# US 10,768,099 B2 · In-vitro biological valve calcification evaluation method and reducing calcium ingredient solution

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- Number: US 10,768,099 B2
- Document ID: US10768099B2
- Status: Granted
- Kind: B2
- Application number: 15/876,908
- Filed: 2018-01-22
- Published: 2018-05-31
- Granted: 2020-09-08
- Earliest claimed: 2015-07-22
- Assignees: Venus Medtech (Hangzhou), Inc.
- Applicants: Venus Medtech (Hangzhou), Inc.
- Inventors: Nan Shao; Dajun Kuang
- Web page: https://patentgrep.com/patent/US10768099B2

## CPC

- G01N 21/31: Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry {(G01N21/72 takes precedence)}
- A61F 2/2433: {using balloon catheter}
- A61L 27/3687: {characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents}
- G01N 1/28: Preparing specimens for investigation {including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q}(mounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 {; laboratory gas handling apparatus B01L5/00})
- G01N 1/38: Diluting, dispersing or mixing samples
- G01N 21/00: Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light (G01N3/00 - G01N19/00 take precedence)
- G01N 1/4044: {by chemical techniques; Digestion; Chemical decomposition}
- G01N 1/44: Sample treatment involving radiation, e.g. heat
- G01N 21/3103: {Atomic absorption analysis}
- G01N 27/30: Electrodes, e.g. test electrodes; Half-cells (G01N27/414 takes precedence)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61F: FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS (dental prosthetics A61C)
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)

## Abstract

An in-vitro biological valve calcification evaluation method, comprising the following steps: preparing calcification evaluation solution comprising glutaraldehyde solution, saline solution, borate buffer solution, reducing calcium ingredient solution and calcium-containing plasma solution, the reducing calcium ingredient solution comprising alcohol, alkaline solution, oil and its derivatives, and buffer solution; preheating the reducing calcium ingredient solution; conducting treatment with the glutaraldehyde solution prior to reducing calcium ingredient solution; conducting treatment with the reducing calcium ingredient solution; preserving the valve samples; conducting in-vitro calcification reaction with the calcium-containing plasma solution; and detecting calcium content by atomic absorption spectrum.

## Claims

### Claim 1

An in-vitro biological valve calcification evaluation method, comprising the steps of:
(1) preparing calcification evaluation solutions, the calcification evaluation solutions comprising: glutaraldehyde solution, saline solution, borate buffer solution, reducing calcium ingredient solution and calcium-containing plasma solution, wherein the reducing calcium ingredient solution comprises an alcohol, an alkaline solution, an oil and/or its derivatives, and a buffer solution;
(2) preheating the reducing calcium ingredient solution by placing the reducing calcium ingredient solution in a 30 to 40° C. thermostat water bath;
(3) conducting treatment prior to reducing calcium ingredient solution by taking a biological valve sample and soaking the biological valve sample in the glutaraldehyde solution completely, and maintaining the biological valve sample at room temperature;
(4) conducting treatment with the reducing calcium ingredient solution by removing the glutaraldehyde solution, and adding the borate buffer solution, immersing the sample therein, then removing the borate buffer solution, and soaking the sample in the reducing calcium ingredient solution completely within a 30 to 40° C. thermostat water bath;
(5) preserving the biological valve samples by removing the reducing calcium ingredient solution from a container and adding the saline solution until the biological valve sample is completely soaked, and storing the sample in the container;
(6) conducting an in-vitro calcification reaction by putting the biological valve sample into the calcium-containing plasma solution in a 30 to 40° C. thermostat water bath for a period of any days within 60 days to let the biological valve sample fully react with the given calcium ions of the solution, such that calcium ions are bonded to the biological valve sample by chemical bonding ;
(7) detecting calcium content measuring the calcium content of the biological reacted valve sample by atomic absorption spectrometry to evaluate the calcium content of the biological valve sample, and comparing it with a standard sample.

