SK48793A3 - Agent for determination aptt, process for its preparation and its use - 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

Field: techniques

The invention relates to an internal clotting initiator, an agent for the determination of activated partial thromboplastin time (aPTT) as well as a process for the manufacture of the agent and optionally the determination of aPTT.

BACKGROUND OF THE INVENTION

The activated partial thromboplastin time of the plasma sample serves as a screening test for the overall detection of clotting factors of internal clotting. Failures during clotting depend on clotting times outside the normal area. Prolonged aPTT as occurs in haemophilia A manifests e.g. lack of clotting factor VIII. In addition, the aPTT assay is often performed to monitor heparin therapy.

The aPTT assay reagent contains an activator for initiating internal clotting up to the formation of activated factor IX. Furthermore, phospholipids will be required for the activation of factor X and prothrombin, therefore these are optionally contained in the aPTT reagent. The reagent is contacted with a plasma sample to which calcium is added after a defined, as short as possible time, and the time to fibrin clump formation is measured.

There are many requirements for the test system and PTT. For routine experiments, simple handling of the aPTT reagent is a prerequisite. Likewise, the stability of the constituents must be very high in order to ensure a constant precipitation time over a longer period of time. This also allows for inexpensive use of the reagent. Appropriate sensitivity to all parameters included is also required for aPTT as an overall assay.

Conventional aPTT reagents contain a surfactant solid as an initiator of internal precipitation. By using a surfactant matrix, factor XII is first activated comparable to physiological conditions. For example, kaolin, celite and glass are used as solid activators. Kaolin is commercially available e.g. in aPTT assay reagents as a suspension (Pathromtin ^R , Behringerwerke (1990) or as PTT reagent, Boehringer Mannheim (1986)). Lyophilized kephalin is used as the phospholipid. However, phospholipids are mixed first after performing the assay with kaolin to a ready-to-use reagent. Separate storage of kaolin and phospholipids is necessary for stabilization reasons.

When lyophilized with lipids by kaolin, the lipids are largely inactivated. Thus, for example, the production of PTT-IMMUNO (Immuno) in the co-lyophilization of kaolin and phospholipids (extracted from pig brain) results in a loss of up to 50% of the phospholipid activity.

activator is also possible ellagic acid and its

As an alternative to solids - use liquid activators such as derivatives or dissolved sulfatides. However, the sensitivity of the heparin assay in the presence of liquid activators is compromised and various additives have been proposed to improve sensitivity. According to WO 91/16453, the heparin-sensitive ellagic acid agent contains dextran sulfate.

A disadvantage of kaolin or solid activators in general and ellagic acid or derivatives thereof is the long activation time of the first internal precipitation phase, which requires a relatively long incubation period of at least 4 minutes for activation. Another disadvantage is the relatively long clotting time in the presence of kaolin. However, a higher concentration of kaolin in the reagent may shorten the activation time. Furthermore, the kaolin deposited from the suspension may lead to prolonged precipitation time or erroneous results when determining the precipitation point using a conventional coagulometer. Care should be taken to keep the concentration of solid-activator as low as possible.

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SUMMARY OF THE INVENTION.

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SUMMARY OF THE INVENTION It is an object of the present invention to provide a standardized reagent (s) of high stability for the determination of aPTT, which is simple to handle and inexpensive and can achieve short activation times as well as high sensitivity to individual clotting cascade factors. . | k ',. '1;

The solution according to the invention is made possible by the initiators of the internal clotting of blood, plasma or blood derivatives, which comprises a mixture of sulfatides and a solid-activator, preferably / kaolin. / ‘&

The agent of the present invention comprises a mixture of sulfatide (e.g. from Pentapharm) and a solid mixture as initiator of both internal and precipitation. .. ·?

activator. It also contains phospholipids. Only the very short activation time that is recorded when you use ’··· -M. : ‘‘ '. Sulf Λ 'sulfatides to activate factor XII will prolong a little. The use of a reagent according to the invention thus allows a short incubation period for pretreatment in routine experiments. At the same time, however, the sensitivity to heparin is also improved by using a solid activator.

The use of the initiator of the present invention in an aPTT reagent also allows for a surprising combination of the advantages of both individual activators, but not their disadvantages.

