FERTILITY AND STERILITY Copyright© 1975 The American Fertility Society

Vol. 26, No. 5, May 1975 Printed in U.S.A.

THE USE OF A RADIORECEPTORASSAY OF HUMAN CHORIONIC GONADOTROPIN FOR THE DIAGNOSIS AND MANAGEMENT OF ECTOPIC PREGNANCY* BRLJ B. SAXENA, PH. D.,t

AND

ROBERT LANDESMAN, M.D.

Cornell University Medical College, New York, New York 10021, and Jewish Memorial Hospital, New York, New York 10040

The early and rapid detection of pregnancy in ectopic gestation continues to be an unsolved clinical problem. 1 It has recently been observed that during the last two decades there has been a rise in the incidence of ectopic pregnancy. 2 In some areas, it has become the most frequent surgical emergency in women. 3 Immunologic agglutination pregnancy tests are reliable only above 700 IU of human chorionic gonadotropin (hCG)/liter of urine4 ; thus they detect only 50% of the ectopic pregnancies 1 and 68% of normal intrauterine pregnancies prior to the 40th day after the last menstrual period. 5 In ectopic pregnancy, especially in reabsorbing ectopic pregnancy, the hCG levels are usually low and, therefore, the hemagglutination tests are less likely to detect an ectopic pregnancy than a normal intrauterine pregnancy of the same duration. 6 Hence, there has been a need for a rapid, simple, and reliable test to establish the diagnosis of ectopic pregnancy. Recently, the use of a radioimmunoassay of the {3 subunit of hCG in blood has been applied to the detection of four ectopic pregnancies. 7 In all four pregnancies the levels of hCG in the blood were below the levels found during normal intrauterine gestation. In order to Received July 18, 1974. *Supported by Grant GA-HS7406 from The Rockefeller Foundation and a grant from the Louis B. Mayer Foundation. tCareer Scientist Awardee, Health Research Council, The City of New York, Contract 1-621.

achieve adequate sensitivity, the performance of the radioimmunoassay requires 36 to 48 hours. 8 An attempt to reduce the incubation period to 2 hours9 yielded 50% false positive results. 9 a Breen10 has studied 654 ectopic pregnancies and has shown that the patients are usually operated upon within 2 hours after admission to the hospital. A practical clinical test for use in ectopic prenancy must, therefore, provide information rapidly in order to be of use, particularly for patients with severe abdominal pain or active bleeding. The radioreceptorassay for hCG in blood 11 • 12 has already demonstrated almost 100% reliability in the detection of pregnancy on day 1 past the missed period in more than 500 cases. Furthermore, because of its high sensitivity, the radioreceptorassay can detect pregnancy as early as day 6 to 8 after conception. The radioreceptorassay can be performed within 1 hour and has the sensitivity and reliability to render it one of the most suitable methods for the diagnosis and management of ectopic pregnancy. We report here our radioreceptorassay studies of a group of 13 patients suspected of having ectopic pregnancies. The results ofthe radioreceptorassay have been compared with other pregnancy tests, pathologic findings, and objective clinical procedures. MATERIALS AND METHODS

