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WO2014194922A1 - Ensemble pour mesurer des paramètres du système d'hémostase dans un échantillon sanguin ou ses composants - Google Patents

Ensemble pour mesurer des paramètres du système d'hémostase dans un échantillon sanguin ou ses composants Download PDF

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Publication number
WO2014194922A1
WO2014194922A1 PCT/EA2014/000012 EA2014000012W WO2014194922A1 WO 2014194922 A1 WO2014194922 A1 WO 2014194922A1 EA 2014000012 W EA2014000012 W EA 2014000012W WO 2014194922 A1 WO2014194922 A1 WO 2014194922A1
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WO
WIPO (PCT)
Prior art keywords
buffer
sample
reagents
kit
blood
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EA2014/000012
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English (en)
Russian (ru)
Inventor
Фазоил Иноятович АТАУЛЛАХАНОВ
Наталья Геннадьевна КОРОТИНА
Ольга Игоревна ХРАПКОВА
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Obschestvo S Ogranichennoy Otvetstvennostyu <<gematologicheskaya Korporatsiya>>
Original Assignee
Obschestvo S Ogranichennoy Otvetstvennostyu <<gematologicheskaya Korporatsiya>>
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Filing date
Publication date
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Publication of WO2014194922A1 publication Critical patent/WO2014194922A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors

Definitions

  • 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.
  • 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. )
  • kits that includes an activator, in particular, a complex of tissue factor with phospholipids, and a calcium chelator.
  • the specified set is added to the blood sample in the form of a solution and mixed with it.
  • a kit for measuring the activity of tissue factor circulating in a plasma sample 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.
  • 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.
  • kits 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.
  • 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.
  • 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.
  • test sample a freshly prepared cell-free blood sample is used.
  • 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.
  • the buffer salt can be selected from the group of buffer salts: HEPES buffer, TRIS buffer, phosphate buffer, carbonate buffer.
  • 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.
  • PVP polyvinylpyrrolidone
  • 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.
  • figure 1 illustrates the appearance of the reagents after freeze drying with the addition of various additives
  • FIG. 2 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;
  • FIG. Figure 3 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 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.
  • 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.
  • 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. ⁇
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 26 shows photographs corresponding to a grown fibrin clot at 30 minutes.
  • 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.
  • 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
  • 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.
  • FIG. 36 shows photographs corresponding to a grown fibrin clot at 30 minutes.
  • 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.
  • kits 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.
  • a buffer salt mainly HEPES
  • 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.
  • 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.
  • Table 4 presents the test data for points of 2 and 9 months of storage, as well as the reference range of values.
  • 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.
  • 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.
  • a coagulation activator such as tissue factor.
  • 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).
  • 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.
  • a carrier such as the inner surface of the cuvette, a separate insert or a microplate well , which makes it very convenient for analysis.
  • 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.
  • Example 1 The use of the claimed kit is further illustrated in the non-exhaustive examples 1 and 2.
  • Example 1 The use of the claimed kit is further illustrated in the non-exhaustive examples 1 and 2.
  • 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.
  • 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 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.
  • 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.

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Abstract

L'invention se rapporte au domaine des biotechnologies et de la médecine, et concerne notamment un ensemble pour mesurer des paramètres du système d'hémostase dans un échantillon sanguin ou ses composants. Le but de la présente invention est d'augmenter la précision de dosage de la quantité de réactifs, d'allonger leur durée de conservation, et d'assurer une commodité d'utilisation pendant le processus de test. Le résultat technique obtenu grâce à la présente solution technique consiste en une plus grande précision lors de la détermination des paramètres du système de coagulation sanguine. Ce résultat est obtenu en préparant un ensemble de réactifs pour diagnostiquer les états du système de coagulation d'un échantillon sanguin ou de ses composants, qui comprend un sel tampon et un sel de calcium, ainsi qu'un inhibiteur d'activation par contact du système de coagulation et un sel de calcium, lesquels se présentent sous forme d'un lyophilisat avec un additif, le sel de calcium consistant en de l'acétate de calcium. L'ensemble de réactifs pour diagnostiquer les états du système de coagulation d'un échantillon sanguin ou de ses composants peut encore comprendre un sel tampon et un sel de calcium, le sel de calcium consistant en de l'acétate de calcium sous forme de lyophilisat avec un additif. On utilise en qualité d'échantillon à étudier un échantillon sanguin fraichement préparé sans cellules. L'inhibiteur d'activation par contact du système de coagulation peut être choisi dans le groupe d'inhibiteurs protéiniques d'activation par contact comprenant : inhibiteur popcorn de facteur Hageman, ses dérivés et ses modifications, de l'infestine-4 et ses dérivés, des inhibiteurs de type Cazal et leurs dérivés, tandis que le sel tampon peut être choisi dans le groupe comprenant un tampon HEPES, un tampon TRIS, un tampon carboné, pu un tampon phosphaté. L'additif peut être choisi dans le groupe comprenant des polymères, des glucides et leurs combinaisons : polyvinylpyrrolidone (PVP) de masse moléculaire diverse, isomalt, mannitol, glucose, saccharose, ainsi que de l'albumine de sérum bovin (ASB) et du polyéthylène glycol (PEG). Cet ensemble peut également comprendre un activateur de coagulation.
PCT/EA2014/000012 2013-06-06 2014-06-04 Ensemble pour mesurer des paramètres du système d'hémostase dans un échantillon sanguin ou ses composants Ceased WO2014194922A1 (fr)

Applications Claiming Priority (2)

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EA201300544 2013-06-06
EA201300544A EA028016B1 (ru) 2013-06-06 2013-06-06 Набор для измерения параметров системы гемостаза в образце крови или ее компонентов

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RU2698206C1 (ru) * 2018-07-02 2019-08-23 Общество с ограниченной ответственностью "Гематологическая Корпорация" (ООО "ГемаКор") Набор реагентов для диагностики степени нейровоспаления

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040037893A1 (en) * 2001-12-21 2004-02-26 Hansen Birthe Lykkegaard Liquid composition of factor VII polypeptides
RU2360970C2 (ru) * 2004-03-31 2009-07-10 Бакстер Интернэшнл Инк. Набор для измерения образования тромбина в образце крови или плазмы пациента

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JP3944267B2 (ja) * 1996-08-09 2007-07-11 財団法人化学及血清療法研究所 プロトロンビンの活性化方法および該方法に基づくトロンビンの製造方法
JP3246749B2 (ja) * 1997-04-23 2002-01-15 インストルメンテーション ラボラトリー エス．ピー．アー． 組換えウサギ組織因子に基づくプロトロンビン時間試薬
RU2256464C1 (ru) * 2004-03-12 2005-07-20 Общество с ограниченной ответственностью "БиоГениус" Способ получения с1-эстеразного ингибитора человека и продукт для использования в медицине

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040037893A1 (en) * 2001-12-21 2004-02-26 Hansen Birthe Lykkegaard Liquid composition of factor VII polypeptides
RU2360970C2 (ru) * 2004-03-31 2009-07-10 Бакстер Интернэшнл Инк. Набор для измерения образования тромбина в образце крови или плазмы пациента

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PEDERSEN L.C. ET AL.: "The corn inhibitor of blood coagulation factor Xlla. Crystallization and preliminary crystallographic analysis.", J MOL. BIOL., vol. 236, no. 1, 11 February 1994 (1994-02-11), pages 385 - 387 *

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EA201300544A1 (ru) 2014-12-30

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