RU2262109C2 - Method for determination of blood plasma antithrombin iii concentration - Google Patents

Abstract

FIELD: medicine, clinical laboratory diagnosis.

SUBSTANCE: method involves using carboxylated polystyrene latex spherical particles suspension with immobilized specific polyclonal antibodies raised against antithrombin III on their surface and wherein milk α-casein (antithrombin III-latest) is added as an inert component. Method for assay involves successive fractional plasma dilutions and the concentration of antithrombin III in the parent sample is calculated by the formula. Method provides rapid determination of antithrombin III concentration in blood plasma. Method can be used for diagnosis of pathological states in the blood coagulation system.

EFFECT: improved assay method.

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Description

The proposed method relates to medicine, namely to methods for quantitative determination of antithrombin III (AT III) in the bloodstream, and can be used to diagnose pathological conditions of the blood coagulation system - various types of thrombophilia, disseminated intravascular coagulation (DIC).

AT III is the main physiological anticoagulant factor in the blood coagulation system, blocking thrombin in the presence of heparin, as well as other serine proteinases involved in blood coagulation. AT III accounts for more than 80% of all anticoagulant blood activity. AT III deficiency leads to the development of various types of thrombophilia and occurs due to a decrease in its synthesis in severe diseases and liver damage, as well as as a result of its intensive consumption during blood coagulation, for example, with DIC, which develops with sepsis, shock, and burn disease, combined injuries, obstetric pathology, malignant neoplasms, infectious diseases, as well as with prolonged treatment with fibrinolytics, heparin. The level of AT III in the blood normally is 235 ± 25 μg / ml, in patients it decreases by 25-50%.

In clinical and laboratory practice, mainly indirect (indirect) methods for determining the content of AT III in blood plasma are used, based on the assessment of its activity, determined by the residual activity of the standard preparation of thrombin after its inactivation by AT III-heparin complex. Known indirect method for determining the content of AT III in blood plasma, in which fibrinogen is used as a substrate for thrombin, the activity of AT III is judged by the time of fibrin formation [Barkagan Z. S., Makarov V.A., Lychov VT. New methods for laboratory diagnosis of disseminated intravascular coagulation (DIC). Methodical recommendations, M., 1989, p.23]. AT III activity is expressed as a percentage; the plasma mixture of healthy donors is taken as 100%. In healthy people, the activity of AT III, determined in this way, ranges from 85-115%. The method is accessible and simple to perform, but cannot be considered reliable enough: being indirect, it does not provide strict specificity of determination, since side factors, for example, drugs circulating in the blood, can influence the time of fibrin formation, in addition to the residual thrombin activity, which leads to a distortion of the determination results. In addition, the lack of sensitivity of the method does not allow to detect the occurrence of AT III deficiency in the early stages of the disease.

Known indirect method for evaluating the activity of AT III in blood plasma, based on the use of strictly specific thrombin low molecular weight chromogenic substrate [J. Fareed et al. "Application of syntetic chromogenic peptid substrate in the evalution of coagulation enzymes and their inhibitors", 1979]. Possessing sufficient sensitivity, this method, as an indirect one, can also give a distorted result due to the influence of side factors. In addition, the application of this method is limited by the need to use expensive special equipment and appropriate sets of reagents.

Direct (direct) methods for the quantitative determination of AT III in blood plasma provide high sensitivity, strict specificity and are characterized by high reliability, however, as a rule, they are difficult to perform, require a lot of labor and time and special equipment and reagents. Known enzyme immunoassay method for direct quantitative determination of AT III in blood plasma, carried out as a multi-stage sequence of immunological and enzymatic reactions [T. Andersson et al. Thrombos. Res., 1996, v. 82, p. 109]. AT III interacts with antibodies specific to it, immobilized on the surface of the holes of polystyrene plates. Quantification of AT III is carried out by spectrometric analysis of the resulting colored products of enzymatic hydrolysis. Despite the high sensitivity and specificity, the method is not used in everyday laboratory practice, because it is difficult to perform and requires expensive equipment and reagents.

A known method of laser nephelometry, in which the formation of immune complexes of AT III with antibodies specific to it, proceeds in solution and is accompanied by an increase in light scattering [K. Akiyata et al. Thrombos. Res., 1993, v. 72, p. 193]. The method provides a quick result, characterized by high sensitivity and specificity, but requires the use of extremely expensive imported equipment, therefore, is not available for use in everyday medical practice.

