Laboratory Suggestions Ristocetin Precipitation Test A New Simple Test for Detection of Fibrin Monomer and Fibrin Degradation Products KIYOAKI WATANABE, M.D., AND JAMES L. TULLIS, M.D.
FIBRIN MONOMERS and fibrin degradation products (fdp) are generated from fibrinogen by the actions of thrombin and plasmin, respectively. The specific detection of these derivatives would be clinically helpful in the diagnosis of disseminated intravascular coagulation and its differentiation from primary fibrinogenolysis without clotting. Since immunologic assays do not differentiate the fibrinogen degradation products (FDP) of plasmin action on fibrinogen from the fdp of plasmin Received July 9, 1976; received revised manuscript August 8, 1977; accepted for publication August 8, 1977. Supported by the Matilda R. Wilson Fund, Detroit, Michigan. The research time of Dr. Tullis was supported by. the Frederick J. Kennedy Memorial Foundation, Inc., Boston, Massachusetts. Presented in preliminary form at the 1976 American Federation for Clinical Research, National Meeting. Address reprint requests to Dr. Tullis: N. E. Deaconess Hospital, 110 Francis Street, Boston, Massachusetts 02215.
Department of Medicine, New England Deaconess Hospital, the Harvard Medical School, and the Center for Blood Research, Boston, Massachusetts
action on fibrin, such technics are of limited value in this clinical separation. Various laboratory methods have been proposed for the measurement of fibrin monomers and fdp.3,6-8-13-u-18,21.23,25 Among them, the ethanol gelation test3-8 and the protamine sulfate test 21,25 are most widely used, due to their simplicity. However, there have been conflicting results with both of the methods.2-5,11-12-19 At least four tests have subsequently been described for the more accurate delineation of fibrin monomers: (1) the indirect demonstration of fibrin monomers through radioimmunoassay for fibrinopeptide A by Nossel and associates 23 ; (2) the incorporation of 14C-glycine ethyl ester into fibrin monomers by activated factor XIII by Kisker and Rush 14 ; (3) the detection of fibrin monomers through agarose chromatography by Fletcher and Alkjaersig6; (4) N-terminal analysis of the fibrinogen fraction. 13 Although these methods have been of considerable value for special studies, they require sophisticated and time-consuming procedures, which are difficult to apply in routine clinical use. 2 1 8 Thus, it is desirable to establish a sensitive and easily performed test for the detection of fibrin monomers and fdp. Recent work from our laboratory showed that ristocetin in low concentrations (1.0-1.5 mg/ml)can precipitate fibrin monomers and fdp from plasma without causing the precipitation of fibrinogen or fibrinogen degradation products (FDP). 28 Based on these data, a rapid, simple procedure for measuring fibrin mono-
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Watanabe, Kiyoaki, and Tullis, James L.: Ristocetin precipitation test. A new simple test for detection of fibrin monomer and fibrin degradation products. Am J Clin Pathol 70: 691-696, 1978. The ristocetin precipitation test was designed as a simplified test to detect fibrin monomers and fibrinogen/fibrin degradation products (FDP/fdp). The ristocetin precipitation test is positive in plasma samples containing either fibrin monomer (>5-10 jug/ml) or early fdp (>50-100 ptg/ml). The ristocetin precipitation test is negative in plasma with fibrinogen concentrations to 1,000 mg/dl or fibrinogen degradation products (FDP) and late fdp to 400 jug/ml. The ristocetin precipitation test is positive in plasmas collected from rabbits after the infusion of thrombin (2.7 u/kg) or thrombin and streptokinase (10,000 u/kg); the test is negative in plasmas from animals treated with streptokinase or saline solution alone. The ristocetin precipitation test is negative in normal human plasmas and plasmas from patients who have primary fibrinogenolysis, but positive in plasmas from patients with disseminated intravascular coagulation. These results suggest that the ristocetin precipitation test can be a useful test for the detection of plasma fibrin monomers and early fdp.
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692
No precipitates
Stippled particles
Small Pellet
Fibrin strands
NEGATIVE
PO S I T I V E
Materials and Methods Reagents Ristocetin (95% ristocetin A and 5% ristocetin B, Lot #59-907-BW) was obtained from Abbott Laboratories* and was dissolved in 0.15 M NaCl solution to yield a concentration of 7.5 mg/ml. Bovine thrombint and streptokinase^ were dissolved in 0.15 M NaCl solution. Purified human fibrinogen (95% clottable protein) was obtained from ImCo Company.§ Heparin (sodium heparin, U.S.P., 10,000 u/ml) was obtained from the Upjohn Company.11 Preparation of Plasma Samples Whole blood (1.7 ml) was collected into a tube containing 0.3 ml of acid-citrate-dextrose solution (ACD, NIH Formula A). The blood was centrifuged at 3,000 x g for 30 minutes at room temperature to obtain the platelet-poor plasma used in the test. Plasma samples were tested within two hours of the blood collection.
