RU2715746C1 - Device for cryopreservation and preparation of thrombocytes for transfusion - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medical equipment, particularly to devices used in transfusiology. A device for cryopreservation and preparation of thrombocytes for trossefusion contains two containers: the first one – for thrombocyte concentrate freezing, storage and thawing, the second one – for plasma connected by tubes with clamps. Second container is connected by tube with clamp to first branch of first quadruple, tube with clamp for connection with container for resuspended thrombocytes, closed from opposite end, connected with second branch pipe, bacterial filter, ending with Luer-Lock connector for connection to container with SSP/SSP+ solution, is connected by tube with clamp to third branch pipe, and the fourth branch pipe is connected to the first branch pipe of the first tee pipe by the tube, the second branch pipe is connected by the tube with the clamp to the input of the speed limiter of the resuspending flow of plasma, the outlet of which is connected to the second branch pipe of the second tee-piece. Third T-branch pipes are interconnected by a bypass tube with a clamp, and the first branch pipe of the second T-joint is connected by a tube with a clamp to the second branch of the second quadruple, the first branch pipe of which is connected by a cryoprotecting tube with a clamp and with a bacterial filter ending with a Luer-Lock connector, third branch pipe is connected by plasma extraction tube with clamp and with check valve terminating with Luer-Lock connector, fourth branch pipe is connected by tube with clamp to first container. Luer-Lock connectors are provided with detachable sealing caps.

EFFECT: technical result consists in ensuring the quality of thrombocytes prepared for transfusion and reducing microbial contamination.

8 cl, 3 dwg

Description

The invention relates to medical equipment, in particular to devices used in transfusion, and is intended for the preparation of platelets of long-term storage, suitable for emergency transfusion.

Known devices for cryopreservation of human and animal cells produced by domestic and foreign firms Viroban, Makofarma, Baxter, Fresenius and others. Such devices contain separate containers connected by tubes with clamps, and are intended for freezing, storage at low temperatures and thawing cells, but not allow in a closed system to carry out technological operations required for cryopreservation of platelets.

Also known is the “Set of products for cryopreservation of single-use erythrocytes, sterile”, manufactured by Synthesis OJSC, consisting of several containers connected by tubes with clamps, one of which contains the blood preservative Glyugitsir. The whole donated blood procured in this system is centrifuged, the plasma is transferred into a separate container, and the container with the erythrocyte mass is connected to the container containing the cryoprotectant solution Kriosin. The connection of the containers is carried out in violation of the tightness and isolation of the system by introducing a perforator located on the tube of the container with the Cryosin solution into the port of the container with an erythrocyte mass. Thus, the risk of microbial contamination of red blood cells due to the conditionally sterile connection of containers increases.

The closest analogues to some structural parts of the proposed device are three devices described in patents RU 169287, 169578, 167874, which due to their design features cannot be combined into a single sterile product during production. Each device is necessary to perform only certain operations of an integrated technological chain of cryopreservation and preparation of platelets for transfusion. These operations are described in separate ways in patents RU 2623081, 2623083, 2623864. Work with devices - analogues is possible only in aseptic conditions in the presence of "clean" rooms, local sterile zones and laminar cabinets that preserve the sterility of the target product - platelet concentrate. In addition, the above methods are associated with the need to use expensive equipment for multiple sterile and not always reliable butt joints of the tubes of each of the three devices. These welded joints are exposed to many physical factors affecting their strength: centrifugation at high overloads, rapid joint freezing and thawing of cells and liquids in a wide range of low and high temperatures.

A single, integral and enclosed device is required, which without violation of sterility at all stages of the technological process can ensure the implementation of the following technological steps:

- transfer to the device of a dose of platelet-rich plasma received from a donor,

- platelet concentration and plasma separation from platelet concentrate,

- extraction of a portion of the plasma and its dilution of the cryoprotectant,

- transfer of the remaining plasma into a separate container,

- introduction according to a specific program (speed, time, concentration, volume) of a portion of the cryoprotectant diluted in plasma into a container with platelet concentrate,

- removal of air from the device,

- freezing in separate containers of plasma and a suspension of platelet concentrate in a cryoprotectant diluted with plasma.

