WO2014194922A1

WO2014194922A1 - Kit for measuring parameters of a hemostasis system in a sample of blood or components thereof

Info

Publication number: WO2014194922A1
Application number: PCT/EA2014/000012
Authority: WO
Inventors: Фазоил Иноятович АТАУЛЛАХАНОВ; Наталья Геннадьевна КОРОТИНА; Ольга Игоревна ХРАПКОВА
Original assignee: Общество С Ограниченной Ответственностью "Гематологическая Корпорация"
Priority date: 2013-06-06
Filing date: 2014-06-04
Publication date: 2014-12-11
Also published as: EA201300544A1; EA028016B1

Abstract

The present invention relates to bioengineering and medicine, in particular to a kit for measuring parameters of a hemostatis system in a sample of blood or components thereof. The problem addressed by the claimed invention consists in increasing the accuracy of metering of a quantity of reagents, extending the shelf life of said reagents, and achieving convenience in use during performance of a test. The technical result which can be achieved when implementing the claimed solution consists in increasing the accuracy of determination of parameters of a blood coagulation system. This result is achieved by the preparation of a kit of reagents for diagnostics in respect of the conditions of the coagulation system of a sample of blood or components thereof, said kit comprising a buffer salt and a calcium salt, wherein said kit comprises an inhibitor of contact activation between the coagulation system and the calcium salt, in the form of a lyophilizate with an additive, and the calcium salt selected is calcium acetate. Alternatively, the technical result consists in that the kit of reagents for diagnostics in respect of the conditions of the coagulation system of a sample of blood or components thereof comprises a buffer salt and a calcium salt, and the calcium salt selected is calcium acetate in the form of a lyophilizate with an additive. Furthermore, the sample used for the investigation is a freshly prepared sample of blood which is free of cells. The inhibitor of contact activation of the coagulation system can be selected from the group of protein contact activation inhibitors consisting of: a corn inhibitor of Hageman factor and derivatives and modifications thereof, infestin-4 and derivatives thereof, Kazal-type inhibitors and derivatives thereof; and the buffer salt can be selected from the group consisting of HEPES buffer, Tris buffer, carbonate buffer, and phosphate buffer. The additive can be selected from the group consisting of polymers, carbohydrates or combinations thereof: polyvinyl polyvinylpyrrolidone (PVP) of varying molecular mass, isomalt, mannitol, glucose, saccharose, and also bovine serum albumin BSA, and polyethyleneglycol PEG. Furthermore, said kit of reagents additionally comprises a coagulation activator.

Description

HEMOSTASIS SYSTEM SET FOR MEASUREMENT OF BLOOD OR ITS COMPONENTS

FIELD OF TECHNOLOGY

The present invention relates to biotechnology and medicine, in particular to a kit for measuring the parameters of the hemostatic system in a blood sample or its components.

BACKGROUND

Reagents are known from the prior art that are used to measure the coagulation parameters of the hemostatic system: contact phase inhibitors, calcium salts, in particular calcium chloride, activating mixtures, additional components (substrates, carriers with antibodies, individual factors, buffer solutions, etc. )

In the publication of American application US2009 / 0061468, cl. C12Q 1/56, publ. 03/05/2009, it is indicated that to measure the coagulation time of a blood sample, a kit is used that includes an activator, in particular, a complex of tissue factor with phospholipids, and a calcium chelator. In this case, the specified set is added to the blood sample in the form of a solution and mixed with it.

In the publication of American application US201 1/0250621, cl. C12Q 1/56, G01N 33/577, publ. 10/13/2011 1, a kit for measuring the activity of tissue factor circulating in a plasma sample is disclosed, including a fibrin polymerization inhibitor, a TFPI inhibitor, purified or recombinant factor VII, mainly lyophilized, purified or recombinant factor X, mainly lyophilized, calcium ions, for example, in the form of calcium chloride CaCl ₂ , a substrate for activated factor X, mainly lyophilized and other reagents.

As the closest analogue of the proposed solution, the set selected is described in the publication of American application US 2008/0268483, cl. C12Q 1/56, G01N 33/86, where a buffer solution, a reagent of one or more coagulation activators, such as tissue factor (TF) and / or thrombin, and one or more activators are used to study thrombus formation and lysis (CloFAL) thrombus lysis, such as tissue plasminogen activator (TPA). The buffer solution may contain TRIS-buffered saline of calcium chloride.

