Case Report
Gestational Trophoblastic Disease Diagnosis Delayed by the Hook Effect Julia Cormano, MD, Gillian Mackay, MD, and Christine Holschneider,
MD
BACKGROUND: A “hook effect” resulting from saturation of antibodies used in pregnancy tests can occur at human chorionic gonadotropin (hCG) levels above 500,000 milliinternational units/mL, resulting in falsely negative values. CASE: A 34-year-old woman, gravida 5 para 3, presented to the emergency department after heavy bleeding. Ultrasonogram revealed a uterine mass, urine pregnancy test result was negative, and endometrial biopsy inconclusive. The patient was discharged and presented 10 days later with recurrent bleeding. Urine pregnancy test result was again negative, but serum hCG was 581 milliinternational units/mL. Serial dilution revealed an actual hCG higher than 5 million milliinternational units/mL. She was diagnosed with gestational trophoblastic disease. CONCLUSION: Awareness of the risk of a false-negative pregnancy test result when hCG levels are extremely high may prevent delayed diagnosis of gestational trophoblastic disease. (Obstet Gynecol 2015;126:811–4) DOI: 10.1097/AOG.0000000000000860
A
bnormal vaginal bleeding is a very common complaint in the emergency department and as a result of its high accuracy and rapid result times, a urine pregnancy test based on a lateral flow chromatographic immunoassay for the qualitative detection of human chorionic gonadotropin (hCG) is used to triage abnormal vaginal bleeding in reproductive-aged women. Understanding the limitations of this test is important to arriving at the correct diagnosis. From the Department of Obstetrics and Gynecology, Olive View-UCLA Medical Center, Sylmar, California. Corresponding author: Christine Holschneider, MD, Olive View-UCLA Medical Center, 14445 Olive View Drive, Sylmar, CA 91342; e-mail: cholschneider@ dhs.lacounty.gov. Financial Disclosure The authors did not report any potential conflicts of interest. © 2015 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0029-7844/15
VOL. 126, NO. 4, OCTOBER 2015
Teaching Points 1. A false-negative or falsely low urine or serum pregnancy test result can occur in the setting of very high human chorionic gonadotropin levels (typically above 500,000 milliinternational units/mL) as a result of a “hook effect.” 2. A “hook effect” can occur when there is so much human chorionic gonadotropin that it independently saturates the capture and tracer antibodies, effectively preventing the sandwich formation needed for a positive test result. 3. If a “hook effect” is suspected, it can be overcome by dilution, which reduces the amount of human chorionic gonadotropin in the test sample.
Commercially available pregnancy tests have an intended use of early pregnancy detection. Urine pregnancy tests used in standard clinical practice have a threshold to detect urine hCG concentrations of 20–50 milliinternational units/mL, whereas serum pregnancy tests typically are positive at hCG levels of 5–10 milliinternational units/mL and ultrasensitive tests detect hCG levels as low as 1–2 milliinternational units/mL.1 At the upper end, most hCG tests are set to the pregnancy range of 27,300 milliinternational units/mL to 233,000 milliinternational units/mL at 8–11 weeks of gestation.2 At hCG levels above 500,000 milliinternational units/mL, erroneous negative test results can occur. We present a case in which a repeatedly falsely negative urine hCG and falsely low serum hCG result led to a delay in diagnosis for a patient with advanced gestational trophoblastic disease.