Procedure for Ristocetin Precipitation Test A 0.1-ml volume of ristocetin solution (7.5 mg/ml) was added to0.4ml of plasma inaglasstube(12 x 75 mm) and mixed thoroughly by shaking with a Vibro Mixer. The mixture was incubated for 30 minutes at 20 C, then centrifuged at 50 x g for five minutes at 20 C. After centrifugation, the appearance of the resultant precipitates in the bottom of the tube was examined. As shown in Figure 1, the test results were classified into five categories. The test was considered positive when fibrin strands or small or large pellets were observed in the bottom of the tube. The test was negative when no precipitate appeared or when only stippled particles were observed. Preparation of Plasma Containing Fibrin Monomer, FDP/fdp, and Variable Concentrations of Fibrinogen Fibrin monomers or early and late FDP/fdp were prepared from purified human fibrinogen as separately described.28 A 0.1-ml volume of fibrin monomer or FDP/fdp was added to 0.9 ml of plasma, followed by the ristocetin precipitation test. Early and late FDP/fdp used in this study contained the following: early FDP, 70% of fragment X; late FDP, 80% of fragment D; early fdp, 60% of fragment X"; late fdp, 70% of fragment D0.28 In the experiments that were designed to examine the effect of fibrinogen concentration on the ristocetin precipitation test, purified human fibrinogen was added to defibrinated plasma, which was prepared, according to the method of Latallo and colleagues,17 to yield a final concentration between 20 and 200 mg/dl for the low-concentration studies and between 200 and 1,000 mg/dl for the high-concentration studies. The ristocetin precipitation test was performed on these fibrinogenadjusted samples after adding fibrin monomer or fdp in a manner similar to that described above. The ethanol gelation test was performed according to the method of Breen and Tullis.3 The serial-dilution
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mers and fdp was designed and tested. Its possible clinical applicability was evaluated by examining its specificity and sensitivity through in-vitro and in-vivo experiments. It was found that the ristocetin precipitation test can specifically and sensitively detect both fibrin monomers and fdp with a clear endpoint and that it is superior to ethanol gelation and serial-dilution protamine sulfate tests as a routine screening procedure. The test is proposed as a useful tool for the diagnosis of in-vivo fibrin formation occurring in disseminated intravascular coagulation or thromboembolic diseases.
* North Chicago, 111. t Parke, Davis and Company, Detroit, Mich. t Lederle Laboratories Division, Pearl River, N. Y. § Stockholm, Sweden. 11 Kalamazoo, Mich.
Large Pellet
FIG. 1. Test results. Schema showing the appearance of the precipitates observed from the bottom of the tube after centrifugation.
protamine sulfate test was carried out according to the method of Niewiarowski and Gurewich. 21 FDP/fdp level was measured by the tanned red cell hemagglutination inhibition test. 20 Fibrinogen level was determined by using the method of Ware and asociates, 26 and platelet count was measured with a Thrombocounter** by the method of Bull and colleagues. 4 Animal Study
Studies
The ristocetin precipitation test was evaluated in human plasma collected from: (1) 20 healthy volunteers ranging in age from 25 to 54 years; (2) three patients ** Coulter Electronics, Inc., Hialeah. Fla.
with primary fibnnogenolysis; (3) 14 patients with disseminated intravascular coagulation. Results In-vitro Specificity and
Sensitivity
Effects of Concentrations of Plasma Fibrinogen, fibrin monomer, FDP, andfdp. The test was performed on plasmas with different concentrations of fibrinogen, fibrin monomer, early FDP and early fdp. The results are shown in Table 1. Positive ristocetin precipitation tests were confined to plasma containing small amounts of fibrin monomers or early fdp. The test was positive at 5-10 /Ag/ml fibrin monomers and at 50-100 /xg/ml early fdp. On the other hand, the test was negative in plasmas containing fibrinogen to 1,000 mg/dl or early FDP to 400 jig/ml. The ristocetin precipitation test was negative in plasmas containing both late FDP and late fdp at concentrations to 400 /xg/ml. The positive ristocetin precipitation test for fibrin monomers and fdp also was not affected by low plasma fibrinogen concentrations between 20 and 200 mg/dl. The addition of heparin to plasma in concentrations to 10 u/ml did not influence the test results. Comparison of the Ristocetin Precipitation Test with the Ethanol Gelation Test and the Serial-dilution Protamine Sulfate Test. The sensitivity of the test to fibrin monomers and fdp was compared with that of the ethanol gelation test and the serial-dilution protamine sulfate test in an in-vitro system (Table 2). On the basis of threshold concentrations of fibrin monomers that can be detected in plasma, the ristocetin precipitation test was 13 times more sensitive than the ethanol gelation test and four times more sensitive than the serialdilution protamine sulfate test. Also, the ristocetin precipitation test was two times more sensitive than the serial-dilution protamine sulfate test to detect early fdp. Sensitivity equal to that of the ristocetin
Table I. Effects of Various Plasma Concentrations of Fibrinogen and Its Derivatives on the Ristocetin Precipitation Test Fibrin Monomer
Early fdp
Fibrinogen
Early FDP
Concentration* (Mg/ml)
Test Resultst
Concentration (Mg/ml)
Test Results
Concentration (Mg/ml)
Test Results
Concentration (mg/dl)
2.5 5 10 20 40
-
25 50 100 200 400
-
+ + + +
25 50 100 200 400
-
20-200 200-400 600 800 1000
* Concentration in plasma. t - = Negative RPT; + = Positive RPT.
+ + + +
Test Results
— -
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New Zealand white rabbits (2.8-3.2 kg) were anesthetized with sodium pentobarbital (Nembutal, Abbott Laboratories), 50 mg/ml. Pentobarbital, 2 ml, was infused slowly into the animal through an ear vein with an automatic infusion pump. An additional dose (0.81.3 ml) was administered as a booster 30 minutes after the initial infusion. Animals were divided into four groups: (I) thrombintreated; (II) thrombin + streptokinase-treated; (III) streptokinase-treated; (IV) saline-treated (control). For Group 1, thrombin (8 u/ml) was infused into an ear vein at a rate of 0.2 ml/min until the total dose of thrombin reached 2.7 u/kg. Streptokinase (30,000 u/ml) was infused into Group HI animals at a rate of 0.5 ml/min for a total amount of 10,000 u/kg. In thrombin + streptokinase-treated animals (Group II), thrombin was given according to the procedure described for Group I animals, followed by streptokinase infusion as administered to Group III. Blood (10 ml) was collected through a polyethylene catheter (PE 190) inserted into a femoral artery. The sample was centrifuged at 2,100 x g for 30 minutes, and the plasma thus obtained was assayed by the ristocetin precipitation test. Human
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Table 2. Comparison of the Ristocetin Precipitation Test with the Ethanol Gelation Test and the Protamine Sulfate Test
Concentration (Mg/ml)*
Ristocetin Precipitation Test (30 Min)
Plasma Fibrin Monomer 100 + 50 + 25 + 12 + 6 + 3 + + + +
+
—
-
(30 Min ) (24 Hours) + + +
—
+ + +
-
+ + + + +
+ + + +
-
* Different plasma concentrations are prepared by adding 0.1 ml fibrin monomer or fdp in 0.9 ml of either ACD (for the ristocetin precipitation test) or citrate plasma (for the ethanol gelation test and the serial dilution protamine sulfate test).
