A Rapid, Quantitative Determination of Clottable Fibrinogen Unaffected by Heparin EDWARD
J.
HERSHGOLD,
M.D.,
AND BARBARA M A R T I N ,
BA (CSLT)
Departments of Medicine and Pathology, University of Utah College of Medicine, Salt Lake City, Utah 84132
ABSTRACT
K N O W L E D G E of the plasma level of clottable fibrinogen is frequently important in following the clinical courses of patients who are also heparinized. Such situations occur in patients being treated with heparin for disseminated intravascular coagulation, in those heparinized for extracorporeal bypass, and particularly in investigations into the possible effects of various prosthetic devices on the blood. In these last two instances relatively high heparin levels may be maintained. Since heparin interferes with the action of thrombin, it may also interfere with assays for clottable fibrinogen.7 Even the use of an excess of thrombin in the assay mixture may be unsatisfactory, since one cannot be certain that all the fibrinogen has been converted to fibrin in this circumstance. However, Reptilase-R is not in-
Received July 22, 1974; received revised manuscript September 5, 1974; accepted for publication September 5, 1974. Supported by a research grant AM-04495 from the National Institutes of Health. Address reprint requests to Dr. Hershgold, University of Utah College of Medicine, 50 N. Medical Dr., Salt Lake City, Utah 84132. 231
hibited by heparin, and this enzyme, derived from the venom of Bothrops atrox, will polymerize fibrinogen also. It has been used as a substitute for thrombin in the study of fibrinogen abnormalities to give a "Reptilase time." 5 We present here a rapid, quantitative turbidimetric assay for clottable fibrinogen based on the substitution of Reptilase for thrombin in the method first described by Ellis and Stransky 4 and subsequently modified by Burmester and associates.2 It is unaffected by heparin or by Pyran copolymer. Also, Reptilase has other advantages when compared with thrombin, including its greater stability, the ease of obtaining a homogeneous mixture, and a more rapid attainment of equilibrium. Experimental Sample: Venous blood is drawn into one tenth its volume of 3.8% sodium citrate. Plasma is separated by centrifugation at 2,250 X g for 5 minutes at room temperature; 2 ml. of plasma are required for the assay. Reagents: Reptilase-R (Abbott Scientific),
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Hershgold, Edward J., and Martin, Barbara: A rapid, quantitative determination of clottable fibrinogen unaffected by heparin. Am. J. Clin. Pathol. 63: 231-236, 1975. A turbidimetric assay for clottable plasma fibrinogen which is not sensitive to heparin or Pyran inhibition is described. The basis of the assay is the substitution of Reptilase-R for the thrombin usually employed. The assay correlates very well with a thrombin turbidimetric method and also has other advantages, including better stability of the clotting enzyme and more rapid attainment of equilibrium. (Key words: Clottable fibrinogen; Heparin; Assay, turbidimetric; Reptilase-R; Thrombin.)