CASE A 34-year-old woman, gravida 5 para 3, presented to the emergency department after an acute vaginal bleeding episode. She also reported 2 weeks of bloating, nausea, vomiting, and breast tenderness. She reported that her home urine pregnancy test result had been negative. Her last regular menstrual period was 8 weeks before presentation, with subsequent, new-onset uterine cramping and irregular vaginal bleeding. Her last pregnancy ended with a first-trimester therapeutic abortion 13 months before presentation. The patient was sexually active. She had been taking combination oral contraceptives, which she had discontinued. On examination, she had normal vital signs and a 14-week-sized, mildly tender uterus. Her hemoglobin level was 10.4 g/dL, and urine pregnancy test result was negative. Pelvic ultrasonography as interpreted by the
OBSTETRICS & GYNECOLOGY
Copyright ª by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
811
radiologist revealed a 13.7-cm complex mass in the uterine cavity with heterogeneous echogenicity and cystic areas but no internal vascularity. There were numerous follicles on the ovaries bilaterally. Given the negative pregnancy test result, endometrial biopsy was performed by the gynecologic consultant, and the patient was discharged home in stable condition with outpatient follow-up in the gynecology clinic within 2 weeks. The patient again presented to the emergency department 10 days later, after another acute bleeding episode. At this time, she reported new-onset shortness of breath, palpitations, and fatigue as well as continued nausea and vomiting. Examination revealed tachycardia, a 20-weeksized uterus, and minimal vaginal bleeding. Her hemoglobin level was 7.1 g/dL. A urine pregnancy test was repeated and remained negative. Because the prior endometrial biopsy result was insufficient, another sample was collected. Brisk bleeding was noted after the biopsy, which resolved spontaneously. On ultrasonography, there was an interval increase to 15 cm of the heterogeneous endometrial mass and bilateral, enlarged multicystic ovaries. Serum hCG level was 581 milliinternational units/mL. Computed tomography of the chest, abdomen, and pelvis revealed more than 10 micronodules with scattered ground glass nodular opacities throughout both lungs and confirmed the pelvic findings seen on ultrasonography. The patient was admitted to the gynecology service and transfused packed red blood cells. As a result of the discrepancy between the urine and serum hCG results and clinical concern for gestational trophoblastic disease, serial dilutions of her urine and serum hCG testing were requested. The urine test result became clearly positive with serial dilutions of 1:100 to 1:10,000, ultimately becoming negative again at a dilution of 1:1,000,000 (Fig. 1). Likewise, her serum hCG dilution of 1:100 revealed “out of range” elevated levels of hCG using the ADVIA Centaur
1:10
1:100
1:1,000
1:10,000
1:100,000 1:1,000,000
Dilution
Fig. 1. Serial dilution of urine human chorionic gonadotropin testing, revealing a hook effect. Dilutions 1:10 and 1:100,000 reveal faintly positive results, whereas dilutions 1:100, 1:1,000, and 1:10,000 reveal strongly positive results. Photo courtesy of the Department of Pathology and Laboratory Medicine, Olive View-UCLA Medical Center. Cormano. False-Negative Pregnancy Test Due to Extremely High hCG. Obstet Gynecol 2015.
812
Cormano et al
XP Immunoassay System. Final quantification by a specialized reference laboratory revealed a serum hCG level of 5,899,478 milliinternational units/mL. The patient’s second endometrial biopsy was consistent with gestational trophoblastic disease. She had laboratory evidence of subclinical hyperthyroidism (thyroid-stimulating hormone less than 0.008 microinternational units/mL; free thyroxine 2.74 ng/dL). Based on her pulmonary metastases, their number, interval since prior pregnancy, pretreatment tumor size, and hCG level, the patient was diagnosed with stage III high-risk gestational trophoblastic disease with a World Health Organization score of 15.3 The patient initially was administered the first cycle of multiagent chemotherapy but began to hemorrhage vaginally requiring an emergency hysterectomy. Findings at surgery included a 20-week-sized boggy uterus with a dilated cervix and bilateral, large, multicystic ovaries consistent with theca lutein cysts. She was promptly restarted on her chemotherapy regimen postoperatively. Pathology showed an invasive hydatidiform mole with superficial myometrial invasion. The patient required a total of nine chemotherapy cycles of two different multiagent regimens to achieve complete remission. Magnetic resonance imaging revealed complete resolution of her lung metastases and theca lutein cysts. The patient has since been in surveillance for 1 year with excellent health and no evidence of disease (all hCG levels less than 2 milliinternational units/mL).
