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A new APTT assay employing a chromogenic substrate and a centrifugal autoanalyzer
RESEARCH 15; 3jl-3j8 Pergamon Press Ltd.1979. Printed in Great Britain
THROMBOSIS
A NEW APTT ASSAY EMPLOYING A CHROMOGENIC SUBSTRATE AND A CENTRIFUGAL AUTOANALYZER
Kane0 YAMAD; and Takashi PEG& *
Department of Pediatrics School of Medicine, Keio University, Tokyo, Japan
**
Research Project of Hematology, Ogikubo Hospital, Tokyo: Japan
(Received 22.2.1979; in revised form 22.3.1979 Accepted by Editor H. Stormorken) ABSTRACT Using the synthetic chromogenic substrate for thrombin, S-2238, a new assay method based on the activated partial thromboplastin test (APTT) was devised with an autoanalyzer,‘Centrifichem system-400. Determinations at maximum optical absorbance of the reaction mixture and at half maximum, revealed good correlations between the values of the present APTT and those of the standard APTT: the correlation coefficients were 0.921 (p
INTRODUCTION Although assays for coagulation factors employing chromogenic substrates have been developed, the activated partial thromboplastin test (APTT) which represents one of the most widely applied screenig tests in coagulation studies, has not previously been adapted for use with chromogenic substrates. Experiments to develop a new method based on the APTTand employing Presentaddress: Departmentof Pediatrics, St. HariannaUniversity School of Medicine, Sugao 2095, Tabatsu-ku,Kawasaki-shi Kanagawa 213, Japan
351
chromogenic substrates for thrombin were therefore carried out. Of the chromogenic substrates for thrombin such as S-2160 (l-5) and S-2238 (6-81, the latter was used in the present work. An autoanalyzer, Centrifichem system-400 was employed for the determinations. By application of this autoanalyzer in the APTT with a chromogenic substrate, a procedure for instant mixing of the reagent and sample was developed. Furthermore, the APTT could be carried out at low cost, in spite of the use of a synthetic chromogenic substrate. MATERIALS AND METHODS Sampling of plasma. Blood was obtained in l/10 its volume of 0.13 mol/l sodium citrate, and then centrifuged at 1500g for 15 min at 4OC. The samples examined were from healthy adults, healthy newborns, cases with hemophilia A, hemophilia B, von Willebrand's disease, factor X deficiency, and congenital afibrinogenemia. Chromogenic substrate. H-D-Phe-Pip-Arg-NH-@-NO2*2IiCl (S-2238, Kabi Vitrum Ltd.), a substrate for thrombin, was used. Twentyfive mg of the substrate was dissolved in 10 ml of diskilled wa+a;;&providing a solution which could be stored at 4 C for 3-6 Prior to use rn the assay , one volume of the solution . was diluted with three volumes of distilled water to obtain a solution containing 1 m mol/l S-2238. Phospholipid.
Activated cephaloplastin (Dade Camp.) was used.
Calcium chloride. was used.
A 100 mmol/lCaC12solution
Buffer solution. Fiftymmol/l ionic strength 0.15 was used.
in distilled water
Tris HCl saline (pH 8.2) buffer of
S-2238 solution, two Reaction reagent. One volumeoflmmol/l volumes of activated cephaloplastin, two volumes of 100 m mol/l CaCl2 and ten volumes of the Tris buffer solution were successively mixed before the assay. Plasmin. Human plasmin (Midori Camp.) was dissolvedindistilled water. Twenty CTA u/ml of plasmin solution was prepared for use. Heparin. water.
