NO310126B1 - Initiator for determination of coagulation time, reagent for determination of aPTT, method of preparation of reagent and use thereof - Google Patents

Abstract

An initiator of the intrinsic coagulation of blood, plasma or blood derivatives is characterised in that it contains a mixture of sulphatides and a solid activator, preferably kaolin. A reagent for the determination of the aPTT of blood or blood derivatives contains a mixture of sulphatides and a solid activator, preferably kaolin, as initiator of intrinsic coagulation and, furthermore, phospholipids.

Description

The invention relates to an initiator for intrinsic coagulation, a reagent for determining the activated partial thromboplastin time (aPTT), a method for producing this reagent or for determination of aPTT, as well as applications of the reagent.

The activated partial thromboplastin time of a plasma sample serves as a "screening" test for global measurement of the activity of the coagulation factor for the intrinsic coagulation. Disturbances in the coagulation process lead to coagulation times that are outside the normal range. The prolongation of aPTT, as it occurs in hemophilia, manifests e.g. a deficiency of coagulation factor VIII. In addition, the aPTT test is often used to monitor heparin therapy.

A reagent for the determination of aPTT contains an activator to initiate the cascade of intrinsic coagulation until the formation of activated factor IX. In addition, phospholipids are needed for the activation of factor X and prothrombin, which is why these are also present in an aPTT reagent. The reagent is brought into contact with a plasma sample, then calcium is added after a defined, as short as possible activation time, and the time until the formation of a fibrin coagulum is measured.

Many requirements are placed on the test system for aPTT. For routine examinations, easy handling of an aPTT reagent is an absolute prerequisite. Likewise, the stability of the ingredients must be very high to ensure a uniform coagulation time over a longer period of time. This also enables a reasonable use of the reagent. For the aPTT as a global test, a uniform sensitivity in relation to all parameters to be measured is also necessary.

Common reagents for determining aPTT contain a surface-active solid as an initiator for intrinsic coagulation. When using a surface-active matrix, factor XII is first activated, comparable to the physiological conditions. As a solid activator, e.g. kaolin, celite and glass. Kaolin exists e.g. in commercial reagents for the determination of aPTT as a suspension ("Pathromtin", company Behringwerke (1990) or "PTT Reagens", company Boehringer Mannheim (1986)). However, the phospholipids are first mixed into a ready-to-use reagent before carrying out the determination with kaolin. The separate storage of kaolin and phospholipids is above all necessary for reasons of stability.

By freeze-drying lipids with kaolin, lipids would be mostly inactivated. Thus, in the production of PTT-IMMUNO (firma Immuno) by joint freeze-drying of kaolin and phospholipids (extracted from pig brain) e.g. a loss of up to 50% of phospholipid activity.

As an alternative to solid activators, liquid activators, such as ellagic acid and derivatives thereof, or dissolved sulfatides can also be used. However, the sensitivity of a test containing liquid activators, in relation to heparin, is worsened, which is why various additions to improve sensitivity are suggested. According to WO 91/16453, a heparin-sensitive reagent based on ellagic acid additionally contains dextran sulfate.

A disadvantage of kaolin or solid activators in general and ellagic acid respectively derivatives, is the long activation time of the first phase of the intrinsic coagulation, whereby a relatively long incubation time of at least 4 min. is required for activation. A further disadvantage consists in the relatively long coagulation time in the presence of kaolin. However, a higher kaolin concentration in the reagent can shorten the activation time. Kaolin that is deposited from the suspension can, however, lead to an extended coagulation time or produce erroneous results when measuring the coagulation end point using ordinary coagulometers. Care must therefore be taken to keep the concentrations of the solid activator as low as possible.

The task of the invention is to provide a standardized reagent with high stability for the determination of aPTT, which is easy to handle and affordable, and which entails both short activation times and a high sensitivity to individual factors of the coagulation cascade.

The solution to this task is made possible according to the invention by means of an initiator for intrinsic coagulation of blood, plasma or blood derivatives, which contains a mixture of sulfatides and a solid activator, preferably kaolin.

The reagent according to the invention contains as an initiator for intrinsic coagulation for determining the coagulation time of blood, plasma or blood derivatives containing a mixture of sulfatides (e.g. from the company Pentapharm) and a solid activator, preferably kaolin. In addition, it contains phospholipids. The short activation time needed when using sulfatides for the activation of factor XII is unlikely to be extended when used together with kaolin. With the use of the reagent according to the invention, a method with a short incubation time that is practicable for routine investigations is thus made possible. At the same time, however, heparin sensitivity is also improved by using a solid activator.