DISCUSSION The general female population has a 0.1% risk of developing gestational trophoblastic disease. Patients often present with continued vaginal bleeding after either a normal or molar pregnancy and an elevated hCG.4 In this case, the patient had compelling clinical findings of increased hCG such as her tender breasts, nausea, and vomiting; however, as a result of erroneous reliance on the negative urine pregnancy test result, the health care providers who saw the patient at initial presentation did not arrive at the diagnosis. This case highlights the need for gynecologists to be familiar with the specific type of hCG tests used in their practices so that they may recognize falsenegative urine or falsely low serum hCG test results when they occur in the presence of extremely high hCG levels such as was observed in our patient in whom the actual hGC level was over 5 million milliinternational units/mL. By increasing this awareness, the gynecologist can adjust the differential diagnoses more accurately and, in a compelling clinical situation, confirm a suspected erroneous result by dilution of the urine and serum sample. Laboratory tests for hCG have close to 100% sensitivity and specificity for trophoblast-related conditions such as pregnancy and gestational trophoblastic disease. All commercially available hCG assays are
False-Negative Pregnancy Test Due to Extremely High hCG
OBSTETRICS & GYNECOLOGY
Copyright ª by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
immunometric assays using two antibodies to distant sites on the b-subunit of the hCG molecule. A first immobilized antibody captures the hCG molecule, to which a second enzymatically or radioactively labeled tracer antibody binds forming a capture antibody-hCG-tracer antibody sandwich. All excess is then washed off and the resulting signal response is directly proportionate to the amount of hCG captured. There are several rare but critically important phenomena, which may result in false-negative urine or serum hCG results. The high-dose “hook effect” results in a falsely low value and can occur when there is an extremely high level of hCG, usually more than 500,000 milliinternational units/mL. There is so much hCG that it independently saturates the immobilized capture antibodies and the free tracer antibodies. This effectively prevents formation of the capture antibodyhCG-tracer antibody sandwich, which is needed for a positive test result, and the saturated tracer antibodies are washed away with the excess material resulting in a falsely low or negative value. The shape of the graph demonstrating the paradox of initially rising but then decreasing signal intensity with extremely high concentration of hCG has a hooklike appearance, leading to the resulting name.5 It is by dilution, which reduces the amount of hCG in the test sample, that the hook effect can be overcome and the correct hCG result obtained. The high-dose hook effect can occur in either serum or urine pregnancy tests. In our case, it occurred in both, resulting in a falsely negative urine hCG test result and a falsely low serum hCG test result. When a high-dose hook effect is suspected, it must be communicated to the laboratory and the hCG test should be repeated at a 1:1,000 dilution with which an accurate value should be obtained. In our case, once the serum dilution was accomplished, the true quantitative value was revealed and found to be consistent with gestational trophoblastic disease. The urine dilution revealed similar positive results (Fig. 1). To complicate the use of immunoassay for the qualitative detection of hCG further, there is also a variant hook effect, which more commonly affects urine hCG tests, which do not necessarily detect all hCG variants that occur with pregnancy or gestational trophoblastic disease. Such hCG variants can bind capture and tracer antibodies independently preventing sandwich formation and leading to a false-negative result. Various commercially available hCG detection kits differ in their susceptibility to the variant hook effect.6 Similar to the high-dose hook effect, the variant hook effect can also be confirmed by dilution.
