|Year : 2015 | Volume
| Issue : 2 | Page : 112-115
False-negative urine human chorionic gonadotropin in molar pregnancy: " The high-dose hook effect" !
Sujata Narendra Datti, Payel Ray, Jayashree Ashok Kumar Dharmavijaya
Department of OBG, MVJ Medical College and Research Hospital, Bengaluru, India
|Date of Web Publication||14-May-2015|
329, 8th Main, HAL 3rd Stage, Bengaluru - 560 075
Source of Support: None, Conflict of Interest: None
Failure to detect pregnancy in the emergency situations can have important consequences. These include missing of ectopic pregnancy (the leading cause of first-trimester pregnancy-related maternal death), administration of medications contraindicated in pregnancy, fetal radiation exposure, and medico legal problems. This in turn has led to the dictum to check for pregnancy in all women of child-bearing age group. Urine pregnancy (human chorionic gonadotropin [hCG]) test is the commonly used test to rule out pregnancy and has been reported by Griffey et al. in their study to achieve 100% sensitivity and 99.2% specificity in a clinical setting, resulting in a positive predictive value of 98.3% and a negative predictive value of nearly 100%. However, the sensitivity is influenced not only by the quantity of β hCG but on its variants that vary with different weeks of pregnancy. β hCG is present in several variant forms that change in their concentrations at different stages of pregnancy. In spite of its high sensitivity, in the presence of molar pregnancy that is associated with very high levels of β hCG it fails to detect the antigen (β hCG). This is explained by the phenomenon known as "high-dose hook effect" which further leads to delay in diagnosis and treatment. This can be overcome by dilution of the sample. In such cases, diagnosis will be made by serum β hCG and ultrasound (USG). Here, we present a case of gravida 2 para 1 living 1 with 2 months amenorrhea with bleeding p/v and pain abdomen of 20 days duration whose urine β hCG was repeatedly negative and diagnosis was made by serum β hCG and USG.
Keywords: False-negative urine β human chorionic gonadotropin, hook effect, molar pregnancy
|How to cite this article:|
Datti SN, Ray P, Dharmavijaya JA. False-negative urine human chorionic gonadotropin in molar pregnancy: " The high-dose hook effect" !. J Sci Soc 2015;42:112-5
|How to cite this URL:|
Datti SN, Ray P, Dharmavijaya JA. False-negative urine human chorionic gonadotropin in molar pregnancy: " The high-dose hook effect" !. J Sci Soc [serial online] 2015 [cited 2020 Dec 4];42:112-5. Available from: https://www.jscisociety.com/text.asp?2015/42/2/112/157049
| Introduction|| |
Gestational trophoblastic disease (GTD) encompasses a spectrum of tumors, including complete and partial hydatidiform mole (molar pregnancy) and locally invasive or disseminated choriocarcinoma. Complete hydatidiform mole produces characteristic clinical features, including vaginal bleeding and uterine size beyond expected gestational age.  Thus, in addition to a complete physical and pelvic examination, complete blood count, blood chemistry and pelvic ultrasound sonography (USG), a hallmark of diagnosing hydatidiform mole is a positive β human chorionic gonadotropin (hCG) assay serum and urine tests. Interestingly, sandwich chromatographic immunoassays, such as qualitative β hCG assays, produce false-negative results in the presence of excessively high antigen concentrations as in molar pregnancy due to a phenomenon known as the "high-dose hook effect."  However, in the literature, descriptions of the hook effect are rare in cases of GTDs.  Hence, we report this case of gravida 2 para 1 living 1 with molar pregnancy with false-negative urine β hCG, which lead to delay in the diagnosis and institution of appropriate treatment.
| Case report|| |
A 24-year-lady, gravida 2 para 1 living 1 with previous one normal delivery 1½ years back, came with amenorrhea of 2½½ months followed by bleeding per vaginum of 20 days duration changing 3 pads/day, with intermittent pain abdomen in the right iliac fossa, history of mass per abdomen of same duration. No history of passing vesicles per vaginum. Her previous cycles were regular with LMP-12.9.13. On examination pallor was present, pulse-84/min, blood pressure-120/70 mmHg respiratory rate-18 cycles/min. On palpation uterus corresponded to 24 weeks size with tenderness in the right iliac fossa, external ballotment absent. On per speculum examination bleeding through os was present. On per vaginal examination uterus corresponded to 24 weeks of gestation with fullness felt in all fornices and parous os. Impression: Gravida 2 para 1 living 1 with 24 weeks of gestation with suspected molar pregnancy.
Hemoglobin-8.8 g/dl, packed cell volume-26.2%, blood group and Rh typing-A+ve, total leukocyte count-8000/cu mm, erythrocyte sedimentation rate-76 mm/1 st h, liver function test and renal function test were normal.
Urine pregnancy test (hCG) was negative. Technical error was suspected and repeated still result was negative by then molar pregnancy was diagnosed based on USG report. Later tests were repeated with dilution of the sample and positive result obtained with 1 in 10 dilution. USG showed uterus enlarged 5.5 cm × 15.8 cm × 8.4 cm with the endometrial cavity demonstrating mixed echogenic lesion with tiny cystic foci. Impression: Complete molar pregnancy with bilateral multiple theca lutein cysts with largest being 3.4 cm × 3 cm in right side and 3.9 cm × 2.2 cm in the left ovary. Serum β hCG-15,000 IU/L. Dilution was not done because of financial constrain. Chest X-ray was found to be normal.
With adequate blood, patient was taken for suction evacuation under general anesthesia. Almost 2750 ml of vesicular mole along with blood and blood clots were removed [Figure 1]. Two units of blood were transfused.
