The invention relates to the collection and processing of fluid samples in a sterile environment. In particular, but not exclusively, it relates to the collection and centrifuging of human blood samples in an operating theatre environment in order to collect platelet material for reconstructive surgery.

Platelet-rich fibrin (PRF) and platelet-rich plasma (PRP) are used in reconstructive surgery for providing a source of cytokines which stimulate bone and soft tissue healing. PRF also provides a reconstruction matrix or scaffold around which and through which cellular material can regenerate. Such surgical techniques can be used for example in the reconnection of tendon or muscle tissue to bone. The can be used in dentistry and in the treatment of sports injuries, for example.

It is essential that the PRF or PRP used during reconstruction be histocompatible with the surrounding tissue, so the platelet material is harvested from the patient's own blood to ensure perfect compatibility. The collection of platelet material can be carried out before the surgery takes place, if it is know in advance that the operation will require the use of PRP or PRF. Alternatively, the PRP or PRF material may be acquired during the operation, in which case blood must be quickly collected and processed. In this latter case, speed is of great importance in order to minimise anaesthesia and open incision exposure time.

The patient's blood is generally collected by syringe and injected into a sterile test-tube or similar sterile vessel. The platelet material can then be separated out from the blood by centrifuging the test-tube so that the platelet material is concentrated in the a layer in the test-tube. If an

anticoagulant such as sodium citrate is added, then this process results in a layer of liquid PRP. If anticoagulants are not added, then the platelet-rich layer begins to polymerise to form a platelet-rich fibrin (PRF) clot. A separated layer of platelet-poor plasma collects in the upper part of the test-tube abobe the platelet-rich layer, and the denser red corpuscles form a layer below the platelet-rich layer. Either or both of the platelet-poor or red corpuscle layers can be drawn off, again by syringe, for example, leaving the platelet-rich material in the test-tube, ready for use by the surgeon. Alternatively, the PRF or PRP material can be withdrawn from the test-tube for use by the surgeon, leaving the platelet-poor plasma and red-corpuscles behind in the test-tube. In the case of PRP, the platelet-rich material is subsequently activated by adding calcium chloride or bovine thrombin, for example, when the liquid is injected into the patient.

The operating region is maintained at a high degree of sterility in order to prevent bacterial contamination of the patient's exposed internal tissue. An aseptic area, known as the sterile field, is created for the operation. Nothing is permitted within the sterile field unless it is known to be sterile. This sterile field may for example be a relatively small zone of around a point of incision.

When the collection of PRF or PRP is performed during the surgery, the PRF or PRP must somehow be transferred into the sterile field where it can be used by the surgeon. The surgeon goes to great lengths to ensure the sterile field around the operation site remains sterile, and he or she must avoid touching any non-sterile objects, or at least change his or her gloves if they come into contact with a non-sterile object. The region outside the sterile zone must be treated as non-sterile, and it is in this region where the processing of the blood must be carried out during surgery in order to provide the required PRF or PRP material. The test-tube in which the separated platelet material is held must therefore also be regarded as non-sterile, and treated accordingly.

Prior art methods of PRP or PRF extraction have focussed on the need to perform the centrifuging or other processing of the blood sample within a sterile environment. US patent application US2007/0131612 (Duffy et al), for example, describes a prior art solution in which the centrifuging and extraction of the required fractions of a blood-sample are carried out by automated mechanical means, inside a sterile environment. This solution is complicated and requires the creation of a separate sterile environment, as well as special handling and sensor mechanisms. The present invention sets out to provide a separation assembly and method which overcome the above and other disadvantages of prior art systems and methods.

In particular, it is an object of the invention to provide a separation vessel assembly for receiving a fluid under sterile conditions and maintaining the sterile status of the fluid during a processing operation on the fluid, the separation vessel assembly comprising: a first vessel for receiving and sealably enclosing the fluid, the first vessel having a first opening, the first opening being sealable with first piercable closure means, the separation vessel assembly comprising a second vessel for sealably enclosing the first vessel, the second vessel having a second opening for introducing or removing the first vessel, the second opening being sealable with second piercable closure means, the inner and outer vessels being arranged such that, when the first vessel is enclosed within the second vessel, the fluid can be introduced into the first vessel from outside the second vessel by injection through both the first and second piercable closure means.

Using this assembly the collection and the processing of the blood sample may be carried out in the first vessel, while it remains protected and sterile inside the second vessel.

According to a variant of the separation vessel assembly of the invention, a separation vessel assembly is provided in which the first and second vessels are arranged such that, when the second piercable closure means is removed from the second vessel, the first vessel can be manually removed from the second vessel by an operator without the operator touching the second vessel. When the processed sample is ready to be handed to the surgeon, the person performing the processing can remove the second closure means from the second (non-sterile) vessel, and the surgeon can take the first (sterile) vessel out of the second vessel and into the sterile field, or zone, without touching the second (non-sterile) vessel. According to a first embodiment of the separation vessel assembly of the invention, the first vessel has an elongated shape and comprises an open proximal end and a closed distal end, the open proximal end being for sealably closing by said first piercable closure means. In this embodiment, the desired PRF, for example, can be removed from the first vessel via the proximal end of the first vessel.