- long-term storage at low subzero temperatures,

- thawing platelet concentrate and plasma,

- resuspension of plasma according to a specific program of platelet concentrate with stirring,

- transfer of resuspended platelets into a gas-permeable container,

- storage until transfusion in a gas-permeable container of resuspended platelets with stirring.

In this regard, the object of the invention is the implementation of the described multi-stage technology of cryopreservation, long-term storage and preparation of platelets for transfusion using a single closed device instead of three, not combined together different devices for each stage of the technology.

The technical result consists in maintaining the quality of thawed platelets prepared for transfusion and reducing the likelihood of their microbial contamination.

An additional technical result is that aseptic conditions are not required for all technological manipulations, multiple sterile attachment of various external functional elements to the device, the work of the staff is simplified and facilitated.

The problem is solved and the technical result is achieved by the fact that the device for cryopreservation and preparation of platelets for transfusion contains two containers connected by tubes with clamps, where the first is for freezing, storing and thawing platelet concentrate, the second for plasma, in which the second container is connected by a tube with a first clamp chetvernika first pipe, tube clamp closed at the opposite end connected to the second pipe, a bacterial filter ending Conneh by Luer-Lock, is connected by a pipe with a clamp to the third pipe, and the fourth pipe is connected by a pipe with the first pipe of the first tee. The second nozzle of this tee is connected by a tube with a clip to the inlet of the speed limiter of the resuspending plasma flow, the output of which is connected to the second nozzle of the second tee. The third nozzles of the tees are interconnected by a bypass tube with a clamp, and the first nozzle of the second tee is connected by a tube with a clamp to the second nozzle of the second quadruple, the first nozzle of which is connected by a tube with a clamp with a bacterial filter ending in the Luer-Lock connector. The third nozzle is connected by a tube with a clamp with a check valve ending in the Luer-Lock connector. The fourth nozzle is connected by a tube with a clamp to the first container, while the Luer-Lock connectors are equipped with removable sealing caps.

Other preferred features and features of the device are indicated in the dependent claims. In particular, an additional technical result is obtained due to the fact that when resuspending thawed and agitated platelets, the hydraulic resistance of the speed limiter of the resuspending plasma flows or SSP / SSP + solutions is adjusted so that the value of this speed is in the range of 10 - 20 ml / min. In this case, containers with thawed plasma or solution should be located 70 - 100 cm above the level of placement of the container with thawed and stirred platelets.

In addition, the speed limiter of the resuspending plasma flows or SSP / SSP + solutions is made in the form of a capillary tube with an inner diameter in the range of 0.2 - 2.0 mm and a length in the range of 10 - 40 mm inserted into the tube connecting the tees. The speed limiter of the resuspended plasma flows or SSP / SSP + solutions can also be made in the form of a tube connecting the tees filled with a spongy open-cell filler.

It was proposed that the containers for plasma and platelets have a nominal volume of 150 - 300 ml and be made of a transparent, flexible, platelet-compatible and gas-permeable polymer material that can withstand centrifugation with an overload of up to 1500 g and freezing at low temperatures - up to minus 196 ⁰ С while maintaining at defrosting the tightness of containers, all elements of the device and their connections.

Conveniently, the clips are made in the form of flat removable or non-removable slotted clips or snap clips.

It is advisable that the bacterial filters have a sterilizing membrane with pores no larger than 0.2 microns in size.

If you hang out a container with thawed plasma or a container with SSP + solution (SSP) at a height of 70-100 cm from the location of the container with thawed and stirred platelets (Fig. 3) and use a capillary tube with an inner diameter as a limiter to the speed of plasma or solution flow in the range of 0.2 - 2.0 mm and a length preferably in the range of 10 - 40 mm, the platelet resuspending plasma or solution flow rate will be in the range of 10 - 20 ml / min. At this speed, platelets do not experience osmotic shock, most platelets retain their structure, shape and functional activity. Lower flow rates significantly extend the resuspension process and adversely affect platelet safety. The best results were obtained when using a capillary with an inner diameter of 0.4 mm and a length of 15 mm. For plasma, the rate of the resuspending flow was 15–18 ml / min, and for the solution SSP + (SSP): 10–14 ml / min.