The main disadvantage of these kits is the need to work with solutions that should be accurately dosed, which affects the quality of the test, in addition, the prepared solutions are stored for a short period of time. And lyophilized reagents often have an unstable form (crumbly), which also needs to be additionally dosed.

SUBSTANCE OF DECLARED DECISION

The problem to which the invention is directed, is to increase the accuracy of dosing the number of reagents, increase their shelf life, achieve ease of use in the process of setting the test.

The technical result that can be obtained by implementing the proposed solution is to increase the accuracy of determining the parameters of the hemostatic system.

This result is provided by preparing a set of reagents for diagnosing the state of the coagulation system of a blood sample or its components, including a buffer salt and a calcium salt, while it contains a contact activation inhibitor - a coagulation system, where the specified inhibitor and calcium salt are made in the form of a lyophilisate with an additive, and as calcium salt, calcium acetate was selected.

Or the fact that a set of reagents for diagnosing the state of the coagulation system of a blood sample or its components includes a buffer salt and a calcium salt, and calcium acetate in the form of a lyophilisate with an additive is selected as a calcium salt.

In this case, as a test sample, a freshly prepared cell-free blood sample is used.

And also by the fact that the contact activation inhibitor of the coagulation system can be selected from the group of protein contact activation inhibitors: the Hageman factor corn inhibitor, its derivatives and modifications, infestin-4 and its derivatives, azal-type inhibitors and their derivatives.

And also the fact that the buffer salt can be selected from the group of buffer salts: HEPES buffer, TRIS buffer, phosphate buffer, carbonate buffer. And also because the additive can be selected from the group of polymers, carbohydrates, or a combination thereof: polyvinylpyrrolidone (PVP) of different molecular weights, isomalt, mannitol, glucose, sucrose; as well as bovine serum albumin BSA, polyethylene glycol PEG.

And also the fact that it additionally contains a coagulation activator.

With the creation of the said set by the authors, the convenience of the formulation and the quality of the test for determining the coagulation states of the test sample containing blood coagulation factors increased. Labeled aliquots of reagents obtained by the authors made it possible to quickly, conveniently and uniformly conduct sample preparation of the test. An accurately dosed amount of reagents in the form of a dry form does not lead to additional dilution of the test sample, leaving it in the most native state. It is not economically feasible to use reagents packaged in the form of solutions of large volumes: as a rule, these packings are designed for long-term use using dispensers and for large volumes of the sample. Such reagents cannot be used in tests to measure the spatial growth parameters of a fibrin clot in a blood sample or its products, since the measurement implies an individual aliquot of a small volume. The additives chosen by the authors provide a long and safe storage of the components of the kit without the threat of bacterial contamination.

The claimed technical solution can be illustrated by the following figures of drawings, where figure 1 illustrates the appearance of the reagents after freeze drying with the addition of various additives;

in FIG. 2 (a, b, c) presents the growth parameters of a fibrin clot in a sample of freshly prepared plasma using an additive for an inhibitor and the corresponding type of reagents;

in FIG. Figure 3 (a, b, c) shows the growth parameters of a fibrin clot in a sample of freshly prepared plasma using calcium additives and the corresponding type of reagents.

The kit proposed by the authors includes, as a first reagent, a lyophilized contact activation inhibitor of the coagulation system with a buffer, and as a second reagent, a lyophilized calcium salt. Set, if necessary, may consist of a lyophilized calcium salt with a buffer salt.

The kit, if necessary, may include a coagulation activator, which may also include auxiliary substances, such as buffer salts, additives, etc.

The used inhibitor of contact activation of the coagulation system is intended, in particular, to suppress the artifact activity of the coagulation system obtained during sampling and manipulation during sample preparation. As the indicated inhibitor, protein contact activation inhibitors can be used, namely, the Hageman factor corn inhibitor, its derivatives and various modifications, infestin-4 and its homologs, Casal-type inhibitors and their derivatives, etc.