VOL. 126, NO. 4, OCTOBER 2015 Cormano et al
The relevance of this hook effect to the use of pregnancy tests has been previously described for urine pregnancy tests in two case reports in the emergency medicine literature,7,8 in a clinical biochemistry journal,9 in the international gynecologic literature,10–13 and in two gynecologic oncology publications,14,15 but not in the American gynecologic literature. Consequently, few American gynecologists are familiar with the hook effect or how it can mislead the differential diagnosis, as is illustrated by our patient and the subsequent treatment delay she incurred. Our patient’s uterine tumor burden was significantly larger by the time the correct diagnosis was made. However, because she underwent hysterectomy, with which the bulk of her disease was resected, it remains unclear whether that delay in diagnosis ultimately prolonged the duration for which she required chemotherapy. Although performance of a serum hCG test may be a good first step when a false-negative urine test result is suspected, our case underscores the importance of dilution of the urine and serum tests samples to obtain the true-positive result. REFERENCES 1. Bastian LA, Brown HL. Clinical manifestations and diagnosis of early pregnancy. Referenced from UpToDate. Available at: http:// www.uptodate.com/contents/clinical-manifestations-and-diagnosisof-early-pregnancy?source5machineLearning&search5pregnancy +test+ranages&selectedTitle51;150§ionRank52&anchor 5H9#H9. Retrieved November 2, 2014. 2. Cole LA. Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites. Clin Chem 1997;43:2233–43. 3. National Cancer Institute at the National Institutes of Health. Gestational trophoblastic disease treatment (PDQÒ). Available at: http://www.cancer.gov/cancertopics/pdq/treatment/gestationaltrophoblastic/HealthProfessional. Retrieved November 2, 2014. 4. Soper J. Gestational trophoblastic disease. Obstet Gynecol 2006;108:176–87. 5. Griffey RT, Trent CJ, Bavolek RA, Keeperman JB, Sampson C, Poirier RF. ‘Hook-like effect’ causes false negative point-of-care pregnancy testing in emergency patients. J Emerg Med 2013; 44:155–60. 6. Nerenz RD, Song H, Gronowski AM. Screening method to evaluate point-of-care human chorionic gonadotropin (hCG) devices for susceptibility to the hook effect by hCG b core fragment: evaluation of 11 devices. Clin Chem 2014;60: 667–74. 7. Mundangepfupfu T, Waseem M. Partial hydatidiform mole with false-negative urine human chorionic gonadotropin test in the emergency department. J Emerg Med 2014;46:348–50. 8. Davidson CM, Kaplan RM, Wenig LN, Burmeister D. Qualitative b-hCG urine assays may be misleading in the presence of molar pregnancy: a case report. J Emerg Med 2004;27:43–7. 9. Wilgen U, Pretorius CJ, Gous RS, Martin C, Hale VJ, Ungerer JP. Hook effect in Abbott i-STAT b-human chorionic point of care assay. Clin Biochem 2014;47:1320–2.
False-Negative Pregnancy Test Due to Extremely High hCG
Copyright ª by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
813
10. Pang YP, Rayesh H, Tan LK. Molar pregnancy with false negative urine hCG: the hook effect. Singapore Med J 2010;51: e58–61. 11. O’Reilly SM, Rustin GJS. Mismanagement of choriocarcinoma due to a false low HCG measurement. Int J Gynecol Cancer 1993;3:186–8. 12. Levavi H, Neri A, Bar J, Regev D, Nordenberg J, Ovadia J. ‘Hook effect’ in complete hydatidiform molar pregnancy: a falsely low level of b-hCG. Obstet Gynecol 1993;82(suppl): 720–1.
13. Flam F, Hambraeus-Jonzon K, Hansson LO, Kjaeldgaard A. Hydatidiform mole with non-metastatic pulmonary complications and false low level of hCG. Eur J Obstet Gynecol Reprod Biol 1998;77:235–7. 14. Wheeler CA, Davis S, Degefu S, Thorneycroft IH, O’Quinn AG. Ovarian choriocarcinoma: a difficult diagnosis of an unusual tumor and a review of the hook effect. Obstet Gynecol 1990;75:547–9. 15. Nodler JL, Kim KH, Alvarez RD. Abnormally low hCG in a complete hydatidiform molar pregnancy: the hook effect. Gynecol Oncol Rep 2011;1:6–7.
