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Human chorionic gonadotropin assay sensitivity on screening for ectopic pregnancy
CORRESPONDENCE Human chorionic gonadotropin assay sensitivity on screening for ectopic pregnancy To the Editors: Romero et a!. report an application of a radioimmunoassay of human chorionic gonadotropin (hCG) as a screening test for ectopic pregnancy (The effect of different human chorionic gonadotropin assay sensitivity on screening for ectopic pregnancy AM J OBsn:T GYNECOL 1985; 153:72-4). The type (monoclonal versus polyclonal). affinity constant, and source of the antibody are not mentioned. Their dialogue included one false negative report in 184 ectopic pregnancies but no information is supplied for false positives. With an hCG quantitative test sensitive to 10 mIU/ml, we would detect many false positives. The use of a polyclonal antibody will sometimes measure this level of hCG in tumor or even in normal tissue extract.' There is a large series of ectopic pregnancies where the presence of hCG could not be detected. Under these circumstances, even elaborate quantitative hCG values by the l3-subunit radioimmunoassay, radioreceptor assays, or sandwichdouble monoclonal techniques have failed to detect the presence of any type of pregnancy. We are surprised that the Yale team uses hCG radioimmunoassay and not a l3-subunit type of test. Any luteinizing hormone (LH) surge or even low levels of LH, 2.4 mIU/ml, could very well interfere with the determination. Among the current pregnancy tests, enzyme-linked immunosorbent assay (ELISA) with use of monoclonal antibodies is relatively accurate and convenient, especially in an emergency situation, for example, an ectopic pregnancy. The ELISA technique employs two monoclonal antibodies recognizing different specific sites on the hCG molecule. ELISA, therefore, is more specific and has a sensitivity of 40 mIU/m!. The ELISA thus recognizes the intact hCG molecule and gives an index of biologic activity of the hormone. Because of the specificity of the antibody there is no interference with LH. The simplicity of these double monoclonal reactions permits a resident to perform a color test in 3 minutes and precludes the need to send a specimen to the laboratory and wait hours for the result from a hospital laboratory. The Romero team states that less specific techniques such as the radioreceptor assay have been used; a 6% false negative rate has been reported. The receptor assay provides a measure ofthe biologic activity ofhCG. The quantitative receptor assay can be accurate to 1 mIU of hCG. The sensitivity of the Biocept-G (radioreceptor assay) has been adjusted to 200 mIU/ml to avoid the remote possibility of interference with LH if in an extremely rare situation a woman ovulates at the time of a missed period. During pregnancy LH remains at a low level of 2 to 3 mIU/m!. In case of the possibility of a delayed LH peak, the receptor assay can be repeated in 24 to 48 hours to discriminate declining LH
levels from rising hCG levels. For example, LH surge declines to basal level where the hCG levels will double every 1.7 days up to the tenth to twelfth week of gestation. 2. " We respectfully submit to our fellow clinicians that to discriminate hCG as a chemical in a solution is not to be compared to the hCG in the circulation as a marker for pregnancy. The development of specific polyclonal and monoclonal antibodies as well as the availability of specific receptor has made it possible to develop competitive binding protein assays to accurately measure as little as 1 mIU of hCG! It should be borne in mind that the levels of hCG at the time of implantation are in normal pregnancy approximately 51 mIU/m!.' Hence a method that measures less than 50 mIUlml of hCG, only confirms the production of hCG prior to or at the time of implantation, since the blastocyst also produces hCG-like material li,7 and does not discriminate between normal or abnormal implantation. The use of more sensitive tests is important in the subsequently declining levels of hCG, which may be the result of a blighted ova syndrome, spontaneous abortion, or missed abortion. In case of an ectopic pregnancy the hCG levels are more or less following the normal pattern in the early stages. Depending on the site of ectopic implantation, the hCG value shows subnormal increases in the repeat serum sample, so it is mandatory to repeat