DIAGNOSIS AND CONTROL OF THE HEMOPHILIOID STATES WITH THE PARTIAL THROMBOPLASTIN TIME (PTT) TEST

Transcription

1 DIAGNOSIS AND CONTROL OF THE HEMOPHILIOID STATES WITH THE PARTIAL THROMBOPLASTIN TIME (PTT) TEST N. F. RODMAN, JR., M.D., EMILY M. BARROW, M.A., AND JOHN B. GRAHAM, M.D. Department of Pathology, University of North Carolina, Chapel Hill, North Carolina In recent years it has become apparent that there are several heritable bleeding disorders in humans other than hemophilia (AHF deficiency). The term "hemophilioid states" has been suggested as the generic name for this group of disorders. 7 The existence of a variety of plasma deficiency states makes urgent the development of simple laboratory tests for specific diagnosis and rational therapy. The purpose of this communication is to describe a procedure which meets this need and to document its utility. It was shown many years ago by Mills 20 that crude cephalin suspensions consistently give longer clotting times with hemophilic than with normal plasma. It has been known since at least 1935, when Quick's prothrombin time test was published, 23 that crude whole brain extract clots hemophilic plasma as rapidly as normal plasma. This dichotomy of effects has given rise to the concept of different types of thromboplastin, "complete" (like whole brain) and "partial" 13 (like cephalin extract). There is little fundamental understanding of the reasons for these differences, but the differences have provided a basis for simple tests of great utility in the diagnosis and control of the hemophilioid diseases. The partial thromboplastin time (PTT, or cephalin clotting time), a 1-stage clotting test in which the crude cephalin extract of brain is used as thromboplastin, was originally described by Langdell and his associates. 13 Plasma in antihemophilic Received, January 27, 1958; revision received, February 19; accepted for publication March 3. Dr. Rodman is Resident Pathologist; Mrs. Barrow is Research Assistant in Pathology; and Dr. Graham is Associate Professor of Pathology. These investigations were supported (in part) by a research grant (H-3140) from the Public Health Service, Department of Health,Education, and Welfare. 525 factor (AHF), plasma thromboplastin component (PTC), factor V (proaccelerin, labile factor), plasma thromboplastin antecedent (PTA), and Stuart factor have been shown previously to be abnormal in this test. 3, n It has also been reported that plasma in Hageman factor has a prolonged partial thromboplastin time 16 and that plasma in SPCA (serum prothrombin conversion accelerator, factor VII, proconvertin) is normal in this test. 5 ' 16 The present communication describes our experience with the PTT in the diagnosis of these deficiencies, in control of therapy, and in detection of circulating anticoagulants. MATERIALS AND METHODS Blood from patients and controls was obtained from the antecubital vein. The blood was mixed either with 1.34 per cent sodium oxalate in a ratio of 9 parts blood to 1 part oxalate solution or with 3.2 per cent sodium citrate in a ratio of 8 parts blood to 1 part citrate solution. The anticoagulants were buffered by dissolving oxalate or citrate powder in 5 per cent imidazole at ph rather than distilled water. Blood was centrifuged for 15 min. at 3200 X g, and the supernatant plasma withdrawn by pipet. Plasmas were usually tested on the day the blood was obtained. They were stored occasionally, however, for several days at -20 C. with little change in the PTT. It appears that the PTT of citrated plasma varies less during storage than that of oxalated plasma, probably because of slower deterioration of AHF 21 and factor V. 4 After several hours at room temperature, the PTT of oxalated plasma sometimes increases significantly while that of citrated plasma usually changes only slightly. The brain extract for use in the prothrombin time test and the cephalin extract for use in the PTT test were prepared as follows: bovine, canine, and human brain have been