This was not supposed. Rather, it was believed that the combination of sulfatide with kaolin as the initiator of internal precipitation in the reagent would result in a decrease in activity. According to the prior art, it would be surprising that the agent according to the invention is well standardizable according to the skilled artisan that difficulties could arise when combining activators with different reaction mechanisms. This is a prerequisite for the high reproducibility of the assay method.

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A low amount of kaolin is maintained in the agent of the invention. Increased deposition of kaolin is prevented and difficulties in the operation of detection devices are eliminated.

The initiator of the invention can be freely lyophilized together with phospholipids to a low-cost agent. Possible negative side effects of individual components are insignificant.

The invention thus also relates to an agent for the determination of aPTT in blood, plasma or blood derivatives, characterized in that it comprises a mixture of sulfatides and a solid-activator, preferably kaolin, as an internal clot initiator and further phospholipids.

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

Preferably, the reagent comprises chemically pure phospholipids in a standardized mixture. It has been shown that this results in an unexpectedly high reproducibility of the test procedure. The phospholipid mixture comprises, for example, phosphatidylserine, phosphatidylcholine and cholesterol.

The mixture preferably consists of 5 to 50% phosphatidylserine, 15 to 60% phosphatidylcholine, 15 to 60% phosphatidylethanolamine, 0 to 20% cholesterol and 5 to 20% phosphatinidic acid, based on the total phospholipid content. For example, 0.2 mg of phosphatidylserine, 0.85 g of phosphatidylcholine, 0.65 mg of phosphatidylethanolamine, 0.15 mg of cholesterol and 0.15 mg of phosphatinidic acid, per ml, optionally diluted, are used as a standardized mixture.

The phospholipids are preferably purified so that unwanted clotting reactions, which could occur in the presence of polybrene, are avoided.

A preferred form of preparation of the agent is characterized in that it comprises an additional oxidation suppressant and a sensitivity enhancer. For example, cysteine, urea, vitamin C or vitamin E are used as an oxidation suppressant to increase the shelf life of the reagent in a lyophilized or liquid state. Additives such as alkali metal salts, optionally together with amino acids, increase the sensitivity of the assay to individual factors. The agent 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, has been shown to be correspondingly dilutions particularly useful for determining aPTT of a single factor deficient plasma sample (Factor VIII).

A further preferred form of preparation of the reagent is characterized in that it comprises an additional chromogenic substrate, which makes it possible, if appropriate, to determine the precipitation time by photometric color development. However, the evaluation of the end product is also possible by photometric determination of the change in light transmittance.

Preferably, the agent further comprises stabilizers, preferably albumin or buffer substances.

The method of making this reagent is simple. The sulfatides are mixed in an aqueous solid-activator slurry with phospholipids and optionally other additives and optionally lyophilized.

However, the preparation of the reagent can also be carried out by mixing the sulfatides, the activator solid and the phospholipids, optionally after lyophilization, optionally together with other additives in a solid state.

The agent according to the invention is also suitable for monitoring heparin therapy, as well as for determining individual factors as well as clotting factors or inhibitors of internal clotting.

The invention further relates to a method for determining the partial thromboplastin time (aPTT) of a blood, plasma or blood derivative sample, which comprises contacting the sample with an agent of the invention, adding calcium ions after a predetermined activation period, and determining precipitation time.

By adding heparin neutralizing substances, the lack of clotting factors can also be determined in the heparinized sample. For example, polybrene neutralizes the heparin present and makes the agent of the invention insensitive to the heparin content of the sample.

Another suitable embodiment of the method according to the invention is also characterized in that the sample is mixed with a heparin neutralizing substance such as polybrene, protamine sulfate, etc. before the addition of the agent according to the invention.

Furthermore, the invention relates to the use of the method of the invention for determining the activity of internal clotting factors and inhibitors thereof, wherein a plasma sample is contacted with an agent of the invention, calcium ions are added after a specified activation period and the clotting time is determined. The result obtained is compared to a standard sample with known factor activity.