The use of the initiator according to the invention in an aPTT reagent thus surprisingly enables the combination of the advantages of both single activators, but not the disadvantages of these.

This could not be predicted. It was rather to assume that the combination of sulfatides with kaolin as an initiator for intrinsic coagulation in the reagent would lead to a reduction in activity. According to the expert's prejudice that a combination of activators with different reaction mechanisms would cause difficulties, it was surprising that the reagent according to the invention is very well standardizable. This is a prerequisite for a high reproducibility of the determination method.

In the reagent according to the invention, the amount of kaolin must be small. The increased deposits of kaolin are prevented and difficulties in the operation of detection devices are avoided.

The initiator according to the invention can be freeze-dried without difficulty together with phospholipids into a reasonable reagent. A possible negative interaction of the individual components is of no importance.

The invention thus relates to a reagent for the determination of aPTT in blood, plasma or blood derivatives, which is characterized in that it contains a mixture of sulfatides and a solid activator, preferably kaolin, as an initiator for the intrinsic coagulation, and also phospholipids.

The concentration of kaolin or sulfatides in the reagent are for kaolin between 0.1 and 0.6% by weight, preferably 0.1 to 0.3% by weight, and for sulfatides 0.001 to 0.03% by weight, preferably 0.002 to 0.01% by weight.

The reagent preferably contains chemically pure phospholipids in a standardized mixture. It has been shown that an unexpectedly high reproducibility of the test procedure is thereby achieved. The mixture of phospholipids contains e.g. phosphatidylserine, phosphatidylcholine and cholesterol.

The mixture preferably contains 5 to 50% phosphatidylserine, 15 to 60% phosphatidylcholine, 15 to 60% phosphate idylethanolamine, 0 to 20% cholesterol and 5 to 20% phosphatidic acid, calculated on the total phospholipid content. A standardized mixture is used, optionally diluted, e.g. 0.2 mg phosphatidylserine, 0.85 mg phosphatidylcholine, 0.65 mg phosphatidylethanolamine, 0.15 mg cholesterol and 0.15 mg phosphatidic acid per ml.

The phospholipids are advantageously purified so that unwanted precipitation reactions, which can occur in the presence of polybrene, are avoided.

A preferred embodiment of the reagent is characterized in that it also contains oxidation inhibitors and sensitivity enhancers. As an oxidation inhibitor to increase the storage stability of the reagent in the freeze-dried or liquid state, e.g. cysteine, uric acid, vitamin C or vitamin E. Additives, such as alkali salts, possibly together with amino acids, increase the test's sensitivity to individual factors. It has been found that a reagent containing cesium chloride (5 to 25 mg/ml) and arginine (2.5 to 20 mg/ml), preferably 16.8 mg CsCl/ml and 10.5 mg arginine/ml, after corresponding dilution is particularly suitable for the determination of aPTT of a plasma sample deficient in individual factors (factor VIII).

A further preferred embodiment of the reagent is characterized by the fact that it also contains a chromogenic substrate with which, if necessary, the coagulation time is determined photometrically by color development. However, the determination of the end product is also possible by photometric determination of the change in light permeability.

Advantageously, the reagent also contains stabilizers, preferably albumin and/or buffer substances.

The procedure for preparing this reagent is simple. Sulfatides are mixed in an aqueous suspension of the solid activator with phospholipids and possibly with further additions, and possibly freeze-dried. However, the preparation of the reagent can also be carried out by mixing sulfatides, the solid activator and phospholipids, possibly after freeze-drying, with further additions in the solid state.

The reagent according to the invention is suitable both for monitoring a heparin therapy and for determining individual factors, i.e. coagulation factors or inhibitors of the intrinsic coagulation.

The invention also relates to the use of the reagent according to the invention, a method for determining the partial thromboplastin time (aPTT) in samples of blood, plasma or blood derivatives, where the sample is brought into contact with a reagent according to the invention that calcium ions are added after a predetermined activation time and that the coagulation time is determined.

By adding heparin neutralizing substances, a coagulation factor deficiency can also be determined in heparinized plasma samples. Polybrene neutralizes e.g. present heparin and thus makes the reagent according to the invention insensitive to a heparin content in the sample.

A further preferred embodiment of the application according to the invention is thus characterized in that the sample is mixed before adding the reagent according to the invention with a heparin neutralizing substance, such as polybrene, protamine sulphate etc.

Furthermore, the invention relates to use according to the invention in the determination of the activity of factors of the intrinsic coagulation and its inhibitors, whereby a plasma sample is brought into contact with the reagent according to the invention, after a predetermined activation time calcium ions are added, and the coagulation time is determined, after which the obtained result is put in relation to a standard sample with known activity of the factors.