Closest to the proposed method for determining the content of AT III in blood plasma is the prototype method of radial immunodiffusion. (Encyclopedia of clinical laboratory tests. / Ed. N. Titsa, trans. From English M., Izv. Lab. Inform., 1997, p.1137]. The method is based on the interaction of AT III with polyclonal antibodies specific to it, placed in a 1% agar gel. Blood plasma samples are poured into the holes punctured in the gel, and then incubated in a wet chamber for 20 hours. The content of AT III is determined by measuring the diameter of the visually observed annular precipitation zones. The method provides high sensitivity, does not require sophisticated equipment, however getting results requires at least 18-20 hours, which limits its use in clinical and laboratory practice and makes unsuitable for rapid analysis. In general, this method is used for research purposes.

The objective of the present invention is to reduce the time for determining the content of AT III in blood plasma while maintaining high sensitivity and low labor costs without the use of special expensive equipment.

The problem is solved using the AT III-lattest immunolatex diagnosticum, which is a suspension of spherical particles of carboxylated polystyrene latex, on the surface of which specific polyclonal antibodies to AT III are immobilized, and milk α-casein is added as a ballast protein. Selective interaction of specific antibodies with AT III leads to visual bonding (agglutination) of latex particles.

"AT III-lattest" is prepared as follows [Application No. 2002113997 of 05.29.2002 for the invention "Immunodiagnosticum based on polystyrene latex"]. An equal volume of α- solution is added to a 2% suspension of spherical particles (0 0.5 μm) of polystyrene carboxylated latex with specific polyclonal antibodies to human AT III immobilized on their surface in 0.1 M glycine buffer (hereinafter referred to as buffer). casein in a buffer (concentration 10 mg / ml) prepared as follows: α-casein is dissolved in the calculated amount of buffer, heated for 30 minutes at 37 ° C, the solution is clarified by centrifugation for 15 minutes at 5000 rpm. The mixture of latex suspension with a solution of α-casein is thoroughly shaken and left at room temperature for 30 minutes, after which it is placed in the refrigerator. After 24 hours, the immunodiagnostic is ready. The sensitivity of the immunodiagnostic is determined by a standard method [Immunological methods. Ed. M. Fremel, M., Mir, 1978] by titration of the AT III preparation with a known initial concentration. The final concentration of the AT III solution, still causing an agglutination reaction of latex particles, is 1 μg / ml, which is the sensitivity of the AT III-lattest immunodiagnostic. "AT III-lattest" can be stored without loss of sensitivity at + 4 ° C for two years.

The proposed method for determining AT III in blood plasma is as follows: the test sample of platelet-free nitrate blood plasma is pre-diluted with a buffer, then 1 drop (10 μl) of each dilution is dispensed onto a glass plate, an equal volume (10 μl) of AT is added to each dose III-lattest "and thoroughly mixed with a glass rod; the agglutination reaction is taken into account visually after 2-3 minutes: with a positive reaction, the latex particles stick together and granularity forms in the drop, with a negative reaction the particles do not stick together and the drop consists of a uniform suspension of latex particles, resembling milk.

The concentration of AT III in the initial plasma is determined by the formula:

C = a × ƒ,

where C is the concentration of AT III in the test sample of blood plasma (μg / ml);

a - sensitivity "AT III-lattest";

ƒ - dilution factor, defined as the value of the last dilution of the investigated plasma, still giving agglutination.

For example: at α = 1 μg / ml and ƒ = 240 C = 1 μg / ml × 240 = 240 μg / ml.

The determination of the content of AT III in a plasma sample by the proposed method takes several minutes, which allows the method to be used in emergency cases. The method does not require the use of special expensive equipment and reagents, therefore it is available for widespread use in clinical and laboratory practice, and if necessary in the field. The high sensitivity of the proposed method allows timely diagnosis of pathological conditions of the blood coagulation system, since it allows you to record small changes in the content of AT III in the bloodstream in the early stages of the disease.

The practical implementation of the proposed method for determining the content of AT III in human blood plasma and the ability to achieve the claimed technical result are illustrated by the following examples:

Example 1. Determination of the content of AT III in the blood plasma of healthy donors.

