DE1943882A1 - Method for the quantitative determination of the fibrinolytic activity in blood plasma and its fractions - Google Patents

Description

dr. W.Schalk dipl.-ing. P. Wirth · dipl.-inc.G. Dannenberg

DR. V. SCHMIED-KOWARZI K · DR. P. WEINHOLD · DR. D. GUDEL

■ - * '6 FRANKFURT AM MAIN

OR. CSCHCNHIIMH STRASf * »9

SK / SK

Baxter Laboratories, Inc. Morton Grove. Ill, 6θ 053 / USA

Method for the quantitative determination of the fibrinolytic activity in blood plasma and its fractions

The present invention relates to a method for determining the fibrinolytic activity, in particular a method for the quantitative determination of the fibrinolytic activity in the blood plasma and fractions of the same

The clotting of the blood is of crucial biological importance It requires a complicated mechanism and depends on the presence and effectiveness of a number of plasma proteins. Of these proteins fibrinogen is of primary importance because the fundamental characteristic of the The coagulation mechanism is the conversion of a fibrinogen solution into a hard, insoluble fibrin clot. This conversion is usually done by mutual action of fibrinogen and thrombin · Thrombin is of its precursor, prothrombin. Conversion of prothrombin to thrombin usually requires the presence of calcium ions and a member of an activating agent known as thromboplastins

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The fibrin clot dissolves over a period of time as a result of an enzymic action. The enzyme responsible for this effect is called Called fibrinolysin (or plasmin). This is proteolytic enzyme in normal blood in the form of its inert precursor, profibrinolysine (or plasminogens) present. The plasminogen can be removed by using certain reagents, such as chloroform, streptokinase, urokinase and others .. such plasminogen activators activated or made proteolytically will. These activators cause the conversion of plasminogen into Plasmin, which attacks the coagulation proteins in the fibrin clot. the inhibitors normally present with plasminogen in the blood delay this Reaction.

The term "fibrinolytic system" used here means that Method of dissolution of the fibrin clot, and the term "fibrinolytic activity" is intended to include the chemical and physical properties described above Include efficacies that come into consideration in this dissolution.

The known methods for determining fibrinolytic activity

usually used the so-called "wet system" reaction. E.g.

by Astrup et al., Arch.Biochem. andBiophys., Vol. * K), pages 346-351 (1952), a wet fibrin plate system for determining piasmin activity in the blood described.

The aim of the present invention is to create a new and improved method for determining the fihrinolytic activity in the blood plasma and fractions of the same.

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Another object of the present invention is to provide - a new method for the quantitative determination of the various "Components of the fibrinolytic system

According to the invention, a stable gel is used as the reaction medium for the fibrinolytic system for the quantitative determination of the fibrinolytic effectiveness created · A fibrinolytic component of the fibrinolytic system from the coagulated fibrinogen is homogeneously suspended in the gel and with an unknown sample of blood plasma or a fraction thereof, the other contains essential components of the fibrinolytic system »reacted for a predetermined time, creating an opaque radial Diffusion zone of the reaction product at the interface of the unknown Sample and gel is formed ". The diameter of the radial diffusion cone is directly proportional to the logarithm of the fibrinolytic Activity in the sample to be tested. The fibrinolytic activity in one unknown sample is determined by comparison with control samples from known fibrinolytic activity.

The term "stable" gel as used herein refers to a gel that is inert or chemically non-reactive with the components of the fibrinolytic system suspended in the gel.

In the preparation of the gel mediuras, about 1.0-10.0% by weight of a gel extractant are dissolved in a heated buffer system and then with a predetermined amount of a coagulated fibrinogen component of the fibrinolytic Systems mixed as described above · Generally about 10 mg- $ to about 2 g- #, preferably about 130 mg- #, of fibrinogen is used in the heated buffer system. This mixture is in a hot state in a flat Low-brimmed container (hereinafter referred to as "plate") in a poured in the amount corresponding to the size of the container. The mixture will gel

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and in the gel cylindrical holes with a diameter of about 1-10 mm are punched out or otherwise formed

Known or unknown samples of blood plasma or a fraction of the same, which contains other essential components of the fibrinolytic system, are then introduced into the open holes in the gel by using a capillary pipette. or similar device and the plate is incubated for a predetermined period at about 20-60 ° C, preferably about 37 ° C. During this incubation, a fibrinolytic reaction occurs when the material in the holes diffuses into the gel medium and becomes opaque forms radial diffusion zone of the reaction product.

