JP7750685B2 - Evaluation specimen and evaluation device - Google Patents

Description

The present invention relates to an evaluation sample and an evaluation device.

Various methods for evaluating the blood compatibility of medical devices have been studied (for example, see Patent Document 1 below). ISO 10993-4 describes methods for evaluating the blood compatibility of medical devices, including in vivo/ex vivo evaluation methods that primarily use animals to simulate clinical use and evaluate the risk of thrombosis at the application site of the medical device through semi-quantitative visual evaluation of the degree of thrombus adhesion to the surface of the medical device, and in vitro evaluation methods that quantitatively evaluate coagulation, platelet activation, complement, and hematological parameters using blood.

In vitro evaluation methods often use the static method, which allows for easier condition setting than the blood circulation method and can be carried out on a small scale. The static method is an evaluation method in which a medical device is immersed in blood and comes into contact with it, and is recommended for evaluating the hemocompatibility of medical devices that come into contact with blood without blood flow, such as blood bags.

WO2003/014394

However, because the static method involves contact between the medical device and blood in a static state, it is not an evaluation method that simulates clinical use for medical devices that come into contact with circulating blood, such as catheters.

For this reason, there is a need for an in vitro evaluation method that simulates clinical use in the blood circulation system.

The inventors therefore conducted various studies with the aim of constructing an evaluation device capable of simultaneously evaluating thrombus formation, coagulation activity, platelet activity, and complement activity using an in vitro evaluation method that simulates clinical use.

In constructing the above-mentioned evaluation device, the inventors focused on the importance of an evaluation sample (positive control substance) that can accurately determine that medical devices that test positive in conventional in vivo evaluations are positive, and thus came up with the present invention.

In other words, the present invention aims to provide an evaluation sample that can be used in an evaluation device that can simultaneously evaluate thrombus formation, coagulation activity, platelet activity, and complement activity in an in vitro evaluation method that simulates clinical use, and that can be appropriately used as a positive control substance.

The present invention also aims to provide an evaluation device for use in evaluation tests using the above-mentioned evaluation samples.

The present invention provides an evaluation sample for use in a test to evaluate the blood compatibility of a medical device. The evaluation sample has a first active part that promotes the coagulation activity and platelet activity of the blood used in the test, and a second active part that promotes the complement activity of the blood. The first active part is at least one selected from the group consisting of polyurethane, latex rubber, and polyurethane elastomer, and the second active part is at least one selected from the group consisting of zymosan, cobra venom, mannan, and agarose .

An evaluation device according to the present invention that achieves the above-mentioned objectives is an evaluation device used in evaluation tests for the blood compatibility of medical devices. The evaluation device has a supply section that supplies blood used in the evaluation test, a circulation path through which the blood circulates, and an insertion section into which an evaluation specimen is inserted, the evaluation specimen having a first activation section that promotes the coagulation activity and platelet activity of the blood and a second activation section that promotes the complement activity of the blood.

With the evaluation sample configured as described above, the first activation unit promotes blood coagulation activity and platelet activity, while the second activation unit promotes blood complement activity. This provides an evaluation sample that can be used in an evaluation device capable of simultaneously evaluating thrombus formation, coagulation activity, platelet activity, and complement activity in an in vitro evaluation method simulating clinical use, and that can be appropriately used as a positive control substance.

Furthermore, the evaluation device configured as described above can be used in evaluation tests using the above-mentioned evaluation samples.

FIG. 1 is a front view showing an evaluation sample according to an embodiment of the present invention. FIG. 2 is a cross-sectional view taken along line 2-2 in FIG. 1 is a schematic diagram illustrating an evaluation device according to an embodiment of the present invention. 1 is a photograph showing a negative control substance after blood circulation when an evaluation test was carried out with an ACT (activated clotting time) of about 200 seconds. This is a photograph showing a negative control substance after blood circulation when an evaluation test was conducted with an ACT of about 300 seconds. 10 is a photograph showing an evaluation sample (positive control substance) after blood circulation when an evaluation test was performed with an ACT of about 200 seconds. 10 is a photograph showing an evaluation sample (positive control substance) after blood circulation when an evaluation test was performed with an ACT of about 300 seconds. 1 is a photograph showing an evaluation sample (positive control substance) when an in vivo thrombosis test was performed.