232
HERSHGOLD AND MARTIN
63
3. Deliver 3.0 ml. of the diluted plasma rapidly into the cuvette, cover with Parafilm, and mix rapidly through four directions for about 15 seconds. 4. Decant the remaining 3.0 ml. of dilute plasma into another cuvette for use as the blank. 5. Set a spectrophotometer, adequate for optical density reading at 300 nm., to that wavelength and adjust to zero absorbance using the blank. 6. Determine the absorbance of the Reptilase-R, dilute plasma sample at 10 minutes, and plot this value against the known fibrinogen concentration, constructing a standard curve. 7. Carry out this same procedure for samples in which fibrinogen levels are to be determined. Use the observed absorbance to read the fibrinogen level from the standard curve. Note that the standard curve can also be constructed using plasma standards in which fibrinogen levels have been determined by other quantitative methods. Correlation between a Thrombin-Turbidimetric Assay of Fibrinogen and the Reptilase Method A blood sample was taken from each of 24 normal individuals and a fibrinogen assay performed on each by the method of Burmester and associates2 and by the Reptilase method described here. Correlation between the two assays was tested by a least-squares fit method employing a standard Olivetti Programma computer program. Correlation coefficient for the results of this experiment for 24 plasma samples was r = 0.95. The coefficient of variation for the method of Burmester and associates2 was 2.6%, and that for the Reptilase method was 2.7%. In 22 normal individuals the range (mean ± 2 S.D.) with the Burmester method was 174-386 mg. per dl. and that with the Reptilase method was 151-375 mg. per dl. We have used the Burmester method as standard for the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
heparin sodium (Upjohn Co. Lot no. NDC9-317-2, from beef lung) and Pyran copolymer (Lot no. XA146-85-2, NSC46015MD, courtesy of Dr. D. S. Breslow, Hercules, Inc., Wilmington, Del.) were used. Preparation of Dialyzed Reptilase: Reptilase as obtained from the manufacturer contains methylparaoxy-benzoate which must be removed prior to use, as this reagent absorbs strongly in the ultraviolet. The Reptilase (enzyme 100 jug., carrier 34 mg.) was reconstituted according to the manufacturer's instructions. Aliquots were then dialyzed against a barbital-glycine-NaCl buffer at pH 7.8 (5.56 Gm. sodium diethyl barbiturate, 11.93 Gm. NaCl, 22 Gm. glycine per liter, titrated to pH 7.8 with 1 N HC1). Five 1-ml. fractions were dialyzed against 1 liter of buffer for 4 hours at room temperature with continuous mixing, the buffer being changed hourly. After dialysis the Reptilase solution had an optical density of 0.025 at 300 nm. The dialyzed aliquots were then delivered into plastic tubes, stoppered well, and stored at - 7 0 C. Prepared in this manner, Reptilase is stable for at least 14 weeks. Preparation of the Standard Curve. The fibrinogen level of normal pooled plasma was first determined by the turbidimetric method of Burmester and associates.2 The plasma was then diluted with barbitalNaCl buffer to provide fibrinogen concentrations from 100% to 10% of the original pool, in 10% decrements. Following addition of Reptilase the optical densities (O.D.) given by these diluted plasma samples were then plotted on linear graph paper, with O.D. on the ordinate and fibrinogen concentration (mg. per dl.) on the abscissa. The procedure is as follows: 1. Dilute 2.0 ml. of plasma in 8.0 ml. of barbital-saline buffer, pH 7.8. 2. Deliver and distribute evenly into the bottom of a cuvette, adequate for reading at 300 nm., 0.1 ml. of dialyzed Reptilase-R.
A.J.C.P.—Vol.
February 1975
ASSAY FOR CLOTTABLE FIBRINOGEN
233
Table 1. Plasma Fibrinogen Measurements by a T h r o m b i n - T u r b i d i m e t r i c and a Reptilase-Turbidimetric Method in the Presence of Various Amounts of Heparin Fibrinogen Concentration Detected in Plasma, mg. per dl. Heparin, 2.0 U. per ml.
Heparin, 3.0 U. per ml.
