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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
  • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
  • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
  • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
  • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
  • G01N2333/96433—Serine endopeptidases (3.4.21)
  • G01N2333/96441—Serine endopeptidases (3.4.21) with definite EC number
  • G01N2333/96447—Factor VII (3.4.21.21)

Definitions

• the present invention relates to a method for measuring the level of activated factor VII (FVIIa) in a sample by using a plasma lacking in FVII and at least one other factor selected from the factor
• Blood coagulation is a mechanism that allows organisms to control blood shedding during vascular lesions and thus prevent bleeding.
• Blood coagulation occurs in cascading steps involving different proenzymes and procofactors in the blood that are converted, via proteolytic enzymes, into their activated form.
• steps (or cascade) of coagulation two pathways are distinguished, called the extrinsic pathway of coagulation and the intrinsic pathway of coagulation.
• prothrombinase consisting of activated factor X (FXa), activated factor V (FVa), phospholipids and calcium. It is prothrombinase that activates prothrombin thrombin allowing the transformation of soluble fibrinogen into insoluble fibrin which forms the clot.
• FVIIa tissue factor
• TF tissue factor
• the FVIIa-FT complex transforms factor X into factor Xa in the presence of calcium ions.
• the FVIIa-FT complex also transforms FIX into FIXa.
• Factors IXa and Xa activate FVII in FVIIa.
• Factor Xa complexed with factor Va and phospholipids (prothrombinase) converts prothrombin to thrombin.
• Thrombin acts on fibrinogen transforming it into fibrin and also has other activities including activation of factor V in Factor Va and FVIII in FVIIIa.
• Thrombin also activates factor XIII in factor XIIIa in the presence of calcium, which allows the consolidation of the fibrin clot.
• FIXa is generated from FIX by the FXIa itself activated by activated factor XII. blood contact with an electronegative surface such as the subendothelium.
• FVIIa a vitamin K-dependent glycoprotein
• the FVIIa has the advantage of being able to act locally in the presence of the tissue factor released after tissue damage causing haemorrhages, even in the absence of Factor VIII or IX. This is why FVIIa has been used for many years to correct some bleeding disorders.
• the first approach was to obtain FVIIa from plasma. But the production of FVIIa from plasma is limited by the availability of the source of supply and this use of plasma presents risks of transmission of pathogens, such as prion and viruses.
• rFVIIa The main therapeutic indication for rFVIIa (in the USA, EU and Japan) is the treatment of spontaneous or surgical bleeding of hemophiliacs A having developed anti-factor VIII antibodies and hemophiliac B having developed anti-factor IX antibodies. In Europe, it is also indicated for use in patients with congenital deficiency of FVII and in patients with Glanzmann thrombasthenia. In addition, numerous publications report the efficacy of rFVIIa in controlling bleeding during surgical procedures in patients who have neither congenital factor deficiency nor thrombasthenia.
• the best known methods for detecting FVIIa activity are the measurement of clotting time, PTT (Partial Thromboplastin Time -
• Partial Thromboplastin Time Partial Thromboplastin Time
• aPTT activate partial thromboplastin time
• Activated thromboplastin TEG (thromboelastograph) and TGT (thrombin generation test). These methods make it possible to detect the activity of FVIIa but do not make it possible so far to directly measure the level of activated FVII in a sample.
• a commercial immunoassay kit of FVIIa is available (IMUBIND Factor VII ELISA kit) but the experimental conditions for the implementation of this technique are difficult to control. Indeed this kit is complex to implement and is characterized by an extremely low dynamic range, a linear range of extremely limited detection and above all requires working at a temperature of + 4 ° C.
• fluorogenic or chromogenic FXa assay methods generated by FVIIa have also proved unsuitable for measuring the concentration of FVIIa because they do not make it possible to differentiate the effect of FVIIa from that of FVIIa.
• thromboelastography is sometimes used. This method consists in measuring the physical properties of whole blood by mechanically analyzing clot formation as a function of time. Depending on the parameters extracted from a graph (called thromboelastogram ® ) generated by the thromboelastograph, the clinician can evaluate the coagulation ability of a patient. Although accurate, this method is tedious, unsuitable for a routine and repetitive analysis, and difficult to apply to multisampling because it requires to be performed within one hour after the blood sample. In addition, this method does not measure the level of activated FVII in a sample.