precipitation test was attained by the serial-dilution protamine sulfate test only when the latter results were evaluated after incubation for 24 hours. In-vivo Specificity and Sensitivity Animal Study. The ristocetin precipitation test was performed on plasma samples collected from four groups of rabbits differently treated with thrombin or streptokinase, or both. A positive ristocetin precipitation test was obtained in plasmas from thrombintreated animals and animals treated with thrombin and streptokinase. In both groups, definitive positive results were obtained 30 and 60 minutes after infusion of the reagents. The tests were uniformly negative in samples collected from animals treated with streptokinase alone and control animals. Human Study. The ristocetin precipitation test was negative in all normal plasma samples from 20 healthy donors. The test was also negative in plasmas from three primary fibrinogenolytic patients, all of whom had hepatic cirrhosis. In this group of patients fibrinogen levels ranged from 125 to 200 mg/dl, and euglobulin lysis times were less than 120 minutes. Serum FDP/fdp levels were increased for each patient, ranging from 9.6 to 38.4 ptg/ml, by the tanned red cell hemagglutination assay.20 Fifteen samples from 14 patients with disseminated intravascular coagulation were tested with the ristocetin precipitation test (Table 3). The clinical diagnosis of disseminated intravascular coagulation was based on variable combinations of a generalized bleeding
tendency in the presence of prolonged prothrombin times and partial thromboplastin times, thrombocytopenia, hypofibrinogenemia, and elevation of FDP/fdp. The ristocetin precipitation test was positive in 15 of the 15 samples. Four of the samples also showed a positive serial-dilution protamine sulfate test after a 30minute incubation, and none showed a positive ethanol gelation test. Plasmas from 11 of the patients showed a positive serial-dilution protamine sulfate test when the test was incubated for 24 hours. Discussion The ristocetin precipitation test described in this paper was designed from our recent observation that ristocetin in low concentrations (1.0-1.5 mg/ml) can selectively precipitate fibrin monomer and fibrin degradation products (fdp).28 The ristocetin precipitation test is positive only in plasmas containing fm or early fdp (fragment X°, 60%) but is negative in normal plasmas and plasmas containing FDP or late fdp (fragment D°, 70%). In animal studies, a positive test is obtained in plasmas from rabbits treated with either thrombin or thrombin plus streptokinase, but not with streptokinase or saline solution alone. For human subjects, the ristocetin precipitation test was positive in plasma samples collected from patients who had disseminated intravascular coagulation and negative in samples from normal persons and hyperfibrinogenolytic patients. Thus, the specificity of the test to fibrin monomer and fdp (fragment X°) is corroborated in the present study both in vitro and in vivo. Samples from all patients with disseminated intravascular coagulation give positive ristocetin precipitation tests, but none of 11 who were tested had a positive ethanol gelation test; four patients had positive serial-dilution protamine sulfate tests at 30 minutes. Although the serial-dilution protamine sulfate test shows sensitivity almost comparable to that of the ristocetin precipitation test with prolonged incubation, 24 hours is necessary in order to obtain such results. Because of the clear-cut endpoint of the ristocetin precipitation test, this test is easily evaluated. Moreover, the test procedure is simple to perform. These results suggest that the ristocetin precipitation test may be superior to either the ethanol gelation test or the serial-dilution protamine sulfate test for the detection of fibrin monomers or fdp. Although the procedure of the ristocetin precipitation test is easily adaptable to routine clinical use, several considerations are important for its proper performance.28 (1) ACD plasma should be used, as this anticoagulant proved uniquely favorable. (2) The test should be carried out at room temperatures between
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Plasma Early fdp 400 200 100 50 25
Ethanol Gelation Test
Serial Dilution Protamine Sulfate Test
A.J.C.P. • October 1978
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Table 3. Results of Ristocetin Precipitation Test for Patients with Disseminated Intravascular Coagulation SDPT* Diagnosis Patient 1 Patient 2
Patient 3 Patient 4 Patient 5
Patient 6 Patient 7
Patient 9 Patient 10 Patient 11 Patient 12 Patient 13 Patient 14
Postdialysis renal failure Toxemia of pregnancy NORMAL RANGE
* Serial dilution protamine sulfate test.
Fibrinogen (mg/dl)
FDP/fdp (Mg/ml)
40.0
+
61,000
180
9.6
45,000
133
19.2 4.64 2.32
100,000
422
150,000400,000
200-400
t Ethanol ge ation test.
20 and 25 C. The precipitation of fibrinogen or FDP may occur at temperatures below 15 C, and the sensitivity of the test is reduced at temperatures as high as 37 C. (3) Complete mixing of the plasma with ristocetin is essential. Since the precipitation.of fibrin monomers and fdp by ristocetin is highly dependent upon the concentration of this antibiotic, nonspecific precipitation may occur when there is unequal distribution in plasma. The speed of centrifugation following ristocetin precipitation was modified from that in earlier report27 to avoid false-positive precipitates. Although the sensitivity to fibrin monomers is commensurately reduced, the test will still distinguish as little as 5-10 yu,g/ml fibrin monomers. It remains insensitive to late fdp. That fibrinogen concentrations ranging from 20 to 200 mg/dl did not affect the test results indicates that the test could be used to detect fibrin monomers or early fdp even in hypofibrinogenemia, which frequently is present in patients who have disseminated intravascular coagulation. Since heparin in concentrations
+
EGTt
-
RPTt
+
—
+
+
+
+
+
+
+
N.D. hosp.
N.D.
+
+
N.D.
+
+
+
N.D.
