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Simplified performance of amidolytic factor X assay
THBNBOSIS RESEARCH 25; 437-440, 1982 0049-3848/82/050437-04$03.00/O Printed in t'neUSA. Cop:Jright(c) 1982 Pergamon Press Ltd. X11 rig'ntsreserved.
METHODS:
MINI-REPORT
SIMPLIFIED PERFOFWNCE OF AMSDOLYTICFACTOR X AS=
N. Egberg and P.A. Heedrran Departmentof Blood Coagulation Disorders,Karolinska Hospital,Stockholm,and Departmentof ClinicalChemistry, CktersundHospital,Ostersund,Sweden.
(Received 2.7.1981; in revised form 15.1.1982. Accepted by A.L. Copley)
for monitoring coumarol The clinical use of factor X assay e.g. treatment (1,2) is partly hamperedby the laboriousanalyticalprocedure (1,3) as comparedto one-stageprothrombinassays. In this report we describe a simplified one-stageprocedurefor factor X determinationusing the Coatest F X reagent. The procedurecould possibly be used in smaller laboratoryunits.
!&&g.& E x ki.& for kinetic, sqectrophotometric determinationof factor X in plasma was a gift from Kabi Diagnostica(Stockholm,Sweden). The followinglot nuxrbers of Russell's viper venom were tested: VR 16 SI'L, 222179, 223/79, 227/49, 240/79. w w substrates specific for factor X were Bz-Ile-Glu-pipiridyl-Gly-Arg-pNA, S 2337 and Bz-Ile-Glu-(gamma-OR)-Gly-Arg -pNA, (whereR is 50% CH31, S 2222. The batches of S 2337 and S 2222 used were: l/79, 4/79, l/80, l/81, and 69 02051, respectively. Stock solutions 1.5 mnol/l were prepared. Bland s.am@&& Standardand factor X deficient plasmas were prepared from blood drawn into citrate (9 ml blood into 1 ml 0.13 M trisodiumcitrate, pH 7.5). Samples from normals and courrarol treatedpatients for comparisonof the standard method and the modifiedmethod were drawn into 5 ml Vacutainer tubes (Be&on-Dicinson,New Jersey, USA, tube No. A 3206 SEW) containing 0.5 ml 0.13 M trisodiumcitrate (pH 7.5). Standard&&rm KEY WORDS:
was prepared by mixing plasmas from at least 20 healthy
FX assay, strates
Coatest FX kits, Chromogenic and Standard plasma. 437
peptide
sub-
vo1.73,
AYIIDOLYTICFX ASSAY PERFOZK'KE
438
so.5
male blooddonors. . . deflclent
gd.ama was obtainedfrom a patientwith knownfactorx a factorX levelof 25%.
deficzwtth
Chromogenicsubstrate solution (1.5 nznol/l) and Bssaye Rw-caC12 solution (35rig/l and 0.05mol/l,respectively) were both prepared accordingto the manufacturer.A reagentwas thenprepared consistingof 1 part buffersolution(Trisbuffer0.05mol./l, pH 7.8,with polybrene20 mg/l), 1 part substratesolutionand 1 part m-C&12 solution. The mixed reagent was preheatedto 37'~. In a measuringcuvette900 ul of reagentsolutionand 15 ul of sampleswere mixed. Immediately aftermixing,the cuvettewas placed in the thennostated(37"C) cuvetteholderof the spectrophotcnreter and the reactionratewas determined at 405 nrn. m PM 2 DL (Zeiss,Lewerkusen, W. Germany)connnectedto a linearrecorder(Servogor SE 210, Goerz,Austria).
* .
The stability of the reagent mixture (Rw-caC12 solutionmixedwith the chromogenic substrate) was investigated by incubation at varioustemperatures for 24 hours. A slew but progressive hydrolysisof the substrate was registered.SubstrateS 2237 appearedto be more susceptible to degradation than S 2222 and the hydrolysis was more rapid at high thanat low temperature (TableI). Stabllltvpf*reaaent.
