RU2270020C2 - Method for production of control plasma with normal level of prothrombin complex - Google Patents

Abstract

FIELD: medicine, in particular production of lyophilized diagnosis reagent, namely, control plasma with normal level of prothrombin complex.

SUBSTANCE: claimed method includes donor blood sampling, plasma separation by centrifugation, blending of donor's plasma, pool centrifugation, followed by canning and lyophilization of finished product. Method is characterized that after the second centrifugation pool is blended with buffer HEPES to finished concentration of 0.004±0.0005 M, then frost and quenched at -38°C or less for 4-6 h. Lyophilization is carried out for 17±1 h at product starting temperature not more than -(54±14°C); during 10±1 h temperature is increased to the positive value; and lyophilization is finished at +(12±3)°C. Diagnosis reagent of present invention is useful in calculation of prothrombin index according to the formula %-PI and/or plotting of calibration curve for calculation of prothrombin complex factor activity.

EFFECT: diagnosis reagent containing normal level of prothrombin complex and being stable for one year.

1 ex, 2 tbl, 1 dwg

Description

The invention relates to medicine and relates to the production of a lyophilized diagnostic reagent - a control plasma with a normal and stable for at least 1 year level of factors of the prothrombin complex used to calculate the prothrombin index (% -PI) according to the formula and / or construct a calibration graph for calculating the activity of factors prothrombin complex.

Currently, there are numerous modifications of the methods for determining these procoagulants, the reproduction of which uses different control reagents to build calibration graphs and analytical calculations according to the formula. This makes it difficult to obtain comparable results. Foreign companies (Boehringer Mannheim, Dade, Sigma, George king bio-medical, Hospitex diagnostics, Human, Grifols, Biomerieux) produce lyophilized reagents: International and National. However, the technology of their manufacture is patented. In addition, imported diagnostics and test systems are expensive.

As a prototype, we used the previously developed "Method for producing a donor standard plasma" (patent N 1592717, authors: G.K. Platonova, L.N. Tarasova). A significant disadvantage of the prototype is that the drying schedule used is quite long and is at least 24 hours; the maximum temperature of the product is not higher than + 5 ° C. In addition, the expensive HEPES reagent is injected directly into the preservative solution before blood collection; its loss occurs during the deposition of shaped elements.

The aim of the present invention is to obtain a diagnostic reagent - control plasma from a fresh donor pool, combined from at least 15 donors, containing a normal level of prothrombin complex, stable for 1 year, to calculate the prothrombin index by the formula (% -PI) and / or construction calibration graph for calculating the activity of factors of the prothrombin complex.

This goal is achieved by the fact that the HEPES buffer substance is introduced into the plasma at the rate of 0.004 ± 0.0005 M after separation of the formed elements, and not into donated blood, and dried by lyophilization according to the following drying schedule: the initial product temperature is not higher than minus 54 ± 14 ° C, reaching a positive temperature after 10 ± 1 hour and completion at a temperature of 12 ± 3 ° C; the duration of the sublimation process is 17 ± 1 hour.

After lyophilization, the loss in mass upon drying is determined, which should not exceed 2%; pH 7.35 ± 0.15; prothrombin index should be within 99 ± 5%, fibrinogen concentration of 2.75 ± 0.75.

METHOD DESCRIPTION EXAMPLE

A control plasma with a normal level of factors of the prothrombin complex is obtained as follows: blood of at least 15 donors is taken in bottles with a preservative (sodium citrate, sodium azide and contracal) in a ratio of 9: 1. Centrifuged at 1800-2000 rpm and a temperature of + (4-6) ° C for 30 minutes. The obtained plasma samples of each donor are separated from the formed elements, mixed, obtaining a pool, and re-centrifuged in the same mode. The supernatant is separated and HEPES buffer substance is added to it to a final concentration of 0.004 ± 0.0005 M. The resulting native control plasma is poured 1 ml into 10 ml siliconized penicillin vials. They are frozen and quenched on a sublimator plate at a temperature of no higher than -38 ° C for 4-6 hours. Freeze-drying is carried out according to the schedule for drying the control plasma (table 2, drawing).