+
N.D.
N.D. +
N.D.
+ + +
—
—
8
— —
~
t Ristocetin precipitation lest.
as high as 10 u/ml did not influence the test, the test could be performed safely during anticoagulant treatment of the patients. Disseminated intravascular coagulation is a multifactorial derangement of hemostasis. It has been suggested by Deykin5 and others that no single test can be diagnostic. The most critical clinical decision in patients with bleeding disorders accompanying acquired hypofibrinogenemia is the differentiation of disseminated intravascular coagulation and secondary fibrinolysis from primary fibrinogenolysis. The sensitivity of the proposed ristocetin precipitation test for the detection of fibrin monomers and fdp and its insensitivity to FDP will assist in making this differentiation possible. Acknowledgments. Miss Phyllis Baudanzaand Mrs. Linda Hewitt for provided assistance.
References 1. Arnenson H, Godal HC, Kierulf P: Ethanol gelation test in acute myocardial infarction. Thromb Res 2:173-176, 1973
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Patient 8
Acute gramnegative sepsis Acute promyelocytic leukemia Renal tumor with metastases Fulminant hepatitis Diffuse lymphoblastic lymphoma Acute renal transplant reject Carcinoma of pancreas Postoperative (L) nephrectomy Early renal rejection Aortic stenosis; multiple systems failure Hepatic failure with sepsis Malnutrition and carcinoma
Platelets (/cu mm)
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WATANABE AND TULLIS
16. Latallo ZS: Formation and detection of fibrinogen derived complexes. Thromb Diath Haemorrh 34:677-685, 1975 17. Latallo ZS, Wegrzynowicz Z, Budzynski A, et al: Effect of protamine sulfate on the solubility of fibrinogen and its derivatives and other plasma protein. Scand J Haemat Suppl 13:151-162, 1971 18. Lipinski B, Worowski K: Detection of soluble fibrin monomer complexes in blood by means of protamine sulfate test. Thromb Diath Haemorrh 20:40-49, 1968 19. MargolisCZ: Ethanol gelation test. New Eng J Med 284:53, 1971 20. Merskey C, Kleiner GJ, Johnson AJ: Quantitative estimation of split products of fibrinogen in human serum, relation to diagnosis and treatment. Blood 28:1-18, 1966 21. Niewiarowski S, Gurewich V: Laboratory identification of intravascular coagulation: The serial dilution protamine sulfate test for the detection of fibrin monomer and fibrin degradation products. J Lab Clin Med 77:665-676, 1971 22. Nossel HL, Butler VP Jr, Canfield RE, et al: Potential use of fibrinopeptide. A measurement in the diagnosis and management of thrombosis. Thromb Diath Haemorrh 33:426434, 1975 23. Nossel HL, Younger LR, Wilner GD, et al: Radioimmunoassay of human fibrinopeptide A. Proc Natl Acad Sci USA 68: 2350-2353, 1971 24. Pilgeram LO, Chee AN, Von den Busshe G: Evidence for abnormalities in clotting and thrombolysis as a risk factor for stroke. Stroke 4:643-657, 1973 25. Seaman AJ: The recognition of intravascular clotting. The plasma protamine paracoagulant test. Arch Intern Med 125:1016-1021, 1970 26. Ware AG, Guest MM, Seegers WH: Fibrinogen: With special reference to its preparation and certain properties of the products. Arch Biochem 13:231-236, 1947 27. Watanabe K: Abstract, American Federation for Clinical Research, 1976 28. Watanabe K, Tullis JL: Precipitation of fibrin monomers and fibrin degradation products by ristocetin. Am J Med Sci 275:337-344, 1978
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2. Bang NV, Chang ML: Soluble fibrin complexes. Semin Thromb Hemost 1:91-128, 1974 3. Breen FA, Tullis JL: Ethanol gelation: A rapid screening test for intravascular coagulation. Ann Int Med 69:1197-1206. 1968 4. Bull BS, Schneiderman MA, Brecher G: Platelet count with the Coulter Counter. Am J Clin Pathol 44:678-688, 1965 5. Deykin D: The clinical challenge of disseminated intravascular coagulation. New Eng J Med 283:636-644, 1970 6. Fletcher AP, Alkjaersig N: Blood hypercoagulability, intravascular coagulation and thrombosis: New diagnostic concept. Thromb Diath Haemorrh Suppl 45:389-394, 1971 7. Fletcher AP, Alkjaersig N: Coagulation studies in acute cerebrovascular disease, Stroke Diagnosis and Management. Edited by Fields W and Moosy F. St. Louis, Warren Green, 1973, p 164 8. Godal HC, Abildgaard U: Gelation of soluble fibrin in plasma by ethanol. Scand J Haemat 3:342-350, 1966 9. Gurewich V, Hume M, Patrick M: The laboratory diagnosis of venous thromboembolic disease by measurement of fibrinogen-fibrin degradation products and fibrin monomer. Chest 64:585-590, 1973 10. Gurewich V, Hutchinson E: Detection of intravascular coagulation by a serial dilution protamine sulfate test. Ann Int Med 75:895-902, 1971 11. Gurewich V, Lipinski B, Lipinska I: A comparative study of precipitation and paracoagulation by protamine sulfate and ethanol gelation tests. Thromb Res 2:539-556, 1973 12. Hender U, Nilsson IM: Parallel determinations of fibrin degradation products and fibrin monomer with various methods. Thromb Diath Haemorrh 28:268-279, 1972 13. Kierulf P, Abildgaard U: Studies on soluble fibrin in plasma. 1. N-terminal analysis of a modified fraction 1 (Cohn) from normal and thrombin-induced plasma. Scand J Clin Lab Invest 28:231-240, 1971 14. Kisker CT, Rush R: Detection of intravascular coagulation. J Clin Invest 50:2235-2241, 1971 15. Konttinen YP, Kemppainen L, Turunen O: Comparison of ethanol and protamine tests in demonstration of soluble fibrin and early products of fibrin degradation. Thromb Diath Haemorrh 28:342-350, 1972
A.J.C.P. • October 1978
FIBRIN MONOMERS and fibrin degradation products (fdp) are generated from fibrinogen by the actions of thrombin and plasmin, respectively. The specific detection of these derivatives would be clinically helpful in the diagnosis of disseminated intravascular coagulation and its differentiation from primary fibrinogenolysis without clotting. Since immunologic assays do not differentiate the fibrinogen degradation products (FDP) of plasmin action on fibrinogen from the fdp of plasmin Received July 9, 1976; received revised manuscript August 8, 1977; accepted for publication August 8, 1977. Supported by the Matilda R. Wilson Fund, Detroit, Michigan. The research time of Dr. Tullis was supported by. the Frederick J. Kennedy Memorial Foundation, Inc., Boston, Massachusetts. Presented in preliminary form at the 1976 American Federation for Clinical Research, National Meeting. Address reprint requests to Dr. Tullis: N. E. Deaconess Hospital, 110 Francis Street, Boston, Massachusetts 02215.