Heparin sodium (Novo Camp.) was diluted in distilled
Autoanalyzer. A Centrifichem system-400 (9) was employed as the instrument of determination in the present APTT. Also, an autopipetter was used in the autoanalyzing system. Plasma wasdiluted automatically with the Tris buffer solution during the autopipetting procedure. Both instruments were products of Union Carbide Company (New York). First, 350 ul of the reaction reagent and 50 ul of the diluted plasma were placed separately on the transfer disc by the autopipetter. The loading transfer disc was then located in the
A NEW AFTT ASSAY
Vo1.15,No.3/4
353
rotor of the autoanalyzer. The temperature of the cuvetteinthe analyzer was adjusted to 37oC, the wavelength of the spectrophotometer was set at 405 nm and the time of checking was set at 30 sec. When the spin button was depressed and the transfer disc rotated, the sample and reagent instantly flowed intothecuvette by centrifugal force. The reaction process was checked every 30 sec. By following this procedure, 28 samples could be measured simultaneously. A prodedure of washing the cuvette followed by dilution of the examined material is required for fluent movement with the present autoanalyzer system. In orderto determine the suitable dilution of plasma to be used, samples at various dilutions were first tested. When the plasma volume within the 50 ul sample fell below 35 ul, the reaction curves showedarapid divergence from the case of 50 ~1 plasma (i.e. without buffer), the APTT being remarkably prolonged. In the present assay, therefore, 40 ul of plasma and 10 l.11 of the Tris buffer were picked up separately by the adjusted autopipetter, mixed, and then loaded onto the transfer disc as the sample. For in vitro experiments to determine the influence of heparin or plasmin on the present method, 5 ~1 of each solution was placed in the groove of the transfer disc. Such pipetting was performed by a manual technique at the time of autopipetting and the materials were separately placed from both the reaction reagent and sample. Five ~1 of saline was used as control.
RESULTS Twenty samples obtained from normal healthy adults gave the reaction curves shown in Fig. 1. In order to select the optimum point for the APTT determination, the following three assessments were made.
FIG. 1 APTT reaction curves bv the oresent methodincaiesofhealthy adults. A: 7 min 22 sec+lmin 13 sec. B: 6ml'n 28sec + 1 min 13 sec. C:>min 30 sec+lmin 20sec. For explanation of A, B and C, see text.
Activation
time (min)
Correlations between results of the present method and the standard method.
TABLE 1 Present APTT and standard APTT results for healthy adults, newborns and cases with various coagulation disorders.
disease
Vol.1 j,XO.3/$
A
SEW APT-TASSAY
355
The average value of the reaction time where the reaction attained the absorbance maximum was estimated. This gave 7 min 22 set + 1 min 13 set, which we termed determination A. Second, the average value of the reaction time where the absorbance of the reaction mixture attained half maximum was estimated. This gave 6 min 28 set + 1 min 13 set, which we termed determination B. Third, the average value of the reaction time wherethe reaction attained an absorbance of 0.025 was estimated. This gave 5 min 30 set + 1 min 20 set, which we termed determination C. The co&elations for each of these three determinations between the values obtained by our method and those obtained by the standrd APTT method are shown in Fig. 2. Both A and 0 correlated well (r-0.921, pcO.01, and r=0.887, pCO.01, respectively),while C correlated less significantly with the standard APTT results. It appeared therefore that A and/or B were useful for APTT determinations by the present method. The following results thus relate to evaluation by A and B. Studies on the APTT by the present and standard methodswere carried outon various subjects (Table 1). The patterns of the reaction curves in some cases are shown in Fig. 3. The patterns were characteristic for different cases. Those in a newborn (which represents a physiologically deficient stage as regards vitamin I(dependent coagulation factors) and a case with factor X deficiency, appeared to be similar, although the former reaction curve became elevated earlier. The patterns in hemophilia A or B were quite different from those in the newborn or factor X deficiency. In the case of congenital afibrinogenemia the present method showed normal results , whereas the standard APTT was extremely prolonged, as expected.
IO
15
Activationtime(min) FIG. 3 APTT reaction curves by the present method in a healthy newborn and in cases with various coagulation disorders.
3%
A
NEW APlY’
ASSAY
Vol.lj,No.3/4
In order to assess the clinical utility of the present met& od more closely, the influence of both heparin and plasminon the assay were observed in vitro. In accordance with increased amounts of heparin in the assay system, APTT resultsby the present method became prolonged, reaching about twice the initial value (no heparin) at a final concentrasion of 1.25u/ml, compared with an immeasurably prolonged value for the standard APTT (Table 2). No significant change was observed in the case of values obtained by the present APTT when plasmin (final concentration 2 CTA u/ml) was added to the assay system (Fig. 4).
