MX2012004513A - Coagulometric method for the antiphospholipid antibody titer. - Google Patents

Abstract

The present invention describes a method for quantifying the serum antiphospholipid antibody (APS) and/or for diagnosing or monitoring the antiphospholipid antibody syndrome (APLS), this being a coagulometric method of the modified thromboplastin time based on the standardization of the phospholipid concentrations required for evidencing the presence of the antibody, where according to the amount of antibody present in the samples and the measurement of the increase of the coagulation time, the thrombotic level would be correlated in the different life stages of patients with APLS.

Description

Coagulometric method for the titration of antiphospholipid autoantibodies Field of the invention.

The present invention corresponds to the field of in vitro diagnostic systems for the diagnosis of antiphospholipid autoantibodies in blood, for example human blood, specifically by coagulometric methods for the identification of alterations in hemostasis derived from the presence of said antiphospholipid autoantibodies.

BACKGROUND OF THE INVENTION The Antiphospholipid Antibody Syndrome (SAAF), also known as Hughes syndrome, is an autoimmune entity with defined clinical characteristics associated with a group of anionic antiphospholipid autoantibodies (AAF).

Antiphospholipid antibodies (AAF) are a wide range of heterogeneous autoantibodies of IgG, IgM and / or IgA type; the most common being anticardiolipin antibodies (ACA) and lupus anticoagulant (AL). Others not so frequently investigated are the anti-beta2 glycoprotein 1 (AB2GP1) antibody and the antiphosphatidylserine (AFS).

The SAAF manifests clinically at different levels: - May affect reproduction causing recurrent spontaneous abortion; second-trimester fetal death and the beginning of the third trimester (the risk of abortion in patients not treated with SAAF can be over 90%); severe intrauterine growth retardation; severe preclampsia of early onset (before 34 weeks); premature birth; alterations in the non-stressful basal record (RBNE), and elevations of alpha-fetoprotein in maternal serum without apparent cause in the middle trimester.

- At the vascular level it can cause arterial or venous thrombosis in any territory but more commonly in the lower extremities, intervellositary thrombosis, intravellositary infarcts and decidual vasculopathy. The onset of symptoms is usually observed during pregnancy or the use of oral contraceptives. A third of patients with thrombotic events have presented at least one pulmonary embolism and recurrent episodes can lead to pulmonary hypertension. Occlusions in the arterial system manifest as transient ischemic attacks, amaurosis fugax, and cerebrovascular accidents.

- Other alterations can manifest in the heart valves, a positive Coombs test, livedo reticularis, migraine, ulcers in the lower extremities, myelopathy, chorea, pulmonary hypertension (in the absence of pulmonary embolisms), avascular necrosis.

The SAAF is usually manifested especially in women (80%), between 20 and 40 years; It occurs frequently in patients with a basic autoimmune disease, usually systemic lupus erythematosus (SLE). In this context, the entity is known as a secondary SAAF. When SAAF is detected without autoimmune pathology underlying is called primary SAAF, which represents more than half of obstetric patients with SAAF. Most women with primary SAAF will not progress to an SLE and may have periods of remission (clinical and laboratory) with little risk of thrombotic manifestations. However, pregnancy imposes a special "stress" being unusual for a patient with SAAF to have a normal reproductive result if it is not treated.

There are two types of tests that can detect antiphospholipid antibodies: the first is the cardiolipin antibody test, which allows detecting several classes of antibodies: IgG, IgM, and / or IgA, (cardiolipin is a very acid phospholipid composed of two molecules of phosphatidic acid, covalently linked by a glycerol molecule, is found mainly in the inner membrane of the mitochondria and in bacteria). The second type of tests are functional and evaluate the lupus anticoagulant (AL) which is a heterogeneous group of autoantibodies of the IgG, IgM and / or IgA isotypes, whose ability to bind to the phospholipids and proteins associated with the cell membrane, prevents coagulation of visible blood in a test tube, this is the basis for the tests for LA, where the time taken for the sample to clot (partial thromboplastin time or TTP) is measured, and require the presence and action of phospholipids for coagulation to occur. Subsequently, other studies of AL confirmation are required, preferably with a method similar to that used in the initial screening stage.

These confirmatory tests and other diagnostic tests may include the RVVT (English, Russell viper venotn time), the PNP (platelet neutralization procedure) and the CT (kaolin clotting timé), and / or the neutralization test of phospholipids (hexagonal) .

Due to the variability in the clinical criteria and laboratory tests for the diagnosis of SAAF, international working groups have been established, which periodically review and update the available information about the elements that make it possible to establish the diagnosis of antiphospholipid syndrome. Thus, the most recent revision proposes the following criteria: Vascular thrombosis: · One or more episodes of arterial or venous thrombosis or, • Thrombosis of small vessels in any organ or tissue confirmed by Doppler imaging or histopathology. By histopathological confirmation, thrombosis should be present without significant evidence of inflammation of the vascular wall.

Morbidity in pregnancy: · Three or more consecutive abortions without explanation, excluded with anatomical, genetic or hormonal studies or, • One or more fetal deaths with normal fetal morphology after 10 weeks of gestation, documented by ultrasound or by direct examination of the fetus or, • One or more preterm births of morphologically normal neonates before week 34 of gestation, associated with severe pre-eclampsia or severe placental insufficiency.

Laboratory criteria: • Anticardiolipin antibodies positive IgG and / or IgM and presence of the medium to high titer or persistent on 2 or more occasions with difference of 6 weeks, • Determination of anti-B2-glycoprotein 1 (AB2GP1), by ELISA dependent on anticardiolipin antibodies, • Determination of lupus anticoagulant (AL), the abnormality present in the plasma on two or more occasions with a difference of six weeks between each determination, • Prolongation of phospholipid-dependent coagulation tests (activated thromboplastin (aPTT), Kaolin time (KCT), Russell's viper venom time (TVdR or RVVT) diluted, diluted prothrombin time, Textarin time, • Failure to correct with normal platelet-poor plasma mixture, • Shortening or correction of trace tests with the addition of an excess of phospholipids (confirmatory), • Exclusion of other clinically evident coagulopathies, such as FVIII inhibitor; FV; heparin effect. People with ACA and LA may present an abnormally high risk to trigger blood clotting. AL is a prothrombotic agent, because the presence of these antibodies produce precipitation and formation of thrombi in vivo; AL interacts with the platelet membrane causing an increase in the adhesion and aggregation of these, thus having its prothrombotic characteristics. Its name, lupus anticoagulant, derives from its properties in vitro, and as mentioned, it manifests as an increase in activated partial thromboplastin time (aPTT), and also in the Russell viper venom time test (TVdR) (Brandt JT, et al, 1995).

In the early 50's, it was observed that some patients with generalized lupus erythematosus (LEG), showed prolongation of aPTT, simulating an anticoagulant effect. Subsequently, in 1972 Feinstein and Rapaport (2) called it lupus anticoagulant, an inappropriate term because it is not exclusively identified in this pathology, but also in other conditions, such as the primary / secondary syndrome of anti-phospholipid-protein antibodies and other diseases. autoimmune, as well as in patients receiving drugs such as phenothiazines, procainamide, quinidine or hydralazine. The presence of LA has also been described in patients with viral infections such as human immunodeficiency virus (HIV) or in acute bacterial infections, in neoplasms, in some neurological diseases (Guillan-Barré syndrome) or in women with recurrent fetal loss , in subjects with thrombocytopenia and patients with unexplained arterial or venous thrombosis (Love and Santoro, 1990, Gharavi AE et al, 1994).

The laboratory tests used in the diagnosis of SAAF are indicated in Table 1.

In the last review of the clinical and laboratory criteria carried out in Sydney Australia, the new parameters were established in the diagnosis of SAAF, which are observed in table 2.

Table 1. Laboratory tests in the identification of the SAAF.

Table 2. Sapporo Criteria - Sidney Review (Miyakis et al., J. Thromb. Haemost., 2006).

Clinical criteria Thrombosis: One or more episodes venous, arterial or small vessels confirmed.

Pregnancy: a) One or more unexplained fetal deaths > 10 sem. b) One or more preclampsias / placental insufficiency < 34 weeks c) 3 or more miscarriages of < 10 consecutive unexplained weeks.

Laboratory criteria Upper-middle titers of anti-CLP Ab IgG or IgM on more than 2 separate occasions at least 12 weeks Ac anti-2GPI: Upper-middle titers of anti-p2GPI IgG and / or IgM on more than 2 separate occasions at least 12 weeks Lupus anticoagulant: 2 altered coagulation tests on 2 separate occasions at least 12 weeks.

To diagnose SAAF, at least one major clinical criterion plus a positive serological test is required (positive AL and / or ACA IgG in moderate or high titres (> 20 GLP units)).

A positive serological test should ideally be repeated after 12 weeks (considering a second laboratory) to obtain confirmation. The clinical significance of a positive result isolated from ACA, whether IgM or IgA, is not definitive: 2% of the normal obstetric population has low ACA titres and these findings do not seem to be associated with an adverse prognosis. The diagnosis of SAAF requires rigorous criteria since the prevention of poor pregnancy outcomes and perinatals are a potentially risky therapy.

The assays that are used in the detection of ACA, or AB2GP 1, are based on immunoenzymatic methods, in which the concentration of antibodies is evaluated when binding to the phospholipid-protein system.

In the study of the clinical problems of haemostasis, such as hemorrhage and thrombosis, a large number of laboratory tests have been incorporated to directly or indirectly assess hemostatic function.