Department of Medicine, New England Deaconess Hospital, the Harvard Medical School, and the Center for Blood Research, Boston, Massachusetts
action on fibrin, such technics are of limited value in this clinical separation. Various laboratory methods have been proposed for the measurement of fibrin monomers and fdp.3,6-8-13-u-18,21.23,25 Among them, the ethanol gelation test3-8 and the protamine sulfate test 21,25 are most widely used, due to their simplicity. However, there have been conflicting results with both of the methods.2-5,11-12-19 At least four tests have subsequently been described for the more accurate delineation of fibrin monomers: (1) the indirect demonstration of fibrin monomers through radioimmunoassay for fibrinopeptide A by Nossel and associates 23 ; (2) the incorporation of 14C-glycine ethyl ester into fibrin monomers by activated factor XIII by Kisker and Rush 14 ; (3) the detection of fibrin monomers through agarose chromatography by Fletcher and Alkjaersig6; (4) N-terminal analysis of the fibrinogen fraction. 13 Although these methods have been of considerable value for special studies, they require sophisticated and time-consuming procedures, which are difficult to apply in routine clinical use. 2 1 8 Thus, it is desirable to establish a sensitive and easily performed test for the detection of fibrin monomers and fdp. Recent work from our laboratory showed that ristocetin in low concentrations (1.0-1.5 mg/ml)can precipitate fibrin monomers and fdp from plasma without causing the precipitation of fibrinogen or fibrinogen degradation products (FDP). 28 Based on these data, a rapid, simple procedure for measuring fibrin mono-
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691
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Watanabe, Kiyoaki, and Tullis, James L.: Ristocetin precipitation test. A new simple test for detection of fibrin monomer and fibrin degradation products. Am J Clin Pathol 70: 691-696, 1978. The ristocetin precipitation test was designed as a simplified test to detect fibrin monomers and fibrinogen/fibrin degradation products (FDP/fdp). The ristocetin precipitation test is positive in plasma samples containing either fibrin monomer (>5-10 jug/ml) or early fdp (>50-100 ptg/ml). The ristocetin precipitation test is negative in plasma with fibrinogen concentrations to 1,000 mg/dl or fibrinogen degradation products (FDP) and late fdp to 400 jug/ml. The ristocetin precipitation test is positive in plasmas collected from rabbits after the infusion of thrombin (2.7 u/kg) or thrombin and streptokinase (10,000 u/kg); the test is negative in plasmas from animals treated with streptokinase or saline solution alone. The ristocetin precipitation test is negative in normal human plasmas and plasmas from patients who have primary fibrinogenolysis, but positive in plasmas from patients with disseminated intravascular coagulation. These results suggest that the ristocetin precipitation test can be a useful test for the detection of plasma fibrin monomers and early fdp.
A.J.C.P. • October 1978
WATANABE AND TULLIS
692
No precipitates
Stippled particles
Small Pellet
Fibrin strands
NEGATIVE
PO S I T I V E
Materials and Methods Reagents Ristocetin (95% ristocetin A and 5% ristocetin B, Lot #59-907-BW) was obtained from Abbott Laboratories* and was dissolved in 0.15 M NaCl solution to yield a concentration of 7.5 mg/ml. Bovine thrombint and streptokinase^ were dissolved in 0.15 M NaCl solution. Purified human fibrinogen (95% clottable protein) was obtained from ImCo Company.§ Heparin (sodium heparin, U.S.P., 10,000 u/ml) was obtained from the Upjohn Company.11 Preparation of Plasma Samples Whole blood (1.7 ml) was collected into a tube containing 0.3 ml of acid-citrate-dextrose solution (ACD, NIH Formula A). The blood was centrifuged at 3,000 x g for 30 minutes at room temperature to obtain the platelet-poor plasma used in the test. Plasma samples were tested within two hours of the blood collection.
