantageously, the liquid component of the one of the lower and higher densities than the density of the specific component which is urged from the third container prior to urging the specific component from the third container is a liquid component of a lower density than the density of the specific component.

Preferably, the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container prior to urging the specific component therefrom is of a lower density than the density of the specific component.

In one embodiment of the invention some of a liquid component of one of a lower density and a higher density than the density of the specific component of the liquid medium is urged from the supply container with the specific component to the third container. Preferably, the quantity of the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is up to 10% by volume of the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium.

In one embodiment of the invention the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is the liquid component of the lower density than the density of the specific component.

In one embodiment of the invention the valving means is operated in the initial state to isolate the second and third containers from the first container and from the inlet port.

In another embodiment of the invention the valving means is operated in the first state to isolate the first and second containers from the third container and the inlet port.

In a further embodiment of the invention the valving means is operated in the second state to isolate the second container and the inlet port from the first and third containers.

In a still further embodiment of the invention the valving means is operated in the third state to isolate the first container and the inlet port from the second and third containers. In one embodiment of the invention the method is adapted for separating a buffy coat from blood.

In another embodiment of the invention the method is adapted for separating a buffy coat from blood of an animal.

Preferably, the buffy coat is rich in stem cells.

Advantageously, the component of the blood of the one of the lower density and the higher density than the density of the buffy coat which is urged from the third container prior to urging of the buffy coat therefrom is blood plasma.

Preferably, the component of the blood of the one of the lower density and the higher density than the density of the buffy coat which is urged from the supply container prior to urging the buffy coat therefrom is blood plasma.

The advantages of the invention are many. Firstly, when the apparatus and the method are used for separating buffy coat from blood, contamination of the buffy coat during harvesting from the blood is minimised, due to the fact that the communicating means of the apparatus connects the inlet port, and in turn the supply container which contains the blood to the first, second and third containers in a communicating system which is effectively a closed system, and thus,

contamination of the buffy coat during transfer from the supply container to the second container is minimised. Secondly, by virtue of the fact that the third container is located in the communicating means to be centrifugable after the buffy coat has been transferred from the supply container to the third container, the buffy coat in the third container can be subjected to a further centrifuge in order to separate out any residual components of the blood from the buffy coat. Thirdly, by virtue of the fact that the valving means is adapted to connect the third container to the first container through the communicating means, any matter, such as blood plasma which has a lower density than the buffy coat can be discharged from the third container to the first container after centrifuging of the third container and prior to discharge of the buffy coat from the third container to the second container. This, thus, minimises the amount and quantity of other components of the blood appearing in the buffy coat when transferred to the second container, and indeed, any components other than the buffy coat remaining in the buffy coat once it has been transferred to the second container are negligible.

Transferring some of the blood plasma and some of the red blood cells with the buffy coat from the supply container to the third container has the advantage that when the third container is subjected to centrifuging, better stratification of the buffy coat from the blood plasma and the red blood cells is obtained than if only the buffy coat were transferred from the supply container to the third container with only small residual amounts of the blood plasma and the red blood cells in the buffy coat. It has been found that transferring up to 10% by volume of the blood plasma and 10% by volume of the red blood cells with the buffy coat from the supply container to the third container provides particularly good results. However, the quantities of the blood plasma and the red blood cells which are transferred with the buffy coat from the supply container to the third container is dependent on the quantities and proportions of the blood plasma and the red blood cells to the buffy coat. The higher the proportions of the blood plasma and the red blood cells to the buffy coat, the smaller will be the percentages of the blood plasma and the red blood cells which will be transferred with the buffy coat from the supply container to the third container. However, in cases where the proportions of the blood plasma and the red blood cells to the buffy coat is relatively low, the percentages of the blood plasma and the red blood cells transferred from the supply container to the third container may be greater than 10% by volume of the blood plasma and the red blood cells, respectively.

A further advantage of the invention is that on completion of the transfer of the buffy coat from the third container to the second container, the components of the blood are separated from each other. The buffy coat is in the second container, the blood plasma is in the first container, and the red blood cells are in the supply container and in the third container. Thus, if it is desired to use the blood plasma and the red blood cells separately from each other, since the three components of the blood are separated, the blood plasma and the red blood cells can be used without further separating being required. In which case, the valving system would be operated to communicate the third container with the supply container, and the red blood cells in the third container would then be transferred back to the supply container.