2 526 RODMAN ET AL. Vol. 29 found to be satisfactory starting material. The meninges were removed, and the brain washed under tap water. About 100 Gm. of wet brain were covered with acetone and allowed to stand for 1 hr. The tissue was gently mashed under acetone in a mortar with a pestle. The supernatant was decanted,.and the process repeated until the tissue became flaky and the acetone remained clear. The tissue was then ground into small particles and spread on a flat pan to dry. Usually 18 to 20 Gm. of dry powder are obtained from 100 Gm. of wet brain tissue. The dry brain extract when resuspended in saline in a concentration of 25 mg. per ml. was used as the thromboplastin in prothrombin time tests. Cephalin was prepared by placing 18 to 20 Gm. of the dry brain extract in an Erlenmeyer flask, covering with ether, and stoppering. The dry material was suspended by shaking and allowed to stand overnight at room temperature. The following morning, the supernatant was filtered until clear, and the clear filtrate evaporated to dryness with a stream of air. The dry residue was washed twice with 40 ml. of acetone at room temperature and twice with 40 ml. boiling acetone, then air-dried. The cephalin obtained from 100 Gm. of fresh brain (18 to 20 Gm. brain extract) usually amounted to 2 to 3 Gm. of pale yellow, waxy material. Of this material 3 Gm. were suspended in 3 ml. of saline, and the volume brought to 100 ml. by the slow addition of saline with careful mixing. In our experience satisfactory material has been white and opaque in the 3 per cent solution. When a yellowish tint has been present, the material has often been inhibitory and unsatisfactory. The 3 per cent stock suspension was stored in lots of 1 ml. at 20 C. Stock suspensions have been stored at 20 C. for 1 yr. or more without significant loss of activity. Before use the 3 per cent stock suspension is diluted to 0.3 per cent with saline. The dilute suspension also has been stored in desired quantities at 20 C. for long periods without serious deterioration. BaSCi adsorbed plasma was prepared as described by Langdell and co-workers. 13 The plasma eluate was prepared from the BaS0 4 "button" from the preceding procedure. The "button" was suspended in a volume of 3.2 per cent sodium citrate equal to that of the original plasma, at 5 C. for 30 min. with occasional stirring. This mixture was centrifuged and the supernatant dialyzed for 3 or more hours against normal saline. Eluates of oxalated serum were pre pared by a similar technic. The partial thromboplastin time.(pit or cephalin clotting time) is defined as the clotting time at 37 C., ph 7.4, of a mixture consisting of ml. citrated or oxalated plasma, ml M CaCl 2, and ml. 0.3 per cent cephalin extract. A convenient way to perform the test is to place test tubes containing, respectively, cephalin extract and 0.02 M CaCl 2 solution in a water-bath at 37 C. The plasma to be tested is kept at room temperature. First, ml. plasma is placed in a clean, dry 10 by 75 mm. test tube. Next, ml. 0.3 per cent cephalin extract is added to the tube; then ml. CaCl 2. A stop watch is started when the CaCU is added. The tube is agitated gently, and the end point consists of the formation of a fibrin clot. When plasma mixtures or dilutions are to be tested, they are mixed or diluted immediately before the addition of cephalin and calcium. Prothrombin times were performed by the method of Quick, 22 using thromboplastin from human brain. In the Stypven-cephalin clotting time a mixture of equal quantities of 0.3 per cent cephalin and Stypven* (Russell's viper venom) was used as the thromboplastin. The Stypven was diluted 1:10,000 prior to mixing with cephalin, using the distilled water supplied with the dried material. Two-stage prothrombin determinations were done by the method of Smith, Warner and Brinkhous as described by Wagner and associates. 24 Prothrombin utilization was determined as described by Graham and co-workers. 8 The thromboplastin generation test of Biggs and Douglas 2 was performed with the modifications described by Graham and Barrow. 6 * Burroughs Wellcome & Company.

3 June 1958 HEMOPHILIOID:STATES 527 Whole blood clotting times were determined by the method of. Lee and White, as described by Jaques. 12. HBSULTS Clinical Experience with the Partial Thromboplastin Time Our experience with the partial thromboplastin time as used routinely in a clinical coagulation laboratory over a period of approximately 1 yr. is summarized in Table I. During this period a single preparation of human brain cephalin was used by 5 different workers. A total of 206 determinations were performed on samples of normal plasma obtained for routine methodologic control of the test, our policy being to use a fresh control plasma in conjunction with each test plasma. Some of the control plasmas were obtained by ourselves in the laboratory, others by medical students and house officers on the wards. It will be seen in the top line of Table 1 that despite the studiedly uncontrolled venipunctures the coefficient of variation was not excessive. Experience with 85 patients known to have one or another of the hemophilioid diseases, but lacking independent evidence of circulating anticoagulants, is summarized similarly in the middle line. It will be noted that the average PTT of the mixture of patient's plasma with control plasma was 8 longer than the average for the controls alone (top line). The smaller coefficient of variation, 11 per cent, may be related to the fact that most of the venipunctures were performed by ourselves under relatively controlled conditions. That the 8- difference between the means of the 2 sets of determinations is real can be shown in either of 2 ways. It can be shown to be statistically significant at the 1 per cent point by a "t" test (t = 5.089, 289 d.f., p < 0.01), or, more crudely, it can be seen from the standard errors of the 2 means that the tails of the 2 distributions overlap only slightly. In individual instances the PTT of the patientcontrol mixture was less than 8 longer than the control, as might have been expected from the variation noted in the last column of Table I. Occasionally, the clotting. -. TABLE 1' CLINICAL EXPERIENCE WITH PARTIAL THROMBO PLASTIN TIME (PTT) TEST 1 Type of Plasma Tested control control + hemophilioid. patient without' circulating anticoagulant' (1:1 mixture)''. Suspected "bleeder,'.' never '. positively: _ _ ' diagnosed.. : PIT (Sec.) Mean S.D S No. of Determinations Coefficient of Variation S.E. Per Cent S time of the' mixture was actually less than that of the control alone. Data on 45 patients suspected on clinical grounds of having a hemorrhagic diathesis, but never definitely established as having 1 of the plasmatic clotting defects, are shown in the bottom line of Table 1. It will be noted that the average PTT of these patients was actually somewhat less than that of the control group with a coefficient of variation of 8 per cent. However, the difference between the means of the control group and the group of suspected "bleeders" was not statistically significant (t = 0.9S99, 249 d.f., v > ). The statistical data can be used to devise a test for the presence of a circulating anticoagulant in a patient known to have a hemophilioid disease. First, an unknown but suspected plasma, a control plasma and a mixture of equal parts of the 2 are tested with the PTT test. If the PTT of the mixture is more than 26 longer than the control (difference between control and mixture equals 8,. plus 2 S. D. about the mixture equals 18), it may be assumed with confidence that the patient has a circulating anticoagulant. Actually, a plasma may contain a low-titered anticoagulant when the prolongation of the PTT of the mixture over that of the control is in the 15 to 25 range.

