WO2020026008A1

WO2020026008A1 - Separation kit for the extraction of platelet-rich plasma and procedure for the use of said kit

Info

Publication number: WO2020026008A1
Application number: PCT/IB2018/056421
Authority: WO
Inventors: Dario VETTORAZZO; Gabriele ANTONINI
Original assignee: VETTORAZZO, Marco
Priority date: 2018-07-30
Filing date: 2018-08-24
Publication date: 2020-02-06
Also published as: IT201800007617A1

Abstract

Fractionation kit for the separation and extraction of autologous serum and platelet- rich plasma from a patient's blood sample, comprising a first separator (1) and a second separator (2), each comprising a receptacle (100, 200) and a moveable closure element (10, 20) inside them. The receptacle (200) of the second separator (2) comprises a concave bottom with a collection chamber (202), that is, a further portion at the bottom for collecting the platelet material which accumulates on the bottom during a centrifuge of enriched plasma obtained from a blood sample contained in the receptacle (200).

Description

SEPARATION KIT FOR THE EXTRACTION OF PLATELET-RICH PLASMA

AND PROCEDURE FOR THE USE OF SAID KIT

DESCRIPTION

The present patent relates to test tubes for biological or chemical fluids and in particular concerns a new separation kit or fractionation kit for the extraction of platelet-rich plasma (PRP) and the method for its use.

Centrifuge tubes are known in the prior art, commonly comprising a cylindrical receptacle, formed by a closed base, a cylindrical wall and an upper opening, with a cap to close the upper opening of the receptacle.

Normally, the receptacle comprises a port to insert syringe needles, so as to allow the insertion or withdrawal of biological samples into or from the receptacle.

This port is usually closed by a self-sealing membrane or other device that prevents the accidental leakage of the contained product.

Centrifuge tubes are commonly used, for example, to separate components of a subject's blood sample.

The use of plasma in medical procedures is well known. The plasma is obtained by the centrifugation of a blood sample.

When a blood sample, contained in a test tube, is centrifuged for a certain period of time at a certain speed, the blood sample is separated into its components and in particular into a layer of red blood cells, which are deposited at the bottom of the tube, a yellow layer that separates blood cells from the plasma and which contains a high concentration of platelets, and finally a layer of plasma, the most liquid fraction that forms the uppermost layer.

To take only the plasma, a syringe needle is then inserted into the tube to aspirate only the supernatant part.

If the objective is to also take the platelets, the needle must be inserted further into the test tube so as to only aspirate the platelet part.

To make these operations easier and more precise, the prior art comprises specially shaped tubes. Document US7976796 relates to a centrifuge tube, comprising a cylindrical receptacle having a flat bottom and a cylindrical wall and an upper cap with a port. A flexible tube connects the port of the cap with the inside of the receptacle.

To ensure the water tightness of the inside of the receptacle, at the end of said flexible tube which is in the receptacle a diaphragm is mounted which can slide inside the cylindrical receptacle in adherence to the cylindrical wall.

Said end of the tube extends through the diaphragm and then communicates with the inside of the centrifuge tube in the space between the diaphragm and the flat bottom. Said diaphragm is initially positioned near the bottom of the tube.

To insert a biological sample inside the receptacle, it is possible to inject it by means of this tube. As the biological sample is introduced into the space between the bottom and the diaphragm, the latter rises, ensuring the complete water tightness of that space and further ensuring that the biological sample contained therein does not come in contact with external agents in any way.

The centrifuge tube can then be placed in a centrifuge, where the separation of the components takes place. The end of the sampling tube is then in contact with the supernatant of the sample, consisting essentially of plasma.

As the fluid is removed from the inside of the tube, the atmospheric pressure acts on the diaphragm pushing it downwards.

Once the interface between the plasma and the platelet layer has been reached, the user must proceed with the aspiration, again by means of the tube.

One drawback of such systems is that, when the diaphragm is in the raised position, because the underlying space has been filled with the biological sample, the tube flexes and bends in the remaining space, between the diaphragm itself and the cap, and said bends can ruin the tube and can also hinder the injection and aspiration of the biological sample.