The advantages which can be achieved from the method and apparatus according to the invention when the method and apparatus are used for separating a specific component from any other liquid medium which contains at least one other component of a density different to the density of the specific component are similar to those discussed above with reference to blood. The invention will be more clearly understood from the following description of some preferred embodiments thereof, which are given by way of example only, with reference to the accompanying drawings, in which:

Fig. 1 is a schematic plan view of apparatus according to the invention for separating a specific component in liquid form from a liquid medium,

Fig. 2 is another schematic plan view of the apparatus of Fig. 1 in use,

Fig. 3 is a view similar to Fig. 1 of apparatus according to another embodiment of the invention for separating a specific component in liquid form from a liquid medium, and

Fig. 4 is a view similar to Fig. 2 of the apparatus of Fig. 3. Referring to the drawings and initially to Figs. 1 and 2, there is illustrated apparatus according to the invention, indicated generally by the reference numeral 1 , for separating a specific component in liquid form from a liquid medium, which comprises at least one other component of a density different to the density of the specific component. In this embodiment of the invention the apparatus 1 is suitable for separating a buffy coat of the type rich in stem cell material from blood, and in particular, although not limited to blood of an animal. The buffy coat in this embodiment of the invention is harvested from the placenta and umbilical cord of an animal, and is subsequently cryogenically stored for use in the treatment of injuries, inflammation and other ailments, particularly musculoskeletal of that animal subsequently through its life.

The apparatus 1 comprises a first container 2, a second container 3 and a third container. In this embodiment of the invention the third container is provided by a syringe 4. The syringe 4 comprises a barrel 5 terminating in an inlet/outlet port 6. A plunger 7 is urgeable through the barrel 5 for urging liquid into and out of the barrel 5 through the inlet/outlet port 6. A communicating means, namely, a system 8 of tubes 9 communicates the first and second containers 2 and 3 with the syringe 4, and with an inlet port 10. The inlet port 10 is adapted for coupling to a supply container 11 in which the liquid medium, namely, the animal blood is contained. In this embodiment of the invention the inlet port 10 is adapted for coupling to a supply container of the type commonly referred to as a blood bag of flexible transparent plastics film material. Such blood bags are provided with at least one inlet/outlet port, namely, an inlet/outlet port 12 of the supply container 11. Additionally, the inlet port 10 is adapted for coupling to the inlet/outlet port 12 of the supply container 11 by heat welding. The plastics film material of the supply container 11 is such as to facilitate discharging the contents thereof manually by applying a peristaltic action to the supply container 11.

The first container 2 is adapted to receive blood plasma of the blood in the supply container 11 after centrifuging of the supply container 11. The first container 2 is of a flexible transparent plastics film material, and is provided with an inlet/outlet port 14. The inlet/outlet port 14 is coupled to one of the tubes 9 of the system 8. The second container 3 is adapted for receiving the buffy coat material from the blood in the supply container 11 , and is of a flexible transparent FEP film material, which is inert to stem cells in the buffy coat material, and thus eliminates any loss of stem cells which would otherwise arise due to adherence to the inner surface of the second container 3. The second container 3 is provided with an inlet/outlet port 15 which is coupled to one of the tubes 9 of the system 8. In this embodiment of the invention the first and second containers 2 and 3 are provided as relatively flat bags of substantially square cross-section, each of which is formed by a pair of flexible panel walls of the relevant plastics film material heat sealed around their peripheral edges.

A first connector element, namely, a first three-way Y-connector 17, connects the first and second containers 2 and 3 to a second connector element, namely, a second three-way Y-connector 18, which in turn is connected to the inlet port 10 and the syringe 4. Tubes 9 of the system 8 communicate the first Y-connector 17 with the inlet port 14 of the first container 2 and with the inlet port 15 of the second container 3. Another one of the tubes 9 of the system 8 connects the first

Y-connector 17 with the second Y-connector 18. Two further tubes 9 of the system 8 connect the second Y-connector 18 to the inlet/outlet port 6 of the syringe 4 and to the inlet port 10.