After the incubation or incubation period, the gel is examined visually, preferably with an illuminated magnifying glass with a sun scale. Measuring the diameter of the radial diffusion zone the fibrinolytic components were formed in the known or unknown samples, makes the fibrinolytic activity in the unknown sample easy to determine

In the preparation of the gel medium, any conventional gelling agent can be used, such as gelatin, pectin, silica gel, starch, polysaccharides from seaweed, such as agar, algin and carrageenan in, synthetic pafemere gelling agents, such as the crosslinked polyacrylamide of the US patent 3 0k6 201 and similar materials. The gelling agent preferably has the physical properties of agar-agar in that it is easily dispersible in water and can form a practically clear hydrogel of sufficient hardness,

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so that the receptacle or plate containing the gel is turned upside down without the risk of the gel falling out

The gelling agent preferred according to the invention is a purified agarose. · 'Agarose is the neutral galactose polymer which is usually obtained from the ■ Agaropectin fraction has been separated from agar (see e.g. the procedures U.S. Patents 3,281,409, 3,335,127 and 3,362,884). This yeast. Medium is preferably used at a concentration of about 2.5% by weight of the gel medium used.

The buffer system commonly used in the gel has a pH of about 6.0 to about 8.5 * preferably about 7.3 * A preferred buffer consists of 0.15 ^. M sodium chloride, 0.04 x M imidazole, and 0.02 M Ethylene diamine tetraacetate. Other buffers that can be used are e.g. tris (hydroxymethyl) aminoraethane, Phosphate and barbital buffers.

For making a plate containing the gel with clotted fibrinogen a suitable clot can be made by solidifying the the The plate containing the gel and fibrinogen is immersed in a thrombin solution for a sufficient time, e.g., about 0.1-15 minutes. Then this is it Gel containing coagulated fibrinogen is prepared in such a way that holes can be punched into it in the manner described above.

A protective membrane, placed and packaged in various ways, leaving the plate ready for subsequent use for determination of fibrinolytic activity is available from hospitals, laboratories, etc., which provide a simplified but accurate determination of fibrinolytic activity in blood plasma samples to need. The packaging for these plates can, for example, be made of plastic

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film or metal foil bags, sacks, etc. are made, which are preferably sterilized and are sealed to prevent the ingress of air, moisture, dirt and other contaminants. Suitable metal foils can be made of, for example, aluminum, etc .; Suitable plastic films can be produced e.g. from vinyl chloride and vinylidene chloride polymers and copolymers, polyvinyl chloride, polyvinyl alcohol, Polyethylene, polypropylene, polystyrene, polycarbonates, polyamides, cellulose acetate and propionate, cellulose triacetate, cellulose acetate butyrate, ethyl po Iycellulose, / fluorocarbons, acrylic plastics such as acrylates and Methacrylates and polyesters, such as the polyesters from the condensation between ethylene glycol and terephthalic acid

The coagulated fibrin plates can also be used in combination with control samples, Buffer materials, capillary tubes and other components for implementation the entire fibrinolytic examination.

According to two preferred embodiments of the present invention are the fibrinolytic component in the gel medium and that in the unknown samples fibrinolytic activity to be determined as follows:

Plate I. Coagulated fibrinogen is suspended in the gel and determinations are made for (a) total plasminogen, (b) available plasmin, (c) active plasmin and (d) fibrinolytic inhibitor in the unknown sample

Plate II . Coagulated fibrinogen and plasminogen are suspended in the gel,

t. and there is a determination for plasminogen activator in the unknown

Sample·

In plate I, a highly purified fibrinogen is used as the fibrinolytic component in the gel

CB - -

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In Plate II, the fibrinolytic component comprises fibrinogen and plasminogen. This component can be prepared using a with a known amount of fibrinogen contaminated with plasminogen or by Use of a highly purified egg brinogen as in Plate I, the a predetermined amount of plasminogen is addedWhen using highly purified fibrinogen, the plasminogen is preferably reduced to a final concentration of about 0.1-20 units / ccm, in particular about 1 unit / ccm, added. The term "1 unit / com plasminogen" used here defines the amount of plasminogen in a combined amount of plasma.

The following examples illustrate the present invention without restricting it. Unless otherwise stated, all parts and percentages are parts by weight and 5 tons by weight. example 1

An ararose gel plate was prepared as follows: preparation of reagents;

A. Agarose gel plate buffer: 0.15 ** M sodium chloride, 0.0 ¹ H * imidazole, and 0.02 M ethylenediaminetetraacetate; pH = 7.30s

Bitrated glycine salt solution: 0.123 M sodium chloride, 0.020 M sodium

citrate and 0.1 M glycine.

