CA2096212A1 - Initiator of the intrinsic coagulationof blood, plasma or blood derivatives, a reagent for determining the aptt of blood, plasma or blood derivatives, a method of producing this reagent and a method of determining the aptt of samples of blood, plasma or blood derivatives - Google Patents
Initiator of the intrinsic coagulationof blood, plasma or blood derivatives, a reagent for determining the aptt of blood, plasma or blood derivatives, a method of producing this reagent and a method of determining the aptt of samples of blood, plasma or blood derivativesInfo
- Publication number
- CA2096212A1 CA2096212A1 CA002096212A CA2096212A CA2096212A1 CA 2096212 A1 CA2096212 A1 CA 2096212A1 CA 002096212 A CA002096212 A CA 002096212A CA 2096212 A CA2096212 A CA 2096212A CA 2096212 A1 CA2096212 A1 CA 2096212A1
- Authority
- CA
- Canada
- Prior art keywords
- reagent
- blood
- plasma
- set forth
- aptt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2405/00—Assays, e.g. immunoassays or enzyme assays, involving lipids
- G01N2405/04—Phospholipids, i.e. phosphoglycerides
Abstract
ABSTRACT OF THE DISCLOSURE
An initiator of the intrinsic coagulation of blood, plasma or blood derivatives is claimed, which is characterised in that it comprises a mixture of sulfatides and a solid activator, preferably kaolin.
Furthermore, a reagent for determining the aPTT of blood or blood derivatives is claimed, which comprises a mixture of sulfatides and a solid activator, preferably kaolin, as the initiator of the intrinsic coagulation, and which furthermore contains phospholipids.
The invention also relates to a reagent for assaying the aPTT of blood, plasma or blood derivatives possibly containing heparin or heparinoid and containing an initiator of the intrinsic coagulation, which is characterised in that it contains a heparin-neutralizing agent and phospholipids. A method of assying the aPTT in such samples as well as the use of this method for assaying the activity of factors of the intrinsic coagulation and the inhibitors thereof is also claimed.
An initiator of the intrinsic coagulation of blood, plasma or blood derivatives is claimed, which is characterised in that it comprises a mixture of sulfatides and a solid activator, preferably kaolin.
Furthermore, a reagent for determining the aPTT of blood or blood derivatives is claimed, which comprises a mixture of sulfatides and a solid activator, preferably kaolin, as the initiator of the intrinsic coagulation, and which furthermore contains phospholipids.
The invention also relates to a reagent for assaying the aPTT of blood, plasma or blood derivatives possibly containing heparin or heparinoid and containing an initiator of the intrinsic coagulation, which is characterised in that it contains a heparin-neutralizing agent and phospholipids. A method of assying the aPTT in such samples as well as the use of this method for assaying the activity of factors of the intrinsic coagulation and the inhibitors thereof is also claimed.
Description
The invention relates to an initiator of the intrinsic coagulation, to a reagent for determining the activated partial thromboplastin time (aPTT) as well as to a method of producing this reagent and of determining the aPTT.
The activated partial tromboplastin time o~ a plasma sample serves as screening test for the overall determination of the activity of the coagulation .
factors of the intrinsic coagulation. Disorders in the coagulation course cause coagulation times that lie beyond the normal range. An increased aPTT, as occurs e.g. in hemophilia A, manifests a deficiency of coagulation factor VIII, e.g.. In addition, the aPTT
test i9 frequently used for monitoring a heparin therapy.
A reagent for determining the aPTT contains an activator to initiate the cascade of the intrinsic coagulation up to the formation of activated factor IX.
Furthermore, phospholipids are required for the activation of factor X and of prothrombin, and there-fore they are also contained in an aPTT reagent. The reagent is contacted with a plasma sample, whereupon calcium is added after a defined activation time which is to be as short as possible, and the time until formation of a fibrin clot is measured.
The aPTT ~est system is subject to a plurality of requirements. For routine check-ups, easy handling of ~. ..
..
. ,: , ~ .
c~ 2 ~ ~
an aPTT reagent is an abolute prerequisite. Also, the stability of the ingredients must be very high to ensure a constant coagulation time over a long period of time. This also enables a low-cost utilisation of the reagent. For the aPTT as a general test, a balanced sentitivity relative to all the pa:rameters to be registered is necessary.
Common reagents *or determining the aPTT contain surface active solid as the initiator of the intrinsic coagulation. By using a surface-active matrix, at first factor XII is activated, comparable to the physiologic conditions. As thA solid activator, e.g. kaolin, celite and glass are used. Kaolin, e.g., is contained as suspension in commercially common reagents for determining the aPTT (Pathromtin~, Behringwerke (1990~, or PTT Reagent, Boehringer Mannheim (1986)). As the phospholipid, a lyophilised lipid extract of human placentas or lyophilised cephalin are used. However, the phospholipids are mixed with kaolin -to a ready-to-use reagent jus-t before the determina-tion is carried out. The separate storage of kaolin and phospholipids is mainly required for stability reasons.
When lyophilizing lipids with kaolin, lipids would become inactivated to a large extent. Thus, in the production of PTT-IMMUN0 (of Immuno) there occurs a loss of up to 50~ of the phospholipid activity at the common lyophilization of kaolin and phospholipids ;
'~ 2 ~ ~
(extracted from porcine brain).
As an al-ternative to solid activators, also liquid activators, such as ellagic acid and its derivatives, or solubilized sulfatides may be used. The sen~itivity of a liquid-activator-containing test relative to heparin is, however, deteriorated, and therefore diverse additives have been suggested to improve its sensitivity. According to WO 91/16453, a heparin-sensitive reagent based on ellagic acid additionally contains dextran sulfate.
A disadvantage of kaolin and of solid activators in general and of ellagic acid or the derivatives thereof is the long activation time of the first phase of the intrinsic coagulation, by which a relatively long incubation time of at least 4 min is necessary for activation. A further disadvantage consists in the relatively long coagulation time in the presence of kaolin. However, a higher kaolin concentration in the reagent can shorten the activation time. Yet kaolin, which deposits from the suspension, may lead to an increased coagulation time or lead to false results when determining the end of coagulation by means of the usual coagulometers. Thus, care must be taken to keep the solid activator concentration as low as possible.
The invention has as its object to provide a standardized rPagent with high stability for determining the aPTT, which is easy to handle and ,', " " ' : - ' ~ " . ' ~? 3~ 9 ~
inexpensive and which involves both short activation times and a high sensitivity relative to individual factors of -the coagulation cascade.
According to the invention, the set object is achieved by an initiator of the intrinsic coagulation of blood, plasma or blood derivatives, which contains a mixture of sulfatides and a solid activator, preferably kaolin.
As initiator of the intrinsic coagulation, the reagent according to -the invention contains a mixture of sulfatides (e.g. of Pentapharm) and a solid activator . Furthermore, phospholipids are contained.
The activation time which is only short when using sulfatides for the activation of factor XII, is hardly increased by their being used together with kaolin.
Thus, by using the reagent according to the invention, a procedure of short incubation time practicable for routine check-ups is made feasible. Simultaneously, however, also the heparin sensitivity is improved by the use of the solid activator.
The use of the initiator according to -the invention in an aPTT reagent thus surprisingly enables a combination of the advantages of both individual activators, yet without a combination of their drawbacks.
This could not be foreseen. Much rather it was to .
~$~
be surmised that the combination of the sulfatides with kaolin as initiator of the intrinsic coagulation would lead to a reduction of ac-tivity in the reagent.
Following the specialists' prejudice that a combination of activators having different reaction mechanisms would create problems, it came as a surprise that the reagent according to the invention can be standardized very well. This is a precondition for a high reproducibility of the determining method.
