CA1250213A - Procedure for the photometric determination of the activated partial thromboplastin time and a reagent for this purpose - Google Patents
Procedure for the photometric determination of the activated partial thromboplastin time and a reagent for this purposeInfo
- Publication number
- CA1250213A CA1250213A CA000450594A CA450594A CA1250213A CA 1250213 A CA1250213 A CA 1250213A CA 000450594 A CA000450594 A CA 000450594A CA 450594 A CA450594 A CA 450594A CA 1250213 A CA1250213 A CA 1250213A
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- reagent
- procedure
- partial thromboplastin
- activated partial
- thromboplastin time
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
- C12Q2337/10—Anilides
- C12Q2337/12—Para-Nitroanilides p-NA
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
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Abstract
Abstract of the disclosure A procedure for the photometric determination of the activated partial thromboplastin time (APTT), in which a chromogenic substrate is used, is described. In addition, a reagent suitable for this procedure is described.
Description
The invention relates to a procedure for the photo-metric determination of the activated partial thromboplastin time tAPTT) and to a reagent sui.able~for this purpose.
Determination of the act;vated partial thrombo-plastin ti~e (APTT) is, in add;tion to the prothroMb;n time (thromboplastin t;me, Quick value), the test of coagu-lation which is carried out most -Frequently. The A?TT per~
mits conclusions to be drawn about the behav;or of the en-dogenous pathway of coagulation. Another important use of this test is for monitoring heparin therapyn The APTT is sens;tive to lligh molecular ~eight kininogen, prekalli-krein and factors XIIr X~ IXr VIII, V and I~ as well as to the ;nhibitors of coagulation, especially antithrombin III
under heparin therapy and fibrinogen cleavage products (antithrombin VI), all of which prolong the hPTT.
Reasents for the determination of the APTT essen-tially contain phospholip;ds and a suitable activator of the "contact phase". By contact activation, factor XII is activatedr and this then activates factor XI and prekalli-krein. Due to the lipids and calcium ions contained in the reagent~ activation of the entire endogenous pathl~ay then occurs, and this terminates in formation of a fibrin clot~
The time equired for this to occur is the variable mea sured.
Inorganic materials are employed as activators of the contact phase, preferably celite or kaolin. Ellag1c - . $
acid is aiso used (U.S. Patent 3,486~981 and German Offen-legungsschrf;t 2,9l5,310). As an optically transparent reagent, ellagic acid has advantages for use in optical measur;ng procedures for detect;ng the formation of the clot. ~owever, according to Bock et al., Biochemistry 20, 7258-7266 (1932), the active species is a complex of me~al ions and ellagic acid, which is insoluble in water. Another activator is dextran sulfate.
i;owever, these activators of the contact phase are non-physiological and are also diff;cult to standardize.
For example, the coagulation time oF a kaolin~activated APTT depends to a quite considerab;e extent on the particle size of the act;va~or. Moreover, some coagulation factors are so strongly adsorbed on surface~active materials of this type, that the latter are used as adsorbents for the prepara~ion of the contact factors. However, in addition~
the colnpos-,tion of the phospholip;d contained in a reagen~
of this type is essential for the result~ Furthermore, the lensth of the preincubation time, after which the actual reaction leading to the forma-ion of the clot is started by "recalcification", plays an inportant part, since activated coagulation factors are inhib;ted by inhibitors in the plasma, especially antithrombin II~.
SuLfatidcs are regarded as being physiological activators of coagulation (Fujikawa e~ al., Biochem. 19 (1980), 1~22-133~, and rans and Griffin~ Blood, 59 (19S2), 69-75). Sulfatides belong to the class of glycosphingo-lipids and they conta;n a suLfate group on the galactose ring~ Various species belong to this group of ~L~;5~ 13 _ 3 compounds, and ~hey differ in the nature of the fatty acid chain. They can be detected in all tissues, in the mem-branes, and in espec;ally large amounts in the bra;n, from which they can be obta;ned in a very pure form.
Sulfatides are more effective than kaolin for the contact activation of plasma.
The end point of conventional determinations of APTT is the measurement of a fibrin clot~ Following the -ForMation of a clot of this type is possible only with technical difficulty. Quite a number of devices have been developed~ and these make use of a variety of mechanical, electrical or optical procedures.
