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Year : 2014  |  Volume : 3  |  Issue : 1  |  Page : 5-7

High-dose hook effect

1 Department of Medical Endocrinology and Metabolism, Narayana Medical College and Superspeciality Hospital, Nellore, Andhra Pradesh, India
2 Department of Medicine, Sri Venkateswara Medical College, Tirupati, Andhra Pradesh, India

Date of Web Publication10-Mar-2014

Correspondence Address:
Rajendra Prasad Namburi
Narayana Medical College and Superspeciality Hospital, Nellore, Andhra Pradesh - 524 003
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2277-8632.128412

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High-dose effect is a common phenomenon documented in various immunological and serological assays, which results in false-negative results. Due to limited amounts of reagent antibodies in the coated wells and extremely high concentrations of analyte in the sample leads to decrease in the formation of antigen-antibody complex and ultimately resulting in false low values of the analyte. High-dose hook effect has important medical implications, and simple methods of sample dilution helps in preventing false low concentrations.

Keywords: Antigen excess, dilution analysis, hook effect

How to cite this article:
Namburi RP, Kancherla V, Ponnala AR. High-dose hook effect. J NTR Univ Health Sci 2014;3:5-7

How to cite this URL:
Namburi RP, Kancherla V, Ponnala AR. High-dose hook effect. J NTR Univ Health Sci [serial online] 2014 [cited 2022 Dec 8];3:5-7. Available from: https://www.jdrntruhs.org/text.asp?2014/3/1/5/128412

  Introduction Top

The high-dose hook effect (pro-zone) describes a low measurement of analytes, which are otherwise present in very high concentration in the specimen. The hook effect is common phenomenon in day-to-day work of a clinical laboratory and should not be neglected. More than 60 years ago, the phenomenon of high-dose hook effect had been documented in serological assays, which resulted in false-negative results. [1]

Ideally, as concentrations of analyte in plasma or serum increase, the response from sandwich immunoassays increases as well. The increase in signal should be linear with concentration of the analyte. However, as the concentration of analyte increases above a certain point, the system gets saturated and the signal begins to decline, the plot of which resembles a 'fish-hook' [Figure 1]. As a result, this phenomenon earned the name "high-dose hook effect." Theoretically, this issue is only applicable to sandwich immunometric assays without a wash step between reagent additions. But, in all sandwich assays, the signal begins to plateau with high concentrations of analyte due to limiting amounts of reagent antibodies and rare samples with extremely high concentrations of analyte can even lead to the hook effect in assays with a wash step.
Figure 1: The hook effect: An excessive quantity of analyte exceeding the binding capacity of the capture antibody

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High-dose hook effect still remains a problem in many assay techniques like one-step immunometric assays, [2],[3],[4],[5],[6],[7],[8],[9] immunoturbidimetric assays, [10] and immunonephelometric assays. [11] The first description of the hook effect in the literature was made by Miles et al., [12] in the early 1970s with a two-site immunoradiometric assays (IRMA) for ferritin. It was later described when hormones were being oversecreted and measured using IRMA. Common analytes subjected to this high-dose hook effect are shown in [Table 1].

Table 1: Common analytes subjected to hook effect

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Published reports document the prevalence of hook effect in immunoassays to be between 0.2 and 2%. [25] In India, the laboratory methods commonly followed are immunometric and enzyme-linked immunosorbent assays where the prevalence of hook effect is more. However, the prevalence of hook effect in India is poorly documented. With the introduction of newer assay techniques like chemiluminescence, high-dose hook effect has only occasionally been observed. [26],[27]

With the wide usage of pregnancy tests in home, emergency departments, and laboratories, false-negative pregnancy tests were reported in ectopic pregnancy, twin pregnancy, triplets, cancer, and gestational trophoblastic diseases. [28],[29] Negative or inconclusive results in patients suspected of pregnancy should be further evaluated by serum quantification of hCG and ultrasonography. [11] Yadav et al.[30] reported a case of false negative qualitative hCG assay with urine pregnancy test kits (a one-step point-of-care), which are widely used at home and laboratories. This false-negative assay was due to "high-dose hook effect," which results in delayed diagnosis and subsequent management.

As with all diagnostic tests, a definitive clinical diagnosis should not be based on the results of a single test, but should only be made after pooling together all relevant clinical and laboratory findings. False-negative results, even if they are extremely rare, may mislead or result in a delayed diagnosis [29] and improper follow up or could have potential medical implications following mismanagement. Also, clinicians should understand the possibility of inaccurate results, and women should be notified of the potential for false-negative or false-positive results wherever possible. [29]

  High-Dose Hook Effect: Its Importance Top

Hook effect is a frequent event that is notoriously difficult to detect in the clinical laboratory. Reporting of an erroneous result can have serious medical implications, and sample pooling is a simple method for detecting falsely low concentrations attributable to the hook effect. Although this screening approach increases reagent costs by 10% and involves additional labor to prepare and analyze pools, it is considerably more cost-effective than analyzing all samples undiluted and after dilution, which doubles reagent costs. Furthermore, this simple hook effect detection method can be adapted to other nephelometric assays with the potential for erroneous results from antigen excess.