The radioreceptorassay of hCG, capable of detecting as little as 0.3 ng or 3

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miU of hCG/ml of plasma, has been described.U· 12 The hCG, containing 12,000 IU/mg, was a gift from Dr. R. E. Canfield (College of Physicians and Surgeons, Columbia University, New York, N. Y.) and Dr. 0. P. Bahl (State University of New York, Buffalo, N.Y.). The hCG was labeled with 125I by lactoperoxidase 13 prepared from milk (Rz = 0. 78; Sigma Chemical Co., St. Louis, Mo.). The specific activity of the labeled hCG was determined by trichloroacetic acid precipitation. Five microliters of the crude reaction mixture were diluted to 5 ml with 0.05 M phosphate buffer (pH 7.5) containing 0.1% bovine serum albumin. To a 200JLl aliquot of this solution were added 400 JLl of trichloroacetic acid, to a final concentration of 10% trichloroacetic acid. The precipitated proteins were recovered by centrifugation. The radioactivity associated with the trichloroacetic acidprecipitable material was considered protein-bound and was used in the calculation of the specific activity of the labeled hormone. 14 Preparation of the Plasma Membrane. Corpora lutea from the ovaries of cows in the first trimester of pregnancy were homogenized in a glass-Teflon homogenizer in 10 volumes of 10 mM Tris-HCl buffer (pH 7 .8) containing 1 mM dithiothreitol, 1 mM MgCl 2 , and 0.25 M sucrose. The homogenate was centrifuged at 480 x g for 20 minutes. The pellet was discarded and the supernatant was centrifuged at 10,000 x g for 1 hour. The pellet was resuspended in the Tris-HCl buffer and layered on top of a discontinuous gradient composed of 50, 40, and 30% sucrose in 1 x 3 inch cellulose nitrate tubes. The tubes were centrifuged in an SW 25 rotor in a Beckman preparative ultracentrifuge (model L265B) at 90,000 x g for 90 minutes. The plasma membranes were recovered from the interface of the sucrose gradient of 30 to 40% and could be stored at -20° C or in lyophilized

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form up to 1 year with little loss of receptor activity. Radioreceptorassay. The radioreceptorassay was performed as follows. Samples of 100 JLl of a standard solution ofhCG in doubling dilution from 100 ng to 0.3 ng/ml in 10 mM Tris-HCl buffer (pH 7.2) containing 1 mM CaCl 2 , 0.1% bovine serum albumin, 10 IU of Trasylol (FBA Pharmaceuticals, New York, N. Y.) in 20 JLl of Tris-HCl buffer, 25 JLg of protein equivalent of plasma membrane obtained from bovine corpora lutea, 14 and approximately 50,000 cpm of 125I-hCG (specific activity, 40 to 50 JLCiiJLg) prepared by the lactoperoxidase method 13 were added to 75 x 100 mm disposable plastic tubes (Falcon Plastics, Greiner Scientific Corporation, New York, N.Y.). Plasma from nonpregnant women (during luteal phase) equivalent to the unknown samples was added to the standard to suppress the competition with basal levels of LH and nonspecific interference by plasma proteins. Both standard and unknown samples were analyzed in duplicate. The reaction mixture was incubated at 37° C for 20 minutes. The tubes were then placed in an ice bath, and 1 ml of chilled Tris-HCl buffer was added to each tube. The contents of the tubes were mixed on a Vortex mixer and the tubes were centrifuged for 10 minutes at 5,000 rpm in a refrigerated centrifuge (model RC2-B, rotor HS 4; Ivan Sorvall, Inc., Norwalk, Conn.). The supernatants were aspirated and the radioactivity bound to the plasma membranes was counted in an Autogamma counter (Packard Instruments, Downers Grove, Ill.) with a 51% efficiency for 1251. The dose-response curve for hCG is presented as logit-log transformation 15 in Figure 1. To establish the validity of the assay, various dilutions of plasma samples from pregnant women were analyzed. The recovery of hCG in the assay was determined by assaying the plasma samples of known hormonal concentrations to which

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ASSAY OF HCG IN ECTOPIC PREGNANCY

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known amounts of hCG had been added prior to the assay. For intra- and interassay reproducibility and quality control, pools of plasma from nonpregnant and pregnant women were analyzed at various dose levels with each assay. The specificity of the assay was established by examining the cross-reactivity of plasma samples from acromegalic and hypothyroid patients as well as from lactating postpartum women. Purified folliclestimulating hormone (FSH), 16 luteinizing hormone (LH), 17 and adrenocorticotropic hormone (ACTH) were also tested for cross-reaction. Patients. Thirteen patients were admitted to the hospital because of suspected ectopic pregnancy. The last menstrual period had occurred 23 to 76 days prior to admission. Lower abdominal LOGIT

BIB

TABLE 1. Comparison of Pregnancy Tests and Pathologic Findings in 13 Suspected Ectopic Pregnancies No. of patients Radiorecel>"' torassay