TABLE 2 Influence of heparin on the present APTT and standard APTT.
I
Heparin (u/mlplSSmS) 0.3
0
0.6
1.25
Presentmethod A
B
10'30" 12'30" 16'30" 23'00" 9'25"
Standardmethod 37.7" ...
10'15" 13' 5" 62.5"
17'45"
180'0" 300"<
withcutadditial withddition
ofpbsmk
Presentmethad A 11.30B 0'45'
11'30 830'
SMdardnuthod90.2'
29.7‘
5 Activation
ofpl& 0 51. "Ml
20
25
time( min)
FIG. 4 Influence of plasmin on the present APTT and standard APTT. ___O___ without addition of plasmin with addition of plasmin (2 CTA u/ml) -o-
A NEW AFIT ASSAY
357
DISCUSSION The primary purpose of the present study was to develop an APTT method using a synthetic substrate for thrombin. Although further studies on the present method in various clinical disorders are necessary for a full evaluation, our results indicate that S-2238 is suitable for APTT measurements. The fact that observations of the reaction curves were possible, represents an additional advantage for screening tests. It must be remembered that the synthetic substrates which have so far been developed are not completely specific for one enzyme. Plasmin has been reported to exert some influence on the substrate for thrombin, S-2238 (10). Although no significant change was observed when determinations were carried out at point A or B in the present assay, the reaction curves with and without plasmin did show some initial differences (Fig. 4). Further experiments are needed tostandardize our APTT method using chromogenic substrates, although a number of problems such as the mixing of the sample and reagent, and the reagent costs appear to have been resolved by the utilization of an autoanalyzer. According to our in vitro results with heparin, the present assay appears to be useful in monitoring heparin therapy. However, further studies in clinical heparin treatment are required.
REFERENCES 1.
SVENDSEN, L., BLOMBACK, B., BLOMBACK, M. and OLSSON, P. I. Synthetic chromogenic substrate for determination oftrypsin, thrombin and thrombin like enzymes. Thromb. Res. 1, 267, 1972.
2.
BLOMGCK, M., BLOMBACK, B., OLSSON, P. and SVENDSEN, L. The assay of antithrombin using a synthetic chromogenic substrate for thrombin. Thromb. Res. 2, 621, 1974.
3.
BERGSTROM, K. and BLOMBXCK, M. Determination of plasma prothrombin with a reaction rate analyzer using a synthetic substrate. Thromb. Res. 6, 223, 1974.
4.
VINAZZER, Ii. Photometric assay of antithrombin IlIwith chromogenic substrate. Hemostasis 4, 101, 1975.
5.
ABILDGAARD, U., LIE, M. and gDEti, 0. R. A simple amidolytic method for the determination of functionally active antithrombin RI. Stand. J. Clin. Lab. Invest. -36, 109, 1976.
6.
GAFFNEY, P. J., LAND, K. and KIRKWOOD, T. B. L. Problems in the assay of thrombin using synthetic peptides as substrates. Thromb. Res. 10, 549, 1977.
7.
ABILDGAARD, U., LIE, M. and IIIDEGARD, 0. R.
Antithrombin
(heparin cofactor) assay with "new chromogenic substrates (S-2238 and Chromozym TH)". Thromb. Res. 11, 549, 1977. 8.
SCUNLY, M. F. and KAKKAR, V. V. Methods for semimicro or automated determination of thrombin, antithrombin, and heparin cofactor using the substrate, H-D-Phe-Pid-Arg-pnitroanilide.2HCl. Clin. Chim. Acta 2, 595, 1976.
9.
ANDERSON, N. G. Analytical techniques for cell fractions, XII. A multiple cuvette rotor for a new microanalytical system. Analyt. Biochem. 28, 545, 1965.