Thrombin Method
Reptilase Method
Thrombin Method
Reptilase Method
Thrombin Method
Reptilase Method
315 279 193 86 42
338 281 191 99 42
32 96 171 83 48
338 275 188 90 45
0 0 83 83 42
338 293 188 90 45
past 3 years. During this time quality control testing with the College of American Pathologists program has shown our results to be within their acceptable range. The Effect of Heparin on the Reptilase Fibrinogen Assay Compared with its Effects on a Thrombin Fibrinogen Assay
method tends to occur with higher fibrinogen levels. Effects ofPyran Copolymer on a Thrombin Turbidimetric Assay of Fibrinogen and on the Reptilase Method
Pyran is a polyanionic, divinyl e t h e r maleic acid copolymer which, besides its other effects, will act as an anticoagulant 6 This was explored by adding heparin with heparin-like effects on thrombin. Beto normal pooled plasma so as to obtain cause of this property it was of interest plasma with final heparin concentrations to determine whether Reptilase would act of 0.6, 2.0, and 3.0 units per ml. These on fibrinogen in the presence of Pyran. concentrations were chosen as those com- Pyran solution, 1 ml., and 0.1 ml. of 40% monly achieved in various clinical situa- sodium citrate were added to 9.0 ml. of tions, as well as to study the effects of whole blood to yield a concentration of different concentrations on the assay. The 0.3 mg. Pyran/ml. of whole blood. This normal, pooled plasma containing 350 mg. concentration was chosen as that reported per dl. of fibrinogen was also diluted with to have anticoagulant effects. T h e fibrinoaged serum to obtain fibrinogen concen- gen concentration in the plasma was detertrations ranging from 350 mg. per dl. mined by the method of Burmester and 2 to 50 mg. per dl. Heparin was then added associates and by the Reptilase method, to each of these samples to obtain the and the results compared with the corresponding assays performed on a control final concentrations stated above. specimen, in which saline solution was subAlthough heparin in concentrations of stituted for Pyran solution. 2.0 units per ml. or more interfered with the thrombin-turbidimetric method, the As with the effects of heparin, Pyran Reptilase method was unaffected. A repre- copolymer did not interfere with the sentative experiment is shown in Table 1, Reptilase method, while it did interfere in which it is also shown that the great- with the thrombin technic. T h e data are est heparin interference in the thrombin shown in Table 2.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Fibrinogen content of test plasma, mg. per dl., prepared by dilution of standard plasma 350 300 200 100 50
Heparin,' 0.6 U. per ml.
234
HERSHGOLD AND MARTIN
Table 2. Effect of Pyran on T h r o m b i n Turbidimetric and Reptilase-Turbidimetric Methods for Plasma Fibrinogen Estimation Fibrinogen Concentration, mg. per dl., in Duplicates Control Plasma with Pyran Added, 0.3 mg. per ml.
Control Plasma Thrombin Method
Reptilase Method
Thrombin Method
Reptilase Method
299 283
283 275
42 54
305 305
Fibrinogen Concentration, mg. per dl. Final Thrombin Concentration .125 U. per ml. .625U.perml. 1.25 U. per ml.
Control Plasma
2.0 U. Heparin Added per ml.
3.0 U. Heparin Added per ml.
343 343 343
J.
HERSHGOLD,
M.D.,
AND BARBARA M A R T I N ,
BA (CSLT)
Departments of Medicine and Pathology, University of Utah College of Medicine, Salt Lake City, Utah 84132
ABSTRACT
K N O W L E D G E of the plasma level of clottable fibrinogen is frequently important in following the clinical courses of patients who are also heparinized. Such situations occur in patients being treated with heparin for disseminated intravascular coagulation, in those heparinized for extracorporeal bypass, and particularly in investigations into the possible effects of various prosthetic devices on the blood. In these last two instances relatively high heparin levels may be maintained. Since heparin interferes with the action of thrombin, it may also interfere with assays for clottable fibrinogen.7 Even the use of an excess of thrombin in the assay mixture may be unsatisfactory, since one cannot be certain that all the fibrinogen has been converted to fibrin in this circumstance. However, Reptilase-R is not in-
Received July 22, 1974; received revised manuscript September 5, 1974; accepted for publication September 5, 1974. Supported by a research grant AM-04495 from the National Institutes of Health. Address reprint requests to Dr. Hershgold, University of Utah College of Medicine, 50 N. Medical Dr., Salt Lake City, Utah 84132. 231
hibited by heparin, and this enzyme, derived from the venom of Bothrops atrox, will polymerize fibrinogen also. It has been used as a substitute for thrombin in the study of fibrinogen abnormalities to give a "Reptilase time." 5 We present here a rapid, quantitative turbidimetric assay for clottable fibrinogen based on the substitution of Reptilase for thrombin in the method first described by Ellis and Stransky 4 and subsequently modified by Burmester and associates.2 It is unaffected by heparin or by Pyran copolymer. Also, Reptilase has other advantages when compared with thrombin, including its greater stability, the ease of obtaining a homogeneous mixture, and a more rapid attainment of equilibrium. Experimental Sample: Venous blood is drawn into one tenth its volume of 3.8% sodium citrate. Plasma is separated by centrifugation at 2,250 X g for 5 minutes at room temperature; 2 ml. of plasma are required for the assay. Reagents: Reptilase-R (Abbott Scientific),
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Hershgold, Edward J., and Martin, Barbara: A rapid, quantitative determination of clottable fibrinogen unaffected by heparin. Am. J. Clin. Pathol. 63: 231-236, 1975. A turbidimetric assay for clottable plasma fibrinogen which is not sensitive to heparin or Pyran inhibition is described. The basis of the assay is the substitution of Reptilase-R for the thrombin usually employed. The assay correlates very well with a thrombin turbidimetric method and also has other advantages, including better stability of the clotting enzyme and more rapid attainment of equilibrium. (Key words: Clottable fibrinogen; Heparin; Assay, turbidimetric; Reptilase-R; Thrombin.)