Agglutination test

Pathology, tubal ectopic

7 positive

7 negative

7 positive

21, 23, 24, 36, 41,51, 76 29,61,65 Unknown 30,48

pain, amenorrhea, frequent vaginal staining, and an adnexal mass were the usual findings. Plasma samples were obtained from each patient prior to surgery and, in one instance, on 4 separate days for the radioreceptorassay ofhCGP Conventional hemagglutination or latex agglutination tests (Pregnosticon Dri-Dot test; Organon, West Orange, N. J.) 18 were

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FIG. 1. Computer output of logit-log transformation of the standard for the radioreceptorassay of HCG. TSH, plasma from hypothyroid patients; HGH, plasma from acromegalic subjects; PRL, plasma from lactating postpartum women. Milli-international units of Second International Reference Preparation equivalent to corresponding nanograms of HCG are indicated on the ordinate. B/Bo is the percentage of total labeled hormone bound specifically at zero concentration of the unlabeled hormone.

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of 1 pregnant subject (8. L.) in whom ovulation had been induced with 10,000 IU of hCG, which was monitored with radioreceptorassay. The hCG rise following the LH peak was first detected RESULTS in the first blood samples obtained, on day 6 to 8 following ovulation, but it might The logit-log transformation of the have occurred earlier. Assays had been standard hCG dose-response curve yieldperformed for three of the ectopic subjects ed a sensitivity of 0.3 ng or 3 miU of prior to the first missed menses, and the hCG/ml (Fig. 1). Various dilutions of a levels of hCG in four were in the range plasma sample from a pregnant woman of normal pregnancy. The other six yielded a slope parallel to that of hCG, ectopic hCG levels were lower than the indicating the validity of the assay. There normal. These observations suggested was no cross-reaction with FSH, thyroidthat the early nidations in the tube, stimulating hormone (TSH), human before rupture and hemorrhage, could growth hormone (hGH), or prolactin in the secrete normal quantities of hCG. Later, assay. There was a 98 to 102% recovery however, when hemorrhage, increased of hCG added to the plasma samples of separation, and reduced blood supply known hormonal concentration. The occurred, the hCG secretion leveled off. intra- and interassay variation in the As shown in Figure 2, in the present estimates of hormonal levels of plasma pools was 6 and 11%, respectively. The series of 13 suspected ectopic pregnancies, radioreceptorassay, however, did not dis- the hCG was 22 and 35 ng/ml or 0.26 and criminate between LH and hCG. This 0.42 IU, respectively, in 2 of the 13 padrawback was circumvented by the ob- tients. The detection of hCG at these low servations and controls: (1) that neither levels aided in the correct management LH nor FSH rises during early pregnancy, of the patients at an earlier stage. The as determined by specific {3 subunit radio- values for the remaining patients were immunoassay of FSH, LH, and hCG in > 100 ng/ml. The levels ofhCG on days 21, the blood 12 ; (2) that the standards contain 24, 30, and 42 of pregnancy (Fig. 2) were the same amount of plasma as that ob- obtained from the same patient. The tained from nonpregnant women during initial test was associated with a negative the luteal phase, during which plasma Pregnosticon test, whereas both tests levels of LH are low; (3) that all unknown were positive in three subsequent detersamples are compared with samples from minations. On the 64th day, abdominal a pool of plasma from nonpregnant women intervention was required because of a and with various dilutions of a pool of ruptured right tube. Goldstein and plasma from pregnant women, analyzed Kosasa8 have reported a level of hCG as routinely with each assay for accuracy high as 87,000 ng/ml in one ectopically and quality control; and, finally, (4) that pregnant patient. during early pregnancy the hCG-LH Table 1 indicates the results obtained levels are 2- to 3-fold higher than basal by the radioreceptorassay and hemaggluLH levels in nonpregnant women during tination tests in 13 suspected ectopic the luteal phase. pregnancies, and, in addition, the time In Figure 2, the hCG levels in 10 from the last menses and the tubal histoectopic pregnancies are compared with logic findings. Seven of the ten ectopic those of 2 subjects of normal intrauterine pregnancies had a positive radioreceptorgravidity (H. D. and R. S.) and with that assay and negative hemagglutination

performed on urine samples at the same time. The results of the pregnancy tests and the pathologic findings are presented in Table 1.

