A Rapid, Quantitative Determination of Clottable Fibrinogen Unaffected by Heparin

Transcription

1 A Rapid, Quantitative Determination of Clottable Fibrinogen Unaffected by Heparin EDWARD J. HERSHGOLD, M.D., AND BARBARA MARTIN, BA (CSLT) Departments of Medicine and Pathology, University of Utah College of Medicine, Salt Lake City, Utah ABSTRACT Hershgold, Edward J., and Martin, Barbara: A rapid, quantitative determination of clottable fibrinogen unaffected by heparin. Am. J. Clin. Pathol. 63: , 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.) KNOWLEDGE 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, Supported by a research grant AM 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 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),

2 232 HERSHGOLD AND MARTIN A.J.C.P. Vol. 63 heparin sodium (Upjohn Co. Lot no. NDC , from beef lung) and Pyran copolymer (Lot no. XA , 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, 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 at 300 nm. The dialyzed aliquots were then delivered into plastic tubes, stoppered well, and stored at -70 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 barbital- NaCl 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 Deliver and distribute evenly into the bottom of a cuvette, adequate for reading at 300 nm., 0.1 ml. of dialyzed Reptilase-R. 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 A blood sample was taken from each of 24 normal individuals and a fibrinogen assay performed on each by the method of Burmester and associates 2 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 = The coefficient of variation for the method of Burmester and associates 2 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 mg. per dl. and that with the Reptilase method was mg. per dl. We have used the Burmester method as standard for the

3 February 1975 ASSAY FOR CLOTTABLE FIBRINOGEN 233 Table 1. Plasma Fibrinogen Measurements by a Thrombin-Turbidimetric and a Reptilase-Turbidimetric in the Presence of Various Amounts of Heparin Fibrinogen Concentration Detected in Plasma, mg. per dl. Heparin,' 0.6 U. per ml. Heparin, 2.0 U. per ml. Heparin, 3.0 U. per ml. Thrombin Reptilase Thrombin Reptilase Thrombin Reptilase Fibrinogen content of test plasma, mg. per dl., prepared by dilution of standard plasma 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 This was explored by adding heparin to normal pooled plasma so as to obtain plasma with final heparin concentrations of 0.6, 2.0, and 3.0 units per ml. These concentrations were chosen as those commonly achieved in various clinical situations, as well as to study the effects of different concentrations on the assay. The normal, pooled plasma containing 350 mg. per dl. of fibrinogen was also diluted with aged serum to obtain fibrinogen concentrations ranging from 350 mg. per dl. to 50 mg. per dl. Heparin was then added to each of these samples to obtain the final concentrations stated above. Although heparin in concentrations of 2.0 units per ml. or more interfered with the thrombin-turbidimetric method, the Reptilase method was unaffected. A representative experiment is shown in Table 1, in which it is also shown that the greatest heparin interference in the thrombin method tends to occur with higher fibrinogen levels. Effects ofpyran Copolymer on a Thrombin Turbidimetric Assay of Fibrinogen and on the Reptilase Pyran is a polyanionic, divinyl ethermaleic acid copolymer which, besides its other effects, will act as an anticoagulant 6 with heparin-like effects on thrombin. Because of this property it was of interest to determine whether Reptilase would act on fibrinogen in the presence of Pyran. Pyran solution, 1 ml., and 0.1 ml. of 40% sodium citrate were added to 9.0 ml. of whole blood to yield a concentration of 0.3 mg. Pyran/ml. of whole blood. This concentration was chosen as that reported to have anticoagulant effects. The fibrinogen concentration in the plasma was determined by the method of Burmester and associates 2 and by the Reptilase method, and the results compared with the corresponding assays performed on a control specimen, in which saline solution was substituted for Pyran solution. As with the effects of heparin, Pyran copolymer did not interfere with the Reptilase method, while it did interfere with the thrombin technic. The data are shown in Table 2.

4 234 HERSHGOLD AND MARTIN A.J.C.P. Vol. 63 Table 2. Effect of Pyran on Thrombin- Turbidimetric and Reptilase-Turbidimetric s for Plasma Fibrinogen Estimation Fibrinogen Concentration, mg. per dl., in Duplicates Control Plasma Control Plasma with Pyran Added, 0.3 mg. per ml. Thrombin Reptilase Thrombin Reptilase Table 3. Effects of Increased Amounts of Thrombin in a Thrombin-Turbidimetric method 2 for Measuring Fibrinogen in Heparinized Plasma Fibrinogen Concentration, mg. per dl. 2.0 U. 3.0 U. Final Heparin Heparin Thrombin Control Added Added Concentration Plasma per ml. per ml..125 U. per ml. 343 < U.perml U. per ml Table 4. Effects of Lipemia on Reptilase of Fibrinogen Determination, and Its Control by Further Dilution of the Plasma Sample* s Lipemic Plasma Sample 1 2 Determining Fibrinogen Concentration, mg. per dl. Burmester etal * Plasma samples from two patients. Reptilase, 1:5 Dil. of Plasma Reptilase, 1:10 Dil. of Plasma Effect of Increasing Thrombin Concentrations on Heparin Interference with the Thrombin- Turbidimetric In order to determine whether increasing the amount of thrombin used as reagent in the method of Burmester and associates would overcome the interference caused by heparin, normal pooled plasma was prepared so as to contain 2.0 and 3.0 units of heparin. The assay was conducted with final concentrations of units and 1.25 units per ml. of thrombin in the mixture rather than the units usually used. These new thrombin concentrations thus represented five- and tenfold increases over the amount usually employed. For higher heparin concentrations (3.0 units per ml.), the addition of as much as 10 times the usual amount of thrombin did not overcome the heparin effect, as shown in Table 3. From these results we deduce that there is probably some range of thrombin addition that would overcome the heparin effect in 3.0 units per ml. concentration since it could be done with lower heparin concentrations. However, it would be necessary to determine this experimentally. Moreover, in such an experiment, it would be important that the thrombin not be added in an amount that would result in too-rapid coagulation. Other Observations on the Reptilase With hyperlipemic test samples we found a poorer correlation between our standard Reptilase method and the method of Burmester and associates (Table 4). This could be improved by diluting the test plasma 1:10 rather than 1:5 as is described. When this modification was made, correlation between the two methods was r = 0.90 (n = 24). We also noted that the Reptilasetest plasma mixture was routinely homogeneous. Occasionally, with inexperienced

