Recovery Test for the Detection of the “High-dose” Hook Effect Phenomenon Using an In-house Developed Immunoradiometric Assay for the Estimation of Serum Thyroglobulin in the Patients with Differentiated Thyroid Cancer

INTRODUCTION

Thyroglobulin (Tg) is a high-molecularweight glycoprotein (~660 kDa), synthesized exclusively by the thyroid follicular epithelial cells and secreted into the colloid of thyroid follicles. As a precursor in the biosynthesis of thyroid hormones (T3 and T4), Tg is crucial in thyroid physiology. More importantly, due to its tissue-specific expression, serum Tg has become an essential biomarker in the management of patients with Differentiated thyroid carcinoma (DTC), particularly papillary and follicular subtypes. Following total thyroidectomy and subsequent radioactive iodine ablation, serum Tg levels ideally fall to undetectable or near-zero concentrations. Therefore, the presence or reappearance of measurable Tg in the serum serves as a sensitive indicator of residual thyroid tissue, local recurrence, or distant metastatic disease, and guides ongoing clinical decision-making.^[1,2]

Despite its diagnostic and prognostic utility, accurate and reliable measurement of serum Tg remains analytically challenging. Multiple factors contribute to variability in Tg assay performance, including inter-assay differences, lack of universal calibration standards, and most notably, interference by endogenous antibodies. One of the most significant confounding factors is the presence of circulating anti Tg autoantibodies (TgAb), which occur in a substantial proportion of patients with autoimmune thyroid disease and a considerable subset of DTC patients. These autoantibodies can interfere with Tg measurement in a variety of immunoassays, mostprominently in sandwich-type immunometric assays (IMAs), leading to falsely low or undetectable Tg values [Figure 1], potentially masking disease persistence or recurrence.^[3,4] Numerous attempts have been made by the researchers to recognize the presence of interference using antibody selection, TgAb measurement, exogenous Tg recovery experiments, and disparity in Tg value by radioimmunoassay (RIA)/IMA methods.^[5]

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Figure 1:

Autoantibody interference in immunometric assays

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While alternative assay formats, such as RIA, are less susceptible to TgAb interference due to their competitive design, they are typically less sensitive, more labor-intensive, and involve the use of radioisotopes, limiting their widespread use in routine laboratories. In recent years, immunoradiometric assays (IRMAs) have emerged as a promising alternative, combining the high sensitivity of IMAs with the enhanced specificity of radioisotopic detection, allowing for improved performance in Tg measurement. However, even advanced assays such as IRMAs are not immune to technical limitations.^[1]

Another critical source of error in Tg assays, which is frequently underrecognized, is the high-dose hook (HDH) effect, also known as the prozone phenomenon [Figure 2]. This phenomenon is typically observed in one-step or sandwich-type immunoassays when extremely high concentrations of analyte (in this case, Tg) saturate both the capture and detection antibodies simultaneously. This saturation prevents the formation of the sandwich complex required for accurate signal generation, resulting in paradoxically low or even undetectable measured values. The hook effect can lead to catastrophic underestimation of Tg levels in patients with extensive tumor burden or aggressive metastatic disease, precisely the cases in which accurate quantification is most critical. Although the reported frequency of the HDH effect ranges from 0.2% to 2%, the clinical implications of missing such cases necessitate its systematic detection and exclusion.^[6]

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Figure 2:

Mechanism of “high-dose” hook effect or prozone phenomenon in immunometric assay

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Given these challenges, quality guidelines such as those issued by the National Academy of Clinical Biochemistry (NACB) recommend that Tg assays be validated not only for analytical sensitivity and precision but also for susceptibility to interference from TgAb and the HDH effect.^[7] Among the suggested strategies, sample dilution and the Tg recovery test have proven useful.^[8] The Tg recovery test involves the addition of a known quantity of exogenous Tg to patient serum and subsequent measurement of the percentage recovered by the assay. A low recovery rate can indicate the presence of TgAb interference or the HDH effect, especially when results improve upon dilution. The test can thus serve as an indirect but informative method to evaluate assay integrity in individual patient samples.

