CS265772B1 - Method of heparin removal from blood in vitro - Google Patents

Abstract

The solution relates to the removal method heparin from blood in vitro. nature a method of removing heparin from the blood, mainly from plasma, in vitro in that k 0.5 to 3 vol. part of blood or blood plasma is added, preferably in the form of a tablet comprising 5 to 150 wt. parts of at least one blood-insoluble polysaccharide derivative tertiary or quaternary amine groups with exchange capacity 0.3 to 1.3 mmol.g-1, swell For at least 1 minute, mix intensively mix or leave it until it is heparin adsorbs, the solid is the blood plasma is used for the assays. A blood addition preparation, or blood plasma, contains an extra microcrystalline cellulose in an amount of up to 12 wt. parts based on 1 wt. part of the derivative polysaccharides. Polysaccharide derivative are preferably cellulose derivatives or starch derivatives. The solution is for use in medicine, especially hematology in investigation patients using heparin on real hemostatic potential. Further wherever simple is required and a rapid method of removing heparin from blood or blood plasma.

Description

The invention relates to a method of removing heparin from blood, in particular blood plasma, in vitro.

In the treatment of thromboembolic diseases with heparin, it is necessary to perform complex haemocoagulation tests in the patient, which characterize the patient's state of health and show the success or failure of the treatment process. However, the heparin present in the collected blood samples distorts these tests considerably. Therefore, a quick and reliable method is required to remove it without any side effects on blood plasma. Heparin is commonly neutralized with protamine in clinical practice [J.G. Allen et al., J. Lab. Clin. Med. 34, 473 (1949);

Perkins et al., J. Lab. Clin. Med. 48, 223 (1956) J, but titration is hampered by the fact that heparin levels are not well known in the collected blood plasma of patients. It follows that the excess of protamine in plasma remains undetected respectively. Heparin remains unneutralized in the blood plasma, which negatively affects the results of hemocoagulation tests. In his work, J. S. Wright et al. have already pointed out these problems [j. S. Wright et al., J. Cardiovas. Surg. 5, 244 (1964). To avoid these problems, Thompson et al. [_A. Thompson, R. B. Counts: J. Lab. Clin. Med. 88, 922 (1976) 3 column chromatography to capture heparin and protamine from blood plasma samples. They used a moderately basic cellulose anion exchanger to capture heparin, and protamine adsorbed on carboxymethylcellulose. The use of column chromatography is time-consuming and requires expertise in chromatography. Furthermore, a portion of the blood plasma is captured on ion exchange columns, which is a drawback in pediatric cases where a small amount of plasma is used for testing.

These disadvantages are substantially eliminated by the in vitro method of removing heparin from blood, in particular blood plasma, according to the invention, which is characterized in that 0.5 to 3 vol. part of the blood or blood plasma is added to the preparation, preferably in the form of a tablet containing 5 to 150 wt. parts of at least one blood-insoluble derivative of polysaccharides with tertiary or quaternary amino groups attached having an exchange capacity of 0.3 to

1.3 nunol-g ^- , swell for at least 1 minute, mix vigorously or allow to stand until the heparin is absorbed, separate the solid and blood plasma is used for the tests. The composition added to the blood or blood plasma contains, in addition, microcrystalline cellulose in an amount of up to 12% by weight. parts based on 1 wt. % of the polysaccharide derivative. The polysaccharide derivative is preferably a cellulose derivative or a starch derivative. The cellulose derivative is selected from: diethylaminoethyl cellulose, dimethylaminoethyl cellulose, trimethylammonium cellulose, diethylaminopropyl cellulose, triethylammonium propyl cellulose, dimethylaminopropyl cellulose, trimethylammonium propyl cellulose, diethylamino-2-hydroxypropyl cellulose, 2-hydroxypropyl cellulose, triethylamine cellulose, triethylamine cellulose, cellulose, cellulose cross-linked with a mixture of epichlorohydrin and diethylamine in an alkaline medium, imidazolium-N, N'-diyl-bis- (2-hydroxy-3-propyl) cellulose ether, dimethylamino-bis- / 2-hydroxy-3-propyl chloride t-cellulose ether, benzyldimethylammonium-2-hydroxypropyl cellulose. The starch derivative is selected from the group: diethylaminoethyl milled starch, dimethylaminoethyl milled starch, triethylammonium propyl milled starch, dimethylaminopropyl milled starch, trimethylammonium propyl Milled starch, diethylamino-2-hydroxypropyl milled starch, polyethyleneimine milled starch, triethylammonium 2-starch, triethylammonium -hydroxypropyl cross-linked starch, trimethylammonium methyl cross-linked starch, diethylaminopropyl cross-linked starch, starch cross-linked with a mixture of epichlorohydrin and diethylamine in an alkaline medium, imidazolium-N, N'-diyl-bis- (2-hydroxy-3-propyl) ether of cross-linked starch, chloride dimethylamino-bis- (2-hydroxy-3-propyl) ether of cross-linked starch, benzyldimethylammonium-2-hydroxypropyl cross-linked starch. The polysaccharide derivative consists of particles having a size of 50 to 300 µm, but it is also possible to use a polysaccharide derivative with a smaller or larger size.