Earn CME Credits for Your Contribution as an Author to Obstetrics & Gynecology In recognition of their time, effort, and expertise, authors of manuscripts for Obstetrics & Gynecology are eligible to receive continuing medical education credits. ACCME Accreditation The American College of Obstetricians and Gynecologists is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. AMA PRA Category 1 Credit(s)™ The American College of Obstetricians and Gynecologists designates this journal-based CME activity for a maximum of 10 AMA PRA Category 1 Credits.™ Physicians should claim only the credit commensurate with the extent of their participation in the activity. College Cognate Credit(s) The American College of Obstetricians and Gynecologists designates this journal-based CME activity for a maximum of 10 Category 1 College Cognate Credits. The College has a reciprocity agreement with the AMA that allows AMA PRA Category 1 Credits™ to be equivalent to College Cognate Credits. First and second authors of articles are eligible to receive 10 AMA PRA Category 1 Credits™ per article for one article per year. Authors should submit a title page to the respective group that will be responsible for providing credits (American Collegeof Obstetricians and Gynecologists or American Medical Association). rev 11/2014
814
Cormano et al
False-Negative Pregnancy Test Due to Extremely High hCG
OBSTETRICS & GYNECOLOGY
Copyright ª by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Gestational Trophoblastic Disease Diagnosis Delayed by the Hook Effect Julia Cormano, MD, Gillian Mackay, MD, and Christine Holschneider,
MD
BACKGROUND: A “hook effect” resulting from saturation of antibodies used in pregnancy tests can occur at human chorionic gonadotropin (hCG) levels above 500,000 milliinternational units/mL, resulting in falsely negative values. CASE: A 34-year-old woman, gravida 5 para 3, presented to the emergency department after heavy bleeding. Ultrasonogram revealed a uterine mass, urine pregnancy test result was negative, and endometrial biopsy inconclusive. The patient was discharged and presented 10 days later with recurrent bleeding. Urine pregnancy test result was again negative, but serum hCG was 581 milliinternational units/mL. Serial dilution revealed an actual hCG higher than 5 million milliinternational units/mL. She was diagnosed with gestational trophoblastic disease. CONCLUSION: Awareness of the risk of a false-negative pregnancy test result when hCG levels are extremely high may prevent delayed diagnosis of gestational trophoblastic disease. (Obstet Gynecol 2015;126:811–4) DOI: 10.1097/AOG.0000000000000860
A
bnormal vaginal bleeding is a very common complaint in the emergency department and as a result of its high accuracy and rapid result times, a urine pregnancy test based on a lateral flow chromatographic immunoassay for the qualitative detection of human chorionic gonadotropin (hCG) is used to triage abnormal vaginal bleeding in reproductive-aged women. Understanding the limitations of this test is important to arriving at the correct diagnosis. From the Department of Obstetrics and Gynecology, Olive View-UCLA Medical Center, Sylmar, California. Corresponding author: Christine Holschneider, MD, Olive View-UCLA Medical Center, 14445 Olive View Drive, Sylmar, CA 91342; e-mail: cholschneider@ dhs.lacounty.gov. Financial Disclosure The authors did not report any potential conflicts of interest. © 2015 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0029-7844/15
VOL. 126, NO. 4, OCTOBER 2015
Teaching Points 1. A false-negative or falsely low urine or serum pregnancy test result can occur in the setting of very high human chorionic gonadotropin levels (typically above 500,000 milliinternational units/mL) as a result of a “hook effect.” 2. A “hook effect” can occur when there is so much human chorionic gonadotropin that it independently saturates the capture and tracer antibodies, effectively preventing the sandwich formation needed for a positive test result. 3. If a “hook effect” is suspected, it can be overcome by dilution, which reduces the amount of human chorionic gonadotropin in the test sample.
Commercially available pregnancy tests have an intended use of early pregnancy detection. Urine pregnancy tests used in standard clinical practice have a threshold to detect urine hCG concentrations of 20–50 milliinternational units/mL, whereas serum pregnancy tests typically are positive at hCG levels of 5–10 milliinternational units/mL and ultrasensitive tests detect hCG levels as low as 1–2 milliinternational units/mL.1 At the upper end, most hCG tests are set to the pregnancy range of 27,300 milliinternational units/mL to 233,000 milliinternational units/mL at 8–11 weeks of gestation.2 At hCG levels above 500,000 milliinternational units/mL, erroneous negative test results can occur. We present a case in which a repeatedly falsely negative urine hCG and falsely low serum hCG result led to a delay in diagnosis for a patient with advanced gestational trophoblastic disease.