4 528 RODMAN ET AL. Vol. 29 Diagnostic Use of the Partial Thromboplastin Time Test The specific diagnosis of a clotting factor deficiency depends at present upon the failure of the unknown plasma or serum to correct the clotting defect of a sample with a known deficiency. We have found that this principle has great utility when the PTT is applied in conjunction with a battery of known, congenitally plasmas. If a test plasma fails to correct the prolonged PTT of a known plasma, the test plasma may be assumed to have the specific deficiency, provided the prolonged PTT of the test plasma is corrected by normal control plasma and, therefore, does not contain an anticoagulant. Conversely, if the test plasma corrects the plasma, it may be assumed to contain the factor known to be. This positive test is a less specific test than the negative one, since contaminants such as traces of thrombin 13 may produce apparent correction of a plasma. The danger of misinterpreting an apparent correction can be minimized by using a normal control plasma for comparison with the unknown test plasma. Similar but less specific tests may be devised by use of mixtures of test plasma with BaS0 4 -adsorbed plasma (rich in AHF and factor V), serum or serum eluate (rich in PTC, Stuart factor, and SPCA), and plasma eluate (rich in prothrombin, PTC, Stuart factor, and SPCA). Patient 1. PTC deficiency. J. J. T. is a 40-year-old white man with a severe bleeding tendency whose maternal uncle and 3 brothers died of hemorrhage. He has had numerous joint hemorrhages, hematuria, epistaxis, bleeding from gums, and prolonged bleeding after tooth extraction. He has a prolonged whole blood clotting time, impaired prothrombin consumption, normal prothrombin time and normal 2-stage prothrombin concentration. His serum is abnormal and his plasma normal in the thromboplastin generation test. Table 2 shows diagnostic data on this patient obtained with the PTT. The patient's clotting time was markedly prolonged, yet corrected to within 1 of the control value by mixing with an equal TABLE 2 DIAGNOSIS OP PLASMA THROMBOPLASTIN COM PONENT DEFICIENCY WITH THE PARTIAL THROM BOPLASTIN TIME TEST Patient Type of Plasma (ml.) BaSOiadsorbed plasma eluate AH Ft PTCf * Partial thromboplastin time test. t Antihemophilic factor. Plasma thromboplastin component. PTT* Sec volume of control plasma. It was not corrected by mixing with BaS0 4 -adsorbed plasma, but was corrected by mixing with the eluate from the same plasma. Further, the patient's plasma corrected AHF- plasma completely, but failed by 366 to correct PTC- plasma. This patient, in PTC, did not have a circulating anticoagulant, as the PTT of the mixture of his plasma and control plasma was only 1 greater than that of the control plasma. Patient 2. AHF deficiency. G. M. T. is a 30-year-old white man who has had bleeding episodes since early childhood. These have included hemorrhages into muscles and major joints, hematuria, and retropharyngeal hematoma. He has a normal prothrombin time, 92 per cent prothrombin by the 2-stage method, impaired prothrombin consumption, and a prolonged whole blood clotting time. Table 3 exemplifies the diagnosis of a case of AHF deficiency with the PTT. The patient's partial thromboplastin time was very long but corrected to within 20 of