A valving means comprising a valving system of five isolating valves 20 are located in the tubes 9 of the system 8 between the first and second Y-connectors 17 and 18 and also in the tubes between the first Y-connector 17 and the inlet ports 14 and 15 of the first and second containers 2 and 3, respectively, and also in the tubes 9 between the second Y-connector 18 and the inlet/outlet port 6 of the syringe 4 and the inlet port 10. The isolating valves 20 in this embodiment of the invention are manually operable valves and are operable between an isolating state isolating the first and second Y-connectors 17 and 18 from each other and the first and second Y-connectors 17 and 18 from the corresponding first and second containers 2 and 3 and the syringe 4 and the inlet port 10, respectively, and an open state

communicating the first and second Y-connectors 17 and 18 with each other and communicating the first Y-connector 17 with the first and second containers 2 and 3 and communicating the second Y-connector 18 with the syringe 4 and the inlet port 10. Alternatively, the isolating valves 20 may be replaced by clamps which would be adjustable between an isolating state tightly clamping the corresponding tube 9 and an open state with the clamp released. The first and second containers 2 and 3, the syringe 4 and the inlet port 10 are connected together by the system 8 of tubes 9 and the first and second

Y-connectors 17 and 18 so that the apparatus 1 can be placed on a jig 25 of a centrifuge (not shown) so that the liquid contents in the supply container 11 and the syringe 4 can be centrifuged. A schematic view of the jig 25 which is carried on a radial arm 26 of the centrifuge (not shown) and which extends radially from a vertically disposed spindle 28 which is rotatably mounted in the centrifuge and driven by a drive motor (not shown). The spindle 28 defines a central vertical rotational axis 29 about which the jig 25 is revolved on the arm 26 as the spindle 28 rotates. Although the spindle 28 has been illustrated as comprising only one single arm extending radially therefrom, it will be readily understood by those skilled in the art that a plurality of arms 26 will be provided extending radially from the spindle 28 equi-spaced apart circumferentially around the spindle 28, and each arm 26 will carry a jig 25.

In this embodiment of the invention the jig 25 is adapted to receive the syringe 4 and the supply container 11 with the inlet/outlet port 6 of the syringe 4 and the inlet/outlet port 12 of the supply container 11 facing inwardly towards the central rotational axis 29 of the spindle 28. Thus during centrifuging of the syringe 4 and the supply container 11 the components of greatest density are urged towards ends 30 and 31 of the syringe 4 and the supply container 1 1 , respectively. Since the plasma is less dense than the red blood cells and the buffy coat, the plasma stratifies adjacent the inlet/outlet port 6 of the syringe 4 and the inlet/outlet port 12 of the supply container 11 , while the red blood cells, being the densest material, is stratified towards the ends 30 and 31. The buffy coat is,stratified between the plasma and the red blood cells.

The use of the apparatus 1 for separating the buffy coat from the blood in the supply container 11 will now be described. Initially, the supply container 11 containing the blood is coupled to the inlet port 10 of the apparatus 1 , and the apparatus 1 and the supply container 11 are placed on the jig 25 of the centrifuge (not shown) with the inlet/outlet ports 6 and 12 of the syringe 4 and the supply container 1 1 facing inwardly towards the spindle 28. The isolating valve 20 in the tube 9 between the inlet port 10 and the second Y-connector 18 is operated into the isolating state. The centrifuge (not shown) is operated to subject the supply container 11 to centrifuging. On completion of centrifuging when the buffy coat is clearly stratified between the blood plasma which is adjacent the inlet/outlet port 12 of the supply container 11 and the red blood cells which are adjacent the end 31 of the supply container 11 , the valving system is configured into an initial state. In the initial state the isolating valves 20 are operated to communicate the first Y-connector 17 with the first container 2 and with the second Y-connector 18, and to communicate the second Y- connector 18 with the inlet port 10. In the initial state, the second container 3 is isolated from the first Y-connector 17 and the syringe 4 is isolated from the second Y-connector 18 by the corresponding isolating valves 20. Thus, in the initial state the supply container 11 is connected directly to and communicating with the first container 2. With the valving system in the initial state, blood plasma is urged from the supply container 11 to the first container 2 through the system 8 of tubes 9 by manual peristaltic action on the supply container 1 1. The manual peristaltic action on the container 1 1 continues until approximately 90% by volume of the blood plasma in the blood has been discharged from the supply container 11 to the first container 2.