Furthermore, it was found that the determination of aPTT in possibly heparin- or heparinoid-containing blood, plasma or blood derivatives can also be carried out without the use of a mixture of sulfatides and solid activator, when in a reagent for the determination of aPTT in addition to an initiator of the intrinsic coagulation, whether it is a solid activator or a liquid activator or a combination thereof, a neutralizing agent and phospholipids are present.

Polybrene, protamine (protamine salts) or heparinase in particular are used as heparin neutralizing agents in the reagent according to the invention.

The concentrations of heparin-neutralizing agents that are used depend in terms of magnitude on the heparin concentration in the sample to be examined, and are usually, e.g. in the case of polybrene, between 1 and 500 mg/ml. In standard procedures, between 2 and 100 mg/ml polybrene is used.

The advantage of a reagent containing a heparin neutralizing agent consists in particular in the fact that the determination of aPTT and the individual factors in a blood or plasma sample can take place regardless of whether or not the sample contains heparin or heparinoids.

The invention also relates to a kit consisting of a) a reagent for determining aPTT according to the invention containing an initiator of the intrinsic coagulation and phospholipids, and b) a heparin neutralizing agent.

Although aPTT is often used to monitor heparin therapy, where heparin sensitivity is desirable, there are still many cases where the intact intrinsic system must be examined regardless of the heparin content.

Namely, should heparin be present in the sample and the aPTT when measured with reagents according to the state of the art thus be delayed, an all too small proportion of coagulation factors would be simulated.

The invention shall be explained in more detail by means of the following examples. 1. Comparison of aPTT using various reagents.

The investigated reagents for the determination of aPTT contained sulfatides (company Pentapharm) and kaolin in different mixing ratios in 200 mM hepes-glycine buffer (pH 6.8) as initiators for the intrinsic coagulation. Also, a mixture of 4.8 µg phosphatidylserine, 20.7 µg phosphatidylcholine, 15.9 µg phosphatidylethanolamine, 3.7 µg cholesterol and 3.7 µg phosphatidic acid per ml in the reagent.

0.1 ml normal plasma (NP) ("Reference Plasma" 100%, company Immuno) or an abnormal plasma (AP) ("Immunocontrol Plasma Abnormal", company Immuno) was mixed with 0.1 ml of reagent. After a 2 min. incubation time at 37°C, 0.1 ml of 0.025 M CaCl2 solution was added and the time until the coagulation point was measured with a Schnitger-Gross coagulometer (company Amelung).

Table 1 shows that the coagulation time for normal plasma (NP) was shortened due to the presence of sulfatides in the reagent. However, kaolin in the reagent also improved the sensitivity, expressed by the ratio of the coagulation times of AP and NP (cf. Table 2).

2. Heparin sensitivity.

Using reagents from 1., normal plasma (cf.

1.) and heparinized normal plasma (0.37 U heparin/ml) investigated. The ratio between aPTT in heparinized normal plasma and normal plasma appears for the different reagents from

table 2. The sensitivity in relation to heparin is improved by an increased kaolin content in the reagent.

3. Sensitivity to factor VIII.

Using reagents from 1., normal plasma (comp. 1.) and "Control Plasma" (company Immuno; contained < 30% factor VIII calculated on factor VIII content in normal plasma (= 100%)) were examined. The ratio between aPTT in "Control Plasma" and normal plasma is shown for the various reagents in Table 4. The sensitivity in relation to factor VIII is improved by means of the sulfatide content in the kaolin-containing reagent.

4. Factor Vlll relationship curve.

To create a reaction curve for factor VIII, "Reference Plasma" 100% (company Immuno) was diluted 1 : 5 with imidazole buffer (3.4 g/l imidazole, 5.85 g/l NaCl, pH 7.4). 0.1 ml of this dilution was mixed with 0.1 ml of phospholipid-kaolin-sulfatide reagent (composition of phospholipids similar to example 1; 0.3% kaolin and 0.01% sulfatides in 200 mM Hepes-glycine buffer , pH 6.8) and incubated for 4 min. at 37°C. Simultaneously with the addition of 0.1 ml of calcium chloride (25 mM), the stopwatch was started and the time until the coagulation point was determined using a Schnitger-Gross coagulometer (company Amelung).

The 1:5 prediluted plasma corresponds to 100% factor VIII. Further dilutions were tested following a geometric dilution series (1:1= 100% to 1 : 128 = 0.78%). The measurement results appear in table 5. 5. aPTT determination with PTT reagents of different composition with and without the addition of polybrene.