• the best known method for measuring the level of activated FVII is to independently measure the concentration of FVII + FVIIa and the concentration of FVIIa in order to deduce the level of activated FVII (ratio FVIIa concentration / concentration of FVII + FVIIa ). Despite the fact that the measurement of FVII + FVIIa concentration is accurate, direct measurements of FVIIa concentration remains imprecise and difficult to implement.
• the Applicant has surprisingly found that the use of a plasma devoid of FVII and at least one other factor chosen from FVIII, FIX and FXI makes it possible to measure the level of activated FVII in a sample in a reliable manner. , reproducible and easy to implement, especially when the sample contains non-activated factor VII and activated factor VII.
• the experimental conditions used in the method of the invention thus make it possible to establish a correlation between the level of activated FVII in a sample and certain characteristic parameters of a thrombin generation test (TGT) and the resulting thrombinogram. .
• TGT thrombin generation test
• Such a correlation makes it possible to determine the level of activated FVII in a sample to be tested, by comparing the thrombinogram parameters of said sample with those of "standard" thrombograms obtained from compositions comprising known levels of activated FVII.
• the present invention therefore relates to a method for measuring the level of activated factor VII in a test sample, comprising the steps of: a) mixing said test sample with a factor VII-deficient plasma (FVII) and lacking in at least one another factor selected from factor VIII (FVIII), factor IX (FIX) and factor XI (FXI), the sample-test + plasma mixture having a final concentration of FVI1 + FVlla ranging from 10 ⁇ M to 80 ⁇ M, - b ) add initiator components of the thrombin generation reaction; c) obtaining a thrombinogram by performing a thrombin generation test (TGT) on the mixture of step b); d) comparing at least one of the thrombinogram parameters of step c) with a homologous parameter of standard thrombograms established on the basis of standard samples whose activated Factor VII level is known and varies between each standard sample ; e) deducing from step d) a measurement of the activated Factor
• the method of the present invention optionally comprises an additional step f) of calculating the concentration of activated factor VII in said test sample from the rate determined in step e).
• the standard thrombograms are obtained by performing a thrombin generation test on a mixture containing (i) a standard sample whose level of activated factor VII is known, (ii) a plasma devoid of FVII and lacking in at least one other factor selected from FVIII, FIX and FXI, the final concentration of the sample-standard + plasma mixture devoid of FVII and lacking in at least one other factor selected from FVIII, FIX and FXI being substantially identical to that of the sample-test + plasma mixture lacking in FVII and devoid of at least one other factor selected from FVIII, FIX and FXI, and (iii) initiator components of the FVIII reaction. thrombin generation.
• the compared thrombinogram parameter is chosen from latency time, peak time and velocity when said plasma is devoid of FVII and FIX, or devoid of FVII and FXI; and among the lag time and the peak time when said plasma is devoid of FVII and FVIII.
• sample-test + plasma and standard-sample + plasma mixtures are made using the same plasma lacking in FVII and lacking in at least one other factor chosen from FVIII, FIX and FXI.
• the initiator components of the thrombin generation comprise a tissue factor (TF), phospholipids, and Ca 2+ , the final concentration of said tissue factor in the sample + plasma mixture + initiator components being in the range of 1 at 10 ⁇ M, the final concentration of said phospholipids in the sample + plasma mixture + initiator components being in the range of 0.1 to 5 ⁇ M and the final concentration of Ca 2+ in the mixture sample + plasma + initiator components being in the range of 14 to 18 mM.
• TF tissue factor
• phospholipids phospholipids
• Ca 2+ the final concentration of said tissue factor in the sample + plasma mixture + initiator components
• the activated factor FVII whose rate is measured is of plasmatic origin (pFVIIa), of recombinant origin (rFVIIa) or of transgenic origin (TgFVIIa).
• test sample is a sample of transgenic mammalian milk or a serum-free cell culture medium.
• the present invention also relates to the use of a FVII-deficient plasma and at least one other factor selected from FVIII, FIX and FXI for measuring the level of activated FVII in a test sample.
• Fractor VII or “FVII” is meant the non-activated factor VII corresponding to the single-chain proenzyme which can not trigger coagulation.