Since the introduction of chromogenic substrates for coaguLation factors, attempts have also been made to emp~oy them for the determinat;on of the coagulation enzymes. The advantage of chromogenic substrates is the poss;b;lity of straightforward standardisation of the low molecular weight substrates~ in contrast to the complexO
h;gh molecular weight natural substrates. The problem of measuring the -format;on of a clot no longer ar;sesO
Moreover, chromogenic substrates have already been employed for carrying out "global tests". Thus, Yamada and Meguro, Thrombos. Res. 15 (1979), 351-358, describe a method for measur;ng the APTT. Th;s is based on activa-tion of the endogenous pathway us;ng ellagic ac;d in the presence of phospholipid~ calcium and the chromogen;c thrombin substrate H-D-Phe-P;p-Arg-pNA (p 2238). The d;sad~arltages of this procedure are the use of a non-physiological activatc,r, the lack of spec;fic;ty of the 3L~ 3 chromogenic substrate~ and the extremely long measurement ti~es (normal figure about 7~4 minutes)~ 9nly incomple~e data has been published on ~he sensitivity to the indivi~
dual cl~tt ing f ~ctors .
Another method is described by P. Aiyappa, Annu r~ew York ~cad. Sci. (1~1), pages ~12-~21. In this tech-n;que of measurement~ plasma is activated by ellagic acid in the presence of phospholipid and calcium ions fcr 5 minutes~ and the thrombin formed in this time is measured using a chromo~enic substrate~ This method can lead to the 'ormation of ,ibrin, and active thrombin undergoes ;nclusion ;n th;s. Moreover, thromb;n can be further deactivated by inhibition. On the other hand, it is possible that~
within the selected ;ncubation time, the activation of patho logical plasmas takes place e;ther not at all or only ;ncom-pletely, although they are actually capable of this~ even though slowlyO The procedure described by Aiyappa cannot be used to measure coagulation in deficient plasmas~
It has now been ~ound~ surprisingly~ that it is poss;ble to elim;nate the essential disadvantages of the described APTT methods with chromogenic substrates by using a sulfatide as the physiological activator in com-bination with a highly specific thromb;n substrate~
Thus the invention relates to a procedure for the determination of the activated partial thromboplastin time using an activator, a coagulation-active phospholip;d or mixture of phospholipids~ calcium ions and a chromogenic substrate ,or thrombin, ~Jh,ch comprises the activator being a sulfa~ide or a mix~ure of sulfatides.
~Z~ 3 The invention also relates to a reagent for the determination of activated partial thromboplastin time~
comprising a sulfatide, a phospl1olip;d~ a soluble calcium salt and a chromogen;~c substrate in the freeze-dried form.
U~ilisable sulfatides are commercially available, for example from Serva, Sigma or Supelco~ It is advan-tageous tc use sulfatides which have~ on thin~layer chroma-tography on silica gel~ an RF of 0.2-003~ preferably 0.25, with a mixture of chloroform/methanol/wa~.er 65:25:4 as the mobile phase and an RF of 0.25~0.35~ preferably 0.31~
using chloroform/methanol 40:15. However, i~ is also pos-s;ble to obtain sulfatides of this type by processes which have been described, for example by Hara and Radin, Anal.
e;ochem. 100, 36~-370 (197~), or from dried acetone extract of brain~ t~lorthwhile test concentrations are betweerl 0~1 and 5Q ~g/ml. The sulfatide can initially be suspended at a concentration of 0.01 g/l in 50 mmol/l of ~EPES buffer, pH
7.6, and this suspension used for prepar;ng the reagent.
The phospholipids which can be used are the animal and veget-7ble lipids employed in conventional reagents~ It is particularly advantageous to use lipids from human plate-lets or an extract from human placenta. It is ;mportant that these lipids have no thromboplastic activity, so that the exogenous path~lay is not also activated.
The chromogenic substrate for thrombin must be speci~i.c for this enzyme to be possible tG employ it in this test, sînce. of course, the intention is to measure thrornbin selective'y in the presence of the other coagulation factors which are likew;se activated~ Chromo~yM tr~
~'~5~ 3 TH (Tos-Gly-Pro-Arg-p~A) and S 2160 (Bz-Phe-Val-Arg-pNA~
are relatively non-specific and also indicate other coagu-lation factors~ such as kallikrein or factors Xa and XIIa.
S 2238 (HD-Phe-Pip-Arg-pNA) is rr,ore suitable, althou~h its spec;ficity for the contact factors is again not very high.
Apart from para-nitroanilides, peptides having other chromo-phores are also suitable~ for example, coumarin derivatives~
where tne hydrolysis is followed by measur;ng the fluores-cence.
The most suitable chrornogenic pep~ides for a global test, such as APTT, are thrombin substrates sucl7 as are des~
cribed in German Patent Application 3,244,~030.8. The chromophoric derivative they contain is 5~amino~2~nitro--henzoic ac;d.
