Vol. 38, No. 5, pp November, 1962 Copyright 1962 by The Williams & Wilkins Co. Printed in U.S.A.

THK AMKHICAN JOURNAL OF CLINICAL PATHOLOGY Vol. 38, No. 5, pp. 473-481 November, 1962 Copyright 1962 by The Williams & Wilkins Co. Printed in U.S.A. THE PARTIAL THROMBOPLASTIN TIME (CEPHALIN TIME) IN ANTICOAGULATION THERAPY GERD P. STRUVER, M.D., AND DONALD L. BITTNER, M.D. Departments of Pathology and Medicine, St. Mary's Hospital, San Francisco, California For a number of years it has been known that tissue extracts can be used to accelerate coagulation of blood. Schmidt, 16 in 1892, recognized that alcoholic tissue extracts accelerate clotting times, and it was believed that the active principle was a lipoid belonging to the lecithins or cephalins. That purification of this substance led to decreased thromboplastin activity was demonstrated by Fischer and Hecht 6 in 1934. It was further revealed that pure cephalin had no thromboplastin activity. The difference in activity between cephalin and tissue thromboplastin was described by Mills, 9 who demonstrated that cephalin preparations yield longer clotting times in hemophiliac persons than tissue thromboplastins, which yield normal clotting times. Bell and Alton, 2 in 1954, modified the extraction process and obtained a more active cephalin preparation. Langdell and associates, 6 in 1953, and Rodman and associates," in 1958, used cephalin to investigate and control hemophiliac disorders, but the clotting times they obtained were long because of submaximal activation. Waaler, 18 in Oslo, and Margolis, 8 in this country, investigated the activation phenomenon of plasma, using Celite and kaolin, respectively, to initiate and accelerate activation. With the use of either of these substances, the reproducibility of the cephalin time (partial thromboplastin time) seemed to be improved. Received, February 10, 1902; revision received, June 29; accepted for publication August 6. Dr. Struver is Assistant Resident in Medicine, Department of Medicine, and Dr. Bittner is Associate Pathologist. This study has been supported in part by a grant-in-aid from Warner-Chilcott Laboratories, Department of Clinical Investigation, General Diagnostics Division. A portion of the study utilized a Platelet Factor Reagent kindly supplied by General Diagnostics Division, Warner-Chilcott. This paper was presented at the regional meeting of The College of American Pathologists, San Francisco, California, December 2, 1901. 473 Very recently Proctor and Rapaport 11 also studied the cephalin time activated with kaolin and demonstrated reproducibility and control similar to that achieved in this study. The factor apparently responsible for the more rapid initiation of the clotting of the cephalin time and for the more reproducible results has been the large surface area supplied by these particulate silicates, which has effectively brought about maximal glass activation. Since 1959, workers in this laboratory have used a Celite-activated cephalin time as 1 study in the laboratory's coagulation investigations. The procedure used has been essentially that outlined by Waaler. 18 Although the reproducibility has been fair, certain variables in the technic used have been demonstrated to influence the results obtained. For this reason an investigation has been made in an attempt to design a more standardized and reproducible partial thromboplastin time that would incorporate maximal glass activation with Celite. It was the final objective in the study described in this paper to observe the behavior of the cephalin time on the plasma of patients receiving anticoagulant drugs, in order to determine the influence of these various drugs on the intrinsic coagulation mechanism. 14 The effect of certain variables in the performance of the partial thromboplastin test are illustrated, and the final method used is outlined. A comparison has been made of the effects of the 1-stage prothrombin time, the Thrombotest, 17 and the partial thromboplastin time on the plasma of those patients on coumarin therapy in this hospital. A separate study deals with the in vitro effect of heparin on the partial thromboplastin time. KEAGENTS AND EQUIPMENT Cephalin was prepared according to the method of Bell and Alton. 2 A 1:100 suspension was prepared by saline dilution of the