• activated Factor VII or “activated FVII”, or FVIIa is meant the double-stranded protein (enzyme), comprising a heavy chain and a light chain linked together by a disulfide bridge, resulting from the cleavage of FVII (proenzyme), and which is able to trigger blood clotting.
• FVII + FVIIa means the sum of the concentrations or the sum of the amounts of FVII and FVIIa present in a sample of interest. The sum of the amounts or concentrations of FVII and FVIIa can be measured, for example, by immunoassay using commercially available kits such as ASSERACHROM® VII: Ag from Diagnostica Stago (Reference 00241).
• Activated Factor VII Level or “FVIIa Level” means the ratio of the amount or concentration of Activated Factor VII (FVIIa) in a sample of interest, and the sum of the amounts or concentrations, respectively, Factor VII and activated Factor VII (FVII + FVIIa) in this same sample of interest.
• the activated FVII level will be equal to 1 (ie 100%) for a sample containing only FVIIa but not containing FVII, this rate will be equal to 0.5 (i.e. 50%) for a sample containing both FVIIa and FVII, and it will be 0 (ie, 0%) for a sample containing only FVII (not containing FVIIa).
• Test sample means a sample containing FVII, activated FVII or a mixture thereof, but the level of activated FVII is not known.
• the test sample is purified from blood or plasma or comes from a body fluid, purified or not, such as, for example, the milk of a mammal, a culture medium or a cell homogenate.
• the test sample is a sample of mammalian milk, particularly a sample of transgenic mammalian milk producing FVII and / or FVIIa in its milk.
• test sample is a serum-free cell culture medium.
• the sample to be tested is a therapeutic sample or not containing plasma FVIIa (pFVIIa), recombinant (rFVIIa) or transgene (TgFVIIa), and FVII, or a mixture thereof in liquid or freeze-dried form.
• pFVIIa plasma FVIIa
• rFVIIa recombinant
• TgFVIIa transgene
• standard sample is meant a sample whose level of activated FVII is known and / or selected, incorporating, for example, appropriate amounts or concentrations of the international standard of FVII (Blood Coagulation Factor VII Concentrate Human, NIBSC reference 97/592) or International Standard for Activated FVII (Blood Coagulation Factor VIIa Concentrate Human, NIBSC Ref 89/688), or a mixture thereof, in a solution of interest, to obtain a desired level of FVII activated.
• the standard sample can also be obtained from blood or plasma, or comes from a body fluid, purified or not, such as, for example, milk, or a culture medium or a cell homogenate.
• Plasma as used in the context of the present invention, is of animal origin, preferably mammalian and preferably human.
• the terms “depleted” or “devoid of” have the same meaning and can be used as alternatives to designate a depletion of a solution of interest (for example a plasma) in one compound (eg a blood coagulation factor), until the presence of the latter is undetectable.
• a solution of interest for example a plasma
• one compound eg a blood coagulation factor
• Plasmid deficient in FVII and in at least one factor chosen from FVIII, FIX or FXI therefore means that the respective concentration of each of these factors FVII, FVIII, FIX or FXI in the plasma in question is less than their detection threshold when their concentration is measured by the assay methods known to those skilled in the art.
• assay methods are those using commercial kits or reagents (for example, ASSERACHROM® VII: Ag from Diagnostica Stago).
• the detection threshold of FVII in plasma is about 1 mIU / ml (0.5 ng / ml), a concentration below which FVII can not be detected.
• the detection threshold of FVIII in the plasma is about 10 mIU / ml (1 ng / ml), a concentration below which FVIII can not be detected.
• the detection threshold of the FIX in the plasma is about 0.2 mIU / ml (1 ng / ml), concentration below which the FIX can not be detected.
• the detection threshold of the FXI in the plasma is about 0.5 mIU / ml (2.5 ng / ml), a concentration below which the FXI can not be detected.
• Plasma depletion techniques as a factor of interest include all techniques known to those skilled in the art. Examples of depletion techniques include immunodepletion, chemical depletion, and the combination thereof.
• Immunodepletion is the use of antibodies specifically directed against an antigen contained in a solution for the purpose of substantially depleting said solution to the antigen of interest.