A cornpound of the general formula I
X-P~o -Arg-Ni~-~f-~o2 ( 1 ) ~here R is C1_5-alkyl or -CH~CH(CH3~2~COOC~l3, and X is H-D-Phe-, Boc- Gly- or Tosyl-Gly-, is preferably ~sed.
The substrate is preferably employed at very low concentration. The best results are obtained with 50 ~umol/l when H-D-Phe-Pro-Ar~ANB~~isopropylamide is used. The calcium concentration should be between 1 and 10 mmol/l preferably 5 mmol/l.
The sulfatide, phospholipid, chromogenic substrate and the calciurn salt, preferably ~a'c1um chloride~ are d;s-solved or suspended at pH 7.2-~.5 in a bu-ffer~ such as HEPES or Tris. IrQidazole, glycyl~lycine or triethat1olalnine buffers can also be used~
The procedure according to the invent;on ;s advan-- -tas~ously carried out at 25 37C b~ m;x;ng a plasma sample w;th the reagent at this temperature in a thermostated cell.
The reaction is followed at 405~410 nm in a photometer.
~ 1ith a typical normal plasma the extinction remains constant for some time and then increases greatly ~lithin a few seconds. The variable measured is the time necessary to reach a spec;f;ed d;fference in ext;nct;on, -ror example 0.1. In principle~ k;netic evaluation of the exponentiai curve is a!so poss;ble~ The reaction of a specified amount of substrate ;s analogous to that in a conventional APTT~
where the activated thrombin reacts with a specified amount of fibrinogen and thus induces the clot. Example 1 shows that the new procedure in the preFerred 'orrn ind;cates cnanges in the activity of each individual factor ;n tne endogenous pathway.
In contrast to the convention APTT~ in ~hich~ a,.er a certain fixed activation time, the actual reaction leading to the clot is started by add;ng CaCl2, it ;s possible for CaCl2 to be added From the st3rt -in the new procedure;
and this leads to there being one pipetting step less (monoreagent). However, in principle~ a test system analo-gous to the procedure used hitherto is also possible with the procedure accordirlg to the invention~ With this ob-jective, the chrornogenic peptide is added~ tc~etller with CaCl2, after a speci~;ed preincubation time. In this case~ it has proved to be favourable to add a little CaCl2 (about 1 l~mol/l~-to the sulfatide reagent everl duril~g æ~3 the pre;ncubat;on time, and to start the actual reaction by addition of more CaCl2 solution~ The optimal final con centration in this case is again 5 mmol/l of CaCl2.
The use o-f sulfatides for the contact activation of plasma, combined with chromogenic substrates, has not hitherto been disclosedO Manuf2cturers of reagents recommend that the sulfatides be stored below 0C. rreeze drying sulfatides together with the other necessary constituents o-f the reagent does not lead to a stable product. The APTT
is always drastically increased.
Surprisingly~ a stable and effective reagent is obtained by addition of serurn albumin, ge1atin or degraded arld che~i-cally crosslinked collage~. A co~c~ntration of only about 0.1-1% ~llows a lyophilisate of high activity to be produced, As with conventional reagents~ it is possible, in combination with deFic;ent plasma to determir)e the indivi~
dual factors of the endogenous pathway. For this purpose, ttle plasma sample w;ll be d;lu.ed ;n order o eliminate the effect of the other coagulation factors, or it is ernployed ;n an excess of the deficient plasma used.
Addition of aminoac;ds, especially glu~amine~
asparagine or glutamic acid, increases the sensitivity of the reagent to heparin.
Abbreviations:
ANBA 5-amino-2-nitrobenzoic acid Arg Arsinine Gly Glycine l-IEPES N-2-hydroxyethylpiperazinyl-N'-2-ethanesulfonic acid ., _ 9 ~
Abrre~iations continued:
Phe Phenylalanine Pip Pipecolic acid Pro Proline Tos Toluenesulfonyl Tris Tr;s(hydro~ymethyl)aminonlethane The invention is illustrated by the Example ~hich follows.
5~2-~
' ~ 10 -Example :
Factor sensit;vity of the new reagent Reagent~ 0.1 ml of 3mmol/l H-D-Phe-Pro Arg-ANB~-isopropyl-~amide chromogen;c substrate (S 82 107) 0.5 ml of Fibraccel R 1:1000 tcoagulation-active homologous phosphol;p;d com-plex, Behr;ngwerke AG) 0.5 ~l of sulfatide solution (supplied by Supelco; RF 0025 with CHCl3/MeOH
H20 6S:25:~ on silica gel) 0~01 g/l 5.0 ml of buffer (HEPES, NaCl, Ca2~: 25, 50, S mmol/l; pH 7~6~ 0~25% HaemaccelR
tGerman patent 1~11S~7~2 and 1,153,134) ixture: 100 ~l of plasma or deficient plasma ~Behr;ngwerke), congen;tal F VII-def;c;ent plasma 1 ml of reagen~
37C, measurement o-F the t;me unt;l E~05 nm = 0~1.