232
HERSHGOLD AND MARTIN
63
3. Deliver 3.0 ml. of the diluted plasma rapidly into the cuvette, cover with Parafilm, and mix rapidly through four directions for about 15 seconds. 4. Decant the remaining 3.0 ml. of dilute plasma into another cuvette for use as the blank. 5. Set a spectrophotometer, adequate for optical density reading at 300 nm., to that wavelength and adjust to zero absorbance using the blank. 6. Determine the absorbance of the Reptilase-R, dilute plasma sample at 10 minutes, and plot this value against the known fibrinogen concentration, constructing a standard curve. 7. Carry out this same procedure for samples in which fibrinogen levels are to be determined. Use the observed absorbance to read the fibrinogen level from the standard curve. Note that the standard curve can also be constructed using plasma standards in which fibrinogen levels have been determined by other quantitative methods. Correlation between a Thrombin-Turbidimetric Assay of Fibrinogen and the Reptilase Method A blood sample was taken from each of 24 normal individuals and a fibrinogen assay performed on each by the method of Burmester and associates2 and by the Reptilase method described here. Correlation between the two assays was tested by a least-squares fit method employing a standard Olivetti Programma computer program. Correlation coefficient for the results of this experiment for 24 plasma samples was r = 0.95. The coefficient of variation for the method of Burmester and associates2 was 2.6%, and that for the Reptilase method was 2.7%. In 22 normal individuals the range (mean ± 2 S.D.) with the Burmester method was 174-386 mg. per dl. and that with the Reptilase method was 151-375 mg. per dl. We have used the Burmester method as standard for the
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
heparin sodium (Upjohn Co. Lot no. NDC9-317-2, from beef lung) and Pyran copolymer (Lot no. XA146-85-2, NSC46015MD, courtesy of Dr. D. S. Breslow, Hercules, Inc., Wilmington, Del.) were used. Preparation of Dialyzed Reptilase: Reptilase as obtained from the manufacturer contains methylparaoxy-benzoate which must be removed prior to use, as this reagent absorbs strongly in the ultraviolet. The Reptilase (enzyme 100 jug., carrier 34 mg.) was reconstituted according to the manufacturer's instructions. Aliquots were then dialyzed against a barbital-glycine-NaCl buffer at pH 7.8 (5.56 Gm. sodium diethyl barbiturate, 11.93 Gm. NaCl, 22 Gm. glycine per liter, titrated to pH 7.8 with 1 N HC1). Five 1-ml. fractions were dialyzed against 1 liter of buffer for 4 hours at room temperature with continuous mixing, the buffer being changed hourly. After dialysis the Reptilase solution had an optical density of 0.025 at 300 nm. The dialyzed aliquots were then delivered into plastic tubes, stoppered well, and stored at - 7 0 C. Prepared in this manner, Reptilase is stable for at least 14 weeks. Preparation of the Standard Curve. The fibrinogen level of normal pooled plasma was first determined by the turbidimetric method of Burmester and associates.2 The plasma was then diluted with barbitalNaCl buffer to provide fibrinogen concentrations from 100% to 10% of the original pool, in 10% decrements. Following addition of Reptilase the optical densities (O.D.) given by these diluted plasma samples were then plotted on linear graph paper, with O.D. on the ordinate and fibrinogen concentration (mg. per dl.) on the abscissa. The procedure is as follows: 1. Dilute 2.0 ml. of plasma in 8.0 ml. of barbital-saline buffer, pH 7.8. 2. Deliver and distribute evenly into the bottom of a cuvette, adequate for reading at 300 nm., 0.1 ml. of dialyzed Reptilase-R.