In the reagent according to the invention the amount of kaolin is kept small. An increased deposit of kaolin is prevented, problems in the operation of de~ection apparatus are avoided.
The initiator of the inven-tion may be lyophilized together with phospholipids without any problems to give a low-cost reagent. Any possible negative interaction of the individual components is without any relevance.
The invention thus relates to a reagent for determining the aPTT of blood, plasma or blood derivatives, which is characterised in that it contains a mixture of sulfatides and a solid activator, preferably kaolin, as initiator of ~he intrinsic coagulation, and further contains phospholipids.
The concentrations of kaolin and sulfatides in the reagent are between 0.1 and 0.6 % by weight, preferably 0.1 to 0.3 % by weight, for k~olin, and between 0.001 2 ~ ~3J
and 0.03 % by weight, preferably 0.002 to 0.01 % by weight, for sulfatides.
The reagent preferably contains chemically pure phospholipids in a standardized mixture. It has been shown that this results in an unexpectedly high reproducibility of the test method. The phospholipid mixture comprises, e.g., phosphatidyl serine, phosphatidyl choline and cholesterol.
The mixture preferably comprises from 5 to 50 phosphatidyl serine, from 15 to 60 ~ phosphatidyl choline, from 15 to 60 ~ phosphatidyl ethanol amine, from 0 to 20 % cholesterol and from 5 to 20 %
phosphatidinic acid, based on the total phospholipid content. As standardized mixture, e.g. 0.2 mg phosphatidyl serine, 0.85 mg phosphatidyl choline, 0.65 mg phosphatidyl ethanol amine, 0.15 mg cholesterol and 0.15 mg phosphatidinic acid per ml, possibly in dilution, are used.
Ad~antageously, the phospholipids are purified in a manner that undesired precipitation reactions, which may occur in the presence of polybrene, are avoided.
A preferred embodiment of the reagent is characterised in that it further contains oxidation inhibitors and sensitivity promoters. ~s the oxidation inhibitors, e.g., cystein, uric acid, vitamin C or vitamin E are utilized so as to increasa the storage stability of the reagent in the lyophilized or liquid .
state. Additives, such as alkali salts, possibly together with amino acids, increasle the sensitivity of the test relative to individual factors. I-t has been shown that a reagent which contains cesium chl~ride (from 5 to 25 mg/ml) and arginin (from 2.5 to 20 mg~ml), preferably 16.8 mg of CsCl/ml and 10.5 mg arginin/ml, after an appropriate dilution, is particularly well suited for determininy the aPTT of a plasma sample with a deficiency of individual factors (factor VIII).
A further preferred embodiment of the reagent is characterised in that it further contains a chromogenic substrate, by which, if desired, the coagulation time can be determined photometrically by color development.
However, the final product can also be determined by a photometric determina~ion of the change in light transmission.
Advantageously, the reagent further contains stabilizers, preferably albumin, and/or buffer substances.
The method of producing this reagent is simple.
Sulfatides are mixed in an aqeuous suspension of the solid activator with phospholipids, and, if desired, with further additives, and, if desired, are lyophilised.
The production of the reagent may, however, also be effected by mixing sulfatides, the so}id activator . . . .
.
and phospholipids, if desired after lyophilizing, possibly with fur~her additives, :in the solid state.
The reagent according to the invention lends itself both to the monitoring of a heparin therapy and to determining individual factors" i.e. coagulation i factors or inhibitors of the intrinsic coagulation.
The invention furthermore re]Lates -to a method of determining the partial thromboplastin time (aPTT) of samples of blood, plasma or blood derivatives, which is characterised in that the sample is contacted with a reagent of the invention, calcium ions are added after a pre-determined activation time, and the coagulation time is determined.
By the addition of heparin-neutralizing substances, a deficiency of coagula-tion factors can also be determined in heparinized plasma samples.
Polybrene, e.g., neutralizes heparin present and thus ; makes the reagent of the invention insensitive relative to a heparin content of the sample.
A further preferred embodiment of the method according to the invention is also characterised in that the sample is admixed with a heparin-neutralizing substance~ such as polybrene, protamin sulfate etc., . .. .
before the reagent of the invention is added.
Furthermore, the invention relates to the use of a method according -to the invention for determinin~ the activity of factors of the intrinsic coagulation and _ g _ , ' the inhibitors thereof, wherein a plasma sample is contacted with a reagent according to the invention, calcium ions are added after a pre-determined activation time, and the coagulation time is determined, whereupon the result obtained is compared to a standard sample with a known activity of the factors.
Furthermore, it was found that assaying of t~e aPTT of blood, plasma or blood derivatives possibly containing heparin or heparinoid, can also be carried out without using a mixture of sulfatides and solid activator, if a heparin-neutralizing agent and phospholipids are contained in a reagent for assaying the aPTT, in addition to an initiator of the intrinsic coagulation, be it a solid activator or a liquid activator or a combination thereof.
As the heparin-neutralizing agents, in particular polybrene, protamin (protamin salts) or heparinase are used in the reagent according to the invention.
As regards the order of magnitude, the concentrations of the heparin-neutralizing agents used depend on the heparin concentration in the sample to be assayed, and usually it ranges between 1 and 500 mg/ml in the case of polybrene. In the standardized method, between 2 and 100 mg/ml polybrene are used.
The advantage of a reagent having a heparin-neutralizing agent consists in particular in that the 3 ~ ~ 2 assaying of the aPTT and of -the individual factors in a blood or plasma sample may be effected irrespective of whether or not the sample contains heparin or heparinoids. , The invention further r~lates to a set comprised of a) a reagent for assaying the aPTT and comprising an initiator of the intrinsic coagulation and phospholipids, and b) a heparin-neutralizing agent.
Although the aPTT is often utilized for monitoring a heparin therapy, wherein a heparin sensitivity is desired, there are also cases in which the intact intrinsic system or factors of the intrinsic system must be assayed independently of the heparin content.
If heparin were contained in the same and thus the aPTT were delayed when measured, too low a portion of coagulation factors would be simulated.
The invention will be explained in more detail by the following examples.
1. Comparison of the aPTT when using diverse reagents As initiator, the assayed reagents for determining the aPTT contained sulfatides (of Pentapharm) and kaolin in various mixing ratios in 200 mM Hepes-glycin-buffer ~pH 6.8). Furthermore, a mixture comprising 4.8 ~g phospha~idyl serine, 20.7 ~g phosphatidyl choline, 15.9 ~g phosphatidyl ethanol amine, 3.7 ~g cholesterol and 3.7 ~g phosphatidinic acid per ml was present in i 2 ~ ~
the reagent.
0.1 ml normal plasma (NP) (Reference Plasma 100 %, Immuno) or abnormal plasma (AP) (Immunocontrol Plasma Abnormal, Immuno) were admixed with 0.1 ml re~gent.
After an incubation time of 2 minutes at 37C, 0.1 ml 0.025 M CaCl2 solution was added, and the time until the point of coaguation was measured by a Schnitger-Gross-coagulometer (of Amelung).
From Table 1 it can be seen that the presence of sulfatides in the reagent shortens the coagulation time of normal pl.asma (NP). Yet kaolin in the reagent also improves the sensitivity, expressed by the ra-tio of the coagulation times of AP and NP (cf. Table 2).
Table 1: aPTT (s) kaolin (%) 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: Ratio of the aPTT
(AP/NP) kaolin (%) 0 0.15 0.30 sulfa-tide (~) 0 X 2.20 2.12 0.005 2.00 2.11 2.21 0.01 2.01 2.15 2.~3 2. Sensitivity to heparin By using the reagents from 1., normal plasma (cf.