  Protocols for Detection of Hook Effect Top

Samples are often tested undiluted and after dilution to detect the hook effect. [9] If the result on dilution is higher than for the undiluted sample, then the undiluted sample most likely exhibited the hook effect. However, this approach increases labor and reagent costs for assays that may only rarely encounter extremely high analyte concentrations. This method prevents the reporting of falsely low results but incurs substantial time and expense.

An alternative approach involves pooling patient samples and measuring the pool and a 10-fold dilution of the pool. [31] Cole et al. reported the effectiveness of this approach, which is simple, inexpensive that uses pooled sample to detect hook effect. [31] In this method, they recommended batching patient samples in groups of 10, forming a pooled sample with each sample diluted 10-fold by the other samples in the batch. In addition, the pooled sample is diluted 10-fold, producing a 100-fold final dilution. "Hook" samples produce a higher result for the 100-fold dilution pool than the 10-fold dilution pool. Each of the 10 samples must then be re-analyzed at a higher dilution to detect the out-of-range result. If one or more of the samples in the pool is falsely low because of the hook effect, then the results from the undiluted and diluted pools (after correcting for the 10-fold dilution) will differ significantly. [31]

In conclusion, high-dose hook effect has serious medical implications, and sample dilution is a simple method for detecting falsely low concentrations. Although modern assay methods have much improved reliability, physicians should still be aware of the potential for false-low due to the high-dose hook effect. In countries like India, screening for all samples increase reagent costs by two-fold and involves labor to prepare and analyze pools. Therefore, clinicians should understand and address the possibility of inaccurate results and order for simple sample dilution technique where possible.