A.J.C.P.—Vol.
February 1975
ASSAY FOR CLOTTABLE FIBRINOGEN
233
Table 1. Plasma Fibrinogen Measurements by a T h r o m b i n - T u r b i d i m e t r i c and a Reptilase-Turbidimetric Method in the Presence of Various Amounts of Heparin Fibrinogen Concentration Detected in Plasma, mg. per dl. Heparin, 2.0 U. per ml.
Heparin, 3.0 U. per ml.
Thrombin Method
Reptilase Method
Thrombin Method
Reptilase Method
Thrombin Method
Reptilase Method
315 279 193 86 42
338 281 191 99 42
32 96 171 83 48
338 275 188 90 45
0 0 83 83 42
338 293 188 90 45
past 3 years. During this time quality control testing with the College of American Pathologists program has shown our results to be within their acceptable range. The Effect of Heparin on the Reptilase Fibrinogen Assay Compared with its Effects on a Thrombin Fibrinogen Assay
method tends to occur with higher fibrinogen levels. Effects ofPyran Copolymer on a Thrombin Turbidimetric Assay of Fibrinogen and on the Reptilase Method
Pyran is a polyanionic, divinyl e t h e r maleic acid copolymer which, besides its other effects, will act as an anticoagulant 6 This was explored by adding heparin with heparin-like effects on thrombin. Beto normal pooled plasma so as to obtain cause of this property it was of interest plasma with final heparin concentrations to determine whether Reptilase would act of 0.6, 2.0, and 3.0 units per ml. These on fibrinogen in the presence of Pyran. concentrations were chosen as those com- Pyran solution, 1 ml., and 0.1 ml. of 40% monly achieved in various clinical situa- sodium citrate were added to 9.0 ml. of tions, as well as to study the effects of whole blood to yield a concentration of different concentrations on the assay. The 0.3 mg. Pyran/ml. of whole blood. This normal, pooled plasma containing 350 mg. concentration was chosen as that reported per dl. of fibrinogen was also diluted with to have anticoagulant effects. T h e fibrinoaged serum to obtain fibrinogen concen- gen concentration in the plasma was detertrations ranging from 350 mg. per dl. mined by the method of Burmester and 2 to 50 mg. per dl. Heparin was then added associates and by the Reptilase method, to each of these samples to obtain the and the results compared with the corresponding assays performed on a control final concentrations stated above. specimen, in which saline solution was subAlthough heparin in concentrations of stituted for Pyran solution. 2.0 units per ml. or more interfered with the thrombin-turbidimetric method, the As with the effects of heparin, Pyran Reptilase method was unaffected. A repre- copolymer did not interfere with the sentative experiment is shown in Table 1, Reptilase method, while it did interfere in which it is also shown that the great- with the thrombin technic. T h e data are est heparin interference in the thrombin shown in Table 2.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Fibrinogen content of test plasma, mg. per dl., prepared by dilution of standard plasma 350 300 200 100 50
Heparin,' 0.6 U. per ml.
234
HERSHGOLD AND MARTIN
Table 2. Effect of Pyran on T h r o m b i n Turbidimetric and Reptilase-Turbidimetric Methods for Plasma Fibrinogen Estimation Fibrinogen Concentration, mg. per dl., in Duplicates Control Plasma with Pyran Added, 0.3 mg. per ml.
Control Plasma Thrombin Method
Reptilase Method
Thrombin Method
Reptilase Method
299 283
283 275
42 54
305 305
Fibrinogen Concentration, mg. per dl. Final Thrombin Concentration .125 U. per ml. .625U.perml. 1.25 U. per ml.
Control Plasma
2.0 U. Heparin Added per ml.
3.0 U. Heparin Added per ml.
343 343 343