It was further invented that the determination of aPTT or heparin or heparinoid-containing blood, plasma or blood derivatives can also be carried out without the use of a mixture and a solid activator, if only the activator or liquid activator or their sulfatides is present in the aPTT reagent. determination of solid combination, heparin neutralizing agent and phospholipids.

In particular, polybrene, protamine (protamine salt) or heparinase in the agent according to the invention are used as heparin neutralizing agents.

The concentration of heparin neutralizing agents is controlled by the concentration of heparin in the sample to be investigated, and is typically, for example, in the range of 1 to 500 mg / ml for polybrene. Between 2 and 100 mg / ml are used in standard methods

polybrene. Γ

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The advantage of an agent comprising heparin neutralizing agent 1 is, in particular, that the determination of aPTT and individual factors in a blood or plasma sample can be performed independently;

whether the sample contains heparin or heparinoids.

The invention further relates to a kit comprising a) an aPTT assay reagent comprising an internal precipitation initiator P

and phospholipids; and b) heparin neutralizing agents.

However, aPTT is often used to monitor t heparin therapy where heparin sensitivity is required, there are many cases in which intact intrinsic

L · system or internal system factors independent of heparin content.

If heparin and hence aPTT should be prolonged in the sample as measured by prior art reagents, only a minor proportion of the clotting factors will be involved. j

The invention is illustrated by the following examples. '· ^; : 4 ·· '·. ··. -6 • ' ^r ''· P

DETAILED DESCRIPTION OF THE INVENTION 4

i. Comparison of aPTT using different reagents

Tested aPTT assay reagents containing sulfatides (Pentapharm) and kaolin as initiator of internal coagulation in various proportions in 200 mM Hepes-glycine buffer (pH 6.8). Next, a mixture of 4.8 μ9 phosphatidylserine, 20.7 μ9 phosphatidylcholine, 15.9 μ9 phosphatidylethanolamine, 3.7 μg cholesterol and 3.7 μg phosphatidinic acid per ml reagent was prepared.

0.1 ml normal plasma (NP) (Reference Plasma 100%, Immuno) or abnormal plasma (AP) (Immunocontrol Plasma

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Abnormal (Immuno) was mixed with 0.1 ml reagent. After

An incubation time of 2 minutes at 37 ° C was added with 0.1 ml of 0.025 M

CaCl ₂ -solution and the time up to the clotting point is measured

Schnitger-Gross-Coagulometer (Amelung).

S Table 1 shows that the presence of sulfatides in the reagent reduces the clotting time of normal plasma (NP). However, kaolin in the reagent also improves the sensitivity, expressed as the ratio of AP and NP clotting times (see Table 2).

Table 1

aPTT (sec) kaolin (WITH) 0 0.15 0.30 sulfatide ( %) NP 0 x 78.1 70.1 NP 0,005 34.4 38.4 38.6 NP 0.01 39.1 41.1 41.8 Table 2 aPTT ratio (AP / NP) kaolin (0) 0 . 0.15 0.30 sulfatide ( %) < 0 X 2.20 2.12 0,005 2.00 2.11 2.21 0.01 2.01 2.15 2.23

2. Sensitivity to heparin

Normal reagents (see 1) and heparinized normal plasma (0.37 µg heparin / ml) were tested using reagents from 1.. aPTT ratio

heparinized normal plasma to normal plasma is the reagents listed in Table 2. Heparin sensitivity for different will improve higher content Table 3 kaolin in the reagent. aPTT ratio (+/- heparin) kaolin (%) 0 0.15 0.30 sulfatides (%) 0 X 1.50 1.66 0,005 1.18 1.28 1.37 0.01 1.23 1.31 1.41

3. Sensitivity to factor VIII

Normal reagents (see 1) and control plasma (Immuno, containing <30% factor VIII relative to factor VIII content in normal plasma (= 100%)) were assayed using reagents from 1. The ratio of control plasma to normal plasma aPTT is shown in Table 4 for various agents. Sensitivity to factor VIII is improved by the sulfatide content of the kaolin-containing agent.

