The Tri-F Titer. Fibrinolysis, Fibrin(ogen) Split Products, and Heparin. Cary J. Lambert, M.D., Alain J. Marengo-Rowe, M.D.,

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1 A Rapid Test for Estimation of Plasma Fibrinogen and Detection of Fibrinolysis, Fibrin(ogen) Split Products, and Heparin Cary J. Lambert, M.D., Alain J. Marengo-Rowe, M.D., James Leveson, Ph.D., Peter Alivizatos, M.D., Gerald F. Geisler, M.D., Maurice Adam, M.D., Ben F. Mitchel, Jr., M.D., and J. Peter Thiele, M.D. ABSTRACT The use of extracorporeal circulation has been associated with operative and postoperative hemorrhage. In patients on the pum.p there are a number of different pathogenetic mechanisms that lead to hemorrhagic disorders. In essence the hemorrhagic diathesis is caused by the increased utilization or destruction of hemostatic factors, the presence of circulating anticoagulants, a reduction in hemostatic factors due to underproduction or dilution by transfused banked blood, or all three. The tri-f titer (TFT) is a new rapid and reproducible test that gives an estimate of the fibrinogen concentration and detects the presence of fibrinolysis, fibrin(ogen) split products, and circulating heparin. The use of the TFT in the diagnosis of various coagulopathies is discussed. The TFT, which depends on the formation and observation of clots in vitro, is considered to have distinct advantages over other tests which rely on immunological, solubility, and other physicochemical phenomena. T he introduction of extracorporeal circulation has been associated with operative and postoperative bleeding. It has been observed that even in patients carefully selected for operation the hemostatic response remains unpredictable [ 101. Nonoperative factors which can predispose to abnormal bleeding include a prolonged pump run; a diminished marrow/liver reserve, resulting in the inability to manufacture the increased quantities of platelets and coagulation proteins imposed by the hemostatic challenge; preoperative medication with drugs such as aspirin, quinidine, and diuretics; and excessive intraoperative heparinization. In each instance a careful assessment of the clinical situation along with the correct interpretation of laboratory tests is essential for accurate diagnosis and successful therapy. In the management of a patient with sudden and unexpected hemorrhage, even a battery of tests such as the platelet count, From the Department of Special Hematology, Baylor University Medical Center, and the Department of Thoracic and Cardiovascular Surgery, Baylor University Medical Center, St. Paul Hospital, and The University of Texas Southwestern Medical School, Dallas, Tex. Presented at the Twentieth Annual Meeting of the Southern Thoracic Surgical Association, Louisville, Ky., Nov. 1-3, Address reprint requests to Dr. Lambert, 3434 Swiss Ave., Suite 404, Dallas, Tex VOL. 18, NO. 4, OCTOBER,

2 LAMBERT ET AL. prothrombin time (PT), partial thromboplastin time (PTT), and fibrinogen level may not provide suffcieiit information on which to base a specific diagnosis. With the exception of the platelet count, all the other tests mentioned can be so markedly afl ected by lysed products of coagulant proteins [5] and by circulating anticoagulants, such as heparin, that their diagnostic usefulness may be severely limited. More elaborate laboratory procedures such as coagulation factor assays, euglobulin clot lysis time (ECLT), and fibrin(ogen) split product (FSP) assays, which give further information, are available. However, these tests take a longer time to perform than the clinical situation allows and do not always correlate with the clinical picture. In this communication we describe a test, the tri-f titer (TFT), in which serial dilutions of the patient s plasma in saline, eaminocaproic acid (EACA), and a protamine sulfate/eaca (PS/EACA) mixture are used to estimate the fibrinogen concentration and detect fibririolytic activity and FSP. This test also detects the presence of circulating heparin. The TFT has proved to be a most valuable test in the diagnosis 2nd management of patients on the pump ivho are in acute hemorrhagic states. This test is rapid and can be performed on the same blood sample that is used for the PT and PTT. The T1.i-F Titer Venous blood is collected into 3.2%) trisodium citrate anticoagulant, and the plasma is separated as for other coagulation studies. A total of 0.9 ml. of the patient s plasma is required for this test. Doubling dilutions of the patient s plasma are made in three separate diluents: the first in saline, the second in EACA (Lederle), and the third in a mixture of PS (Eli Lilly) and EACA. All the tubes are then placed in a 37 C. incubator and thrombin is added. The end-point of each titration is taken as the last tube in which a visible clot is formed. The purpose of EACA is to inhibit fibrinolysis, while that of PS is to negate the anticoagulant effects of FSP [I I]. The layout of the three rows of tubes is shown in Figure 1. Sodium chloride (0.3 ml. of 0.15 M) is added to each tube in the first row: 0.3 ml. of M EACA in 0.15 M sodium chloride solution is added to each tube in the second row; 0.3 ml. of 40 7, mg. per 100 ml. PS in 0.15 M sodium chloride is added to the first tube only of the third row, and 0.3 ml. of M EACA in 20 mg. per 100 ml. PS (prepared by mixing the M EACA and 40 mg. per 100 ml. PS, in equal volumes) is added to the remainder of the tubes in the third row. Using a pipette, 0.3 ml. of the patient s plasma is added to the first tube in the first row. The contents of the tube are mixed, using the pipette, and 0.3 ml. is then transferred to the second tube; doubling dilutions are continued to the eighth and last tube in the first row, from which 0.3 ml. of the contents is discarded. Serial doubling dilutions of 358 THK ANNALS OF THORACIC SURGERY

3 PLA MA 1:2 1:4 1:s 1:16 1:32 1:64 1: FIG. 1. Layout of tubes in tri-f titer test. (PS = protamine sulfate; EACA = -aminocapoic acid; SAL = saline.) the patient s plasma are performed in an identical manner in the second row, which contains EACA, and then again in the third row, which contains the PS/EACA mixture. A control is performed in similar fashion using plasma containing a standardized amount of fibrinogen (Hyland). Thrombin (Parke-Davis), 0.02 ml. at 25 units per ml., is added to all 24 tubes. The test and control dilutions are then incubated in a water bath at 37 C. for 5 to 15 minutes. Each tube is then inspected individually for the presence of a clot by a gentle tilting action, and the last tube in which a gelatinous mass is seen is recorded as the end-point of the titration. In the PS/EACA dilution the clots are unlike those seen in the other two rows. Instead of the usual gelatinous mass, a small, coherent, retracted clot forms, and the highest dilution in which such a clot is seen is taken as the endpoint. The subsequent tube in this row may contain loose strands of fibrin and should be ignored. The sensitivity of the tri-f titer can be increased by reexamining all tubes at 30 minutes and noting any changes in the titrations. It is most convenient and timesaving to arrange the tubes with the reagents in them in the sequence described and store them at -20 C. When required, the tubes may then be warmed to 37 C. while the patient s blood is being centrifuged, and the results can be available within 30 minutes of obtaining the patient s blood sample. Results and Interpretation of Tri-F Titer A group of 224 patients undergoing cardiopulmonary bypass over a period of five years were surveyed and are included in this study. All patients were examined and hematologic/hemostatic profiles obtained. In each instance blood smears were assessed and platelet counts obtained. The bleeding time was estimated according to Ivy [3]. The PT was determined using thromboplastin and control plasma (Metrix). The PTT with kaolin was performed using kaolin,/cephalin and control reagents (Hyland). The fibrinogen concentration was estimated by the method of VOL. 18, NO. 4, OCTOBER, !)

4 LAMBERT ET AL. COMPARISON BETWEEN THE TITER IN THE PS/EACA MIXTURE AND THE FIBRINOGEN LEVEL Fibrinogen Level (mg./ 100 ml.) Titer Value Range Mean No. of Samples 1:2 1 :4 1:8 1:16 1:32 1:64 1 : 128 1:256 1:512 1 : 1, Ratnoff and Menzie [9], and FSP were quantitated by tanned red cell hemagglutination inhibition (TRCHI) technique [8] using commercial reagents (Burroughs and Welcome). The ECLT was measured according to von Kaulla and Schultz [13], and fibrin plates were prepared with human fibrinogen as described by Astrup and Mullertz [l]. 'The TFT was performed in all cases, and, when required, heparin assays were carried out as described by Dacie and Lewis [2]. The interpretation of the TFT is as follows. The fibrinogen level is directly related to the titer in the PS/EACA mixture: the lower the titer in the mixture, the lower the fibrinogen level in the patient, and vice versa. The normal range is 1:128 to 1:256, which is equivalent to a fibrinogen concentration of 300 mg. per 100 ml. or more (Table, Fig. 2). Statistical analysis cannot be performed directly on the data in the Table since graphically we are plotting a continuous function (fibrinogen level) against I 1 I I I I I FIG. 2. TITER Correlation belwcen the tri-f titer and the fibrinogen level. 360 THE ANNALS OF THORACIC SURGERY

5 a discontinuous function having increasing interval (titer value in protamine sulfate). An approximation can be made by plotting loglo (fibrinogen level in milligrams per deciliter) against log:! (fibrinogen titer in PS/EACA). Thus the titer now has integer values, and although it is still a discontinuous function, it is a reasonable assumption that it behaves as a continuous function since its intervals are now constant. Analyzing the data in this way gave a correlation coefficient of and the relationship: loglo (fibrinogen level) = log, (titer value in protamine sulfate). Comparison of the titer in PS/EACA with that in EACA alone detects the presence of FSP. If the titer is shorter in EACA than in the PS/EACA mixture, one can conclude that FSP are present in the plasma in sufficient amounts to interfere with clot formation. In correlation experiments on plasma samples treated with streptokinase, the presence of FSP was quantitated by TRCHI and detected by the TFT. It was observed that a two-tube difference in titer between the EACA and PS/EACA rows indicated the presence of FSP in amounts greater than 75 pg. per milliliter at a fibrinogen concentration of 160 mg. per 100 ml. (or less). In patients specimens chosen at random, however, no correlation was possible between the tri-f titer and the TRCHI assay. This is hardly surprising, since the TRCHI measures the presence of fibrin split products immunologically in serum while in the tri-f titer test they are detected as they interfere with coagulation in plasma. In cardiac surgery, two commonly used tests-the staphylococcal clumping assay for FSP and the TRCHI-do not quantitatively agree. Recently Leavelle and associates [6] have shown that while the staphylococcal clumping test and the TRCHI both measure fibrin split products, they do not measure the same types and hence cannot correlate in all situations. In samples of heparinized blood it was noted that no fibrin clots formed in the lower dilutions of the patient s plasma in saline and EACA, while clots did form in the equivalent dilutions in PS/EACA. In the saline and EACA rows the increasing dilutions of the patient s plasma reduce the concentration of heparin to a level at which it becomes inadequate to neutralize the added thrombin; in the PS/EACA row the effect of heparin is neutralized by the protamine sulfate in all tubes. The TFT is extremely sensitive to heparin and can detect its presence at levels below 0.1 unit per milliliter. It should be emphasized that whereas this test does not measure the heparin concentration in units per milliliter (because the fibrinogen level in the patient affects this assay), it nevertheless can easily detect the presence of heparin down to levels of less than 0.1 unit per milliliter. In 200 specimens analyzed during thoracic operations, we have found that in titers in which the first four tubes contaitiing saline or EACA did not clot, the patient had in excess of 3 units of heparin per milliliter of blood. Patient specimens VOL. 18, NO. 4, OCTOBER, 1974 $1

6 LAMBERT ET AL. which did not clot in the first tube of the saline and EACA rows had a heparin level of 0.1 to 1.0 unit per milliliter of blood. Finally, it was not possible to observe any consistent correlation between excessive fibrinolytic activity as detected by the ECLT and as estimated by the TFT. However, fibrinolysis detected by TFT was found to correlate much more closely with the clinical situation. Comment In the investigation of a bleeding patient, the TFT enables one to obtain rapidly four pieces of relevant information, namely, the level of fibrinogen and the presence of fibrinolysis, FSP, and circulating heparin. Since the PT and PTT themselves are dependent upon normal fibrinogenfibrin conversion, the TFT also acts as a guide to interpretation of the intrinsic and extrinsic mechanisms of blood coagulation. The relative ease and speed with which this test can be performed makes it particularly suitable for urgent situations. Previously the thrombin time, corrected with protamine sulfate, had been used to detect the presence of heparin. However, in our hands this test has given rather inconclusive results and does not differentiate between the presence of heparin and FSP. In the presence of a high level of heparin-which completely eliminates clotting in the EACA and saline rows-it is not possible to detect fibrin split products; but in a patient who is bleeding, clearly the detection of circulating heparin is of paramount importance. Administration of protamine sulfate to the patient would then permit the detection of circulating fibrin(ogen) split products if that were still clinically necessary. Although it is conceivable that patients specimens with an extremely low fibrinogen level could have no clots in the saline and EACA rows due to the presence of large quantities of fibrin(ogen) split products, no such example has been found to date. The TFT is interpreted by comparing the respective titers of the patient s plasma diluted in saline, EACA, and PS/EACA, clotted with thrombin. In the test system, the function of EACA is to inhibit fibrinolysis and that of protamine sulfate is to neutralize heparin and overcome the inhibitory effects of any FSP present on fibrinogen-fibrin conversion [ 121. The titer patterns of heparin and FSP are quite different and therefore can easily be differentiated from one another. Close correlation was obtained between the levels of fibrinogen and FSP as estimated by the TFT and other, more conventional and lengthy techniques. However, a consistent correlation was not found in the detection of excessive fibrinolysis by the tri-f titer and by the ECLT. This is hardly surprising since these two tests are not strictly comparable. In the tri-f titer, whole plasma is used and the inhibitory effect of EACA on any fibrinolytic activity that may be present is observed. In the ECLT only the euglobulins (activators, plasminogen, and fibrinogen) are allowed to participate in the 962 THE ANNALS OF THORACIC SURGERY