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ABBAY OF HCG IN ECTOPIC PREGNANCY

Vol. 26, No.5 10,000-

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FIG. 2. Comparison of the plasma levels of HCG during 2 normal pregnancies (H. D., R. S.) and 1 induced ovulation intrauterine pregnancy (S. L.) with the HCG levels of 10 ectopically pregnant patients. The plasma levels of HCG during early induced or natural pregnancy were similar. 19 • 20 The day of pregnancy was based on the last menstrual period reported by the patients. In one of the 10 patients (& · · · ·&), the radioreceptorassay was performed on days 21, 24, 30, and 42 of pregnancy. 'rhe positive radioreceptorassays on days 21 and 24 were associated with negative Pregnosticon tests.

pregnancy tests. One false positive hemagglutination test was obtained, but no false positive radioreceptorassays. There was a wide range for the duration of pregnancy, between 23 and 76 days past the last missed period. The levels of

hCG obtained were not always related to the duration of gravidity. The following three suspected ectopic pregnancies are summarized to demonstrate the clinical value of the radioreceptorassay. Patient 1, a 40-year-old gravida 2, para

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1, whose last regular menses had occurred on November 23, 1973, was admitted to the hospital 7 weeks later because of continuous spotting of 2 weeks' duration and moderate lower abdominal pain for 12 hours. The hemagglutination test (UCG test; Wampole and Princeton Laboratories, Princeton, N. J.), performed on January 13, 1974, was negative. Because of little change in the abdominal signs and lack of any definitive adnexal mass, observations were continued. Two days later, on January 15, 1974, the agglutination test was again negative, but the radioreceptorassay was positive. Surgery revealed a normal uterine cavity without chorionic tissue. Culdocentesis showed unclotted blood in the peritoneal cavity, and laparoscopy demonstrated a right ruptured tube which was removed at subsequent laparotomy. Histologic examination confirmed the diagnosis of ectopic pregnancy. Patient 2, a 28-year-old gravida 4, para 5, with four living children, was admitted for elective laparoscopic tubal ligation on February 8, 1974. The last menstrual period occurred on January 9, 1974. At operation, a 5-cm left adnexal mass was palpated but the curettage revealed a normal cavity. Laparoscopy demonstrated a hematoma at the left fimbria which had the appearance of an aborting left ectopic pregnancy. Although the gross diagnosis was ectopic pregnancy, histology revealed an organized hematoma of the fimbriated end of the left tube but no evidence of chorionic tissue. The radioreceptorassay for hCG was negative, confirming the microscopic findings. Patient 3, a 23-year-old nulligravida, was admitted with right lower quadrant pain of 5 days' duration. The time of the last menses was unknown. Her pulse was 150, temperature 39° C, and the abdomen was normal except for the right lower quadrant tenderness and moderate rebound. Pelvic examination revealed a right lower quadrant 5-cm mass; the

May 1975

uterus was not enlarged. Later, an agglutination test for pregnancy was positive; however, the radioreceptorassay was negative. Exploratory laparotomy revealed a necrotic appendix and cecum; an appendectomy and right colectomy were performed. Uterus, tubes, and ovaries were normal. Pathologic examination demonstrated acute ulcerative colitis of the appendix and cecum. The first case demonstrated the value of the radioreceptorassay over the hemagglutination technique. Two of the latter tests were negative, whereas the radioreceptorassay was positive. The radioreceptorassay is 500-fold more sensitive and could detect hCG levels in a concentration of 3 miU/ml. In the other two cases, the radioreceptorassay excluded the . diagnosis of pregnancy by the absence of hCG in the blood specimens. DISCUSSION