Table 4

control plasma aPTT ratio (<30% factor VLIII / Normal plasma) kaolin (%, 0 0.15 0.30 sulfatides (%) 0 X 1.48 1.51 0,005 1.66 1.75 1.74 0.01 1.71 1.72 1.70  - J

4. Factor VIII - calibration curve

To determine the calibration curve for Factor VIII, the 100% reference plasma (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 is mixed with 0.1 ml of phospholipid-kaolin-sulfatide reagent (phospholipid composition as in Example 1, 0.3% kaolin and 0.01% sulfatides in 200 mM Hepes-glycine buffer, pH 6, 8) and incubated at 37 ° C for 4 min. Simultaneously with the addition of 0.1 ml of calcium chloride (25 mM), the stem is started and the time up to the point of precipitation is determined by means of a Schin-Gross coagulometer (Amelung).

1: 5 diluted plasma corresponds to 100% factor VIII. Other dilutions made in the geometric series (1: 1 = 100% to 1: 128 = 0.78%) were tested. Table 5 shows the measurement results.

Table 5

Factor VIII (%) precipitation time (%) 100 78.5 50 91.0 25 101.7 12.5 115.3 6.25 128.9 3.13 139.9 1.56 148.6 - 0,78 159.4

5. aPTT assay with PTT reagents of different composition and with and without polybrene

In the examples, five different reagents were used, both with and without the addition of heparin-neutralizing agents, for the determination of aPTT.

These reagents have the following composition (% by weight):

Reagent A: 0.075 kaolin and 0.01% sulfatide (Pentapharm) as initiator, high purity phospholipids (17% phosphatidylserine, 44% phosphatidylcholine,% phosphatidylethanolamine, 5% cholesterol and 5% phosphatidinic acid).

For use, the reagent is prepared by reconstitution with 2 ml dest. water (phospholipid concentration: 65 μά / π »1).

Reagent B: as reagent A but only 0.0017% sulfatide Reagent C: 0.5% kaolin, pig mucosa lipid extract (Tachostyptan ^R , Hormonochemie).

For use, the reagent is prepared by reconstitution with 2 ml of distilled water.

Reagent D: Actin FS ^R Baxter (contains ellagic acid as an initiator, and phospholipids from soybeans)

Reagent E: Behring Pathrombin ^R (contains 0.5% kaolin in liquid suspension as initiator and lipid placenta lipid extract [lyo]).

For use, the agent is prepared by dissolving the lipid extract in a kaolin suspension.

Normal plasma (NP, Immunocotrolplasma Normal from Immuno) or heparinized normal plasma with 0.4 µl heparin / ml (HP1) or 1.0 heparin / ml (HP2) are used as plasma samples. 0.1 ml of the plasma sample is mixed with 0.1 ml of reagent. After an incubation time of 2 minutes at 37 ° C, 0.1 ml of 0.025 M CaCl ₂ solution is added and the time is measured for the time of precipitation by Schnitger-Gross coagulometer (Amelung).

Table 6 shows the results obtained, with row 1 showing the clotting time (in seconds) of normal plasma (NP), lines 2 and 3 clotting time of heparinized plasma (HP1 and HP2) and rows 4 and 5 between clotting times of heparinized plasma ä normal plasma.

Table 6

Reagent A Reagent C Reagent D Reagent E b. half . 25mg / 1 P · b. half . 25mg / 1 P- b. half . 25mg / 1 p. b. half . 50mg / 1 p. NP (s) 34.0 32.4 52.4 68.0 43.1 65.9 * 34.1 46.6 HP (S) 52.9 36.1 123.1 75.8 99.8 67.6 76.1 50.9. HP 2 (s) 214.1 39.1 253.4 80.3 283.4 59.1 210.1 96.2 HP1 / NP 1.56 1.11 2.35 1.11 2.32 1.03 2.23 1.09 HP2 / NP 6.30 1.21 4.84 1.18 6.58 0.90 6.16 2.06

b.pol = without polybrene p. = polybrene

The results clearly show that aPTT in heparinized plasma in the absence of polybrene is significantly prolonged and thus distorted or erroneous. The presence of polybrene also allows the determination of aPTT using all tested reagents. in heparinized plasma. The ratio between clotting time of heparinized plasma and normal plasma, which is ideally equal to 1 (ie clotting time is exactly the same), deviates completely from the ideal value of 1 in tests with polybrene-free reagents; in the presence of polybrene is closer to 1.