  References Top

1.Landsteiner K. The specificity of serological reactions. Cambridge, MA: Harvard University Press; 1946. p. 240-52.  Back to cited text no. 1
2.Brensing AK, Dahlmann N, Entzian W, Bidlingmaier F, Klingmuler D. Underestimation of LH and FSH hormone concentrations in a patient with a gonadotropin secreting tumor: The high dose "hook effect" as a methodological and clinical problem. Horm Metab Res 1989;21:697-8.  Back to cited text no. 2
3.Haller BL, Fuller KA, Brown WS, Koenig JW, Evelend BJ, Scott MG. Two automated prolactin immunoassays evaluated with demonstration of a high-dose "hook effect" in one. Clin Chem 1992;38:437-8.  Back to cited text no. 3
4.Petakov MS, Damjanovic SS, Nikolic-Durovic MM, Dragojlovic ZL, Obradovic S, Gilgorovic MS, et al. Pituitary adenomas secreting large amounts of prolactin may give false low values in immunoradiometric assays. The hook effect. J Endocrinol Invest 1998;21:184-8.  Back to cited text no. 4
5.Flam F, Hambraeus-Jonzon K, Hansson LO, Kjaeldgaard A. Hydatidiform mole with nonmetastatic pulmonary complications and a false low level of hCG. Eur J Obstet Gynecol Reprod Biol 1998;77:235-7.  Back to cited text no. 5
6.Zweig MH, Csako G. High-dose hook effect in a two site IRMA for measuring thyrotropin. Ann Clin Biochem 1990;27:494-5.  Back to cited text no. 6
7.Vaidya HC, Wolf BA, Garrett N, Catalona WJ, Clayman RV, Nahm MH. Extremely high values of prostate-specific antigen in patients with adenocarcinoma of the prostate; demonstration of the "hook effect". Clin Chem 1988;34:2175-7.  Back to cited text no. 7
8.Pesce MA. "High-dose hook effect" with the Centocar CA 125 assay. Clin Chem 1993;39:1347.  Back to cited text no. 8
9.Saryan JA, Garrett PE, Kurtz SR. Failure to detect extremely high levels of serum IgE with an immunoradiometric assay. Ann Allergy 1989;63:322-4.   Back to cited text no. 9
10.Jury DR, Mikkelsen DJ, Dunn PJ. Prozone effect and the turbidimetric measurement of albumin in urine. Clin Chem 1990;36:1518-9.   Back to cited text no. 10
11.Van Lente F. Light scattering immunoassays. In: Rose NR, de Macario EC, Folds JD, Lane HC, Nakamura RM, editors. Manual of clinical laboratory immunology. 5 th ed. Washington, DC: ASM Press; 1997. p. 13-9.  Back to cited text no. 11
12.Miles LE, Lipschitz DA, Bieber CP, Cook JD. Measurement of serum ferritin by a 2-site immunoradiometric assay. Anal Biochem 1974;61:209-24.  Back to cited text no. 12
13.Barkan AL, Chandler WF. Giant pituitary prolactinoma with falsely low serum prolactin: The pitfall of the "high-dose hook effect:" Case report. Neurosurgery 1998;42:913-5.  Back to cited text no. 13
14.Comtois R, Robert F, Hardy J. Immunoradiometric assays may miss high prolactin levels. Ann Intern Med 1993;119:173.  Back to cited text no. 14
15.Delgrange E, de Hertogh R, Vankrieken L, Maiter D. Potential hook effect in prolactin assay in patients with giant prolactinoma. Clin Endocrinol (Oxf) 1996;45:506-7.  Back to cited text no. 15
16.Frieze TW, Mong DP, Koops MK. "Hook effect" in prolactinomas: Case report and review of literature. Endocr Pract 2002;8:296-303.  Back to cited text no. 16
17.Pakzaban P. Giant prolactinoma and hook effect. Neurology 2000;55:1415-6.  Back to cited text no. 17
18.Killeen AA, Ramey ML, Dean JJ. High-dose hook effect in an immunoluminometric thyrotropin assay: The open-faced sandwich artefact. Ann Clin Biochem 1993;30:413-4.   Back to cited text no. 18
19.Garcia-Webb P, Watson FE, Whiteside N. High-dose "hook" effect in measurement of somatotropin by two-site immunoradiometric assay. Clin Chem 1986;32:2102.  Back to cited text no. 19
20.Leboeuf R, Langlois MF, Martin M, Ahnadi CE, Fink GD. "Hook Effect" in Calcitonin Immunoradiometric Assay in Patients with Metastatic Medullary Thyroid Carcinoma: Case Report and Review of the Literature. J Clin Endocrinol Metab 2006;9:361-4.  Back to cited text no. 20
21.Güran T, Yeþil G, Güran Ö, Cesur S, Bosnalý O, Celayir A, et al . A giant ovarian cyst in a neonate with classical 21-hydroxylase deficiency with very high testosterone levels demonstrating a High-Dose Hook Effect. J Clin Res Pediatr Endocrinol 2012;4:151-3.   Back to cited text no. 21
22.Levavi H, Neri A, Bar J, Regev D, Nordenberg J, Ovadia J. "Hook effect" in complete hydatidiform molar pregnancy: A falsely low level of β-HCG. Obstet Gynecol 1993;82:720-1.   Back to cited text no. 22
23.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.  Back to cited text no. 23
24.Dahlmann N, Brensing KA, Klingmuller D, Bidlingmaier F. "Hookeffect" in a patient with a gonadotropin-secreting tumor. Clin Chem 1990;36:168.   Back to cited text no. 24
25.Musher DM, Hamill RJ, Baughn RE. Effect of human immunodeficiency virus (HIV) infection on the course of syphilis and on the response to treatment. Ann Intern Med 1990;113:872-81.  Back to cited text no. 25
26.Unnikrishnan AG, Rajaratnam S, Seshadri MS, Kanagasapabathy AS, Stephen DC. The 'hook effect' on serum prolactin estimation in a patient with macroprolactinoma. Neurol India 2001;49:78-80.  Back to cited text no. 26
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27.Agarwal M, Ananya D, Singh AS. High-dose hook effect in prolactin macroadenomas: A diagnostic concern. J Hum Reprod Sci 2010;3:160-1.  Back to cited text no. 27
[PUBMED]  Medknow Journal  
28.Hunter CL, Ladde J. Molar pregnancy with false negative β-hCG urine in the emergency department. West J Emerg Med 2011;12:213-5.  Back to cited text no. 28
29.Tabas JA, Strehlow M, Isaacs E. A false negative pregnancy test with a hydatidi form molar pregnancy. New Engl J Med 2003;349:2172-3.  Back to cited text no. 29
30.Yadav YK, Fatima U, Dogra S, Kaushik A. Beware of "hook effect" giving false negative pregnancy test on point-of-care kits. J Postgrad Med 2013;59:153-4.  Back to cited text no. 30
[PUBMED]  Medknow Journal  
31.Cole TG, Johnson D, Eveland BJ, Nahm MH. Cost-effective method for detection of "hook effect" in tumor marker immuometric assays. Clin Chem 1993;39:695-6.  Back to cited text no. 31


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  [Table 1]

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