Method for increasing quality in keeping raw material activity to manufacture complete soluble thromboplastin reagent before and during sublimation

FIELD: medicine.

SUBSTANCE: raw material obtained out of gray substance homogenate should be supplemented with ionol (2.6-di-tret-butyl-4-methylphenol) at quantity necessary to prepare 0.0001 M final solution. Addition of this substance to raw material enables to keep its high activity being equal to 12.5-16.0 sec and enables to prolong its storage terms at (-40 ± 2) C up to 10 wk before carrying out sublimation and prevent the decrease of activity during sublimation process.

EFFECT: higher efficiency.

1 ex, 2 tbl

 

The invention relates to medicine and relates to a method of improving the quality of conditions of activity of the raw materials for the manufacture of reagent thromboplastin full-soluble, used to determine the prothrombin time (PT, C) plasma (blood), before and during sublimation.

Reflecting the state of the external path of blood clotting, prothrombin time widely used in clinical diagnostic laboratories for screening studies of the coagulation system, monitoring of therapy with anticoagulants of indirect action, diagnosis of liver disease, lack of vitamin K. the Principle of the method is based on the activation of coagulation cascade tissue thromboplastin - analogue of tissue factor.

As the prototype used in our patent No. 2161975 “a Method for preserving the activity of the raw materials for the manufacture of thromboplastin full instant before and during freeze-drying” (1).

A significant disadvantage of the prototype is that it is necessary to use several protective substances that retains the activity of the raw materials. These are antioxidants, cryoprotectants, and buffer systems. Preparation and mixing them with the obtained raw material, four-time monitoring of pH and activity (in seconds) with control plasma (a mixture of fresh plasma at least 10 donors) are procedures is mi long and laborious requiring four substances, as well as preparation of the buffer.

The aim of the present invention is to improve the quality and duration of preservation of raw materials from gray matter cadaver brain to 10 weeks before freeze-drying and during its implementation.

This goal is achieved by the fact that as a protective substances use only one substance, BHT (2,6-di-tert-butyl-4-METHYLPHENOL), which, according to the literature(2, 3, 4, 5), stabilizes coagulation activity of thromboplastin and increases resistance to thermal denaturation during freeze-drying. It also prevents oxidative modification of protein and lipid parts of the device during storage. Therefore, BHT acts as antioxidant, cryoprotectant and buffer systems simultaneously after mixing the substrate with this protective substance to be tested raw material once through pH control and activity (MF, C). During storage of raw materials at (-40±2)°to sublimation, the pH values are in the range of 6.5 to 7.4, and the activity does not exceed 14 C. the storage Time of frozen raw materials for which the activity is not reduced, increasing to 10 weeks. This is important when creating the production technology of the biological reagent is soluble thromboplastin.

WHEN THE EP DESCRIPTIONS of the WAY

Highly active raw materials for the manufacture of soluble thromboplastin derived from gray matter cadaver brain according to our patent No. 2260111 "a Method to obtain high raw material for the manufacture of thromboplastin full-soluble" (6). The homogenate gray matter twice frozen at -40°C and thawed at +36°With, then twice diluted with equal volumes of purified water at +36°and centrifuged at 600G and a temperature of +5°C for 30 minutes, obtaining the supernatant raw material for the manufacture of soluble thromboplastin high thromboplastin activity. To it add BHT for the preparation of 0.0001 M of the final solution. Check pH and activity (MF, C) with control plasma immediately after cooking, then as needed after 4 and 10 weeks of storage at (-40±2)°C. If the received values satisfy (pH of 6.4 to 7.4, and the activity is not more than 14 (C), the mixture is ready for sublimation. After freeze-drying have the same control.

EXPERIMENTAL STUDIES

conducted on the basis of the laboratory of biochemistry of blood and NGOs GU Kirov research Institute of Hematology and blood transfusion.

Cooked 5 series thromboplastin reagent soluble without the use of protective substances and 7 series with a protective substance. Stability of the reagent with protective vasectomies method accelerated degradation during storage for one month at +37° C. Degradation in these conditions is equivalent to 1 year of storage of the reagent at 4-8°C. the Results are shown in tables 1 and 2.

