Method for the Determination of Coagulation Parameters

The invention concerns a method for the determination of coagulation parameters in which inhibitors of the activation of F XIII, F XIII.sub.A or the fibrin cross-linking activity of F XIII.sub.A are added.

The determination of coagulation parameters in sample material usually involves a reaction cascade (cf. Haemostasis 20 (suppl. 1) 14-29, 1990). In a multitude of determinations the last step is the formation of a fibrin clot catalyzed by thrombin. In this manner it is for example possible to determine PT (prothrombin time), PTTa (partial thromboplastin time activated), fibrinogen, factors II, V, VII, VIII, IX, X, XI, XII, protein C, protein S and APC resistance (see for example Bergmeyer (editor) (1988): Methods of Enzymology, Vol. V, 3rd edition; Guglumone and Vides (1992): Thromb Haemostas 67, 46-9; Preda et al (1990): Thromb Res. 60, 19-32 or Dahlback et al (1993): Proc Natl. Acad. Sci. USA 90, 1004-8). These references are herein incorporated by reference into the present application. Since the fibrin clot which forms cannot in practice be removed from the measuring vessels without a considerable amount of manual work, the measuring vessels are usually used only once in the determination of coagulation parameters and are subsequently discarded. Apart from the costs which this causes, such tests cannot be applied to instruments on which cuvettes are used several times. Special coagulation analysers equipped with disposable cuvettes are relatively expensive since they are only manufactured in low numbers.

The invention eliminates these disadvantages.

The invention comprises a method for the determination of coagulation parameters in sample material via a reaction cascade during which a thrombin-catalyzed formation of a fibrin clot occurs and the formation of the fibrin clot is measured which is characterized in that the determination is carried out in the presence of an inhibitor of the activation of F XIII, F XIII.sub.A or of the fibrin cross-linking activity of F XIII.sub.A.

If the determination of a coagulation parameter is carried out in the presence of such an inhibitor it is subsequently possible to dissolve the fibrin clot under conditions which abolish the mutual affinity of the fibrin molecules and to remove it from the measuring vessel by rinsing processes without having an adverse effect on further determinations of coagulation parameters.

Any inhibitor of the activation of F XIII, F XIII.sub.A itself or of the fibrin cross-linking activity of F XIII.sub.A is potentially suitable.

These are for example: hydroxylamine, benzothiophene, iodoacetic acid, N-ethylmaleinimide, lanthanide ions, cefuroxime, L-arginine, L-lysine, peptide fragments of F XIII.sub.A or fibrin as well as antibodies. Examples of these inhibitors are described in the following state of the art:

Feddersen J., Gormsen J. (1976): Thromb. Haemostasis 36.

Achyuthan, E. A., Enghild, J. J., Greenberg C. S., (1991): Thromb. Haem. 65, 901.

Achyuthan, E. A., Mary and C. S. Greenberg (1989): Biochem. J. 257:331-8.

Glover C. J., L. V. McIntire, C. H. Brown and E. A. Natelson (1975): J. Lab. Clin. Med. 86:644-56.

Jiang, S. T. and J. J. Lee (1992): J. Agr. Food. Chem. 40: 1101-7.

Lee, K. N., L. Fesus S. T., Yanbcey, J. E. Girard and S. I. Chung (1985): J. Biol. Chem. 260: 14 689-94.

Samara M., J. Soria, C. Soria, M. Maamer and A. M. Otero (1979): Prog. Chem. Fibrinnolysis Thrombolysis 4: 235-40.

Lukacova, D., Matsueda, G. R., Haber, E. and Reed, G. L. (1991): Biochemistry 30: 10164-70.

Coysne C. P., Smith, J. E. and De Bowers R. M. (1992): Am. J. Vet. Res 53:695-705.

Achyuthan, K. E., Slaughter, T. F., Santiago M. A., Enghild, J. J. and Greenberg, C. S. (1993): J. Biol. Chem. 268: 21284-92.

Any solution is suitable for dissolving the clot which abolishes the mutual affinity of the fibrin molecules.

These are for example acids or bases, solutions of chaotropic ions such as KSCN or Na salicylate, urea or arginine.