Histopathological examination report: Complete molar pregnancy
Histopathological examination shows trophoblastic tissue [Figure 2]. It confirms complete molar pregnancy. Serum β hCG done after 1-week-15,000 IU/L. Thinking of incomplete evacuation and USG was repeated. USG showed cavity empty with bilateral large theca lutein cysts. Chest X-ray was normal. This again can be explained by hook effect leading to false low serum β hCG. Dilution is the key to accurate testing. Subsequent reports showed declining trend of β hCG. Patient was discharged, with advice for weekly serum β hCG till it returned to normal. Contraceptive advice during this period was given. She was also advised to visit doctor, if the levels were plateauing or increasing or if she became symptomatic. At 10 weeks, postevacuation patient was asymptomatic and repeat serum β hCG was 321 IU/L. On further follow-up at 12 weeks β hCG was 3.7 IU/L.
| Discussion|| |
Gestational trophoblastic disease demonstrates marked geographic and ethnic differences, with the highest incidence in Southeast Asia. The rates are 12/1000 pregnancies in India, Indonesia, and Turkey. It is an uncommon cause of abdominal pain and vaginal bleeding that may lead to serious disseminated disease and death if left untreated. It is most commonly associated with pregnancy in the early (15-20 years old) and late (>35 years old) reproductive periods.  β hCG is commonly used as the marker of GTD. It is detected by urine pregnancy test and serum β hCG.
Urine pregnancy tests are widely used in emergency departments as the first screening test for patients of reproductive age presenting with gynecological problems in order to rule out pregnancy.  False-negative results can occur when an extremely high level of substrate overwhelms the assay system. If the concentration of the antigen is sufficiently high to saturate, both the solid migratory phase and fixed detection antibodies independently, it prevents the same molecule from binding the two antibodies and forming a "sandwich." This is so called "high-dose hook effect," which prevents the formation of color change and leads to a false-negative test. This can be overcome by dilution of the sample. The hook effect was first described by Miles et al in 1976 and is more commonly seen in assays for prolactin and thyroid stimulating hormone.
The β hCG is also present in several variant forms that change in their concentrations at different stages of pregnancy. Some assays include an antibody that does not recognize certain variants present in later stages of pregnancy. The β subunit exists in several forms, including hyperglycosylated hCG (H-hCG), nicked hCG, free β subunit, the core fragment of β hCG (hCG-βcf), and others. The relative fractional concentrations of these forms change throughout pregnancy. For example, H-hCG is the primary, if not sole form of hCG produced after implantation, and accounts for up to as much as 60% of β hCG found at 4 weeks, dropping to <5% in the second and third trimesters. By contrast, hCG-βcf is high in mid-pregnancy urine. Some assays may include an antibody that does not recognize certain variants present in later stages of pregnancy. When this variant is in excess, it can bind one antibody avidly and the other not at all, resulting in a false-negative test (hook-like phenomenon).  Because of the difference in antibody specificity in various commercial automated immunoassays of hCG, discordant results may be obtained by laboratories using different hCG assays, with a falsely low or negative result obtained if the assay does not recognize the hCG variants produced from the trophoblastic tissue. 
Complete hydatidiform moles present with abnormally high levels of hCG; >40% of patients have levels >100,000.  Serum hCG may be misreported in cases of extremely high levels due to the "hook effect," where falsely low or negative results occur from oversaturation of the signaling antibodies employed to detect hCG by the testing equipment. Serum testing is performed using two antibodies to the β subunit of hCG molecules. When hCG is present, it is immobilized by a capture antibody, and labeled by a tracer antibody, resulting in an immobilized antibody-hCG-tracer sandwich. When hCG levels are high, both the capture and tracer antibodies saturate, and the signal response is decreased. The "hook effect" occurs when nonsandwiched tracer antibodies are washed away with the excess material resulting in a falsely low or negative test that happened in our patient.  In most of the kits, this effect occurs when concentration >1,000,000 IU/L. However, in the literature, descriptions of the hook effect are rare in cases of GTDs. , Laboratory errors can occur with β hCG levels higher than 500,000 IU/L.
According to the manufacturer's information of the Beckman access 2 analyzer, a hook effect will occur when the serum β hCG concentration exceeds 1,000,000 IU/L. The manufacturer's information for the ACON urine hCG one step pregnancy device (format: FHC-102) does not provide any information about a hook effect, but hook effects occurring in a qualitative urine and a quantitative serum β hCG assay have both been reported recently. However ACON laboratories state an accuracy of 99%, without any mention of sensitivity and specificity. On the basis of our findings, we suggest that, when hydatidiform mole is suspected, the urine sample should be diluted at least 1:10, particularly when using the ACON, one step pregnancy device, which is used in our hospital, to avoid inaccurate urine hCG results. However urine and serum hCG concentrations vary widely during pregnancy, with urine concentrations measuring approximately half that of the corresponding serum fractions, depending on the hydration status of the patient. Management includes suction evacuation with postevacuation follow-up. Follow-up includes baseline physical examination, pelvic examination, chest radiographs (to check for lung metastasis) and β hCG level surveillance that has to be continued till β hCG becomes normal.
| Conclusion|| |
Clinicians need to be aware of and familiarize themselves with the limitations, of the specific type of qualitative hCG tests used in their practice and also of limitations of laboratory measurements of extremely high levels of hCG, understanding that false low or negative tests may arise due a hook effect particularly in situations where clinical scenario may not correspond to false low laboratory value. Also negative or false low results in patients with a high suspicion of molar pregnancy should be further evaluated by appropriate sample dilution.
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[Figure 1], [Figure 2]