The valving system is then configured into a first state with the supply container 11 communicating with the syringe 4 for discharging the buffy coat from the supply container 1 1 to the syringe 4. In the first state of the valving system the isolating valves 20 are operated to communicate the second Y-connector 18 with the syringe 4 and with the inlet port 10, and to isolate the second Y-connector 18 from the first Y-connector 17. Once the valving system has been operated into the first state, the buffy coat along with the remaining approximately 10% by volume of the blood plasma and approximately 10% by volume of the red blood cells in the blood is urged from the supply container 1 1 into the syringe 4 by manual peristaltic action on the supply container 11. As the buffy coat, the blood plasma and the red blood cells are being urged from the supply container 11 to the syringe 4, the plunger 7 of the syringe 4 is urged in a direction outwardly of the barrel 5 by the pressure of the buffy coat, blood plasma and red blood cells entering the barrel 5. Once all the buffy coat, the 10% of the blood plasma and the 10% of the red blood cells has been urged from the supply container 11 to the barrel 5 of the syringe 4, the apparatus 1 with the supply container 11 connected to the inlet port 10 is placed on the jig 25, and is again centrifuged, thereby subjecting the buffy coat, the blood plasma and the red blood cells in the barrel 5 of the syringe 4 to centrifuging. This centrifugal action stratifies the buffy coat, the blood plasma and the red blood cells in the syringe 4 so that blood plasma is stratified towards the inlet/outlet port 6 of the syringe 4 and the red blood cells are stratified towards the end 30 of the barrel 5. The buffy coat is stratified between the blood plasma and the red blood cells. The valving system is then configured into a second state. In the second state the isolating valves 20 are operated to communicate the first Y-connector 17 with the first container 2 and with the second Y-connector 18, and to communicate the second Y-connector 18 with the syringe 4. In the second state of the valving system the first Y-connector 17 is isolated from the second container 3 and the second Y-connector 18 is isolated from the inlet port 10. With the valving system in the second state, the plunger 7 of the syringe 4 is depressed into the barrel 5 for discharging blood plasma from the barrel 5 to the first container 2.

When all the blood plasma has been discharged from the barrel 5 of the syringe 4, the valving system is configured into a third state. In the third state the valves 20 are operated to communicate the first Y-connector 17 with the second container 3 and with the second Y-connector 18, and to communicate the second Y-connector 8 with the syringe 4. In the third state of the valving system, the first Y-connector 17 is isolated from the first container 2 and the second Y-connector 18 is isolated from the inlet port 10. With the valving system configured into the third state, the plunger 7 is further depressed into the barrel 5 of the syringe 4 to discharge the buffy coat in the barrel 5 to the second container 3. During depressing of the plunger 7 into the barrel 5 of the syringe 4, care is taken to avoid discharging any stratified red blood cells which are stratified adjacent the buffy coat on the side of the buffy coat facing the end 30 of the syringe 4.

With the buffy coat now safely in the second container 3, the inlet port 15 of the second container 3 is sealed by heat sealing or other suitable means. The buffy coat in the second container 3 may then be transferred into a cryogenic container having a plurality of discrete pouches whereby small quantities of the buffy coat with a cryogenic protective solution and stored in the discrete pouches for subsequent freezing and storing under cryogenic conditions.

Referring now to Figs. 3 and 4, there is illustrated apparatus according to another embodiment of the invention, indicated generally by the reference numeral 40, for separating a specific liquid component from a liquid medium comprising at least one other component of density different to that of the specific component. In this embodiment of the invention the apparatus 40 is also suitable for separating buffy coat from blood of an animal. The apparatus 40 is substantially similar to the apparatus 1 and similar components are identified by the same reference numerals. The only difference between the apparatus 40 and the apparatus 1 is that in the apparatus 40 the syringe 4 has been replaced by a third container 41 in the form of a relatively flat bag of transparent FEP film material. In this embodiment of the invention the third container 41 is formed by two panel walls 42 sealed around the peripheral edge 44 thereof by heat welding or other suitable sealing means.

However, in this embodiment of the invention the panel walls 42 are tapered so that the third container 41 tapers at 45 towards an inlet/outlet port 46, which in this case is formed by an elongated tubular portion 47 of the third container 41. The system 8 of tubes 9 is adapted for connecting the first and second containers 2 and 3, the third container 41 and the inlet port 10 so that the apparatus 40 can be placed on a suitable jig 48 of a centrifuge (not shown) for centrifuging buffy coat, blood plasma and red blood cells in the third container 41 and in the supply container 11. A schematic view of the apparatus 40 mounted on a jig 48 of a centrifuge (not shown) is illustrated in Fig. 4. The jig 48 is carried on a radial arm 49 which extends radially from a vertically extending spindle 50 of the centrifuge. The arm 49 and spindle 50 are substantially similar to the arm and spindle 26 and 28, respectively, of the centrifuge which has been described with reference to the apparatus 1 , and further description of the centrifuge should not be required.