The correct understanding of these tests will act positively in the evaluation of patients by becoming a useful tool in diagnostic support, as well as in therapeutic monitoring and prognosis.

Pregnant patients with recurrent miscarriages, even without clinical data or confirmatory serological tests of connective or immunological tissue diseases, must undergo the tests indicated to detect LA, since their diagnosis requires applying the suggested treatment, which would guarantee the Proper completion of pregnancy.

The methodological control of the variables involved in the effectiveness and efficiency of haemostasis tests is based on the stratification of the variables in relation to the moment in which the technical procedure of the laboratory study is carried out in a preanalytical, analytical and post-analytic manner. . This systematization in the study of the variables allows to provide the clinician with a laboratory result with known and controlled scopes and limitations, to incorporate it in the evaluation of the patient with alterations in hemostasis (Table 3).

Table 3. Studying phases of hemostasis The diagnostic techniques of AAF or SAAF described in patent applications are based on the use of antiphospholipid antibodies (WO / 2008/078193 and US7053 178), where patent US7053178 describes the use of intermediate peptides that inhibit the binding between antibodies AB2GP1 and the phospholipids, while the patent US7252959 describes the use of annexin V that inhibits the binding between the phospholipids and the antibodies, so their inclusion in coagulometric methods helps to confirm the diagnosis.

The existing reagents for the determination of the anti-phospholipid antibodies by coagulometric (AL) methods, are made up of 2 reagents with different concentrations of phospholipid, one of lower concentration, which allows to show the antibody, expressed by prolonging the coagulation time and another reagent with concentration of excess phospholipids that corrects the time of coagulation, using as normal control the times obtained with a pool of healthy donors, therefore "only express the positivity to the test", by means of a mathematical index that is obtained by determining the difference of the coagulation times between the prolongation and the correction.

The application WO / 2003/083490 describes a method based on the measurement of coagulation in time by measurement of transmittance, where the result of the sample to be analyzed is compared with a control sample (normal blood), and another sample is included with excess phospholipids as confirmation of the phospholipid dependence.

All the above tests are qualitative and confirm the diagnosis of AAF or SAAF in the reaction mixture, however none of them measures the activity of the antibody (quantitative).

The aPTT test consists of taking a blood sample in a vacuum test tube in the presence of oxalate or citrate to stop coagulation by binding to calcium, then activating the intrinsic pathway of coagulation by adding phospholipids and an activator such as silica, kaolin or ellagic acid and calcium (to reverse the anticoagulant effect of oxalate) that are mixed in the plasma sample. Time is measured until a clot or thrombus forms.

In coagulation tests it must be taken into account that the inhibition of coagulation by AL is greater in the system with low phospholipid concentrations and the abnormal prolongation of said tests is corrected by the addition of phospholipids to the test system (Feinstein DI , et al 1972).

It should also be considered that because AL is a heterogeneous group of acquired autoantibodies or inhibitors, it is clear that the tests used to detect them show variable results when used in different groups of patients and even in the same patient or group. healthy population (Exner T, et al 1991).

Given the dilemma of a specific test, it has been decided to use a defined process, which includes various tests and allows to establish the presence of the AL or the indirect detection of it by means of inhibition tests. In the algorithms most used in the systematic study of LA, they divide the techniques into those that allow the LA to be traced (table 4) and those used to confirm their presence (table 5), or integrated from the initial result in the determination of the APTT.

Coagulation assays for detecting AL may be those based on prothrombin time, either those measuring the extrinsic pathway of coagulation (coagulation factor FVII, FX, FV and prothrombin), activated partial thromboplastin time (aPTT) ) which measure the intrinsic pathway of coagulation (FXII, FXI, FIX, FVIII, FX, FV and prothrombin) and the Russell viper venom time assay, which directly activates FX and measures the common coagulation pathway ( FX, FV and prothrombin), each of these assays has its own sensitivity and specificity for AL.

The tests of AL are made more standardized and according to guidelines that the laboratories follow, however improvements are still required; since the samples of plasmas that present much lupus anticoagulant can be confused with the diagnosis of other anticoagulants or with the presence of heparin; likewise, the presence of low levels of AL also gives rise to false negatives. The concentrations of AL above 15% can be corrected if the samples are measured immediately after the reaction mixture, that is, without prior incubation, since the increase in pH that occurs in the incubation lengthens the clotting time artificially . The technical principles for each test differ substantially, but can be summarized according to Table 4.

The coagulation factors are plasma proteins that promote coagulation, while coagulation inhibitors are directed against some binding site in the process of blood coagulation, which can be natural those that are part of plasma proteins, own of blood and which are responsible for inhibiting coagulation as anti-thrombin III, and specific those that are directed against a specific factor of coagulation as antibodies against factor V or the case of the lupus anticoagulant that is directed against phospholipids .

In principle, any test that measures the coagulation reactions where the conversion of fibrinogen to fibrin occurs by thrombin is suitable for the detection of AL, however, it should be considered that the results among the laboratories that perform them must coincide, for this they should only reagents with adequate quality control (such as commercial ones); Another consideration is that the assay be robust and highly reproducible, since many of the assays are often affected by the variable presence of phospholipids in the sample.

The detection of LA can be problematic in patients receiving oral anticoagulants or unfractionated heparin because the coagulation times of such patients appear longer in both the screening studies and in the combined studies, giving false positive results; in these cases the trials require combining the studies with normal plasma and performing a confirmatory test. When using normal plasma the studies require more manipulation and therefore are exposed to more variations in the results.

Importance of the diagnosis of SAAF.

The early detection of AL in the blood plasma of women with recurrent abortions is of great importance because the rapid application of a treatment based on prednisone, aspirin or both, allows in many cases to achieve the completion of pregnancy. Soulier & Boffa and Luke et al. (16, 24) suggest the combination of doses of immunosuppressive agents, such as prednisone (40-60 mg / day) and small doses of aspirin. Jaffe et al., Found with this treatment a significant reduction in spontaneous abortions of affected women (31).

The success of this therapy depends on the suppression of LA and the normalization of coagulation tests. There are reported cases of patients who do not respond adequately to this treatment (16, 24); Lubbe et al. reported failure in two cases, which did not correct their coagulation tests and whose placentas presented infarction and thrombosis (16). In this situation, a more aggressive treatment is recommended, and for 3 to 8 weeks, with doses of prednisone that return the normal values of TTP and CT.

Another important aspect in relation to the treatment is to start it as soon as the diagnosis of pregnancy is confirmed, preferably before twelve weeks. Prednisone, at a dose of 40 mg / day, is continued for one month, until the TTP and KCT tests are normal, and then reduced to 10 or 15 mg / day, enough to keep the tests at the lower limit of the normal field (30.31). In patients who do not respond to such treatment within a month, it is recommended to increase the dose of prednisone and add azathioprine or cyclophosphamide from 1.0 to 1.5 mg / kg (6, 24).

Table 4. Trace tests in the identification of lupus anticoagulant * The Rosner index (IR) is useful to evaluate the results in the correction with normal plasmas mix. IR = b-c xl00 / a, where a = coagulation time of the patient's plasma, b = coagulation time of the mixture, c = coagulation time of the normal control or control plasma. The IR equal to or less than 15 means correction.

Table 5. Confirmatory tests in the identification of lupus anticoagulant In general, once a patient is diagnosed with SAAF, the anticoagulant treatment should be administered for a period of time greater than that which could be indicated in the absence of these antibodies. For this reason, it is imperative that the assays or tests used to detect antiphospholipid antibodies be of sufficient quality to ensure an optimal diagnosis.

On the other hand, the existing laboratory tests that evaluate the anti-phospholipid antibody (AAF) by coagulometric methods are dependent on analytical variables that have to do with the composition of the reagent, since there are various commercial reagents with which the lupus anticoagulant (AL). All these tests are NOT quantitative, nor do they directly measure the antiphospholipid antibody concentration; besides showing a wide variability in the characteristics of thromboplastins.

Among the analytical variables that most influence the sensitivity and specificity of the tests used in the detection of LA, are: a) The activator used to accelerate the reaction. Among the most used are kaolin, silica dioxide and aluminum oxide as insoluble activators and ellagic acid and Russell's viper venom as a soluble activator, insolubles being more sensitive for this effect. b) The main variable that affects the sensitivity of the reagent to detect small amounts of antibody is the concentration at which each of the components that make up the reagent is present. c) The concentration of phospholipids turns out to be critical, since in test systems with a reduced concentration of phospholipids (aPTT, TVdR, kaolin time) offers the possibility of increasing the sensitivity in AL detection; in this regard also the use of platelet-free plasma is included. d) Another variable is the type of activator used in the APTT, to accelerate the contact phase; thus ellagic acid does not turn out to be an adequate activator or accelerator, when compared with the silica particles (3).

Due to the high concentrations of phospholipids and / or activator that is used in most commercial reagents with which the AL study is performed, they do not allow the antiphospholipid antibody to be demonstrated, because it neutralizes the action of the antibody, such Thus, the coagulation time under study does not extend significantly with respect to the control, making this method less sensitive to detect low antibody concentrations.

A more sensitive detection alternative consists in the dilution of the thromboplastin, decreasing the concentration of phospholipids present in the reagent, thus an increase in the sensitivity of the detection is observed but the concentration of the activator is also altered. With these two variables that modify the clotting time, the problems to interpret the patient's results with respect to the control increase.

As described in detail below, the present invention relates to a more sensitive AL detection method and with the possibility of determining the concentration thereof, regardless of the presence of high concentrations of endogenous phospholipids in the sample.