Def;cient plasma Seconds Factor II 1,200 Factor V 1,200 Factor VIII 378 Factor IX 498 Factor X ~48 Fac'.or XI 396 Factor XII 336 HMWK (high molecular ~eight kin1nogen) 396 Kailikrein 720 Pooled plasma 170
Determination of the act;vated partial thrombo-plastin ti~e (APTT) is, in add;tion to the prothroMb;n time (thromboplastin t;me, Quick value), the test of coagu-lation which is carried out most -Frequently. The A?TT per~
mits conclusions to be drawn about the behav;or of the en-dogenous pathway of coagulation. Another important use of this test is for monitoring heparin therapyn The APTT is sens;tive to lligh molecular ~eight kininogen, prekalli-krein and factors XIIr X~ IXr VIII, V and I~ as well as to the ;nhibitors of coagulation, especially antithrombin III
under heparin therapy and fibrinogen cleavage products (antithrombin VI), all of which prolong the hPTT.
Reasents for the determination of the APTT essen-tially contain phospholip;ds and a suitable activator of the "contact phase". By contact activation, factor XII is activatedr and this then activates factor XI and prekalli-krein. Due to the lipids and calcium ions contained in the reagent~ activation of the entire endogenous pathl~ay then occurs, and this terminates in formation of a fibrin clot~
The time equired for this to occur is the variable mea sured.
Inorganic materials are employed as activators of the contact phase, preferably celite or kaolin. Ellag1c - . $
acid is aiso used (U.S. Patent 3,486~981 and German Offen-legungsschrf;t 2,9l5,310). As an optically transparent reagent, ellagic acid has advantages for use in optical measur;ng procedures for detect;ng the formation of the clot. ~owever, according to Bock et al., Biochemistry 20, 7258-7266 (1932), the active species is a complex of me~al ions and ellagic acid, which is insoluble in water. Another activator is dextran sulfate.
i;owever, these activators of the contact phase are non-physiological and are also diff;cult to standardize.
For example, the coagulation time oF a kaolin~activated APTT depends to a quite considerab;e extent on the particle size of the act;va~or. Moreover, some coagulation factors are so strongly adsorbed on surface~active materials of this type, that the latter are used as adsorbents for the prepara~ion of the contact factors. However, in addition~
the colnpos-,tion of the phospholip;d contained in a reagen~
of this type is essential for the result~ Furthermore, the lensth of the preincubation time, after which the actual reaction leading to the forma-ion of the clot is started by "recalcification", plays an inportant part, since activated coagulation factors are inhib;ted by inhibitors in the plasma, especially antithrombin II~.
SuLfatidcs are regarded as being physiological activators of coagulation (Fujikawa e~ al., Biochem. 19 (1980), 1~22-133~, and rans and Griffin~ Blood, 59 (19S2), 69-75). Sulfatides belong to the class of glycosphingo-lipids and they conta;n a suLfate group on the galactose ring~ Various species belong to this group of ~L~;5~ 13 _ 3 compounds, and ~hey differ in the nature of the fatty acid chain. They can be detected in all tissues, in the mem-branes, and in espec;ally large amounts in the bra;n, from which they can be obta;ned in a very pure form.
Sulfatides are more effective than kaolin for the contact activation of plasma.
The end point of conventional determinations of APTT is the measurement of a fibrin clot~ Following the -ForMation of a clot of this type is possible only with technical difficulty. Quite a number of devices have been developed~ and these make use of a variety of mechanical, electrical or optical procedures.
Since the introduction of chromogenic substrates for coaguLation factors, attempts have also been made to emp~oy them for the determinat;on of the coagulation enzymes. The advantage of chromogenic substrates is the poss;b;lity of straightforward standardisation of the low molecular weight substrates~ in contrast to the complexO
h;gh molecular weight natural substrates. The problem of measuring the -format;on of a clot no longer ar;sesO
Moreover, chromogenic substrates have already been employed for carrying out "global tests". Thus, Yamada and Meguro, Thrombos. Res. 15 (1979), 351-358, describe a method for measur;ng the APTT. Th;s is based on activa-tion of the endogenous pathway us;ng ellagic ac;d in the presence of phospholipid~ calcium and the chromogen;c thrombin substrate H-D-Phe-P;p-Arg-pNA (p 2238). The d;sad~arltages of this procedure are the use of a non-physiological activatc,r, the lack of spec;fic;ty of the 3L~ 3 chromogenic substrate~ and the extremely long measurement ti~es (normal figure about 7~4 minutes)~ 9nly incomple~e data has been published on ~he sensitivity to the indivi~
dual cl~tt ing f ~ctors .
