DE1189763B - Reagent for controlling the coagulability of the blood during anticoagulant therapy and method for the preparation of the reagent - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. CL:

GOIn

German KL: 421-3 / 54

Number: 1189 763

File number: O6747IXb / 421

Filing date: April 30, 1959

Opening day: March 25, 1965

Oral anticoagulants (dikumarol and dikumarol derivatives, Phenylindandione and similar substances) reduce the ability to clot of the blood. They come in great measure in the treatment and prevention of thrombotic Done for application. The thrombosis prophylaxis takes place more and more in arteriosclerotic Conditions (coronary infarction, angina pectoris, cerebral and peripheral arterial thrombosis) general Use.

Effective treatment with anticoagulants requires regular monitoring of the ability to clot of the blood. It is necessary to customize the dosage; because on the On the one hand it is important to avoid bleeding complications caused by overdosing, on the other hand On the other hand, the treatment is ineffective if the dose is too small.

With the control methods described earlier it was not possible to determine the coagulability of the blood individually enough to determine each case with sufficient certainty to identify the difficulties in the To solve dosage. There is therefore an urgent need for a method of determination that allows precise control makes possible.

From the following statement of the invention it is apparent how the present problem with the help of a special reagent developed for this purpose is dissolved in a satisfactory manner.

In order to make the invention easier to understand, a brief overview of the mechanism of action should first be given the anticoagulants are given.

Blood coagulation depends on two partially separate coagulation systems, the external and the internal system. The peroral appli-

Reagent to control the coagulability of the blood during anticoagulant therapy and methods of making the reagent

Applicant:

Dr. med. Paul Arnor Owren,

Blommenholm, Baerum (Norway)

Representative:

Dr. F. Zumstein,

Dipl.-Chem. Dr. rer. nat. E. Assmann and Dipl.-Chem. Dr. R. Koenigsberger, Patent Attorneys, Munich 2, Bräuhausstr. 4th

Named as inventor:

Dr. med. Paul Arnor Owren,

Blommenholm, Baerum (Norway)

Claimed priority:

Norway dated May 16, 1958 (128 104)

anticoagulants reduce the following blood coagulation factors: prothrombin, Stuart factor, proconvertin and antihaemophilia B factor. Of these factors, they are prothrombin and the Stuart factor involved in both systems. There are also on both systems Proaccelerin and fibrinogen involved. Proconvertin is only in the external system and the antihaemophilia B-factor only effective in the internal system. This shows the following scheme:

Systems of blood clotting

The "internal" system ("Kephalin period")

Blood platelet factor 3 ("Kephalin") antihaemophilia A factor (A. H. G.) Antihaemophilia B factor (P. T. C.) Antihaemophilia C factor (P. T. A.) "Hageman" factor

"Stuart" factor protombin

The "external" system
("Thromboplastin Time")

Tissue thromboplastin
Proconvertin

Proaccelerin

Fibrinogen

So far there has been no viable method that would allow the full extent of the reduction in four important factors for the whole coagulation process to be recorded quantitatively, i.e. both in the internal as well as external system.

509 520/34 *

The most common methods so far, namely the Quick method and the so-called PP method (Prothrombin-Proconvertin-Method), only show changes in the external system. You wise therefore, when used to monitor anticoagulant treatment, only one Decrease in proconvertin, Stuart factor and prothrombin after. A decrease in antihaemophilia B-factor is not recorded at all by Quick's method and only to a very small extent with the PP method.

The reagent according to the invention for controlling the coagulability of the blood during anticoagulant therapy is characterized in that it is a thromboplastin with weak but constant Activity in combination with a Kepha-Hn or Kephalin substituent with very high activity to human plasma.

The reagent preferably also contains bovine plasma, from which prothrombin, Stuart factor, Proconvertin and antihaemophilia B-factor are removed. Furthermore, it advantageously also contains coagulation factors caused by the oral anticoagulants not be diminished, in great concentration. The reagent also preferably contains calcium chloride in an amount that after redissolution of the reagent and addition of the test plasma one ensures optimal concentration for coagulation.