According to a second embodiment of the separation vessel assembly of the invention, the first vessel has an elongated shape and comprises an open proximal end and an open distal end, the open proximal end being sealably closable by said first piercable closure means and the open distal end being sealably closable by third piercable closure means. In this embodiment, the desired PRP, for example, can be removed from the first vessel via the distal end of the first vessel.

According to another variant of the separation vessel assembly of the invention, the second piercable closing means is sealably secured to the second vessel by means of cooperating screw-threads.

According to another variant of the separation vessel assembly of the invention, the second and/or the third piercable closure means is made from an elastic material, such that the second and/or third piercable closure means can be resealably pierced by a cannula, and such that the second and/or third piercable closure means is sealably held in position at the respective open distal or proximal end by elasticity deformation of the elastic material.

According to another variant of the separation vessel assembly of the invention, the distal ends of the first and second vessels are separated by a load-transferring element for bearing forces of centrifuging at 600g.

It is also an object of the invention to provide a separation vessel kit comprising a plurality of separation vessel assemblies as described above. According to a variant of the invention, the separation vessel kit comprises at least one separation vessel assembly in which the first vessel has an elongated shape and comprises an open proximal end and a closed distal end, the open proximal end being for sealably closing by said first piercable closure means, and at least one separation vessel assembly in which the first vessel has an elongated shape and comprises an open proximal end and a open distal end, the open proximal end being sealably closable by said first piercable closure means and the open distal end being sealably closable by third piercable closure means. The advantage of this separation vessel kit variant is that the kit offers the surgeon the choice of two types of separation vessel assembly, to harvest PRF and/or PRP, for example, as required.

It is a further object of the invention to provide a method of collecting a sample of fluid in a medical operating environment, the medical operating environment having a sterile zone and a non-sterile zone, the method including processing the sample of fluid in the non-sterile zone, and transferring the processed sample to an operator working in the sterile zone, the method comprising a first step of providing a separation vessel assembly as described above, a second step of introducing the sample of fluid into the first vessel of the separation vessel assembly by injection through both the first and second piercable closure means, a third step of processing the sample of fluid in the non-sterile zone such that at least a portion of the fluid remains in the inner vessel, a fourth step of removing the second piercable closure means from the second vessel, a fifth step wherein the operator removes the first vessel from the second vessel without touching the second vessel and transfers the first vessel into the sterile zone, and a sixth step of withdrawing at least a portion of the contents of the first vessel. In this way, the first vessel is maintained under sterile conditions during processing, such as centrifuging, in a non-sterile zone.

According to a variant of the method of the invention, the sixth step comprises removing the first piercable closure means in order to withdraw the at least a portion of the contents of the first vessel. This step may be required, for example, if the first vessel contains a PRF clot or matrix which must be removed whole. According to a variant of the method of the invention, the third step comprises one or more of centrifuging, precipitation, coagulation,

sedimentation and fractionation.

According to a variant of the method of the invention, the separation vessel assembly is a separation vessel assembly in which the first vessel has an elongated shape and comprises an open proximal end and a closed distal end, the open proximal end being for sealably closing by said first piercable closure means, the fluid is blood, and the third step includes processing the blood such that a body of platelet-rich fibrin is separated out from the rest of the fluid, and wherein the sixth step comprises removing platelet-poor plasma from above the body of separated platelet-rich fibrin.

According to a variant of the method of the invention, the separation vessel assembly is a separation vessel in which the first vessel has an elongated shape and comprises an open proximal end and a open distal end, the open proximal end being sealably closable by said first piercable closure means and the open distal end being sealably closable by third piercable closure means, the fluid is blood, and the third step includes centrifuging the blood such that a layer of platelet-rich plasma is separated in the fluid, and wherein the sixth step comprises removing the separated platelet-rich plasma via the third piercable closure means.

According to a variant of the method of the invention, the third step includes centrifuging the fluid at 600g. It has been found that centrifuging at approximately 600g produces PRF material having the maximum concentration of non-lysed leukocytes.

The invention will now be described in more detail with reference to the accompanying drawings, in which:

Figure 1 shows in sectional, schematic view, a separation vessel assembly suitable for collecting PRF material, according to a first embodiment of the invention. Figure 2 shows in sectional, schematic view, a separation vessel assembly suitable for collecting PRP material, according to a second

embodiment of the invention.

Figures 3 and 4 show perspective and sectional views respectively of a separation vessel kit suitable for collecting PRF or PRP material.