Another method is described by P. Aiyappa, Annu r~ew York ~cad. Sci. (1~1), pages ~12-~21. In this tech-n;que of measurement~ plasma is activated by ellagic acid in the presence of phospholipid and calcium ions fcr 5 minutes~ and the thrombin formed in this time is measured using a chromo~enic substrate~ This method can lead to the 'ormation of ,ibrin, and active thrombin undergoes ;nclusion ;n th;s. Moreover, thromb;n can be further deactivated by inhibition. On the other hand, it is possible that~
within the selected ;ncubation time, the activation of patho logical plasmas takes place e;ther not at all or only ;ncom-pletely, although they are actually capable of this~ even though slowlyO The procedure described by Aiyappa cannot be used to measure coagulation in deficient plasmas~
It has now been ~ound~ surprisingly~ that it is poss;ble to elim;nate the essential disadvantages of the described APTT methods with chromogenic substrates by using a sulfatide as the physiological activator in com-bination with a highly specific thromb;n substrate~
Thus the invention relates to a procedure for the determination of the activated partial thromboplastin time using an activator, a coagulation-active phospholip;d or mixture of phospholipids~ calcium ions and a chromogenic substrate ,or thrombin, ~Jh,ch comprises the activator being a sulfa~ide or a mix~ure of sulfatides.
~Z~ 3 The invention also relates to a reagent for the determination of activated partial thromboplastin time~
comprising a sulfatide, a phospl1olip;d~ a soluble calcium salt and a chromogen;~c substrate in the freeze-dried form.
U~ilisable sulfatides are commercially available, for example from Serva, Sigma or Supelco~ It is advan-tageous tc use sulfatides which have~ on thin~layer chroma-tography on silica gel~ an RF of 0.2-003~ preferably 0.25, with a mixture of chloroform/methanol/wa~.er 65:25:4 as the mobile phase and an RF of 0.25~0.35~ preferably 0.31~
using chloroform/methanol 40:15. However, i~ is also pos-s;ble to obtain sulfatides of this type by processes which have been described, for example by Hara and Radin, Anal.
e;ochem. 100, 36~-370 (197~), or from dried acetone extract of brain~ t~lorthwhile test concentrations are betweerl 0~1 and 5Q ~g/ml. The sulfatide can initially be suspended at a concentration of 0.01 g/l in 50 mmol/l of ~EPES buffer, pH
7.6, and this suspension used for prepar;ng the reagent.
The phospholipids which can be used are the animal and veget-7ble lipids employed in conventional reagents~ It is particularly advantageous to use lipids from human plate-lets or an extract from human placenta. It is ;mportant that these lipids have no thromboplastic activity, so that the exogenous path~lay is not also activated.
The chromogenic substrate for thrombin must be speci~i.c for this enzyme to be possible tG employ it in this test, sînce. of course, the intention is to measure thrornbin selective'y in the presence of the other coagulation factors which are likew;se activated~ Chromo~yM tr~
~'~5~ 3 TH (Tos-Gly-Pro-Arg-p~A) and S 2160 (Bz-Phe-Val-Arg-pNA~
are relatively non-specific and also indicate other coagu-lation factors~ such as kallikrein or factors Xa and XIIa.
S 2238 (HD-Phe-Pip-Arg-pNA) is rr,ore suitable, althou~h its spec;ficity for the contact factors is again not very high.
Apart from para-nitroanilides, peptides having other chromo-phores are also suitable~ for example, coumarin derivatives~
where tne hydrolysis is followed by measur;ng the fluores-cence.
The most suitable chrornogenic pep~ides for a global test, such as APTT, are thrombin substrates sucl7 as are des~
cribed in German Patent Application 3,244,~030.8. The chromophoric derivative they contain is 5~amino~2~nitro--henzoic ac;d.
A cornpound of the general formula I
X-P~o -Arg-Ni~-~f-~o2 ( 1 ) ~here R is C1_5-alkyl or -CH~CH(CH3~2~COOC~l3, and X is H-D-Phe-, Boc- Gly- or Tosyl-Gly-, is preferably ~sed.