• the antibodies used to carry out an immunodepletion may be polyclonal and / or monoclonal derived from one or different cellular clones.
• the antibodies used can be directed directly against the antigen that it is desired to eliminate or against a protein that binds to this antigen.
• Plasma depleted in FVII, FIX or FXI can thus be obtained by using respectively anti-FVII, anti-FIX or anti-FXI antibodies.
• Plasma free of FVIII may be obtained using anti-FVHI antibodies or antibodies against von Willebrand factor, a plasma protein that transports FVIII to the blood.
• FVIII-depleted plasma can also be obtained by chemical depletion, using EDTA (ethylene-diamine-tetraacetic acid) since FVIII is a Ca 2+ dependent factor. EDTA is then removed by methods well known to those skilled in the art, for example by dialysis.
• EDTA ethylene-diamine-tetraacetic acid
• the plasma used to implement the present invention is a plasma lacking factor VII and at least one other factor selected from FVIII, FIX and FXI.
• the plasma used to implement the present invention is prepared from a hemophiliac plasma of type A, naturally free of FVIII, from a hemophiliac plasma of type B, naturally free of FIX, or from a plasma from patients with a complete deficiency of factor XI, said plasmas naturally free of FVIII, FIX or FXI are then depleted in FVII, by an immunological or chemical method such as those described above.
• the plasma used is prepared from a normal plasma which is, initially, spoiled in FVII and then, in a second stage, depleted of FVIII and / or FIX and / or FXI.
• initiator components of the thrombin generation reaction or “initiator components” means the essential components for starting the thrombin generation from prothrombin.
• the initiator components of the thrombin generation reaction essentially comprise a source of calcium ion (Ca 2+ ), a phospholipid agent and tissue factor (FT), in concentrations sufficient to trigger the thrombin generation reaction.
• a source of calcium ions suitable for the purpose of the present invention is any source of biologically compatible calcium ions, such as CaCl 2 .
• the source of Ca 2+ can be added to the sample / plasma mixture extemporaneously with the other initiator components of the thrombin generation reaction or in a delayed manner, ie after the addition of the other initiating components of the generation reaction. thrombin.
• a final concentration of calcium ions in the sample + plasma + initiator components mixture in the range of 14 to 18 mM, and in particular equal to 16, 7 mM.
• Phospholipid agents suitable for use in the present invention may be in the form of a concentrate or lyophilizate and preferably comprise a mixture comprising a major amount of phosphatidylcholine and phosphatidylserine or containing exclusively phosphatidylcholine and phosphatidylserine.
• a final concentration of phospholipidic agents in the sample + plasma + initiator components mixture in the range from 0.1 to 5 ⁇ M, in particular from 0, 5 to 2 ⁇ M, and more particularly equal to 1 ⁇ M.
• Tissue factor (FT) suitable for use in the present invention may be selected from the group consisting of any native, plasmatic, recombinant or transgenic tissue factor, any modified tissue factor, including any truncated tissue factor that has lost its function. activation of Factor VIIa Factor VIIa, provided that said modified Tissue Factor has retained, even partially, its ability to act as a cofactor of factor VIIa enzymatic activity.
• An appropriate modified tissue factor may, for example, be deleted from its transmembrane domain such as the tissue factor of the STACLOT kit of Diagnostica Stago (Reference 00281) available commercially.
• tissue factor concentration in the sample + plasma + initiator components mixture is in the range from 1 to 10 ⁇ M, in particular from 4 to 6 ⁇ M. , and more particularly equal to 5 ⁇ M.
• the test sample, the standard sample, the plasma devoid of FVII and at least one other factor chosen from FVIII, FIX or FXI, and / or the initiator components of the generation reaction Thrombin can be in liquid or freeze-dried form. When they are in freeze-dried form, these compounds can advantageously be resuspended, prior to the implementation of the process according to the invention, in a suitable aqueous solvent, such as purified water for injection (PPI).
• PPI purified water for injection
• the Applicant has therefore developed a method for measuring the level of activated factor VII in a test sample based on specific experimental conditions that make it possible to overcome the disadvantages resulting from the presence of non-activated FVII in the test sample. .