CASE A 34-year-old woman, gravida 5 para 3, presented to the emergency department after an acute vaginal bleeding episode. She also reported 2 weeks of bloating, nausea, vomiting, and breast tenderness. She reported that her home urine pregnancy test result had been negative. Her last regular menstrual period was 8 weeks before presentation, with subsequent, new-onset uterine cramping and irregular vaginal bleeding. Her last pregnancy ended with a first-trimester therapeutic abortion 13 months before presentation. The patient was sexually active. She had been taking combination oral contraceptives, which she had discontinued. On examination, she had normal vital signs and a 14-week-sized, mildly tender uterus. Her hemoglobin level was 10.4 g/dL, and urine pregnancy test result was negative. Pelvic ultrasonography as interpreted by the
OBSTETRICS & GYNECOLOGY
Copyright ª by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
811
radiologist revealed a 13.7-cm complex mass in the uterine cavity with heterogeneous echogenicity and cystic areas but no internal vascularity. There were numerous follicles on the ovaries bilaterally. Given the negative pregnancy test result, endometrial biopsy was performed by the gynecologic consultant, and the patient was discharged home in stable condition with outpatient follow-up in the gynecology clinic within 2 weeks. The patient again presented to the emergency department 10 days later, after another acute bleeding episode. At this time, she reported new-onset shortness of breath, palpitations, and fatigue as well as continued nausea and vomiting. Examination revealed tachycardia, a 20-weeksized uterus, and minimal vaginal bleeding. Her hemoglobin level was 7.1 g/dL. A urine pregnancy test was repeated and remained negative. Because the prior endometrial biopsy result was insufficient, another sample was collected. Brisk bleeding was noted after the biopsy, which resolved spontaneously. On ultrasonography, there was an interval increase to 15 cm of the heterogeneous endometrial mass and bilateral, enlarged multicystic ovaries. Serum hCG level was 581 milliinternational units/mL. Computed tomography of the chest, abdomen, and pelvis revealed more than 10 micronodules with scattered ground glass nodular opacities throughout both lungs and confirmed the pelvic findings seen on ultrasonography. The patient was admitted to the gynecology service and transfused packed red blood cells. As a result of the discrepancy between the urine and serum hCG results and clinical concern for gestational trophoblastic disease, serial dilutions of her urine and serum hCG testing were requested. The urine test result became clearly positive with serial dilutions of 1:100 to 1:10,000, ultimately becoming negative again at a dilution of 1:1,000,000 (Fig. 1). Likewise, her serum hCG dilution of 1:100 revealed “out of range” elevated levels of hCG using the ADVIA Centaur
1:10
1:100
1:1,000
1:10,000
1:100,000 1:1,000,000
Dilution
Fig. 1. Serial dilution of urine human chorionic gonadotropin testing, revealing a hook effect. Dilutions 1:10 and 1:100,000 reveal faintly positive results, whereas dilutions 1:100, 1:1,000, and 1:10,000 reveal strongly positive results. Photo courtesy of the Department of Pathology and Laboratory Medicine, Olive View-UCLA Medical Center. Cormano. False-Negative Pregnancy Test Due to Extremely High hCG. Obstet Gynecol 2015.
812
Cormano et al
XP Immunoassay System. Final quantification by a specialized reference laboratory revealed a serum hCG level of 5,899,478 milliinternational units/mL. The patient’s second endometrial biopsy was consistent with gestational trophoblastic disease. She had laboratory evidence of subclinical hyperthyroidism (thyroid-stimulating hormone less than 0.008 microinternational units/mL; free thyroxine 2.74 ng/dL). Based on her pulmonary metastases, their number, interval since prior pregnancy, pretreatment tumor size, and hCG level, the patient was diagnosed with stage III high-risk gestational trophoblastic disease with a World Health Organization score of 15.3 The patient initially was administered the first cycle of multiagent chemotherapy but began to hemorrhage vaginally requiring an emergency hysterectomy. Findings at surgery included a 20-week-sized boggy uterus with a dilated cervix and bilateral, large, multicystic ovaries consistent with theca lutein cysts. She was promptly restarted on her chemotherapy regimen postoperatively. Pathology showed an invasive hydatidiform mole with superficial myometrial invasion. The patient required a total of nine chemotherapy cycles of two different multiagent regimens to achieve complete remission. Magnetic resonance imaging revealed complete resolution of her lung metastases and theca lutein cysts. The patient has since been in surveillance for 1 year with excellent health and no evidence of disease (all hCG levels less than 2 milliinternational units/mL).