To determine the content of AT III, a platelet-free citrate blood plasma is used, prepared by adding 1 part of a 0.11 M sodium citrate solution to 9 parts of the blood, followed by centrifugation for 20 minutes at 2000 rpm. The test sample is diluted with a working buffer with a pH of 8.2, containing per liter of a solution of 0.1 M glycine, 0.15 M NaCl and 0.02% sodium azide. A sample for analysis is prepared by mixing 0.1 ml of platelet-free citrate blood plasma prepared as described above with 15.9 ml of working buffer, i.e. dilute the original sample 160 times. Then make serial fractional dilutions: 1: 176, 1: 192, 1: 208, 1: 224, 1: 240, 1: 256, etc. Then, 10 μl of each dilution is transferred to a glass plate and 10 μl of an AT III-lattest suspension is added to each sample, each sample is thoroughly mixed with a glass rod and the agglutination reaction is observed for 2 minutes, continuously shaking the plate. As a control, a deliberately negative sample is used in which the plasma sample is replaced with a working buffer. After the reaction is completed, the value of the dilution factor ƒ is determined - the last dilution, still giving agglutination, and the concentration of AT III in the test sample is calculated using the above formula.

The results of determining the content of AT III in the plasma of 5 healthy donors are shown in table 1. For comparison, the table shows the results of determining AT III in the same samples obtained indirectly using a commercial kit "Bechring AT III" to determine the activity of AT III on chromogenic the substrate, as well as with the results of direct quantitative determination of the content of AT III in blood plasma by radial immunodiffusion, taken as a prototype.

Table 1.
Comparison of the results of the determination of AT III in the blood plasma of healthy donors in various ways Healthy blood donor plasma samples "AT III-lattest" Concentration of AT III, µg / ml "Bechring AT CH (on a chromogenic substrate) AT III activity,% Radial immunodiffusion AT III concentration, μg / ml Donor 1 256 112 240 Donor 2 224 96 230 Donor 3 208 92 200 Donor 4 256 117 255 Donor 5 240 99 235 Mean 237 ± 21 103 ± 12 232 ± 23 Test lead time 2-3 min More than 1 hour 18 hours

Given that 100% of the activity of AT III corresponds to the content of 235 μg / ml AT III in blood plasma, the data indicate a good correlation of the results of determining AT III by the proposed method and known methods used in clinical laboratory and research practice. At the same time, the time taken to obtain the result of the proposed method is incomparably less than the time required to conduct an analysis of the methods currently used.

Example 2. Determination of the content of AT III in the blood plasma of patients suffering from DIC.

Plasma samples of patients in whom DIC developed on the background of purulent meningitis (patients 1 and 2), during traumatic surgery (patient 3), during obstetric-gynecological pathology (patients 4 and 5), and during prolonged therapy with fibrinolytics and heparin are examined (patients 6-8). In all cases, DIC was verified by known clinical and laboratory criteria.

The determination of AT III is carried out in a platelet-free citrate blood plasma prepared as in Example 1. The initial plasma dilution is 1:80, the subsequent ones are 1:88, 1:96, 1: 104, 1: 112, 1: 120, etc. The determination process is similar to that described in example 1. Comparative results obtained by the same methods as in example 1 are presented in table 2.

Table 2.
Determination of AT III in the blood plasma of patients suffering from DIC. Blood plasma samples of patients with DIC "AT III-lattest" Concentration of AT III, µg / ml "Bechring AT III" (on a chromogenic substrate) AT III activity,% Radial immunodiffusion AT III concentration, μg / ml Patient 1 160 71 170 Patient 2 152 65 155 Patient 3 176 74 180 Patient 4 136 57 140 Patient 5 128 55 135 Patient 6 192 70 180 Patient 7 160 62 155 Patient 8 144 55 140 Healthy donors 238 ± 21 103 ± 12 232 ± 23

The data show that determined by the proposed method, the content of AT III in the blood plasma of patients is significantly lower than normal and correlates well with the amount of AT III determined by known methods. Thus, the proposed method allows you to reliably diagnose pathological conditions of the blood coagulation system.

The above data show that, in contrast to the known currently used methods, the proposed method for determining antithrombin III in blood plasma combines high sensitivity and reliability with availability, simplicity and speed of execution. For its implementation, special equipment is not required, and the lattest used in the AT-III method has a long shelf life and can be used without additional training. Thus, the method can be used in any laboratory, allows determination in express mode, and, if necessary, in the field.

Claims (1)

A method for determining the concentration of antithrombin III (AT III) in blood plasma, comprising the interaction of AT III with its specific polyclonal antibodies, characterized in that they use a suspension of spherical particles of carboxylated polystyrene latex, on the surface of which specific polyclonal antibodies to AT III are immobilized, and as ballast protein added milk α-casein (AT III-latest), in this case, serial fractional dilutions of plasma are used for determination, and the concentration of AT III in the initial sample is calculated Pipeline according to the formula C = a where C is the concentration of AT III in the test sample of blood plasma (μg / ml); a - sensitivity AT III-lattest; ƒ - dilution factor, defined as the value of the last dilution of the investigated plasma, still giving agglutination.