Five different reagents were used in the present examples both with and without the addition of heparin-neutralizing agents for the determination of aPTT. These reagents have the following composition (% is weight%).

Reagent A: 0.075% kaolin and 0.01% sulfatide (company Pentapharm) as initiator, highly purified phospholipids (17% phosphatidylserine, 44% phosphatidylcholine, 29% phosphatidylethanolamine, 5% cholesterol and 5% phosphatidic acid). The ready-to-use reagent is prepared by reconstitution with 2 ml Aqua dest. (concentration of the phospholipids: 65 ug/ml.

Reagent B: as reagent A, but only 0.0017% sulfatide.

Reagent C: 0.5% kaolin, lipid extract from porcine mucus ("Tachostyptan", company Hormonchemie).

The ready-to-use reagent is prepared by reconstitution with 2 ml Aqua dest.

Reagent D: "Actin FS" from the company Baxter (contains ellagic acid as initiator and phospholipids from soybeans).

Reagent E: "Pathromtim" from the company Behring (contains 0.5% kaolin in liquid suspension as initiator and lipid extract from human placenta (lyo)).

The ready-to-use reagent is prepared by dissolving the lipid extract in the kaolin suspension.

A normal plasma (NP; Immunocontrolplasma Normal from the company Immuno) or heparinized normal plasma with 0.4 U heparin/ml (HP1) or 1.0 U heparin/ml /HP2) was used as plasma samples. 0.1 ml plasma sample was mixed with 0.1 ml reagent. After a 2 min. incubation time at 37°C, 0.1 ml of 0.025 M CaCl2 solution was added and the time until the coagulation point was measured with a Schnitger-Gross coagulometer (company Amelung).

Table 6 shows the results obtained, where in line 1 is indicated the coagulation time (in seconds) for normal plasma (NP), in lines 2 and 3 the coagulation time for the heparinized plasma (HP1 and HP2) and in lines 4 and 5 the ratio between the coagulation times for the heparinized plasma and normal plasma. The results clearly show that aPTT in heparinized plasma without the presence of polybrene is clearly prolonged and thus falsified respectively. be distorted. In the presence of polybrene, aPTT with all tested reagents can also be determined for heparinized plasma. The ratio between the coagulation times for heparinized plasma and normal plasma, which in the ideal case is equal to 1 (i.e. the coagulation time is exactly the same), deviates considerably from the ideal value of 1 in the tests with reagents without polybrene, on the other hand, the ratio of the coagulation times in the presence of polybrene is close to 1 6. Factor VIII relationship curve with PTT reagents of different compositions, with and without the addition of polybrene.

A reference plasma ("Reference Plasma" 100% from the company Immuno) was used to produce these reaction curves. This reference plasma was dissolved in 1 ml Aqua dest.

(RP) or with 1 ml Aqua dest., containing 1 U/ml heparin (Immuno) (HP).

The plasmas were prediluted 1:5 with imidazole buffer (3.4 g/l imidazole, 5.85 g/l NaCl, pH 7.4); this corresponds to 100% factor VIII. Further dilutions were prepared following a geometric dilution series (1:1 = 100% to 1:128 = 0.78%). 0.1 ml of the sample was mixed with 0.1 ml of reagent and incubated for 4 min. at 37°C. Then 0.1 ml of 0.025 M CaCl2 solution was added, and the time until the coagulation point was measured with a Schnitger-Gross coagulometer.

The results are shown in tables 7 to 10.

The results show that normally (plasma without heparin; RP, reagents without polybrene) there is an unequivocal correlation between the coagulation time and the factor VIII concentration: The lower the concentration of factor VIII, the longer the coagulation time.

The measurement of heparin-containing samples (HP, reagents without polybrene) shows no clear correlation, the measurement gives an unusable factor VIII relation curve which does not enable any linear assignment of coagulation time and factor VIII concentration.

First, the presence of polybrene in the reagents enables a determination of the factor VIII concentration in heparin-containing samples (HP, reagents with 12.5/25/50 mg/ml polybrene). The results of the measurements do not deteriorate in the presence of heparin or polybrene when using the reagent according to the invention. This is shown both by comparing the measurements of RP with reagents with and without polybrene, and by comparing the coagulation times of RP and HP in the presence of polybrene, whose quotient is always close to 1. 7. aPTT determination with reagent B with and without addition of heparinase.

"Immunocontrolplasma Normal" (firma Immuno) was dissolved in 1 ml Aqua dest., which contained 0, 0.5 or 1 U/ml heparin.