1.) and heparinized normal plasma (0.37 U of heparin/ml) were assayed. In Table, 2, the rat'io of the aPTT of heparinized normal plasma to normal plasma is indicated for the different reagents. The sensitivity relative to heparin is improved by a higher kaolin content in the reagent.
Table 3: Ratio of the aPTT
~ 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 relative to factor VIII
:
By using the reagents from 1., normal plasma (cf.
1.) and Control Plasma (of Immuno, contained < 30~ of factor VIII, based on the factor VIII content of normal plasma (= 100 ~)) were assayed. In Table 4, the ratio of the aPTT of the Control Plasma to normal plasma is indicated for the different xeagents. The sensitivity relative to factor VIII is improved by the content of sulfatides in the kaolin-containing raagent.
' . ,:
`` ,.
2 ~
Table 4: Ratio of the aPTT
Control Plasma (< 30 % of factor VIII/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 4. Factor VIII-calibration curve To draw a calibration curve for factor VIII, Reference Plasma 100 ~ (of Immuno) was diluted 1 : 5 with imidazole buffer 3.4 g/l imidazole, 5.85 g/l NaCl, pH 7.4). 0.1 ml o~ this dilution were mixed with 0.1 ml of a phospholipid-kaolin-sulfatide reagent (aomposition of the phospolipids as in Example 1; 0.3 ~ kaolin and 0.01 % sulfatides in 200 mM Hepes-glycin-buffer, pH
6.8) and incubated for 4 min at 37C. Simultaneously with the addition of 0.1 ml of calcium chloride (25mM), the stop watch was started, and the time until thè end of coagulation was determined by means of a Schnitger-Gross-coagulometer (of Amelung).
The prediluted plasma (1:5) corresponds to lO0 %
of factor VIII. Further dilutions were tested according to a geometric dilution series ( 1 : l = 100 ~ to 1 :
128 = 0.78 %). In Table 5, the results of the measurements are indicated.
.~ 1'1 - ~ . . .
, : ~ : -::- , , : :, , ,, , .
: ~
~:, -~ ` 2~9~2 ~ 2 Table 5:
- Factor VIII (%) Coagulation Time (s) lOO 78.5 : 50 91.~ , :L01.7 12.5 :Ll5.3 6.25 :L28.9 3.13 .L39.g :~ 1.56 148.6 :~ 0.7~ 159.4 5. aPTT assaying with PTT reagents of different compositions, with and without the addition of ~; polybrene.
Five different reagents are used in the examples : or assaying the aPTT, both with and without the J addition of heparin-neutralizing agents.
These reagents have the following compositions (in by weight):
Reagent A: 0.075 % kaolin and 0.01 % sulfatide (of :~ Pentapharm) as the initiator, highly purified phospholipids (17 ~ phosphatidyl serine, 44 %
phosphatidyl choline, 29 % phosphatidyl ethanol : amine, 5 ~ cholesterol and 5 % phosphatidinic acid).
~ The ready-to-use reagent is prepared by ;~ reconstitution with 2 ml of aqua dest.
. , (concentration of the phospholipids: 65 ~g/ml~.
:: ;`
~ ' ~ - 15 -.
~' . : , .
J :~ ~
Reagent B: like reagent A, yet only 0.0017 % sulfatide.
Reagent C: 0.5 % kaolin, lipid extract fro~ porcine mucosa (Tachostyptan~, of Hormonchemie)., The ready-to-use reagent is prepared by reconstitution with 2 ml of aqua dest.
Reagent D: Aktin FS~, of Baxter (contains ellagic acid as the initiator and phospholipids from soy bean).
Reagent E: Pathromtin~, of Behring (contains 0.5 ~
kaolin in liquid suspension as the initiator and lipid extract from human placenta (lyo)).
The ready-to-use reagent is prepared by dissolving the lipid extract in the kaolin suspension.
A normal plasma (NP; Immunocontrolplasma Normal, of Immuno) or heparini~ed normal plasma containing 0.4 U heparin/ml (HPl) or 1.0 U heparin/ml (HP2) were used as the plasma samples. 0.1 ml plasma sample was admixed with 0.1 ml reagent. After an incubation time of two minutes at 37C, 0.1 ml 0.025 M CaC12 solution was admixed, and the time up to the point of coagulation was measured with a Schnitger-Gross coagulometer (of Amelung~.
~ .
`:
- .
, .
Table 6 shows the results obtained, line 1 indicating the coagulation time (in seconds) of normal plasma (NP), lines 2 and 3 indicating the coagulation times of heparinized plasmas (HP1 and HP~), and lines 4 and 5 indicating the ratio between the coagulation times of the heparinized plasmas and of normal plasma.
Table 6 7 Reagent C Reagent D Reagent E
w.o_pQlyh~ene 25mg/l P. ~.o.poly}~rene 25mg/l P. 7.o.Eolyb~ene 25mg/l P. ~1.o.polybrene SOmgl~P
NPsec. 34.0 32.4 52.4 68.0 43.1 65.9 34 1 46.6 HP1 sec. 52.9 36.1 123.1 75.8 99.8 67.6 76.1 50.g ¦HP2 sec. 214.1 39.1 253.4 80.3 283.4 59.1 210.1 96.2 Igatio HP1/NP 1.56 1.11 2.35 1.11 2.32 1.03 2.23 1.09 .. ¦~atio HP2/NP 1 6.30 1.21 4.84 1.18 6.58 0.90 6.16 2.06 w.o . = w ithout P. = polybrene The results clearly indicate that in heparinized plasmas without the presence of polybrene the aPTT is clearly increased and thus falsified or distorted. In the presence of polybrene, also the aPTT of heparinized plasmas could be assayed with all the reagents tested.
The ratio between the coagulation times of the heparinized plasmas and of normal plasma, which ideally equals 1 (i.e., the coagulation times are exactly the same) deviates considerably from the ideal value of 1 :-~;
, , :. .' ~J ~
in the tests with the reagents without polybrene, whereas the ratio of the coagulation -times in the presence of polybrene is close to 1.
The activated partial tromboplastin time o~ a plasma sample serves as screening test for the overall determination of the activity of the coagulation .
factors of the intrinsic coagulation. Disorders in the coagulation course cause coagulation times that lie beyond the normal range. An increased aPTT, as occurs e.g. in hemophilia A, manifests a deficiency of coagulation factor VIII, e.g.. In addition, the aPTT
test i9 frequently used for monitoring a heparin therapy.
A reagent for determining the aPTT contains an activator to initiate the cascade of the intrinsic coagulation up to the formation of activated factor IX.
Furthermore, phospholipids are required for the activation of factor X and of prothrombin, and there-fore they are also contained in an aPTT reagent. The reagent is contacted with a plasma sample, whereupon calcium is added after a defined activation time which is to be as short as possible, and the time until formation of a fibrin clot is measured.
The aPTT ~est system is subject to a plurality of requirements. For routine check-ups, easy handling of ~. ..
..
. ,: , ~ .
c~ 2 ~ ~
an aPTT reagent is an abolute prerequisite. Also, the stability of the ingredients must be very high to ensure a constant coagulation time over a long period of time. This also enables a low-cost utilisation of the reagent. For the aPTT as a general test, a balanced sentitivity relative to all the pa:rameters to be registered is necessary.
Common reagents *or determining the aPTT contain surface active solid as the initiator of the intrinsic coagulation. By using a surface-active matrix, at first factor XII is activated, comparable to the physiologic conditions. As thA solid activator, e.g. kaolin, celite and glass are used. Kaolin, e.g., is contained as suspension in commercially common reagents for determining the aPTT (Pathromtin~, Behringwerke (1990~, or PTT Reagent, Boehringer Mannheim (1986)). As the phospholipid, a lyophilised lipid extract of human placentas or lyophilised cephalin are used. However, the phospholipids are mixed with kaolin -to a ready-to-use reagent jus-t before the determina-tion is carried out. The separate storage of kaolin and phospholipids is mainly required for stability reasons.