Since in the presence of contact with atmospheric air, С0 ₂ evaporation occurs in a blood sample (blood plasma), this leads to an increase in the pH of the test sample. This effect may affect the measurement results. To ensure a constant pH value of the sample during the test, it is advisable to use buffer solutions, in particular, stabilizing solutions of buffer salts. Adding buffer salt to the composition of one of the reagents eliminates the need for additional manipulations in the formulation of the test. The concentration and pH of the buffer were selected so that from the pH range of the test sample 7. (H8.0, which can be achieved by an in vitro test, the buffer returns it to the range 7.2 ^ -7.6. The buffer can be selected from buffer salts with buffer properties at pH 7.CH-9.0, which corresponds to physiological pH values, for example TRIS-buffer or HEPES-buffer. ·

So the authors found that high concentrations of HEPES can inhibit coagulation, the final buffer concentration sufficient to stabilize the pH during the experimental time should not exceed 80 mm. Standard reagents for conducting blood coagulation tests, used in the form of solutions, such as a solution of calcium chloride or calcium acetate, a solution of a coagulation inhibitor, can increase the measurement error due to inaccuracies when dosing into the reaction mixture. In addition, solutions of these reagents further dilute the test sample itself. The authors have suggested not only the use of of these reagents in lyophilized form, but also due to selected additives, it became possible to form a physically stable form in the form of a tablet, which helps to visually control the transition of the substance into the reaction mixture. This, in turn, also allowed to increase the stability of the reagents, to improve their properties and the duration of storage. The mechanical strength of the resulting tablet can be of great importance during transportation of the kit, since the tablet is able to withstand significant mechanical loads, for example, up to 10 taps of a microtube with a reagent on a hard surface without crumbling.

To form this dry form of reagents, special additives and their compositions were tested. Polyvinylpyrrolidone PVP 40000, PVP 360000, bovine serum albumin (BSA), glycine, mannitol, sucrose, etc. were chosen as such additives. These additives were selected in the concentration range of 0.1 -4% (m / m). In FIG. 1 illustrates the appearance of an inhibitor with the addition of HEPES (FIG. 1 a); with the addition of HEPES and 1% (m / m) PVP 360000 (Fig. 1 b), as well as calcium salts: calcium chloride with the addition of 0.4% (m / m) isomalt (Fig. 1 c), calcium acetate with the addition of 1% (m / m) PVP 360000 (Fig. 1 g). The growth parameters of a fibrin clot in a sample of freshly frozen plasma, determining the choice of additives for the first reagent, are illustrated in Table. 1 and are illustrated by graphs in FIG. 2 a.

Table 1.

So, column 3 shows the ranges of values for the control formulation of the test, where an inhibitor with the addition of HEPES buffer (for this specific example, the final concentration of 30 mM) and calcium chloride with the addition of 0.4% (m / m) isomalt were used. Columns 4 and 5 show the values the parameters of a typical study using 2% (m / m) glycine with 1% (m / m) albumin and 1% (m / m) PVP 360000, respectively. From the obtained data it is seen that, when using the first variant of the additive, the value of the stationary velocity does not correspond to the reference values.

In FIG. 2a shows graphs of the dependence of the size of the fibrin clot on time for the conditions indicated in table 1, i.e. for the control formulation of the test, as well as with the addition of 2% (m / m) glycine with 1% (m / m) albumin and 1% (m / m) PVP 360000, respectively. In FIG. 26 shows photographs corresponding to a grown fibrin clot at 30 minutes. In FIG. 2c shows photographs illustrating the appearance of the tablet form of the reagents. When 1% (m / m) PVP is added, the tablet is formed in the best way.

It was found that in order to obtain a stable and high-quality dry form of the first reagent, the ratio of the amount of contact activation inhibitor of the coagulation system to the amount of PVP can lie in the range: Inhibitor HDL = 1: 1.25-1: 25 (m / m).

Instead of the standard calcium chloride used, it was proposed to use calcium acetate, such a replacement of calcium salt allowed to improve the hygroscopic properties of the reagent: in this form, this reagent gains less moisture, which contributes to a longer storage and preservation of the properties of the reagent. This also made it possible to obtain a stable tablet form (see FIG. 1).

Various calcium salts have been tested: calcium chloride, calcium acetate, calcium formate. Additives or their composition were used: isomalt, glucose, sucrose, mannitol, polyvinylpyrrolidone (PVP 40000, PVP 360000, PVP 24000), polyethylene glycol (PEG), bovine serum albumin (BSA). These additives were selected in the concentration range of 0.1-4% (m / m). The growth parameters of a fibrin clot in a sample of freshly prepared plasma, determining the choice of additives for the second reagent, are illustrated in Table 2 and are illustrated by the graphs in FIG. 3 a.