DISCUSSION The general female population has a 0.1% risk of developing gestational trophoblastic disease. Patients often present with continued vaginal bleeding after either a normal or molar pregnancy and an elevated hCG.4 In this case, the patient had compelling clinical findings of increased hCG such as her tender breasts, nausea, and vomiting; however, as a result of erroneous reliance on the negative urine pregnancy test result, the health care providers who saw the patient at initial presentation did not arrive at the diagnosis. This case highlights the need for gynecologists to be familiar with the specific type of hCG tests used in their practices so that they may recognize falsenegative urine or falsely low serum hCG test results when they occur in the presence of extremely high hCG levels such as was observed in our patient in whom the actual hGC level was over 5 million milliinternational units/mL. By increasing this awareness, the gynecologist can adjust the differential diagnoses more accurately and, in a compelling clinical situation, confirm a suspected erroneous result by dilution of the urine and serum sample. Laboratory tests for hCG have close to 100% sensitivity and specificity for trophoblast-related conditions such as pregnancy and gestational trophoblastic disease. All commercially available hCG assays are
False-Negative Pregnancy Test Due to Extremely High hCG
OBSTETRICS & GYNECOLOGY
Copyright ª by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
immunometric assays using two antibodies to distant sites on the b-subunit of the hCG molecule. A first immobilized antibody captures the hCG molecule, to which a second enzymatically or radioactively labeled tracer antibody binds forming a capture antibody-hCG-tracer antibody sandwich. All excess is then washed off and the resulting signal response is directly proportionate to the amount of hCG captured. There are several rare but critically important phenomena, which may result in false-negative urine or serum hCG results. The high-dose “hook effect” results in a falsely low value and can occur when there is an extremely high level of hCG, usually more than 500,000 milliinternational units/mL. There is so much hCG that it independently saturates the immobilized capture antibodies and the free tracer antibodies. This effectively prevents formation of the capture antibodyhCG-tracer antibody sandwich, which is needed for a positive test result, and the saturated tracer antibodies are washed away with the excess material resulting in a falsely low or negative value. The shape of the graph demonstrating the paradox of initially rising but then decreasing signal intensity with extremely high concentration of hCG has a hooklike appearance, leading to the resulting name.5 It is by dilution, which reduces the amount of hCG in the test sample, that the hook effect can be overcome and the correct hCG result obtained. The high-dose hook effect can occur in either serum or urine pregnancy tests. In our case, it occurred in both, resulting in a falsely negative urine hCG test result and a falsely low serum hCG test result. When a high-dose hook effect is suspected, it must be communicated to the laboratory and the hCG test should be repeated at a 1:1,000 dilution with which an accurate value should be obtained. In our case, once the serum dilution was accomplished, the true quantitative value was revealed and found to be consistent with gestational trophoblastic disease. The urine dilution revealed similar positive results (Fig. 1). To complicate the use of immunoassay for the qualitative detection of hCG further, there is also a variant hook effect, which more commonly affects urine hCG tests, which do not necessarily detect all hCG variants that occur with pregnancy or gestational trophoblastic disease. Such hCG variants can bind capture and tracer antibodies independently preventing sandwich formation and leading to a false-negative result. Various commercially available hCG detection kits differ in their susceptibility to the variant hook effect.6 Similar to the high-dose hook effect, the variant hook effect can also be confirmed by dilution.
VOL. 126, NO. 4, OCTOBER 2015 Cormano et al
