Beware of "hook effect" giving false negative pregnancy test on point-of-care kits

YK Yadav¹, U Fatima¹, S Dogra², A Kaushik²,  
¹ Department of Pathology, Integral Institute of Medical Sciences and Research, Lucknow, Uttar Pradesh, India
² Safdarjung Hospital and Vardhman Mahavir Medical College, New Delhi, India

Correspondence Address:
Y K Yadav
Department of Pathology, Integral Institute of Medical Sciences and Research, Lucknow, Uttar Pradesh
India

Full Text

Urine pregnancy test kits, a one step point-of-care (POC) are widely used both at home and in laboratories. We report in this paper, a case of false-negative qualitative human chorionic gonadotropin (hCG) assay which was caused by the "high-dose hook effect" leading to delay in diagnosis and subsequent management.

A 30-year-old gravida 3 para 2, female with 16 weeks amenorrhea, presented to the emergency department with vaginal spotting, lower abdominal pain, nausea, and vomiting. On examination, her heart rate and blood pressure were elevated. The abdomen was diffusely tender. Pelvic examination revealed a closed cervical os and an 18-week sized uterus with adenexal tenderness. The complete blood count and biochemical profile were within normal limits. Results of urine tests for the beta subunit of human chorionic gonadotropin (β-hCG) (tested with one step pregnancy test device by Abon Biopharm Company, China, sensitivity of 25 milli-international units per milliliter (mIU/ml) for β-hCG) was negative. An ultrasonography revealed an enlarged uterus with a heterogeneous mass with multiple cystic areas measuring 10.6 cm × 6.2 cm × 6.4 cm in the corpus. A differential diagnosis of vesicular mole and fibroid with degeneration was suggested. Subsequently, on clinical suspicion the test for β-hCG was performed after a 1:10 dilution and it showed weak positivity. On increasing the dilution up to 1:100, the test was strongly positive [Figure 1]a. Quantitative assay after multiple dilutions and repeated testing of the serum showed a β-hCG level of 1,430,000 mIU/ml (normal <5-200,000 mIU/ml). Thus a final diagnosis of molar pregnancy was made. The patient immediately underwent dilation and curettage. Histopathological examination confirmed a complete hydatidiform mole [Figure 1]b.

Gestational trophoblastic disease (GTT) demonstrates marked geographic and ethnic differences, with the highest incidence in Southeast Asia. The rates are 12 per 1,000 pregnancies in India, Indonesia, and Turkey. [1] There have been reports of false-negative urine, serum, and both urine and serum β-hCG pregnancy tests in hydatidiform mole. [2],[3] The different screening methods including different kits for home pregnancy tests based on chromatographic sandwich immunoassays in which two antibodies directed to different portions (the α and β subunits) of the hCG molecule "sandwich", a single antigen to produce a color change.{Figure 1}

Falsely low or 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. [4] The signal increases progressively until the hormone concentration exceeds the binding capacity. Subsequently, instead of a proportional response to the increase in hCG concentration, the output signal decreases creating a fish hook effect [Figure 2] which happened in our patient.

The hook effect can be overcome by dilution as described. [4] Apart from β-hCG, other immunoassays in which high dose hook effect has been documented include prolactin, immunoglobulin E (IgE), ferritin, thyrotropin, prostate-specific antigen, albumin, and tumor markers (e.g., cancer antigen (CA-125), α-fetoprotein (AFP)). [5] In India as the incidence of gestational trophoblastic disease among the highest in world. Thus, physicians should always be aware of false-negative urinary and serum β-hCG due to the "high dose hook effect", as this will delay diagnosis and can precipitate complications.{Figure 2}

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