Fig. 3 (a, b, c) illustrates an example of a study of the possibility of using different calcium salts and additives in comparison with the growth parameters of a fibrin clot in a control formulation. The data determining the choice of additives are illustrated in Table 2 and are illustrated by the graphs in FIG. 3 a. table 2

So column 3 shows the ranges of values in the control statement of the test, columns 4 and 5 show the parameter values when using calcium acetate with the addition of 1% (m / m) sucrose and calcium acetate with the addition of 0.5% (m / m) PVP, respectively.

In FIG. The time dependences of the size of the fibrin clot are given for the conditions indicated in table 2, i.e. for the control formulation, as well as using calcium acetate with the addition of 1% (m / m) sucrose and calcium acetate with the addition of 0.5% (m / m) PVP, respectively. In FIG. 36 shows photographs corresponding to a grown fibrin clot at 30 minutes. In FIG. Sv are photographs illustrating the appearance of the form of the reagents with the corresponding additives. The tablet forms best when 0.5% (m / m) PVP is added.

It was found that to obtain a stable and high-quality dry form of the second reagent, the ratio of calcium acetate to PVP can lie in the range: Calcium acetate: PVP = 1: 0.07-1: 1.4 (m / m).

According to the results of the study, it was decided that it is advisable to use an inhibitor with the addition of HEPES and PVP 360000, and calcium acetate with the addition of PVP 360000, while PVP 360000 should be used in an amount of 0.1% - 2% by weight.

It should be noted that the authors prepared calcium chloride as a second reagent in tableted lyophilized form. However, in its properties, it is inferior to the claimed version of the kit and its active hygroscopicity does not provide reliable storage. For the use of calcium chloride in lyophilized form, a special requirement is the special conditions and method of packaging, which additionally increases the cost of the reagent.

In a special case, it is allowed to use a kit comprising a tabletted calcium acetate lyophilisate with a buffer salt, mainly HEPES, i.e. variants of the claimed kit. Namely, in the case of testing a freshly prepared platelet-free blood sample (platelet-free blood plasma (pfp)) and using a cuvette to conduct a test having a surface in contact with the sample,. perfectly smooth and made of a material with minimal activating contact activation properties.

While the kit comprising the first reagent (inhibitor) can be used for blood samples or its components for samples of any type, not just freshly prepared pfp.

The growth parameters of a fibrin clot in a sample of freshly prepared pfp plasma using an alternative kit (reduced to a second reagent with the addition of a buffer) are illustrated in Table 3.

Table 3.

So, column 3 presents the ranges of values in the control formulation of the test using freshly prepared platelet-free blood plasma as the test sample, columns 4 and 5 show the average values of the parameters when using the first and second variants of the proposed sets, namely, tablet lyophilized contact inhibitor activation with a buffer solution and calcium acetate, as well as salts of a tabletted lyophilized calcium acetate with the addition of a buffer solution, respectively.

The data obtained allow us to conclude that it is possible to use both variants of the set.

In addition, it was found that for the second variant of the claimed kit, the ratio of calcium acetate to additive and to the buffer can lie in the range of Calcium acetate: PVP: HEPE8 = 1: 0.07: 2.0-1: 1, 4: 2.0 ( m / m).

To study the changes in the properties of the claimed kit options over time, their samples were packed in ceflen bags (12 PET T & 9 AL & 60LDP) with the addition of silica gel (packaged silica gel, Sorbis) and stored at 4 ° C and normal humidity.

At set intervals, individual samples were taken and checked according to the parameters indicated in table 4, the rest continued to be stored in the described conditions. The study was conducted by direct storage (in real time). The control was also carried out visually for changes in the shape of the reagent tablet.

We monitored changes in the activity of the first reagent during its storage by means of the test “Activated Partial Thromboplastin Time (APTT)”, using the diagnostic kit “coagulo-test PG-6”, “Renam”. All measurements were carried out using control (lyophilized) human blood plasma. The concentration of the second reagent (calcium salt) was measured by the colorimetric method using the diagnostic kit Calcium-vital, Agate.