It is relatively simpler to produce a resuspending flow rate limiter in the form of a hollow elastic cylinder made of otrotoporous polyurethane foam with a diameter of 3.1 - 3.2 mm, a length of 20 - 25 mm, a porosity of 90 - 95% with an average pore size of 0.2 mm, which is inserted into a tube with 3.0 mm inner diameter connecting the tees of the device. In this case, the rates of the resuspending plasma flow or SSP + solution (SSP) are almost the same as those using the capillary flow rate limiter. Unlike device analogs, personnel do not need to precisely control the plasma flow with a roller clamp, visually counting the number of drops per unit time, which is difficult, because it is necessary to count 3 - 4 drops per second. The use, like an analog, of a flow rate regulator from an Exadrop infusion system, requires a sterile connection of the tubes and the presence of a heat-sealing polymer tube apparatus CompoDock (Fresenius) or similar devices.

The nominal volume of containers of 150-300 ml is selected taking into account the small volume of platelet concentrate in a portion of plasma received from the donor and the small volume of the glass of the centrifuge rotor, in which it is necessary to place not only the containers, but the entire device. For the same reason, it is advisable to use small-sized flat slotted tube clamps or snap clips.

The use of bacterial filters with a hydrophilic membrane for solutions with pores of not more than 0.2 μm in the device allows cold sterilization of the solutions introduced into the device.

Preferred features of the invention are described in the dependent claims, and also follow from the following specific, but not limiting example embodiment of the device with reference to the accompanying drawings.

Figure 1 - diagram of a device for cryopreservation and preparation of platelets for transfusion. The device of FIG. 1 consists of a platelet container - K1, a plasma container K2, clamps 1 - 9, Luer-Lock connectors with removable sealing caps 11 - 13, bacterial filters 14, 15, the first quadruple 16, the second quadruple 17, the first tee 18, the second tee 19, check valve 20, speed limiter for plasma or SSP + (SSP) solutions 21. The listed device elements are connected by tubes with clamps using welded and adhesive technologies, and the tube with clamp 2 is closed at one end.

FIG. 2 is a diagram of the attachment to the device of a container with an initial platelet concentrate - K4, obtained from a donor, a container with a solution of SSP + (SSP) - K3 (in case of technological necessity), a syringe 22 for sampling a portion of plasma (shown on the left in FIG. 2) and injection into the device using the same syringe 22 of a cryoprotectant diluted with plasma (shown on the right in Fig. 3). The arrows indicate the flow directions of the initial platelet concentrate, plasma separated by centrifugation from the platelet concentrate, cryoprotectant diluted with plasma, and also the defrosted plasma stream at the stage of resuspension of thawed platelets or joint solution with SSP + (SSP) solution in case of technological need.

 FIG. 2 also explains to which connectors of the device containers K4, K3 and syringe 22 are connected. Two dotted lines, arrows next to them and the letter “C” show the end-to-end sealing place of the thermal joint of the tubes. Dotted lines A1 - A3 indicate the location and sequence of sealing the tubes and hermetically disconnecting the overlying parts of the device.

FIG. 3 - a variant of the mutual arrangement of containers with thawed plasma, a solution of SSP + (SSP) and platelet concentrate at the stage of resuspension and stirring of platelets after thawing.

All components of the device are made of non-toxic platelet-compatible, hypoallergenic and pyrogen-free polymeric materials. The device is sterilized by radiation with a dose of 20 +/- 5 kGy.

In accordance with the proposed technical solution, the claimed device is designed and manufactured. By means of this device and modern methods of cryopreservation and preparation for platelet transfusion described in patents RU 2623081, 2623083, 262386412, 12 portions of platelets were prepared. The procedure for working with the device, the sequence of manipulations with its elements, indicators of the cryopreservation process and the preparation of platelets for transfusion, as well as quality indicators of platelets prepared for transfusion, are given as an example of a specific embodiment of the invention.