The fact that AL is actually a very heterogeneous group of antibodies, limits the use of a single test to identify it, there is even controversy about the specific binding site of the antibodies of the AL; since it has been pointed out that it is directed against different epitopes of the anionic phospholipidic components (phosphatidylserine, phosphatidyl-inositol, phosphatidic acid and diphosphatidyl glycerol or cardiolipin), or neutral phospholipids (phosphatidylethanolamine in hexagonal form), or against the neoepitopes of the phospholipid-protein binding such as FVII-tissue factor, diezae or prothrombinase complexes with negative net charge (phosphodiester group) as well as in other endothelial complexes thrombomodulin-thrombinaproteins C and S, high molecular weight kininogen, PGI2, platelet or b2 glycoprotein. When the LA is fixed to each of its epitopes, it prevents its proper functioning, which translates into some or some of the clinical manifestations that appear in this syndrome. In vitro, phospholipid-dependent coagulation times are prolonged by making the catalytic surface inaccessible (7,8). The most mentioned mechanisms are described in table 6.

The main consequences of such heterogeneity are first, that there is no gold standard of control tests with plasma to contrast, in order to determine the cut-off values and evaluate the reagents, and second, the results of coagulation times, for a given LA trial, they are variable among themselves, which has led to the creation of numerous trials and diagnostic algorithms to express the results. Until now it is considered that the diagnosis of AL is difficult and the accuracy of the AL tests It is far from being safe. After an optimal pre-analytical step that includes the scrupulous preparation of platelet-free plasma, the diagnosis of LA includes four steps: a) a screening with at least 2 very high sensitivity tests, b) the demonstration of the inhibitory activity by studies with plasma mixture, c) the demonstration that the inhibitor is phospholipid-dependent, d) the exclusion of another coagulopathy, especially deficiencies due to coagulation factors or anti-factor antibodies (Revue Francophone des Laboratoires, volume 2006, no 382, May 2006, pp. 33-38).

Table 6. Sites of action of the lupus anticoagulant.

The existing reagents for the determination of the anti-phospholipid antibodies by coagulometric method (AL), consist of 2 reagents with different concentrations of phospholipid, one of lower concentration that allows to show the antibody, expressed by prolongation of the coagulation time and another Reagent with excess concentration of phospholipids that corrects the clotting time, using as normal control the times obtained with a pool of healthy donors, therefore "only express the positivity to the test", by means of a mathematical index that is obtained when determining the difference of coagulation times between prolongation and correction.

Due to the low sensitivity of the aPTT reagents to detect low levels of AL, it is difficult to establish a general criterion for their detection, besides that the coagulometric techniques, although they are relatively simple in their execution, do not quantify the concentration of the antibody.

Therefore, the present invention provides a coagulometric method to demonstrate the presence of the antibody, regardless of the concentration to which it is found in each of the patients, which allows to evaluate its titer and relate it to the clinical conditions of the patient, such and as described in detail in the following sections.

Modalities of the invention.

The main purpose of the present invention is to provide a method for the titration of antiphospholipid antibodies, as well as to establish the presence and titer of the antibody, by using different concentrations of phospholipid in the modified APTT test.

The coagulometric method of the invention enables the determination and titration of antiphospholipid antibodies in a plasma sample or in a series of plasma samples, whereby the results obtained by the method of the invention aid in the diagnosis of diseases associated with the presence of said antibodies, such as AAF and SAAF.

The method of the invention comprises the controlled addition of phospholipids in the APTT test maintaining the activator at a constant concentration, thus achieving that only one variable has an effect on the coagulation time, which allows to determine the presence of the antiphospholipid antibody, even in low concentrations in the sample.

Part of the embodiments of the invention consists of the possibility of measuring the concentration of phospholipids necessary to demonstrate the presence of the antibody, by prolonging the aPTT, and on the other hand it allows us to measure the concentration of the antibodies through the addition of amounts defined of phospholipids.

In one of its embodiments, the present invention provides reagents comprising phospholipids at known and controlled concentrations, which vary their concentration in each of the determinations, while the concentration of the activator necessary for the test remains constant for all determinations.

The method of the present invention allows to demonstrate the presence of antiphospholipid antibodies in the analyzed sample, as well as to quantify its titer regardless of the concentration to which it is found in each patient, and then to relate the titer obtained with the clinical conditions of the patient, way that allows to monitor the disease and relate the concentrations of such antibodies with the evolution or treatment of diseases associated with these antibodies.

Another embodiment of the invention relates to a kit or diagnostic kit for performing the titration of antiphospholipid antibodies by coagulometry (TITFOS) in samples from suspected patients, which makes it possible to determine precisely the presence and quantity of said antibodies.

In another of its modalities, the present invention provides a test that enables an accurate and quantifiable diagnosis of diseases associated with the presence of antiphospholipid antibodies, such as example AAF or SAAF, so that the patient diagnosed can receive an effective treatment with fewer side effects, as well as being monitored during treatment to be able to quantify the presence of such antibodies (evolution of the disease), so the method of invention provides an added value for individualized clinical research, as well as for populations where you want to apply the test.

In one of its modalities, the method of the present invention is able to detect 100% of patients positive for ACA IgG + AB2G-1 associated with thrombosis and recurrent fetal loss, detect at least 86% of patients positive for ACA IgG, and at least 84% of patients positive for AB2GP-1, detect at least 67% of patients positive for LA, detect at least 83% of patients positive for AAF and detect at least 99% of patients positive for SAAF .

Brief description of the figures.

Figure 1. The titration method of anti-phospholipid antibodies of the invention and the graphic representation of the presence of the antibody as well as the evidence of the activity thereof are shown.

Figure 2. The ROC curves for ACA (A), ABP2GP (B) and AL (C) are shown, where the diagonal segments are produced by the ties.

Figure 3. A graph of the cut-off values of the anti-phospholipid antibody titration method of the invention is shown. The cut-off values are established through the graphic union of the line that passes through all the points.

Figure 4. A graph of the titration method of anti-phospholipid antibodies of the invention is shown where the line represents the cut-off values and the points represent the 5 values obtained for each reagent of the patient no. 32 Detailed description of the invention.

As already mentioned, the presence of AL in plasma alone or in combination with the presence of antiphospholipid antibodies in solid phase such as ACA or AB2GP1 is an important prerequisite for the diagnosis of SAAF.

Known routine coagulation tests (prothrombin time, partial thromboplastin time or cephalin time) to determine AL are less sensitive tests, while techniques such as activated partial thromboplastin time (aPTT) or cephalin-kaolin, of inhibition of tissue thromboplastin The dilute diluted Viper viper venom, kaolin clay and Exner's modified kaolin are more sensitive, but none of them determines the concentration of antiphospholipid antibodies, that is, they are merely qualitative tests.

The present invention proposes a standardized method for diagnosing SAAF comprising a system of reagents for the determination and titration of anti-phospholipid antibodies for the determination of AL, which comprises different concentrations of phospholipid keeping the activator (silica dioxide, Russell's viper venom and / or ellagic acid) constant, that is to say that the only variable that affects the coagulation time of each one of the determinations is the capacity that the antibody has to inhibit coagulation, where said quality of the standard system allows: - To demonstrate the presence of the antiphospholipid antibody by means of the mathematical evaluation of the normal behavior of samples of healthy subjects in the standardized system of the invention, with respect to the behavior of samples of subjects with the presence of the antiphospholipid antibody, that is, unlike the reactants and conventional methods, the reagent system of the invention, not only establishes the prolongation and correction of coagulation times as a criterion of positivity, but also is based on the evaluation of the behavior of coagulation times in the reagent system, to establish your positivity.

- Unlike conventional reagents that only use 2 concentrations of phospholipids (one of lower concentration and one of excess), the system of the invention uses a wider range of these, for example a range of 5 variations in phospholipid concentrations , which allow the antibody to be demonstrated even when it is present in very small or asymptomatic quantities, which gives the method of the present invention greater sensitivity.

- Unlike conventional reagents and methods, the reagent system of the invention allows the titre of the antibody present in the sample of subjects with this disease to be quantified by determining the capacity of inhibition that the antibody has to prolong the coagulation times in the reagent system and the evaluation of its mathematical behavior.

- Unlike conventional reagents and procedures, the reagent system allows measuring the activity of the antiphospholipid antibody in subjects with the disease, which has a great diagnostic value, since the activity of the antibody is intimately related to the thrombotic risk.

- Establish the antiphospholipid antibody titer, by the ability or activity that the antibody has to inhibit the action and binding sites of the phospholipids present in the system of integrating reagents of the method of the invention.

- Establish the antiphospholipid antibody titre individually for each patient, by comparative analysis of the activity of the antibody, with respect to normal behavior (healthy population), in the reagent system of the invention.

- Obtain results of the antibody titer, which represents its activity and not necessarily its concentration.

- Represent the antibody titer with arbitrary units, defined as for example Units of inhibition of active phospholipids (UNIFAC).

- Demonstrate the presence or absence of antiphospholipid antibodies, by graphically plotting the coagulation times obtained with the reagent system of the invention, which make up said test.

- Represent graphically the individual mathematical behavior of antiphospholipid antibodies for each patient under study.

- Determine by means of the analysis of the behavior of the antibody, the activity of said antibody at different times of life for patients with SAAF, which allows to monitor the evolution of the disease and / or its treatment.