The substrate is preferably employed at very low concentration. The best results are obtained with 50 ~umol/l when H-D-Phe-Pro-Ar~ANB~~isopropylamide is used. The calcium concentration should be between 1 and 10 mmol/l preferably 5 mmol/l.
The sulfatide, phospholipid, chromogenic substrate and the calciurn salt, preferably ~a'c1um chloride~ are d;s-solved or suspended at pH 7.2-~.5 in a bu-ffer~ such as HEPES or Tris. IrQidazole, glycyl~lycine or triethat1olalnine buffers can also be used~
The procedure according to the invent;on ;s advan-- -tas~ously carried out at 25 37C b~ m;x;ng a plasma sample w;th the reagent at this temperature in a thermostated cell.
The reaction is followed at 405~410 nm in a photometer.
~ 1ith a typical normal plasma the extinction remains constant for some time and then increases greatly ~lithin a few seconds. The variable measured is the time necessary to reach a spec;f;ed d;fference in ext;nct;on, -ror example 0.1. In principle~ k;netic evaluation of the exponentiai curve is a!so poss;ble~ The reaction of a specified amount of substrate ;s analogous to that in a conventional APTT~
where the activated thrombin reacts with a specified amount of fibrinogen and thus induces the clot. Example 1 shows that the new procedure in the preFerred 'orrn ind;cates cnanges in the activity of each individual factor ;n tne endogenous pathway.
In contrast to the convention APTT~ in ~hich~ a,.er a certain fixed activation time, the actual reaction leading to the clot is started by add;ng CaCl2, it ;s possible for CaCl2 to be added From the st3rt -in the new procedure;
and this leads to there being one pipetting step less (monoreagent). However, in principle~ a test system analo-gous to the procedure used hitherto is also possible with the procedure accordirlg to the invention~ With this ob-jective, the chrornogenic peptide is added~ tc~etller with CaCl2, after a speci~;ed preincubation time. In this case~ it has proved to be favourable to add a little CaCl2 (about 1 l~mol/l~-to the sulfatide reagent everl duril~g æ~3 the pre;ncubat;on time, and to start the actual reaction by addition of more CaCl2 solution~ The optimal final con centration in this case is again 5 mmol/l of CaCl2.
The use o-f sulfatides for the contact activation of plasma, combined with chromogenic substrates, has not hitherto been disclosedO Manuf2cturers of reagents recommend that the sulfatides be stored below 0C. rreeze drying sulfatides together with the other necessary constituents o-f the reagent does not lead to a stable product. The APTT
is always drastically increased.
Surprisingly~ a stable and effective reagent is obtained by addition of serurn albumin, ge1atin or degraded arld che~i-cally crosslinked collage~. A co~c~ntration of only about 0.1-1% ~llows a lyophilisate of high activity to be produced, As with conventional reagents~ it is possible, in combination with deFic;ent plasma to determir)e the indivi~
dual factors of the endogenous pathway. For this purpose, ttle plasma sample w;ll be d;lu.ed ;n order o eliminate the effect of the other coagulation factors, or it is ernployed ;n an excess of the deficient plasma used.
Addition of aminoac;ds, especially glu~amine~
asparagine or glutamic acid, increases the sensitivity of the reagent to heparin.
Abbreviations:
ANBA 5-amino-2-nitrobenzoic acid Arg Arsinine Gly Glycine l-IEPES N-2-hydroxyethylpiperazinyl-N'-2-ethanesulfonic acid ., _ 9 ~
Abrre~iations continued:
Phe Phenylalanine Pip Pipecolic acid Pro Proline Tos Toluenesulfonyl Tris Tr;s(hydro~ymethyl)aminonlethane The invention is illustrated by the Example ~hich follows.
5~2-~
' ~ 10 -Example :
Factor sensit;vity of the new reagent Reagent~ 0.1 ml of 3mmol/l H-D-Phe-Pro Arg-ANB~-isopropyl-~amide chromogen;c substrate (S 82 107) 0.5 ml of Fibraccel R 1:1000 tcoagulation-active homologous phosphol;p;d com-plex, Behr;ngwerke AG) 0.5 ~l of sulfatide solution (supplied by Supelco; RF 0025 with CHCl3/MeOH
H20 6S:25:~ on silica gel) 0~01 g/l 5.0 ml of buffer (HEPES, NaCl, Ca2~: 25, 50, S mmol/l; pH 7~6~ 0~25% HaemaccelR
tGerman patent 1~11S~7~2 and 1,153,134) ixture: 100 ~l of plasma or deficient plasma ~Behr;ngwerke), congen;tal F VII-def;c;ent plasma 1 ml of reagen~
37C, measurement o-F the t;me unt;l E~05 nm = 0~1.