6. Factor VIII - calibration curve with reagents of different composition with and without polybrene.

A reference plasma (Reference Plasma 100% from Immuno) was used to determine these relationship curves. This reference plasma is dissolved in 1 ml dest. water containing 1 J / ml heparin (Immuno) (HP).

Plasma was pre-diluted with 1: 5 imidazole buffer (3.4 g / l imidazole, 5.85 g / l NaCl, pH 7.4, equivalent to 100% factor VIII; further dilutions were made in a geometric series (1: 1 = 100% to 1: 128 = 0.78%) 0.1 ml of the sample is mixed with 0.1 ml reagent and incubated at 37 ° C for 4 minutes, then 0.1 ml of 0.025 M CaCl ₂ solution is added and measured the time up to the point of precipitation with a Schnitger-Gross coagulometer.

The results are shown in Tables 7-10.

Table 7 Reagent A without polybrene 25 mg / ml polybrene

F Wine: (%) RP HP RP HP

100 54.7 82.0 40.0 1 42.2 50 '65, 2 78.6 48.6 47.6 25 73.5 82.1 52.0 52.1 12.5 81.7 86.6 58.3 58.0 6.25 90.7 92.6 64.5 64.6 3.13 98.1 104.1 70.6 71.6 1.56 107.2 108.6 78.6 78.6 0.78 115.1 117.6 83.6 83.6

Table 8 Reagent C

F VIII (%) without polybrene 12.5 mg / ml polybrene RP HP RP HP 100 81.6 129.1 90.1 87.6 50 91.3 105.6 99.6 99.6 25 104.5 102.1 113.1 110.6 12.5 110.6 109.1 123.1 127.6 6.25 121.5 120.1 138.1 134.6 3.13 130.5 13 2,1 150.1 151.1 1.56 141.6 143.1 163.1 163.1 0.78 150.5 151.6 177.5 175.1

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Table 9 Reagent D without polybrene 25 ng / ml polybrene

F VIII (%)

100 60.6 107.2 70.1 65.6 50 71.7 84.1 25 79.0 82.6 100.1 93.6 12.5 88.1 89.6 6.25 98.1 100.1 3.13 109.1 109.6 140.6 138.6 1.56 118.1 118.2 0.78 127 126.6 164 160.5

Table 10 Reagent E

F VIII (%) without polybrene 50 mg / ml polybrene RP HP RP HP 100 54.9 127.5 72.1 68.1 50 61.2 98.5 78.4 75.1 25 67.4 96.4 87.9 82.6 12.5 74.2 102.4 94.9 92.1 6.25 83.4 112.6 106.2 105.1 3.13 89.9 120.6 113.2 115.6 1.56 97.1 131.6 121.1 125.5 0.78 102.4 140.9 127.6 131.6 The results show that in normal event (plasma without

heparin, RP, polybrene-free agents) there is a clear relationship between clotting time and factor VIII concentration: the lower the factor VIII concentration, the longer the clotting time.

and

There is no clear relationship when measuring samples containing heparin (HP, polybrene-free reagent), providing an unusable factor VIII relationship curve that does not allow any linear assignment of clotting time and factor VIII concentration.

Only the presence of polybrene in the reagent makes it possible to determine the concentration of factor VIII in samples containing heparin (HP, reagent with 12.5 / 25/50 mg / ml polybrene). Results of measuring the use of the agents of the invention. This is evident both when comparing RP measurements with polybrene and polybrene-free reagents, but also when comparing RP and HP precipitation times in the presence of polybrene, the proportion of which is always quite close to 1.

7. Reagent B aPTT assay with and without heparinase f

Immunocontrolplasma Dissolve normal (Immuno) in 1 ml dest. water containing 0, 0.5 or 1 J / ml heparin.

The reagent is dissolved in 1 ml of distilled water containing 0, 0.74, 0.22, 0.07 and 0.02 U / ml heparinase (from IBEX, Canada).

PTT determination is performed as in Example 5.

The results of the measurement are given in Table 11.