- Determine the thrombotic risk of patients with SAAF, since the method of the invention measures the activity of said antibodies, by means of the capacity of inhibition of the phospholipids, which has a great diagnostic value, since the activity of the antibody, is closely related with the thrombotic risk.

During the development of the invention it was possible to verify that by the controlled addition (dosing) of phospholipids in the coagulation time test by keeping the activator (silica dioxide, Russell's viper venom and / or ellagic acid) at a constant concentration, a single variable is obtained that has an effect on the coagulation time, with which it is possible to determine the presence of the antiphospholipid antibody, even at low concentrations in the samples analyzed.

The main aspects of the reagent system of the method of the invention are conferred to the use of different concentrations of phospholipids, such as low, normal and high, taking as reference concentration the optimum amount of phospholipids to obtain a TTP of healthy donors in a seconds, that is to say that from this "normal" concentration, 2 dilutions of phospholipids are made at 3.125% and 6.25% (lower) and the phospholipids are concentrated at 200% and 300%.

Regardless of the number of phospholipid concentrations that could vary in the standardized system of the invention, comprising for example 5 different concentrations, the result obtained is the same. It is demonstrated that the behavior of the anti-phospholipid antibody is related to the ability to inhibit the clotting time, which is a function of evaluating its behavior with different concentrations of phospholipid, present in the reagent system of the invention, so which independently of the number of concentrations, the resulting behavior is the same. Likewise, the origin and composition of the phospholipids that could vary in the reagent system of the invention, does not affect the result obtained therein, which represents a great advantage, since phospholipids can be used from various sources without altering the result obtained according to what is described for the method of the present invention.

In nature, different types of phospholipids and their isotypes are present; because the anti-phospholipid antibody is directed against these, and not necessarily against any of them in specific, regardless of the type or types of phospholipids that make up the reagent system of the invention, the result is the same.

Similarly, the composition of the coagulation promoter that forms part of the reagent system of the method of the invention, whatever the origin or quantity of this, the rationale is to keep it constant, to establish as the only variable of the coagulation time at phospholipids, so the result is the same, regardless of the amount and composition of the activator.

It can be considered that an abnormal TTP result (too prolonged) may be due not only to the presence of LA but to any of the following causes, for example cirrhosis, disseminated intravascular coagulation (DIC), factor XII deficiency, hemophilia A, hemophilia B , hypofibrinogenemia, malabsorption, vitamin K deficiency and von Willebrand disease; The TTP test is only an indication of AL, so in addition to the qualitative test, confirmatory tests are required.

Although the APTT test is very sensitive, the reagents used for it do not allow detecting low levels of AL, so establishing a general criterion for its detection is difficult; besides, the known coagulometric techniques, although they are relatively simple in their embodiment, do not quantify the concentration of the antibody. Precisely one of the objects of the present invention relates to a method for determining the titration of the anti-phospholipid antibody by the coagulometric test. Even the confirmatory techniques mentioned in Table 5 do not allow the LA to be quantified.

The known assays for AL are by definition dependent on phospholipids, and are carried out in the presence of limited amounts of phospholipids since the presence of high concentrations of them can mask the presence of AL antibodies, in addition to ensure optimal detection of AL , the content of endogenous phospholipids in the sample must be low. With all these technical elements, the developers of the present invention present a method with which it is possible to determine the presence of the antiphospholipid antibody, even in the presence of low concentrations of said antibody and which is based on the controlled addition of phospholipids in the test of APTT maintaining the activator (silica dioxide, Russell viper venom and / or ellagic acid) at a constant concentration, so that with the method of the invention there is only one variable that affects the coagulation time. With the method of the invention it is possible to measure the concentration of phospholipids necessary to demonstrate the presence of the antiphospholipid antibody, by prolonging the coagulation time, and on the other hand it allows to measure the activity of the antiphospholipid antibodies through the addition of defined amounts. of phospholipids, so that the title of the antiphospholipid antibody is obtained through this coagulometric test.

The diagnostic method of the present invention confirms the dependence on phospholipids, which is why it is also a confirmatory test for LA, besides that it is not affected by the concentration of phospholipids that originally appear in the sample. With the method of the present invention, false positives caused by the presence of other anticoagulants in the blood of the patient treated with them are eliminated, as well as by deficiencies in the coagulation factors.

One of the objects of the present invention is to provide a coagulometric method designed in such a way that it allows not only the detection but also the titration of serum antiphospholipid antibodies, so that their presence and concentration in the plasma sample of the patient is confirmed. analysis. The composition of the reagent of the invention containing the standardized phospholipid concentrations is specified in table 7.

Table 7. Reagent for titration of antiphospholipid antibodies in a coagulometric method The active elements of the reagent of the invention are kept constant in the reaction mixture for all determinations, except the phospholipid concentrations since their concentration varies in each of the determinations.

The reagent of the invention was contrasted with a battery of tests (AL, ACA, AB2GP 1, ANA, aPTT, IROS, NEUT, FVIII), by various techniques (micro-ELISA or coagulometry), which have been validated for diagnosis of the SAAF.

In the method of the invention, thromboplastin can come from rabbit brain extract, plant phospholipid extract (soybean), egg yolk extract, lung extract, or mixtures thereof, or from natural sources containing amounts significant and / or useful phospholipids.

For purposes of the present invention, the tests used during the development of the invention are defined as follows.

Regarding the corrections with normal plasma, it is a coagulometric test that serves to determine if the prolongation of the aPTT is caused by some deficiency of factors that intervene in the intrinsic pathway or by some inhibitor, which is expressed in seconds.

The neutralization with hexagonal phospholipids refers to a coagulometric determination confirming the presence of antiphospholipid antibodies, by adding phospholipids arranged in hexagonal phase, which is expressed in seconds.

LA1 and LA2 refers to the detection of lupus anticoagulant (screening and confirmatory) by Russell's venom. It is a coagulometric test that determines the presence of lupus anticoagulant (presence of antiphospholipid antibodies), by means of the LA1 / LA2 ratio, using as a direct activator of factor X and V, Russell's viper venom, where in the case of LA1 there is a decreased concentration of phospholipids, whereas in LA2 an excess of these.

Corrections with normal plasma (IROS), refer to a coagulometric test used to determine if prolongation of aPTT is caused by a deficiency of factors involved in the intrinsic pathway or by an inhibitor, which is expressed in an index of correction.

The detection of lupus anticoagulant (screening and confirmatory) by Russell's venom, (LA1 and LA2), refers to a coagulometric test that determines the presence of lupus anticoagulant (presence of antiphospholipid antibodies), by means of the LA1 / LA2 ratio, using as a direct activator of the factor X and V, viper venom Russell, where in the case of LA1 there is a decreased concentration of phospholipids, while in LA2 an excess of these.

The method of the present invention is a coagulometric test that establishes the presence of antiphospholipid antibodies, by the controlled addition (dosing) of phospholipids in the activated partial thromboplastin time test, keeping the activators (silica dioxide, venom viper venom). Russell and / or ellagic acid) at a constant concentration, which results in a single variable that affects the coagulation time, evidencing the presence of the anti-phospholipid antibody in the sample analyzed, even when it is present in low concentrations; it also allows to show the graphic representation of the activity of the antibody and the title of this activity through units called U IFAC (Units of inhibition of active phospholipids).

The types and isotypes of phospholipids present in nature are very varied, such as for example phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, and others. Part of the characteristics of the method of the present invention is the use of a controlled phospholipid composition, where the type and proportion of phospholipids are determined and which conform to the reagent of the invention already defined in table 7.

The method of the invention makes it possible to detect the presence of antiphospholipid antibodies and at the same time determine their titer, independently of the concentration in each patient and it is then possible to relate the titer obtained with the clinical conditions of the patient, so that using this method can be established an effective treatment with fewer side effects for the patient, as well as monitoring during treatment, so it has value in individualized clinical research and in populations as well.

The reagent system of the method of the invention establishes that by the controlled tion (dosing) of phospholipids in the activated partial thromboplastin time test, keeping the activators (silica dioxide, Russell's viper venom and / or ellagic acid) At a constant concentration, a single variable is obtained that affects the coagulation time. Thus, it is possible to demonstrate the presence of the anti-phospholipid antibody, even in the presence of low concentrations of said antibody.

According to the present invention, the reagent system of the method of the invention comprises: a) Various compositions of phospholipids which decrease their concentration gradually. Said compositions, for example, comprise 5 different phospholipid compositions obtained from various sources, for example natural, which have for example the composition and concentrations shown in table 8.

Table 8 * μg / mL.

Stabilized and preserved with HEPES pH 7.6 buffer.

Y b) A coagulation activating composition, which comprises silicon dioxide, ussell viper venom, ellagic acid and mixtures thereof, conforming for example to table 9.

Table 9 * μg / mL.

Stabilized and preserved in saline buffer.

For purposes of the invention, the method of detection and titration of antiphospholipid antibodies of the invention, eliminates the possibility of obtaining false positive results, caused by alterations caused by deficiency of coagulation factor or by anticoagulant effect with heparin, which allows to equip the method of the invention of a high reliability in the obtained results.

The technique used in the method of the present invention allows to show the presence of the antibody, even when the patient with SAAF is under anticoagulant treatment with heparin, since with the same technique a correction of the anticoagulant effect is made when incorporating the anticoagulant. Test a mixture of normal plasma. Likewise, with the tion of normal plasma to the method of the invention, the coagulation times are corrected in the deficiencies of coagulation factors, so that the only variable that affects the coagulation times to be detected in the method of the invention is the presence of anti-phospholipid antibodies.