When lyophilizing lipids with kaolin, lipids would become inactivated to a large extent. Thus, in the production of PTT-IMMUN0 (of Immuno) there occurs a loss of up to 50~ of the phospholipid activity at the common lyophilization of kaolin and phospholipids ;
'~ 2 ~ ~
(extracted from porcine brain).
As an al-ternative to solid activators, also liquid activators, such as ellagic acid and its derivatives, or solubilized sulfatides may be used. The sen~itivity of a liquid-activator-containing test relative to heparin is, however, deteriorated, and therefore diverse additives have been suggested to improve its sensitivity. According to WO 91/16453, a heparin-sensitive reagent based on ellagic acid additionally contains dextran sulfate.
A disadvantage of kaolin and of solid activators in general and of ellagic acid or the derivatives thereof is the long activation time of the first phase of the intrinsic coagulation, by which a relatively long incubation time of at least 4 min is necessary for activation. A further disadvantage consists in the relatively long coagulation time in the presence of kaolin. However, a higher kaolin concentration in the reagent can shorten the activation time. Yet kaolin, which deposits from the suspension, may lead to an increased coagulation time or lead to false results when determining the end of coagulation by means of the usual coagulometers. Thus, care must be taken to keep the solid activator concentration as low as possible.
The invention has as its object to provide a standardized rPagent with high stability for determining the aPTT, which is easy to handle and ,', " " ' : - ' ~ " . ' ~? 3~ 9 ~
inexpensive and which involves both short activation times and a high sensitivity relative to individual factors of -the coagulation cascade.
According to the invention, the set object is achieved by an initiator of the intrinsic coagulation of blood, plasma or blood derivatives, which contains a mixture of sulfatides and a solid activator, preferably kaolin.
As initiator of the intrinsic coagulation, the reagent according to -the invention contains a mixture of sulfatides (e.g. of Pentapharm) and a solid activator . Furthermore, phospholipids are contained.
The activation time which is only short when using sulfatides for the activation of factor XII, is hardly increased by their being used together with kaolin.
Thus, by using the reagent according to the invention, a procedure of short incubation time practicable for routine check-ups is made feasible. Simultaneously, however, also the heparin sensitivity is improved by the use of the solid activator.
The use of the initiator according to -the invention in an aPTT reagent thus surprisingly enables a combination of the advantages of both individual activators, yet without a combination of their drawbacks.
This could not be foreseen. Much rather it was to .
~$~
be surmised that the combination of the sulfatides with kaolin as initiator of the intrinsic coagulation would lead to a reduction of ac-tivity in the reagent.
Following the specialists' prejudice that a combination of activators having different reaction mechanisms would create problems, it came as a surprise that the reagent according to the invention can be standardized very well. This is a precondition for a high reproducibility of the determining method.
In the reagent according to the invention the amount of kaolin is kept small. An increased deposit of kaolin is prevented, problems in the operation of de~ection apparatus are avoided.
The initiator of the inven-tion may be lyophilized together with phospholipids without any problems to give a low-cost reagent. Any possible negative interaction of the individual components is without any relevance.
The invention thus relates to a reagent for determining the aPTT of blood, plasma or blood derivatives, which is characterised in that it contains a mixture of sulfatides and a solid activator, preferably kaolin, as initiator of ~he intrinsic coagulation, and further contains phospholipids.
The concentrations of kaolin and sulfatides in the reagent are between 0.1 and 0.6 % by weight, preferably 0.1 to 0.3 % by weight, for k~olin, and between 0.001 2 ~ ~3J
and 0.03 % by weight, preferably 0.002 to 0.01 % by weight, for sulfatides.
The reagent preferably contains chemically pure phospholipids in a standardized mixture. It has been shown that this results in an unexpectedly high reproducibility of the test method. The phospholipid mixture comprises, e.g., phosphatidyl serine, phosphatidyl choline and cholesterol.
The mixture preferably comprises from 5 to 50 phosphatidyl serine, from 15 to 60 ~ phosphatidyl choline, from 15 to 60 ~ phosphatidyl ethanol amine, from 0 to 20 % cholesterol and from 5 to 20 %
phosphatidinic acid, based on the total phospholipid content. As standardized mixture, e.g. 0.2 mg phosphatidyl serine, 0.85 mg phosphatidyl choline, 0.65 mg phosphatidyl ethanol amine, 0.15 mg cholesterol and 0.15 mg phosphatidinic acid per ml, possibly in dilution, are used.
Ad~antageously, the phospholipids are purified in a manner that undesired precipitation reactions, which may occur in the presence of polybrene, are avoided.
A preferred embodiment of the reagent is characterised in that it further contains oxidation inhibitors and sensitivity promoters. ~s the oxidation inhibitors, e.g., cystein, uric acid, vitamin C or vitamin E are utilized so as to increasa the storage stability of the reagent in the lyophilized or liquid .
state. Additives, such as alkali salts, possibly together with amino acids, increasle the sensitivity of the test relative to individual factors. I-t has been shown that a reagent which contains cesium chl~ride (from 5 to 25 mg/ml) and arginin (from 2.5 to 20 mg~ml), preferably 16.8 mg of CsCl/ml and 10.5 mg arginin/ml, after an appropriate dilution, is particularly well suited for determininy the aPTT of a plasma sample with a deficiency of individual factors (factor VIII).
A further preferred embodiment of the reagent is characterised in that it further contains a chromogenic substrate, by which, if desired, the coagulation time can be determined photometrically by color development.
However, the final product can also be determined by a photometric determina~ion of the change in light transmission.
Advantageously, the reagent further contains stabilizers, preferably albumin, and/or buffer substances.
The method of producing this reagent is simple.
Sulfatides are mixed in an aqeuous suspension of the solid activator with phospholipids, and, if desired, with further additives, and, if desired, are lyophilised.
The production of the reagent may, however, also be effected by mixing sulfatides, the so}id activator . . . .
.
and phospholipids, if desired after lyophilizing, possibly with fur~her additives, :in the solid state.
The reagent according to the invention lends itself both to the monitoring of a heparin therapy and to determining individual factors" i.e. coagulation i factors or inhibitors of the intrinsic coagulation.
The invention furthermore re]Lates -to a method of determining the partial thromboplastin time (aPTT) of samples of blood, plasma or blood derivatives, which is characterised in that the sample is contacted with a reagent of the invention, calcium ions are added after a pre-determined activation time, and the coagulation time is determined.
By the addition of heparin-neutralizing substances, a deficiency of coagula-tion factors can also be determined in heparinized plasma samples.
Polybrene, e.g., neutralizes heparin present and thus ; makes the reagent of the invention insensitive relative to a heparin content of the sample.
A further preferred embodiment of the method according to the invention is also characterised in that the sample is admixed with a heparin-neutralizing substance~ such as polybrene, protamin sulfate etc., . .. .
before the reagent of the invention is added.
Furthermore, the invention relates to the use of a method according -to the invention for determinin~ the activity of factors of the intrinsic coagulation and _ g _ , ' the inhibitors thereof, wherein a plasma sample is contacted with a reagent according to the invention, calcium ions are added after a pre-determined activation time, and the coagulation time is determined, whereupon the result obtained is compared to a standard sample with a known activity of the factors.
Furthermore, it was found that assaying of t~e aPTT of blood, plasma or blood derivatives possibly containing heparin or heparinoid, can also be carried out without using a mixture of sulfatides and solid activator, if a heparin-neutralizing agent and phospholipids are contained in a reagent for assaying the aPTT, in addition to an initiator of the intrinsic coagulation, be it a solid activator or a liquid activator or a combination thereof.