Document US2002/0185457 relates to a centrifuge tube comprising a cylindrical receptacle with a pointed or tapered bottom, a cylindrical wall, and a cap to close the upper opening. There are at least two ports on said cap in which two needles having different lengths are inserted, and which can then draw at different heights inside the receptacle. One of these needles has such a length that its draw end is near the lowest point of the tapered bottom.

The cylindrical wall of the receptacle is also graduated so as to be able to evaluate the volume of sample contained and the volume of sample drawn.

In the event that the tube is filled with centrifuged blood, the blood will separate into its components as described above.

The component of blood cells that settles on the bottom can thus be aspirated completely by the longer needle.

After aspirating this component, a further centrifugation can be carried out, obtaining, in a starting sample of about 50 ml, a lower layer normally comprised of 4-5 ml of platelets and an upper layer of plasma.

To extract only the plasma, the shorter needle can be used, which by custom is of such a length that its draw end is positioned slightly higher than the level corresponding to 5 ml.

What remains after the plasma aspiration is a product with a very high concentration of platelets, with about 1 ml of plasma and 4 ml of platelets.

A device of this type has the disadvantage of not guaranteeing precision in the sampling, since the drawing level is established by the length of the tube.

Furthermore, if a test tube of this type is used in the double centrifugation procedures for the extraction of platelet-rich plasma, after the extraction of the red blood cells deposited in the lower part of the test tube by means of the longer tube, the walls of the test tube remain contaminated by this material, compromising the purity of the substance to be subsequently drawn, that is, the platelet-enriched plasma that settles on the bottom with the second centrifugation.

To overcome all the aforementioned drawbacks, a new fractionation kit for the extraction of platelet-rich plasma has been designed and produced.

The main object of the present invention is to ensure the complete sterility of the inside of the test tubes, thanks to particular closure elements that slide inside the test tubes. This ensures a closed and sterilized environment.

Another important object of the present invention is to ensure the maximum precision in the extraction of the product of interest from inside the test tube, minimizing the risk of the presence of impurities in the product drawn.

Yet another object of the present invention is to ensure maximum extraction efficiency of the product concerned, that is, of platelet-rich plasma.

A further object of the present invention is to be easy to use and not to have tubes positioned internally which can bend and break.

These and other aims, direct and complementary, are achieved by the new fractionation kit for the extraction of platelet-rich plasma comprising in its main parts a first separator, in turn comprising:

- at least one first tube or receptacle with a closed bottom, side wall and open upper end,

- at least one closure element mounted in the first receptacle, designed to delineate a closed volume between the bottom of the receptacle and the closure element itself, and where the closure element comprises a port for the engagement of an injection/aspiration valve;

- at least one valve installed on the closure element, through which to inject or extract, by means of a syringe, a fluid from or inside the closed volume of the receptacle,

and where the lower surface of the closure element, facing the bottom of the receptacle, is in the shape of an inverted funnel, that is, conical with vertex upwards and communicating with the port in which the valve is installed.

Said first closure element is configured to move inside the receptacle maintaining water tightness with the side wall, so that, as aspiration from inside the receptacle occurs, the first closure element slides towards the bottom of the receptacle.

The kit also comprises at least a second separator, in turn comprising a second test tube or receptacle, the bottom of which comprises at least one collection chamber, that is, a lowered portion for the collection of the platelet material which, during a centrifugation, accumulates at the bottom of the test tube.

Said second separator comprises a second closure element mounted in the second receptacle and delineating a closed volume comprised between the bottom of the receptacle and the second closure element. Said second closure element can move inside the receptacle, maintaining water tightness with the receptacle's walls, and comprises a port in which an injection/aspiration valve is installed.

Said second closure element further comprises a pipe communicating with the port and projecting downwards, in an axial position with respect to the collection chamber, so that, by lowering the closure element, the pipe is inserted into the collection chamber so as to be able to draw all the material contained in the chamber itself.

At least said second receptacle suitably comprises a graduated scale on its side wall. Said first and second separators are disposable.

The kit also suitably comprises anticoagulants and all the accessories for the collection of blood, such as syringes, needles, tubes, etc.

Said separators are made of polymers suitable for medical use, silicone, acrylic, polyester and borosilicate.

The materials used for syringes, needles, tubes and separators are composed of polymers suitable for medical use, elastomers, stainless steel.

All the components of the kit are properly sterilized.