According to the present invention, the method can be carried out according to the following: a) To obtain the plasma, one part of sodium citrate (0.129 mol / L) is mixed with 9 parts of freshly extracted venous blood. For the collection, handling and conservation of the plasma it is recommended to follow the indications of the document H21 -A4 of the CLSI.5 (Clinical and Laboratory Standards Institute / NCCLS, Collection, Transport and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays, Fourth Edition, CLSI / NCCLS Document H21 -A4; Vol. 23 No. 35). b) 50 microliters of the patient's plasma to be analyzed in a plastic tube and keep at 37 ° C. 5 different tubes are prepared from said mixture. c) Add in a separate tube, 50 microliters of normal plasma from a pool of healthy donors or a normal commercial control. 5 different tubes are prepared from said mixture. d) Add to each of the above mixtures, 50 microliters of phospholipid reagent (individually, ie, from a single phospholipid reagent composition of the reagent system, reagent 1 to reagent 5) and 50 microliters of the activator composition ( reagent 6 of the reagent system). e) After the addition of the activator incubate for 300 seconds at 37 ° C, to subsequently add 0.025 molar calcium chloride, previously heated to 37 ° C. Immediately afterwards, measure the time until the formation of the clot, f) Obtain the results of the corresponding coagulation time for each of the reagents 1 to 5, both in the plasma samples of the patient and those of the control plasma, g) Perform a regression analysis, for example, logarithmic regression, independent of the data of each sample analyzed, to obtain the equation of the line that defines the mathematical behavior of the antibody present in each patient, and h) Determine the presence and the corresponding title of antiphospholipid antibodies in the sample.

The above method can be observed in simplified form in table 10.

The mathematical algorithm derived from the method of the invention demonstrates the presence of the antibody and makes it possible to measure the activity, by means of the capacity of inhibition of the antibodies that are present in each patient sample detected by the reagent system, which translates into a representation graph of the description of the behavior and antibody titre. This allows an analysis of the values or coagulation times obtained with the reagent system in a standardized and reproducible way, establishing the presence, the title and the behavior of the antiphospholipid antibodies in the samples analyzed.

To obtain the individual logarithmic regression, for example, each phospholipid reagent composition corresponds to a value determined in seconds, which corresponds to the coagulation time obtained (see table 1 1).

After each phospholipid reagent, the values indicated in Table 12 are assigned Table 10 Table 11. Values obtained.

* It refers to the 5 determinations of each one of the reagents of the reagent system of the titration test of anti-phospholipid antibodies for each patient under study, for which reason they are not fixed values.

Table 12 The values indicated in Table 12 correspond to the logarithm of the concentration of each reagent and are obtained from the validation procedure of the anti-phospholipid antibody titration test (Tables 26 and 27).

Once the values have been assigned, the slope (m) is determined considering the assigned fixed values for the axis of the ordinates (y), while the time taken in seconds for each reagent for the axis of the abscissa (x); subsequently, the intersection of the order of origin (b) is determined. From the above parameters, we obtain the equation of the line (1) that defines the mathematical behavior of the antibody: y = m (x) + b (1) where, m = slope, b = constant, x = known value, and and - incognito In the previous formula, the values considered are substituted for the values of X i, X2, X.}. , X4, X¡ each of the times obtained in seconds, obtaining 5 points of y, that is yi, y2, yi, y4, yi. The 5 points obtained in semi-logarithmic paper are plotted, that is to say: point 1 (x¡, y¡), point 2 (¾ y2), point 3 (xj, y¡), point 4. { x4, y4) and point 5 (jtj, y5), drawing a straight line that matches the 5 points obtained for each patient, which graphically represents the presence or absence of the anti-phospholipid antibodies (figure 1).

The equation of the line that determines the cut-off value is defined by: Y = M (X) + B (2) Y (Y 1, Y ... = 7.228 (X, X ... J + (79,677) where, M = Pending (7.228), B = Intersection to the axis (79.677), X¡ - = Values assigned from X¡ to Xn, ?? - y ..... n = Values obtained in the equation for Yi to Yn.

Note: if the value of the slope is negative, multiply by -1 so that the graph is interpolated with positive values.

Subsequently, the values of X are substituted in the previous equation (for example, from X = 20 to X = 150), to obtain the corresponding values from Y2o to Y¡50. The values obtained for each point are interpolated in semi-logarithmic paper and the straight line joining all the points is drawn, where said line corresponds to the cut value (figures 3 and 4). To determine the presence of antiphospholipid antibodies in the plasma samples of the subjects under study, the following condition must be met: The result is positive if and only if, the 5 points are above the equation of the line that defines the cut value.

Finally the antibody titer is determined by equating the equations of the patient's line, with respect to the equation of the line of the cut-off value, substituting in both equations the value of the coagulation time obtained in the concentration of reagent No. 5, according to the following: m (pac) (b (pac)) + x (pac) = M (B) + x (pac) (3) m (pac) (b (paci) + (pac) = 7-228 (79,677) + Title = m (pac) (b (pac)) + x (pac) / 7.228 (79,677) + x (pac) where, itifpac) = The value of the slope, obtained in part b), b (pac) = The value of the intersection to the axis, obtained in part e) X (pac) = Time of coagulation of the patient obtained with reagent no. 5, M = 7.228, and 5 = 79,677 According to the present invention, the compositions comprising the reagent system of the method of the invention can contain preservatives known in the art, such as for example sodium azide, pronobol or gentamicin, in concentrations such that they allow the preservation of their components, such as, for example, less than 1 gr. per liter.

The reagent system of the method of the invention can be contained in a kit or diagnostic kit for detecting and titrating antiphospholipid antibodies by coagulometry, which can basically contain: • Means to obtain a blood sample and keep it in an uncoagulated vacuum test tube, which may include those that are commonly used for such purposes.

· Support media where the suspicious samples to be analyzed can be mixed with the system of reagents described above, such as, for example, ELISA plates, or by manual procedures.

• The reagent system described above comprising the compositions with different concentrations of phospholipids, as well as the activating coagulation composition, and · An instruction for the realization of the test, which includes the steps of the method that will be carried out by a technician or person with knowledge in the field; how to handle the sample to be analyzed, as well as the interpretation of the results obtained based on a standard curve.

According to one of the embodiments of the present invention, the method of detection and titration of antiphospholipid antibodies described herein can be carried out by means of the diagnostic kit or kit described above, by means of which several samples can be analyzed at the same time, which would allow studies to be carried out. of detection and titration of said antibodies when required, as for example in epidemiological studies of open populations.

The phospholipids (phosphatidyl-serine, phosphatidyl-choline, phosphatidyl-inositol, phosphatidyl-glycerol, phosphatidyl-ethanolamine and / or mixtures thereof) contained in the reagent system of the method of the invention can be obtained and purified from natural sources such as example from extracts of rabbit brain, egg yolk and / or soybeans, by the following method: a) Add an adequate amount of water to the natural source of phospholipids, to later add an organic solvent that allows the disintegration and dissolution of a large part of the natural source of phospholipids. Preferably it is made from 100 g of the natural source, 50 ml of bidistilled water and 50 ml of acetone, b) Homogenize the previous mixture, let stand and filter to eliminate the insoluble material in the mixture. In this case, homogenization is carried out by means of a magnetic stirrer and for the time necessary to dissolve the source of phospholipids in the mixture, c) Recover the mixture and distill it, with which the solvent is eliminated and an aqueous solution of different lipids, fatty acids and phospholipids is obtained, d) To the above mixture, add a mixture of non-polar solvents, such as, for example, methanol-chloro in a 2: 1 ratio, wherein the amount of chloroform is equal to the amount of solution obtained in the distillation. Homogenize the mixture until forming an emulsion and centrifuge later to break said emulsion obtaining 2 liquid phases, e) Recover the chloroform liquid phase (yellow color), eliminate the chloroform by evaporation and add acetone to the resulting extract. Subsequently the mixture is stirred and the resulting solution is centrifuged, removing the acetone by decantation, f) The precipitate obtained, which corresponds to the purified phospholipids from the natural source, is subsequently separated by HPLC and stabilized with a buffer solution of HEPES pH 7.6, and g) Finally, the concentration of the phospholipids obtained is quantified, for example, by spectrophotometry.

The phospholipids obtained according to the above method are subsequently mixed in the aforementioned proportions to obtain the different reagent compositions of the reagent system of the method of the present invention.

With the reagent of the invention, the quantitative diagnostic test that also conforms to the invention has been validated. This validation consists in contrasting a battery of tests (lupus anticoagulant, anticardiolipin antibodies, anti-B2-glycoprotein-1 antibodies, ANA, aPTT, FVIII), by means of diverse techniques (micro-ELISA, coagulometry), which in turn have been previously validated for the diagnosis of antiphospholipid antibody syndrome.

The following examples describe the technical support of the invention and are only illustrative of their quality but not limiting their scope.

Example 1. Extraction of phospholipids. The phospholipids were extracted from rabbit brain, according to the method of Bell and Anton (13) modified. To the brains washed with saline, they added 400 mL of acetone in agitation for one hour, the acetone was filtered and distilled at 56 ° C, chloroform was added in an approximate concentration of 50 mL for every 1 gram of the material obtained in distillation, plus NAL solution 9 g / L in 20 mL and methanol QP 25 mL; The solution was subsequently mixed for 30 minutes and a white emulsion formed, which is separated by centrifugation at 2500 rpm. During 10 minutes, the chloroform fraction (which is yellowish) was obtained by evaporating between 56 and 60 ° C. added acetone at 5 ° C, forming a white precipitate, centrifuged and decanted and resuspended in 0.9% saline, and diluted in HEPES solution to find the optimal concentration for a 35-second APTT assay. Once extracted the phospholipids were stabilized with a buffer solution of HEPES pH 7.6 which was prepared as follows: HEPES 14 gr, NaCl 2 gr, sodium azide 0.5 gr and distilled water 1000 mL. With this solution all the dilutions of the phospholipids were made.