A platelet-rich portion of donor plasma of 200 +/- 20 ml with a cell content of (1-1.5) x ^{10 9} in ml is taken on a Trima Accel apheresis apparatus into a K4 container (Fig. 2), which is used as a PL- platelet storage container 2410 (Baxter, Fenval) with a nominal volume of 1000 ml. In the device, containers CS150N (Cryostor) and Maco Biotech Freezing - EVA bag, both with a nominal volume of 250 ml, are used as containers K1 and K2. Close all clamps 1-9 on the device tubes. The tube of the container K4 and the tube with clamp 2 are sterilely connected to each other by means of a CompoDock heat sealing apparatus (Frezenius). Clips 2, 4, 7, 9 are opened and the contents of the container K4 are transferred to the container K1. Clips 2 and 9 are closed, container K1 is placed in one section, and the rest of the device is in the other section of the rotor cup of the Sorvall RC 3C Plus centrifuge. Centrifuge the device for 7-12 minutes with a centrifugal acceleration of 1100-1400 g. 1-3 ml of a sterile combined endo- and exocytic cryoprotectant containing 55% DMSO and 5% dextran 40 are collected from a 5 ml vial into a two-component syringe 22 (Fig. 2) with a volume of 12 ml. Having removed the cap from connector 12, a syringe is attached, clamps 6 and 9 are opened, plasma is taken from container K1, which after centrifugation is contained in the upper part of the container in the form of supernatant, and the concentration of DMSO in the syringe is adjusted to 10-15%. The non-return valve 20 prevents accidental backfilling of the conditionally sterile contents of the syringe into the device. Using the apparatus, the Gecko B brew the pipe along line A2 and separate it.

Clamps 9 and 3 are opened and, by manually compressing or using a Jouan plasma extractor, container K1, the plasma is transferred from it to container K2. Clamps 3 and 7 are closed.

Having removed the cap from connector 13, attach a syringe, open clamp 8 and, through the sterilizing bacterial filter 15, platelets remaining in container K1 are resuspended in 2 ml portions of cryoprotectant plasma at intervals of 2 minutes with constant stirring of platelets on the thrombomixer. The concentration of DMSO cryoprotectant is reduced to 4-6%. At the end of platelet resuspension, the tube is brewed along line A3 and separated.

Container K1 respectively resuspended in a cryoprotectant platelets and plasma container K2 is placed together with the other elements of the device in metal or cardboard cartridge and frozen in a freezing chamber at a rate ^of 1-3 / min at a temperature of 85-100 ^° C minus apparatus with frozen platelets and the plasma stored at minus 85-196 ^{° C} up to 2 years.

Thaw frozen platelets and plasma, respectively container K1 and K2 in the programmable device defroster Barkey Plasmatherm for 2-10 minutes at a temperature of 37-40 ° ^C.

The container K2 with the plasma is suspended on a typical transfusion rack with a height of 170-190 cm at a level of 70-100 cm from the location of the container K1, which is placed on a weighing machine located on the laboratory bench (figure 3). Clamp clamp 4 on the bypass tube, open clamps 3, 5, 7, and 9. Plasma flows from the container K2 under gravity into the container with resuspended and continuously stirred platelets through the flow restrictor 21, which, when assembling the device, is adjusted so that it sets the flow rate in the range of 10-20 ml / min. The volume of plasma resuspended platelets prepared for transfusion, on average, is 150 +/- 15 ml. In this case, approximately 10-fold plasma dilution of the same donor of thawed platelets and a decrease in the concentration of DMSO cryoprotectant to 0.4-0.6% occur.

Clamps 3 and 5 are closed, the container K1 is suspended on a transfusion rack, clamps 2 and 4 are opened. The platelets resuspended in plasma under gravity flow from the container K1 into the container K4, which is permeable to oxygen and carbon dioxide (PL-2410 (Baxter, Fenval)) made of platelet-compatible hydrophilic and low platelet adhesion material. A line with a clamp 2 is brewed along line A1 and the container K4 is separated from the device. Thawed platelets stored in R4 container at a temperature of 20-24 ^{° C} and constant agitation is not more than 4 hours before transfusion.

Resuspension of thawed platelets by thawed plasma of the same donor from container K2 is carried out, in particular, to solve some of the clinical problems of urgent surgery associated with large blood loss and the need to correct severe thrombocytopenia. Solutions SSP + (SSP) from an external container K3 together with plasma from a container K2 or separately used for prophylactic transfusions, for example, in oncohematology. In this case, the container K3 with the SSP + solution (SSP) is connected to the connector 10 using the connector 10 (FIG. 2), similarly to the connection of the syringe 22 to the connector 13. Thawed and agitated platelets are subsequently reperfused, as shown in FIG. 3, first with plasma from the container 2 through flow restrictor 21, then with SSP + solution through the same flow restrictor 21, observing the ratio (20% plasma + 80% SSP +), and for the SSP solution the ratio (30% plasma + 70% SSP).