5 February 1975 ASSAY FOR CLOTTABLE FIBRINOGEN 235 technologists, a thrombin-test plasma mixture produces non-homogeneous, strand-like opacities. In our experience, changes in absorbance following the addition of thrombin to the plasma sample may continue slowly for as long as an hour. This was also noted by the originators of the method. 2 After the addition of Reptilase, however, there is no change in absorbance of the sample after the first 5 minutes (Fig. 1) Assays for clottable plasma fibrinogen have generally employed thrombin as the clotting enzyme. In the assay described here, Reptilase-R is substituted for thrombin, and definite benefits are gained thereby. These include the avoidance of heparin interference in situations where clottable fibrinogen is to be determined in the presence of that inhibitor, greater stability of the reagent, Reptilase, compared with thrombin, and a shorter equilibration time. In addition, Pyran copolymer, which may also inhibit thrombin-mediated fibrinogen determinations, does not affect the Reptilase assay. Clottable fibrinogen determinations may be of three types: chronometric, in which the time to fibrin formation is determined; turbidimetric, such as those utilized in our experiments in which changes in absorbance produced by fibrinogen polymerization are measured; and direct collection followed by some measurement of the weight of clot formed. Funk and colleagues have shown that a chronometric Reptilase assay for fibrinogen has unsatisfactory reproducibility. 5 This assay, based on the method of von Gauss, 3 is described in the manufacturers' brochure as a way of measuring fibrinogen in the presence of heparin. Even when chronometric methods are performed using thrombin, only semiquantitative re- I.I- ^ ^-p «VReptilase turbidimetric method Discussion Time (min.l FIG. 1. Times to equilibrium needed by a thrombinturbidimetric method for plasma fibrinogen assay. suits are obtained. The methods that depend on collection of the actual fibrin clot formed are least sensitive to the action of heparin, 7 probably because relatively longer periods are allowed for clot formation. Assays of this type, however, are considerably more time-consuming and complicated than are turbidimetric methods. The assay we describe here is based on the Burmester modification of the original method of Ellis and Stransky. 4 In our laboratory the thrombin-turbidimetric method has proven to be rapid and quantitative, and in quality control programs has been routinely satisfactory. The substitution of Reptilase for thrombin has not altered the reproducibility of the method and has even improved certain aspects. Although we used the Burmester method to determine the fibrinogen concentration from which we constructed the standard curve for the Reptilase method, any other quantitative method could have been used for this calibration. Degradation products of fibrinolysis will also inhibit the thrombin-fibrinogen reac-

6 236 HERSHGOLD AND MARTIN A.J.C.P. Vol. 63 tion. In this respect, Reptilase has apparently either no advantage or only a slight one over the use of thrombin in fibrinogen assays. It was earlier reported that Reptilase was less subject to such inhibition by fibrinolytic split products. 8 A more recent report suggests that there is no difference between the susceptibilities of thrombin clotting activity and Reptilase clotting activity to these inhibitors. 1 In other studies, not reported here, we have observed no advantage of the Reptilase assay in this regard. A disadvantage of the use of Reptilase is that at the present time it is significantly more expensive than thrombin. This work shows that a Reptilaseturbidimetric assay for plasma fibrinogen is not affected by heparin or by Pyran copolymer at concentrations usually encountered. This quantitative method has a reproducibility comparable to other turbidimetric assays, and certain technical advantages including better reagent stability, more homogeneous mixing, and more rapid attainment of equilibrium. References 1. Arnesen H, Kierulf P, Godal HC: The influence of fibrinogen degradation products on the reptilase-time of plasma. Scand J Haematol 11: , Burmester HBC, Aulton K, Horsfield GI: Evaluation of a rapid method for the determination of plasma fibrinogen. J Clin Pathol 23:43-46, Clauss A: Gerinnungsphipiologiache Schnellmethode zur Bestimmung des Fibrinogens. Acta Haematol 17: , Ellis BC, Stransky A: A quick and accurate method for the determination of fibrinogen in plasma. J Clin Pathol 23:43-46, Funk C, Gmur J, Herold R, et al: Reptilase-R A new reagent in blood coagulation. Br J Haematol 21:43-52, Leavitt TJ, Merigan TC: Hemolytic-uremic-like syndrome following polycarbocylate interferon induction. Am J Dis Child 212:45-47, Stevens DJ, Sanfelippo MJ: Evaluation of three methods for plasma fibrinogen determination. Am J Clin Pathol 59: , Straub PW, Funk C: The Reptilase time in presence of fibrinogen degradation products. Scand J Haematol suppl 13: , 1971