The procedure for preparing the reagent according to the invention is as follows: The thromboplastin prepared in a manner known per se is mixed with the cephalin suspension, likewise prepared in a manner known per se, and prothrombin, Stuart factor, proconvertin and antihaemophilia B factor are produced from bovine plasma repeated absorption of insoluble heavy metal salts, especially barium sulfate, and subsequent separation of the salt by filtration or centrifugation removed, the resulting depleted plasma solution is dialyzed, and the dialyzed plasma solution is added to the thromboplastin-cephalin mixture, the pH of the mixture is adjusted to 6 adjusted to 8, and finally the CaCl _{2 is} added.

The fundamental problem with making the reagent is that it is a mixture of enzymatically active proteins is involved in their separation and purification from the accompanying ones Proteins everything must be avoided that could lead to denaturation. Isolating and purifying a protein is almost always an empirical task since the known fractionation methods, such as. B. fractionation with salts, especially ammonium sulfate, precipitation with organic solvents such as alcohol and acetone, absorption on cellulose, activated charcoal, Aluminum oxide, ion exchangers, etc., all are unspecific and their appropriate composition and succession is unpredictable. Another requirement is a corresponding powerful test that checks the results of the separation processes used can be.

The production of the thromboplastin and the cephalin suspension is based on known methods older processes, which also go back to the inventor. The production of an enzymatic active and stable bovine plasma, from prothrombin, Stuart factor, proconvertin and antihaemophilia B factor were removed, succeeded for the first time, surprisingly the repeated absorption of insoluble heavy metal salts to Goal led. Heavy metal ions are known to be strong enzyme poisons and usually also have in Traces result in inactivation of the proteins, so that usually the solutions so-called Heavy metal scavenger, such as B. complexing agents are added to odei in trace amounts from vessels to remove leached ions from other devices. The following procedural steps were also by no means obvious, since z. B. dialysis also in many cases inactivating the enzyme As a result, it is usually only done carefully and to remove high salt concentrations like them z. B. occur in magnesium sulfate precipitation is applied.

In the drawing, F i g. 1 the sensitivity of the three methods discussed to declining Amounts of antihaemophilia B-factor. The "concentration" of the antihaemophilia B-factor in the Compared to the other factors, the percentage of the norm is plotted on the abscissa. The clotting time is plotted on the ordinate in seconds (20 to 160 seconds).

The study was carried out by mixing antihaemophilia B plasma carried out with normal plasma in various proportions.

Curve A shows the sensitivity of the new method to the reduction of the antihaemophilia B factor.

Curve B shows the sensitivity of the PP method and curve C that of Quick's method to reduce the antihaemophilia B factor.

The differences shown in the curves can be explained by the fact that with the PP method and Quick's method, a very active tissue thromboplastin is added to the plasma or blood will.

With the Quick method one uses a rabbit brain extract and with the PP method one Human brain extract. These very active tissue thromboplastins cause only one Coagulation takes place via the external system, since the coagulation process takes place very quickly here, long before the internal system is fully activated. Therefore, the normal clotting time is Plasma after the addition of active thromboplastin, as with Quick's method, only 12 to 13 seconds. The internal system, on the other hand, needs even after adding an optimal amount of Kepha-Iin at least 50 seconds for the formation of thrombin, which in a further step fibrinogen for coagulation brings. The addition of such active thromboplastin bypasses the internal system fully. Quick's method of coagulation time determination (also »prothrombin time« or “Thromboplastin time”) shows even in the complete absence of the antihaemophilia B factor completely normal values (see Fig. 1).

It should also be noted that there are methods of controlling the internal system, who use cephalin in place of thromboplastin. Depending on their type, these methods can be used for the determination of the various antihaemophilicity factors can be used. Methods of this kind However, for two reasons they were never

used to monitor anticoagulant therapy. First, such "cephalin times" are very sensitive against a number of external influences. Second, they don't talk about change at all in the external coagulation system, with fluctuations in the proconvertin concentration in particular can be very meaningful.

The present invention represents a method which makes it possible in one examination at the same time to determine the coagulability of the blood in the internal and external system. With With the help of the new reagent, the coagulation process in the internal and external system is simultaneously in Set in gear, and in such a way that they proceed at approximately the same speed. Through this it is achieved that both systems have approximately the same influence on the coagulation time of the determination system exercise.