5 June 1958 HEMOPHIMOID STATES 529 TABLE 3 DIAGNOSIS OF ANTIHEMOPHILIC FACTOR DEFI CIENCY WITH THE PARTIAL THROMBOPLASTIN Patient TIME Type of Plasma (ml.) BaSOjadsorbed plasma eluate TEST PTCt Deficient AHFJ Deficient * Partial thromboplastin time test, t Plasma thromboplastin component. % Antihemophilic factor. PTT* Sec the control plasma by mixing with the latter. The patient's prolonged PTT was corrected almost as well by the BaSOu-adsorbed plasma as by whole normal plasma, but was not corrected by the eluate from adsorption of this plasma. (The partial correction of the patient's PTT by the eluate may have resulted from a trace of thrombin in this eluate, as on other occasions there has been no correction by similar eluates.) His plasma corrected PTC- plasma but not AITF- plasma, thus indicating specific AHF deficiency. The fact that his PTT was not completely corrected by any of the above reagents suggests a mild circulating anticoagulant, a distinct possibility in view of the hundreds of transfusions he has received. However, it will be noted that the PTT of the patient-control mixture was only 20 longer than the control plasma, thus not falling outside our limit for specific diagnosis of anticoagulants. Patient S. SPCA (factor VII, proconvertin) deficiency. Through the courtesy of Dr. Harold A. Wurzel of the University of TABLE 4 DIAGNOSIS OF SPCA* DEFICIENCY WITH THE PROTHROMBIN TIME TEST, THE PARTIAL THROMBOPLASTIN TIME TEST, AND STYPVEN PLUS CEPHALIN Type of Plasma (ml.) Patient Stuart plasma eluate I-Stage Clotting Tests (Sec.) Type of thromboplastin Brain extract S S BaSOiadsorbed Cephalin S S7 S4 5S Stypven plus cephalin 5.S * Serum prothrombin conversion accelerator. Pennsylvania we obtained plasma from a patient, previously unreported. She is a 23-year-old white woman who has been a "bleeder" since infancy. She has been considered in SPCA, since her prothrombin time has been consistently prolonged, not corrected by BaSCi-adsorbed plasma but corrected by the eluate, and her prothrombin by a 2-stage assay has been normal. Both her plasma and serum are normal in the thromboplastin generation test. Table 4 shows data obtained in the prothrombin time, the partial thromboplastin time, and the Stypven-cephalin clotting time. The prothrombin time of the patient's plasma is prolonged; it is corrected by mixing with Stuart factor- plasma and by plasma eluate, but not by the supernatant of BaSO^adsorbed plasma. The partial thromboplastin time and Stypven-cephalin times of every mixture containing this patient's plasma are normal. Patient 4. Stuart defect. A. G., a patient previously reported by Dr. Charles Barnett of Fredericksburg, Virginia, 1 as having factor

6 530 RODMAN ET AL. Vol. 29 VII deficiency, is a 32-year-old Negro man who has had transfusions for excessive bleeding following tooth extraction and laceration of the right forearm. Minor cuts have always been difficult to stanch. Table 5 illustrates diagnostic data using the prothrombin time, PTT, and the Stypvencephalin time. It will be noted that his clotting time was prolonged in each of these tests. The patient's plasma failed in the prothrombin time to correct known Stuart plasma but did correct SPCA plasma. The patient's prolonged clotting time in all 3 of these tests was not corrected by the BaSOvadsorbed plasma. Although the specific data are not shown, the patient's clotting tests were corrected by plasma eluate. Patient 5. Hageman deficiency. L. C, a 17-year-old white boy, has had no bleeding episodes despite tonsillectomy at age 5, appendectomy at age 15, and a tooth extraction. Minor cuts have not been troublesome, and he does not bruise easily. He was found by routine study prior to operation to have TABLE 5 DIAGNOSIS OF STUART FACTOR DEFICIENCY WITH THE PROTHROMBIN TIME TEST, THE PARTIAL THROMBOPLASTIN TIME TEST, AND STYPVEN PLUS CEPHALIN Type of Plasma (ml.) Patient Stuart l-stage Clotting Tests Type c f thromboplastin Brain extract SPCA* BaSO (- adsorbed Cephalin 55 > >300 Stypven plus cephalin S S * Scrum prothrombin conversion accelerator. a prolonged whole blood clotting time (29 rain.). He has a normal bleeding time, negative tourniquet test, impaired prothrombin consumption, abnormal thromboplastin generation test, and normal prothrombin by 2-stage assay. Table 6 shows diagnostic data using the partial thromboplastin time. In this test the patient's PTT was greatly prolonged, but corrected by control plasma as well as by both the BaSCVadsorbed plasma and the eluate. The patient's plasma corrected plasmas specifically in AHF, PTC, Stuart factor, and SPCA. His plasma failed by 78 to correct plasma known to be specifically in the Hageman factor. Thus, this patient was established specifically as having the Hageman trait solely by the PTT test. The Partial Thromboplastin Time Test for Evaluation of Therapy The PTT test has been found very helpful in day-to-day evaluation of the status of patients receiving transfusion therapy. The following examples demonstrate its usefulness. PTC deficiency. The clinical history of J. J. T., patient 1, has been described above, and the diagnostic data were shown in Table 2. Because of severely carious teeth and gum disease it was thought desirable to extract all his teeth. For the first stage, he was admitted to the hospital for preparation by transfusion and extraction of 6 teeth. Figure 1 illustrates daily partial thromboplastin time data, with the patient, control, and the patient plus control (equal parts mixture) values, during a period of about 1 month while 6 teeth were being removed. Extractions were performed on 3 separate occasions, as shown. A single tooth was extracted on March 14 and 20, and on March 26, 4 teeth were removed. He was given plasma before extraction on each occasion, with a significant shortening of the PTT. The extractions were not undertaken until after it had been determined by the PTT that a good response had been obtained from transfusion. During and after each procedure there was minimal bleeding. Only after the 3rd extraction, involving 4