Mishell and Davajan, 4 using the hemagglutination inhibition method described by Wide and Gemzell, 21 were able to recognize hCG in titers of more than 600 to 700 IU of hCG/liter of urine. Of 24 ectopic pregnancies, 22 had positive tests and hCG titers of more than 1000 IU/liter of urine, 2 patients had negative tests, and 9 others showed titers of more than 900 IU/liter of urine. Brody and Carlstrom, 6 also using a hemagglutination inhibition technique with a sensitivity of 2 IU of hCG/ml of serum, studied a group of 17 patients. Eight of these had negative reactions and nine showed subnormal levels or levels which rapidly declined from the normal. Kosasa et al. 7 used antisera specific to the f3 subunit ofhCG22 and demonstrated hCG levels far below normal levels in four early ectopic pregnancies. The sensitivity of the radioimmunoassay using antisera to the f3 subunit of hCG was 6 to 15 miU/ml of serum, which is approximately 200 times greater than the conventional hemagglutination inhibition test for pregnancy,

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ASSAY OF HCG IN ECTOPIC PREGNANCY

in which latex particles or hCG-coated red blood cells are analyzed. In our patients, the routine hemagglutination tests were negative. The hCG levels ranged from 10 to 134 miU/ml of serum, which are significantly lower than the hCG levels found during normal pregnancy of the same duration. However, to achieve the desired sensitivity and reliability, the radioimmunoassay of hCG requires 36 to 48 hours. An attempt to adapt the radioimmunoassay for quicker detection of pregnancy may result in loss of sensitivity and subsequent false positive indication of pregnancy.9 The radioreceptorassay has demonstrated almost 10CY}b reliability in detecting normal pregnancy of various duration, from as early as 1 week following conception, in 1000 patients and in the present series of 13 patients with suspected ectopic pregnancy. The radioreceptorassay has a sensitivity of more than 500 times that of conventional hemagglutination tests, and the diagnosis of pregnancy can be made within 1 hour without loss of sensitivity or reliability. As indicated in Figure 2, the plasma hCG levels during ectopic pregnancy may vary from low to normal levels, compared with those found during normal intrauterine pregnancy. In three of the ectopic pregnancies, radioreceptorassay determinations were positive before the first missed cycle. The levels of hCG were higher than those of the normal intrauterine controls. Douglas3 indicated that 14% of the tubes in 47 tubal pregnancies had ruptured before the first missed cycle. The high values of hCG in the earlier detected cases may be related to normal tubal chorionic growth, while later disruption of the implantation, with hemorrhage and separation, result in lower hCG levels. The rapid, early diagnosis of pregnancy by the radioreceptorassay, with frequently a low level of hCG, may alert the physician to the likely presence of an ectopic pregnancy prior to rupture.

From the present study of 13 suspected ectopic pregnancies, it appears promising that the routine use of the radioreceptorassay could provide a quick, convenient, and reliable clinical test for the diagnosis and management of ectopic pregnancy. This assay may completely eliminate false positive or negative diagnoses of pregnancy which frequently result in unnecessary or delayed surgical procedures. With the availability of reagents through a pharmaceutical company, the radioreceptorassay can be done in any laboratory equipped to perform radioimmunoassay. The 1-hour assay period, including incubation time, renders the possibility of 24-hour coverage of service in an emergency situation. After further simplification of the procedure and perhaps with the use of nonradioactive markers, the radioreceptorassay may be performed in the future in a physician's office, without unusually skilled laboratory personnel. SUMMARY

The radioreceptorassay of human chorionic gonadotropin (hCG), with a sensitivity of 50 pg or 3 miU/ml of plasma, has provided almost 100% reliability in detecting pregnancy after the first missed cycle. This test may be performed within 1 hour and is ideally suited to the clinical detection of ectopic pregnancy, especially in patients who require immediate surgical intervention. Thirteen patients with suspected ectopic pregnancy were evaluated by the radioreceptorassay, one of whom was followed with four separate determinations. The results of the assay were subsequently compared with those of hemagglutination pregnancy tests, clinical symptoms, and pathologic findings. All of the patients were diagnosed accurately by the radioreceptorassay, even when hemagglutination tests yielded a false indication of pregnancy. By this assay, the hCG levels during ectopic pregnancies are generally lower than those