Table 4 presents the test data for points of 2 and 9 months of storage, as well as the reference range of values.

Table 4

Parameter Unit Control 2 months 9 months Reference

(metering (day 0) storage storage range

1 Delay min 0.8 0.9 [0.4-1, 2]

clot growth 1.0

2 Beginner

μm / min 57 52

speed [52-64]

58

clot growth

3 Stationary speed µm / in 32 29 [29-37]

29th

clot growth four

APTT sec 191 193 no less than 105

195

(the first

reagent)

5 oncentration

I calcium μM 19 1 8 [1 6-5-22]

eighteen

(second

reagent)

6 Visual Appearance Appearance Appearance Thick

control view has not changed the tablet has not changed

has changed

From the presented data it can be seen that there were no deviations from the reference values of the fibrin clot growth parameters (see lines 1-3), the activity of the first reagent (see line 4) and the concentration of calcium ions (see line 5) also did not change. Appearance (dense tablet) did not change during the indicated time points.

As mentioned earlier, when using tableted lyophilized calcium chloride, an increase in its shelf life could be achieved only by additional storage conditions, in particular, packaging in moisture-tight bags, in a strictly dry atmosphere with the mandatory addition of a desiccant and subsequent storage at 4 ° C in normal humidity , which leads to higher storage costs.

To measure the parameters of the coagulation system in an in vitro test sample, it was proposed to use a special test developed by the applicant, where the spatiotemporal dynamics of blood coagulation was initiated by a localized coagulation activator under conditions similar to in vivo blood coagulation.

The sample (in the form of whole blood or its components (plasma, etc.)) can be freshly prepared, or it can be frozen, for example, in the case of samples that do not contain cells, or in a lyophilized form (control plasma). The sample may also contain mixtures of purified natural, synthetic or recombinant proteins and / or other preparations / reagents with hemostatic, fibrinolytic or antifibrinolytic activity. The sample can be obtained from healthy subjects or from subjects that may or may not suffer from a blood clotting disorder. This test takes into account the spatial heterogeneity of the processes that occur during blood coagulation and is carried out without mixing in a thin layer of plasma. To conduct it, blood plasma samples are mixed with the first and second reagents after a set period of time and placed in the channels of the measuring cell, and then the test sample is brought into contact with a coagulation activator, such as tissue factor. In the case of testing a sample of freshly prepared platelet-free plasma, the first reagent may not be used, and the second reagent additionally contains a buffer salt. The process of occurrence and growth of a fibrin clot is recorded by a digital video camera in scattered light. The resulting series of images gives detailed information on the dynamics of blood coagulation in time and space (Fig. 2 b and 3 b). Based on the obtained photographs, the numerical parameters of the spatiotemporal dynamics of the growth of the fibrin clot are calculated: the time delay of clot growth, the rate of clot growth, the presence of spontaneous clots formation far from the activator, shown in Tables 1 and 2.

An additional reagent - a coagulation activator in the form of, in particular, tissue factor (TF) used in the test, due to the specificity of the test, is brought into contact with the test sample, being immobilized on a carrier, such as the inner surface of the cuvette, a separate insert or a microplate well , which makes it very convenient for analysis.

It should be noted that the proposed kit can be used not only in the test for measuring spatial growth parameters of a fibrin clot in a blood sample or its components proposed by the applicant, but also in any other coagulological test for determining blood coagulation parameters.

The inventive set has the same sensitivity when determining the growth parameters of a fibrin clot for different forms of the sample: freshly prepared, frozen, freeze-dried plasma. This additionally allows you to diagnose the general status of the patient’s hemostatic system in different situations, for example, in a delayed version.

The use of the claimed kit is further illustrated in the non-exhaustive examples 1 and 2. Example 1

Unified test performance. Growth parameters of a fibrin clot in different plasma samples.

The fibrin clot growth parameters in qualitatively different samples were obtained by the above method: freshly prepared and freshly frozen blood plasma of healthy donors, and a freeze-dried pool of freshly prepared blood plasma of healthy donors (at least 6 donors in the pool) was used as a control sample. To conduct a test, 120 microliters of a blood plasma sample is mixed with ready-made, precisely metered lyophilized aliquots (in the form of tablets) of the first and second claimed reagents and placed in a measuring cell, after which it is brought into contact with an activator insert - a plastic plate with a coagulation activator applied to the end of the plate . A fibrin clot begins to grow from the activating surface deep into the plasma, the growth parameters of which are calculated after testing with the help of software developed by the applicant.