Spontaneous hydrolysis of S 22Ed'S 2337 in the reagentmixture. Absorbanceat 405 nm S 2237
s 2222 Incubation time,hours 0
4z?c 0.069
: 24
0.120 0.092 0.198
Tenp 20" c
Temp 44i"c
0.069
0.028
0.214 0.139 0.379
0.152 0.083 0.366
Temp 20" c
0.028
0.134 0.296 0.723 ---
To obtaina roughestimateof to what extent various RW preparations would hydrolysethe substrate,five differentbatchesof RW were used for preparation of five reagentmixtures all containingS 2337. The reagent mixtureswere incubated at + 4OC for 22 hours. The resultsare shownin Table foundafter22 h (batch222/79)was calculatedto II. The maxtil absorbance representa degradationof about 11% of the substrate present(pm E405 10000). Four batchesof substrateS 2337 were incubatedfor 24 hours with the most and the least active Rw batches(c.f. TableII, 222/79and 240179 Therewas no differencewith regard to susceptability to respectively). cleavagebetweenthe differentsubstratebatches.
b-01.25,
so.5
It39
AHIDOLYTIC FX ASSAY PEF'..FOELWCE
Hydrolysisof S 2337 by various R’W batches in the reagent mixture. Incubation at +4’ C. Absorbanceat 405 nm after: 22 hours 0 hours 6 hours
RVVBatch No. VP 16 SIz 222/79 223/79 227/79 240/79
0.037 0.037 0.034 0.035 0.034
0.146 0.244 0.132 0.200 0.102
0.326 0.584 0.298 0.487 0.220
Tnfluencepf~substrate~MtianalvsisQf factor x. Two batches of FNV were used, one with high and one with low activityagainst the substrateS-2337 (c.f. above). The factor x values obtained after different times of storage of the reagent mixtures are essentially the same (Table III) in spite of the fact that there is a considerable background absorbance in the reagentmixture after 22 hours storage at +4'C (c.f. Table II).
Results of factor X analyses of plasma samples from one patient with congenital factor X deficiency and two coumarol treated patients. l'Vo differentreagentmixtures have been used and analyseshave been performed of newly thawn plasma samplesat zero time and after 6 and 22 hours incubationof the reagentmixture at +4'C. Factor X concentration% of standardplasma
Plasma from --X def. pat. Patient 1 Patient 2
KW 222/79 stored at +4' C for 0 hrs 6 hrs 22 hrs 26.7 72.3 41.6
27.0 75.6 39.1
25.0 71.9 41.3
RJV 240/79 stored at +4O C for 0 hrs 6 hrs 22 hrs 25.6 25.5 22.5 70.3 68.9 75.8 39.3 42.3 46.1
Npfresultsobtainedtitimodifiednnd !di.th Lb52 standard Twenty-sixsamples from coumaroland non-courrarol treatedpatientswere kit. analyzedfor factor X with the standardkit method and the modified method presentedhere. A good correlationwas found between the two methods as shown in Fig. 1.
iGO
.AXIDOLYTICFX ASSAY PERFOR.'.'AXCE
FX mod of norm
%
150-
Fig. 1 Correlation betweenthe standard kit methodand the tiified methodin a patientmaterial. Pointswithinparenthesis were excludedin the calculation of the regression line (r,0.992).
0
50
100
FX ki:t* % of norm
The experience we have had so far with the modifiedprocedurefor factor X determinations suggeststhat it is possibleto performthe assayessentially as a one-stage prothrombin assay i.e. mixinga preheated reagent with the sample and read the result. The demonstrated RW inducedcleavageof the chromogenic substratedoes not seem to be extensive.The increased background absorbancein the reagent mixture does not seem to reducethe accuracyof factorX determinations as long as the substratedegradationis limited to about 10% of available substrate. According to our resultsthe reagent mixturecouldbe storedat +4OC at least8-12 hours (during a working day) even if the RVV batchhas a high hydrolytic capacitytowardsthe substrate. If the manufacturer limitsthe enclosed RW to batches to those of low hydrolyticpotential or purifiesthe factorx activating enzymethe reagent couldprobablybe stored at room temperaturefor one working day. The eguipnentneeded for this analysis is availableat most laboratories and simplified instruments suitablefor this type of analysiswill possibly soon be available. REFERENCES 1. BERGSl'RW,K.and EGBERG,N. Determination of vitaminK sensitive coagulation factorsin plasma. Studieson threemethodsusingsynthetic chromogenic substrates.Thrombosis Res. lJ, 531-547,1978. 2. LAMMLE,B., BCXTNAMEAUX, H., MARBET,G.A.,EICHLISBERGER, R. and DUCKEKY., of oral anticoagulation by an amidolytic factorX assay. F. Monitoring A long-termstudyin 42 patients. Thromb.Haemost.44, 150-153,1980. P., KARLSSON,G. and CLAESON,G. A new sensitive 3. AURELL,L., FRIBERGER, and highlyspecificchromogenic peptidesubstratefor factorXa. Thrombosis Res. II, 595609, 1977.