the pregnancy test in cases of highrisk patients for ectopic pregnancy or suspected ectopic pregnancy with close clinical correlation of symptoms. The foregoing makes it obligatory for serum hCG levels of> 50 mIUlml of hCG to be corroborated with clinical circumstances to establish a state of normal gestation. It has been amply demonstrated that even in a highrisk pregnancy destined to be ablated, the level of hCG at the time of implantation is the same as in a normal pregnancy which is destined to reach normal maturity. The name of the game is to catch the declining levels or subnormal increase of hCG during the period intervening from the time of implantation until after the missed period by repeat assays (hCG levels at missed period is <200 mIU/ml). Of course, we need the most sensitive but also specific test to measure intact hCG molecules. The l3-subunits of hCG produce relatively more specific antibody, but it is biologically inert when not linked with the a-subunit. Notwithstanding highly reliable monoclonal two-site capture (sandwich-type enzyme assays) and highly immunologically specific radioassays using antibodies to specific l3-subunit has provided confusion in the establishment of early pregnancy. Current sensitive radioimmunoassay tests for hCG have not been validated quantitatively by comparison with a radioligand receptor assay of hCG which measures the biologically active form of hCG. Hence the sensitivity of an immunologic test of hCG could possibly be also due to the discordant production of
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hCG and its free subunits, especially in the case of an abnormal and thereby a high-risk pregnancy.s This, in our opinion, is the reason for false results in so-called more improved and highly sensitive radioimmunoassays. In a molecule-like hCG the immunological specificity is conformation dependent. Even antibodies from monoclonal hybridomas may recognize epitopes that have no bearing on the specific biologic activity of hCG. In summary, most of the current pregnancy tests have a high degree of reliability at the time of a missed period. However, for the purposes of diagnosis and management of ectopic pregnancy, repeat determinations of hCG by the aid of a reliable test and more importantly a clinical judgment are essential ingredients to control this morbidity, which is as high as one in 40 women. Finally, it is obligatory for the manufacturers of pregnancy tests not only to provide data comparing the detection of pregnancy but also to show that these tests have high correlation with radioligand receptor assay for the measurement of hCG. It is advisable to use a kit with a sensitivity >50 mIU because this level of hCG is obtained at the time of implantation. The hCG tests with greater sensitivity are suited to the monitoring of declining levels of hCG after rupture of ectopic pregnancy, spontaneous and induced abortion, chemotherapy, or surgical ablation of hCG-producing carcmoma. Robert Landesman, M.D. Mukul Singh, M.D. Brij B. Saxena, Ph.D., D.Sc. Division of Reproductive Endocrinology Department of Obstetrics and Gynecology The New York Hospital-Cornell Medical Center 525 East 68th Street New York, New York 10021
REFERENCES I. Hussa RO. Clinical utility of human chorionic gonad-
otropin and ex-subunit measurements. Obstet Gynecol 1982;60:1-12.
2. Warkentin DL. From A to hCG in pregnancy testing. Diag Med 1984;7:35-43. 3. Balzer FR, Schlaff S, Goldfarb AF, et al. Serial ~-subunit human chorionic gonadotropin doubling due as a prognosticator of pregnancy outcome in an infertile population. Fertil Steril 1981;35:307-12. 4. Saxena BB, Hasan SH, Haour F. Radioreceptor assay of human chorionic gonadotropin: detection of early pregnancy. Science 1974;184:793-5. 5. Saxena BB, Singh M. Current pregnancy tests and their clinical applications. In: Langer A, Iffy L, eds. Extrauterine pregnancy. Littleton, Massachusettes: PSG Publishing Company, Inc., 1986. 6. Catt KJ, Dufau ML, Vaitukaitis JL. Appearance of hCG in pregnancy plasma following initiation of implantation of the blastocyst. Clin Endocrinol Metab 1975;40:537-40. 7. Asch RH, Fernandez EO, Magnas LA, Pauerstein CJ. Demonstration of a chorionic gonadotropin-like substance in rabbit morulae. Ferti! Steril 1978;29:444-6. 8. Hussa RO, Rinke ML, Schweitzer PG. Discordant human chorionic gonadotropin results: causes and solutions. Obstet Gynecol 1985;65:211-9.