474 STKUVER AND BITTNER Vol. 38 stock suspension, which was kept at 20 C. Aliquots of the 1:100 cephalin suspension were stored in the refrigerator. A saline-celite suspension was prepared with Celite 315 (Johns-Mansville, San Francisco). This material is a once-calcined, diatomaceous silicate with 65 per cent of the particles ranging in diameter from 1.0 to 4.0 fi. The silicate surface area of 1 Gm. of Celite 315 is approximately 5 sq. m. Celite, 0.7 Gm., was suspended in normal saline solution to yield a final volume of 100 ml. This suspension represents approximately 1 volume of Celite to 20 volumes of saline solution, or 3.5 sq. m. of silicate surface area per 100 ml. One advantage of this particulate silicate as compared with other silicates we have used is that the settling rate of the particle is slow. When settling has occurred, resuspension is easily achieved by shaking the suspension momentarily before use. The calcium reagent was 25 mm calcium chloride solution. All blood was obtained with new Vacutainer test tubes No. 3204W (Becton, Dickinson & Co., Columbus, Nebraska) containing 0.5 ml. of 3.8 per cent sodium citrate. The negative pressure in this tube is adjusted to yield a final volume of 5.0 ml. The accuracy of the volume delivered was tested on several occasions, and no significant discrepancy was observed. The pipets used were blow-out, 0.2-mL, rapid flow, glass pipets graduated at 0.1 ml. and 0.2 ml. Clean glass test tubes of the type used for the collection of blood were used for the actual performance of the partial thromboplastin time. All glassware was washed with chromic acid for 1 to 3 hr. and was finally rinsed with 2 changes of double-distilled water. The glassware was used only for this coagulation study. The water bath used was maintained at a constant temperature between 37 and 37.5 C. STUDY OF TEST VARIABLES Pooled normal plasma was obtained from 5 or more apparently normal donors who had no history of bleeding disorder and who were not receiving drugs that would influence coagulation activity. In general, each test was performed in triplicate not later than 2 hr. after collection. Plasma that was not used immediately was stored at 4 C. In order to determine the roles of platelets and of centrifuge speed on the partial thromboplastin time, pooled normal blood was divided into 4 aliquots, which were centrifuged at varying speeds ranging from 1600 to 3800 r.p.m. for 10 min. On the plasma collected from these aliquots, which presumably contained varying concentrations of platelets, a plasma recalcification time was made after the method of Waaler, 18 and then a partial thromboplastin time was performed. It can be seen (Fig. 1) that, after the addition of cephalin reagent, clotting times of all 4 aliquots were identical. This result has demonstrated to us that platelet concentration does not influence the partial thromboplastin time by our method and that centrifuge speed within the range of our experiment is not critical in regard to the final results. In contrast, the plasma recalcification time is greatly altered by changes in centrifuge speed or platelet concentration, and a comparison of the plasma recalcification time with the cephalin time rapidly and effectively demonstrates the presence of a platelet deficiency or functional defect. Inasmuch as the relative concentration of the activating particle has been demonstrated by others 8,18 to alter the partial thromboplastin time, we have attempted to devise a method of adding accurately the optimal amount of Celite for our method. Although others 18 have added the dry particle, we have found that this is somewhat awkward and does not always produce consistent results unless the powder is accurately weighed before it is added to individual tubes. For this reason, we have made suspensions of varying concentrations of Celite (Fig. 2) in saline solution in order to demonstrate the effect of Celite concentration on our final method. The results of this study have revealed that high concentrations of Celite tend to lengthen the partial thromboplastin times, presumably

Nov. 1962 CEPHALIN TIME ANTICOAGULATION 475 100-90- r~ 80-70- ^60- Q 50- u Ul </> 40-30- & ^j CEPHALIN TIME rime 2 3 < CEPH Q Z SECC 20-10 ' 1 1 1 _1 1600 2300 3300 3800 1:5 1:10 1:20 1:40 CENTRIFUGE SPEED (rpm) RATIO OF CELITE TO SALINE V./v. FIG. 1 (left). The role of centrifuge speed FIG. 2 (right). The effect of concentration of Celite on the basis of mechanical interference with the clotting process, as well as to yield poor visualization of the end point. Low concentrations of Celite also tend to lengthen the clotting time, presumably on the basis of submaximal glass activation of the system. We have found that the concentration of Celite yielding the shortest clotting times by our final method is 1:20, volume for volume with saline solution. This concentration was regarded as the optimal one and was subsequently used throughout our study. Our investigation of the various saline dilutions of the stock cephalin preparation of Bell and Alton has demonstrated that only slight variations in the partial thromboplastin time result from the use of saline dilutions of 1:25 to 1:200. Although the results obtained with various dilutions were found to differ slightly with different preparations of the concentrate, adjustment of the cephalin solution to yield the desired time with pooled normal plasma is readily accomplished by trying the test on serial dilutions of the concentrate. We have used a saline dilution of 1:100 of the cephalin concentrate as suggested by Bell and Alton. 2 This mixture has manifested good stability characteristics when stored in the refrigerator. A separate study of the stability of this reagent at room temperature, when no special precautions are taken to prevent bacterial contamination, demonstrates that activity remains fairly consistent for a period of approximately 2 weeks. The concentration of calcium used with the partial thromboplastin time does not seem to be critical, so long as enough is present to allow rapid generation of the thromboplastin complex. We have chosen to use 25 mm calcium chloride, believing that this represents a 2-fold excess of calcium under the conditions of citrate volume and concentration used for blood collection. The effect of various means of agitation, both hand and mechanical, during the activation phase of our partial thromboplastin time was studied. Although hand agitation has been suggested by others, 18 we have found agitation more consistent and uniform when a mechanical apparatus such as the Vortex Jr. Mixer (Scientific Industries, Springfield, Mass.) is used, although the use of such an instrument is a matter of convenience rather than necessity. After 1 min. of hand agitation or 15 sec. of mechanical agitation, the maximal effect of agitation is gained, although maximal production of the activation product of citrated plasma is not obtained until approximately 5 min. later. The effect of temperature on plasma storage has also been studied (Fig. 3).