METHODS:
MINI-REPORT
SIMPLIFIED PERFOFWNCE OF AMSDOLYTICFACTOR X AS=
N. Egberg and P.A. Heedrran Departmentof Blood Coagulation Disorders,Karolinska Hospital,Stockholm,and Departmentof ClinicalChemistry, CktersundHospital,Ostersund,Sweden.
(Received 2.7.1981; in revised form 15.1.1982. Accepted by A.L. Copley)
for monitoring coumarol The clinical use of factor X assay e.g. treatment (1,2) is partly hamperedby the laboriousanalyticalprocedure (1,3) as comparedto one-stageprothrombinassays. In this report we describe a simplified one-stageprocedurefor factor X determinationusing the Coatest F X reagent. The procedurecould possibly be used in smaller laboratoryunits.
!&&g.& E x ki.& for kinetic, sqectrophotometric determinationof factor X in plasma was a gift from Kabi Diagnostica(Stockholm,Sweden). The followinglot nuxrbers of Russell's viper venom were tested: VR 16 SI'L, 222179, 223/79, 227/49, 240/79. w w substrates specific for factor X were Bz-Ile-Glu-pipiridyl-Gly-Arg-pNA, S 2337 and Bz-Ile-Glu-(gamma-OR)-Gly-Arg -pNA, (whereR is 50% CH31, S 2222. The batches of S 2337 and S 2222 used were: l/79, 4/79, l/80, l/81, and 69 02051, respectively. Stock solutions 1.5 mnol/l were prepared. Bland s.am@&& Standardand factor X deficient plasmas were prepared from blood drawn into citrate (9 ml blood into 1 ml 0.13 M trisodiumcitrate, pH 7.5). Samples from normals and courrarol treatedpatients for comparisonof the standard method and the modifiedmethod were drawn into 5 ml Vacutainer tubes (Be&on-Dicinson,New Jersey, USA, tube No. A 3206 SEW) containing 0.5 ml 0.13 M trisodiumcitrate (pH 7.5). Standard&&rm KEY WORDS:
was prepared by mixing plasmas from at least 20 healthy
FX assay, strates
Coatest FX kits, Chromogenic and Standard plasma. 437
peptide
sub-
vo1.73,
AYIIDOLYTICFX ASSAY PERFOZK'KE
438
so.5
male blooddonors. . . deflclent
gd.ama was obtainedfrom a patientwith knownfactorx a factorX levelof 25%.
deficzwtth
Chromogenicsubstrate solution (1.5 nznol/l) and Bssaye Rw-caC12 solution (35rig/l and 0.05mol/l,respectively) were both prepared accordingto the manufacturer.A reagentwas thenprepared consistingof 1 part buffersolution(Trisbuffer0.05mol./l, pH 7.8,with polybrene20 mg/l), 1 part substratesolutionand 1 part m-C&12 solution. The mixed reagent was preheatedto 37'~. In a measuringcuvette900 ul of reagentsolutionand 15 ul of sampleswere mixed. Immediately aftermixing,the cuvettewas placed in the thennostated(37"C) cuvetteholderof the spectrophotcnreter and the reactionratewas determined at 405 nrn. m PM 2 DL (Zeiss,Lewerkusen, W. Germany)connnectedto a linearrecorder(Servogor SE 210, Goerz,Austria).
* .
The stability of the reagent mixture (Rw-caC12 solutionmixedwith the chromogenic substrate) was investigated by incubation at varioustemperatures for 24 hours. A slew but progressive hydrolysisof the substrate was registered.SubstrateS 2237 appearedto be more susceptible to degradation than S 2222 and the hydrolysis was more rapid at high thanat low temperature (TableI). Stabllltvpf*reaaent.
