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. 
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.
High-dose hook effect still remains a problem in many assay techniques like one-step immunometric assays,  immunoturbidimetric assays,  and immunonephelometric assays.  The first description of the hook effect in the literature was made by Miles et al.,  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.
Published reports document the prevalence of hook effect in immunoassays to be between 0.2 and 2%.  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. 
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.  Negative or inconclusive results in patients suspected of pregnancy should be further evaluated by serum quantification of hCG and ultrasonography.  Yadav et al. 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  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. 
HIGH-DOSE HOOK EFFECT: ITS IMPORTANCE
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
Samples are often tested undiluted and after dilution to detect the hook effect.  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.  Cole et al. reported the effectiveness of this approach, which is simple, inexpensive that uses pooled sample to detect hook effect.  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. 
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.
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