10.
S-2238
Kabi Vitrum Ltd., Data sheet, 1976.
THROMBOSIS
A NEW APTT ASSAY EMPLOYING A CHROMOGENIC SUBSTRATE AND A CENTRIFUGAL AUTOANALYZER
Kane0 YAMAD; and Takashi PEG& *
Department of Pediatrics School of Medicine, Keio University, Tokyo, Japan
**
Research Project of Hematology, Ogikubo Hospital, Tokyo: Japan
(Received 22.2.1979; in revised form 22.3.1979 Accepted by Editor H. Stormorken) ABSTRACT Using the synthetic chromogenic substrate for thrombin, S-2238, a new assay method based on the activated partial thromboplastin test (APTT) was devised with an autoanalyzer,‘Centrifichem system-400. Determinations at maximum optical absorbance of the reaction mixture and at half maximum, revealed good correlations between the values of the present APTT and those of the standard APTT: the correlation coefficients were 0.921 (p
INTRODUCTION Although assays for coagulation factors employing chromogenic substrates have been developed, the activated partial thromboplastin test (APTT) which represents one of the most widely applied screenig tests in coagulation studies, has not previously been adapted for use with chromogenic substrates. Experiments to develop a new method based on the APTTand employing Presentaddress: Departmentof Pediatrics, St. HariannaUniversity School of Medicine, Sugao 2095, Tabatsu-ku,Kawasaki-shi Kanagawa 213, Japan
351
chromogenic substrates for thrombin were therefore carried out. Of the chromogenic substrates for thrombin such as S-2160 (l-5) and S-2238 (6-81, the latter was used in the present work. An autoanalyzer, Centrifichem system-400 was employed for the determinations. By application of this autoanalyzer in the APTT with a chromogenic substrate, a procedure for instant mixing of the reagent and sample was developed. Furthermore, the APTT could be carried out at low cost, in spite of the use of a synthetic chromogenic substrate. MATERIALS AND METHODS Sampling of plasma. Blood was obtained in l/10 its volume of 0.13 mol/l sodium citrate, and then centrifuged at 1500g for 15 min at 4OC. The samples examined were from healthy adults, healthy newborns, cases with hemophilia A, hemophilia B, von Willebrand's disease, factor X deficiency, and congenital afibrinogenemia. Chromogenic substrate. H-D-Phe-Pip-Arg-NH-@-NO2*2IiCl (S-2238, Kabi Vitrum Ltd.), a substrate for thrombin, was used. Twentyfive mg of the substrate was dissolved in 10 ml of diskilled wa+a;;&providing a solution which could be stored at 4 C for 3-6 Prior to use rn the assay , one volume of the solution . was diluted with three volumes of distilled water to obtain a solution containing 1 m mol/l S-2238. Phospholipid.
Activated cephaloplastin (Dade Camp.) was used.
Calcium chloride. was used.
A 100 mmol/lCaC12solution
Buffer solution. Fiftymmol/l ionic strength 0.15 was used.
in distilled water
Tris HCl saline (pH 8.2) buffer of
S-2238 solution, two Reaction reagent. One volumeoflmmol/l volumes of activated cephaloplastin, two volumes of 100 m mol/l CaCl2 and ten volumes of the Tris buffer solution were successively mixed before the assay. Plasmin. Human plasmin (Midori Camp.) was dissolvedindistilled water. Twenty CTA u/ml of plasmin solution was prepared for use. Heparin. water.