As the heparin-neutralizing agents, in particular polybrene, protamin (protamin salts) or heparinase are used in the reagent according to the invention.
As regards the order of magnitude, the concentrations of the heparin-neutralizing agents used depend on the heparin concentration in the sample to be assayed, and usually it ranges between 1 and 500 mg/ml in the case of polybrene. In the standardized method, between 2 and 100 mg/ml polybrene are used.
The advantage of a reagent having a heparin-neutralizing agent consists in particular in that the 3 ~ ~ 2 assaying of the aPTT and of -the individual factors in a blood or plasma sample may be effected irrespective of whether or not the sample contains heparin or heparinoids. , The invention further r~lates to a set comprised of a) a reagent for assaying the aPTT and comprising an initiator of the intrinsic coagulation and phospholipids, and b) a heparin-neutralizing agent.
Although the aPTT is often utilized for monitoring a heparin therapy, wherein a heparin sensitivity is desired, there are also cases in which the intact intrinsic system or factors of the intrinsic system must be assayed independently of the heparin content.
If heparin were contained in the same and thus the aPTT were delayed when measured, too low a portion of coagulation factors would be simulated.
The invention will be explained in more detail by the following examples.
1. Comparison of the aPTT when using diverse reagents As initiator, the assayed reagents for determining the aPTT contained sulfatides (of Pentapharm) and kaolin in various mixing ratios in 200 mM Hepes-glycin-buffer ~pH 6.8). Furthermore, a mixture comprising 4.8 ~g phospha~idyl serine, 20.7 ~g phosphatidyl choline, 15.9 ~g phosphatidyl ethanol amine, 3.7 ~g cholesterol and 3.7 ~g phosphatidinic acid per ml was present in i 2 ~ ~
the reagent.
0.1 ml normal plasma (NP) (Reference Plasma 100 %, Immuno) or abnormal plasma (AP) (Immunocontrol Plasma Abnormal, Immuno) were admixed with 0.1 ml re~gent.
After an incubation time of 2 minutes at 37C, 0.1 ml 0.025 M CaCl2 solution was added, and the time until the point of coaguation was measured by a Schnitger-Gross-coagulometer (of Amelung).
From Table 1 it can be seen that the presence of sulfatides in the reagent shortens the coagulation time of normal pl.asma (NP). Yet kaolin in the reagent also improves the sensitivity, expressed by the ra-tio of the coagulation times of AP and NP (cf. Table 2).
Table 1: aPTT (s) kaolin (%) 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: Ratio of the aPTT
(AP/NP) kaolin (%) 0 0.15 0.30 sulfa-tide (~) 0 X 2.20 2.12 0.005 2.00 2.11 2.21 0.01 2.01 2.15 2.~3 2. Sensitivity to heparin By using the reagents from 1., normal plasma (cf.
1.) and heparinized normal plasma (0.37 U of heparin/ml) were assayed. In Table, 2, the rat'io of the aPTT of heparinized normal plasma to normal plasma is indicated for the different reagents. The sensitivity relative to heparin is improved by a higher kaolin content in the reagent.
Table 3: Ratio of the aPTT
~ 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 relative to factor VIII
:
By using the reagents from 1., normal plasma (cf.
1.) and Control Plasma (of Immuno, contained < 30~ of factor VIII, based on the factor VIII content of normal plasma (= 100 ~)) were assayed. In Table 4, the ratio of the aPTT of the Control Plasma to normal plasma is indicated for the different xeagents. The sensitivity relative to factor VIII is improved by the content of sulfatides in the kaolin-containing raagent.
' . ,:
`` ,.
2 ~
Table 4: Ratio of the aPTT
Control Plasma (< 30 % of factor VIII/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 4. Factor VIII-calibration curve To draw a calibration curve for factor VIII, Reference Plasma 100 ~ (of Immuno) was diluted 1 : 5 with imidazole buffer 3.4 g/l imidazole, 5.85 g/l NaCl, pH 7.4). 0.1 ml o~ this dilution were mixed with 0.1 ml of a phospholipid-kaolin-sulfatide reagent (aomposition of the phospolipids as in Example 1; 0.3 ~ kaolin and 0.01 % sulfatides in 200 mM Hepes-glycin-buffer, pH
6.8) and incubated for 4 min at 37C. Simultaneously with the addition of 0.1 ml of calcium chloride (25mM), the stop watch was started, and the time until thè end of coagulation was determined by means of a Schnitger-Gross-coagulometer (of Amelung).
The prediluted plasma (1:5) corresponds to lO0 %
of factor VIII. Further dilutions were tested according to a geometric dilution series ( 1 : l = 100 ~ to 1 :
128 = 0.78 %). In Table 5, the results of the measurements are indicated.
.~ 1'1 - ~ . . .
, : ~ : -::- , , : :, , ,, , .
: ~
~:, -~ ` 2~9~2 ~ 2 Table 5:
- Factor VIII (%) Coagulation Time (s) lOO 78.5 : 50 91.~ , :L01.7 12.5 :Ll5.3 6.25 :L28.9 3.13 .L39.g :~ 1.56 148.6 :~ 0.7~ 159.4 5. aPTT assaying with PTT reagents of different compositions, with and without the addition of ~; polybrene.
Five different reagents are used in the examples : or assaying the aPTT, both with and without the J addition of heparin-neutralizing agents.
These reagents have the following compositions (in by weight):
Reagent A: 0.075 % kaolin and 0.01 % sulfatide (of :~ Pentapharm) as the initiator, highly purified phospholipids (17 ~ phosphatidyl serine, 44 %
phosphatidyl choline, 29 % phosphatidyl ethanol : amine, 5 ~ cholesterol and 5 % phosphatidinic acid).
~ The ready-to-use reagent is prepared by ;~ reconstitution with 2 ml of aqua dest.
. , (concentration of the phospholipids: 65 ~g/ml~.
:: ;`
~ ' ~ - 15 -.
~' . : , .
J :~ ~
Reagent B: like reagent A, yet only 0.0017 % sulfatide.
Reagent C: 0.5 % kaolin, lipid extract fro~ porcine mucosa (Tachostyptan~, of Hormonchemie)., The ready-to-use reagent is prepared by reconstitution with 2 ml of aqua dest.
Reagent D: Aktin FS~, of Baxter (contains ellagic acid as the initiator and phospholipids from soy bean).
Reagent E: Pathromtin~, of Behring (contains 0.5 ~
kaolin in liquid suspension as the initiator and lipid extract from human placenta (lyo)).
The ready-to-use reagent is prepared by dissolving the lipid extract in the kaolin suspension.
A normal plasma (NP; Immunocontrolplasma Normal, of Immuno) or heparini~ed normal plasma containing 0.4 U heparin/ml (HPl) or 1.0 U heparin/ml (HP2) were used as the plasma samples. 0.1 ml plasma sample was admixed with 0.1 ml reagent. After an incubation time of two minutes at 37C, 0.1 ml 0.025 M CaC12 solution was admixed, and the time up to the point of coagulation was measured with a Schnitger-Gross coagulometer (of Amelung~.
~ .
`:
- .
, .
Table 6 shows the results obtained, line 1 indicating the coagulation time (in seconds) of normal plasma (NP), lines 2 and 3 indicating the coagulation times of heparinized plasmas (HP1 and HP~), and lines 4 and 5 indicating the ratio between the coagulation times of the heparinized plasmas and of normal plasma.