Previous methods of monitoring the
Anticoagulant Therapy

As previously reported, two main methods have been used to monitor anticoagulant therapy used:

I. Quick's method.
II. The PP method.

I. Quick's method
(AJ Quick: Biol. Chem. J., 109, LXXIII [1935])

Quick's method was originally used as a method for determining prothrombin in blood has been introduced. The Proconvertin, the Stuart Factor and the Proaccelerin were still there at the time unknown. With this method, the concentrations of calcium and thromboplastin become constant held.

This method has undergone minor changes.

The execution is as follows in brief.

Reagents:

1. Thromboplastin suspension: acetone-dried rabbit brain extract.

2. Calcium chloride solution 0.01 molar.

Determination: 0.1 ml of oxalate plasma is mixed with 0.1 ml of thromboplastin solution and placed in a water bath held at 37 ° C. After adding 0.1 ml of a 0.01 molar calcium chloride solution, the clotting time or “thromboplastin time” (also called “Quick prothrombin time”).

Assessment: The method only covers the external coagulation system (see scheme). As already stated, the internal coagulation system has no influence on this determination.

Only the thromboplastin and calcium are kept constant in Quick's method. the Change in any of the other factors in the external system: prothrombin, proconvertin, Stuart factor, Proaccelerin and fibrinogen, would therefore the described Quick's "thromboplastin time" can influence. It is believed that during treatment with anticoagulants, proaccelerin and fibrinogen remain largely constant. Any extension of Quick's "thromboplastin time" is thus aimed at a reduction in proconvertin, Stuart factor and / or prothrombin be due. On the other hand, there will be a decrease in antihaemophilia B-factor not detected at all with this method. In hemophilia B, Quick's "thromboplastin time" is normal (see Fig. 1). This method is therefore used to control the dosage in the course If used in anticoagulant therapy, there is a risk of bleeding complications as a result

ίο a particularly strong reduction in the antihaemophilia B factor, since such a change cannot be detected with the help of this method. With the quantitative evidence of the various factors involved in specific determination systems has been found to be the antihaemophilia B-factor is often much more diminished than any other factor. This can be seen from the F i g. 2 see. These results apply to phenylindandione, but dikumarol and its derivatives basically have the same effect.

On the abscissa, the numbers 8-30 indicate the date in March 1957 when these investigations were carried out became. The investigations were completed on April 2, 1957.

On the ordinate is the percentage (this is not absolute, 100% means: normal »Concentration«) of prothrombin, proconvertin and antihaemophilia B-factor, which is related to the Days found in the blood.

Curve D shows the percentage of prothrombin in the blood; Curve E shows the percentage of proconvertin and curve F the percentage of antihaemophilia B-factor.

As can be seen from this, the level of the three factors mentioned was approximately 100 ^fl / o at the start of the investigation. On March 9, 1957, the administration of the anticoagulant phenylindandione was started. The daily dose is recorded in the upper part of the figures. The vertical line to the left of this portion means 100 mg of phenylindanediones. The size of each daily dose, plotted down on the vertical line, appears at the level of the relevant date recorded on the abscissa. On March 25, the administration of the anticoagulant was stopped. The percentage of the factors then increased rapidly.

It is believed that the fact that Quick's method is insensitive to unity Decrease in antihaemophilia B-factor is one of the causes of the relatively common bleeding complications when using Quick's method Control used for anticoagulant therapy is.

Among other disadvantages of this method it should be emphasized that it is insensitive against changes in all three factors involved, provided that these are in the range of 50 to 100% move of the normal is. Furthermore, the calcium concentration used is when the hematocrit value of the blood is not normal, not optimal. In addition, the "thromboplastin time" is through the Affects the pro-accelerin concentration in the test plasma. The Proaccelerin is unstable and becomes during the Storage of oxalate plasma affected. The Proaccelerin is unstable and becomes during storage inactivated by oxalate plasma. Because of this, they are relatively fresh for this method Blood samples required.