The advantage of the proposed method of removing heparin from the blood being examined, resp. In vitro blood plasma is that it uses a progressive method of the test pill as a quick and simple method to completely remove clinical levels of heparin from blood plasma to determine true haemostatic potential and monitor other coagulation disorders in the patients under investigation. A further advantage is that the test preparation with adsorbed heparin is completely separated in the plasma, so that it cannot affect subsequent haemocoagulation tests in contrast to the previous methods.

Example 1 <·

Κ 0.5 ml of blood plasma in a tube (12x95 mm) is added a tablet containing 5 mg imidazolium-N, N'-diyl-bis (2-hydroxy-3-propyl) cellulose ether chloride with an exchange capacity of 1.3 mmol g ¹ and 6Ό mg of microcrystalline cellulose and swell for 0,5 min at 20 ° C. The contents of the tube are then vigorously mixed on a vibrator until the tablet particles are evenly distributed in the plasma and stirred for another 10 minutes at 20 ° C on a mixer. The heparin-adsorbed tablet substance is centrifuged in a laboratory centrifuge (1200 x g, 5 min) and appropriate plasma aliquots are used for hemocoagulation assays.

Example 2

Κ 1 ml of blood plasma in a tube (12x95 mm) is added a tablet containing 20 mg of imidazolium-N, N'-diyl-bis (2-hydroxy-3-propyl) cellulose ether with a capacity of 0.7 mmol.g ¹ and 60 mg of microcrystalline cellulose and allowed to swell at 22 ° C for at least 1 min. Then the contents of the tube are vigorously mixed on a vibrator until the tablet substance is evenly distributed in the plasma and mixed further on a magnetic stirrer for 10 min at 22 ° C. The heparin-adsorbed tablet substance is centrifuged in a laboratory centrifuge (1200 x g, 5 min) and appropriate plasma aliquots are used for hemocoagulation assays.

Example 3

To 3 ml of blood plasma in a tube (12 x 95 mm) is added a tablet containing 30 mg of imidazolium-N, N'-diyl-bis (2-hydroxy-3-propyl) cellulose ether with a capacity of 1.3 mmol.g ¹ and 150 mg of microcrystalline cellulose and allowed to swell at 22 ° C for 2 min. Then the contents of the tube are vigorously mixed on a vibrator until the tablet substance is evenly distributed in the plasma and allowed to stand at 22 ° C for 15 min. The heparin-adsorbed tablet substance is centrifuged in a laboratory centrifuge and the appropriate plasma aliquots are used for hemocoagulation tests.

attaches

The procedure is as in Example 3 except that a 150 mg tablet of imidazolium-N, N'-diyl-bis (2-hydroxy-3-propyl) -cellulose ether with a capacity of 0.3 mmol.g ^{1 is used.} .

Example 5

The procedure is as in Example 1 except that a tablet containing 5 mg of diethylaminoethyl cellulose with an exchange capacity of 1 mmol.g ^-1 and 60 mg of microcrystalline cellulose is added.

Example 6

The procedure is as in Example 2, except that a tablet containing 20 mg of trimethylammonium methyl cellulose with an exchange capacity of 0.7 mmol.g ^-1 and 60 mg of microcrystalline cellulose is added.

Example 7

The procedure is as in Example 3 except that a tablet containing 30 mg of triethylammonium propyl cellulose with an exchange capacity of 0.7 mmol.g ^-1 and 150 mg of microcrystalline cellulose is added.

Example 8

The procedure is as in Example 4 except that the 150 mg tablet was added diethylamino-2-hydroxypropyl cellulose having an exchange capacity of 0.3 mmol per gram ^1st

The procedure is as in Example 1 except that a tablet containing 5 mg of the active substance prepared by reacting cellulose, epichlorohydrin and diethylamine in an alkaline medium with a capacity of exchange of 1.3 mmol.g ¹ and 60 mg of microcrystalline cellulose is added.

Example 9

Example

The procedure is as in Example 2 except that the addition tablet containing 20 mg of polyethyleneimine cellulose having an exchange capacity of 0.6 mmol per gram ¹ and 60 mg of microcrystalline cellulose.

Example

The procedure is as in Example 3 except that a tablet containing 30 mg of dimethylaminoethyl cellulose with a capacity of 0.7 mmol / ^l and 150 mg of microcrystalline cellulose is added.