• the first step of this method is to mix a test sample containing an unknown level of activated FVII with a FVII-deficient and FVIII-deficient plasma, and / or FIX and / or FXI, so that the final FVII concentration + FVIIa in the resulting mixture is in the range of 10 ⁇ M to 80 ⁇ M. It is this specific combination of properties related to the nature of the plasma and the concentration range used which makes it possible to implement the method for measuring the level of activated FVII according to the invention.
• Initiating components of the thrombin generation reaction are then added to the test-sample + plasma mixture in order to trigger the cleavage reaction which leads to the generation of thrombin from the prothrombin contained in the plasma.
• a thrombin generation test is then performed.
• TGT thrombin generation test
• the thrombin generation test begins when the sample of interest (or a solution comprising it) is contacted with initiator components of the reaction. This initial time corresponding to the beginning of the thrombin generation test is called t 0 .
• the generated thrombin is then revealed by the use of a developing agent, preferably by the use of a fluorogenic agent, whose degradation by thrombin causes the appearance of a fluorescent compound, or by the use of a chromogenic agent.
• a developing agent preferably by the use of a fluorogenic agent, whose degradation by thrombin causes the appearance of a fluorescent compound, or by the use of a chromogenic agent.
• the fluorogenic agent or the chromogenic agent are added to the sample + plasma mixture at the same time as the initiator components of the thrombin generation reaction.
• the fluorescence resulting from the degradation of the fluorogenic agent by the newly generated thrombin is detected by a measuring device such as a fluorometer.
• a measuring device such as a fluorometer.
• the fluorimeter used is provided with recording means or means for tracing the variation of fluorescence over time.
• the data collected by the fluorimeter make it possible to establish the curve of variation of fluorescence over time, called thrombinogram.
• the peak height expressed in nM thrombin, corresponds to the maximum concentration of thrombin generated at a time t max during the reaction
• the latency expressed in minutes, is the time elapsed between the start of the test
• the peak time expressed in minutes
• the velocity expressed in nM thrombin formed / min, corresponds to the peak height divided by the difference between the peak time t max and the lag time.
• these parameters are directly provided by the device used to measure the formation of thrombin.
• standard thrombograms are obtained from standard samples containing a known level of activated FVII. As described above for the test sample, at least two standard samples containing different levels of activated FVII are mixed with FVII-free plasma and at least one factor selected from FVIII, FIX and FXI. . Initiator components of the thrombin generation reaction are then added to the sample-standard + plasma mixture to begin the Thrombin Generation Test and to obtain standard thrombograms corresponding to each standard sample.
• the concentration of FVII + FVIIa of the sample-standard + plasma mixture is between 10 ⁇ M and 80 ⁇ M.
• the concentration of FVII + FVIIa of the sample-standard + plasma mixture is substantially identical to the concentration of FVII + FVIIa of the sample-test + plasma mixture.
• the plasma mixed with the standard samples is identical to that which has been mixed with the test sample.
• the interpolation can be of linear, geometric, cubic, polynomial, Lagrangian or Newtonian type.
• the interpolation performed corresponds to the variation of the latency time as a function of the activated FVII level in the standard samples, to the variation of the peak time as a function of the activated FVII level in the standard samples or to the variation velocity versus FVII activated in standard samples.
• the level of activated FVII contained in the test sample is determined by plotting at least one of the parameters derived from the thrombinogram of this test sample on a corresponding interpolation made from standard thrombograms. If the reported parameter is the lag time, the interpolation used will correspond to the variation of the lag time as a function of the factor VII level activated in the standard samples. If the reported parameter is the peak time, the interpolation used will correspond to the change in peak time as a function of the factor VII level activated in the standard samples. Finally, if the reported parameter is the velocity, the interpolation used will correspond to the variation of the velocity as a function of the factor VII level activated in the standard samples.
• the factor VII level determined from the interpolation thus corresponds to the activated factor VII level of the test sample.
• the method of the invention may comprise an additional step of calculating the concentration of activated factor VII in the test sample from the activated factor VII level determined from the interpolation.