The use of the apparatus 40 for harvesting the buffy coat into the second container 3 is similar to the use of the apparatus 1 , with the exception that the buffy coat together with the 10% by volume of the blood plasma in the blood and the 10% by volume of the red blood cells in the blood is transferred from the supply container 11 to the third container 41 after centrifuging of the supply container 11 and after approximately 90% by volume of the blood plasma in the blood has been transferred from the supply container 1 1 to the first container 2. When the buffy coat with the 10% of the blood plasma and 10% of the red blood cells has been transferred into the third container 41 , the apparatus is placed on the jig 48 of the centrifuge and the third container 41 with the buffy coat, the blood plasma and the red blood cells therein is centrifuged. On completion of centrifuging , the valving system is configured into the second state and the blood plasma is transferred from the third container 41 to the first container 2. On transfer of the blood plasma, the valving system is configured into the third state and the buffy coat is transferred from the third container 41 to the second container 3. Transfer of the blood plasma from the third container 41 to the first container 2 and transfer of the buffy coat from the third container 41 to the second container 3 is carried out by a manual peristaltic action on the third container 41. The advantage of providing the third container 41 tapering towards the inlet/outlet port 46 is that it permits the boundary between the plasma and the buffy coat to be defined with higher resolution as the transverse cross-sectional area of the tapering portion 45 decreases. Thus, as the blood plasma is being transferred from the third container 41 , as the boundary between the blood plasma and the buffy coat approaches the outlet port 46, the resolution with which the boundary can be identified is significantly increased, and thus, all the blood plasma can be discharged from the third container 41 with minimum loss of buffy coat. Similarly, when the buffy coat is being transferred from the third container 41 to the second container 3, the resolution with which the boundary between the buffy coat and the red blood cells is defined similarly increases as the boundary approaches the outlet port 46, and thus, the buffy coat can be transferred to the second container 3 without red cell material and with minimum loss of stem cells from the buffy coat.

It is envisaged that the supply container 1 1 may be provided with a tapering portion, similar to the tapering portion 45 of the third container 41 , which in the case of the supply container would taper towards the inlet/outlet port 12 of the supply container 11 in order to facilitate a more precise identification of the boundary between the plasma and buffy coat, and the boundary between the buffy coat and the red blood cells.

While the apparatus and method according to the invention have been described for separating a buffy coat from animal blood, the apparatus and the method may be used for separating any specific liquid component from a liquid medium which comprises at least one other liquid component of a different density to that of the specific component.

Additionally, while the apparatus and the method according to the invention have been described for separating buffy coat from animal blood, it will be readily apparent to those skilled in the art that the apparatus and method according to the invention may also be used for separating buffy coat from human blood, and needless to say, the animal or human blood need not necessarily be derived from the placenta and the umbilical cord of an animal or a human, the blood may be derived directly from an animal or a human.

While the valves of the valving means have been described as being provided by isolating valves, which are manually operated, it is envisaged that the valves of the valving means may be suitable for being operated by a control system, which would automatically operate the valves into the respective initial, first, second and third states sequentially. Such valves may, for example, be solenoid operated valves or motor driven valves, which could be driven by, for example, an electrically powered rotary motor or an electrically powered linear motor. While the valving means of the apparatus has been described as being operable in an initial state, and the method with respect to its use in the separation of buffy coat from blood has been described as including the initial step of urging blood plasma from the supply container to the first container, it is envisaged that the method according to the invention need not necessarily include this initial step. For example, the blood plasma or the appropriate amount of blood plasma may be discharged from the supply container prior to coupling the supply container to the inlet port of the apparatus. Alternatively, the supply container may be provided with an additional and separate container communicating with the supply container, which would permit transfer of the blood plasma or the appropriate amount of blood plasma to be discharged into the additional container, either before or after the supply container is connected to the inlet port of the apparatus. In such cases, the apparatus could be provided without the valving means being configurable into the initial state, and the valving means would be configurable into the first, second and third states only. Needless to say, where the apparatus and method are used for separating a specific component from other liquid media, in certain cases, for the same reason, the initial step of the method may be omitted, and the apparatus may be provided with a valving means which is configurable into the first, second and third states only.