Example

The procedure is as in Example 4 except that the 150 mg tablet was added triethyl-2-hydroxypropyl cellulose having an exchange capacity of 0.3 mmol per gram ^1st

Example 13 was followed as in Example 1 except that a tablet containing 5 mg of trimethylammonium propyl cellulose with a capacity of 0.6 mmol.g ^-1 and 60 mg of microcrystalline cellulose was added.

Example 14

The procedure is as in Example 2 except that a tablet containing 20 mg of dimethylamino-bis (2-hydroxy-3-propyl) cellulose ether with a capacity of 0.6 mmol.g ^-1 and 60 mg of microcrystalline cellulose is added. .

Example 15

The procedure is as for Example 3 except that the addition tablet containing 30 mg of benzyldimetylamónium-2-hydroxypropyl cellulose having an exchange capacity of 0.6 mmol per gram, and ¹ 150 mg of microcrystalline cellulose.

Example 16;

viv

The procedure is as in Example 1 except that a tablet containing 5 mg of imidazolium-N, N'-diyl-bis (2-hydroxy-3-propyl) ether of cross-linked starch with a capacity of 1.3 mmol is added. g ^-1 and 60 mg microcrystalline cellulose.

Example 17

The procedure is as in Example 2 except that a tablet containing 10 mg of diethylaminoethyl cellulose and 10 mg of diethylaminohydroxypropyl cellulose with an exchange capacity of 0.7 mmol.g ^-1 and 60 mg of microcrystalline cellulose is added.

Example 18

The procedure is as in Example 3 except that a tablet containing 15 mg of imidazolium-N, N'-diyl-bis- (2-hydroxy-3-propyl) cellulose ether and 15 mg of imidazolium--chloride is added, Starch di -diyl-bis (2-hydroxy-3-propyl) ether with a capacity of 1.3 mmol.g ³ and 150 mg microcrystalline cellulose.

Example 19

The procedure is as in Example 2 except that a tablet containing 10 mg of diethylaminoethyl starch and 10 mg of diethylaminohydroxypropyl starch with an exchange capacity of 0.7 mmol.g ^-1 and 60 mg of microcrystalline cellulose is added.

The invention has utility in medicine, in particular hematology, in the examination of patients using heparin for real hemostatic potential. Furthermore, wherever a simple and rapid method of removing heparin from blood or blood plasma is required.

Claims (5)

A method of removing heparin from blood, in particular from plasma in vitro, characterized in that 0.5 to 3 vol. a portion of blood or blood plasma is added to the composition, preferably in the form of a tablet containing 5 to 150 wt. parts of at least one blood-insoluble polysaccharide derivative having pendant tertiary amine groups with a quaternary exchange capacity from 0.3 to 1.3 mmol per gram ^1, allowed to swell for at least 1 minute, the mixture was vigorously stirred and allowed to stand until the adsorbed heparin, the solid was collected and blood plasma was used for the tests. 2. A method according to claim 1, wherein the preparation added to the blood or blood plasma contains, in addition, microcrystalline cellulose in an amount of up to 12% by weight. parts per 1 wt. % of the polysaccharide derivative. 3. A method according to claim 1, wherein the polysaccharide derivative is preferably a cellulose derivative or a starch derivative. 4. The method according to claim 3, wherein the cellulose derivatives are selected from the group: diethylaminoethyl cellulose, dimethylaminoethyl cellulose, trimethylammonium methyl cellulose, dimethylaminopropyl cellulose, trimethylammonium propyl cellulose, diethylamino-2-hydroxypropyl cellulose, polyethylenimine cellulose, triethylenimine cellulose, triethylenimine cellulose, triethylenimine cellulose. -hydroxypropyl cellulose, dimethylamino-2-hydroxypropyl cellulose, cellulose Combined with a mixture of epichlorohydrin and diethylamine in an alkaline medium, imidazolium-N, N'-diyl-bis- (2-hydroxy-3-propyl) cellulose ether, dimethylamino-bis chloride (2-hydroxy-3-propyl) cellulose ether, benzyldimethylammonium-2-hydroxypropyl cellulose, diethylaminopropyl cellulose, triethylammonium propyl cellulose. 5. The method according to claim 3, wherein the starch derivative is selected from the group: diethylaminoethyl starch, dimethylaminoethyl starch, triethylammonium propyl starch, dimethylaminopropyl starch, trimethylammonium propyl starch, diethylamino-2-hydroxypropyl starch. polyethyleneimine Cross-linked starch, triethylammonium-2-hydroxypropyl cross-linked starch, dimethylamino-2-hydroxypropyl cross-linked starch, starch cross-linked with a mixture of epichlorohydrin and diethylamine in an alkaline medium, imidazolium · N, N'-diyl-bis (2-hydroxy-3-propyl) cross-linked starch ether, dimethylamino-bis- (2-hydroxy-3-propyl) cross-linked starch ether, benzyldimethylammonium-2-hydroxypropyl cross-linked starch, trimethylammonium ethyl cross-linked starch, diethylaminopropyl cross-linked starch.