Despite improvements in assay technologies, false-negative and falsely low Tg results remain a clinical concern. These inaccuracies can delay diagnosis, mislead therapeutic decisions, or result in inappropriate reassurance to patients.^[9,10] Moreover, most commercial kits offer limited flexibility in modifying protocols to troubleshoot interferences, highlighting the value of in-house assay development where assay conditions can be customized and interference mitigation strategies implemented more rigorously.

In light of these complexities, the present study focuses on the development and validation of a recovery test protocol for the detection of the HDH effect using an in-house developed IRMA specifically optimized for serum Tg measurement in patients with DTC.^[1] Our IRMA method aims to combine the sensitivity and specificity required for accurate Tg quantification while minimizing common interferences. Through parallel assessment of TgAb interference and HDH effect, this study contributes to the broader goal of enhancing assay reliability and improving the quality of postoperative monitoring in thyroid cancer patients. The results of this study are intended to inform both clinical practice and future assay development efforts.

MATERIAL AND METHODS

RESULTS

A total of 2,389 routine patient samples were analyzed from the TLM database to assess Tg concentrations and evaluate potential assay interference using the Tg recovery test. Two main types of interference were identified: HDH effect and TgAb interference.

As shown in Figure 3, the HDH effect was detectedin 91 samples, representing 3.8% of the total cohort. In contrast, TgAb interference was identified in 24 samples (1.0%). The frequency of HDH was significantly higher than that of TgAb interference as determined by the recovery test (3.8% vs. 1.0%; P < 0.05).

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Figure 3:

Prevalence of high-dose “hook effect” using thyroglobulin recovery test in DTC patients, DTC: Differentiated thyroid carcinoma

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In cases affected by the HDH effect, undiluted serum Tg concentrations were found to range between 14 and 300 ng/ ml [Table 1]. Initially, these samples exhibited abnormally low Tg recovery values, suggesting assay interference. However, upon dilution of samples (typically 1:10) with concentrations up to 200 ng/ml, Tg concentrations increased significantly, indicating the presence of the HDH effect. Notably, while the HDH phenomenon was still observed at Tg levels exceeding 200 ng/ml, its impact on assay results was comparatively less pronounced [Figure 4].

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Figure 4:

Bar diagram illustrating the statistically significant (P < 0.05) reduction in measured thyroglobulin concentrations upon dilution in TgAb-Tg autoantibody positive samples, consistent with antibody interference

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For the TgAb-positive samples, Tg concentrations measured in diluted samples (C) were consistently and significantly lower than the corresponding undiluted values (A), even aft er multiplying with the dilution factor (D), as shown in Table 2. This pattern indicates assay interference leading to falsely low Tg readings, particularly evident upon dilution, likely due to altered antibody-to-analyte ratios affecting assay accuracy.

Clinical implications: Table 3 depicts the interpretation of the Tg recovery test results

• 80%–130% recovery is considered acceptable, indicating that the assay is performing reliably and is likely free of significant interference.

• <70% recovery strongly suggests analytical interference, most commonly due to:

• TgAb binding, which interferes with assay antibody recognition.

• HDH effect, causing falsely low Tg levels due to antigen excess saturating assay antibodies.

• >130% may indicate nonspecific signal enhancement, although less common and generally of lower clinical concern.

DISCUSSION

The clinical utility of Tg is oft en compromised by assay interferences, which can result in falsely elevated or suppressed Tg values. This interference is highly prevalent in IMAs, which is preferred for its sensitivity and user-friendly presentation.