7 r June 1958 HEMOPHILIOID STATES 531 TABLE 6 DIAGNOSIS OF HAGEMAN DEFICIENCY WITH PARTIAL THROMBOPLASTIN TIME TEST Type of Plasma (ml.) Sec. Patient BaSO.,- adsorbed plasma eluate AHK* PTCt Stuart SPCAt Hageman Partial thromboplastin time Prothrombin time 72 30S So 77 SI S S * Antihemophilic factor. t Plasma thromboplastin component. t Scrum prothrombin conversion accelerator. Kindly supplied by Dr. Oscar Ratnoff. teeth, were additional postoperative plasma transfusions required to control oozing. It will be noted also that this was the only period during which there was immediate and significant postoperative prolongation of the PTT. It should be noted, finally, that the clotting times of the mixtures of patient's with control plasma were never more than 26 longer than the control clotting time alone, indicating no significant inhibitory activity generated by the patient in response to transfusion. AHF deficiency. W. W. R. is a 26-year-old white man with classic hemophilia whose first severe bleeding was into the jaw at 6 months of age. There have been numerous subsequent episodes of bleeding including hemarthroses, melena, a retropharyngeal hematoma, and bleeding after tooth extraction. He was admitted to the North Carolina Memorial Hospital on July 18, 1957, with hemorrhage into the left hip. By assay on a previous admission he had had 2 per cent AHF. 17 Figure 2 shows daily PTT data on the patient's plasma, control plasma and a mixture of equal parts of the 2. Following admission on July 18, he received 6 units of fresh plasma, and the PTT dropped from 256 to 94. On less intensive plasma therapy his PTT rose gradually to 230 on the 8th hospital day, July 25, a phenomenon which has been demonstrated in dogs to be the result of loss of injected AHF. 14 Physical therapy was begun July 24 but stopped on July 26 when pain recurred in the left hip and extended into the lumbosacral region. An abscess developed in the

8 532 RODMAN ET Ah. Vol J.J.T. PTC-DEFICIENCY A Patient x Control e Mixture 1:1 PTT 180 (Sec.) x---x» * \ A x x- o-i x-x ^ - ' -&-< «4 X Units «Plasma ml/unit i I3'l4 15" 16" MARCH Fio. 1. Daily partial thromboplastin times of patient's plasma, control plasma, and the mixture of equal parts of patient's and control plasma. In each instance the date of each tooth extraction is designated by an appropriate symbol..the first 2 extractions involved single teeth, and the third, on March 26, involved 4 teeth. deltoid region July 29 following an injection of Demerol, and therapy by transfusion was resumed. The abscess was treated conservatively and allowed to drain spontaneously. When the abscess had healed, transfusions were stopped and the patient's PTT lengthened into its usual range. This man never showed evidence of a circulating anticoagulant, since the PTT of the mixture of his plasma with control plasma was always within a few seconds of the control value. Demonstration of an Inhibitor with the Partial Thromboplastin Time Test P. W. B. is a 13-year-old white boy with a severe deficiency of PTC. He has a lifelong history of hemorrhages, including epistaxis, hemarthroses, hematuria, and prolonged bleeding with tooth extraction. He has a normal prothrombin time and prolonged PIT. His plasma corrects AHF-defieient plasma but not PTC- plasma in the partial thromboplastin time test. He has been described previously as a PTC- patient with a circulating anticoagulant specific against PTC. 18 He was admitted to the North Carolina Memorial Hospital in September 1956 with increasingly severe headache and drowsiness following a mild blow to the head. Despite the fact that he had been shown a year previously to have a circulating anticoagulant, he appeared on admission to have at most a very weak inhibitor. It was decided to treat him vigorously with fresh frozen plasma. On this therapy the patient's PTT, the daily determinations of which are shown in Figure 3, became much shorter for several days. During this period there was improvement in his level of consciousness. On the 7th day after institution of plasma therapy, the patient's PTT became markedly pro-

9 owne 1958 HEMOPHILIOID STATES PTT 220 (Sec.) W.W.R. AHF- DEFICIENCY A Patient x Control o Mixture 1: x.^q "X^ "" &» -; "!( ' x- -x' "^ 60H 6 Units 5- Plasma 3 " 250 ml/unit 2 H x.-x ' I JULY AUGU9T FIG. 2. Daily partial thromboplastin times of patient's plasma, control plasma, and a patientcontrol plasma mixture. longed, as did the PTT of the patient-control mixture. This prolongation of the mixture was interpreted as indicating development of a strong inhibitor. During this period and subsequently the patient improved gradually, and several months later the only residual defect was a mild speech disorder. On July 31,1957, he was readmitted in mild shock from a retroperitoneal hemorrhage which had reduced his packed cell volume to 13 per cent. Figure 4 shows daily PTT data during this admission. The first PTT on his plasma was markedly prolonged, but there was correction to within 15 of the control value by mixing with control plasma. This indicated that the previously demonstrated inhibitor had largely disappeared. The patient's PTT was much shorter after vigorous transfusion therapy until the reappearance of the inhibitor on August 7. With the development of this potent inhibitor it was felt that neither plasma nor whole blood transfusions would be of further benefit. Fortunately the bleeding had ceased, and he was discharged on the 10th hospital day. The thromboplastin generation test has been considered a reliable method for demonstrating a circulating anticoagulant. 10 Table 7 contains the thromboplastin generation test data on this patient, and at the time (September 24, 1956) the PTT clearly showed inhibitory activity. Both his plasma and serum were defective in the thromboplastin generation test, the serum being more defective than the plasma. In addition, the patient's serum in an equal quantity inhibited the action of control serum. Plasma from this patient the same day showed inhibitory activity detectable in high dilution by the PTT test (Table 8). If an increase greater than 26 over the control diluted with saline is taken as the end point for clear demonstration of inhibitory activity, it would appear that the titer at this time lay between 1:100 and 1:1000. Relative Sensitivities of the PTT and the Thromboplastin Generation Test in Mild Deficiency States We have found that the PTT is very sensitive in demonstrating mild plasma clotting defects. The following examples demon-