METHOD

The fractionation kit thus configured is particularly useful for the separation and extraction of autologous serum and platelet-rich plasma from a patient's blood sample, by implementing the following method:

- use of said first separator;

- injection of the blood sample into said first separator using a syringe inserted into the valve;

- first centrifugation in which the separation of the two components of the blood sample is obtained where the lower layer is composed of red blood cells, and the upper layer of plasma and platelets; - first extraction of the upper plasma layer, which occurs by inserting a syringe in said valve and performing the aspiration resulting in the lowering of said first movable closure element up to the upper level of the plasma. Normally, during the aspiration operations, it is preferable to use a syringe with an extension to facilitate the operation itself. At this point the syringe is emptied of any air and re-inserted into the valve to draw the plasma. As a result of the plasma aspiration operation, the level of the closure element lowers as the plasma is drawn. During the plasma extraction, the plasma surface tends to adhere to the inverted funnel surface. The extraction continues until the lower edge of the lower surface of the closure element rests on the interface between the enriched plasma and the red cells. In this way it is possible to control with precision the extraction of the plasma alone, without accidentally drawing red blood cells that would compromise the purity of the material drawn. Thus the red blood cells remain in the first separator which at this point can be disposed of;

- injection of the material drawn from the first receptacle into the second separator, wherein the bottom of the receptacle is equipped with a collection chamber;

- second centrifugation, in which the separation between the huffy coat deposited on the bottom and the platelet-poor plasma forming a top layer takes place;

- extraction of the platelet-poor plasma which occurs by inserting a syringe in the valve and performing the aspiration resulting in the lowering of said second movable closure element up to the upper level of the plasma. At this point the syringe is emptied of any air and re-inserted into the valve to draw the plasma. As a result of the plasma aspiration, the second closure element is lowered to a certain volume, visible on the graduated scale of the second receptacle, so that inside only the huffy coat and a limited amount of plasma remains; - mixing of the contents remaining in said second separator which results in a very platelet-rich product, where the small amount of plasma will act as a vehicle for the subsequent infusion of the product into the patient;

- platelet-rich plasma extraction, which occurs by inserting an additional syringe in the valve and performing the aspiration such that due to the aspiration effect, said second closure element progressively lowers, as the product is drawn. The product is aspirated completely, thanks to the pipe that is inserted inside the collection chamber, thus allowing all the platelets which have accumulated inside the chamber to be collected.

The characteristics of the fractionation kit will be better clarified by the following description with reference to the drawings attached by way of non-limiting example. Figure 1 shows an exploded view of the first extractor (1).

Figures 2a and 2b show respectively a three-dimensional view and a side view of a closure element (10) of the first extractor (1).

Figure 3 shows the stop ring (103) of the first extractor (1).

Figure 4 shows an exploded view of the second extractor (2).

Figure 5 shows only the receptacle (200) of the second extractor (2), where the conical bottom (201) with the chamber (202) is visible.

Figures 6a and 6b show respectively a three-dimensional view and a side view of a closure element (20) of the second extractor (2).

This is a fractionation kit specially designed for the separation and extraction of autologous serum and platelet-rich plasma from a patient's blood sample.

The fractionation kit includes two separators, a first separator (1), to be used for the first centrifugation of the blood sample, where the separation into red blood cells and plasma takes place, and a second separator (2), to be used in the second centrifugation of the plasma and the "huffy coat" taken from the first separator (1).

Said first separator (1) comprises a first test tube or receptacle (100) with a closed bottom (101), a side wall (104) and an upper open end (102).

Said first separator (1) also comprises a closure element (10) suited to define a closed volume (11) comprised between the bottom (101) of the receptacle (100) and the closure element (10) itself.

The closure element (10) comprises a port (12) for the connection of an injection/aspiration valve (3) and, preferably, a vent valve (18).

Said valve (3) installed on the closure element (10) is suitable for the injection and aspiration of a fluid by means of a syringe, into or from the inside of the closed volume (11) of the receptacle (100).

The lower surface (13) of said closure element (10), facing towards the bottom (101) of the receptacle (100), is in the shape of an inverted funnel, that is, conical with an upwards vertex (14) which communicates with the port (12) in which the valve (3) is installed.

The lower annular edge (17) of the lower surface (13) of the first closure element (10) is suited to rest on the surface of the plasma layer to be aspirated.