Two major sources of Tg assay interference are the presence of TgAb and the HDH effect. Routine TgAb testing is typically performed alongside Tg assays to flag this potential interference.^[11] In contrast, the HDH effect is a less commonly discussed but equally critical analytical challenge. The standard TgAb testing does not detect the HDH effect, which may go unnoticed unless specifically evaluated by assay dilution protocols.^[12] Nevertheless, Association for Diagnostics & Laboratory Medicine. ADLM (formerly National Academy of Clinical Biochemistry (NACB)/ American Association for Clinical Chemistry (AACC)) recommends using highly sensitive Tg assays with concurrent TgAb measurement for monitoring DTC, and alternative methods like liquid chromatography– tandem mass spectrometry (LC-MS/MS) may be used in TgAb-positive cases to ensure reliable interpretation.^[13]

In this study, we evaluated the frequency and impact of these interferences on Tg measurement in a cohort of 2,389 screened routine patient samples. The results demonstrate that both HDH and TgAb interference are significant sources of variability in Tg measurement. specifically, theHDH effect was identified in 91 (3.8%) samples, whereasTgAb interference was present in 24 (1.0%) samples.

The frequency of the HDH effect was significantly higher than that of TgAb interference (P < 0.05), underscoring its importance as a common source of assay interference, highlighting the importance of recognizing and differentiating between HDH and TgAb interference. These findings are consistent with previous reports that have highlighted the HDH effect as a potential challenge in immunoassays for high-concentration analytes. Up to the present, there are no cost-effective algorithms available for the safe detection of the prozone effect.^[12]

To detect the HDH effect, two conventional strategies have been described. The first involves testing all the patient samples in both undiluted and appropriately diluted forms, observing for a nonlinear increase in Tg concentration upon dilution, which is indicative of HDH. This approach is reliable but labor-intensive and cost-prohibitive when applied to all routine samples. The second approach^[12] involves pooling multiple patient samples and comparing Tg concentrations before and aft er a 10-fold dilution.^[14] While this method is more cost-effective for large-scale screening, it lacks the specificity to identify affected individual samples and may miss clinically significant cases of interference.

We incorporated a Tg recovery assay into routine IRMA testing to simultaneously assess TgAb interference and the HDH effect. Significantly reduced recovery in TgAb-negative samples, especially with increased Tg upon 1:10 dilution, indicates HDH, whereas poor recovery (<70%) in TgAb-positive samples reflects TgAb interference, which limits accurate Tg quantification. However, our proposed Tg recovery method, which offers a comprehensive check for assay interference, is cost-effective and can be easily integrated into routine workflows without significant increases in labor or reagent costs. It allows flexible batch processing and improves efficiency. In contrast, assays for Tg antibodies, while sensitive, are expensive and require a separate setup.

Mitigating the HDH effect analytically involves assay design improvements, such as transitioning to two-step immunoassays that incorporate a wash step between sample and tracer addition to remove excess analyte before detection. In addition, emerging approaches, such as artificial neural networks trained to analyze kinetic data during immunoassay reactions, show promise for detecting anomalous signal patterns indicative of HDH interference.^[15] However, these advanced technologies may not yet be widely available or economically feasible for all clinical laboratories.

While TgAb-related discrepancies are identified when Tg and TgAb results are reviewed, HDH effects oft en remain unnoticed until inconsistent Tg trends or imaging results prompt repeat testing. Testing in a different laboratory can introduce interlaboratory and intermethod variability, complicating patient monitoring and delaying decisions. Hence, integrating Tg recovery tests into routine assays allows early detection of TgAb-and HDH-related interferences, improving diagnostic accuracy, minimizing repeat testing and interlaboratory variability, and supporting timely clinical decisions through careful review of assay results.

Finally, the in-house Tg IRMA kit, while cost-effective and convenient, has limitations, including potential batch-to-batch variability, limited standardization, suboptimal sensitivity at very low Tg levels, and possible interference from high Tg concentrations or anti-Tg antibodies. Despite these limitations, careful standardization allows the kit to reliably track serum Tg trends in a clinical setting.

CONCLUSION

The integration of the Tg recovery assay into routine clinical practice provides an effective and efficient approach for identifying assay interferences early in the testing process. This method supports timely clinical decision-making, optimizing patient outcomes by facilitating the rapid and accurate detection of disease recurrence or metastasis in DTC patients.