### Claim 2 (depends on 1)

The in-vitro biological valve calcification evaluation method according to claim 1, wherein the concentration of the glutaraldehyde solution is from 0.25% to 5.0% and compositions and contents of each 5 L volume of the glutaraldehyde solution comprises: 1.0 to 3.0 g of magnesium sulfate; 1.0 to 3.0 g of potassium chloride; 3.0 to 5.0 g of disodium hydrogen phosphate; 0.6 to 1.2 g of potassium dihydrogen phosphate; 15 to 30 g of sodium chloride; 150 to 250 ml of 25% glutaraldehyde; 500 to 1000 ml of isopropanol; and pure water to complete to 5 L total volume.

### Claim 3 (depends on 1)

The in-vitro biological valve calcification evaluation method according to claim 1, wherein the concentration of the saline solution is 0.9%, the compositions and contents of each 10 L volume of the saline solution comprises: 90 g±0.1 g of sodium chloride; and pure water to complete to 10 L total volume.

### Claim 4 (depends on 1)

The in-vitro biological valve calcification evaluation method according to claim 1, wherein the compositions and contents of each 1 L volume of the borate buffer solution comprises: 3.0 to 15.0 g of sodium chloride; 2.0 to 5.0 g of sodium tetraborate; and pure water to complete to 1 L total volume, with a pH of 8.5 to 9.5.

### Claim 5 (depends on 1)

The in-vitro biological valve calcification evaluation method according to claim 1, wherein the compositions and contents of each 1 L volume of the reducing calcium ingredient solution comprises: 150 to 300 ml of reagent A, wherein reagent A is an alcohol; 10 to 100 ml of reagent B, wherein reagent B is an alkaline solution; 0.5 to 9.0 g of reagent C, wherein-the reagent C is an oil or a derivative thereof; 690 to 820 ml of reagent D with a pH of 8.5 to 9.5, wherein reagent D is a buffer solution.

### Claim 6 (depends on 1)

The in-vitro biological valve calcification evaluation method according to claim 1, wherein the calcium-containing plasma is a plasma substitute, the compositions and contents of each 1 L volume of the calcium-containing plasma comprises: 10 to 15 ml of 10% CaCl2 solution; and 900 to 1000 ml of hydroxyethyl starch sodium chloride injection.

### Claim 7 (depends on 5)

The in-vitro biological valve calcification evaluation method according to claim 5, wherein reagent A comprises any one of ethanol, isopropanol, and amyl alcohol.

### Claim 8 (depends on 5)

The in-vitro biological valve calcification evaluation method according to claim 5, wherein reagent B comprises KOH or NaOH.

### Claim 9 (depends on 5)

The in-vitro biological valve calcification evaluation method according to claim 5, wherein reagent C comprises one or more of tetraenoic acid, linoleic acid, eicosatrienoic acid, linolenic acid, aminoleic acid, lauric acid, heptanoic acid, palmitic acid, stearic acid, oleic acid, and derivatives thereof.

### Claim 10 (depends on 5)

The in-vitro biological valve calcification evaluation method according to claim 5, wherein reagent D is buffer solution comprising phosphate buffer solution, borate buffer solution or glycine hydrochloride buffer solution.

### Claim 11

A reducing calcium ingredient solution, comprising:
reagent A;
reagent B;
reagent C; and
reagent D,
wherein reagent A is an alcohol, comprising any one of ethanol, isopropanol and amyl alcohol; reagent B is an alkaline solution, comprising KOH or NaOH; reagent C is an oil and/or its derivatives, comprising one or more of tetraenoic acid, linoleic acid, eicosatrienoic acid, linolenic acid, aminoleic acid, lauric acid, heptanoic acid, palmitic acid, stearic acid, and derivatives thereof; and reagent D is a buffer solution with a PH of 8.5 to 9.0 comprising phosphate buffer, borate buffer or glycine hydrochloride buffer.