C. Throebine Solution - Parke-Davis Topical Thrombin .from cattle; 5000 units / The ampoule was filled with cold distilled water. Water diluted to 50 units / cc, the The pH was adjusted to 5.30 with cold 1.0 ON acetic acid, the same as the one obtained The precipitate is centrifuged in a cooled centrifuge and the supernatant solution is decanted and lyolphilized. Before use, the lyophilized product was again made up to its original volume with sterile distilled water brought·

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Procedure ; ' _t A highly purified fibrinogen solution was prepared as follows *

combined human plasma was frozen and then slowly reduced to ^ f ⁰ C.

thawed. The cryogenic bottom was removed by centrifugation and

dissolved in l / lO of the original plasma volume of the saline-glycine-citrate solution. The pH of the solution was adjusted to 6.5 with 1, ON acetic acid. To the above solution, then slowly 3.5 & been - $ ^ OCQ polyethylene glycol (molecular weight about 4000) is added; precipitation was carried out by stirring for 15 minutes at room temperature. The obtained precipitate became centrifuged off and dissolved in the above saline-glycine-citrate solution (1/10 of the original PlasraaYoiutaens). The pH of the solution "" was adjusted to 6.88 with i.ON sodium hydride, whereupon the solution was cooled to 9 ° C. Glycine was slowly added to a final glycine concentration of 1.8 M to the solution Precipitation was carried out by stirring for 45 minutes at 5 ° C. The resulting precipitate was centrifuged off and dissolved in normal physiological saline to give a concentration of about 2.5 g fibrinogen Darco G-60 charcoal treated. each adsorption successes for 30 minutes at Zimmertenperatur, wherein the solid charcoal in> .- any method was .entfernt by centrifugation. the final solution was prepared by appropriate dilution with normal saline at 260 ng $ fibrinogen set and stored in the frozen state.

An agarose solution was prepared as follows: 2.5 g of agarose mouth full. The agarose gel plate buffer described above is constantly dissolved

Then, a congealed fibrous sheet was made as follows '.'

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- ■ · The agarose solution prepared above was brought to 53-56 ⁰ C., and 1 volume. the solution was mixed homogeneously with 1 volume of the above-described, highly purified fibrinogen solution brought to room temperature. ··; The mixture was then poured into a plate about 7 »5 * 2.5 cm and 6 mm deep and allowed to solidify. Then the plate with the solidified gel was immersed in the above-described thrombin solution for an appropriate period (about 30 seconds) and removed. The plate was correspondingly long (about 30 seconds) in a bath of dist. Submerged in water and then removed. Then 6 holes each having a diameter of 2 mm were punched in the gel in the coagulated plate at the same distance from one another. Then the plate was provided with a protective membrane, packed and stored at 2-8 ⁰ C..

The clotted fibrin plate was used with plasma or equivalent plasma formulations to make the following quantitative determinations:. Test I. Total plasminogen was determined in a streptokinase-activated euglobulin fraction of the plasma.

Test II . Available plasmin was determined in a streptokinase-activated plasma.
Test III . Active plasmin was determined in the plasma.

The above three determinations were made according to the following procedure:

I «Total plasminogen test . ·. ·

1. Preparation of a fraction activated with streptokinase (SK-E fraction). The citrated plasma of the patient was with dist. Water diluted from 5 ^° C. to 1:18, the pH value was adjusted to 5.3 with 1, ON acetic acid, after which it was centrifuged for 20 minutes in a cooled centrifuge. The precipitate was diluted again to the original plasma volume with sufficient A ^ aroe gel plate buffer, whereupon streptokins were added to this euglobulin fraction in a ratio of 0.1 ecm to 1.0 ecm.

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2. An activated euglobulin control sample was prepared by addition of 1.0 ecm of euglobulin control with known plasainogen activity to 0.1 ecm streptokinase.

3 · To a total of 6 test tubes, the following amounts of agaric sailplate buffer were added admitted:

Pipe flr. ecm agarose gel plate buffer

1 no

~ 2 0.25

3 0.4

V no

5 0.25

6 ', 0.4

4. The SK-E fraction prepared above, the activated euglobulin control and the 6 tubes of agaro flattening buffer were placed at 37 ° C for 10 minutes incubated.

5 · The following amounts of incubated SK-E fraction and activated euglobulin control added and well mixed:

Pipe no, SK-E fraction; ecm activated euglobulin control; ecm

1 0.5 -

2 0.25 -

3 0.1 -

4 - 0.5

5 - o ', 25

6 - 0.1

6 · The clotted fibrin plate was opened and the protective membrane from the Agar surface removed and discarded

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7. Three holes of the clotted fibrin plate were made with the dilutions activated euglobulin control and agarose plate buffer and the three * remaining holes with the dilutions from SK-E fraction and agarose gel plates Buffer Filled · The tip of each capillary tube was brought to the bottom of the hole, whereupon the solution was allowed to flow in by gravity «to • ie filled the hole to the level of the agar surface.