Claims

1. Apparatus for separating a specific component in liquid form from a liquid medium comprising the specific component and at least one other liquid component of a density different to the density of the specific component, the apparatus comprising a first container, a second container and a third container, a

communicating means communicating the first, second and third containers to each other and to an inlet port, the inlet port being adapted for coupling to a supply container comprising the liquid medium, and a valving means located in the communicating means, the valving means being selectively operable in respective ones of a first state communicating the inlet port with the third container, a second state communicating the third container with the first container, and a third state communicating the third container with the second container, so that when the valving means is in the first state, the specific component is urgeable from the supply container through the inlet port to the third container, when the valving means is in the second state any liquid component of one of a lower density and a higher density than the density of the specific component in the third container, which may have been transferred from the supply container with the specific component, is urgeable from the third container to the first container, and when the valving means is in the third state the specific component is urgeable from the third container to the second container.

2. Apparatus as claimed in Claim 1 in which the valving means is selectively operable in an initial state communicating the inlet port with the first container, so that when the valving means is in the initial state, a liquid component of one of a lower density and a higher density than the density of the specific component of the liquid medium is urgeable from the supply container through the inlet port to the first container.

3. Apparatus as claimed in Claim 1 or 2 in which the apparatus is adapted for placing in a centrifuge so that the liquid in the third container is centrifugable in the centrifuge.

4. Apparatus as claimed in any preceding claim in which a first connector element couples the first and second containers to a second connector element, and the second connector element couples the first connector element to the inlet port and to the third container.

5. Apparatus as claimed in Claim 4 in which the valving means is adapted to selectively communicate the first connector element with the first container, the second container and the second connector element.

6. Apparatus as claimed in Claim 4 or 5 in which the valving means is adapted to selectively communicate the second connector element with the third container, the inlet port and the first connector element.

7. Apparatus as claimed in any of Claims 4 to 6 in which the valving means is adapted to communicate the second connector element with the inlet port and with the first connector element, and to communicate the first connector element with the first container when the valving means is operated in the initial state.

8. Apparatus as claimed in any of Claims 4 to 7 in which the valving means is adapted to isolate the second connector element from the third container and to isolate the first connector element from the second container when the valving means is operated in the initial state.

9. Apparatus as claimed in any of Claims 4 to 8 in which the valving means is adapted to communicate the second connector element with the inlet port and with the third container when the valving means is operated in the first state.

10. Apparatus as claimed in any of Claims 4 to 9 in which the valving means is adapted to isolate the second connector element from the first connector element when the valving means is operated in the first state.

11. Apparatus as claimed in any of Claims 4 to 10 in which the valving means is adapted to communicate the second connector element with the third container and with the first connector element, and to communicate the first connector element with the first container when the valving means is operated in the second state.

12. Apparatus as claimed in any of Claims 4 to 11 in which the valving means is adapted to isolate the second connector element from the inlet port and to isolate the first connector element from the second container when the valving means is operated in the second state.

13. Apparatus as claimed in any of Claims 4 to 12 in which the valving means is adapted to communicate the second connector element with the third container and with the first connector element, and to communicate the first connector element with the second container when the valving means is operated in the third state.

14. Apparatus as claimed in any of Claims 4 to 13 in which the valving means is adapted to isolate the second connector element from the inlet port and to isolate the first connector element from the first container when the valving means is operated in the third state.

15. Apparatus as claimed in any preceding claim in which the third container comprises an inlet/outlet port.

16. Apparatus as claimed in Claim 15 in which the third container comprises a syringe.

17. Apparatus as claimed in Claim 16 in which the syringe comprises an elongated barrel terminating at one end in the inlet/outlet port, and a plunger urgeable through the barrel for urging liquid alternately into and out of the barrel through the inlet/outlet port.

18. Apparatus as claimed in Claim 17 in which the barrel of the syringe is of a translucent material.

19. Apparatus as claimed in Claim 17 or 18 in which the barrel of the syringe is of a transparent material.

20. Apparatus as claimed in Claim 15 in which the third container is adapted to facilitate urging of the liquid therefrom by a peristaltic action on the third container.