476 STRUVER AND BITTNEB Vol. 38 T 1 y 40-30- 37t 0 30 60 120 180 MINUTES AFTER ACTIVATION FIG. 3. The influence of time and temperature after activation. After maximal production of the activation product was obtained, aliquots of pooled normal plasma were stored on ice (4 C), at room temperature, and in the water bath at 37 C. At both room temperature and 37 C, there was a conspicuous to great increase in the partial thi'omboplastin time with increasing incubation periods. In contrast, storage of the activated plasma at 4 C. demonstrated reasonably consistent clotting time over a period of 3 hr. after maximal glass activation was obtained. SUMMARY OF FINAL METHOD 1. In a glass test tube containing 0.5 ml. of 3.8 per cent sodium citrate are put 4.5 ml. of blood obtained by a clean venipuncture. The contents of the tube are centrifuged for 10 min. at 3300 r.p.m., and the plasma is separated and transferred to another clean glass test tube. 2. A mixture of 1 volume of plasma and 1 volume of saline-celite reagent is agitated on the Vortex Jr. Mixer for 15 sec. 3. To a 0.2-ml. aliquot of plasma-celite mixture, a 0.1-ml. aliquot of 1:100 partial thromboplastin (cephalin) reagent is added, and the mixture is allowed to incubate for at least 6.0 min. (Plasma-Celite mixtures not used immediately should be stored on ice.) 4. After the incubation phase, 0.1 ml. of 25 mm calcium chloride solution is blown into the plasma-celite-cephalin mixture and the stop watch is started. The contents of the tube are then mixed, and the tube is returned to the 37 C. water bath. After 30 sec. the tube is removed from the water bath and rolled gently between the fingers in adequate light over a black background in anticipation of an end point at 40 sec. The end point that we have recorded is the first appearance of a scalloped fibrin web at the margin of the plasma mixture. We have not used the initial granulation or the final solid clot as our end point, believing that the formation of the rolled "web" end point is more striking and instantaneous. In order to determine the normal partial thromboplastin time by this method, we have performed this test on 20 apparently normal laboratory personnel in replicate. None of these volunteers gave a history suggestive of a coagulation defect, and all had normal prothrombin times. The mean cephalin time in this group was 40.7 sec. with a coefficient of variation of less than 2.0 per cent. Using pooled plasma from this group, we have attempted to determine the correlation of the test with coagulation activity and to compare this correlation with those of Thrombotest and the 1-stage prothrombin time (Fig. 4). Dilutions of the pooled normal plasma were made with barium sulfate-absorbed plasma prepared after the method of Quick. 12 The barium sulfate-adsorbed plasma was adsorbed twice, and the final prothrombin time on this plasma was greater than 3 min. STUDY OF PLASMA FROM PATIENTS ON COUMA RIN TH EHA PY An initial study comparing the partial thromboplastin time with the 1-stage prothrombin time on patients having received coumarin drugs involved 60 patients at this hospital. Although the plasma of these patients revealed varying percentages of coagulation activity, the results of this study demonstrated that coagulation ac-

