AU637136B2 - A stabilizer for improving the recovery of coagulation factors in blood samples - Google Patents

Description

637136 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Clas Application Number: Lodged: Form ;s Complete Specification Lodged: Accepted: Published: SPriority: Related Art SName of Applicant: Address of Applicant: BEHRINGWERKE AKTIENGESELLSCHAFT D-3550 Marburg, Federal Republic of Germany Actual Inventor: THCMAS STIEF and NORBERT HEIMBURGER Address for Service WATERMARK PATENT TRADEMARK ATTORNEYS.

LOCKED BAG NO. 5, HAWTHORN, VICTORIA 3122, AUSTRALIA Complete Specification for the invention entitled: A STABILIZER FOR IMPROVING THE RECOVERY OF COAGULATION FACTORS IN BLOOD SAMPLES The following statement is a full description of this invention, including the best method of performing it known to us BEHRINGWERKE AKTIENGESELLSCHAFT HOE 89/B 037 Ma 696 Dr. Pfe/Zi Description A stabilizer for improving the recovery of coagulation factors in blood samples The present invention relates to an agent for stabilizing the functional activities of coagulation factors in samples of biological fluids, especially to the stabilization of plasminogen activators.

The functional detection of the activity of coagulation factors is of great clinical importance, both with regard So* to therapy and in respect of diagnosis.

00 However, there are difficulties in detecting the activi- .5 ties of coagulation factors because specific and nonspecific inhibitors present in the blood depress the activities in vitro irreversibly as a function of the time the removed sample of blood has stood.

Thus, for example, the functional detection of plasmino- 20 gen activators on the one hand of the urinary (u- PA), and on the other hand of the tissue (t-PA) type, is of great clinical importance, both with regard to monitoring fibrinolytic therapy and in respect of the diagnosis of a predisposition to thrombotic events.

Up to 80 90% of the PA activity can be lost in vitro even with the minimum elapse of time, i.e. even after the shortest time required for removing and working up the sample (centrifugation).

The object on which the present invention is based was thus to find an agent which can be used as medium, both for removing the sample and for incubation, which stabilizes the activity of the coagulation factors over the e r o period necessary for reliable determination of the coagulation factors. It has now been found, surprisingly, that a medium which, besides the sample, contains at least either a diamino monocarboxylic acid, a substance with chaotropic activity, an oxidizing agent or a combination of at least two of these compounds, in conjunction with an appropriate anticoagulant such as, for example, EDTA or citrate, permits almost 100% recovery of PA activity after a standing time at room temperature of about 1 hour.

A chaotropic substance is defined as a substance that stabilizes the conformation of macromolecules, by impairing development of the H 2 0 cage structure necessary for solvation, alleviating the transfer of apolar molecules from non-aqueous to aqueous phases during distribution. Examples of chaotropic substances are ammonium sulphate, thiocyanate, perchlorate and cesium chloride.

Thus the invention relates to an agent for stabilizing the functional activity of coagulation factors in samples of biological fluids, where the agent contains, besides one of the customary anticoagulants such as, for example, ED'A or citrate, at least one compound from one of the following classes of substances: diamino monocarboxylic acids (II) chaotropic agents (II) oxidizing agents It is preferred in this connection to use an oxidizing agent which preferably converts methionine into methionine sulfoxide at pH 7.5 9.

It is particularly preferred to use arginine as diamino monocarboxylic acid, CsCI as chaotropic agent and chloramine or one of its derivatives of HOCI as oxidizing agent. The arginine concentration in this mixture should be 25 150, preferably 40 80 mmol/l, the CsCI concentration should be 50 200, preferably 80 150 mmol/l, -and the chloramine concentration should be 1 preferably 2 10 mmol/l.

It is furthermore preferred to use arginine and chloramines and a combination of these substances.

It is very particularly preferred to use the medium for stabilizing plasminogen activator activity.

3 It is very particularly preferred to use the medium for stabilizing plasminogen activator activity.

The invention furthermore relates to the use of the medium as medium for removing the sample and, more particularly, a method for blood sampling, which comprises collecting the blood in a vessel into which a concentrated solution of the agent according to the invention has been initially introduced in a volume such that, after the blood sampling, the desired concentration of the active substances in the agent is attained. It is preferred to introduce initially one part of a ten-fold concentrated solution and to make up with 9 10 parts of patient's blood.