Procedure for Ristocetin Precipitation Test A 0.1-ml volume of ristocetin solution (7.5 mg/ml) was added to0.4ml of plasma inaglasstube(12 x 75 mm) and mixed thoroughly by shaking with a Vibro Mixer. The mixture was incubated for 30 minutes at 20 C, then centrifuged at 50 x g for five minutes at 20 C. After centrifugation, the appearance of the resultant precipitates in the bottom of the tube was examined. As shown in Figure 1, the test results were classified into five categories. The test was considered positive when fibrin strands or small or large pellets were observed in the bottom of the tube. The test was negative when no precipitate appeared or when only stippled particles were observed. Preparation of Plasma Containing Fibrin Monomer, FDP/fdp, and Variable Concentrations of Fibrinogen Fibrin monomers or early and late FDP/fdp were prepared from purified human fibrinogen as separately described.28 A 0.1-ml volume of fibrin monomer or FDP/fdp was added to 0.9 ml of plasma, followed by the ristocetin precipitation test. Early and late FDP/fdp used in this study contained the following: early FDP, 70% of fragment X; late FDP, 80% of fragment D; early fdp, 60% of fragment X"; late fdp, 70% of fragment D0.28 In the experiments that were designed to examine the effect of fibrinogen concentration on the ristocetin precipitation test, purified human fibrinogen was added to defibrinated plasma, which was prepared, according to the method of Latallo and colleagues,17 to yield a final concentration between 20 and 200 mg/dl for the low-concentration studies and between 200 and 1,000 mg/dl for the high-concentration studies. The ristocetin precipitation test was performed on these fibrinogenadjusted samples after adding fibrin monomer or fdp in a manner similar to that described above. The ethanol gelation test was performed according to the method of Breen and Tullis.3 The serial-dilution
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
mers and fdp was designed and tested. Its possible clinical applicability was evaluated by examining its specificity and sensitivity through in-vitro and in-vivo experiments. It was found that the ristocetin precipitation test can specifically and sensitively detect both fibrin monomers and fdp with a clear endpoint and that it is superior to ethanol gelation and serial-dilution protamine sulfate tests as a routine screening procedure. The test is proposed as a useful tool for the diagnosis of in-vivo fibrin formation occurring in disseminated intravascular coagulation or thromboembolic diseases.
* North Chicago, 111. t Parke, Davis and Company, Detroit, Mich. t Lederle Laboratories Division, Pearl River, N. Y. § Stockholm, Sweden. 11 Kalamazoo, Mich.
Large Pellet
FIG. 1. Test results. Schema showing the appearance of the precipitates observed from the bottom of the tube after centrifugation.
protamine sulfate test was carried out according to the method of Niewiarowski and Gurewich. 21 FDP/fdp level was measured by the tanned red cell hemagglutination inhibition test. 20 Fibrinogen level was determined by using the method of Ware and asociates, 26 and platelet count was measured with a Thrombocounter** by the method of Bull and colleagues. 4 Animal Study
Studies
The ristocetin precipitation test was evaluated in human plasma collected from: (1) 20 healthy volunteers ranging in age from 25 to 54 years; (2) three patients ** Coulter Electronics, Inc., Hialeah. Fla.
with primary fibnnogenolysis; (3) 14 patients with disseminated intravascular coagulation. Results In-vitro Specificity and
Sensitivity
Effects of Concentrations of Plasma Fibrinogen, fibrin monomer, FDP, andfdp. The test was performed on plasmas with different concentrations of fibrinogen, fibrin monomer, early FDP and early fdp. The results are shown in Table 1. Positive ristocetin precipitation tests were confined to plasma containing small amounts of fibrin monomers or early fdp. The test was positive at 5-10 /Ag/ml fibrin monomers and at 50-100 /xg/ml early fdp. On the other hand, the test was negative in plasmas containing fibrinogen to 1,000 mg/dl or early FDP to 400 jig/ml. The ristocetin precipitation test was negative in plasmas containing both late FDP and late fdp at concentrations to 400 /xg/ml. The positive ristocetin precipitation test for fibrin monomers and fdp also was not affected by low plasma fibrinogen concentrations between 20 and 200 mg/dl. The addition of heparin to plasma in concentrations to 10 u/ml did not influence the test results. Comparison of the Ristocetin Precipitation Test with the Ethanol Gelation Test and the Serial-dilution Protamine Sulfate Test. The sensitivity of the test to fibrin monomers and fdp was compared with that of the ethanol gelation test and the serial-dilution protamine sulfate test in an in-vitro system (Table 2). On the basis of threshold concentrations of fibrin monomers that can be detected in plasma, the ristocetin precipitation test was 13 times more sensitive than the ethanol gelation test and four times more sensitive than the serialdilution protamine sulfate test. Also, the ristocetin precipitation test was two times more sensitive than the serial-dilution protamine sulfate test to detect early fdp. Sensitivity equal to that of the ristocetin
Table I. Effects of Various Plasma Concentrations of Fibrinogen and Its Derivatives on the Ristocetin Precipitation Test Fibrin Monomer
Early fdp
Fibrinogen
Early FDP
Concentration* (Mg/ml)
Test Resultst
Concentration (Mg/ml)
Test Results
Concentration (Mg/ml)
Test Results
Concentration (mg/dl)
2.5 5 10 20 40
-
25 50 100 200 400
-
+ + + +
25 50 100 200 400
-
20-200 200-400 600 800 1000
* Concentration in plasma. t - = Negative RPT; + = Positive RPT.
+ + + +
Test Results
— -
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New Zealand white rabbits (2.8-3.2 kg) were anesthetized with sodium pentobarbital (Nembutal, Abbott Laboratories), 50 mg/ml. Pentobarbital, 2 ml, was infused slowly into the animal through an ear vein with an automatic infusion pump. An additional dose (0.81.3 ml) was administered as a booster 30 minutes after the initial infusion. Animals were divided into four groups: (I) thrombintreated; (II) thrombin + streptokinase-treated; (III) streptokinase-treated; (IV) saline-treated (control). For Group 1, thrombin (8 u/ml) was infused into an ear vein at a rate of 0.2 ml/min until the total dose of thrombin reached 2.7 u/kg. Streptokinase (30,000 u/ml) was infused into Group HI animals at a rate of 0.5 ml/min for a total amount of 10,000 u/kg. In thrombin + streptokinase-treated animals (Group II), thrombin was given according to the procedure described for Group I animals, followed by streptokinase infusion as administered to Group III. Blood (10 ml) was collected through a polyethylene catheter (PE 190) inserted into a femoral artery. The sample was centrifuged at 2,100 x g for 30 minutes, and the plasma thus obtained was assayed by the ristocetin precipitation test. Human
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Table 2. Comparison of the Ristocetin Precipitation Test with the Ethanol Gelation Test and the Protamine Sulfate Test
Concentration (Mg/ml)*
Ristocetin Precipitation Test (30 Min)
Plasma Fibrin Monomer 100 + 50 + 25 + 12 + 6 + 3 + + + +
+
—
-
(30 Min ) (24 Hours) + + +
—
+ + +
-
+ + + + +
+ + + +
-
* Different plasma concentrations are prepared by adding 0.1 ml fibrin monomer or fdp in 0.9 ml of either ACD (for the ristocetin precipitation test) or citrate plasma (for the ethanol gelation test and the serial dilution protamine sulfate test).