Nov. 1962 CEPHALIN TIME IN ANTICOAGULATION 477 150 140 130 120 110 100 90 - * * Thertipeuhc» J t f- Range / i / / m 80 Q O 70 O ui </> 60 50 40 30 _ THROMBOTEST^^ if=:^^== :::::=:::: CEPHAUH TIME ^*^>^ < 20 ""PROTHROMBIN TIME t i 10. 1. 1. 1 i 1 i 1. 1 i 1 1 1 100 50 20 10 PERCENT COAGULATION ACTIVITY FIG. 4. Correlation curves tivity judged by the partial thromboplastin time was approximately 4 per cent lower than coagulation activity judged by the 1- stage prothrombin time. There was no manifestation of spontaneous bleeding in these patients, and the partial thromboplastin time seemed to express the true coagulation status of the patient on coumarin therapy equally as well as did the 1-stage prothrombin time. Inasmuch as we have regarded the partial thromboplastin time as an expression of the status of the intrinsic coagulation system, and the 1-stage prothrombin time as an expression of the status of the extrinsic system, these observations would tend to confirm those of others 7 ' 10 that certain factors in the intrinsic coagulation mechanism are depressed with coumarin therapy. In this study the intrinsic mechanism seems to have been depressed to a level equal to the extrinsic system and sometimes more severely. In a second portion of our study on the plasma of patients receiving coumarin drugs, a comparison was made of the 1-stage prothrombin time and the partial thromboplastin time with the Thrombotest 10 in order to visualize the effects of coumarin drugs on the extrinsic and intrinsic systems combined, as well as on these systems individually. The plasmas of 45 patients on coumarin therapy were studied by means of all 3 tests performed in replicate. These 45 patients represented only those manifesting less than 50 per cent coagulation activity by means of all 3 tests. It was our belief that this group would represent patients who had received sufficient coumarin therapy to have reached a stable base level of coagulation activity. The 1-stage pro-

478 STRUVER AND BITTNER Vol. SS 140 IN VITRO HEPARIN CONCENTRATION mg. per ml. 0.0005 0.001 0.002 0.003 0.004 sr;-; >,, >' ; ' 1 1 ;i -: > "'' 71 I UJ \- 2 t-^ < ~\ u o CQ ^ o or T \- ~j <r K- Qr $ 1 CO O ^ o s> Ul CO 130 120 110 100 90 80 /O 60 50 40 /> i i [THERAPEUTIC RANGE''. 2"7' ><'--S-'I:I>^<IJIJ\ / / / J_LL II 1 i I 1 I-'.Ml y\" 100 70 50 30 20 10 5 4 % COAGULATION ACTIVITY ( BaS0 4 ADSORPTION ) FIG. 5 (upper). Average level of coagulation activity in coumarin-treated patients. The 45 patients all manifested less than 50 per cent coagulation activity, by means of all 3 tests. FIG. 6 (lower). Partial thromboplastin time correlation data

Nov. 1962 CEPHALIN TIME IN ANTICOAGULATION 479 thrombin time (Warner-Chilcott) and the Thrombotest (Nyegaard & Co., Oslo) were performed according to the specific directions of the manufacturer. The partial thromboplastin time Avas performed as indicated above. The results of this study demonstrated an average coagulation activity of 23 per cent as judged by the 1-stage prothrombin time, an average coagulation activity of 19 per cent as judged by the partial thromboplastin time, and a coagulation activity of 9 per cent as judged by the Thrombotest (Fig. 5). There was no spontaneous hemorrhage in this group. One episode of recurrent thrombosis was evident. This patient was within the accepted range of coagulation activity by the prothrombin time and the cephalin time, and yet the patient was in the potential bleeding range as judged by the Thrombotest. IN VITRO EFFECT OF HEPARIN ON THE PARTIAL THROMBOPLASTIN TIME During the course of this investigation, we noted that the plasmas from patients who had received heparin manifested evidence of being in the accepted therapeutic range of anticoagulation activity as judged by the partial thromboplastin time. These same plasmas revealed only slight to moderate evidence of depression of coagulation activity as judged by the 1-stage prothrombin time and by Thrombotest. In view of the known antithromboplastin activity of heparin, 3 it seemed feasible that the effect of heparin, which most noticeably alters the intrinsic coagulation mechanism, could be demonstrated with the partial thromboplastin time. In order to visualize the effect of heparin on this test, we added aqueous heparin to normal plasma to yield final concentrations of heparin of 5 X 10~ 4 and 1, 2, 3, and 4 X 10-3 mg. per ml. of plasma. The partial thromboplastin time was then performed on the plasmas containing dilutions of heparin. The results of this study (Fig. 6) clearly indicate that the partial thromboplastin time may be correlated with in vitro heparin concentrations and that the effect of heparin on the coagulation mechanism in vitro may be expressed in coagulation activity as judged by the partial thromboplastin time. In conjunction with this study, we have calculated that 100 mg. of aqueous heparin administered to a 70-kg. patient correspond to approximately 2.4 X 10~ 3 mg. of heparin per ml. of plasma. This dosage also corresponds to approximately 10 per cent coagulation activity as judged by the partial thromboplastin time. Clinical studies have further demonstrated to us that Lee-White clotting times of 35 min. closely correspond to 10 per cent coagulation activity as judged by our partial thromboplastin time. Additional studies in the control of heparin anticoagulation therapy with the partial thromboplastin time are in progress in this laboratory and will constitute the basis of a future report. DISCUSSION The prothrombin time of Quick and coworkers 13 and its various modifications have been used almost universally to control anticoagulation with coumarin therapy. Other tests have been designed that are reported to be more sensitive to factors not tested by the prothrombin time. 10 Under some circumstances 10 it has been said that coumarin anticoagulation is better controlled with Thrombotest, which measures the anticoagulation status of the intrinsic system and the extrinsic system simultaneously. Others believe that depression of Factor VII is responsible for anticoagulation and that this factor should be closely controlled. 10 It is clear that the question of which factor or factors are most important in the prevention of thrombosis or spontaneous hemorrhage has not yet been answered to everyone's satisfaction. Our study suggests that at least a portion of the thromboplastin complex is depressed with coumarin drugs and that, if one looks at a cross section of the patients in this hospital who are thought to be in the therapeutic range of coumarin therapy, the mean coagulation activity as determined by the 1-stage prothrombin time is essentially similar to that obtained with the partial thromboplastin time. In this same group Thrombotest suggests that plasma coagula-