Def;cient plasma Seconds Factor II 1,200 Factor V 1,200 Factor VIII 378 Factor IX 498 Factor X ~48 Fac'.or XI 396 Factor XII 336 HMWK (high molecular ~eight kin1nogen) 396 Kailikrein 720 Pooled plasma 170
Claims (9)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A procedure for the determination of the activated partial thromboplastin time (APTT) using an activator, phospholipid, calcium ions and a chromogenic substrate for thrombin, which comprises the activator being a sulfa-tide or mixture of sulfatides.
2. The procedure as claimed in claim 1, wherein the chromogenic substrate is a compound of the formula I
(I) where R is C1-5-alkyl or -CH[CH(CH3)2]COOCH3, and X is H-?-Phe-, Boc-Gly- or tosyl-Gly-.
(I) where R is C1-5-alkyl or -CH[CH(CH3)2]COOCH3, and X is H-?-Phe-, Boc-Gly- or tosyl-Gly-.
3. A reagent for the determination of the activated partial thromboplastin time, which is composed of a sulfatide, phospholipid, a soluble calcium salt and a chromogenic sub-strate for thrombin.
4. The reagent as claimed in claim 3, which contains a buffer of pH 7.2-8.5.
5. The reagent as claimed in claim 3, which contains HEPES buffer of pH 7.2-8.5.
6. The reagent as claimed in claim 3, which contains an aminoacid.
7. The reagent as claimed in claim 3, which contains serum albumin, gelatin or a degraded and chemically cross-linked collagen.
8. The reagent as claimed in claim 3, which is in the freeze-dried form.
9. A process for determining a factor of the endogenous pathway using a reagent as claimed in claim 3 combined with a plasma deficient in the factor.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3311287.8 | 1983-03-28 | ||
| DE19833311287 DE3311287A1 (en) | 1983-03-28 | 1983-03-28 | METHOD FOR PHOTOMETRICALLY DETERMINING THE ACTIVATED PARTIAL THROMBOPLASTIN TIME AND REAGENT TO IT |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1250213A true CA1250213A (en) | 1989-02-21 |
Family
ID=6194903
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000450594A Expired CA1250213A (en) | 1983-03-28 | 1984-03-27 | Procedure for the photometric determination of the activated partial thromboplastin time and a reagent for this purpose |
Country Status (14)
| Country | Link |
|---|---|
| EP (1) | EP0123883B1 (en) |
| JP (1) | JPS59187800A (en) |
| AT (1) | ATE67520T1 (en) |
| AU (1) | AU582534B2 (en) |
| CA (1) | CA1250213A (en) |
| DE (2) | DE3311287A1 (en) |
| DK (1) | DK162179C (en) |
| ES (1) | ES8505115A1 (en) |
| GR (1) | GR81852B (en) |
| IE (1) | IE58463B1 (en) |
| IL (1) | IL71370A (en) |
| NO (1) | NO164443C (en) |
| NZ (1) | NZ207626A (en) |
| ZA (1) | ZA842240B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU4158785A (en) * | 1984-03-26 | 1985-11-01 | International Health Services | A method of determining the clotting time of blood and particulate reagents therefor |
| GB8426004D0 (en) * | 1984-10-15 | 1984-11-21 | Ortho Diagnostic Systems Inc | Coagulation monitoring |
| DE3504405A1 (en) * | 1985-02-08 | 1986-08-14 | Boehringer Mannheim Gmbh, 6800 Mannheim | DETERMINATION OF PROKALLIKREIN |
| SE8702556D0 (en) * | 1987-06-18 | 1987-06-18 | Kabivitrum Ab | DETERMINATION OF PROTEOLYS ACTIVE COMPONENTS |
| US4865984A (en) * | 1988-02-08 | 1989-09-12 | Mount Sinai School Of Medicine Of The City University Of New York | Dynamic continuous flow enzyme reactor |
| DE4006634A1 (en) * | 1990-03-03 | 1991-09-05 | Behringwerke Ag | FUNCTIONAL TEST TO DETERMINE PROTEIN S ACTIVITY |
| JP3180824B2 (en) * | 1991-08-30 | 2001-06-25 | シスメックス株式会社 | Blood coagulation reagent |