It should be noted that the drawings are provided merely as an aid to understanding the invention, and should not be interpreted as limiting the definition of the invention, which is set out in the appended claims. Where the same reference signs are used in different drawings, it should be understood that these refer to the same or corresponding features.

The invention is described with reference to the example of centrifuging PRF or PRP platelet material from a sample of human blood, but it will be appreciated that the same apparatus and techniques described here can also be used for other processes where fluids may need to be processed under non-sterile conditions and then transferred to a sterile field for use. The invention may also be used in veterinary medicine, for example. Test-tubes are cited as examples of vessels to be used for carrying out the invention, but other vessels may also be used.

Figure 1 shows a cross-section of an example separation vessel assembly 1 . It comprises an inner test-tube 1 1 and an outer test-tube 10. Outer test-tube 10 is shown fitted with a screw-on piercable cap 13 which can be unscrewed and removed to allow the inner test-tube to be inserted or removed. Screw-on function is afforded by mutually cooperating corresponding thread pair 17. The screw-on piercable cap 13 is provided with a piercable membrane and a canal 15 through which a cannula can be inserted. The inner test-tube 1 1 is also provided with a piercable cap 14. In the example shown, the piercable cap 14 is made of an elastic material, such as rubber or plastics, which can simply be pushed on to the open proximal end of the inner test-tube 1 1 as shown in figure 1 , where it is held in position by its own elasticity. Shoulders 18 are provided on the face of the cap 14 facing the inner surface of the inner test- tube 1 1 . These shoulders 18 provide improved mechanical and sealing contact between the cap 14 and the walls of the tube 1 1 . As with the piercable cap on the outer test-tube, the inner piercable cap 14 also has a piercable membrane and a channel through which a cannula 16 can be introduced. The channels 15 and 16 of the inner 14 and outer 13 piercable caps are aligned so that a single cannula can be inserted through both cap channels 15 and 16, piercing both membranes, so as to introduce fluid to or remove fluid from the interior of the inner test-tube 1 1 .

The inner test-tube 1 1 and its cap 14 are fully enclosed within the outer test-tube 10 and sealed in by the outer cap 13. The inner vessel is sterilised inside and out before being sealed inside the outer vessel. As will be described later, the fluid sample can be injected into the inner vessel 1 1 using a sterile needle piercing both the inner 14 and outer 13 caps, without introducing contaminants into the inner vessel 1 1 . The outer cap can then be removed, once the sample processing is complete, for example, and the sterile inner vessel can then be withdrawn by an operator without touching the outer parts of the outer vessel. The screw cap 13 may be provided with knurls or grooves (not shown) on its outer face for easy gripping and turning. A surgical assistant may need to open the cap 13 with one hand, and this knurling allows the assistant to hold the assembly 1 in his or her hand, while turning the cap 13 with the thumb and forefinger of the same hand. The dimensions of the screw- cap 13 may also me chosen to be large enough to facilitate such one-handed removal.

When the outer cap 13 is removed from the outer test-tube 10, a part of the inner test-tube 10 and/or the inner piercable cap 14 protrudes above the end of the outer test-tube 10 so that it is possible for an operator (the surgeon, for example), to grasp the sterile inner vessel 1 1 (with its processed contents) and remove it from the outer vessel 10 without touching any of the non-sterile surfaces of the outer vessel 10.

A suitably-shaped support spacer 12 is shown moulded into the inner wall of the outer test-tube. This space 12 serves to support the bottom of the inner test-tube during high-speed centrifuging. Without this support, it is likely that the centrifuging forces, could cause the bottom of the inner test-tube to rupture.

Figure 2 shows a cross-section of another example separation vessel assembly 2, according to a second embodiment of the invention. The illustrated assembly is identical to the assembly shown in figure 1 except in that the bottom end of the inner test-tube 21 is provided with a piercable cap 24. Bottom piercable cap 24 is provided for drawing off liquid from below the require centrifuged layer. In the collection of PRP, for example, the PRP layer is between a lower layer of red blood corpuscles and an upper layer of platelet- poor plasma. The red corpuscle layer can be drawn off from underneath the PRP layer, using a syringe for example introduced through the bottom

piercable cap 24.

Inner vessel 21 is shown having a narrowed portion 29 towards its lower end. This narrowing is provided in order to provide space for fitting the bottom piercable cap 24. The piercable cap 24 may be shaped such that it rests on projection 12 during centrifuging, or the support for the inner test-tube may alternatively be afforded, as shown in figure 2, by the contact between the piercable cap 24 and the inner wall of the outer test-tube 10. Acquiring the PRP can require centrifuging at 2000g or more, so it is important that the forces are transferred between the test-tubes in a distributed fashion, such as by the resilient material of the piercable cap 24.