480 STRUVER AND BITTNER Vol. 38 tion activity is actually lower than anticipated from a clinical impression based on the lack of spontaneous bleeding and the use of customary coumarin dosages. When the partial thromboplastin time has been used in conjunction with the 1- stage prothrombin time, we have noted that there is often a difference between the coagulation activities of the intrinsic and extrinsic systems during the initial phases of coumarin therapy. After a more stable level of coagulation activity is reached, the percentages of activity of the two systems are generally comparable. This is not invariably the case, however, for we have seen at least 1 case of spontaneous urinary bleeding in which the partial thromboplastin time has exposed an unexpected and severe depression of the intrinsic system. This was not revealed with the 1-stage prothrombin time. With these latter observations in mind, we have been impressed with the apparent independence of these 2 clotting mechanisms in coumarin therapy and with the additional knowledge that is gained by testing the 2 systems independently. The 1-stage prothrombin time has seemed to offer adequate control in most instances, whereas the partial thromboplastin time has seemed a more appropriate means of investigation of the status of the factors in the thromboplastin complex that may also be depressed with coumarin therapy. The partial thromboplastin time has also served our purpose of observing the summation of effects of heparin and coumarin drugs on the coagulation mechanism during the beginning phases of anticoagulation, when therapy has been initiated with heparin. As was demonstrated by Langdell and associates 6 and Rodman and associates 14 and again by Proctor and Rapaport, 11 this test is quite useful for the control and detection of first stage coagulation defects. Because of its technical simplicity, it would be easily adapted to use as a screening test for the detection of mild hemophilias, and it has greater reliability than customary bleeding and clotting times. 1 ' 4 SUMMARY This paper deals with the description of a method of performing a simple and reliable partial thromboplastin time (cephalin time). Celite is used to obtain and standardize maximal glass activation. Some of the physical and chemical variables in the method are studied and illustrated. The test has been applied to the study of plasmas from coumarin-treated patients in order to demonstrate the effect of coumarin drugs on the intrinsic coagulation mechanism. The results of the study are compared with the mean values obtained for the 1-stage prothrombin time (extrinsic system) and for Thrombotest (combined systems) performed on the same plasmas. The in vitro effect of heparin on the partial thromboplastin time has also been investigated, and the results suggest that the in vitro and in vivo effects of heparin are comparable, and that this test may be used as a sensitive method of detecting activity of heparin. SUMMARIO IN INTERLINGUA Iste communication presenta le description de un methodo, que es simple e fidel, pro determinar le tempore de thromboplastina partial (tempore de cephalina). Es usate Celite in establir e standardisar le maximo del activation causate per le vitro. Plure variabiles physic e chimic in le methodo es studiate e illustrate. Le test ha essite applicate al studio de plasmas ab patientes tractate con coumarina con le objectivo de demonstrar le effecto de drogas coumarinic super le intrinsec mechanismo coagulatori. Le resultatos del studio es comparate con le velores medie obtenite pro le monophasic tempore de prothrombina (systema extrinsec) e pro Thrombotest (systemas combinate) effectuate in le mesme plasmas. Le effecto in vitro de heparina super le tempore de thromboplastina partial ha etiam essite investigate, e le resultatos suggere que le effectos de heparina in vitro e in vivo es comparabile e que iste test pote esser usate como un sensibile methodo pro deteger le activitate de heparina.

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