Table 6 7 Reagent C Reagent D Reagent E
w.o_pQlyh~ene 25mg/l P. ~.o.poly}~rene 25mg/l P. 7.o.Eolyb~ene 25mg/l P. ~1.o.polybrene SOmgl~P
NPsec. 34.0 32.4 52.4 68.0 43.1 65.9 34 1 46.6 HP1 sec. 52.9 36.1 123.1 75.8 99.8 67.6 76.1 50.g ¦HP2 sec. 214.1 39.1 253.4 80.3 283.4 59.1 210.1 96.2 Igatio HP1/NP 1.56 1.11 2.35 1.11 2.32 1.03 2.23 1.09 .. ¦~atio HP2/NP 1 6.30 1.21 4.84 1.18 6.58 0.90 6.16 2.06 w.o . = w ithout P. = polybrene The results clearly indicate that in heparinized plasmas without the presence of polybrene the aPTT is clearly increased and thus falsified or distorted. In the presence of polybrene, also the aPTT of heparinized plasmas could be assayed with all the reagents tested.
The ratio between the coagulation times of the heparinized plasmas and of normal plasma, which ideally equals 1 (i.e., the coagulation times are exactly the same) deviates considerably from the ideal value of 1 :-~;
, , :. .' ~J ~
in the tests with the reagents without polybrene, whereas the ratio of the coagulation -times in the presence of polybrene is close to 1.
6. Factor VIII-calibration cu:rve with PTT~reagents of diferent compositions with and without -the addition of polybrene.
To draw these calibration curves, a reference plasma (Reference Plasma 100 ~ of :Immuno) was used.
This Reference Plasma was dissolved in 1 ml of aqua dest. (RP) and in 1 ml of aqua des-t. containing 1 U/ml heparin (of Immuno) (HP).
The plasmas were pre-diluted 1:5 with imidazol buffer (3.4 g/l imidazol, 5.85 g/l NaCl, pH 7.4); this corresponds to 100 % of Factor VIII. Further dilutions were prepared according to a geometric dllution series (1:1 = 100 % to 1:128 = 0.78 %). 0.1 ml sample were mixed with 0.1 ml reagent and incubated for 4 min at 37~C. Subsequently, 0.1 ml 0.025 M CaCl2 solution was admixed, and the time up to the coagulation point was measured with a Schnitger-Gross-coagulometer.
The results are indicated in Tables 7 to 10.
:
-~ :
- .
2~
j Table 7 Reageni A
=- __ __ ,~J. o. Polybrene 25 mg/ml P~ che F.VIII (%) RP HP RP HP
100 54.7 82.0 40 0 42.2 S0 65.2 78.6 48.6 47.6 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
r----------~ ~ o PolybtenC 1 Z S mg/ml F~r~
F.VIII (%) RP HP RP
5000 891 63 12095 6 999 6 979.66 104.5 102.1 113.1 110.6 12.5 110.6 109.1 123 1 127.6 6.25 121.5 120.1 138.1 134.6 141.6 143 1 j 150.1 151.1 Table 9 Reagent D
_ == ~J.o Polybrene 25 mg/ml Po~;~b~ i :.: F.VIII (%) ~P HP RP HP
100 60.6 107.2 70.1 65.6 S0 79 o 82 9 100.1 93.6 3 13 109.1 109 6 140.6 138.6 . ~ ¦ 0 79 118 1 118 2 164 160.5 ' "
:' `' .
?~ b~
Table 10 Reagent E
__ ~.o. ~olyb;~n~ S0 mg/ml P~
F.VIII (%) RP --HP RP HP
100 54.9 127.S 72.1 68.1 61.2 98.5 78.4 75.1 2S 67.4 96.4 87.9 82 6 : 12 S 74.Z 102.4 94.9 92.1 6.2S 83.4 112.6 106.2 10S.1 3.13 89,9 120.6 113.2 115.6 1.56 97.1 131.6 121.1 12S.S
~ 0.78 102.4 140.9 ~ 127.6 131.6 :~
~ ~ .
The results indicate that in the normal case (plasma without heparin; RP, reagents without polybrene) there clearly exists a correlation between the coagulation time and the factor VIII concentration:
; the lower the concentration of factor VIII, the longer the coagulation time.
When measuring heparin-containing samples (HP, reagents without polybrene), no clear correlation appears, the measurement gives a useless factor VIII-calibration curve which does not allow for a linear attributing of coagulation time and factor VIII
concentration~
Only the presence of polybrene in the reagents allows for a determination of the factor VIII
concentration in heparin-containing samples (HP, ::
- reagents with 12.5/25/5Q mg/ml polybrene). The results . ., ;~;` of the measurements are not falsified by the presence ~. of heparin and also of polybrene when using the reagent :
~ - 20 -:'~
;'' .. ~ : , : .
; ' ` . ~ ' , .: :': ' . . ~: , ., 2 ~ ~
according to the invention. This becomes apparent both when comparing the measurements of RP with reagents with and without polybrene, and when comparing the coagulation times of RP and HP in the presencé,of polybrene, whose quotient always is quite close to l.
To draw these calibration curves, a reference plasma (Reference Plasma 100 ~ of :Immuno) was used.
This Reference Plasma was dissolved in 1 ml of aqua dest. (RP) and in 1 ml of aqua des-t. containing 1 U/ml heparin (of Immuno) (HP).
The plasmas were pre-diluted 1:5 with imidazol buffer (3.4 g/l imidazol, 5.85 g/l NaCl, pH 7.4); this corresponds to 100 % of Factor VIII. Further dilutions were prepared according to a geometric dllution series (1:1 = 100 % to 1:128 = 0.78 %). 0.1 ml sample were mixed with 0.1 ml reagent and incubated for 4 min at 37~C. Subsequently, 0.1 ml 0.025 M CaCl2 solution was admixed, and the time up to the coagulation point was measured with a Schnitger-Gross-coagulometer.
The results are indicated in Tables 7 to 10.
:
-~ :
- .
2~
j Table 7 Reageni A
=- __ __ ,~J. o. Polybrene 25 mg/ml P~ che F.VIII (%) RP HP RP HP
100 54.7 82.0 40 0 42.2 S0 65.2 78.6 48.6 47.6 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
r----------~ ~ o PolybtenC 1 Z S mg/ml F~r~
F.VIII (%) RP HP RP
5000 891 63 12095 6 999 6 979.66 104.5 102.1 113.1 110.6 12.5 110.6 109.1 123 1 127.6 6.25 121.5 120.1 138.1 134.6 141.6 143 1 j 150.1 151.1 Table 9 Reagent D
_ == ~J.o Polybrene 25 mg/ml Po~;~b~ i :.: F.VIII (%) ~P HP RP HP
100 60.6 107.2 70.1 65.6 S0 79 o 82 9 100.1 93.6 3 13 109.1 109 6 140.6 138.6 . ~ ¦ 0 79 118 1 118 2 164 160.5 ' "
:' `' .
?~ b~
Table 10 Reagent E
__ ~.o. ~olyb;~n~ S0 mg/ml P~
F.VIII (%) RP --HP RP HP
100 54.9 127.S 72.1 68.1 61.2 98.5 78.4 75.1 2S 67.4 96.4 87.9 82 6 : 12 S 74.Z 102.4 94.9 92.1 6.2S 83.4 112.6 106.2 10S.1 3.13 89,9 120.6 113.2 115.6 1.56 97.1 131.6 121.1 12S.S
~ 0.78 102.4 140.9 ~ 127.6 131.6 :~
~ ~ .
The results indicate that in the normal case (plasma without heparin; RP, reagents without polybrene) there clearly exists a correlation between the coagulation time and the factor VIII concentration:
; the lower the concentration of factor VIII, the longer the coagulation time.