II. PP method (Prothrombin proconvertin method)

Reagents:

1. Thromboplastin suspension: shemales extract.

Watery men

2. Bovine plasma absorbed.

3. Calcium chloride solution in optimal concentration (normally 0.025 molar).

Execution: 0.2 ml of absorbed bovine plasma are mixed with 0.2 ml of thromboplastin suspension and 0.2 ml of a 1:10 dilution of the test plasma. This incubation mixture is kept at 37 ° C. in a water bath. After adding 0.2 ml of calcium chloride solution, the clotting time is measured.

Assessment: As in the case of Quick's method, the PP method is a determination of the thromboplastin time, ie it is primarily a method for determining the activity in the external coagulation system. The main differences between the PP method and Quick's method are as follows:

1. A new reagent, absorbed bovine plasma, is introduced to stabilize proaccelerin and fibrinogeal concentrations. The determination is therefore independent of the concentration of these factors in the test plasma. Since the factors that are reduced in the course of anticoagulant treatment are relatively stable during storage of blood or plasma, this method can also be used with stored blood samples .

2. The test plasma is diluted 1:10. This increases the sensitivity of the method, especially in the range between 50 and 100%. The dilution of the test plasma also has the advantage that with different hematocrit values the risk of an inoptimal calcium concentration is far lower than with Quick's method . The dilution also eliminates the effects of small amounts of heparin on the test plasma.

Like Quick's method , the PP method is also sensitive to reductions in prothrombin, Stuart factor and proconvertin.

The thromboplastin (watery human brain extract) used in the PP method has a cephalic effect on the internal coagulation system in addition to its thromboplastic effect. However, this effect is not optimal. As a result of this cephalic effect and the test plasma dilution , it can be demonstrated that the PP method has a certain, albeit extremely low, sensitivity to a strong reduction in the antihaemophilia B factor if the external coagulation system is already largely disturbed in the course of an anticoagulant treatment (see Fig. 1). This sensitivity of the PP method to a reduction in antihaemophilia B-factor is, from the standpoint of clinical practice, far too low to offer reliable protection against bleeding complications as a result of strong reductions in antihaemophilia B-factor.

Over time, other modifications of Quick's method have been recommended, e.g. B. the application other thromboplastin preparations - from. Rabbit lung or human brain, some in acetone-dried form, and others in lyophilized form. Test plasma dilutions with the intention of It was also possible to increase the sensitivity of the method, especially in the range from 50 to 100% to use. The change in calcium concentration in relation to the hematocrit value of the Blood has also been recommended to ensure optimal recalcification. However, has not these modifications changed the principle of the method, and none was able to, the lack of sensitivity the method towards a reduction in the antihaemophilia B factor.

The reagents for the PP method - the absorbed bovine plasma and the thromboplastin suspensions - were also produced in lyophilized form. Of course, this change had no effect the lack of sensitivity of the method to a reduction in the antihaemophilia B factor the end.

Method of using the reagent according to the invention for monitoring the coagulability of the blood during anticoagulant therapy

From the above description it can be seen that none of the previously available tools is a represents satisfactory control in anticoagulant therapy. The preparation according to the invention allows, however, a satisfactory control of this therapy.

This process is based on an entirely new principle, namely that the determination of the Activity in the internal and external coagulation system carried out simultaneously and in combination will. So far there has been no method for such a combined determination. The two coagulation systems were always examined separately. The method is based on a reagent that both coagulation systems at the same time and in such a way that both use almost the same amount of time. In order to meet this condition, the internal coagulation system were optimal conditions for greatest speed given by kephalin from human brain or other cephalins, which have the same activity, to which reagent has been added at an optimal concentration. Under these Conditions alone, the internal coagulation system becomes complete in about 50 seconds Lead to coagulation.

At the same time, the reagent contains a thromboplastin, which activates the external coagulation system puts. This thromboplastin is of such a nature that in optimal concentration there is a low and a has constant activity. This has the consequence that the speed in the external coagulation system compared to the normal rate of active thromboplastin types such as those in Quick's and PP method are used, is significantly reduced. The thromboplastin, that is used in this method should be in optimal concentration when using give normal plasma a thromboplastin time of 35 to 50 seconds.