Table 11

heparin in plasma (U / mL) 0 0.02 heparinase j / ml 0.07 0.22 0.74 0.0 33.6 33.6 33.1 3 3,6 38.3 0.5 52.4 45.8 41.8 37.7 41.4 1.0 85.8 65.6 50.8 4 7.3 42.3

The results show that the heparinase-containing agent is also i

perfectly suited for aPTT-determination of heparinized plasma

Claims (19)

PV PATENT CLAIMS An internal clotting agent of blood, plasma or blood derivatives, characterized in that it comprises a mixture of sulfatides and a solid activator, preferably kaolin. 2. Reagent for the determination of aPTT, plasma or blood derivatives, characterized in that it contains a mixture of sulphatides and a solid-activator, preferably kaolin, as an initiator of internal clotting and further phospholipids. Reagent according to claim 2, characterized in that the phospholipids contained are chemically pure phospholipids in a standardized mixture. Agent according to Claim 3, characterized in that the phospholipid mixture comprises, based on the total phospholipid content, 5 to 50% phosphatidylserine, 15 to 60% phosphatidylcholine; 60% phosphatidylethanolamine, 0-20% cholesterol and 5-20% phosphatidinoic acid. Agent according to one or more of claims 2 to 4, characterized in that it further comprises an oxidation inhibiting agent. And / or a sensitivity enhancer. Agent according to claim 5, characterized in that it contains an alkali metal salt, optionally together with amino acids. Reagent according to one or more of Claims 2 to 6, characterized in that it further comprises a chromogenic substrate, with which it is possible to determine the precipitation time by photometric color development. Agent according to one or more of Claims 2 to 7, characterized in that it further contains stabilizers, preferably albumin and / or buffer substances. '·'. 'Z-' ^: ..ú Agent according to one or more of claims 2 to 8, characterized in that it is in lyophilized form. Process for the preparation of an agent according to one or more of claims 2 to 9, characterized in that the sulfatides in the aqueous suspension of the activator solid are mixed with phospholipids and optionally other substances and optionally lyophilized. Process for the preparation of an agent according to one or more of claims 2 to 9, characterized in that the sulfatides, the solid activator and the phospholipids are mixed in the solid state, if necessary after lyophilization or with other additives. Method for determining the partial triple megoplastin time (aPTT) of blood, plasma or blood derivative samples, characterized in that the sample is contacted with the reagent according to one or more of claims 2 to 9, after which time calcium ions are added and the time is determined clotting. Method according to claim 12, characterized in that the sample is mixed with a heparin neutralizing substance before the addition of the agent according to one of claims 2 to 9. Use of a method according to claim 12 or 13 for determining the activity of internal clotting factors and their inhibitors, characterized in that the plasma sample is contacted with an agent according to one of claims 2 to 9, calcium ions are added after a predetermined activation period and the clotting time is determined, and the result obtained is compared to a standard sample with known activity of the factors. ^! . A reagent for the determination of aPTT or heparin or heparinoids containing blood, plasma ¹ or blood derivatives, comprising an internal clotting initiator, characterized in that it comprises a heparin-neutralizing agent and a phospholipid. 16. The agent of claim 15, wherein the heparin neutralizing agent is polybrene, a protamine salt, or heparinase. The agent according to claim 15 or 16, wherein the phospholipid component comprises, based on the total phospholipid content, 5 to 50% phosphatidylserine, 15 to 60% phosphatidylcholine, 15 to 60%; phosphatidylethanolamine, 0-20% cholesterol, and 5-20% phosphatidinic acid. Method for determining partial thromboplastin time (aPTT) of samples, optionally containing heparin - or heparinoid, blood, plasma or blood derivatives, characterized in that the sample is contacted with an agent according to one or more of claims 15 to 17, after predetermined calcium ions are added during the activation period and the clotting time is determined. ¹ , jr '· Use of a method according to claim 18 for determining the activity of internal clotting factors and inhibitors thereof, characterized in that a plasma sample, optionally containing heparin or heparinoid, is contacted with an agent according to one of claims 15 to 17 after a predetermined activation period. calcium ions are added and the clotting time is determined and the result obtained is compared to a standard sample of known factor activity. 20. Set consisting of (a) reagents for the determination of aPTT containing an internal clotting initiator and phospholipids; and b) a heparin neutralizing agent