10 534 RODMAN ET AL. Vol. 29 P.W.B. PTC-DEFICIENCY WITH INHIBITOR J PTT (Sec)" u o o- <? / \ a / 80- X' * / X' v^ -x',* x-..x--x---x^,-x- / -x' 60- x 5- Units 4- Plosmo *" 250ml/unH ' l'22'23'24'25'26'27'28 29'30l SEPTEMBER OCTOBER FIG. 3. Daily partial thromboplastin times of patient's plasma, control pla asma, and the mixture of patient's and control plasma in equal parts. The great prolongation of the mixture indicates the appearance of the inhibitor. strate this in patients in antihemophilic factor (AHF) and plasma thromboplastin component (PTC). Mild AHF deficiency. Recently a mild hemophiliac with a negative history 1 of a 9 family described several years ago," was observed in connection with a plastic operation on the kidney. For several weeks after this operation he bled persistently from the operative site. Prior to operation the patient's plasma showed only a 1 prolongation over control BaSCi-adsorbed plasma in the thromboplastin generation test. This difference might have been attributed to laboratory error had we not known independently that he was mildly in AHF. (An assay several years earlier had revealed 35 per cent AHF.) By comparison with control plasma in the PTT test the patient's plasma repeatedly failed to correct plasma severely in AHF, the differences ranging up to 93 At the same time in the PTT the patient's plasma corrected PTC- plasma almost as well as did the control plasma and never showed evidence of a circulating anticoagulant. Mild PTC deficiency. Similarly we have had the opportunity of studying a patient with a mild deficiency in PTC by both methods. In the thromboplastin generation test the patient's serum was only 1.8 longer than control serum. The patient's PTT, nevertheless, was 18 longer than the control, and correction of plasma known to be very low in PTC was 34 better by control than by the patient's plasma. At the same time the patient's plasma corrected AHF- plasma as well as did the control and showed no evidence of an anticoagulant. He was prepared by plasma

11 June 1958 HEMOPHIMOII) STATES 535 TABLE S DEMONSTRATION OF INHIBITOR IN HIGH TITER IN PTC* DEFICIENT PATIENT WITH THE PARTIAL THROMBOPLASTIN TIME PTC-DEFICIENCY WITH INHIBITOR ISOO- p W B transfusions preoperatively and underwent hemorrhoidectomy without complications. If the thromboplastin generation test alone had been used to determine the pres- I250- A Patient x Control o Mixture 1:1 Clotting Mixtures Control Control and saline (1:1) Control plus patient's plasma (1:11 Patient's Plasma Diluted with Saline 1:10 1:100 1:1,000 1:10,000 1:100,000 * Plasma thromboplastin component. t Preincubation for 15 min. at 37 C. PTTt (Cephalin) > ence or absence of a deficiency in the last 2 patients, we might well have concluded that both were normal. Using the PTT and severely plasmas as test substrates, there was never doubt of the presence or the nature of the defects. FIG. 4. Daily partial thromboplastin times of patient's plasma, control plasma, and an equal parts mixture. DISCUSSION The partial thromboplastin time is a very useful clinical test because it is simple, versatile, and sensitive. While determinations vary somewhat from day to day, there TABLE 7 THROMBOPLASTIN GENERATION TEST DEMONSTRATING INHIBITORY ACTIVITY IN PTC* DEFICIENCY AFTER TRANSFUSION Incubation time (min.) l Control plasma plus control serum Patient's plasma plus patient's scrum Control plasma plus patient's serum Patient's plasma plus control serum Control plasma plus control serum and patient's serum (equal parts) \ 30.0 > > IS.4 IS S J S.6 15.S * Plasma thromboplastin component. f Clotting time. I Italicized numbers represent minimum clotting times.