Table 1

Characteristics of the reagent prepared without protective substances during storage
the number of seriesTimes (weeks)
pHPV(C)
Source410after sublimationsource410after sublimation
0104036,407,007,158,2516,518,019,521,0
020403the 7.658,058,108,6018,320,520,522,0
0404037,187,70the 7.858,1015,018,5of 21.922,5
0504037,22the 7.85of 7.908,3014,519,020,022,0
0704037,12the 7.858,05 8,3515,018,019,520,0
HSR7,11of 7.697,818,3215,8618,80to 20.2821,50
±m0,200,180,170,080,690,460,440,45

The data in table 1 suggests that the storage of raw materials, prepared without the use of protective substances, called it a significant alkalization; pH changed from 7.11 to 8.32. Such changes of acid-base balance has led to unacceptable reduction in the activity of the raw materials; the values of PV (s) changed from 15,86 up of 21.50.

Table 2

Characteristics of the reagent prepared with protective substances during storage
the number of seriesTime studies
pHPV(C)
to sublimationafter sublimationafter degradationto sublimationafter sublimationafter degradation
3103036,456,50 6,6012,5to 12.015,5
3203036,606,506,5512,5to 12.016,0
4003036,556,606,60to 12.014,518,0
8103036,506,556,5512,514,015,5
8203036,606,556,6512,514,016,5
1004036,856,85to 6.8013,514,0of 17.0
1104036,856,906,9013,014,014,5
HSR6,636,646,6612,6413,5016,14
±m0,060,060,050,180,390,43

The results in table 2 show the changes of pH values and activity (MF, C) 7 series of soluble thromboplastin reagent prepared using as a protective substance of ionol (2,6-di-tert-butyl-4-METHYLPHENOL). When using the AI pH values did not change and was equal to 6,63 and 6,64, which helped to maintain the high activity of the substrate equal 12,64 and 13.50 for 10 weeks prior to freeze-drying and after it. After accelerated degradation, equivalent to one year of storage of the reagent, the pH and activity (MF, C) has changed slightly.

Thus, we have developed a way to keep the activity of the raw materials to freeze-drying for 10 weeks at (-40±2)°and during sublimation and subsequent storage of the received reagent. It will be used when creating the production technology of soluble thromboplastin reagent.

LITERATURE

1. Patent No. 2161975 from 20.01.2001, the method of preserving the activity of the raw materials for the manufacture of thromboplastin full instant before and during lyophilization /GU KNIG and PC, Repnikova E.M., Tarasova, L.N., Savinykh EJ, Gonin L.N., Reshetnikova NR

2. Vartanyan PS, Gurevich, S.M. the Effect of BHT on the metabolism of superoxide radicals in the liver of mice /Questions of medical chemistry. -1999.-so 45.-4.-s-320.

3. Samuilov V.D., Barsky EL, Samuilov VD Interaction of the antioxidant 2,6-di-tert-butyl-4-METHYLPHENOL with photosystem 2 of chloroplasts /Biochemistry. -1995. -so 60. -VIP. -s-1859.

4. Barsky EL, Kravtsova TR, Samuilov V.D., Samuilov AD Action of phenolic antioxidants nature on photosynthetic transfer is of elektronov in chloroplasts /Biochemistry. -1992. -so 57. -9. -s-1431.

5. Zubairov D.M. Molecular basis of blood coagulation and clot formation. -Kazan: FENG. -2000, 49-51.

6. Patent No. 2260111 from 10.12.2000, the Method to obtain high raw material for the manufacture of thromboplastin complete soluble /GU KNIG and PC, Tarasova, L.N., Repnikova E.M., Savinykh EJ, Reshetnikova NR, Gonin L.N.

The way to improve the quality of preservation activity raw materials for the manufacture of soluble thromboplastin reagent before and during freeze-drying, characterized in that obtained from the gray matter cadaver brain raw material is introduced BHT (2,6-di-tert-butyl-4-METHYLPHENOL) in an amount necessary for the preparation of 0.0001 M of the final solution, and get the target product, retaining a high level of activity equal to 12.5-16,0 C, at pH 6.4-7.4 for 10 weeks storage at (-40±2)°and during freeze-drying.



 

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