For the quantification of the concentration of the phospholipids, it was taken as 100% of phospholipids at the concentration that establishes the average, from a normal time in 35 seconds, for a total of 100 samples of normal plasmas, distributed in 10 normal control of 10 samples each.

Here we include two plasmas of commercial origin of different origin of purification and normal concentrations of proteins and inhibitors of coagulation.

The phospholipids are quantified according to the phosphomolybdate method, starting from a standard phospholipid solution, elaborating a concentration curve against absorbance.

Example 2. Determination of the concentration of phospholipids that allows to obtain a normal coagulation time in 35 seconds. To obtain the normal coagulation times for each known concentration of phospholipids, aPTT tests were performed with variation in the phospholipid concentration determining 100 samples of normal reference plasmas that were distributed in batches of 10 samples of normal reference plasmas, daily ( control, normal, witness).

The variation in phospholipid concentrations was made from the optimal concentration that establishes a time of 35 seconds.

The variation in the amounts of phospholipids was established as shown in 5 determinations of the table 13.

Table 13. Optimum concentration (35 sec) of antiphospholipid antibodies.

Example 3. Adjustment of blood samples for coagulometric study. The blood sample for the coagulometric study conforms to widely validated procedures, to avoid preanalytical errors. (10) The plasma for the study of AL should be centrifuged, but not subjected to ultracentrifugation, because platelets can break down and release their phospholipids into the medium, causing errors in the analytical phase.

The plasma must be platelet poor, that is, it must not have more than 1x10 platelets / L (10,000 platelets per ml), this can be obtained by centrifuging the sample 10-20 minutes at 1500-2500 g (12).

If the plasma is frozen for further studies, it should be done before 2 hours at -70 ° C, it is recommended that it be previously filtered through a membrane of 0.22 microns in diameter to prevent the passage of platelets and be destroyed by the effect of freezing and thawing, which should be done at 37 ° C for 10 minutes maximum to avoid denaturing labile factors. It is recommended to use non-frozen plasma, within the first four hours of extraction (1 1, 8).

For the evaluation of the quality of the techniques carried out, the following criteria were used: Internal quality assessment: It is based on the use of a daily record control sheet (Levey-Jennings chart) to obtain the graphical control of the standard deviation (SD) and calculate the coefficient of variation (CV) of each type of study carried out through a calibrator or control, DE and CV are indicators of accuracy.

Preparation of the control chart: The chart was made by doing at least 20 times the parameter to be determined, with these results the mean and the standard deviation needed to construct the graph are calculated. In the vertical axis, a baseline is drawn in the value of the average X and three lines that represent +/- 3 DE above and below the average, the permitted limits are +/- 2 SD.

The following guidelines can help decide if the test is out of control: 1) A value falls completely outside the control limits of +/- 2 DV (warning limits). 2) Several consecutive values show a tendency to increase. 3) Several consecutive values show a tendency to decrease. 4) Several consecutive values fall on one side of the average. 5) Two or more values of 20 measurements fall on the line of +/- 2 DV.

Normal and abnormal control (low and high), with a pre-assigned concentration, were used. It was prepared from a mixture of normal plasmas as normal control (control).

External evaluation of quality. It consists in the evaluation of the precision of the methods used in the different laboratories, by means of the sending of controlled samples from a Reference Center. In general terms, the CV should not be greater than 10%.

In order to avoid bias in carrying out the study phases of this protocol. The studies of the technical stage of extraction and preparation of rabbit brain phospholipids were always carried out by the same researcher (researcher 1), not knowing the serological results (AL, ACA and AB2GP1) of the patients with SAAF. In the same way, the tests that allow identifying each patient with SAAF were always carried out by another researcher (researcher 2) with no knowledge of the technical stage.

Example 4. Validation of the method of the invention. a) Standardization of the reagent system. The standardization procedure of the reagent system, which forms the anti-phospholipid antibody titration test (TICFOS) of the invention, has as a fundamental principle, to obtain the optimum concentrations of phospholipids and activators, necessary for On the one hand, the antibody is expressed by prolonging the clotting time and on the other, the shortening is achieved, which establishes the principle of the methodology; that is; by the controlled addition (dosing) of phospholipids in the activated partial thromboplastin time test, keeping the activators (silica dioxide, Russell's viper venom and ellagic acid) at a constant concentration, a single variable is obtained that affects the coagulation time, evidencing the presence of the anti-phospholipid antibody.

Through an experimental prospective study, 50 plasma samples from healthy donors were used to establish the concentrations required for each of the reagents, using the procedure described below: The optimal concentrations of phospholipids and activators that allow us to establish a normal clotting time of 35 seconds are taken as reference. From this concentration of phospholipids, which is called 100% (reagent 3), 2 dilutions of phospholipids are made, at 6.25% (reagent 4) and 3.125% (reagent 5) and 2 phospholipid concentrations are carried out, 200% (reactive 2) and 300% (reactive 1) (table 14).

Table 14 * μ / mL.

The concentration of activators is maintained permanently at a constant concentration (table 15).

Table 15 * μg / mL.

The coagulometric technique for the titration of anti-phospholipid antibodies of the invention is described below and is shown in summary in Table 16. a) To obtain the plasma, mix 1 part of sodium citrate (0.129 mol / L), with 9 parts of venous blood freshly extracted. For the collection, handling and conservation of plasma follow the recommendations of document H21 -A4 of CLS1.5 (Clinical and Laboratory Standards Institute / NCCLS Collection, Transport and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays, Fourth Edition, CLS1 / NCCLS Document H21-A4; Vol. 23 No. 35. b) Add 50 microliters of plasma to a plastic tube and hold at 37 degrees centigrade. c) Add 50 microliters of normal plasma from a pool of healthy donors or a normal commercial control. d) Addition of 50 microliters of phospholipid reagent to the plasma (individually, that is, from a single bottle of phospholipids from the reagent system of the anti-phospholipid antibody titration assay of the invention) e) Add 50 microliters of the activating reagent to the mixture (reagent 6 of the reagent system). f) After the addition of the activator measure 300 seconds. g) After the time elapsed, add 0.025 molar calcium chloride, previously heated to 37 ° C. h) Immediately after, measure the time to clot formation (see table 16). i) Perform the technique described for each of the reagents (1 to 5) of different concentration of phospholipids from subsection b), to subsection g), obtaining 5 results, one for each phospholipid reagent.

Table 16. Coagulometric technique of the TICFOS test. 75 determinations were made in duplicate, for each of the reagents of the reagent system, using plasma from healthy donors, in order to establish the average, the coefficient of variation (CV) and the standard deviation (SD), to observe the reproducibility of the reagent system (table 17). b) Validation of the test. The method of the invention (TICFOS) is able to show the antibody, by means of the graphic representation of the mathematical behavior of the antibody, in the reagent system with respect to the cut-off value, which results in the observation of the expression and inhibition of the anti-phospholipid antibody, in the reagent system, which will be able to detect more than 90% of the samples of patients with SAAF, established with the traditional tests.

Table 17. Reproducibility of the reagent system of the titration test of anti-phospholipid antibodies (TICFOS).

Note: 5 different batches of the TICFOS reagent system were used Through a prospective and experimental study, the method of the present invention was contrasted against the tests used in the determination of SAAF: lupus anticoagulant (AL), specific anti-cardiolipin antibodies (ACA) and specific anti-p2 glycoprotein 1 antibodies (AB2GP 1 ) to establish the correlation between the degree of antiphospholipid antibodies determined by the test of the invention with respect to those determined by traditional tests.

Plasma collection of patients from the positive cases studied in the Perinatal Hematology Laboratory of the National Institute of Perinatology was carried out for the tests under study (table 18).

Table 18. Dependent variables The universe under study was made up of 148 patient samples with the reactivity to the traditional tests for the determination of SAAF (table 19).

In particular, the classification for each of the tests is shown in Table 20.

To each of the 148 samples of patients, the specific tests for anti-cardiolipin antibodies, anti- 2 glycoprotein 1 antibodies and the lupus anticoagulant with Russel viper venom were determined, where the stratification of the samples is described in Table 21 and consists of positive cases demonstrated experimentally, for each of the dependent variables.

In the same way for the 148 samples of the universe under study, the determination was made by the method of the invention, obtaining the results of table 22 for the reagent system.

Table 19. Universe in study.

Table 20 Table 21. Dependent variables under study.

Because finally; The objective of the method of the present invention (TICFOS) is to demonstrate an expression value of the antibodies in the reagent system and to avoid the controversy of the contrary conditions of sensitivity and specificity (when one moves the other moves in reverse direction), it was decided to establish the critical limit value most suitable for the diagnostic test by constructing the ROC (Charasteristic Operator Receiver, COR) curves for the evaluation of the reliability of diagnostic tests, based on representing the curve defined for each possible limit value according to the sensitivity of the test (S) on the axis of the ordinates and the percentage of false positives for the test (1 -E) on the abscissa axis, using as a reference measure the positive values for the test of : anti-cardiolipin antibodies (ACA), anti-32 glycoprotein 1 antibodies (AB2GP 1) and Lupus anticoagulant (AL) (figures Table 22. Universe under study and the determination of the TICFOS test.