12 536 RODMAN ET AL. Vol. 29 is little variation with repeated determinations on a single plasma or on a series of plasmas during a single day. Preparations are simple, as little time is required for thawing the cephalin, eluate, serum, and known plasmas. The most time-consuming step is the preparation of fresh BaS0 4 -adsorbed plasma, when it is to be used. When performing daily tests on patients previously diagnosed, it is a simple matter of preparing the plasma from whole blood and thawing the cephalin. The actual determinations in such an instance, which always include the PTT of the mixture of patient's with control plasma for detection of inhibitory activity, usually require only 10 min. The versatility of the partial thromboplastin time in diagnosis has been exemplified in detail. Plasma in all of the blood coagulation factors mentioned above with the single exception of SPCA have been shown to have prolonged partial thromboplastin times. By the use of mixtures with prototype plasmas a conclusive diagnosis can be made easily. For this purpose we maintain a battery of lyophilized, known plasmas. If such plasmas are not available, a presumptive diagnosis can be made by use of the PTT and prothrombin time with BaS0 4 -adsorbed plasma and eluate. For example, in a congenital male bleeder whose prothrombin time is normal, it should be suspected that the patient is probably in either AHF or PTC. If his partial thromboplastin time is prolonged, and is corrected by mixing with BaS0 4 -adsorbed plasma, AHF deficiency is almost certainly present. Conversely if this patient's prolonged partial thromboplastin time is not corrected by BaSCvadsorbed plasma but is corrected by mixing with the eluate of either plasma or serum, PTC deficiency probably exists. It might be argued that the PTT is of greatest diagnostic value when used in conjunction with a battery of prototype plasmas. This is true. It is our policy to use such plasmas routinely rather than the BaS0 4 -adsorbed plasma and eluate since specific diagnosis ultimately requires plasma mixtures anyhow. It might seem that this policy introduces a difficulty which the ordinary laboratory can not overcome. There is no problem, however, once a prototype patient has been diagnosed. If he has been properly handled, he will be willing to donate his own plasma whenever it is needed. We call in our prototype patients at intervals for donations, and lyophilize the plasma obtained. Thus there is always on hand a sufficient supply of the complete battery. Most severe bleeders who will be encountered are men with either AHF or PTC deficiency, and there is little drain on the stocks of Stuart, SPCA and Hageman plasmas once obtained. Furthermore, a diagnostic laboratory has no need for the more recondite types until 1 of these rare types is encountered. When this occurs, laboratories such as ours are glad to furnish small samples to others. In the past year many workers in other parts of the world have been assisted in diagnosing the rarer defects by receiving small samples of lyophilized plasma. As a result, a number of SPCA- and Stuart factor- patients have been specifically identified and have themselves become local prototypes. As a guide to therapy the PTT helps determine the status of each of 2 distinct aspects of the patient's coagulation mechanism. First, the disappearance of the clotting factor supplied by transfusion can be readily followed. This disappearance is manifested by the gradual prolongation of the patient's PTT after transfusion is decreased or stopped. This phenomenon is illustrated in Figures 1 and 2. Secondly, by testing mixtures of a patient's plasma with control plasma, the presence or absence of a circulating anticoagulant can be determined readily. This is seen most strikingly (see Figs. 3 and 4) when such daily mixtures suddenly show significant prolongation over the control PTT. The sensitivity of the partial thromboplastin time in detecting mild deficiencies of AHF and PTC is well illustrated by the last 2 patients. In each instance symptoms were either mild or inapparent. Had the thromboplastin generation test alone been relied upon, we probably would have decided that no significant deficiency existed.

13 June 1958 HEMOPHILIOID STATES 537 By testing the ability of each patient's plasma to correct plasmas with known, severe deficiencies, however, the defects were clearly demonstrated. It has not yet been established that the partial thromboplastin time is as sensitive in detecting mild deficiencies of PTA, Hageman, and Stuart factors as detecting deficiencies of AHF and PTC, but our data suggest that such is the case. SUMMARY 1. The partial thromboplastin time (PTT) test is described in detail, including preparation of the reagents. 2. The partial thromboplastin times of plasmas in antihemophilic factor (AHF), plasma thromboplastin component (PTC), Stuart factor, and Hageman factor are shown to be prolonged. It is also known that such is also the case in factor V (proaccelerin, labile factor) deficiency. The conclusive diagnosis in each instance can be demonstrated by experiments in which test plasmas fail to correct the clotting time of known plasmas. 3. Plasma in serum prothrombin conversion accelerator (SPCA) is shown to have a normal partial thromboplastin time. 4. The PTT test is shown to have considerable value in observing daily changes in a patient's coagulation status. 5. Circulating anticoagulants can be diagnosed by a simple modification of the test, in which unknown plasma is mixed with a normal control plasma. The titer of the anticoagulant can also be determined if desired, by mixing the plasmas in various ratios and incubating prior to testing. 6. The sensitivity of the PTT test in detecting mild deficiencies of AHF and PTC is compared with another widely used method. It would appear that the PTT test is not only simpler to perform but also more sensitive. SUMMARIO IN INTEIILINGUA 1. Le test del tempore thromboplastinic partial (TTP) es describite in detalio, incluse le preparation del reagentes. 2. Le tempores thromboplastinic partial de plasmas e in factor antihemophilic (FAH), componente de thromboplastina de plasma (CTP), factor Stuart, e factor Hageman esseva determinate e trovate prolongate. II es etiam cognoscite que le mesmo vale in caso de ia de factor V (proaccelerina, factor labile). Le diagnose conclusive pote esser establite in le caso individual per experimentos in que le plasma sub investigation non corrige le tempore de coagulation de un plasma a ia cognoscite. 3. Esseva trovate que plasma e in accelerator del conversion de prothrombina serai (ACPS) ha un normal tempore thromboplastinic partial. 4. Es monstrate que le test de TTP es de alte valor in le observation de alterationes diurne in le stato coagulatori del patiente individual. 5. Anticoagulantes circulante pote esser diagnosticate per un simple modification del test, in que le non-cognoscite plasma es miscite con un normal plasma de controlo. Etiam le titro del anticoagulante pote esser determinate, si desirate, per miscer le plasmas in varie proportiones e per incubar los ante le test. 6. Le sensibilitate del test de TTP in le detection de leve grados de ia de FAH e de CTP es comparate con un altere methodo que es usate extensemente. II pare que le test de TTP es non solmente plus simple in su execution sed etiam plus sensibile in su resultatos. REFERENCES 1. BARNETT, C. P.: Hemorrhagic diathesis clue to factor VII deficiency. A. M. A. Arch. Int. Med., 99: 2S0-2S4, BIGGS, R., AND DOUGLAS, A. S.: The thromboplastin generation test. J. Clin. Path., 6: 23-29, BWNKHOUS, K. M., LANGDELL, R. D., PENICK, G. D., GRAHAM, J. B., AND WAGNER,R. H.: Newer approaches to the study of hemophilia and the hemophilioid states. J. A. M. A., 154: 4S1-4S6, FAHEY, J. L., WARE, A. G., AND SEEGERS, W. H.: Stability of prothrombin and ac-globulin ' in stored human plasma as influenced by conditions of storage. Am. J. Physiol., 154: , CONYEA, L. M., HJORT, P., AND OWREN, P. A.: A study of the relationship of concentrations of prothrombin, proconvertin, and proaccelerin to three methods for measuring "prothrombin time." J. Lab. <fc Clin. Med., 48: , 1956.