The invention additionally relates to a method for the determination of PA, where either the sample has already been taken up in the medium according to the invention, or the medium according to the invention is added to the blood to which an anticoagulant has been added, before it is investigated 15 for PA activity. In this connection, the determination of PA activity can in .principle be carried out by one of the methods known to the person skilled in the art. It is preferable to use the method described in EP-A-0,297,597.

i The examples which follow describe specific embodiments of the invention and are not intended to restrict the invention in any way.

Example 1 Recovery of u-PA in blood and plasma .200 p l of substance (see Table 1) were added to 2 ml of citrated blood. Then S100 IU of u-PA or, as control, physiological NaCI solution were added, and, after incubation at room temperature for 60 min and centrifugation at 2000 x g for 20 min, the plasma was investigated for PA activity by the method described by Stief et al. in EP-A-0,297,597. For this purpose, 200 Itl of plasminogen CTA-U/ml in: 100 mmol/l TRIS, 100 mmol/l NaCI, 1% polygeline, 0.1% Triton X100, pH 8.4 (TNPT buffer)) and 200 p l of 8 mmol/I chloramine T, 3 mmol/l tranexamic acid were added to 50 pl of plasma, which was then incubated at 37 0 C for 5 min. Then 500 pl of 0.6 mmol/l 4 Nva-CHA-Lys-pNA in 480 mmol/l NaC1, 50 mmol/1 CsCl (CS solution) were added and, after incubation at 37 0 C for 2 min, the reaction was stopped with 100 1p of 8.4 mol/1 acetic acid, and the resulting extinction was determined at 405 nm.

Blank: plasminogen solution was replaced by TNPT buffer.

See Table 1 for result.

Table 1 Addition 10 *0S 0* *0 *0 0 1 *1 Plasmin activity measured Notes

(A)

1. phys. NaCl solution 0.153 2. ZnClz 0.1 mmol/l 0.161 slightly hemolyt.

3. ZnCl 2 0.2 mmol/l 0.137 hemolytic 4. CsCl 100 mmol/l 0.491 CsC1 50 mmol/l 0.353 6. KC1 200 mmol/l 0.362 7. NaCl 240 mmol/l 0.141 slightly hemolyt.

8. Chloramine T 5 mmol/l 1.216 9. Chloramine T 10 mmol/l 1.312 Chloramine T 20 mmol/1 1.524 slightly hemolyt.

11. Arginine 60 mmol/l 1.130 12. Arginine 200 mmol/l 0.325 It is evident that u-PA activity is recovered to a high degree on addition of chloramine T, arginine or CsCl to citrated blood, whereas, for example, addition of physiological NaCl solution leads to almost complete loss of PA activity. However, addition of more than 10 mmol/l chloramines results in a tendency to hemolysis of the erythrocytes in the blood.

Example 2 Recovery of u-PA in blood and Plasma as a function of the standing time The assay was carried out as in Example 1 but, this time, u-PA was added to blood on the one hand and to plasma on the other hand, and the samples were left to stand for one or two hours. 100% recovery: addition of u-PA to normal plasma followed by immediate assay of PA activity.

See Table 2 for results.

*e*STable 2 Addition Plasmin activity decrease/h after storage at RT *lh 2 h M% 1 a) Blood 1. phys. NaCl 0.154 0.093 2. CsCl 100 mmol/l 0.624 0.472 3. CsC1 200 mmol/l 0.821 0.671 18 4. Chioramine T 5 nimol/l 1.341 1.029 23 5. Arginine 60 mmol/l 1.234 1.118 9 6. Arginine 120 nimol/1 1.166 1.046 19 7. Arginine CsCl 1.395 1.236 11 (60/100 mmoi/l) b) Plasma 1. phys. NaCl 0.093 0.057 2. CsC1 100 mmol/1 0.262 0.175 33 3. CsCl 200 rnmol/l 0.382 0.288 4. Chloramine T 5 mmol/l 0.764 0.689 5. Arginine 60 xnmol/l 0.804 0.749 7 6. Arginine 120 mmol/l 0.832 0.810 3 7. Arginine CsCI 0.866 0.862 1 (60/100 rnrolll) 100% control: 0.884 6 It is evident that arginine, chloramine and CsCl, and especially the combination of arginine and CsCl, lead to a large increase in the recovery rate and diminution in the decrease rate of PA activity in blood and plasma.