Table 22 (continued) Table 22 (continued) Table 22 (continued) Table 22 (continued) 2A to 2C).

The line joining the points S = l and 1-E = 1 is then drawn. The point where the curves previously described with the line are cut constitutes the most appropriate critical limit value to be used, which can be established between the asymptotic 95% confidence interval for each test (tables 23, 24 and 25).

With the data obtained through the ROC curves, for the "lupus anticoagulant" test, the cut-off value is established in seconds for each of the reagents considering the 0.800 susceptibility (sensitivity), since this value is found in the 95% confidence interval (between the lower limit value and the upper limit value), which is valid for all reagents in the system.

The values in seconds corresponding to the 0.800 susceptibility (sensitivity), for each reagent of the system, define the cut-off point of the method of the present invention (TICFOS) (table 26).

Table 23. Confidence interval established by the ROC curve for the ACA test.

Note: it can be seen that for reagents 1, 2 and 3 the 95% confidence interval is less than 0.800.

Table 24. Confidence interval established by the ROC curve for the AB2GP test.

Note: it can be seen that for all the reagents the confidence interval at 95% is less than 0.800.

Table 25. Confidence interval established by the ROC curve for the lupus anticoagulant test.

Note: for all reagents, the value of 0.800 is within the 95% confidence interval Table 26. Cut-off values in seconds for each system reagent.

Once the desired sensitivity values for each of the reagents of the reagent system of the method of the present invention (TICFOS) have been established, the equation of the line defining the cut-off value for the test is determined, where; the axis of the ordinates (y) corresponds to the concentration and the axis of the abscissas (x) corresponds to the values in seconds, obtained by means of the ROC curves (sensitivity of 0.800), same values that are described in table 27.

Table 27. Cut values.

Note. For each concentration of the system reagents corresponds a value in seconds With the data described in Table 27, different linear regression models are constructed to determine the equation of the line that defines the mathematical behavior of the anti-phospholipid antibodies in the reagent system of the method of the invention (TICFOS) (Table 28 ).

Table 28. Model summary and parameter estimates.

The equation of the line defined by the logarithmic regression model is established, as the equation that defines the mathematical behavior of the anti-phospholipid antibodies, although at any moment it can be replaced by the equation that obtains the correlation coefficient R square, more next to 1.0 or any other that you wish to observe. In such a way that the line is defined by the following equation: y = m (x) + b y = 7.228 (x) + (79,677) where, m = Pending, x = Value assigned in seconds, b = Constant, and y = Incognita Note: the value of the slope is multiplied by - 1 so that the graph interpolates with positive values. "X" values are assigned, which correspond to the time values in "seconds", to obtain the values of "y" corresponding to the values of the "concentration", for example from 20 to 150 (table 29).

Table 29 The graphical representation of the mathematical behavior of the cut-off values (cut-off point) is determined by interpolation of the values described in Table 29 (Figure 3).

Once the cut-off values are established, the values obtained in seconds for each of the reagents of the system of the method of the invention (TICFOS) and for the 148 samples of patients are analyzed with the same mathematical algorithm; that is to say: 1. The values are determined in seconds for each of the phospholipid reagents and for each of the patients. A table is constructed of the logarithm of concentration and time in seconds (table 30).

Table 30. Example of values in seconds, obtained for the patient 32. 2. The equation of the line defining the individual behavior of the anti-phospholipid antibody is determined by means of the logarithmic regression model. y = m (x) + b y = 8.47 (x) + 79.8 where, m = Pending, Constant, Value obtained in seconds, and Incognito (concentration).

The corresponding values of the logarithm of the concentration are obtained by substituting in the previous equation the values in seconds of each reagent (table 31).

Table 31. Example of the logarithm of the concentration, obtained for the patient 32. 4. The values obtained in Table 31 are interpolated, considering the values in seconds on the axis of the abscissas (x) and on the axis of the ordinates (y) the logarithm of the concentration, in order to establish and graphically observe the mathematical behavior of the anti-phospholipid antibody of patient 32, in the reagent system of the method of the invention (TICFOS), taking into account the cut-off value (figure 4). 5. To establish the positivity of the method of the invention (TICFOS), the following condition must be met: "If and only if, it is positive when each and every one of the values obtained for the 5 phospholipid reagents, are above the cut-off values" (greater than 1 of the antibody titer). c) Determination of the antiphospholipid antibody titer. The objective of the determination of the antibody titer is to assign a value corresponding to the activity of the antibody, by describing the mathematical behavior of the antibody in the reagent system of the method of the invention (TICFOS), which is established by the capacity that the antibody has to inhibit the phospholipids of the reagent system and results in the prolongation of the coagulation times, with respect to the cut-off values.

The anti-phospholipid antibody titer is established by matching the equations of the cut-off value and the individual equation obtained from each of the samples with the reagent system of the method of the invention (TICFOS), by means of the following mathematical algorithm, where The equations of the cutoff value and the patient, obtained with the reagent system, are equalized: m (pac) (X (pac)) + > (pac) = M (x (pac)) + B m (pac) (X (Pac)) + b (pac) = 7.228 (x (pac)) + 79,677 Title = m (pac) (x (pac)) + b (pac) / 7.228 (x (pac)) + 79,677 where, mfrac) = The value of the slope, obtained for each patient, Xfpac) ~ The coagulation time in seconds obtained with reagent no. 5, b (ac) = Constant, M - 7.228 (Slope of the equation of the line of the cut-off value), and B = 79.677 (Constant of the equation of the line of the cut-off value).

Because the reagent does not. 5 contains the lowest amount of phospholipids, therefore corresponds the maximum value of antibody expression in the reagent system of the method of the invention (TICFOS) and this value is considered for obtaining the anti-phospholipid antibody titer ( see the following example): The values obtained, for example, with the sample of patient 32 and the cut-off values, as well as the time obtained with reagent 5 for sample no, are substituted in the equation. 32 Patient equation = 8.47 (x) + 79.8 Equation of the cutoff value = 7.228 (x) + (79.677) Time obtained with reagent no. 5 of the patient sample (x) = 76.1 Title = 8.47 (76.1) + 79.8 / 7.228 (76.1) + (79.677) Title = 724.36 / 629.76 Title = 1.15 To establish the positivity of the method of the invention (TICFOS), the following condition must be met: "Yes and only yes, it is positive when the antibody titer is greater than 1.00" d) Determination of the sensitivity and specificity of the method of the invention. The determination of sensitivity and specificity was made by applying the Bayes theorem (table 32), as applicable gold test, the method of the invention (TICFOS) and contrasting against the dependent variables (table 33 and 34).

Table 32. Bayes theorem.

A Number of true positives B Number of false positives C Number of false negatives D Number of true negatives Sensitivity (%) 100 * A / (A + C) Specificity (%) 100 * D / (B + D) Positive predictive value 100 * A / A + B) Negative predictive value 100 * D / (C + D) Table 33. Bayes theorem. TICFOS vs. dependent variables.

Table 34. Sensitivity and specificity of the TICFOS test.

From the Bayes theorem applied to the method of the invention (TICFOS), we can establish the following: 1 . The method of the invention (TICFOS) has a specificity equal to or greater than 90% for the antibodies specific for ACA IgG and AB2GP1 IgG, which is of great significance since these antibodies have been related to the thrombotic risk. 2. The method of the invention (TICFOS) predicts positive values for ACA IgG, ACA IgA and AbB2GP IgG in more than 85 samples of every 100 patients positive for these specific tests. e) Statistical correlation of the positivity of the method of the invention against the dependent variables. To check if there is dependence between the variables under study and the method of the invention, the Chi-square Pearson coefficient is applied (table 35).

From the results obtained by applying the Chi square coefficient, we can establish the following: 1 . For the ACA IgG, ACA IgM, ACA IgA, AB2GP IgG and AL tests, the value obtained is < 0.05, so the null hypothesis is rejected and it is established that there is a relationship or dependency between the variables and the method of the invention (TICFOS). 2. In contrast, in the AB2GP IgM and IgA tests a coefficient is obtained > 0.05, for which the null hypothesis is accepted and the non-relationship between these variables and the method of the invention is established (TICFOS).

Table 35. Pearson Chi-square coefficient.

In order to determine the type of relationship that exists between the dependent variables and the method of the invention (TICFOS), association measures are applied for ordinals (table 36).

Table 36. Symmetric measurements According to the results obtained for the symmetric association measures, we can establish that: 1. For the ACA IgG, ACA IgA, AB2GP IgG and AL tests, the Gamma symmetric association measure obtained indicates a strong relationship between these variables and the method of the invention (TICFOS). 2. While for the ACA IgM test, the Gamma association indicates a moderate relationship with the method of the invention (TICFOS).