■ 8 · The cover of the plate was put back on and the plate, preferably in a moisture bottle, for about 2.5 hours or until clear Reaction probes for all control dilutions appear incubated

9 · Dam a traction curve was produced in the manner described below ·

Jl * Test for available plasmin

1 · To produce activated plasma, 1.0 ecm of the plasma was dated

0.1 ecm streptokinase added to patients

2 · The procedure was then as in step 2-7 of total plasminogen test 1.

3 · The cover of the plate was put back on and the plate, preferably in a humidity chamber, for at least k hours or until clear

at 37 ° C. Reaction probes for all activated plasma dilutions appeared / incubated.

4- · Then a traction curve was made in the manner described below.

!!!. Test for active plasmin

1 · To produce the plasma, 1.0 ecm of the patient's plasma was su 0.1 ecm

Agaroe gel plate buffer added

2 · Then the procedure was as in step 2.7 of the oesate plasminogen test I.

3 · The cover of the plate was put on again and .the plate, preferably in a humidity chamber, for at least 10 hours or until sum Clear reaction sonic phenomena for all plasma dilutions at 37%. incubated.

/ f. As shown below, a traction curve was established as soon as it ran.

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Creation of a reference curve . . '"' - ■". ,,.

ί. After incubation of the clotted fibrin plate, the diameter of each reaction zone was measured by placing the plate under a Hyland "Immunoplate" device after removing the cover. After aligning one side of the zone with the zero mark on the grid the diameter "'-" of the reaction zone has been measured to the nearest 0.1 mm. A dissecting microscope with a step micrometer or a wire mesh on the eyepiece can also be used ■ ■ ·. - .. ·.: ..

2. With X "2-CyCIe") semilogarithraic sootf data paper ^:. \

. . . - ■ '* "■' ■■

the diameters of the reaction zones of the three control samples on the horizontal (arithmetic) scale and the percentage activity of the corresponding control plotted on the vertical (logarithmic) scale Then * the best fitting straight line is drawn .. ','

3 · The determination of total plasminogenic, available plasain or active plaenin in the patient's sample takes place in relation to the ί formed by the controls dete line. The value of the fibrinolytic inhibitor can be calculated by dividing the available plasmin from the total plasminogen concentration '

subtracted. '.;' - "

B e i s ρ i el 2 · '' -. :;

As in Example 1, a coagulated fibrin plate was produced »but with ·

a predetermined amount of 1 unit / ecm of plasminogen for extremely high levels. purified fibrind gene solution was added. Then the clotted fibrin plate was used for a quantitative determination for plasminogen activator. The method for this determination was similar for test H and in Example III \ i 1, but the control sample of combined human "euglobulin. ■ '

additionally contained plasminogen activator streptokinae of a known activity, ·.

The plasminogen activator in the test sample was as in Example 1 by Ver,. . "Γ '

equal to ¹ control sample determined ·. '

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Claims (1)

- 13 - Claims Method for the quantitative «« * determination of the fibrinolytic tables. Activity in 'Blood plasma and fractions thereof, marked by the fact that one fibrinolytic component of the fibrinolytic system from coagulated fibri-. nogen with an unknown sample of blood plasma or a fraction thereof, which contains other essential components of the fibrinolytic system, '-in a stable gel medium for a predetermined period of time to form a, translates opaque radial diffusion zone of the reaction product, the Measures the diameter of the radial diffusion zone and compares it with control samples of known fibrinolytic activity. 2 · - Method according to claim 1, characterized in that the gel medium contains about 1.0-10 wt .- # of a gelling agent 3 · - Method according to claim 1 and 2, characterized in that the gelation agent Agarose is k, - Method according to claim 3 »characterized in that the gel medium contains about 2.5 wt .- ^ agarose. .5 · - The method according to claims 1 to 4-, characterized in that the gel medium contains about 10 mg # to 2 g % fibrinogen. 6.- The method according to claim 1 to 5 »characterized in that the gel medium additionally contains a predetermined amount of plasminogen. 7.- The method according to claim 1 to 6, characterized in that the gel medium contains the coagulated fibrinogen on a plate and that the other, Containing essential components of the Jibrinolytic System 009815/1296 .- ■. / i 194383-2 * - The sample is brought into contact with the coagulated fibrinogen by This reaction takes place by diffusion from the holes punched through the gel surface. 8.- A plate for the quantitative determination of the fibrinolytic activity in blood plasma and fractions of the same * consisting of a plate and a, agarose gel medium containing coagulated fibrinogen The patent attorney: 009815/1296