precipitation test was attained by the serial-dilution protamine sulfate test only when the latter results were evaluated after incubation for 24 hours. In-vivo Specificity and Sensitivity Animal Study. The ristocetin precipitation test was performed on plasma samples collected from four groups of rabbits differently treated with thrombin or streptokinase, or both. A positive ristocetin precipitation test was obtained in plasmas from thrombintreated animals and animals treated with thrombin and streptokinase. In both groups, definitive positive results were obtained 30 and 60 minutes after infusion of the reagents. The tests were uniformly negative in samples collected from animals treated with streptokinase alone and control animals. Human Study. The ristocetin precipitation test was negative in all normal plasma samples from 20 healthy donors. The test was also negative in plasmas from three primary fibrinogenolytic patients, all of whom had hepatic cirrhosis. In this group of patients fibrinogen levels ranged from 125 to 200 mg/dl, and euglobulin lysis times were less than 120 minutes. Serum FDP/fdp levels were increased for each patient, ranging from 9.6 to 38.4 ptg/ml, by the tanned red cell hemagglutination assay.20 Fifteen samples from 14 patients with disseminated intravascular coagulation were tested with the ristocetin precipitation test (Table 3). The clinical diagnosis of disseminated intravascular coagulation was based on variable combinations of a generalized bleeding
tendency in the presence of prolonged prothrombin times and partial thromboplastin times, thrombocytopenia, hypofibrinogenemia, and elevation of FDP/fdp. The ristocetin precipitation test was positive in 15 of the 15 samples. Four of the samples also showed a positive serial-dilution protamine sulfate test after a 30minute incubation, and none showed a positive ethanol gelation test. Plasmas from 11 of the patients showed a positive serial-dilution protamine sulfate test when the test was incubated for 24 hours. Discussion The ristocetin precipitation test described in this paper was designed from our recent observation that ristocetin in low concentrations (1.0-1.5 mg/ml) can selectively precipitate fibrin monomer and fibrin degradation products (fdp).28 The ristocetin precipitation test is positive only in plasmas containing fm or early fdp (fragment X°, 60%) but is negative in normal plasmas and plasmas containing FDP or late fdp (fragment D°, 70%). In animal studies, a positive test is obtained in plasmas from rabbits treated with either thrombin or thrombin plus streptokinase, but not with streptokinase or saline solution alone. For human subjects, the ristocetin precipitation test was positive in plasma samples collected from patients who had disseminated intravascular coagulation and negative in samples from normal persons and hyperfibrinogenolytic patients. Thus, the specificity of the test to fibrin monomer and fdp (fragment X°) is corroborated in the present study both in vitro and in vivo. Samples from all patients with disseminated intravascular coagulation give positive ristocetin precipitation tests, but none of 11 who were tested had a positive ethanol gelation test; four patients had positive serial-dilution protamine sulfate tests at 30 minutes. Although the serial-dilution protamine sulfate test shows sensitivity almost comparable to that of the ristocetin precipitation test with prolonged incubation, 24 hours is necessary in order to obtain such results. Because of the clear-cut endpoint of the ristocetin precipitation test, this test is easily evaluated. Moreover, the test procedure is simple to perform. These results suggest that the ristocetin precipitation test may be superior to either the ethanol gelation test or the serial-dilution protamine sulfate test for the detection of fibrin monomers or fdp. Although the procedure of the ristocetin precipitation test is easily adaptable to routine clinical use, several considerations are important for its proper performance.28 (1) ACD plasma should be used, as this anticoagulant proved uniquely favorable. (2) The test should be carried out at room temperatures between
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Plasma Early fdp 400 200 100 50 25
Ethanol Gelation Test
Serial Dilution Protamine Sulfate Test
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Table 3. Results of Ristocetin Precipitation Test for Patients with Disseminated Intravascular Coagulation SDPT* Diagnosis Patient 1 Patient 2
Patient 3 Patient 4 Patient 5
Patient 6 Patient 7
Patient 9 Patient 10 Patient 11 Patient 12 Patient 13 Patient 14
Postdialysis renal failure Toxemia of pregnancy NORMAL RANGE
* Serial dilution protamine sulfate test.
Fibrinogen (mg/dl)
FDP/fdp (Mg/ml)
40.0
+
61,000
180
9.6
45,000
133
19.2 4.64 2.32
100,000
422
150,000400,000
200-400
t Ethanol ge ation test.
20 and 25 C. The precipitation of fibrinogen or FDP may occur at temperatures below 15 C, and the sensitivity of the test is reduced at temperatures as high as 37 C. (3) Complete mixing of the plasma with ristocetin is essential. Since the precipitation.of fibrin monomers and fdp by ristocetin is highly dependent upon the concentration of this antibiotic, nonspecific precipitation may occur when there is unequal distribution in plasma. The speed of centrifugation following ristocetin precipitation was modified from that in earlier report27 to avoid false-positive precipitates. Although the sensitivity to fibrin monomers is commensurately reduced, the test will still distinguish as little as 5-10 yu,g/ml fibrin monomers. It remains insensitive to late fdp. That fibrinogen concentrations ranging from 20 to 200 mg/dl did not affect the test results indicates that the test could be used to detect fibrin monomers or early fdp even in hypofibrinogenemia, which frequently is present in patients who have disseminated intravascular coagulation. Since heparin in concentrations
+
EGTt
-
RPTt
+
—
+
+
+
+
+
+
+
N.D. hosp.
N.D.
+
+
N.D.
+
+
+
N.D.
+
N.D.
N.D. +
N.D.