Theoretically, such a weak thromboplastin could be produced by diluting an active thrombo-

9 10

plastins (such as it is for the Quick's and PP indexes. The decisive difference compared to

Method is used). In such earlier mechodes, as already described,

chem procedure, however, one obtains an unstable and the sensitivity to the new method

practically useless reagent, since small changes - a decrease in antihaemophilia B-factor.

runes vary considerably in their activity. , τ.

cause genes in the timekeeping. In the invention composition of the reagent

According to the reagent there is therefore a thromboplastin The new reagent has the following composition

for the application that has a stable and lower settlement:

Has activity at optimal concentration and 1. Thromboplastin. This thromboplastin has in

a relatively wide range of optimal concentration io optimal concentration low activity and

owns. shows a thromboplastin time with normal plasma

As a starting material for the production of a sol- from 35 to 50 seconds. This thromboplastin

Chen thromboplastins are made of certain animal organs, because of their animal organs

types used. The thromboplastin produced is species-specific only slowly with human procon-

species-specific and only reacts slowly with humans. Examples are: cattle,

lichem proconvertin. Thromboplastins, produced horse and pig brain.

from cattle, pig and horse brains have this 2nd cephalin. This cephalin is charac-

Characteristic. terized that it is intense with human plasma

This combination in one reagent: a throme reacts. It can be made from human brain

boplastin with weak human activity. Also other kephalin-lecithin preparations that

human plasma, together with a human, can react intensively with human plasma

Kephalin (or cephalin substituents), both of which can be used in opti-, e.g. B. a cephalin preparation from

painter concentration, to mix, the result is that soybeans.

the internal and external coagulation system nearly 3. Absorbed ox plasma. This is in the

exert the same influence on the clotting time. 25 type manufactured so that there is a high and constant

Hence, this method is both sensitive to the amount of all of the coagulation factors caused by

about a decrease in antihaemophilia-B- did not change the anticoagulant treatment

Factor in the internal system as well as being opposite to one, while at the same time it is free from the

Reduction of the proconvertin in the external system. the following four factors prothrombin, Stuart factor,

Also, of course, the Stuart factor and 30 show proconvertin and antihaemophilia B factor's.

the prothrombin, which intervene in both systems, 4. calcium chloride. Calcium chloride is the reagent

their effect. added in an amount after the addition of the

Fig. 1 shows the clear sensitivity of this test plasma an optimal concentration for the

Method towards a large reduction in coagulation.

Antihaemophilia B-Factor, in contrast to the 35 All of the four proportions listed are in one

the very poor sensitivity of Quick's reagent. This reagent is so stable that

Method and the very weak sensitivity it even in solution after 24 hours of storage

the PP method. It follows from this that the basic room temperature shows unchanged activity.

Inadequacies of the previously used Me- This stability is achieved, among other things, by

methods and reagents in all essential points 40 that the abovementioned four coagulation

th were abolished. to completely eliminate factors. Of course it was

The invention is also based on the recognition - earlier possible, thromboplastin and calcium in

nis that the determined coagulation times can be combined independently of a reagent. However, it wasn't

gig of changes in all of the coagulation factors- possible after adding calcium to an absor-

ren which did not breathe plasma in or without the presence of thrombus during anticoagulant treatment

should be affected. That will allow boplastin to get this as a stable reagent,

borne by all of these factors in high and this "all-in-one" reagent is ampouled

constant amount included in the reagent are brought in lyophilized form for distribution. the

the. For this purpose, a carefully absorbed ox- examinations clearly showed the optimal loading

plasma, similar to the PP method. 50 conditions for the stability of the dried preparation

This absorbed bovine plasma contains great rates, particularly as regards the ionic strength, which

Amount of anti-haemophilia A factor, anti-haemo- concentration of anticoagulants, etc.