When measuring heparin-containing samples (HP, reagents without polybrene), no clear correlation appears, the measurement gives a useless factor VIII-calibration curve which does not allow for a linear attributing of coagulation time and factor VIII
concentration~
Only the presence of polybrene in the reagents allows for a determination of the factor VIII
concentration in heparin-containing samples (HP, ::
- reagents with 12.5/25/5Q mg/ml polybrene). The results . ., ;~;` of the measurements are not falsified by the presence ~. of heparin and also of polybrene when using the reagent :
~ - 20 -:'~
;'' .. ~ : , : .
; ' ` . ~ ' , .: :': ' . . ~: , ., 2 ~ ~
according to the invention. This becomes apparent both when comparing the measurements of RP with reagents with and without polybrene, and when comparing the coagulation times of RP and HP in the presencé,of polybrene, whose quotient always is quite close to l.
7. aPTT assaying with reagent B with and without addition of heparinase.
Immunocontrolplasma Normal (oE Immuno) was dissolved in l ml of aqua dest. containing 0, 0.5 or 1 U/ml heparin.
Reagent B was dissolved in 1 ml of aqua dest.
containing 0, 0.74, 0.22, 0.07 and 0.02 U/ml heparinase (of IBEX, Canada).
The PTT assay was carried out as described in Example S.
The results of the measurements carried out are indicated in Table 11.
Table ll heparin in plasma j heparinase U/ml I
U/ml 0 _0.02 0.07 0.22 0.74 '! . I 0.0 33.6 33.6 33.1 33.6 38.3 0.5 52.4 45.8 41.8 37.7 41.4 1.0 85.8 65.6 50.8 47.3 42.3 , __-, These results show that also a heparinase-containing reagent is excellently suited for -the aPTT
assay of heparinized plasma.
;,.
, . ' , ' .
Immunocontrolplasma Normal (oE Immuno) was dissolved in l ml of aqua dest. containing 0, 0.5 or 1 U/ml heparin.
Reagent B was dissolved in 1 ml of aqua dest.
containing 0, 0.74, 0.22, 0.07 and 0.02 U/ml heparinase (of IBEX, Canada).
The PTT assay was carried out as described in Example S.
The results of the measurements carried out are indicated in Table 11.
Table ll heparin in plasma j heparinase U/ml I
U/ml 0 _0.02 0.07 0.22 0.74 '! . I 0.0 33.6 33.6 33.1 33.6 38.3 0.5 52.4 45.8 41.8 37.7 41.4 1.0 85.8 65.6 50.8 47.3 42.3 , __-, These results show that also a heparinase-containing reagent is excellently suited for -the aPTT
assay of heparinized plasma.
;,.
, . ' , ' .
Claims (34)
1. An initiator of the intrinsic coagulation of blood, plasma or blood derivatives comprising a mixture of sulfatides and a solid activator.
2. An initiator as set forth in claim 1, wherein said solid activator is kaolin.
3. A reagent for determining the aPTT of blood, plasma or blood derivatives comprising a mixture of sulfatides and a solid activator as initiator of the intrinsic coagulation, and furthermore comprising phospholipids.
4. A reagent as set forth in claim 3, wherein said solid activator is kaolin.
5. A reagent as set forth in claim 3, wherein said phospholipids are chemically pure phospholipids in a standardized mixture.
6. A reagent as set forth in claim 5, wherein said phospholipid mixture, based on the total phospholipid content, comprises 5 to 50 % of phosphatidyl serine, 15 to 60 % of phosphatidyl choline, 15 to 60 % of phosphatidyl ethanol amine, 0 to 20 % of cholesterol and 5 to 20 % of phosphatidinic acid.
7. A reagent as set forth in claim 3, further comprising at least one of oxidation inhibitors and sensitivity promoters.
8. A reagent as set forth in claim 3, further comprising alkali salts.
9. A reagent as set forth in claim 8, further comprising amino acids.
10. A reagent as set forth in claim 3, further comprising a chromogenic substrate enabling a photometric determination of the coagulation time by colour development.
11. A reagent as set forth in claim 3, further comprising at least one of stabilizers and buffer substances.
12. A reagent as set forth in claim 11, wherein among said stabilizers is albumin.
13. A reagent as set forth in claim 3 in the lyophilized form.
14. A method of producing a reagent for determining the aPTT of blood, plasma or blood derivatives, said method comprising providing sulfatides, providing an aqueous suspension of a solid activator, providing phospholipids, mixing said sulfatides with said phospholipids in said aqueous suspension of said solid activator.
15. A method as set forth in claim 14, further comprising providing further additives, and mixing said further additives with said sulfatides in said aqueous suspension of said solid activator.
16. A method as set forth in claim 14, further comprising lyophilizing said reagent.
17. A method of producing a reagent for determining the aPTT of blood, plasma or blood derivatives, said method comprising providing sulfatides, providing a solid activator, providing phospholipids and , mixing said sulfatides, said phospholipids, and said solid activator in the solid state.
18. A method as set forth in claim 17, further comprising lyophilizing said sulfatides, said solid activator and said phospholipids before mixing them.
19. A method as set forth in claim 17, further comprising providing further additives, and admixing said further additives.
20. A method of determining the partial thromboplastin time (aPTT) of samples of blood, plasma or blood derivatives, said method comprising providing a sample, providing a reagent comprised of a mixture of sulfatides and a solid activator as initiator of the intrinsic coagulation, and of phospholipids, contacting said sample with said reagent for a pre-determined activation time, admixing calcium ions after said pre-determined activation time, and determining the coagulation time of said sample.
21. A method as set forth in claim 20, further comprising providing a heparin-neutralizing substance, and admixing said heparin-neutralizing substance with said sample before contacting said sample with said reagent.
22. A method as set forth in claim 20 to be used for determining the activities of factors of the intrinsic coagulation and inhibitors thereof, wherein a plasma sample is contacted with said reagent, calcium ions are admixed after a pre-determined activation time, and the coagulation time is determined so as to give a certain result, and said certain result is compared to the result of a standard sample of known activity of the factors.
23. In a reagent for determining the aPTT of blood, plasma or blood derivatives containing an initiator of the intrinsic coagulation, the improvement wherein said reagent contains a heparin-neutralizing agent and a phospholipid component.
24. A reagent as set forth in claim 23, wherein said blood, plasma or blood derivatives contain heparin.
25. A reagent as set forth in claim 23, wherein said blood, plasma or blood derivatives contain heparinoid.
26. A reagent as set forth in claim 23, wherein said heparin-neutralizing agent is selected from the group consisting of polybrene, a protamin salt or heparinase.
27. A reagent as set forth in claim 23, wherein said phospholipid component, based on the total phospholipid content, comprises from 5 to 50 % phosphatidyl serine, from 15 to 60 % phosphatidyl choline, from 15 to 60 %
phosphatidyl ethanol amine, from 0 to 20 % cholesterol and from 5 to 20 % phosphatidinic acid.
phosphatidyl ethanol amine, from 0 to 20 % cholesterol and from 5 to 20 % phosphatidinic acid.
28. In a method of determining the partial thromboplastin time (aPTT) of samples of blood, plasma or blood derivatives, the improvement comprising providing a sample, providing a reagent containing an initiator of the intrinsic coagulation, a heparin-neutralizing agent and phospholipids, contacting said sample with said reagent, providing calcium ions, admixing said calcium ions, and determining the coagulation time of said sample.
29. A method as set forth in claim 28, wherein said sample of blood, plasma or blood derivatives contains heparin.
30. A method as set forth in claim 28, wherein said sample of blood, plasma or blood derivatives contains heparinoid.
31. A method as set forth in claim 28 to be used for determining the activities of factors of the intrinsic coagulation and inhibitors thereof, wherein a sample of blood, plasma or blood derivatives is contacted with said reagent, calcium ions are admixed after a pre-determined activation time, and the coagulation time is determined so as to give a certain result, and said certain result is compared with the result of a standard sample of known activity of said factors.