After lyophilization, the mass loss upon drying of the diagnostic reagent, the prothrombin index (% -PI), and the concentration of fibrinogen are determined. As a control, when calculating% -PI, a pool of fresh plasma of at least 20 donors is used. If this indicator is within 99 ± 5%, and the fibrinogen concentration (F) is not less than 2.0 g / l, the reagent is used as a control plasma for calculating the prothrombin index and / or activity of the prothrombin complex factors when conducting studies with plasma samples of patients and / or healthy individuals.

EXPERIMENTAL RESEARCH

(conducted at the Kirov Research Institute of Hematology and Blood Transfusion and the Republican Blood Transfusion Station at the Institute).

The developed method was used to obtain 5 series of control plasma. In the manufacture of series 040901, 010302, 020602, 030902 and 041202, blood of 17, 17, 16, 18 and 20 donors were used, respectively. The loss in mass upon drying of the reagent of all the series obtained did not exceed 2%. Determination of pH, prothrombin index and fibrinogen of control plasma samples was carried out after lyophilization, after 6 and 12 months. The research results are shown in table 1. They indicate that the reagent can be used as a control plasma with a normal and stable level of prothrombin complex factors for at least 1 year and used to calculate the prothrombin index (% -PI) by the formula and / or constructing a calibration graph for calculating the activity of the prothrombin complex.

The developed method will find application in the creation of production technology for the production of domestic control donor plasma, the use of which is convenient in clinical diagnostic and specialized hemostasis laboratories, allows to obtain comparable research results; in addition, the reagent is much cheaper than foreign analogues.

Table 1 Some indicators of the control plasma during storage Series No. Shelf life (months) After lyophilization 6 12 pH % PI F g / l pH % PI F g / l pH % PI F g / l 040901 7.35 98.5 2.75 7.38 97.0 3.00 7.40 96.5 3.00 010302 7.25 99.0 3.00 7.28 98.5 2,50 7.30 98.0 2.00 020602 7.35 99.0 3,50 7.40 98.5 3.00 7.43 100.0 2.75 030902 7.29 99.0 2,50 7.32 98.0 2.75 7.35 97.5 2.75 041202 7.30 99.5 2,50 7.31 98.0 3.00 7.30 97.0 3.00

table 2 DRYING SCHEDULE Control plasma series 020602 Serial number Lyophilization process registration time (hour) Cartridge temperature (in degrees C) Product temperature (in degrees C) one 19 -68 -46 2 twenty -67 -44 3 21 -26 -33 four 22 -25 -29 5 23 -fifteen -24 6 24 -fifteen -23 7 one 0 -eighteen 8 2 +4 -fifteen 9 3 +5 -8 10 four +5 0 eleven 5 +6 +3 12 6 +6 +4 13 7 +7 +6 fourteen 8 +7 +7 fifteen 9 +8 +8 16 10 +9 +9 17 eleven +10 +10

Claims (1)

A method of obtaining a lyophilized control plasma with a normal level of the prothrombin complex, including taking blood from donors in vials with a preservative, separating the plasma by centrifugation at 1800-2000 rpm for 30 minutes, separating it from the cells, pooling the donor plasma, centrifuging the pool under the same conditions, bottling and lyophilization, characterized in that after repeated centrifugation, HEPES buffer substance is added to the plasma pool to a final concentration (0.004 ± 0.0005) M, then it is frozen and quenched t at a temperature of no higher than -38 ° C for 4-6 hours, lyophilization is carried out starting from the product temperature not higher than (54 ± 14) ° C with reaching a positive temperature after (10 ± 1) h and completion of the lyophilization process at temperature + (12 ± 3) ° С, using a drying schedule of (17 ± 1) h.