Philie C factor and Hageman factor (from the in- The lyophilized reagent is used for the capillary

internal system) and Proaccelerin and Fibrinogen (the method or the venous blood method in distilled

act on both systems). Furthermore, it is dissolved in water or a calcium chloride solution in such a way that

processed so that there are no detectable amounts of stability: After storing the ampoules over

Proconvertin, antihaemophilia B factor, Stuart- 30 days at 47 ° C showed the reagent unchanged

Factor and prothrombin (just the four during activity.

anticoagulant treatment reduced fac-. _, _

gates). 60 Preparation of the reagent

It follows that the reagent is specific for and.

sensitive to all the four new reagents belonging to a manure.

Anticoagulant treatment reduced factors Thromboplastin: Bovine brain was used because,

is. At the same time, changes in the cattle brain thromboder, as already described above Coagulation factors not affected. The 65 plastin apply sluggishly with human proconvertin

The coagulation times achieved by this method react because of their species specificity. Beef brain extract

len therefore gives one of the full effects of the anti- in optimal concentration with normal

coagulants on blood clotting equivalent human plasma a thromboplastin time of

approximately 35 seconds. It is prepared in the same way as the human brain thromboplastin (P.A. Owren, J. Elin. & Lab. Invest., I, p. 81 [1949]).

"Raw" cephalin suspension: This is made according to the recently published method Human brain produced (P. Hjort, S. I. Rapaport and P. A. Owren, J. Lab. Elin. Medicine, 46, Pp. 89 to 97 [1955]).

The amount of thromboplastin and cephalin (or cephalin substituents) is coordinated in such a way that that their concentration in the final reagent is optimal.

Absorbed ox plasma

1. Blood and plasma are treated in such a way that the absorbed plasma, in addition to the antihaemophilia A factor, Hageman factor, proaccelerin and fibrinogen have high concentrations of antihaemophilia C factor contains in non-activated form. The decisive factor of this circumstance was used in the past not understood.

2. Absorption is carried out with barium sulfate, in such an amount and by repeated use Absorption that the substrate is completely free of prothrombin, Stuart factor, proconvertin and antihaemophilia B factor is.

3. Extra care must be taken to completely remove the suspended barium sulfate centrifuged.

4. As a new principle, dialysis is used to prepare this reagent to achieve the following:

a) Removal of the plasmatic carbonic acid-bicarbonate buffer system. This is for precise adjustment and stabilization of the pH in the final, dried preparation necessary.

b) Removal of the oxalate. This is necessary firstly for the stability of the Proaccelerin, secondly to the precipitation of calcium oxalate with the associated absorption of coagulation factors during the execution of the test, and thirdly, to prevent the use of citrate test plasma during the determinations to allow.

5. After dialysis, the pH is carefully adjusted (to a value between pH 6 and 8 in order to achieve a to achieve an optimal and stable pH after the redissolution of the dried preparation. this has decisive influence on the constant quality of the reagent and on the solubility of the fibrinogen? at the redissolution).

Thromboplastin, cephalin (or cephalin substituent) and calcium are absorbed by ox plasma added in a concentration that is optimal for the coagulation process taking place in the test.

The preparation thus obtained is freeze-dried.

Instructions for use

When using the preparation to control the coagulability of the blood, the same procedure is followed in the following way:

After dissolving the reagent in distilled water or a calcium chloride solution (see above) 0.5 ml of it with the help of a pipette into small test tubes (diameter 8 to 9 mm, length 60 mm). A measured amount of the plasma or blood to be tested (see below) is added and the clotting time is measured with a stopwatch. The time received brings the Effect of anticoagulant treatment on the coagulation process expresses itself. The determination is carried out in a water bath at 37 ° C.

ίο The coagulation time obtained is based on the percentage transferred expressed relative activity on the basis of a correlation curve. Examples of correlation curves are given in FIG. 3. These correlation curves show the ratio of the achieved Coagulation time in the two systems for coagulation activity in percent.

On logarithmic paper, the concentration of normal plasma is shown in percent on the Abscissa and the clotting time in seconds

ao of the ordinate. The curve K shows the capillary blood method which uses an amount of 0.10 ml. Curve H shows the method used for citrated whole blood, amount 0.10 ml, and for citrated plasma in the following dilution: 3 parts of plasma to 2 parts

As physiological saline solution, amount 0.10 ml. Curve G shows the method for citrated plasma in a dilution of 1: 5 and with a plasma amount of 0.20 ml.