• the Activated factor VII concentration meets the following formula:
• FVIIa concentration FVIIa rate x FVII concentration + FVIla
• FIG. 1 Standard thrombograms obtained in the presence of a final concentration of FVII + FVIla of 50 ⁇ M in the sample-standard / plasma mixture, with a plasma devoid of FVII and FVIII, and for activated FVII levels ranging from 0 to 100% (with 5 ⁇ M Tissue Factor and 1 ⁇ M
• Figures 2 and 2bis Variations in lag time and peak time, respectively, as a function of the level of FVII activated in the standard sample, with a plasma deficient in FVII and FVIII, for a final concentration of FVII + FVIla 50 pM
• FIG. 7 Standard thrombograms obtained in the presence of a final concentration of FVII + FVIIa of 50 ⁇ M in the sample-standard / plasma mixture, with a plasma deficient in FVII and in FXI (with 5 ⁇ M Tissue Factor and 1 ⁇ M Phospholipids).
• Figures 8, 9, 10 and 11 Latency, peak time, peak height, and velocity variations, respectively, as a function of FVII level in the standard sample, with a plasma devoid of FVIII and FXI, for a final concentration of FVII + FVIIa of 50 ⁇ M (with 5 ⁇ M Tissue Factor and 1 ⁇ M Phospholipids).
• FIG. 12 Standard thrombograms obtained in the presence of a final FVII + FVIIa concentration of 10 ⁇ M in the sample-standard / plasma mixture, with a plasma deficient in FVII and in FVIII (with 5 ⁇ M tissue factor and 1 ⁇ M
• Figures 13, 14, 15 and 16 Latency, peak time, peak height, and velocity variations, respectively, as a function of FVII level in standard sample, with plasma devoid of FVII and FVIII, for a final FVII + FVIIa concentration of 10 ⁇ M (with 5 ⁇ M Tissue Factor and 1 ⁇ M Phospholipids).
• Figure 17 Standard thrombograms obtained in the presence of a final concentration of FVII + FVIIa of 80 ⁇ M in the sample-standard / plasma mixture, with a plasma deficient in FVII and FVIII (with 5 ⁇ M Tissue Factor and 1 ⁇ M Phospholipids).
• Figures 18, and 19 Variations in lag time and peak time, respectively, as a function of the level of FVII activated in the standard sample, with a plasma deficient in FVII and FVIII, for a final concentration of FVII + FVIIa of 80 pM
• the column was regenerated by eluting the fixed FVII with 20 mL of regeneration buffer (50 mM NaCl, 0.1 M glycine pH 2.4) and then the column was rebalanced with 20 mL of equilibration buffer (10 mM citrate). 0.15 M NaCl pH 7.4).
• the appropriate volume of an international standard sample of FVII (SI-FVII) provided by NIBSC and / or international standard of FVIIa (SI-FVIIa) also provided by NIBSC is taken to obtain a standard sample containing a known activated FVII, which is added to 80 ⁇ L of FVII- and FVIII-deficient plasma (plasma deficient in FVIII from the company Diagnostica Stago or being a plasma of type A hemophiliacs, which was then depleted in FVII as in Example 2), in order to obtain a mixture which contains a known fixed activated FVII level of between 0% and 100% for a concentration of FVII + FVIIa of between 10 ⁇ M and 80 ⁇ M.
• initiator factors of the thrombin generation reaction (Ca 2+ , phospholipids and FT) are added to the mixture comprising the plasma and the sample, at final concentrations of 5 ⁇ M in FT, 1 ⁇ M in phospholipids, (reagent Diagnostica Stago 86195 diluted 1/4) and 16.7 mM Ca 2+ , and 20 ⁇ L of agent specific fluorogenic thrombin (Fluca reagent kit Diagnostica Stago 86197).
• Standard TGT curves (standard thrombograms) were established for fixed and known levels of activated FVII between 0% and 100%, in order to obtain standard thrombograms providing the various parameters (latency, peak height, time at peak and velocity).
• the thrombograms are established using a fluorimetric device for measuring the thrombin formation time (Fluoroskan - Thermo Electron) equipped with software for establishing thrombinograms (Thrombinoscope BV Company), for a wavelength of excitation of 390 nm and an emission wavelength of 460 nm.
• FIG. 1 represents standard thrombograms obtained in the presence of 50 ⁇ M of final FVII + FVIIa in the plasma / sample mixture and for variable activated FVII levels ranging from 0 to 100%.