32. A method as set forth in claim 31, wherein said sample of blood, plasma or blood derivatives contains heparin.
33. A method as set forth in claim 31, wherein said sample of blood, plasma or blood derivatives contains heparinoid.
34. A set comprised of a) a reagent for assaying the APTT and comprising an initiator of the intrinsic coagulation and phospholipids, and b) a heparin-neutralizing agent.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ATA998/92 | 1992-05-15 | ||
| AT99892A AT398983B (en) | 1992-05-15 | 1992-05-15 | Determn. of activated partial thromboplastin time - using mixt. of sulphatide(s) and kaolin as coagulation initiator |
| ATA841/93 | 1993-04-30 | ||
| AT84193A AT402074B (en) | 1993-04-30 | 1993-04-30 | Reagent for determination of the aptt |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2096212A1 true CA2096212A1 (en) | 1993-11-16 |
Family
ID=25594074
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002096212A Abandoned CA2096212A1 (en) | 1992-05-15 | 1993-05-13 | Initiator of the intrinsic coagulationof blood, plasma or blood derivatives, a reagent for determining the aptt of blood, plasma or blood derivatives, a method of producing this reagent and a method of determining the aptt of samples of blood, plasma or blood derivatives |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP0570356B1 (en) |
| JP (1) | JPH0643169A (en) |
| AT (1) | ATE176822T1 (en) |
| CA (1) | CA2096212A1 (en) |
| CZ (1) | CZ87993A3 (en) |
| DE (1) | DE59309374D1 (en) |
| DK (1) | DK0570356T3 (en) |
| ES (1) | ES2130244T3 (en) |
| FI (1) | FI932195A (en) |
| HU (1) | HU216882B (en) |
| MX (1) | MX9302831A (en) |
| NO (1) | NO310126B1 (en) |
| SK (1) | SK48793A3 (en) |
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| US6448024B1 (en) | 2000-10-03 | 2002-09-10 | Roche Diagnostics Corporation | Method, reagent, cartridge, and device for determining fibrinogen |
| US6699718B1 (en) | 1999-09-03 | 2004-03-02 | Roche Diagnostics Corporation | Method, reagent and test cartridge for determining clotting time |
| US8357539B2 (en) | 2009-12-25 | 2013-01-22 | Sysmex Corporation | Activated partial thromboplastin time measuring reagent, activated partial thromboplastin time measuring method, and determination method for determining presence or absence of blood coagulation inhibitor |
| CN107356769A (en) * | 2017-06-23 | 2017-11-17 | 宁波艾科生物科技有限公司 | A kind of detection reagent of liquid instant activated partial thromboplastin time |
| CN107356768A (en) * | 2017-06-23 | 2017-11-17 | 宁波艾科生物科技有限公司 | A kind of liquid instant prothrombin time detection reagent |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4337278C2 (en) * | 1993-11-02 | 1996-09-05 | Wolf Dr Bertling | Kit for individual control of anticoagulant therapy |
| GR1002776B (en) * | 1996-12-02 | 1997-09-26 | . | Anticoagulant effect of the complex attiii/heparin : new screening test for thrombophilic tendency. |
| AT405692B (en) * | 1997-03-27 | 1999-10-25 | Immuno Ag | REAGENT FOR DETERMINING THE APTT |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1213198A (en) * | 1982-09-29 | 1986-10-28 | James E. Hughes | Diagnostic activated partial thromboplastin reagent |
| DE3311287A1 (en) * | 1983-03-28 | 1984-10-04 | Behringwerke Ag, 3550 Marburg | METHOD FOR PHOTOMETRICALLY DETERMINING THE ACTIVATED PARTIAL THROMBOPLASTIN TIME AND REAGENT TO IT |
| CH678892A5 (en) * | 1989-04-04 | 1991-11-15 | Pentapharm Ag | Heparin-sensitive reagent for measuring activated thromboplastin - contains cephalin, sulphate and additive, e.g. lidocaine, modifying sensitivity to heparin or lupus anticoagulant |
| ATE140034T1 (en) * | 1990-04-17 | 1996-07-15 | Analytical Control Syst Inc | COAGULATION ASSAY AND REAGENTS |
| US5314695A (en) * | 1990-11-13 | 1994-05-24 | Corvas International, Inc. | Tissue factor based prothrombin time reagent |
-
1993
- 1993-05-12 DE DE59309374T patent/DE59309374D1/en not_active Expired - Fee Related
- 1993-05-12 DK DK93890099T patent/DK0570356T3/en not_active Application Discontinuation
- 1993-05-12 EP EP93890099A patent/EP0570356B1/en not_active Expired - Lifetime
- 1993-05-12 AT AT93890099T patent/ATE176822T1/en not_active IP Right Cessation
- 1993-05-12 ES ES93890099T patent/ES2130244T3/en not_active Expired - Lifetime
- 1993-05-12 CZ CZ93879A patent/CZ87993A3/en unknown
- 1993-05-13 CA CA002096212A patent/CA2096212A1/en not_active Abandoned
- 1993-05-14 FI FI932195A patent/FI932195A/en unknown
- 1993-05-14 JP JP5112783A patent/JPH0643169A/en active Pending
- 1993-05-14 SK SK487-93A patent/SK48793A3/en unknown
- 1993-05-14 HU HU9301400A patent/HU216882B/en not_active IP Right Cessation
- 1993-05-14 MX MX9302831A patent/MX9302831A/en unknown
- 1993-05-14 NO NO931774A patent/NO310126B1/en not_active IP Right Cessation
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6699718B1 (en) | 1999-09-03 | 2004-03-02 | Roche Diagnostics Corporation | Method, reagent and test cartridge for determining clotting time |
| US6448024B1 (en) | 2000-10-03 | 2002-09-10 | Roche Diagnostics Corporation | Method, reagent, cartridge, and device for determining fibrinogen |
| US8357539B2 (en) | 2009-12-25 | 2013-01-22 | Sysmex Corporation | Activated partial thromboplastin time measuring reagent, activated partial thromboplastin time measuring method, and determination method for determining presence or absence of blood coagulation inhibitor |
| CN107356769A (en) * | 2017-06-23 | 2017-11-17 | 宁波艾科生物科技有限公司 | A kind of detection reagent of liquid instant activated partial thromboplastin time |
| CN107356768A (en) * | 2017-06-23 | 2017-11-17 | 宁波艾科生物科技有限公司 | A kind of liquid instant prothrombin time detection reagent |
Also Published As
| Publication number | Publication date |
|---|---|
| NO310126B1 (en) | 2001-05-21 |
| JPH0643169A (en) | 1994-02-18 |
| HU216882B (en) | 1999-09-28 |
| FI932195A (en) | 1993-11-16 |
| MX9302831A (en) | 1994-05-31 |
| SK48793A3 (en) | 1993-12-08 |
| HUT75667A (en) | 1997-05-28 |
| CZ87993A3 (en) | 1994-02-16 |
| ATE176822T1 (en) | 1999-03-15 |
| DE59309374D1 (en) | 1999-03-25 |
| EP0570356A1 (en) | 1993-11-18 |
| EP0570356B1 (en) | 1999-02-17 |
| ES2130244T3 (en) | 1999-07-01 |
| NO931774D0 (en) | 1993-05-14 |
| NO931774L (en) | 1993-11-16 |
| DK0570356T3 (en) | 1999-09-20 |
| HU9301400D0 (en) | 1993-09-28 |
| FI932195A0 (en) | 1993-05-14 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |