Prothrombin Time after Heparin Removal Application to Monitoring Simultaneous Anticoagulation with Heparin and Coumarins HERMAN E. BRANSON, M.D., LEWIS M. SLATER, M.D., MARIE G. ANDERSON, M. T. (ASCP)SH, AND HUBERT PIRKLE, M.D.

RECENT EVIDENCE indicates that certain vitamin K-mediated reactions necessary for carboxylating and possibly sialylating the precursors of factors II, VII, IX and X are altered in the presence of coumarins, resulting in the circulation of proteins with impaired procoagulant activity, reflected functionally as defective binding capacity for calcium, barium, and phospholipid.4'7,8" Thompson and Counts described a simple and effective chromatographic technic for the removal as much as 300 u/ml heparin from citrateanticoagulated plasma employed in routine coagulation screening and specific factor assays.12 In the original procedure the acidic anticoagulant was adsorbed from an aqueous milieu containing presumably functionally competent clotting proteins. As patients frequently receive concurrent anticoagulant treatment with coumarins, or indanediones, and Received May 22, 1978; accepted for publication June 14, 1978. Address reprint requests to Dr. Branson: Department of Pathology 40, U.C.I. Medical Center, Bldg. 10, 101 City Drive South, Orange, California 92668.

Departments of Pathology and Internal Medicine, University of California, Irvine, Irvine, California

heparin, it was important to establish whether the coumarin effect significantly altered the chromatographic removal of heparin. Despite the almost universal adoption of more regimented protocols for heparin administration, on an individual basis, this inhibitory effect varies considerably due to differences in drug preparations, metabolic life span, antithrombin IN, and platelet factor 4 levels. The heparin effect, with its minor fluctuations, unfortunately obscures the actions of the vitamin K antagonists, thereby prohibiting the accurate clinical assessment of such anticoagulation by means of the one-stage prothrombin time test. 313 Special dilutional prothrombin time reagent systems were developed specifically for this application, which effectively eliminated heparin's influence while surrendering some of the sensitivity of the simpler test.' !,1° In the present study heparin-removal chromatography, in combination with the one-stage prothrombin time, was found to be a practical alternative to these dilutional technics of dual anticoagulant monitoring. Materials and Methods Epichlorohydrin triethanolamine cellulose (ECTEOLA) cellulose anion exchanger, capacity 0.33 mEq/g, medium mesh, was obtained from Sigma Chemical Company, St. Louis, Missouri. Heparin sodium salt from Rilker Laboratories, Northridge, California, was employed for simultaneous studies. Commercial prothrombin time reagent was obtained from Ortho Diagnostics, Raritan, New Jersey. All prothrombin times were determined by the fibrin switch semiautomated technic.*5-6 Following equilibration in 0.05 M Tris-HCl {pH 7.5) * Fibrometer, BBL, Baltimore, Maryland.

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Branson, Herman E., Slater, Lewis M., Anderson, Marie G., and Pirkle, Hubert: Prothrombin time after heparin removal. Application to monitoring simultaneous anticoagulation with heparin and coumarins. Am J Clin Pathol 71: 665-667, 1979. Coumarin-anticoagulated plasmas from 31 subjects comprising two study groups were passed through epichlorohydrin triethanolamine cellulose (ECTEOLA) chromatography columns. Group I plasmas, which were run with nothing added, had mean prothrombin times of 21.6 ± 5.4 sec prior to and 22.4 ± 5.6 sec following exposure to these columns (r = 0.9449; / = 1.8307, P < 0.100). The mean prothrombin time for Group II, 18.2 ± 4.9 sec, lengthened with 5 u/ml heparin to 35.5 ± 16.2 sec, and returned to 19.2 ± 5.0 (r = 0.9763) after chromatography. Therefore, it appears that coumarin anticoagulation had no significant influence upon the capacity of ECTEOLA effectively to remove heparin in therapeutic doses. This means that virtually all prothrombin time reagent systems can be employed to monitor concurrent heparin and coumarin anticoagulation. In addition, quality control of the combined technic is simpler, and the technic more sensitive to low levels of fibrinogen and factor V. (Key words: Prothrombin in time after heparin removal; Dual anticoagulant therapy; Owren prothrombin times.)

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Table I. Therapeutic Indices for Group I Plasmas (Conmarin Effect Alone) Given as Number of Times Baseline

Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Pre-column

Post-column

2.3 2.8 2.1 1.5 2.1 1.7 1.5 1.1 1.4 1.5 1.6 1.6 2.0 1.4 2.3

2.3 3.0 2.1 1.5 2.1 1.9 1.8 1.1 1.4 1.8 1.7 1.5 2.0 1.7 2.3

Therapeutic range = 1.5- 2.5 x baseline pro

containing 0.1 M NaCl (Tris-saline solution) ECTEOLA was poured into modified disposable one-piece polyethylene pipettes without plasticizer.t Column retention supports were fashioned of polyester surgical dressing. The final packed column height was between 0.5 and 2.0 cm. Columns were stored immersed in Trisbuffered saline solution (/?H 7.4, 172 = 0.12), sealed with Parafilm,® and maintained at 4 C until used. Samples of blood from patients being treated with coumarins alone were collected in Vacutainers® containing 1/9 vol 3.8% sodium citrate. Samples were centrifuged at 4 C for 10 min at 10,000 x g. Coumarinized and coumarinized-heparinized samples t Beral Enterprises, Inc., Arleta, California.

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FIG. 2. Prothrombin clotting times at three experimental intervals for 16 patients in Group II. N, baseline clotting times (mean [x] = 18.2 ± 4.9 sec). H, times following addition of 0.5 u/ml heparin (mean [x] 35.5 ± 16.2 sec). C, times following heparin removal (mean [x] = 19.2 ± 5.0 sec).

were applied to ECTEOLA columns immediately after the efflux of Tris-saline buffer. Six or seven drops of the initial amber effluent were discarded before assaying. Plasma showing a coumarin effect (3 sec more than control) were admitted randomly to Groups I and II. In Group I, prothrombin times were performed on coumarin-anticoagulated plasmas before and after chromatography. Group II coumarin-anticoagulated plasmas after initial assay had heparin added to a final concentration of approximately 5 u/ml. These speciTable 2. Pre-column and Post-column Therapeutic Indices for Group II Plasmas (Dual Anticoagulant Effect)

Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Pre-column

Post-column

1.3 1.4 1.1 1.5 1.1 2.1 1.4 2.4 1.7 1.8 1.2 1.9 1.1 1.1 1.9 1.2

1.4 1.5 1.2 1.6 1.2 2.3 1.6 2.3 1.8 1.9 1.2 2.0 1.2 1.2 2.0 1.3

Therapeutic range = 1.5-2.5 x baseline pr

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FIG. 1. Prothrombin clotting times prior to (x) and following (y) ECTEOL A chromatography for 15 patients comprising Group 1. The linear regression is described by: y = 1.3579 + 0.9772x (r = 0.9449). The mean ( + ) for the regression line is depicted ±1 SD (x = 21.6 ± 5.4 sec, y = 22.4 ± 5.6 sec).

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PROTHROMBIN TIME AFTER HEPARIN REMOVAL

mens were reassayed, passed through the exchange column, and assayed a third time. Verification of coumarin anticoagulation alone was secured from the requesting physician for patients in the study group. Results

Chromatography in the Presence of Heparin The addition of the 5 u/ml heparin to the plasma of 16 patients being treated with coumarin anticoagulants in Group II resulted in a mean prolongation of 17.3 sec from 18.2 ± 4.9 sec to 35.5 ± 16.2 sec. Heparin removal yielded a mean prothrombin time of 19.2 ± 5.0 sec, which correlated well with the prechromatography value (r = 0.9763) (Fig. 2). The fact that the chromatographic procedure did not alter the invitro measurement of the coumarin anticoagulant effect in the presence of heparin was verified despite a small but significant difference (/ = 3.6802; P < 0.010) between the paired samples. As shown in Table 2, the therapeutic indices were not significantly altered. Discussion The combination of heparin-removal chromatography with the prothrombin time has several advantages in monitoring patients simultaneously receiving anticoagulant therapy with heparin and coumarin. The utility of the technic rests in its applicability to virtually all one-stage prothrombin time reagents, thereby eliminating the need for the prothrombin and proconvertin method, Thrombotests®, or Two-Seven-Ten® reagents.1-810 While these specialized prothrombin times have been effectively and extensively employed for dual-anticoagulant monitoring, they represent a significant investment in terms of standardization and maintenance.2 In addition, these special reagents contain bovine factor V and fibrinogen in the form of adsorbed plasma to enhance the reproducibility of the diluted specimens. This, however, renders them insensitive to low levels of these factors, unlike the

References 1. Denson KWE: An investigation of thrombotest and the preparation of a similar combined reagent. J Inst Med Lab Tech 18:257, 1961 2. Denson KWE, Biggs R: Laboratory diagnosis, tests of clotting function and their standardization, Human Blood Coagulation, Haemostasis and Thrombosis. Edited by Bigg R. Oxford, Blackwell Scientific Products, 1976, p 565 3. Douglas, AS, McNicol GP: Anticoagulant therapy, Human Blood Coagulation Haemostasis and Thrombosis. Edited by Biggs R. Oxford, Blackwell Scientific Products, 1976, p565 4. Frenlund P, Stenflo J, Suttie JW, et al: Vitamin K and the biosynthesis of prothrombin. V. y-Carboxyglutamic acids, the vitamin K dependent structures in prothrombin. J Biol Chem 250:6125-6133, 1975 5. Lusted D, Bartlett RC, Dawson J: Evaluation of a semiautomatic prothrombin time determination. Am J Clin Pathol 43:393-395, 1965 6. Miale JB: The fibrometer system for routine coagulation tests. Am J Clin Pathol 43:475-480, 1965 7. Morris HR, Thompson MR, Dell A: Synthesis and proof of structure of a new amino acid in prothrombin. Biochem Biophys Res Commun 62:856-861, 1975 8. Morrissey JJ, Jones JP, Olson RE: Isolation and characterization of isoprothrombin in the rat. Biochem Biophys Res Commun 54:1075-1082, 1973 9. Owren PA: Thrombotest. A new method for controlling anticoagulant therapy. Lancet 2:754-758, 1959 10. Owren PA, Aas K: The control of dicumarol therapy and the quantitative determination of prothrombin and proconvertin. Scand J Clin Lab Inv 3:201, 1951 11. Stenflo J, Fernlund P, Egan W, et al: Vitamin K-dependent modifications of glutamic acid residues in prothrombin. Proc Natl Acad. Sci (USA) 71:2730-2733, 1974 12. Thompson AR, Counts RB: Removal of heparin and protamine from plasma. J Lab Clin Med 88:922-929, 1976 13. Wessler S, Gitel S: Control of heparin therapy. Prog Hemostasis Thromb. 3:311-329, 1976

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Chromatography without Heparin Fifteen randomly selected samples (Group I) were chromatographed to assess the effect of ECTEOLA upon the prothrombin time. The mean for the group prior to passage was 21.6 ± 5.4 sec; 22.4 ± 5.6 sec following exposure to the anion exchange cellulose it was (Fig. 1). The correlation coefficient (r) was 0.9449. The differences between samples before and after treatment were not significant according to the Student / test for paired data (/ = 1.8307; P < 0.100). Survey of the therapeutic indices disclosed no significant postchromatography difference warranting alteration of therapy (Table 1).

normal prothrombin reagent. Because both fibrinogen and factor V are unaffected by exposure to such a mild anion exchanger, the prothrombin time with heparin removal retains its responsiveness to alterations in the levels of these factors. Furthermore, the coumarin anticoagulant effect does not seem to interfere with the removal of heparin in the upper therapeutic range of concentrations (5 u/ml). In addition, no patient showed an alteration in therapeutic indices that necessitated an increased dosage of vitamin K antagonists. Although three of 12 "hypocoumarinized" patients were moved into the therapeutic range following chromatography, these shifts were within the range of variation (±1.5 sec) for running a one-stage prothrombin time in most laboratories. It is also conceivable that when precise quantitation of heparin is desired, in situations of simultaneous anticoagulation, elution from the cellulosic exchanger and assay may be possible. In conclusion, it appears that the prothrombin time with heparinremoval chromatography is a sensitive and flexible, yet simple, alternative to the dilutional tests for monitoring heparin-coumarin therapy.

Prothrombin time after heparin removal. Application to monitoring simultaneous anticoagulation with heparin and coumarins.

Prothrombin Time after Heparin Removal Application to Monitoring Simultaneous Anticoagulation with Heparin and Coumarins HERMAN E. BRANSON, M.D., LEWI...
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