Embodiments of the present invention will now be described with reference to the accompanying drawings. Please note that the following description does not limit the technical scope or meaning of the terms described in the claims. Also, the dimensional proportions in the drawings have been exaggerated for the sake of explanation and may differ from the actual proportions.

The evaluation sample 1 according to this embodiment will now be described with reference to Figures 1 and 2. Figure 1 is a front view showing the evaluation sample 1 according to this embodiment. Figure 2 is a cross-sectional view taken along line 2-2 in Figure 1.

Evaluation sample 1 is used in evaluation tests for the blood compatibility of medical devices. In evaluation tests, it is important to be able to distinguish between negative and positive results. Evaluation sample 1 is used as a positive control substance to properly determine positive results in a medical device blood compatibility evaluation test system. As used herein, "positive" means that the sample significantly promotes thrombus formation, coagulation activity, platelet activity, and complement activity in a medical device blood compatibility evaluation test system compared to medical devices with a proven track record of clinical use.

As shown in Figures 1 and 2, the evaluation specimen 1 has a first active section 10 consisting of a main body extending in the axial direction, and a second active section 20 consisting of a coating section arranged to cover part of the outer surface of the main body.

The first active part 10 promotes blood coagulation activity and platelet activity. As shown in Figures 1 and 2, the first active part 10 is a string with a circular cross section.

The diameter of the first active part 10 is not particularly limited, as long as it can be inserted through the insertion part of the evaluation device described below, blood flows around the first active part 10 when inserted, and it is not significantly different from the diameter of the medical device being evaluated. The axial length of the first active part 10 is also not particularly limited, but is generally between 100 and 300 mm.

The surface of the first active portion 10 is textured. There are no particular limitations on the width of the grooves in the textured surface.

The material constituting the first active section 10 is not particularly limited, and polymeric materials, metal materials, and the like can be used as appropriate. Examples of polymeric materials include polyolefins (e.g., polyethylene, polypropylene, polybutene, ethylene-propylene copolymer, ethylene-vinyl acetate copolymer, ionomers, and mixtures thereof), polyolefin elastomers, crosslinked polyolefins, polyvinyl chloride, polyamides, polyamide elastomers, polyesters, polyester elastomers, polyurethanes, polyurethane elastomers, latex rubber, fluororesins, polycarbonates, polystyrenes, polyacetals, polyimides, polyetherimides, ethylene-tetrafluoroethylene copolymers (ETFE), tetrafluoroethylene-hexafluoropropylene copolymers (FEP), polyetheretherketones (PEEK), and polyvinylidene fluoride (PVDF). Examples of metal materials include stainless steel (SUS) such as SUS304, SUS316, SUS316L, SUS420J2, and SUS630, gold, platinum, silver, copper, nickel, cobalt, titanium, iron, aluminum, tin, or nickel-titanium (Ni-Ti) alloy, nickel-cobalt (Ni-Co) alloy, cobalt-chromium (Co-Cr) alloy, and zinc-tungsten (Zn-W) alloy. From the perspective of enhancing thrombus formation, blood coagulation, and/or platelet activation performance, the first active portion 10 preferably contains at least one of polyurethane, latex rubber, and polyurethane elastomer, and more preferably is composed of at least one of polyurethane, latex rubber, and polyurethane elastomer. These materials may be used alone or in combination.

The second active portion 20 promotes complement activation in the blood. The axial length of the region R where the second active portion 20 is provided is not particularly limited, but is generally between 30 and 50 mm.