• a decrease in lag time and thrombin formation time at the peak is observed as a function of the increase in the level of activated FVII in the sample.
• the peak time reaches a limit that can be estimated at 14 minutes for an activated factor level of 100%. It is noted that a plasma deficient in FVII and FVIII without the addition of FVII or FVIIa does not induce the formation of thrombin.
• Figures 2 and 2bis show the variations in latency and peak time, respectively, as a function of the rate of activated FVII in the sample. The results obtained show that it is possible to establish a correlation between the level of activated FVII in the sample and the various parameters deduced from thrombograms obtained for a concentration of FVII + FVIIa of 50 ⁇ M in the plasma / sample mixture.
• Example 4 Preparation of a standard thrombinogram from a FVII and FIX-free plasma, for a concentration of FVII + FVIIa of 50 ⁇ M The experiment of Example 3 is repeated, but using a reactive plasma lacking in FIX, from the company Diagnostica Stago, which was then depleted of FVII according to the procedure of Examples 1 and 2.
• FIG. 3 represents the thrombograms obtained in the presence of 50 ⁇ M of FVII + FVIIa in the plasma / sample mixture, said plasma being devoid of FVII and of FIX.
• a decrease in lag time and thrombin formation time at the peak is observed as a function of the increase in the level of activated FVII in the sample.
• the peak time reaches a limit that can be estimated at 16 minutes for an activated FVII level of 100%. It is noted that plasma deficient in FVII and FIX without the addition of FVII or FVIIa does not induce thrombin formation.
• FIGS. 4, 5, and 6 respectively show latency, peak time, and velocity variations as a function of the rate of activated FVII in the sample.
• the results obtained show that it is possible to establish a correlation between the level of activated FVII and the various parameters deduced from the thrombograms obtained in the presence of 50 ⁇ M of FVII + FVIIa in the plasma / sample mixture, said plasma being devoid of FVII. and FIX.
• EXAMPLE 5 Preparation of a Standard Thrombinogram from a Plasma Free of FVII and FXI for a FVII + FVIIa Concentration of 50 ⁇ M
• the experiment of Example 3 is repeated, but using a reactive plasma free of FXI, from the company Diagnostica Stago, which was then spoiled in FVII according to the procedure of Examples 1 and 2.
• Figure 7 shows the thrombograms obtained in the presence of 50 ⁇ M FVII + FVlla in the plasma / sample mixture, said plasma being devoid of FVII and FXI.
• a decrease in lag time and thrombin formation time at the peak is observed as a function of the increase in the level of activated FVII in the sample.
• the peak time reaches a limit that can be estimated at 12 minutes for an activated FVII level of 100%. It is noted that a plasma lacking FVII and FXI without the addition of FVII or FVIIa does not induce thrombin formation.
• Figures 8, 9, 10, and 11, respectively, show variations in latency, peak time, peak height, and velocity as a function of the rate of activated FVII in the sample.
• the results obtained show that it is possible to establish a correlation between the level of activated FVII and the various parameters deduced from the thrombograms obtained in the presence of 50 ⁇ M of FVII + FVIIa in the plasma / sample mixture, said plasma being devoid of FVII. and in FXI.
• Example 6 Preparation of a standard thrombinogram from a FVII and FVIII-deficient plasma, for a concentration of FVII + FVIIa of 10 ⁇ M The experiment of Example 3 is repeated, but using a final concentration FVII + FVIIa of 10 ⁇ M.
• FIG. 12 represents thrombograms obtained in the presence of 10 ⁇ M of FVII + FVIIa in the plasma / sample mixture, said plasma being devoid of FVII. and in FVIII.
• a decrease in lag time and thrombin formation time at the peak is observed as a function of the increase in the level of activated FVII in the sample.
• the peak time reaches a limit that can be estimated at 21 minutes for an activated FVII level of 100%. It is noted that a plasma deficient in FVII and FVIII without the addition of FVII or FVIIa does not induce the formation of thrombin.
• Figs. 13, 14, 15, and 16 show variations in latency, peak time, peak height, and velocity, respectively, as a function of the rate of activated FVII in the sample.