Said closure element (10) is configured to move inside the receptacle (100) maintaining water tightness with the side wall (104), so that, as aspiration from inside the receptacle (100) takes place, the first closure element (10) slides towards the bottom (101) of the receptacle (100).

In order to ensure water tightness, at least one seal, for example an O-ring (15), is mounted on the closure element (10) which, by adhering to the side wall (104) of the receptacle (100), maintains the seal.

Said seal (15) is, for example, mounted on the closure element (10) which has a substantially cylindrical shape with at least one recess (16) to engage the gasket (15). On said open upper end (102) of the receptacle (100) a stop ring (103) is preferably mounted to prevent the closure element (10) installed inside the receptacle (100) from coming out.

The kit also comprises at least one second separator (2), in turn comprising a second test tube or receptacle (200), the bottom of which (201) is preferably concave, for example conical, and comprises at least one collection chamber (202), that is, a lowered portion, preferably but not necessarily in a central position. Said second receptacle (200) has its upper end (203) open.

Said second separator (2) also comprises a closure element (20) mounted in said second receptacle (200) and defining a closed volume (21) comprised between said bottom (201) of the receptacle (200) and said second closure element (20).

Said second closure element (20) comprises a port (22) in which an injection/aspiration valve (3) is installed.

Said second closure element (20) also preferably comprises a further port (23) in which a vent valve (24) is installed.

Said second closure element (20) can move inside said receptacle (200), maintaining water tightness with the receptacle's (200) walls (204) by means of at least one O-ring gasket (25) fitted on the cylindrical body of the closure element (20), being for example engaged in a suitable seat or recess (26).

Said second closure element (20) further comprises a pipe (27) projecting from the lower surface (28) of the closure element (20), that is, towards the bottom (201) of the receptacle (200), and communicating with said port (22) of the closure element (20) itself.

Said pipe (27) is in an axial position with respect to said collection chamber (202) on the bottom (201) of the receptacle (200) so that, by lowering said closure element (20) towards the bottom (201) of the receptacle (200), the pipe (27) is inserted in the collection chamber (202), until it touches the bottom.

Said second separator (2) also suitably comprises a stop ring (205) suited to be mounted on the upper open end (203) of the second receptacle (200), to prevent the closure element (20) installed inside of the receptacle (200) itself from coming out. These specifications are sufficient for the expert person to make the invention, as a result, in the practical application there may be variations without prejudice to the substance of the innovative concept.

Therefore, with reference to the preceding description and the attached drawings the following claims are made.

Claims

1. Fractionation kit for the separation and extraction of autologous serum and platelet-rich plasma from a patient's blood sample, characterized in that it comprises at least one separator (2), or second separator, in turn comprising at least one test tube or receptacle (200), the bottom of which (201) comprises at least one collection chamber (202), that is, a lowered portion for collecting the platelet material which accumulates on the bottom during the centrifuge of enriched plasma contained in said receptacle (200) and obtained from a blood sample.

2. Fractionation kit, according to claim 1, characterized in that said second separator (2) comprises a closure element (20), or second closure element, mounted on said second receptacle (200) and defining a closed volume (21) comprised between said bottom (201) of the receptacle (200) and said second closure element (20) itself, and where said second closure element (20) can move inside said receptacle (200), maintaining water tightness with the walls (204) of the receptacle (200), and comprises a port (22) in which a valve (3) is installed for the injection/aspiration of material into or from the interior of the receptacle (200).

3. Fractionation kit, according to claim 2, characterized in that said second closure element (20) further comprises a pipe (27) projecting at least partly from the lower surface (28) of the closure element (20), that is, towards the bottom (201) of the receptacle (200), and communicating with said port (22) of the closure element (20), and where said pipe (27) is in an axial position with respect to said collection chamber (202) on the bottom (201) of the receptacle (200).

4. Fractionation kit, according to claim 1, characterized in that said bottom (201) of said second receptacle (200) is concave and said chamber (202) is positioned substantially in the middle of said bottom (201).