The obtained quantitative indicators of the parameters of typical studies (a typical example of a single study of the indicated test of the applicant), as well as the ranges of reference values for each type of plasma are summarized in table 5. As can be seen from table 5, the ranges of the values of the corresponding parameters differ, and the values of the parameters characteristic of freshly prepared plasma , as a rule, lower (see for initial and stationary bunch growth rates) than for frozen and control samples, which is explained by the increased status activity of the latter as a result of cold activation and cooking features, respectively.

Table 5

Freshly prepared Freshly frozen Control plasma plasma plasma parameter result reference result result reference result

Delay 0.9 [0.8-1.5] 1 [0.7-1.9] 1 [0.4-1.2] clot growth,

min

Initial 46 [39.1-54.6] 56 [47-59] 53 [52-64] speed

clot growth

μm / min

Stationary 25 [20.5-30] 31 [25-33] 33 [29-37] speed clot growth

μm / min

Size 921 [833-1173] 1 183 [971-1295] 1234 1 155-1399] 30 clot

min, microns

The indicators given in the table indicate that the reagents described above can be used to test different types of the test sample, while the set of reagents is unified, that is, when changing the type of sample, the set remains the same, and in the case of a freshly prepared blood plasma sample, as described above, can be reduced to lyophilized calcium acetate with a buffer solution. The tablet form of the reagents increases the convenience of their use, as well as the accuracy of the test, by eliminating the stage of additional dilution of the test sample. In addition, there is no stage of dosing small volumes of the reagent, if the reagent is in liquid form.

Example 2

Unified test, freshly prepared blood plasma. The fibrin clot growth parameters are normal and pathological.

The inventive kit of reagents was used to test samples of freshly prepared plasma of patients with weakened status (hemophilia) and increased status (hypercoagulation) of the hemostasis system.

The preparation of the test sample and the test was carried out as in Example 1.

The quantitative characteristics obtained as a result of typical studies, as well as the range of reference values for freshly prepared plasma, are summarized in Table 6. As can be seen from the table, the parameter values for pathological conditions differ from the norm to a smaller and greater side, respectively, unambiguously separating hypo- and hyper-states . Stationary speed and clot size for 30 min. for a hypercoagulable sample were not determined due to the transient transition of the plasma to a gel state over the entire volume. Table 6

The above described example confirms that the inventive kit of reagents can be used to test pathological samples. It also confirms the convenience of their use (tablet form) and the accuracy of determining the test parameters due to pre-aliquoted reagents.

Claims

CLAIM

1. A set of reagents for diagnosing the state of the coagulation system of a blood sample or its components, including a buffer salt and a calcium salt, characterized in that it contains a contact activation inhibitor of the coagulation system, where the indicated inhibitor and calcium salt are made in the form of a lyophilisate with an additive, and As a calcium salt, calcium acetate was selected.

2. The reagent kit according to claim 1, characterized in that the contact activation inhibitor of the coagulation system can be selected from the group of protein contact activation inhibitors: Hageman factor corn inhibitor, its derivatives and modifications, infestin-4 and its derivatives, Casal-type inhibitors and their derivatives.

3. A set of reagents for diagnosing the state of the coagulation system of a blood sample or its components, including a buffer salt and a calcium salt, characterized in that calcium acetate in the form of a lyophilisate with an additive is selected as a calcium salt.

4. The reagent kit according to claim 3, characterized in that as a test sample, a freshly prepared cell-free blood sample is used.

5. The reagent kit according to p. 1 and 3, characterized in that the buffer salt can be selected from the group: HEPES buffer, TRIS buffer, carbonate buffer, phosphate buffer.

6. The reagent kit according to p. 1 and 3, characterized in that the additive can be selected from the group of polymers, carbohydrates or a combination thereof: polyvinyl pyrrolidone (PVP) of different molecular weights, isomalt, mannitol, glucose, sucrose; as well as bovine serum albumin BSA, polyethylene glycol PEG.

7. The reagent kit according to p. 1 and 3, characterized in that it further comprises a coagulation activator.