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9. Kosasa TS, Levesque LA, Goldstein DP, Taymor found during a normal intrauterine pregML: Clinical use of a solid phase radioimmunonancy; in addition, pregnancy may be deassay of human chorionic gonadotropin. Am J tected much earlier (prior to the rupture) Obstet Gynecol 119:784, 1974 than is possible by hemagglutination 9a. Goldstein DP: Personal communication tests. Furthermore, the diagnosis of 10. Breen JL: A 21-year-old survey of 654 ectopic pregnancies. Am J Obstet Gynecol 106:1004, ectopic pregnancy may be excluded for pa1970 tients admitted to the hospital with acute 11. Saxena BB, Hasan SH, Haour F, Gollwitzer MS: abdominal emergencies. Radioreceptorassay of human chorionic gonadoAcknowledgment. Our thanks are due to Miss Nancy Moore for excellent technical assistance in the performance of the assays.

12.

REFERENCES 1. Reid DE, Ryan KJ, Benirschke K: Principles and Management of Human Reproduction. Philadelphia, W B Saunders Co, 1972 2. Webster HD, Barclay CL, Fischer CK: Ectopic pregnancy. A 17-year review. Am J Obstet Gynecol 92:23, 1965 3. Douglas CP: Tubal ectopic pregnancy. Br Med J 2:838, 1963 4. Mishell DR Jr, Davajan V: Quantitative immunologic assay of human chorionic gonadotropin in normal and abnormal pregnancy. Am J Obstet Gynecol 96:231, 1966 5. Brenner W, Edelman DA, Davis GLR, Kessel E: Suction curettage for "menstrual regulation." Presented at the American Association of Planned Parenthood Physicians, Houston, Tex, April 11 to 13, 1973, p 1 6. Brody S, Carlstrom G: Human chorionic gonadotropin in abnormal pregnancy. Serum and urinary findings using various immunoassay techniques. Acta Obstet Gynecol Scand 44:32, 1965 7. Kosasa TS, Taymor ML, Goldstein DP, Levesque L: Use of radioimmunoassay specific for human chorionic gonadotropin in the diagnosis of early ectopic pregnancy. Obstet Gynecol42:868, 1973 8. Goldstein DP, Kosasa TS: The beta subunit radioimmunoassay for HCG---dinical application. In Progress in Gynecology, Vol 6, Edited by ML Taymor, TH Green. New York, Grune and Stratton, Inc, 1974

13.

14.

15.

16.

17.

18.

19.

20. 21. 22.

tropin: early detection of pregnancy. Science 184:793, 1974 Saxena BB, Gollwitzer KS: Determination of protein hormones by radioimmuno- and radioreceptorassays in human plasma with emphasis during normal pregnancy. In Hormonal Investigations in Human Pregnancy, Edited by R Scholler. Paris, Editions SEPE, 1974 Miyachi Y, Vaitukaitis JL, Nieshlag E, Lipsett MB: Enzymatic radioiodination of gonadotropin. J Clin Endocrinol Metab 34:23, 1972 Haour F, Saxena BB: Characterization and solubilization of gonadotropin receptor of bovine corpus !uteum. J Bioi Chern 249:2195, 1974 Rodbard D, Rayford PL, Cooper JA, Ross GT: Statistical quality control of radioimmunoassays. J Clin Endocrinol Metab 28:1412, 1968 Saxena BB, Rathnam P: Purification of folliclestimulating hormone from human pituitary glands. J Bioi Chern 242:3769, 1967 Rathnam P, Saxena BB: Subunits of luteinizing hormone from human pituitary glands. J Bioi Chern 246:7087, 1971 Horwitz CA, Garmezy L, Lyon F, Hensley M, Desmond B: A comparative study of five immunologic pregnancy tests. Am J Clin Pathol 58:305, 1972 Kosasa TS, Levesque LA, Goldstein DP, Taymor ML: Early detection of implantation using a radioimmunoassay for human chorionic gonadotropin. J Clin Endocrinol Metab 36:622, 1973 Saxena BB: Unpublished observations Wide L, Gemzell CA: An immunological pregnancy test. Acta Endocrinol (Kbh) 35:261, 1960 Vaitukaitis JL, Braunstein GD, Ross GT: A radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone. Am J Obstet Gynecol 113:751, 1972