Reagent B was dissolved in 1 ml Aqua dest., which contained 0.0.74, 0.22, 0.07 and 0.02 U/ml heparinase (company IBEX, Canada).

The PTT determination was carried out as described in example 5.

The results of the measurements carried out are shown in table 11.

The results show that a reagent containing heparinase is also preferably suitable for the aPTT determination of heparinised plasma.

Claims (20)

1. Initiator for intrinsic coagulation for determining the coagulation time of blood, plasma or blood derivatives, characterized in that it contains a mixture of sulfatides and a solid activator, preferably kaolin. 2. Reagent for determination of aPTT for blood, plasma or blood derivatives, characterized in that it contains a mixture of sulfatides and a solid activator, preferably kaolin, as initiator for the intrinsic coagulation, and also phospholipids. 3. Reagent as stated in claim 2, characterized in that the present phospholipids are chemically pure phospholipids in a standardized mixture. 4. Reagent as stated in claim 3, characterized in that the phospholipid mixture, calculated on the total phospholipid content, contains 5 to 50% phosphatidylserine, 15 to 60% phosphatidylcholine, 15 to 60% phosphatidylethanolamine, 0 to 20% cholesterol and 5 to 20% phosphatidic acid. 5. Reagent as stated in one or more of claims 2 to 4, characterized in that it also contains oxidation inhibitors and/or sensitivity improvers. 6. Reagent as stated in claim 5, characterized in that it contains alkali salts, possibly together with amino acids. 7. Reagent as specified in one or more of claims 2 to 6, characterized in that it also contains a chromogenic substrate with which the coagulation time can possibly be determined photometrically by color development. 8. Reagent as specified in one or more of claims 2 to 7, characterized in that it also contains stabilizers, preferably albumin and/or buffer substances. 9. Reagent as specified in one or more of claims 2 to 8, characterized in that it is available in freeze-dried form. 10. Method for producing the reagent according to one or more of claims 2 to 9, characterized in that sulfatides are mixed in an aqueous suspension of the solid activator with phospholipids and possibly further additions and possibly freeze-dried. 11. Method for the preparation of the reagent according to one or more of claims 2 to 9, characterized in that sulfatides, the solid activator and phospholipids, optionally after freeze-drying, are optionally mixed with further additions in the solid state. 12. Use of a reagent according to one of claims 2 to 9 when determining the partial thromboplastin time (aPTT) in samples of blood, plasma or blood derivatives, where the sample is brought into contact with a reagent according to one or more of claims 2 to 9, that that after a predetermined activation time, calcium ions are added and that the coagulation time is determined. 13. Use as stated in claim 12, where the sample is mixed with a heparin neutralizing substance before adding a reagent according to one of claims 2 to 9. 14. Application according to claim 12 or 13 in determining the activity of factors of the intrinsic coagulation and its inhibitors, where a plasma sample is brought into contact with a reagent according to claims 2 to 9, that calcium ions are added after a predetermined activation time and that the coagulation time is determined, after which the obtained result is compared to a standard sample with known activity of the factors. 15. Reagent according to one of claims 2 to 9 for determination of aPTT of possibly heparin- or heparinoid-containing blood, plasma or blood derivatives, containing an initiator of the intrinsic coagulation, characterized in that it contains a heparin neutralizing agent and phospholipids. 16. Reagent as stated in claim 15, characterized in that it contains polybrene, a protamine salt or heparinase as a heparin neutralizing agent. 17. Reagent as stated in claim 15 or 16, characterized in that the phospholipid components, calculated on the total phospholipid content, contain 5 to 50% phosphatidylserine, 15 to 60% phosphatidylcholine, 15 to 60% phosphatidylethanolamine, 0 to 20% cholesterol and 5 to 20% phosphatidic acid. 18. Use of a reagent according to one of claims 15 to 17 when determining the partial thromboplastin time (aPTT) in samples of possibly heparin- or heparinoid-containing blood, plasma or blood derivatives, where the sample is brought into contact with a reagent according to one or more of the claims 15 to 17, that after a predetermined activation time, calcium ions are added and the coagulation time is determined. 19. Use according to claim 18 when determining the activity of factors of the intrinsic coagulation and its inhibitors, by bringing a possible heparin- or heparinoid-containing plasma sample into contact with a reagent according to claims 15 to 17, that after a predetermined activation time is added calcium ions and the coagulation time are determined, after which the result obtained is compared with a standard sample with known activity of the factors. 20. Kit, characterized in that it consists of a) a reagent for determining aPTT according to one of claims 2 to 9 containing an initiator of the intrinsic coagulation and phospholipids, and b) a heparin neutralizing agent.