21. Apparatus as claimed in Claim 20 in which the third container comprises a bag formed of a flexible film material for facilitating the ^"peristaltic action.

22. Apparatus as claimed in Claim 20 or 21 in which the film material of the bag of the third container comprises a translucent material.

23. Apparatus as claimed in any of Claims 20 to 22 in which the film material of the bag of the third container comprises a transparent material.

24. Apparatus as claimed in any of Claims 15 to 23 in which the third container is of varying transverse cross-sectional area adjacent the inlet/outlet port.

25. Apparatus as claimed in any of Claims 15 to 24 in which the transverse cross-sectional area of the third container reduces progressively towards the inlet/outlet port.

26. Apparatus as claimed in any preceding claim in which the second container comprises a film material.

27. Apparatus as claimed in Claim 26 in which the film material of the second container comprises fluorinated ethylene-propylene (FEP).

28. Apparatus as claimed in any preceding claim in which the communicating means comprises a system of tubes.

29. Apparatus as claimed in any preceding claim in which the valving means comprises a plurality of isolating valves.

30. Apparatus as claimed in Claim 29 in which the valving means comprises an isolating valve located between the first and second connector elements and respective isolating valves located between the first connector element and the first and second containers, respectively, and between the second connector element and the inlet port and the third container, respectively.

31. Apparatus as claimed in any preceding claim in which the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the third container prior to urging the specific component therefrom is of a lower density than the density of the specific component.

32. Apparatus as claimed in any preceding claim in which the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container prior to urging the specific component therefrom is of a lower density than the density of the specific component.

33. Apparatus as claimed in any preceding claim in which some of a liquid component of one of a lower density and a higher density than the density of the specific component of the liquid medium is urged from the supply container with the specific component to the third container.

34. Apparatus as claimed in Claim 33 in which the quantity of the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is up to 10% by volume of the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium.

35. Apparatus as claimed in Claim 33 or 34 in which the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is the liquid component of the lower density than the density of the specific component.

36. Apparatus as claimed in any of Claims 33 to 35 in which the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is the liquid component of the higher density than the density of the specific component.

37. Apparatus as claimed in any of Claims 33 to 36 in which some of the liquid components of both the lower and higher densities than the density of the specific component of the liquid medium are urged from the supply container with the specific component to the third container.

38. Apparatus as claimed in any preceding claim in which the apparatus is adapted for separating a buffy coat from blood.

39. Apparatus as claimed in any preceding claim in which the apparatus is adapted for separating a buffy coat from blood of an animal.

40. Apparatus as claimed in Claim 38 or 39 in which the buffy coat is rich in stem cells.

41. Apparatus as claimed in any of Claims 38 to 40 in which the component of the blood of the one of the lower density and the higher density than the density of the buffy coat which is urged from the third container prior to urging of the buffy coat therefrom is blood plasma.

42. Apparatus as claimed in any of Claims 38 to 41 in which the component of the blood of the one of the lower density and the higher density than the density of the buffy coat which is urged from the supply container prior to urging the buffy coat therefrom is blood plasma.

43. Apparatus as claimed in any of Claims 38 to 42 in which the component of the blood of the lower density than the density of the buffy coat which is urged from the supply container with the buffy coat to the third container is blood plasma.

44. Apparatus as claimed in any of Claims 38 to 43 in which the component of the blood of the higher density than the density of the buffy coat which is urged from the supply container with the buffy coat to the third container is red blood cells.

45. A method for separating a specific component in liquid form from a liquid medium comprising at least one other liquid component of a density different to the density of the specific component, the method comprising providing first, second and third containers, providing a communicating means communicating the first, second and third containers with each other and with an inlet port whereby the inlet port is adapted for coupling to a supply container comprising the liquid medium, and providing a valving means in the communicating means, the valving means being selectively operable in respective ones of a first state communicating the inlet port with the third container, a second state communicating the third container with the first container, and a third state communicating the third container with the second container, the method further comprising coupling a supply container containing the liquid medium with the inlet port, operating the valving means in the first state, urging the specific component from the supply container through the inlet port to the third container, operating the valving means in the second state and urging any liquid component of one of a lower density and a higher density than the density of the specific component in the third container, which may have been transferred from the supply container with the specific component, from the third container to the first container, and operating the valving means in the third state and urging the specific component from the third container to the second container.