The analysis of the quality of thawed platelets in each of the 12 portions prepared for transfusion using the proposed device, carried out by the method for assessing the morphofunctional status of platelets described in patent RU No. 2485502, showed that the biological usefulness (structural and functional) of platelets was practically not violated. Thus, the adhesive activity of thawed platelets by the end of 4 hours was at least 90% of the initial adhesive activity, which indicated that the basic functional quality characteristic of thawed platelets remained at a fairly high level. The total number of thawed platelets averaged 90 +/- 5% of the initial number. The structural usefulness of platelets also remained at a high level: the number of platelets with granules averaged 60 +/- 10%, which also indicated that the basic structural quality characteristic of thawed platelets remained at a fairly high level.

 Tests conducted in accordance with GOST EN 556-1-2011 did not reveal a violation of the sterility of cryopreserved and thawed platelets prepared for transfusion using the proposed device, which testified to the achievement of a technical result: a low probability of contamination of the target product of cryopreservation technology - resuspended thawed platelets. The whole multi-stage technology of cryopreservation and preparation of platelets for transfusion was carried out in one closed device; it did not require several different devices for each technology stage, a large number of sterile end-to-end tube thermal connections, aseptic conditions, and “clean” production facilities compared to analog devices.

Claims (8)

1. A device for cryopreservation and preparation of platelets for transfusion, containing two containers: the first for freezing, storing and thawing platelet concentrate, the second for plasma connected by tubes with clamps, characterized in that the second container is connected by a tube with a clamp to the first nozzle of the first quadruple, tube with a clamp for connecting to a container for resuspended platelets, closed from the opposite end, connected to the second nozzle, a bacterial filter ending in connector m Luer-Lock for connecting to the container with the SSP / SSP + solution, is connected by a pipe with a clamp to the third pipe, and the fourth pipe is connected by a pipe to the first pipe of the first tee, the second pipe is connected by a pipe with a clip to the inlet of the speed limiter of the resuspending plasma flow, the output of which with the second nozzle of the second tee, the third nozzles of the tees are connected by a bypass tube with a clamp, and the first nozzle of the second tee is connected by a pipe with a clamp with the second nozzle of the second quadruple, the first pa the cabin of which is connected by a cryoprotectant supply tube with a clamp and with a bacterial filter ending with a Luer-Lock connector, the third nozzle is connected by a plasma sampling tube with a clamp and with a check valve ending by a Luer-Lock, the fourth nozzle is connected by a clamp with the first container, while the Luer-Lock connectors are equipped with removable sealing caps. 2. The device according to claim 1, characterized in that the speed limiter of the resuspending plasma flows or SSP / SSP + solution during resuspension of thawed and agitated platelets has the ability to adjust the hydraulic resistance to maintain a speed in the range of 10 - 20 ml / min. 3. The device according to claim 1, characterized in that the speed limiter of the resuspending plasma flows or SSP / SSP + solution is made in the form of a capillary tube with an internal diameter of 0.2 - 2.0 mm and a length of 10 - 40 mm inserted into the tube, connecting tees. 4. The device according to claim 1, characterized in that the speed limiter of the resuspending plasma flows or the SSP / SSP + solution is made in the form of a tube connecting the tees filled with a spongy open-cell filler. 5. The device according to claim 1, characterized in that when resuspending thawed and agitated platelets by plasma flows or an SSP / SSP + solution created by gravity, the containers with the plasma or SSP / SSP + solution are located 70 - 100 cm above the level of the location of the platelet container . 6. The device according to p. 1, characterized in that the containers for plasma and platelets have a nominal volume of 150 to 300 ml and are made of transparent, elastic, platelet-compatible and gas-permeable polymer material. 7. The device according to p. 1, characterized in that the clamps are made in the form of a flat removable or non-removable slotted clips or snap clips. 8. The device of claim 1, characterized in that the bacterial filters contain a sterilizing membrane with pores no larger than 0.2 microns in size.

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