The reagent system of the method of the present invention allows the antibody to be demonstrated, by using 5 reagents of different concentration of phospholipids and keeping the activators of the sixth reagent in constant concentration, so that the only variable that affects the time of coagulation, is due to the expression of the antibody, for this reason we can establish that: 1. If different concentrations of phospholipids and activators are used or even different types of these, to those established in the reagent system, the result is the same as established by the method of the invention, since the behavior of the antibody is established by the dosage of the phospholipids in the test, observing the expression and inhibition of the same, by means of the representation graph of the straight line that passes through the 5 points of different concentration, so that if higher or lower concentrations were used, the straight line would inevitably pass through these points. 2. The mathematical algorithm or methodology of the method of the invention, could have variants regarding the establishment of the cut values, because these are established by the construction of ROC curves, allowing to choose a sensitivity or specificity different to that used in the validation procedure and in the same way the model could vary of linear regression for the analysis of the samples, all this in order to give the test a greater sensitivity or specificity, depending on the population in which the method of the invention is going to be used. But it is demonstrated that the method of the invention and its variants allow to determine the presence of the antibody as well as to establish the mathematical behavior of the expression and inhibition of the antibody, by means of the graphic representation of the action of the same in the reagent system and finally can quantify the activity of the antibody, by determining the "phospholipid inhibition" titre (units of inhibition of active phospholipids, UNIFAC).

References. 1. Brandt JT, Barna L, Triplett DA. Laboratory identification of Lupus Anticoagulants: Results of The second international Workshop for identification of Lupus Anticoagulants. Tromb Haemostat 1995; 74: 1597-1603 2. Feinstein DI, Rappaport SI. Acquired inhibitors of blood coagulation Prog. Tromb Haemostat 1972; 10: 75-95 3. Love PE, Santoro SA. Antiphospholipid antibodies: Anticardiolipin and the lupus anticoagulant in systemic Lupus erythematosus (SLE) and in non SLE disorders Ann Int Med 1990; 1 12: 682-99 4. Gharavi AE, Sammaritano LR, Wen J, Miyawaki N, Morse JH, Zarabi MH, Lockshin MD.

Characteristics of Human immunodeficiency virus and chlorpromazine induced antiphospholipid antibodies: effect of beta 2-glycoprotein 1 and bindingto phospholipid. J Rheumatol 1994; 21: 94-9 5. Harris EN, Plerangeli SS, Antiphospholipid Antibodies: Nature and Mechanism of Action. In: Teodorescu Metal, Editors. Advanced Immunoassays in Rheumatology. Boca Ratón: CRC Press 1994; 1 12: 682-99 6. Brandt JT, Barna LK, Triplett DA, Alving B, Scarrer I. Criteria for the Diagnosis of Lupus Anticoagulants: An Update. Thromb Haemostat 1995; 74: 1185-90 7. Howarth S. Activated pardal thromboplastin time reagents: an evaluation. Br J Biomed Sci 1993; fifty: 109-13 8. Exner T, Douglas A, Triplett DA, Taberner D, Machín SJ. Scientifics and Standardization Committee Communications. Thromb Haemostat 1991; 65: 320- 22 . Michael D, Locksshin MD. Pregnancy Loss in the Ant iphospholipid Syndrome. Thromb Haemostat 1999; 82: 641-48 10. NCCLS Document h3-A3: Procedures for the collection of diagnostic blood samples by venipunture.

Approved standard 1991; 11:10 1 1. Sletres KE; Gravmen, Wisloff. Preparation of plasma for the detection of lupus anticoagulants and antiphospholipid antibodies Thromb Res 1992; 66: 43-53 12. Domínguez GV, Rosenfeld MF, Baptist GHA. Laboratory studies in hemostasis. Principles and Quality Control. In: Martínez-Murillo C, Quintana-González S, editors. Manual of hemostasis and thrombosis. Mexico: Prado 1996; 27: 413-42 13. Bell WN, and Alton HG, A brain extract as substitute for platelet suspension in the thromboplastin generation test. Nature (London) 1954: 174: 880

Claims (1)

Claims 1. A coagulometric method for determining the presence and titer of the activity of the anti-phospholipid antibody (AAF) in a plasma sample, characterized in that it evidences the antibody, by means of the expression and inhibition thereof when determining the coagulation times of five different phospholipid concentrations, keeping the activator at a constant concentration at all times, the only possibility of altering the coagulation times is the presence of the antibody and determining the titer of the activity of the antibody, by evaluating the mathematical behavior of said antibody to the compare it with the "normal" behavior (cut-off value) in the reagent system that varies in five concentrations of phospholipids, keeping the activator constant, in addition a graph is obtained by interpolating the values obtained in each phospholipid concentration, represented by a straight line, the expression of the co behavior of the anti-phospholipid antibody for each sample. 2. A kit of reagents characterized in that it allows to obtain the results according to claim 1 and that is constituted by 5 bottles of different concentrations of phospholipids and 1 bottle of a single concentration of activators. 3. The method of claim 2, wherein the reagent composition of the first phospholipid bottle contains: 625 μg / ml phosphatidylcholine, 625 μg / ml phosphatidylserine, 625 μg / ml phosphatidylethanolamine, 525 g / ml phosphatidylinositol, 375 μg phosphatidyl glycerol. / ml. 4. The method of claim 2, wherein the reagent composition of the second phospholipid bottle contains: 450 μg / ml phosphatidylcholine, 450 μg / ml phosphatidylserine, 450 μg / ml phosphatidylethanolamine, 350 μg / ml phosphatidylinositol, 250 g / ml phosphatidylinosilicate. μg / ml. 5. The method of claim 2, wherein the reagent composition of the third phospholipid bottle contains: 250 μg / ml phosphatidylcholine, 250 μg / ml phosphatidylserine, 250 μg / ml phosphatidylethanolamine, 175 μg / ml phosphatidylinositol, 125 μg / ml phosphatidylglycerol . 6. The method of claim 2, wherein the reagent composition of the fourth bottle of phospholipids contains: phosphatidylcholine 14.1 μg / ml, phosphatidylserine 14.1 μg / ml, phosphatidylethanolamine 14.1 μg / ml, phosphatidylinositol 10.9 μ / p ?1, phosphatidylglycerol 7.8 μg / ml. 7. The method of claim 2, wherein the reagent composition of the fifth phospholipid bottle contains: 7.0 μg / ml phosphatidylcholine, 7.0 μg / ml phosphatidylserine, 7.0 μg / ml phosphatidylethanolamine, 5.5 μg / ml phosphatidylinositol, 3.9 μg / ml phosphatidylglycerol . 8. The method of claim 2, wherein the reagent composition of the activator bottle contains: Silica dioxide (Si02) 3.0 μg / ml, Russell viper venom 0.15 μg / ml, ellagic acid 0.15 μg / ml. 9. The method of claim 2, wherein the composition and characteristics of the reagents and materials to be used are described by means of an instruction manual, as well as the precautionary and use measures of the same, the diagnostic value, the graphics for the interpolation of results obtained, the interpretation thereof and the cut-off values (normal values) in accordance with claim 1. 0. The method of in vitro application of the reagent system of claim 2, characterized by the coagulometric technique to determine the coagulation times of each sample using the reagent bottles described in claims 3, 4, 5, 6, 7 and 8 , which consists of the following steps: a) A sample of platelet-poor plasma is obtained, by venous puncture, at least one blood sample to be analyzed is taken and aliquoted in test tubes in the presence of oxalate or citrate. sodium, to stop coagulation by binding to calcium, centrifuging them for 10-20 minutes at 1500-2500 g, so that samples are poor in platelets, that is, with no more than 1 x 10 platelets / L, b) 50 micro-liters of the plasma sample are added in a test tube, c) 50 micro-liters of normal plasma or pool of healthy donors are added to the same test tube and incubated for 1 minute at 37 degrees centigrade, d) subsequently 50 micro-liters of reagent of the activating bottle, described in claim 8, is added to the plasma mixture. e) immediately after, 50 micro-liters of reagent from the phospholipid bottle number one, described in claim 3, is added to the test tube. f) is kept in incubation at 37 degrees centigrade for 5 minutes, g) Finally, 100 micro-liters of 0.025 molar calcium chloride reagent, previously heated to 37 degrees Celsius, are added, h) and immediately afterwards the chronometer is activated and stopped when the first fibrin threads are formed. i) Carry out the same procedure, replacing in "item e", each of the two, three, four and five phospholipid bottles, described in claims 4, 5, 6 and 7 respectively, in such a way that 5 results of coagulation times of the sample under study, one for each phospholipid concentration, as follows: j) Phospholipid vial one, described in claim 3 = time 1 k) Phospholipid vial two, described in claim 4 = time 2 1) Three bottle of phospholipids, described in claim 5 = time 3 m) Phospholipid four bottle, described in claim 6 = time 4 n) Flask phospholipid five bottle, described in claim 7 = time 5

1. The method for determining the presence and titration of the activity of anti-phospholipid antibodies in a plasma sample, according to claim 1, characterized by the comparative mathematical analysis of the coagulation times obtained in claim 10, between the values of the plasma samples under study and the normal or cut-off values, which include: a) A linear regression analysis, which defines the equation of the individualized line for each sample, determined by the concentration of each of the phospholipid reagents and the coagulation times obtained for each of these, b) where to determine the positivity of the test for each sample, the individualized line must be greater than the equation of the line that defines the cutoff value. c) The antibody titer is determined by the difference between the value of the time obtained for each sample in the phospholipid reagent described in claim 7, interpolated in the equation of the individualized line and the same time value, but interpolated in the equation of the line of the cut-off value, d) The equation of the line that defines the cut-off value is provided in the claim method 2, both in its mathematical formula, and graphically in semi-logarithmic paper, in order to comply with claim 9 e) in such a way that a titre of antibody activity is obtained, for positive samples, equal to or greater than one. f) With the coagulation times obtained in the claim method 10, a concentration vs. time in semi-logarithmic paper, provided in the reagent kit of the claim method 2 and to comply with claim 9, which defines the graphic behavior of the antibody activity, whereby it is possible to observe the activity of the antibody and compare it with respect to the cutoff value.