| DE59309374D1 (en) * | 1992-05-15 | 1999-03-25 | Immuno Ag | Reagent for determining the activated partial thromboplastin time (aPTT) |
| AT402074B (en) * | 1993-04-30 | 1997-01-27 | Immuno Ag | Reagent for determination of the aptt |
| AT398983B (en) * | 1992-05-15 | 1995-02-27 | Immuno Ag | Determn. of activated partial thromboplastin time - using mixt. of sulphatide(s) and kaolin as coagulation initiator |
| ES2131543T3 (en) * | 1993-06-30 | 1999-08-01 | Stiftung Fur Diagnostische For | MEASURING THE TIME OF ACTIVATED PARTIAL THROMBOPLASTINE (APTT) IN A SINGLE-STAGE REACTION. |
| US5780255A (en) * | 1995-06-09 | 1998-07-14 | Instrumentation Laboratory, S.P.A. | Protein C pathway screening test |
| ATE398775T1 (en) | 2001-05-09 | 2008-07-15 | Axis Shield Asa | TEST APPARATUS |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3486981A (en) * | 1965-03-15 | 1969-12-30 | Roy E Speck | Substances relating to testing of blood-coagulation |
| DE2915310A1 (en) * | 1979-04-14 | 1980-10-30 | Behringwerke Ag | NEW PARTIAL THROMBOPLASTINE AND THEIR USE |
| CA1161432A (en) * | 1980-02-12 | 1984-01-31 | Lars G. Svendsen | Tripeptide derivatives and their application in assaying enzymes |
| DE3244030A1 (en) * | 1982-11-27 | 1984-05-30 | Behringwerke Ag, 3550 Marburg | CHROMOGENIC COMPOUNDS, METHOD FOR THEIR PRODUCTION AND THEIR USE |
| US4487060A (en) * | 1983-05-18 | 1984-12-11 | Glenayre Electronis, Ltd. | Railway brake pressure monitor |
-
1983
- 1983-03-28 DE DE19833311287 patent/DE3311287A1/en not_active Withdrawn
-
1984
- 1984-02-27 DK DK109084A patent/DK162179C/en active
- 1984-03-22 DE DE8484103163T patent/DE3485067D1/en not_active Expired - Fee Related
- 1984-03-22 AT AT84103163T patent/ATE67520T1/en not_active IP Right Cessation
- 1984-03-22 EP EP84103163A patent/EP0123883B1/en not_active Expired - Lifetime
- 1984-03-26 NZ NZ207626A patent/NZ207626A/en unknown
- 1984-03-26 ES ES530983A patent/ES8505115A1/en not_active Expired
- 1984-03-26 GR GR74205A patent/GR81852B/el unknown
- 1984-03-27 IL IL71370A patent/IL71370A/en not_active IP Right Cessation
- 1984-03-27 CA CA000450594A patent/CA1250213A/en not_active Expired
- 1984-03-27 AU AU26211/84A patent/AU582534B2/en not_active Ceased
- 1984-03-27 JP JP59057507A patent/JPS59187800A/en active Granted
- 1984-03-27 IE IE74384A patent/IE58463B1/en not_active IP Right Cessation
- 1984-03-27 ZA ZA842240A patent/ZA842240B/en unknown
- 1984-03-28 NO NO841231A patent/NO164443C/en unknown
Cited By (2)
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| DE3485067D1 (en) | 1991-10-24 |
| DK162179C (en) | 1992-03-02 |
| NO841231L (en) | 1984-10-01 |
| IE58463B1 (en) | 1993-09-22 |
| ATE67520T1 (en) | 1991-10-15 |
| EP0123883A3 (en) | 1988-02-10 |
| DK109084A (en) | 1984-09-29 |
| IE840743L (en) | 1984-09-28 |
| JPS59187800A (en) | 1984-10-24 |
| EP0123883B1 (en) | 1991-09-18 |
| NZ207626A (en) | 1987-05-29 |
| JPH0365958B2 (en) | 1991-10-15 |
| ES530983A0 (en) | 1985-05-01 |
| DK162179B (en) | 1991-09-23 |
| AU2621184A (en) | 1984-10-04 |
| IL71370A (en) | 1989-06-30 |
| DK109084D0 (en) | 1984-02-27 |
| AU582534B2 (en) | 1989-04-06 |
| ES8505115A1 (en) | 1985-05-01 |
| EP0123883A2 (en) | 1984-11-07 |
| ZA842240B (en) | 1984-10-31 |
| NO164443C (en) | 1990-10-03 |
| DE3311287A1 (en) | 1984-10-04 |
| NO164443B (en) | 1990-06-25 |
| IL71370A0 (en) | 1984-06-29 |
| GR81852B (en) | 1984-12-12 |
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| MKEX | Expiry |