September 1986 Am J Obstet Gynecol
Reply
To the Editors: While we shall endeavor to address the comments made in the letter of Landesman et al., it must be said at the outset that we fail to see exactly what point(s) these authors were trying to make, since their letter struck us as a melee of disjointed observations (some true, some false), largely disconnected from the point of the article at which the letter was directed. For example, the remarks regarding the properties and uses of the Biocept-G assay which echo what was written about this assay over 10 years ago hardly need saying and warrant no reply. The remarks about the detectability of hCG in various malignant conditions in a letter about a paper on ectopic pregnancy surely fall into the same category. What puzzled us even more, however, was the authors' allusion to the fact that serial hCG determinations can be used to evaluate further the significance of a positive hCG assay result, since, to the best of our knowledge, our group was the first to describe in detail how serial hCG measurements and grayscale sonography can be combined to the best advantage in evaluating patients for a suspected ectopic pregnancy.'-7 Therefore we cannot reply to these authors in the coherent manner we would have liked and can only comment in a piecemeal fashion on the points they have chosen to air. 1. We use an antibody (prepared in our own laboratory some 10 years ago) raised in rabbits to a specific ~-subunit « 1% a-subunit in the antigen), as clearly stated in our original paper,' which was referenced in the article under discussion. The referees of that original manuscript insisted, somewhat pedantically, that we desist from referring to the substance being measured in our assay as "~-hCG," since it is the intact hCG molecule in the circulation that is being measured by the antibody, even though this is by way of its ~-subunit (since there are vanishingly small amounts of free ~ subunit in circulation). Therefore, we have consistently referred to the substance measured in our assay as "hCG." However, in our opinion, our publications have left no room for doubt about what type of antibody is used in our laboratory. Certainly, to our knowledge, no one other than Dr. Landesman and his colleagues have ever been under the misimpression that we use an "hCG radioimmunoassay and not a ~-subunit type of test." Had the authors read our papers with care, they would have spared themselves this misunderstanding and us a dissertation about how hCG antibodies that are not ~-subunit-specific cross-react with luteinizing hormone; hardly news to us or your readers. 2. We are fully aware of the clinical potential of the ELISA technique as reported by Professor Chard's group from St. Bartholomew's Hospital in London,S and it may well be that radioimmunoassays will become a thing of the past regarding "emergency" pregnancy testing. However, these techniques need to be evaluated in the specific context under discussion, and much more work is needed before we can determine the reliability/efficacy of serial hCG measurements with this
levels from rising hCG levels. For example, LH surge declines to basal level where the hCG levels will double every 1.7 days up to the tenth to twelfth week of gestation. 2. " We respectfully submit to our fellow clinicians that to discriminate hCG as a chemical in a solution is not to be compared to the hCG in the circulation as a marker for pregnancy. The development of specific polyclonal and monoclonal antibodies as well as the availability of specific receptor has made it possible to develop competitive binding protein assays to accurately measure as little as 1 mIU of hCG! It should be borne in mind that the levels of hCG at the time of implantation are in normal pregnancy approximately 51 mIU/m!.' Hence a method that measures less than 50 mIUlml of hCG, only confirms the production of hCG prior to or at the time of implantation, since the blastocyst also produces hCG-like material li,7 and does not discriminate between normal or abnormal implantation. The use of more sensitive tests is important in the subsequently declining levels of hCG, which may be the result of a blighted ova syndrome, spontaneous abortion, or missed abortion. In case of an ectopic pregnancy the hCG levels are more or less following the normal pattern in the early stages. Depending on the site of ectopic implantation, the hCG value shows subnormal increases in the repeat serum sample, so it is mandatory to repeat the pregnancy test in cases of highrisk patients for ectopic pregnancy or suspected ectopic pregnancy with close clinical correlation of symptoms. The foregoing makes it obligatory for serum hCG levels of> 50 mIUlml of hCG to be corroborated with clinical circumstances to establish a state of normal gestation. It has been amply demonstrated that even in a highrisk pregnancy destined to be ablated, the level of hCG at the time of implantation is the same as in a normal pregnancy which is destined to reach normal maturity. The name of the game is to catch the declining levels or subnormal increase of hCG during the period intervening from the time of implantation until after the missed period by repeat assays (hCG levels at missed period is <200 mIU/ml). Of course, we need the most sensitive but also specific test to measure intact hCG molecules. The l3-subunits of hCG produce relatively more specific antibody, but it is biologically inert when not linked with the a-subunit. Notwithstanding highly reliable monoclonal two-site capture (sandwich-type enzyme assays) and highly immunologically specific radioassays using antibodies to specific l3-subunit has provided confusion in the establishment of early pregnancy. Current sensitive radioimmunoassay tests for hCG have not been validated quantitatively by comparison with a radioligand receptor assay of hCG which measures the biologically active form of hCG. Hence the sensitivity of an immunologic test of hCG could possibly be also due to the discordant production of
681
682
Correspondence
hCG and its free subunits, especially in the case of an abnormal and thereby a high-risk pregnancy.s This, in our opinion, is the reason for false results in so-called more improved and highly sensitive radioimmunoassays. In a molecule-like hCG the immunological specificity is conformation dependent. Even antibodies from monoclonal hybridomas may recognize epitopes that have no bearing on the specific biologic activity of hCG. In summary, most of the current pregnancy tests have a high degree of reliability at the time of a missed period. However, for the purposes of diagnosis and management of ectopic pregnancy, repeat determinations of hCG by the aid of a reliable test and more importantly a clinical judgment are essential ingredients to control this morbidity, which is as high as one in 40 women. Finally, it is obligatory for the manufacturers of pregnancy tests not only to provide data comparing the detection of pregnancy but also to show that these tests have high correlation with radioligand receptor assay for the measurement of hCG. It is advisable to use a kit with a sensitivity >50 mIU because this level of hCG is obtained at the time of implantation. The hCG tests with greater sensitivity are suited to the monitoring of declining levels of hCG after rupture of ectopic pregnancy, spontaneous and induced abortion, chemotherapy, or surgical ablation of hCG-producing carcmoma. Robert Landesman, M.D. Mukul Singh, M.D. Brij B. Saxena, Ph.D., D.Sc. Division of Reproductive Endocrinology Department of Obstetrics and Gynecology The New York Hospital-Cornell Medical Center 525 East 68th Street New York, New York 10021
REFERENCES I. Hussa RO. Clinical utility of human chorionic gonad-
otropin and ex-subunit measurements. Obstet Gynecol 1982;60:1-12.