Heparin sodium (Novo Camp.) was diluted in distilled
Autoanalyzer. A Centrifichem system-400 (9) was employed as the instrument of determination in the present APTT. Also, an autopipetter was used in the autoanalyzing system. Plasma wasdiluted automatically with the Tris buffer solution during the autopipetting procedure. Both instruments were products of Union Carbide Company (New York). First, 350 ul of the reaction reagent and 50 ul of the diluted plasma were placed separately on the transfer disc by the autopipetter. The loading transfer disc was then located in the
A NEW AFTT ASSAY
Vo1.15,No.3/4
353
rotor of the autoanalyzer. The temperature of the cuvetteinthe analyzer was adjusted to 37oC, the wavelength of the spectrophotometer was set at 405 nm and the time of checking was set at 30 sec. When the spin button was depressed and the transfer disc rotated, the sample and reagent instantly flowed intothecuvette by centrifugal force. The reaction process was checked every 30 sec. By following this procedure, 28 samples could be measured simultaneously. A prodedure of washing the cuvette followed by dilution of the examined material is required for fluent movement with the present autoanalyzer system. In orderto determine the suitable dilution of plasma to be used, samples at various dilutions were first tested. When the plasma volume within the 50 ul sample fell below 35 ul, the reaction curves showedarapid divergence from the case of 50 ~1 plasma (i.e. without buffer), the APTT being remarkably prolonged. In the present assay, therefore, 40 ul of plasma and 10 l.11 of the Tris buffer were picked up separately by the adjusted autopipetter, mixed, and then loaded onto the transfer disc as the sample. For in vitro experiments to determine the influence of heparin or plasmin on the present method, 5 ~1 of each solution was placed in the groove of the transfer disc. Such pipetting was performed by a manual technique at the time of autopipetting and the materials were separately placed from both the reaction reagent and sample. Five ~1 of saline was used as control.
RESULTS Twenty samples obtained from normal healthy adults gave the reaction curves shown in Fig. 1. In order to select the optimum point for the APTT determination, the following three assessments were made.
FIG. 1 APTT reaction curves bv the oresent methodincaiesofhealthy adults. A: 7 min 22 sec+lmin 13 sec. B: 6ml'n 28sec + 1 min 13 sec. C:>min 30 sec+lmin 20sec. For explanation of A, B and C, see text.
Activation
time (min)
Correlations between results of the present method and the standard method.
TABLE 1 Present APTT and standard APTT results for healthy adults, newborns and cases with various coagulation disorders.
disease
Vol.1 j,XO.3/$
A
SEW APT-TASSAY
355
The average value of the reaction time where the reaction attained the absorbance maximum was estimated. This gave 7 min 22 set + 1 min 13 set, which we termed determination A. Second, the average value of the reaction time where the absorbance of the reaction mixture attained half maximum was estimated. This gave 6 min 28 set + 1 min 13 set, which we termed determination B. Third, the average value of the reaction time wherethe reaction attained an absorbance of 0.025 was estimated. This gave 5 min 30 set + 1 min 20 set, which we termed determination C. The co&elations for each of these three determinations between the values obtained by our method and those obtained by the standrd APTT method are shown in Fig. 2. Both A and 0 correlated well (r-0.921, pcO.01, and r=0.887, pCO.01, respectively),while C correlated less significantly with the standard APTT results. It appeared therefore that A and/or B were useful for APTT determinations by the present method. The following results thus relate to evaluation by A and B. Studies on the APTT by the present and standard methodswere carried outon various subjects (Table 1). The patterns of the reaction curves in some cases are shown in Fig. 3. The patterns were characteristic for different cases. Those in a newborn (which represents a physiologically deficient stage as regards vitamin I(dependent coagulation factors) and a case with factor X deficiency, appeared to be similar, although the former reaction curve became elevated earlier. The patterns in hemophilia A or B were quite different from those in the newborn or factor X deficiency. In the case of congenital afibrinogenemia the present method showed normal results , whereas the standard APTT was extremely prolonged, as expected.
IO
15
Activationtime(min) FIG. 3 APTT reaction curves by the present method in a healthy newborn and in cases with various coagulation disorders.
3%
A
NEW APlY’
ASSAY
Vol.lj,No.3/4
In order to assess the clinical utility of the present met& od more closely, the influence of both heparin and plasminon the assay were observed in vitro. In accordance with increased amounts of heparin in the assay system, APTT resultsby the present method became prolonged, reaching about twice the initial value (no heparin) at a final concentrasion of 1.25u/ml, compared with an immeasurably prolonged value for the standard APTT (Table 2). No significant change was observed in the case of values obtained by the present APTT when plasmin (final concentration 2 CTA u/ml) was added to the assay system (Fig. 4).