The relevance of this hook effect to the use of pregnancy tests has been previously described for urine pregnancy tests in two case reports in the emergency medicine literature,7,8 in a clinical biochemistry journal,9 in the international gynecologic literature,10–13 and in two gynecologic oncology publications,14,15 but not in the American gynecologic literature. Consequently, few American gynecologists are familiar with the hook effect or how it can mislead the differential diagnosis, as is illustrated by our patient and the subsequent treatment delay she incurred. Our patient’s uterine tumor burden was significantly larger by the time the correct diagnosis was made. However, because she underwent hysterectomy, with which the bulk of her disease was resected, it remains unclear whether that delay in diagnosis ultimately prolonged the duration for which she required chemotherapy. Although performance of a serum hCG test may be a good first step when a false-negative urine test result is suspected, our case underscores the importance of dilution of the urine and serum tests samples to obtain the true-positive result. REFERENCES 1. Bastian LA, Brown HL. Clinical manifestations and diagnosis of early pregnancy. Referenced from UpToDate. Available at: http:// www.uptodate.com/contents/clinical-manifestations-and-diagnosisof-early-pregnancy?source5machineLearning&search5pregnancy +test+ranages&selectedTitle51;150§ionRank52&anchor 5H9#H9. Retrieved November 2, 2014. 2. Cole LA. Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites. Clin Chem 1997;43:2233–43. 3. National Cancer Institute at the National Institutes of Health. Gestational trophoblastic disease treatment (PDQÒ). Available at: http://www.cancer.gov/cancertopics/pdq/treatment/gestationaltrophoblastic/HealthProfessional. Retrieved November 2, 2014. 4. Soper J. Gestational trophoblastic disease. Obstet Gynecol 2006;108:176–87. 5. Griffey RT, Trent CJ, Bavolek RA, Keeperman JB, Sampson C, Poirier RF. ‘Hook-like effect’ causes false negative point-of-care pregnancy testing in emergency patients. J Emerg Med 2013; 44:155–60. 6. Nerenz RD, Song H, Gronowski AM. Screening method to evaluate point-of-care human chorionic gonadotropin (hCG) devices for susceptibility to the hook effect by hCG b core fragment: evaluation of 11 devices. Clin Chem 2014;60: 667–74. 7. Mundangepfupfu T, Waseem M. Partial hydatidiform mole with false-negative urine human chorionic gonadotropin test in the emergency department. J Emerg Med 2014;46:348–50. 8. Davidson CM, Kaplan RM, Wenig LN, Burmeister D. Qualitative b-hCG urine assays may be misleading in the presence of molar pregnancy: a case report. J Emerg Med 2004;27:43–7. 9. Wilgen U, Pretorius CJ, Gous RS, Martin C, Hale VJ, Ungerer JP. Hook effect in Abbott i-STAT b-human chorionic point of care assay. Clin Biochem 2014;47:1320–2.
False-Negative Pregnancy Test Due to Extremely High hCG
Copyright ª by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
813
10. Pang YP, Rayesh H, Tan LK. Molar pregnancy with false negative urine hCG: the hook effect. Singapore Med J 2010;51: e58–61. 11. O’Reilly SM, Rustin GJS. Mismanagement of choriocarcinoma due to a false low HCG measurement. Int J Gynecol Cancer 1993;3:186–8. 12. Levavi H, Neri A, Bar J, Regev D, Nordenberg J, Ovadia J. ‘Hook effect’ in complete hydatidiform molar pregnancy: a falsely low level of b-hCG. Obstet Gynecol 1993;82(suppl): 720–1.
13. Flam F, Hambraeus-Jonzon K, Hansson LO, Kjaeldgaard A. Hydatidiform mole with non-metastatic pulmonary complications and false low level of hCG. Eur J Obstet Gynecol Reprod Biol 1998;77:235–7. 14. Wheeler CA, Davis S, Degefu S, Thorneycroft IH, O’Quinn AG. Ovarian choriocarcinoma: a difficult diagnosis of an unusual tumor and a review of the hook effect. Obstet Gynecol 1990;75:547–9. 15. Nodler JL, Kim KH, Alvarez RD. Abnormally low hCG in a complete hydatidiform molar pregnancy: the hook effect. Gynecol Oncol Rep 2011;1:6–7.
Earn CME Credits for Your Contribution as an Author to Obstetrics & Gynecology In recognition of their time, effort, and expertise, authors of manuscripts for Obstetrics & Gynecology are eligible to receive continuing medical education credits. ACCME Accreditation The American College of Obstetricians and Gynecologists is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. AMA PRA Category 1 Credit(s)™ The American College of Obstetricians and Gynecologists designates this journal-based CME activity for a maximum of 10 AMA PRA Category 1 Credits.™ Physicians should claim only the credit commensurate with the extent of their participation in the activity. College Cognate Credit(s) The American College of Obstetricians and Gynecologists designates this journal-based CME activity for a maximum of 10 Category 1 College Cognate Credits. The College has a reciprocity agreement with the AMA that allows AMA PRA Category 1 Credits™ to be equivalent to College Cognate Credits. First and second authors of articles are eligible to receive 10 AMA PRA Category 1 Credits™ per article for one article per year. Authors should submit a title page to the respective group that will be responsible for providing credits (American Collegeof Obstetricians and Gynecologists or American Medical Association). rev 11/2014
814
Cormano et al
False-Negative Pregnancy Test Due to Extremely High hCG
OBSTETRICS & GYNECOLOGY
Copyright ª by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