The material constituting the second active part 20 is not particularly limited, and examples include immunoglobulins such as IgA, IgD, IgE, IgM, IgG1, IgG2, and IgG3, C polysaccharide-CRP conjugates, polymer DNA, bacterial lipopolysaccharide, dextran, dextran sulfate, polyanion-polycation conjugates, polymeric substances such as zymosan (the pellicle membrane of yeast and gram-negative bacteria), mannan, bacterial wall peptidoglycan, inulin, and agarose, proteolytic enzymes such as trypsin and plasmin, microorganisms such as Trypanosoma, Entamoeba histolytica, and Schistosoma, cells such as virus-infected cells and tumor cells, uric acid crystals, cardiac muscle mitochondria, retroviruses, and cobra venom. Among these, the complement activator preferably includes at least one of zymosan (particularly zymosan A), cobra venom, mannan, and agarose, and more preferably at least one of zymosan (particularly zymosan A), cobra venom, mannan, and agarose. Note that these complement activators may be used alone or in combination of two or more.

From the viewpoint of exerting sufficient complement activation performance, the amount of the second active part 20 is more preferably 132.7 g/ ^m2 or more per unit surface area of the region R where the second active part 20 is arranged. There is no particular upper limit to the amount of the complement activating substance, but it is usually 265.4 g/ ^m2 or less.

<Evaluation Device 90>
Next, the configuration of an evaluation device 90 used in an evaluation test for the blood compatibility of a medical device will be described with reference to Fig. 3. Fig. 3 is a schematic diagram showing the evaluation device 90 according to an embodiment of the present invention.

As shown in Figure 3, the evaluation device 90 has a supply unit 91 that supplies blood used in the evaluation test, a blood bag 92 to which the supply unit 91 is connected, a circulation path 93 through which the blood circulates, an insertion unit 94 into which the evaluation specimen 1 is inserted, a collection unit 95 that is connected to the circulation path 93 and collects the blood used in the evaluation test, and a pump 96 for circulating the blood within the circulation path 93.

The supply section 91 is a three-way stopcock located above the blood bag 92. The circulation path 93 is a tube. A known sheath can be used as the insertion section 94. The insertion section 94 is provided with a valve (not shown) to prevent backflow of blood. The collection section 95 is a three-way stopcock. The pump 96 can be, for example, a roller pump.

<Evaluation method using evaluation device>
Next, an evaluation method using the evaluation device 90 will be described.

First, each evaluation sample (negative control substance, positive control substance) 1 is inserted through the insertion portion 94 connected to the circulation path 93 and placed at the sample insertion point on the circulation path 93. Each evaluation sample is exposed approximately 10 cm from the tip of the insertion portion 94.

Next, 50 mL of saline is injected into the circulation path 93 from the collection portion 95 to prime it. Priming is performed so that air bubbles in the circulation path 93 are collected in the blood bag 92.

Next, use a syringe to remove the air from inside the blood bag 92 through the supply part 91 at the top of the blood bag 92.

Next, 100 mL of test blood is injected from the supply port 91 at the top of the blood bag 92. After injection, the syringe is again used to remove the air from the blood bag 92.

Next, the pump 96 is turned on, with the flow rate of the evaluation sample portion in the circulation path 93 set to 200 mL/min, to begin circulating the blood.

Next, blood samples for measurement are taken 5 minutes, 90 minutes, and 180 minutes after the start of circulation. The blood samples are collected from the collection unit 95 using a syringe.

Next, a blood sample for measurement is taken 180 minutes after the start of circulation, and then the pump 96 is stopped to end the circulation. After the pump 96 is stopped, the evaluation sample is removed.

After the evaluation sample is removed, it is visually observed to measure clot formation. Additionally, the thrombin-antithrombin III complex (TAT) is measured to measure blood coagulation activity, β-thromboglobulin (β-TG) and platelet count are measured to measure platelet activation, and SC5b-9 is measured to measure complement activation.

<Example>
Examples will be described below.

First, a Radifocus Introducer IIH Fr. 9 (Terumo Corporation: sheath only) was prepared as a negative control.

Next, as a positive control substance (corresponding to the evaluation specimen in this embodiment), a urethane round cord with a textured surface (manufactured by Fuso Rubber Industries Co., Ltd., diameter 3 mm) was prepared by adhering zymosan A (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The urethane round cord corresponds to the first active part 10, and zymosan A corresponds to the second active part 20.