46. A method as claimed in Claim 45 in which the valving means is selectively operable in an initial state, and the valving means is operated in the initial state and a liquid component of one of a lower density and a higher density than the density of the specific component of the liquid medium is urged from the supply container through the inlet port to the first container prior to urging the specific component from the supply container to the third container.

47. A method as claimed in Claim 45 or 46 in which the third container is subjected to centrifuging after the specific component has been urged into the third container from the supply container.

48. A method as claimed in Claim 47 in which the third container is centrifuged so that the one of the liquid component of one of a lower density and a higher density than the density of the specific component in the third container is located adjacent an inlet/outlet port of the third container.

49. A method as claimed in any of Claims 45 to 48 in which the third container is subjected to centrifuging when the third container is connected to the first and second containers and to the inlet port.

50. A method as claimed in any of Claims 45 to 49 in which the third container is subjected to centrifuging when the third container is connected to the first and second containers and to the supply container through the inlet port.

51. A method as claimed in any of Claims 45 to 50 in which the supply container is subjected to centrifuging prior to urging the specific component from the supply container to the third container.

52. A method as claimed in any of Claims 45 to 51 in which the supply container is subjected to centrifuging prior to urging the one of the liquid component of one of a lower density and a higher density than the density of the specific component of the liquid medium from the supply container to the first container.

53. A method as claimed in any of Claims 45 to 52 in which the supply container is subjected to the centrifuging when connected to the inlet port.

54. A method as claimed in any of Claims 45 to 53 in which the liquid component of the one of the lower and higher densities than the density of the specific component which is urged from the third container prior to urging the specific component from the third container is a liquid component of a lower density than the density of the specific component.

55. A method as claimed in any of Claims 45 to 54 in which the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container prior to urging the specific component therefrom is of a lower density than the density of the specific component.

56. A method as claimed in any of Claims 45 to 55 in which some of a liquid component of one of a lower density and a higher density than the density of the specific component of the liquid medium is urged from the supply container with the specific component to the third container.

57. A method as claimed in Claim 56 in which the quantity of the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is up to 10% by volume of the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium.

58. A method as claimed in Claim 56 or 57 in which the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is the liquid component of the lower density than the density of the specific component.

59. A method as claimed in any of Claims 56 to 58 in which the liquid component of the one of the lower and higher densities than the density of the specific component of the liquid medium which is urged from the supply container with the specific component to the third container is the liquid component of the higher density than the density of the specific component.

60. A method as claimed in any of Claims 56 to 59 in which some of the liquid components of both the lower and higher densities than the density of the specific component of the liquid medium are urged from the supply container with the specific component to the third container.

61. A method as claimed in any of Claims 45 to 60 in which the valving means is operated in the initial state to isolate the second and third containers from the first container and from the inlet port.

62. A method as claimed in any of Claims 45 to 61 in which the valving means is operated in the first state to isolate the first and second containers from the third container and the inlet port.

63. A method as claimed in any of Claims 45 to 62 in which the valving means is operated in the second state to isolate the second container and the inlet port from the first and third containers.

64. A method as claimed in any of Claims 45 to 63 in which the valving means is operated in the third state to isolate the first container and the inlet port from the second and third containers.

65. A method as claimed in any of Claims 45 to 64 in which the method is adapted for separating a buffy coat from blood.

66. A method as claimed in any of Claims 45 to 65 in which the method is adapted for separating a buffy coat from blood of an animal.

67. A method as claimed in Claim 65 or 66 in which the buffy coat is rich in stem cells.

68. A method as claimed in any of Claims 65 to 67 in which the component of the blood of the one of the lower density and the higher density than the density of the buffy coat which is urged from the third container prior to urging of the buffy coat therefrom is blood plasma.

69. A method as claimed in any of Claims 65 to 68 in which the component of the blood of the one of the lower density and the higher density than the density of the buffy coat which is urged from the supply container prior to urging the buffy coat therefrom is blood plasma.

70. A method as claimed in any of Claims 65 to 69 in which the component of the blood of the lower density than the density of the buffy coat which is urged from the supply container with the buffy coat to the third container is blood plasma.

71. A method as claimed in any of Claims 65 to 70 in which the component of the blood of the higher density than the density of the buffy coat which is urged from the supply container with the buffy coat to the third container is red blood cells.