5. Fractionation kit, according to claim 1, characterized in that it comprises a first separator (1), in turn comprising:

- at least one first test tube or receptacle (100), with a closed bottom (101), side wall (104), and open upper end (102); - at least one closure element (10) mounted in said first receptacle (100), suited to define a closed volume (11) comprised between the bottom (101) of the receptacle (100) and the closure element (10) itself, and wherein said closure element (10) comprises a port (12);

and wherein the lower surface (13) of said closure element (10), facing towards the bottom (101) of the receptacle (100), is in the shape of an inverted funnel, that is, conical with the vertex (14) pointing upwards and communicating with said port (12).

6. Fractionation kit, according to claim 5, characterized in that said first separator (1) comprises at least one valve (3) installed in said port (12) of said closure element (10), through which to inject or extract, by means of a syringe, a fluid from or inside said closed volume (11) of said receptacle (100).

7. Fractionation kit, according to claim 5, characterized in that said first closure element (10) of said first separator (1) is configured to move inside said receptacle (100) maintaining water tightness with the side wall (104) so that, as aspiration from inside the receptacle (100) occurs, said first closure element (10) slides towards the bottom (101) of the receptacle (100).

8. Fractionation kit, according to claim 1, characterized in that said second receptacle (200) has an open upper end (203).

9. Fractionation kit, according to claim 1, characterized in that said first and second closure elements (10, 20) also comprise a further port (18, 23) in which a vent valve (24) is installed.

10. Fractionation kit, according to the preceding claims, characterized in that on said closure elements (10, 20) of said first and second receptacles (100, 200) one or more seals (15, 25) are mounted to maintain water tightness with the side wall (104, 204) of the receptacles (100, 200).

11. Fractionation kit, according to the preceding claims, characterized in that on said open upper end (102, 203) of said first and second receptacles (100, 200) a stop ring (103, 205) is mounted to prevent the closure element (10, 20) installed inside the receptacles (100, 200) from coming out.

12. Fractionation kit, according to the preceding claims, characterized in that at least said second receptacle (200) comprises a graduated scale on its side wall (204).

13. Fractionation kit, according to the preceding claims, characterized in that said first and second separators (1, 2) are disposable.

14. Method for the separation and extraction of autologous serum and platelet-rich plasma from a patient's blood sample, using the kit according to the preceding claims, characterized in that it comprises the following steps:

- use of said first separator (1);

- injection of the blood sample into said first separator (1) by means of a syringe inserted in said valve (3) on said first closure element (10);

- initial centrifugation in which the separation of the blood sample into the components takes place where the lower layer is composed of red blood cells, and the upper layer of plasma and platelets;

- first extraction of the upper layer of platelet enriched plasma, which occurs by inserting a syringe in said valve (3) on said first closure element (10) and starting an aspiration cycle which involves lowering said movable first closure element (10) to the upper level of the plasma; removal of the air drawn in by the syringe and re-insertion of the syringe into said valve (3) to proceed with the aspiration of the plasma, and wherein, due to the effect of the aspiration, said closure element (10) is lowered as the plasma is aspirated, and wherein the extraction proceeds until the lower edge (17) of the lower surface (13) of the closure element (10) rests on the interface with the red blood cells;

setting aside of said first separator (1);

- injection of the material taken from said first separator (1) into said second separator (2);

second centrifugation, in which the platelet material settles on the bottom (201) and in said collection chamber (202) of the second receptacle (200), and the platelet-poor plasma forms a top layer; - extraction of said poor platelet plasma which occurs by inserting a syringe in said valve (3) of said second closure element (20) of said second separator (2) and starting an aspiration cycle which involves lowering said movable second closure element (20) to the upper level of the plasma; emptying the syringe and re- inserting it in said valve (3) of the second closure element (20) to proceed with the aspiration of the plasma, and wherein, due to the aspiration of the plasma, said second closure element (20) is lowered up to a certain volume, so that the huffy coat and a reduced volume of plasma remains inside it;

- mixing of the contents remaining in said second separator (2), obtaining a product very rich in platelets, wherein the minimal amount of plasma will serve as a vehicle for the subsequent infusion of the product into the patient;

extraction of the platelet-rich plasma, which occurs by inserting an additional syringe into said valve (3) of the second closure element (20) and aspirating in such a manner that, due to the effect of the aspiration, said second closure element (20) is lowered progressively, as the product is aspirated and wherein the product is completely aspirated by means of said pipe (27) of the second closure element (20) which is inserted inside said chamber (202).