Example 3 Recovery of t-PA activity in whole blood and plasma First 200 Ap of various substances and subsequently 200 IU/ml single-chain t-PA or physiol. NaCI (control) were added to 2 ml portions of whole blood and plasma I, and, after having been left to stand at room temperature for 1 hour, the blood was centrifuged at 2000 x g for min plasma II). 50 pl samples of plasma I and plasma II were investigated for their t-PA activity by incubating 50 &l of sample with 200 pl of plasminogen (3 CTA-U 5 in TNPT buffer), 100 pl of 16 mmol/l chloramine T and 100 tl of fibrinogen degradation products (200 pg, obtained as described by Stief et al., Thromb.Res. 48, 603, 1987) or 100 pi of TNPT buffer as blank at 37 0 C for min, adding 500 pl of CS solution, incubating at 37°C for a further 5 min, and stopping the substrate conversion by addition of 100 pl of 8.5 mol/l acetic acid. 100% control: addition of t-PA to plasma and immediate measurement. See Table 3 for result.

Table 3 Addition Plasmin activity in a) Plasma I b) Plasma II (whole blood) phys. NaCl 0.087 0.110 CsCl 100 mmol/l 0.103 0.212 Chloramine T (CT) 5 mmol/l 0.309 0.658 Arginine (Arg) 60 mmol/l 0.201 0.575 100% control: 0.332 I 1*II i" 7 It is evident that addition of chloramine T or arginine leads to considerably improved PA activity recovery rates.

Example 4 Recovery of PA activity in plasma, effect of combinations 200 pl of various substances with 40 IU/ml u-PA or 200 IU/ml single-chain t-PA were added to 2 ml portions of plasma, and the PA activity was determined immediately and after one hour as in Example 1 (for u-PA) and Example 2 (for t-PA). Final incubation for u-PA: 2 1/4 min, t-PA> min. See Table 4 for result.

Table 4 e**8 Addition u-PA recovery t-PA recovery S* Plasmin activity after 1 h 15 37 0 C of initial activity) phys. NaCj 21 CsCl 100 mmnol/1 48 72 CT 5 mmol/l 103 100 :20 Arg 60 mmol/l 104 100 CsCl CT (100/5 mmol/l) 106 82 S' CsCl Arg (100/60 mmol/l) 100 110 CT Arg (5/60 mmol/l) 110 115 CsC1 CT Arg (100/5/60 mmol/1) 110 100 Arginine and chloramine T are suitable and preferred for quantitative detection of PA activities even after the sampler have stood for a lengthy time.

Claims (6)

1. An agent for stabilizing the functional activity of coagulation factors in samples of biological fluids, containing an appropriate anticoagulant and at least one compound selected from the following classes of subo'ances: diamino monocarboxylic acids (II) chaotropic agents (as hereinbefore defined) oxidizing agents.

2. Ar agent as claimed in claim 1 in which said one compound is an oxidizing agent which preferably converts methionine into methionine sulfoxide at pH 7.5 9.

3. An agent as claimed in claim 1, wherein said diamino monocarboxylic acid is arginine, wherein said chaotropic agent is CsCI, and said oxidizing agent is HOCI or a chloramine, preferably chloramine T, and the arginine concentration in the mixture is, after addition of the sample, 25 150, preferably 40 80 mmol/l, the CsCI concentration is 50 200, preferably 80 150 mmol/l, and the chloramine conceriation is 1 20, preferably 2 10 mmol/l.

4. An agent as claimed in claim 1 for stabilizing plasminogen activator activity.

A method for blood sampling, which comprises collecting the blood in a vessel into which a concentrated solution of the agent as claimed in claim 1 has been initially introduced in a volume such that, after the blood sampling, the desired concentration of the active substances in the agent is attained. 9

6. In a method for the determination of PA, the improvement comprising: taking up the sample in the agent as claimed in claim 1, or (II) adding the agent as claimed in claim 1 to the blood to which an anticoagulant has been added before it is investigated for PA activity. DATED this 25th day of February, 1993. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA o* DBM/MED/ML SDOC 030 AU6208790.WPC