+ + +
—
—
8
— —
~
t Ristocetin precipitation lest.
as high as 10 u/ml did not influence the test, the test could be performed safely during anticoagulant treatment of the patients. Disseminated intravascular coagulation is a multifactorial derangement of hemostasis. It has been suggested by Deykin5 and others that no single test can be diagnostic. The most critical clinical decision in patients with bleeding disorders accompanying acquired hypofibrinogenemia is the differentiation of disseminated intravascular coagulation and secondary fibrinolysis from primary fibrinogenolysis. The sensitivity of the proposed ristocetin precipitation test for the detection of fibrin monomers and fdp and its insensitivity to FDP will assist in making this differentiation possible. Acknowledgments. Miss Phyllis Baudanzaand Mrs. Linda Hewitt for provided assistance.
References 1. Arnenson H, Godal HC, Kierulf P: Ethanol gelation test in acute myocardial infarction. Thromb Res 2:173-176, 1973
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Patient 8
Acute gramnegative sepsis Acute promyelocytic leukemia Renal tumor with metastases Fulminant hepatitis Diffuse lymphoblastic lymphoma Acute renal transplant reject Carcinoma of pancreas Postoperative (L) nephrectomy Early renal rejection Aortic stenosis; multiple systems failure Hepatic failure with sepsis Malnutrition and carcinoma
Platelets (/cu mm)
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16. Latallo ZS: Formation and detection of fibrinogen derived complexes. Thromb Diath Haemorrh 34:677-685, 1975 17. Latallo ZS, Wegrzynowicz Z, Budzynski A, et al: Effect of protamine sulfate on the solubility of fibrinogen and its derivatives and other plasma protein. Scand J Haemat Suppl 13:151-162, 1971 18. Lipinski B, Worowski K: Detection of soluble fibrin monomer complexes in blood by means of protamine sulfate test. Thromb Diath Haemorrh 20:40-49, 1968 19. MargolisCZ: Ethanol gelation test. New Eng J Med 284:53, 1971 20. Merskey C, Kleiner GJ, Johnson AJ: Quantitative estimation of split products of fibrinogen in human serum, relation to diagnosis and treatment. Blood 28:1-18, 1966 21. Niewiarowski S, Gurewich V: Laboratory identification of intravascular coagulation: The serial dilution protamine sulfate test for the detection of fibrin monomer and fibrin degradation products. J Lab Clin Med 77:665-676, 1971 22. Nossel HL, Butler VP Jr, Canfield RE, et al: Potential use of fibrinopeptide. A measurement in the diagnosis and management of thrombosis. Thromb Diath Haemorrh 33:426434, 1975 23. Nossel HL, Younger LR, Wilner GD, et al: Radioimmunoassay of human fibrinopeptide A. Proc Natl Acad Sci USA 68: 2350-2353, 1971 24. Pilgeram LO, Chee AN, Von den Busshe G: Evidence for abnormalities in clotting and thrombolysis as a risk factor for stroke. Stroke 4:643-657, 1973 25. Seaman AJ: The recognition of intravascular clotting. The plasma protamine paracoagulant test. Arch Intern Med 125:1016-1021, 1970 26. Ware AG, Guest MM, Seegers WH: Fibrinogen: With special reference to its preparation and certain properties of the products. Arch Biochem 13:231-236, 1947 27. Watanabe K: Abstract, American Federation for Clinical Research, 1976 28. Watanabe K, Tullis JL: Precipitation of fibrin monomers and fibrin degradation products by ristocetin. Am J Med Sci 275:337-344, 1978
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2. Bang NV, Chang ML: Soluble fibrin complexes. Semin Thromb Hemost 1:91-128, 1974 3. Breen FA, Tullis JL: Ethanol gelation: A rapid screening test for intravascular coagulation. Ann Int Med 69:1197-1206. 1968 4. Bull BS, Schneiderman MA, Brecher G: Platelet count with the Coulter Counter. Am J Clin Pathol 44:678-688, 1965 5. Deykin D: The clinical challenge of disseminated intravascular coagulation. New Eng J Med 283:636-644, 1970 6. Fletcher AP, Alkjaersig N: Blood hypercoagulability, intravascular coagulation and thrombosis: New diagnostic concept. Thromb Diath Haemorrh Suppl 45:389-394, 1971 7. Fletcher AP, Alkjaersig N: Coagulation studies in acute cerebrovascular disease, Stroke Diagnosis and Management. Edited by Fields W and Moosy F. St. Louis, Warren Green, 1973, p 164 8. Godal HC, Abildgaard U: Gelation of soluble fibrin in plasma by ethanol. Scand J Haemat 3:342-350, 1966 9. Gurewich V, Hume M, Patrick M: The laboratory diagnosis of venous thromboembolic disease by measurement of fibrinogen-fibrin degradation products and fibrin monomer. Chest 64:585-590, 1973 10. Gurewich V, Hutchinson E: Detection of intravascular coagulation by a serial dilution protamine sulfate test. Ann Int Med 75:895-902, 1971 11. Gurewich V, Lipinski B, Lipinska I: A comparative study of precipitation and paracoagulation by protamine sulfate and ethanol gelation tests. Thromb Res 2:539-556, 1973 12. Hender U, Nilsson IM: Parallel determinations of fibrin degradation products and fibrin monomer with various methods. Thromb Diath Haemorrh 28:268-279, 1972 13. Kierulf P, Abildgaard U: Studies on soluble fibrin in plasma. 1. N-terminal analysis of a modified fraction 1 (Cohn) from normal and thrombin-induced plasma. Scand J Clin Lab Invest 28:231-240, 1971 14. Kisker CT, Rush R: Detection of intravascular coagulation. J Clin Invest 50:2235-2241, 1971 15. Konttinen YP, Kemppainen L, Turunen O: Comparison of ethanol and protamine tests in demonstration of soluble fibrin and early products of fibrin degradation. Thromb Diath Haemorrh 28:342-350, 1972
A.J.C.P. • October 1978