The preparation of the positive control substance is explained in more detail below. Frozen zymosan A was allowed to return to room temperature. A 20 cm length of urethane round cord was then cut and marked 5 cm and 8 cm from the tip. 0.0375 grams of zymosan A was then weighed out on an electronic balance and crushed. Cemedine 3000 Gold (jelly-like) (manufactured by Cemedine Co., Ltd.) was then applied to the entire area between the marks to allow the zymosan A to adhere, and the cord was then allowed to air dry for approximately two hours.

Next, we confirmed whether the prepared negative and positive control substances could be determined as negative and positive, respectively, when evaluated using the evaluation device 90.

Since the evaluation device 90 was designed as a control for a medical device that comes into short-term contact with circulating blood, the ACT (activated clotting time) was adjusted to a target of 200-300 seconds, based on the ACT during clinical PCI. Therefore, the evaluation confirmed whether it was possible to distinguish between negative and positive results under two conditions: approximately 200 seconds, which is near the lower limit, and approximately 300 seconds, which is near the upper limit.

In the evaluation device 90, the flow rate of circulating blood per minute using the pump 96 was controlled to 200 mL ± 15%.

Under the above conditions, evaluation tests were conducted on the negative and positive control substances using the evaluation method described above.

After the evaluation test was completed, macroscopic observation was performed to measure thrombus formation, and thrombin-antithrombin III complex (referred to as TAT) was measured to measure blood coagulation activity, β-thromboglobulin (β-TG) and platelet count to measure platelet activation, and SC5b-9 was measured to measure complement activation.

When macroscopic observation was performed to evaluate thrombus formation, it was graded according to the grades in Table 1 below.

Figure 4 shows the negative control substance after blood circulation when an evaluation test was performed with an ACT of approximately 200 seconds, Figure 5 shows the negative control substance after blood circulation when an evaluation test was performed with an ACT of approximately 300 seconds, Figure 6 shows evaluation sample 1 (positive control substance) after blood circulation when an evaluation test was performed with an ACT of approximately 200 seconds, and Figure 7 shows evaluation sample 1 (positive control substance) after blood circulation when an evaluation test was performed with an ACT of approximately 300 seconds.

As can be seen from Figure 4, at an ACT of 200 seconds, no thrombus adhesion was observed on the surface of the negative control material (thrombus adhesion grade 0). As can be seen from Figure 5, even at an ACT of 300 seconds, no thrombus adhesion was observed on the surface of the negative control material (thrombus adhesion grade 0). As can be seen from Figure 6, at an ACT of 200 seconds, thrombus adhesion was observed on the surface of the positive control material (thrombus adhesion grade 4). As can be seen from Figure 7, at an ACT of 300 seconds, slight thrombus adhesion was observed on the surface of the positive control material (thrombus adhesion grade 1).

Table 2 below shows the results of macroscopic observation, as well as the measurement results of TAT, β-TG, platelet count, and complement activation.

Under conditions of an ACT of approximately 200 seconds, the thrombus adhesion grade was 0 for the negative control substance and 4 for the positive control substance. Furthermore, when comparing the values for each parameter, TAT, β-TG, and platelet count, 180 minutes after the start of circulation with the values for the negative control substance, a significant increase in TAT and β-TG was observed with the positive control substance, and a significant decrease in platelet count was observed. For SC5b-9, a rapid increase was observed with the positive control substance starting 5 minutes after the start of circulation, and clear differences were observed between the negative and positive control substances at all time points.

Under conditions of an ACT of approximately 300 seconds, the thrombus adhesion grade was 0 for the negative control substance and 1 for the positive control substance. Furthermore, when comparing the values for the negative control substance and the positive control substance for TAT and β-TG 180 minutes after the start of circulation, a significant increase was observed for the positive control substance. On the other hand, no difference was observed in platelet counts. For SC5b-9, a rapid increase was observed with the positive control substance starting 5 minutes after the start of circulation, and clear differences were observed between the negative and positive control substances at all time points.