2. Warkentin DL. From A to hCG in pregnancy testing. Diag Med 1984;7:35-43. 3. Balzer FR, Schlaff S, Goldfarb AF, et al. Serial ~-subunit human chorionic gonadotropin doubling due as a prognosticator of pregnancy outcome in an infertile population. Fertil Steril 1981;35:307-12. 4. Saxena BB, Hasan SH, Haour F. Radioreceptor assay of human chorionic gonadotropin: detection of early pregnancy. Science 1974;184:793-5. 5. Saxena BB, Singh M. Current pregnancy tests and their clinical applications. In: Langer A, Iffy L, eds. Extrauterine pregnancy. Littleton, Massachusettes: PSG Publishing Company, Inc., 1986. 6. Catt KJ, Dufau ML, Vaitukaitis JL. Appearance of hCG in pregnancy plasma following initiation of implantation of the blastocyst. Clin Endocrinol Metab 1975;40:537-40. 7. Asch RH, Fernandez EO, Magnas LA, Pauerstein CJ. Demonstration of a chorionic gonadotropin-like substance in rabbit morulae. Ferti! Steril 1978;29:444-6. 8. Hussa RO, Rinke ML, Schweitzer PG. Discordant human chorionic gonadotropin results: causes and solutions. Obstet Gynecol 1985;65:211-9.
September 1986 Am J Obstet Gynecol
Reply
To the Editors: While we shall endeavor to address the comments made in the letter of Landesman et al., it must be said at the outset that we fail to see exactly what point(s) these authors were trying to make, since their letter struck us as a melee of disjointed observations (some true, some false), largely disconnected from the point of the article at which the letter was directed. For example, the remarks regarding the properties and uses of the Biocept-G assay which echo what was written about this assay over 10 years ago hardly need saying and warrant no reply. The remarks about the detectability of hCG in various malignant conditions in a letter about a paper on ectopic pregnancy surely fall into the same category. What puzzled us even more, however, was the authors' allusion to the fact that serial hCG determinations can be used to evaluate further the significance of a positive hCG assay result, since, to the best of our knowledge, our group was the first to describe in detail how serial hCG measurements and grayscale sonography can be combined to the best advantage in evaluating patients for a suspected ectopic pregnancy.'-7 Therefore we cannot reply to these authors in the coherent manner we would have liked and can only comment in a piecemeal fashion on the points they have chosen to air. 1. We use an antibody (prepared in our own laboratory some 10 years ago) raised in rabbits to a specific ~-subunit « 1% a-subunit in the antigen), as clearly stated in our original paper,' which was referenced in the article under discussion. The referees of that original manuscript insisted, somewhat pedantically, that we desist from referring to the substance being measured in our assay as "~-hCG," since it is the intact hCG molecule in the circulation that is being measured by the antibody, even though this is by way of its ~-subunit (since there are vanishingly small amounts of free ~ subunit in circulation). Therefore, we have consistently referred to the substance measured in our assay as "hCG." However, in our opinion, our publications have left no room for doubt about what type of antibody is used in our laboratory. Certainly, to our knowledge, no one other than Dr. Landesman and his colleagues have ever been under the misimpression that we use an "hCG radioimmunoassay and not a ~-subunit type of test." Had the authors read our papers with care, they would have spared themselves this misunderstanding and us a dissertation about how hCG antibodies that are not ~-subunit-specific cross-react with luteinizing hormone; hardly news to us or your readers. 2. We are fully aware of the clinical potential of the ELISA technique as reported by Professor Chard's group from St. Bartholomew's Hospital in London,S and it may well be that radioimmunoassays will become a thing of the past regarding "emergency" pregnancy testing. However, these techniques need to be evaluated in the specific context under discussion, and much more work is needed before we can determine the reliability/efficacy of serial hCG measurements with this