14 53S HODMAN ET AL. Vol GRAHAM, J. B., AND BARROW, E. M.: The pathogenesis of hemophilia; an experimental analysis of the anticephalin hypothesis in hemophilic dogs. J. Exper. Med., 106: , GRAHAM,.1. B., AND BRINKHOUS, K. M.: Christmas disease. Brit. M..)., 2: 97, GRAHAM, J. B., LANG DELL, R. D., AND BRINK HOUS, K. M.: Estimation of the rate of prothrombin utilization. /wthe Coagulation of Blood: Methods of Study, ed. by L. M. Tocantins. New York: Grime & Stratton Inc., 1955, pp GRAHAM, J. 13., MCLENDON, W. W., AND BRINKHOUS, K. M.: Mild hemophilia: an allelic form of the disease. Am..1. M. Sc, 225: 46-53, HOUOIE, C.: Circulating anticoagulants. Brit. M. Bull., 11: 16-20, J HOUOIE, C, BARROW, E. M., AND GRAHAM, J..13.: Stuart clotting defect. I. Segregation of an hereditary hemorrhagic state from the heterogeneous group heretofore called "stable factor" (SPCA, proconvertin, factor VII) deficiency. J. Clin. Invest., 36: , JAQUES, L. 13.: Determination of the clotting time of whole blood. In The Coagulation of Blood: Methods of Study, ed. by L. M. Tocantins. New York: Grune & Stratton, Inc., 1955, pp LANGDELL, R. D., WAGNER, R. H., AND BRINK HOUS, K. M.: Effect of antihemophilic factor on one-stage clotting tests. J. Lab. & Clin. Med., 41: , LANGDELL, R. D., WAGNER, R. H., AND BRINK HOUS, K. M.: Antihemophilic factor (AHF) levels following transfusions of blood, plasma and plasma fractions. Proc. Soc. Exper. Biol. <fc Med., 88: , LARRIEU, M. J., SOULIER, J. P., AND CULOT, Y.: Ddficit en facteur Hagcman. Sang, 28: , LARRIEU, M. J., AND WEILLAND, C: Utilisation de la "odphalino" dans les tests do coagulation. Rev. hdmat., 12: , LEWIS, J. H., AND FERGUSON, J. H.: Unpublished data. IS. LEWIS, J. H., FERGUSON, J. H., AND ARENDS, T.: Hemorrhagic disease with circulating inhibitors of blood clotting: anti-ahf and anti-ptc in eight cases. Blood, 11: S46- S55, MERTZ, E. T., AND OWEN, C A.: Imidazole buffer: its use in blood clotting studies. Proc. Soc. Exper. Biol. & Med., 43: , MILLS, C. A.: Blood clotting studies in hemophilia. Am. J. Physiol., 76: , PENIOK, G. D., AND BRINKHOUS, K. M.: Relative stability of plasma antihemophilic factor (AHF) under different conditions of storage. Am. J. M. Sc, 232: , QUICK, A. J.: The clinical application of the hippuric acid and the prothrombin tests. Am. J. Clin. Path., 10: , QUICK, A. J., STANLEY-BROWN, M., AND BANCROFT, F. W.: A study of the coagulation defect in hemophilia and in jaundice. Am. J. M. Sc, 190: , WAGNER, R. H., GRAHAM, J. B., PENICK, G. D., AND BRINKHOUS, K. M.: Estimation of prothrombin by the two-stage method. In The Coagulation of Blood: Methods of Study, ed. by L. M. Tocantins. New York: Grune & Stratton, Inc., 1955, pp