The inhibitors are added at such a concentration which allows the clot to be dissolved by addition of the cleaning solution such as e.g. 1 M hydrochloric acid within a maximum of 5-10 minutes to such an extent that it can be completely removed from the measuring vessel. In the case of hydroxylamine, iodoacetic acids, N-ethyl-maleinimide this preferred concentration range is between 0.5 and 20 mmol/1.

In order to remove the fibrin clot from the measuring vessel it is preferably incubated with an acidic solution (pH<2, preferably .ltoreq.1) for a period of between 10 seconds and 10 minutes. Hydrochloric acid is preferably used for this. Subsequently it is rinsed once to three times with water or aqueous buffer solution. Afterwards the fibrin clot is removed to such an extent that further determinations of coagulation parameters are not impeded.

As an alternative to an acidic solution, it is also possible to use one of the aforementioned substances alone or in combination.

Procedure for a PT determination on a Hitachi 717

Sample

20 .mu.l citrate plasma (contains ca. 5.5 g/l fibrinogen)

Reagent 1 (R1):

300 .mu.l, 100 mmol/l Tris buffer, pH 7.0

10 mmol/l potassium chloride

2.5 mmol/l hydroxylamine and

3.75 mg/ml rabbit brain thromboplastin.

Reagent 2 (R2):

300 .mu.l, 1 mol/l HCl

The PT determination is carried out by mixing 20 .mu.l plasma and 300 .mu.l R1 and measuring the time until the clot forms.

After the clot has formed, 300 .mu.l R2 is added, mixed at 37.degree. C. and aspirated after a further 100 seconds - for 10 minutes. It is washed once to three times with water.

An absorbance check in the measuring vessel after carrying out the determination and cleaning the measuring vessel with hydrochloric acid/water showed that the absorbance of the blank before and after the determination was not particularly different (max. ca. 0.1 mA from 50 measurements).

In order to examine which substances are suitable as an inhibitor of factor XIII.sub.A activity, a PT determination is carried out on a coagulometer from the H. Amelung Company, Lemgo, Germany (type KC 4A) (100 .mu.l plasma +200 .mu.l Neoplastin.RTM. Plus-reagent). Two minutes after the clotting has taken place, 200 .mu.l 1 mol/l hydrochloric acid is added and the time until the ball can move freely again is measured. The results are shown in Table 1.

In order to examine which solutions are suitable for dissolving the clot after the measurement, a PT determination is carried out on a coagulometer from F.H. Amelung, Lemgo, Germany, type (KC 4A) (100 .mu.l plasma + 200 .mu.l Neoplastin.RTM.-Plus+hydroxylamine, final concentration 2.5 mM). Two minutes after the clotting has taken place, 200 .mu.l dissolution agent is added and the time until the ball can move freely again is measured. The results are shown in Table 2.

Procedure for a PTTa determination

The transferability of the data obtained for the PT determination was validated on a KC-4A as follows:

10 .mu.l 100 mM hydroxylamine

100 .mu.l plasma

100 .mu.l PTTa reagent (Boehringer Mannheim GmbH)

3 minutes incubation

100 .mu.l CaCl2

Measurement of the clotting time

2 minutes additional incubation

200 .mu.l 1 M HCl

Measurement of the time until the ball moves again freely.

The results are shown in Table 3.

Procedure for a protein C determination

The transferability of the data obtained for the PT determination was validated on a KC-4A as follows:

10 .mu.l 100 mM hydroxylamine

10 .mu.l plasma

90 .mu.l Owrens buffer (Boehringer Mannheim)

100 .mu.l R1 protein C reagent (Boehringer Mannheim)

100 .mu.l R2 protein C reagent (Boehringer Mannheim)

3 minutes incubation

100 .mu.l CaCl2 solution

Measurement of the clotting time

2 minutes additional incubation

200 .mu.l 1 M HCl

Measurement of the time until the ball moves freely again.

The results are shown in Table 4.

Procedure for a thrombin time determination

The transferability of the data obtained for the PT determination was validated on a KC-4A as follows:

20 .mu.l 100 mM hydroxylamine

200 .mu.l plasma

2 minute incubation

200 .mu.l thrombin reagent (Boehringer Mannheim Order No. 126 594)

Measurement of the clotting time

2 minutes additional incubation

300 .mu.l 1 M HCl

Measurement of the time until the ball moves freely again.

The results are shown in Table 5.