Spontaneous hydrolysis of S 22Ed'S 2337 in the reagentmixture. Absorbanceat 405 nm S 2237
s 2222 Incubation time,hours 0
4z?c 0.069
: 24
0.120 0.092 0.198
Tenp 20" c
Temp 44i"c
0.069
0.028
0.214 0.139 0.379
0.152 0.083 0.366
Temp 20" c
0.028
0.134 0.296 0.723 ---
To obtaina roughestimateof to what extent various RW preparations would hydrolysethe substrate,five differentbatchesof RW were used for preparation of five reagentmixtures all containingS 2337. The reagent mixtureswere incubated at + 4OC for 22 hours. The resultsare shownin Table foundafter22 h (batch222/79)was calculatedto II. The maxtil absorbance representa degradationof about 11% of the substrate present(pm E405 10000). Four batchesof substrateS 2337 were incubatedfor 24 hours with the most and the least active Rw batches(c.f. TableII, 222/79and 240179 Therewas no differencewith regard to susceptability to respectively). cleavagebetweenthe differentsubstratebatches.
b-01.25,
so.5
It39
AHIDOLYTIC FX ASSAY PEF'..FOELWCE
Hydrolysisof S 2337 by various R’W batches in the reagent mixture. Incubation at +4’ C. Absorbanceat 405 nm after: 22 hours 0 hours 6 hours
RVVBatch No. VP 16 SIz 222/79 223/79 227/79 240/79
0.037 0.037 0.034 0.035 0.034
0.146 0.244 0.132 0.200 0.102
0.326 0.584 0.298 0.487 0.220
Tnfluencepf~substrate~MtianalvsisQf factor x. Two batches of FNV were used, one with high and one with low activityagainst the substrateS-2337 (c.f. above). The factor x values obtained after different times of storage of the reagent mixtures are essentially the same (Table III) in spite of the fact that there is a considerable background absorbance in the reagentmixture after 22 hours storage at +4'C (c.f. Table II).
Results of factor X analyses of plasma samples from one patient with congenital factor X deficiency and two coumarol treated patients. l'Vo differentreagentmixtures have been used and analyseshave been performed of newly thawn plasma samplesat zero time and after 6 and 22 hours incubationof the reagentmixture at +4'C. Factor X concentration% of standardplasma
Plasma from --X def. pat. Patient 1 Patient 2
KW 222/79 stored at +4' C for 0 hrs 6 hrs 22 hrs 26.7 72.3 41.6
27.0 75.6 39.1
25.0 71.9 41.3
RJV 240/79 stored at +4O C for 0 hrs 6 hrs 22 hrs 25.6 25.5 22.5 70.3 68.9 75.8 39.3 42.3 46.1
Npfresultsobtainedtitimodifiednnd !di.th Lb52 standard Twenty-sixsamples from coumaroland non-courrarol treatedpatientswere kit. analyzedfor factor X with the standardkit method and the modified method presentedhere. A good correlationwas found between the two methods as shown in Fig. 1.
iGO
.AXIDOLYTICFX ASSAY PERFOR.'.'AXCE
FX mod of norm
%
150-
Fig. 1 Correlation betweenthe standard kit methodand the tiified methodin a patientmaterial. Pointswithinparenthesis were excludedin the calculation of the regression line (r,0.992).
0
50
100
FX ki:t* % of norm
The experience we have had so far with the modifiedprocedurefor factor X determinations suggeststhat it is possibleto performthe assayessentially as a one-stage prothrombin assay i.e. mixinga preheated reagent with the sample and read the result. The demonstrated RW inducedcleavageof the chromogenic substratedoes not seem to be extensive.The increased background absorbancein the reagent mixture does not seem to reducethe accuracyof factorX determinations as long as the substratedegradationis limited to about 10% of available substrate. According to our resultsthe reagent mixturecouldbe storedat +4OC at least8-12 hours (during a working day) even if the RVV batchhas a high hydrolytic capacitytowardsthe substrate. If the manufacturer limitsthe enclosed RW to batches to those of low hydrolyticpotential or purifiesthe factorx activating enzymethe reagent couldprobablybe stored at room temperaturefor one working day. The eguipnentneeded for this analysis is availableat most laboratories and simplified instruments suitablefor this type of analysiswill possibly soon be available. REFERENCES 1. BERGSl'RW,K.and EGBERG,N. Determination of vitaminK sensitive coagulation factorsin plasma. Studieson threemethodsusingsynthetic chromogenic substrates.Thrombosis Res. lJ, 531-547,1978. 2. LAMMLE,B., BCXTNAMEAUX, H., MARBET,G.A.,EICHLISBERGER, R. and DUCKEKY., of oral anticoagulation by an amidolytic factorX assay. F. Monitoring A long-termstudyin 42 patients. Thromb.Haemost.44, 150-153,1980. P., KARLSSON,G. and CLAESON,G. A new sensitive 3. AURELL,L., FRIBERGER, and highlyspecificchromogenic peptidesubstratefor factorXa. Thrombosis Res. II, 595609, 1977.