TABLE 2 Influence of heparin on the present APTT and standard APTT.
I
Heparin (u/mlplSSmS) 0.3
0
0.6
1.25
Presentmethod A
B
10'30" 12'30" 16'30" 23'00" 9'25"
Standardmethod 37.7" ...
10'15" 13' 5" 62.5"
17'45"
180'0" 300"<
withcutadditial withddition
ofpbsmk
Presentmethad A 11.30B 0'45'
11'30 830'
SMdardnuthod90.2'
29.7‘
5 Activation
ofpl& 0 51. "Ml
20
25
time( min)
FIG. 4 Influence of plasmin on the present APTT and standard APTT. ___O___ without addition of plasmin with addition of plasmin (2 CTA u/ml) -o-
A NEW AFIT ASSAY
357
DISCUSSION The primary purpose of the present study was to develop an APTT method using a synthetic substrate for thrombin. Although further studies on the present method in various clinical disorders are necessary for a full evaluation, our results indicate that S-2238 is suitable for APTT measurements. The fact that observations of the reaction curves were possible, represents an additional advantage for screening tests. It must be remembered that the synthetic substrates which have so far been developed are not completely specific for one enzyme. Plasmin has been reported to exert some influence on the substrate for thrombin, S-2238 (10). Although no significant change was observed when determinations were carried out at point A or B in the present assay, the reaction curves with and without plasmin did show some initial differences (Fig. 4). Further experiments are needed tostandardize our APTT method using chromogenic substrates, although a number of problems such as the mixing of the sample and reagent, and the reagent costs appear to have been resolved by the utilization of an autoanalyzer. According to our in vitro results with heparin, the present assay appears to be useful in monitoring heparin therapy. However, further studies in clinical heparin treatment are required.
REFERENCES 1.
SVENDSEN, L., BLOMBACK, B., BLOMBACK, M. and OLSSON, P. I. Synthetic chromogenic substrate for determination oftrypsin, thrombin and thrombin like enzymes. Thromb. Res. 1, 267, 1972.
2.
BLOMGCK, M., BLOMBACK, B., OLSSON, P. and SVENDSEN, L. The assay of antithrombin using a synthetic chromogenic substrate for thrombin. Thromb. Res. 2, 621, 1974.
3.
BERGSTROM, K. and BLOMBXCK, M. Determination of plasma prothrombin with a reaction rate analyzer using a synthetic substrate. Thromb. Res. 6, 223, 1974.
4.
VINAZZER, Ii. Photometric assay of antithrombin IlIwith chromogenic substrate. Hemostasis 4, 101, 1975.
5.
ABILDGAARD, U., LIE, M. and gDEti, 0. R. A simple amidolytic method for the determination of functionally active antithrombin RI. Stand. J. Clin. Lab. Invest. -36, 109, 1976.
6.
GAFFNEY, P. J., LAND, K. and KIRKWOOD, T. B. L. Problems in the assay of thrombin using synthetic peptides as substrates. Thromb. Res. 10, 549, 1977.
7.
ABILDGAARD, U., LIE, M. and IIIDEGARD, 0. R.
Antithrombin
(heparin cofactor) assay with "new chromogenic substrates (S-2238 and Chromozym TH)". Thromb. Res. 11, 549, 1977. 8.
SCUNLY, M. F. and KAKKAR, V. V. Methods for semimicro or automated determination of thrombin, antithrombin, and heparin cofactor using the substrate, H-D-Phe-Pid-Arg-pnitroanilide.2HCl. Clin. Chim. Acta 2, 595, 1976.
9.
ANDERSON, N. G. Analytical techniques for cell fractions, XII. A multiple cuvette rotor for a new microanalytical system. Analyt. Biochem. 28, 545, 1965.
10.
S-2238
Kabi Vitrum Ltd., Data sheet, 1976.