• the results obtained show that it is possible to establish a correlation between the level of activated FVII and the various parameters deduced from the thrombograms obtained in the presence of 10 ⁇ M of FVII + FVIIa in the plasma / sample mixture, said plasma being devoid of FVII. and in FVIII.
• Example 7 Preparation of a Standard Thrombinogram from a Plasma Free of FVII and FVIII, for a Concentration of FVII + FVIIa of 80 ⁇ M
• FIG. 17 shows the thrombograms obtained in the presence of 80 ⁇ M of FVII + FVIIa in the plasma / sample mixture, said plasma being devoid of FVII and of FVIII.
• a decrease in thrombin formation time at the peak is observed as a function of the increase in the level of activated FVII in the sample.
• the peak time reaches a limit that can be estimated at 12 minutes for an activated FVII level of 100%. It is noted that a plasma devoid of FVII and FVIII without the addition of FVII or FVIIa does not induce thrombin formation.
• Figures 18, and 19 show the variations in latency and peak time respectively as a function of the rate of activated FVII in the sample.
• Example 8 Measurement of the activated FVII level of a rabbit milk sample containing FVII The test sample has an unknown rate of
• Activated FVII and its concentration of FVII + FVIIA is 500 ⁇ M.
• a volume of 8 .mu.l of this sample to be tested was mixed with 72 .mu.l of plasma lacking FVII and FVIII as described above. This gives a test mixture whose volume is 80 .mu.L and whose concentration of FVII + FVIIA is 50 .mu.M.
• thrombin generation reaction 20 ⁇ l of the initiator components of the thrombin generation reaction (Phospholipids and FT) were added to final concentrations of 5 ⁇ M FT, 1 ⁇ M phospholipids, (Diagnostica Stago reagent 86195 diluted 1/4), and 20 ⁇ l of a thrombin-specific calcium fluorogenic agent (final concentration of 16.7 mM Ca 2+ ) (Fluca kit Diagnostica Stago 86197 reagent)
• FVII and FVIII as previously described in order to obtain a concentration of FVII + FVIIa of 50 ⁇ M.
• initiator components of the thrombin generation reaction were added to the sample / plasma mixture to achieve final concentrations of 5 ⁇ M FT and 1 ⁇ M phospholipids (Diagnostica Stago 86195 reagent diluted 1 / 4).
• 20 .mu.l of a fluorogenic calcium-specific thrombin agent final concentration of 16.7 mM Ca 2+
• Fluca kit Diagnostica Stago 86197 reagent were added to the previous mixture.
• Standard thrombogram parameters were measured to plot standard curves for each of the parameters as a function of the -log of the activated FVII level (see Figures 2 and 2bis and Table 1).
• the values of the parameters obtained from the thrombinogram of the mixture containing the test sample were plotted on the different standard standard curves, which makes it possible to deduce a measurement of the level of activated FVII in the sample to be tested.
• a latency time of 6 minutes and 30 seconds and a time at the peak of 18 minutes are obtained, by plotting these values on the different standard curves, a measurement of the level of activated FVII in the sample to be tested equal to 20 is deduced. %.
• Example 9 Influence of Rabbit Milk on the Thrombin Generation Test
• the experiment of Example 3 is repeated, but by prediluting the standard sample containing a known level of activated FVII in Owren-Koller buffer containing 1% human serum albumin (Tp OK - 1% HSA) or in Tp OK - 1% SAH containing rabbit milk.
• Figure 20 shows standard thrombograms obtained in the presence of 50 ⁇ M of final FVII + FVIIa in the plasma / sample mixture and for 0% and 100% activated FVII levels.
• a perfect superposition of the thrombograms of the prediluted sample in OK-1% SAH Tp and prediluted sample in OK-1% SAH Tp containing rabbit milk is observed, which allows to deduce that rabbit milk has no influence on TGT. It is noted that a plasma deficient in FVII and FVIII without addition of FVII or FVIIa does not generate thrombin formation.
• Figures 21, 22 and 23 show thrombograms, latency and peak time variations, respectively, as a function of activated FVII level in the prediluted Tp OK-1% SAH sample containing rabbit milk for known fixed activated FVII level between 0% and 100%.
• Latency 11 8, 83 7, 50 6, 50 5, 33 4, 83 4, 50 4, 17 (min) 50

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