The method can be used for capillary blood (whole blood), which does not contain any added anticoagulants, as well as used for citrated blood or citrated plasma will. The reagent is provided with an optimal calcium concentration for capillary blood and can therefore not easily be used for citrated blood or citrated plasma, since the calcium concentration for this is not optimal.

When using citrated blood or citrated plasma, the dried reagent must therefore with a 3.2 mmol calcium chloride solution, which is contained in the to the amount of citrate in the blood sample to be tested is compensated for.

1. Capillary blood. A skin incision is made in the fingertip or earlobe, which is sufficient Blood leakage guaranteed. 0.10 ml of the whole blood are drawn into a pipette and immediately mixed with 0.5 ml of the reagent already stored in a water bath at 37 ° C. the The clotting time is then determined.

2. Citrated blood. Venous blood is made with 3.13% sodium citrate dihydrate solution (in a ratio of 1 part sodium citrate solution to 9 parts blood). Example: 0.2 ml of sodium citrate solution is poured into a 2 ml syringe raised. From a venipuncture, the syringe is filled with blood up to an amount of 2 ml filled up. The blood sample is then filled into a test tube, 0.10 ml of citrated blood are made with the aid removed from a pipette and mixed with 0.5 ml of reagent. The clotting time is described in the above Way determined.

3. Citrated plasma. Citrated blood is drawn in the same way as in point 2. After this Centrifugation, 0.6 ml citrated plasma are transferred to a test tube, 0.4 ml physiological Saline solution added and both mixed.

With the help of a pipette, 0.10 ml of the diluted plasma are removed and immediately with 0.5 ml des Reagent mixed. The clotting time is determined in the manner indicated above.

The new »all-in-one KK reagent according to the Invention makes it possible to carry out a combined determination of the coagulation activity of both the internal as well as the external coagulation system.

The preparation of the reagent is based on principles not previously applied.

The reagent exhibits a number of advantages over previous reagents:

1. It is very stable at room temperature and thus simplifies distribution and storage.

2. It can withstand heating up to 47 ° C for at least 30 days and can therefore also be used in tropical areas Regions are used.

3. Since only one reagent is required, the production and the execution are simplified of the test is faster than previous methods.

20th

4. It is the only previously known method that uses oral anticoagulants caused coagulation defect can be detected in a sufficiently reliable manner. In contrast to earlier methods show this against a decrease in the antihaemophilia B-factor sensitive.

5. As a result of the special features described above, this method grants a far greater one Protection against bleeding complications than other methods.

6. the method is time-saving.

7. shows it due to the stability of the reagent

reliable and reproducible results in redissolved form.

Claims (6)

Patent claims: 1. Reagent to control the coagulability of the blood during anticoagulant therapy, characterized in that it is a thromboplastin with weak but constant Activity in combination with a cephalin or cephalin substituent with very large Contains activity towards human plasma. 2. Reagent according to Claim 1, characterized in that it is also bovine plasma contains, from which prothrombin, Stuart factor, proconvertin and antihaemophilia B factor are removed are. 3. Reagent according to claim 2, characterized in that the bovine plasma used all coagulation factors that are not reduced by the oral anticoagulants in contains great concentration. 4. Reagent according to one of the preceding claims, characterized in that it is calcium chloride in an amount that will be obtained after the reagent has been redissolved and the test plasma has been added ensures an optimal concentration for coagulation. 5. Process for the preparation of the reagent according to one of the preceding claims, characterized in that the thromboplastin prepared in a manner known per se is mixed with the cephalin suspension also prepared in a manner known per se, and that prothrombin, Stuart factor and proconvertin are also prepared from bovine plasma and antihaemophilia B-factor removed by repeated absorption of insoluble heavy metal salts, especially barium sulfate, and subsequent separation of the salt by filtration or centrifugation, the depleted plasma solution obtained is dialyzed and the dialyzed plasma solution is added to the thromboplastin-cephalin mixture, the pH of the Adjust the mixture to 6 to 8 and finally add the CaCl ₂ . 6. The method according to claim 5, characterized in that the product obtained is lyophilized will. 1 sheet of drawings