7 test, and the effects of natural inhibitors and other plasma fractions are eliminated and therefore ignored [4]. From our clinical studies we were able to confirm the earlier observation of Sharp and Eggleton [ll] that the differential fibrinogen titer in saline and EACA is a useful laboratory procedure for the detection of excessive fibrinolysis. The diagnosis of bleeding disorders is an aspect of clinical medicine in which laboratory tests are chosen and interpreted in light of the clinical findings. In essence, nonoperative bleeding in patients on the pump can be brought about by several mechanisms. The increased utilization or destruction of hemostatic factors (abnormal coagulation/fibrinolysis), the presence of circulating anticoagulants (heparin), and the reduction in circulating hemostatic factors due to underproduction or dilution by transfused banked blood (platelets, factors VIII and V) have individually or in combination given rise to hemorrhagic syndromes [7]. A battery of tests such as the PT, the PTT, and an assessment of the peripheral smear and platelet count is essential for diagnosis. The addition of the TFT provides further relevant information and serves to assist in the interpretation of the clinical and laboratory findings. References 1. Astrup, T., and Mullertz, S. Fibrin plate method for estimating fibrinolytic activity. Arch. Biochem. 40:346, Dacie, J. V., and Lewis, S. M. Practical Haematology (4th ed.). London: Churchill, 1968, P Ivy, A. C., Nelson, D., and Bucher, G. The standardization of certain factors in cutaneous venostasis bleeding time technique. J. Lab. Clin. Med. 26: 1812, Katz, J., Lurie, A., Becker, D., and Metz, J. The euglobulin lysis time test: An ineffectual monitor of the therapeutic inhibition of fibrinolysis. J. Clin. Pathol. 23:529, Kowalski, E. Fibrinogen derivatives and their biologic activities. Semin. Hematol. 5:45, Leavelle, D. E., Bowie, E. J. W., Merten, B. F., McDuffie, F. C., and Owen, C. A. Assay of fibrin split products: Comparison of staphylococcal clumping and hemagglutination-inhibition tests. J. Lab. Clin. Med. 77:993, Marengo-Rowe, A. J., and Leveson, J. E. Developments in transient hemorrhagic disorders. Tex. Med. 69:72, Merskey, C., Kleiner, G. J., and Johnson, A. J. Quantitative estimation of split products of fibrinogen in human serum: Relation to diagnosis and treatment. Blood 28: 1, Ratnoff, 0. D., and Menzie, C. A new method for the determination of fibrinogen in small samples of plasma. J. Lab. Clin. Med. 37:316, Salzman, E. W., and Britten, A. Hemorrhage and Thrombosis. Boston: Little, Brown, P Sharp, A. A., and Eggleton, M. J. Haematology of the extracorporeal circulation. J. Clin. Pathol. 16:551, Stewart, G. J., and Niewiarowski, S. Aggregation of fibrinogen and its degradation products by basic proteins. Thromb. Diath. Haemorrh. 25:566, von Kaulla, K. N., and Schultz, R. L. Methods for the evaluation of human fibrinolysis: Studies with two combined technics. Am. J. Clin. Pathol. 29: 104, VOL. 18, NO. 4, OCTOBER,