The outer vessel 10 of figure 2 is advantageously the same as the outer vessel 10 of figure 1 . In this way, only one type of outer vessel is required, which can be used to enclose either type, 1 1 or 21 , of inner vessel.

Figures 3 and 4 show perspective and sectional views respectively of a separation kit comprising at least one of the separation vessel assemblies 1 , 2 shown in figures 1 and 2. Line A-A in figure 4 shows the position of the section shown in figure 3. Holder 3 is provided with recesses 4, or other suitable vessel-holding geometries, into which one or more separation vessel assemblies 1 , 2, or separation vessels 10, 1 1 , 21 , can be placed such that they are held substantially upright. One or more of each type of separation vessel assembly 1 , 2 may be provided, as shown in figure 4, in order to allow the same kit to be used for either PRF or PRP collection. A PRF collection harpoon 5 may also be provided, resting or fixed in a suitable recessed of the body of the holder 3. The PRF harpoon is an aid to retrieving the PRF matrix from the inner test-tube.

An example will now be given of how the separation vessel assemblies of figures 1 and 2 can be used. In the example, reconstructive surgery is being performed on a patient, and the surgeon requires some PRF matrix material to implant into the reconstruction area, in order to stimulate regrowth of body tissues. The surgeon is working in a sterile field, using sterile tools and materials and wearing sterile surgical gloves. He asks his assistant to prepare some PRF matrix material. The assistant, who is outside the sterile field, swabs the patient with aseptic fluid and takes a sample of blood from the patient using a sterile needle. He then takes a separation vessel assembly 1 as shown in figure 1 , and, using a sterile needle or cannula, inserts the sterile cannula through the first and second piercable caps and injects the blood into the inner vessel 1 1 . The collection of the blood sample could equally be performed by the surgeon and handed to the assistant, for example. Note that the inner vessel is advantageously prepared with a vacuum inside, so that the injection of liquid into the inner vessel does need to displace any gas.

At this point, the outside of the outer vessel is non-sterile, but everything inside the outer vessel is still sterile.

The sample is then centrifuged at, for example, 600g. This figure refers to a centrifugal acceleration equivalent to 600 times the Earth's gravitational acceleration.

Table centrifuges such as are commonly used for separating PRF or PRP are unsuitable for use in a sterile surgical zone, therefore the centrifuging is usually carried out by the assistant outside the sterile zone. The separation vessel assembly 1 is centrifuged complete. In other words, the sample is centrifuged contained and sealed within the inner vessel 1 1 , which is contained and sealed within the outer vessel 10. No anticoagulant is added to the inner test-tube 1 1 , so a PRF fibrin clot polymerises in the inner test-tube 1 1 during and after centrifuging. The assistant then takes the assembly 1 out of the centrifuge, removes the cap 13 of the outer tube 10 without touching the inner tube 1 1 , and offers the assembly 1 to the surgeon, who grasps the protruding inner tube 1 1 with sterile, gloved fingers and pulls it out of the outer tube 10 without touching the outer tube 10. The outer tube 10 then remains outside the sterile zone, while the surgeon works with the inner test-tube 1 1 in the sterile zone. He removes the cap 14 from the tube 1 1 , removes the PRF matrix from the tube 1 1 (using the PRF harpoon provided with the kit, for example) and implants the PRF matrix into the reconstruction zone of the patient.

If the surgeon wishes to inject PRP into the reconstruction zone of the patient, he asks the assistant to prepare a quantity of PRP. The process is the same as the process for extracting PRF except that the assistant may use separation vessel assembly 2 (ie with an inner vessel 21 having piercable closures 14 and 24 at the top and the bottom) instead of separation vessel assembly 1 . As with the PRF collection method described above, the inner vessel is advantageously prepared with a vacuum inside.

A further difference is that an anticoagulant such as sodium citrate is present in the inner vessel 21 before the blood sample is introduced. This prevents the blood from clotting and allows a layer of platelet-rich plasma to be formed during centrifuging, between an upper layer of platelet-poor plasma and a lower layer of red corpuscles.

When the surgeon takes the inner tube 21 from the outer tube 10, he can then draw off the red blood corpuscles by means of a syringe cannula inserted through the bottom piercable cab 24. He can then separately draw off the liquid PRP layer in the same fashion. It would in theory be possible to extract the PRP layer via the top piercable cap 14, but this would be more difficult and would result in a less complete extraction of the PRP liquid.

Following the extraction of the PRP from the inner vessel 21 , the PRP can be activated and injected into the patient in known fashion. In this way, the surgeon can quickly and reliably obtain a sterile sample of PRF or PRP material for immediate surgical use, without complex sterilisation systems or additional sterile environments.

The method is not limited to the use of PRF and PRP, however, but is applicable to any situation where a sample of liquid must be centrifuged or otherwise processed in a non-sterile environment before being transferred to a sterile environment.