From the above, under conditions where the ACT was approximately 200 seconds, clear differences were observed between the negative control substance and the positive control substance in all evaluation items, making it possible to distinguish between negative and positive results. Furthermore, under conditions where the ACT was approximately 300 seconds, no significant differences were observed in visual observation or platelet count, but clear differences were observed in TAT, β-TG, and SC5b-9, making it possible to distinguish between negative and positive results for blood coagulation ability and complement activation using the measurement results of these sensitive parameters. From the above, it was concluded that when these negative and positive control substances were evaluated using the evaluation device 90, it was possible to determine whether they were negative or positive.

Next, the suitability of Evaluation Sample 1 as a positive control substance used in Evaluation Device 90 was evaluated by confirming whether it would produce thrombogenic results in the NAVI model (Non-Anticoagulated Venous Implant Model), which has a proven track record as an in vivo thrombosis test. Furthermore, the results verified the consistency between Evaluation Device 90, an in vitro evaluation system, and the in vivo thrombosis test.

The test was conducted in accordance with ISO 10993-4:2017. One test sample per dog was inserted approximately 12-13 cm into the jugular vein of two hound dogs, toward the heart, and left in place for four hours. To prevent blood coagulation after euthanasia, 500 IU/kg of heparin was administered after the four-hour period to heparinize the blood throughout the body. Five to 15 minutes later, each dog was euthanized.

The jugular vein at the placement site was removed and the blood vessel was incised longitudinally. After the incision, thrombus formation was confirmed around the blood vessel and in evaluation specimen 1.

In both animals, thrombus formation was confirmed around the entire circumference of Evaluation Specimen 1 (see Figure 8). Thrombus formation was observed regardless of whether or not zymosan A was attached. According to the criteria of ISO 10993-4, the highest grade was Grade 4.

Since significant thrombus formation was observed in the positive control substance after placement, it was determined that Evaluation Sample 1 also functions as a positive control substance in the in vivo thrombosis test. Therefore, it was concluded that Evaluation Sample 1 is appropriate as a positive control substance. Furthermore, it was found that the evaluation results of Evaluation Sample 1 were consistent between the in vitro evaluation system, Evaluation Device 90, and the in vivo thrombosis test.

The evaluation specimens according to the present invention have been described above through embodiments and modifications, but the present invention is not limited to the configurations described in the embodiments and can be modified as appropriate based on the claims.

1. Evaluation specimen,
10 first active part,
20 second active part,
90 Evaluation device,
91 Supply Department,
93 Circulation route,
94 Insertion part.

Claims (6)

An evaluation sample used in an evaluation test for the blood compatibility of a medical device,
a first activation part that promotes the coagulation activity and platelet activity of blood used in the evaluation test;
and a second active part that promotes complement activity in the blood,
The first active portion is at least one selected from the group consisting of polyurethane, latex rubber, and polyurethane elastomer,
The evaluation specimen, wherein the second active portion is at least one selected from the group consisting of zymosan, cobra venom, mannan, and agarose . a main body made of a polymer;
a covering portion disposed to cover a portion of the outer surface of the main body portion,
the first active portion is composed of the main body portion,
The evaluation specimen according to claim 1 , wherein the second active portion is formed from the covering portion. 3. The evaluation specimen according to claim 1, wherein the amount of the second active portion is 132.7 to 265.4 g/m ² per unit area of the region where the second active portion is disposed. The evaluation specimen described in any one of claims 1 to 3, wherein the first active portion is a string having a circular cross section. The evaluation specimen according to any one of claims 1 to 4, wherein the surface of the first active portion is textured. An evaluation device used in an evaluation test for blood compatibility of a medical device,
a supply unit to which blood used in the evaluation test is supplied;
a circulation path through which the blood circulates;
an evaluation specimen having a first active part that promotes blood coagulation activity and platelet activity and a second active part that promotes complement activity of the blood , or an insertion part into which the medical device is inserted;
The first active portion is at least one selected from the group consisting of polyurethane, latex rubber, and polyurethane elastomer,
The evaluation device, wherein the second active part is at least one selected from the group consisting of zymosan, cobra venom, mannan, and agarose .