Diagnostic pitfalls in assessment of ferritin following CAR-T-cell therapy: Understanding the hook effect.

The hook effect is a well-described but clinically underappreciated immunoassay interference, where a falsely lowered result is caused by analyte excess. We describe a situation in which ferritin immunoassay results from a 27-year-old female with immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome were more than 1000 times lower at a reference laboratory than those determined in-house after dilution. This case underscores the importance for clinical care providers to be aware of the impact of the hook effect on ferritin measurements, and to promptly communicate with the laboratory when there are discrepancies between clinical symptoms and test results.

Diagnosis of hyperprolactinemia in women: A Position Statement from the Brazilian Federation of Gynecology and Obstetrics Associations (Febrasgo) and the Brazilian Society of Endocrinology and Metabolism (SBEM).

Hyperprolactinemia is a frequent cause of menstrual irregularity, galactorrhea, hypogonadism, and infertility. The most common etiologies of hyperprolactinemia can be classified as physiological, pharmacological, and pathological. Among pathological conditions, it is essential to distinguish prolactinomas from other tumors and pituitary lesions presenting with hyperprolactinemia due to pituitary stalk disconnection. Proper investigation considering clinical data, laboratory tests, and, if necessary, imaging evaluation, is important to identify the correctcause of hyperprolactinemia and manage the patient properly. This position statement by the Brazilian Federation of Gynecology and Obstetrics Associations (Febrasgo) and Brazilian Societyof Endocrinology and Metabolism (SBEM) addresses the recommendations for measurement of serum prolactin levels and the investigations of symptomatic and asymptomatic hyperprolactinemia and medication-induced hyperprolactinemia in women.

Hook-effect in MAGLUMI immunoassay for serum anti-GAD antibodies in neurological disorders: When "wrong" matrix is the right choice.

Antibodies against glutamic acid decarboxylase (anti-GAD) are a valuable diagnostic tool to detect severe autoimmune conditions as type 1 diabetes mellitus (T1DM) and anti-GAD related neurological disorders, having the latter more often anti-GAD concentrations in serum multiple times higher than in the former. Automated immunoassays, either with ELISA or chemiluminescent technology, are validated for diagnostic use in serum with analytical ranges suitable for T1DM diagnosis. In a patient presenting with a suspected autoimmune ataxia, anti-GAD testing on an automated chemiluminescent immunoassay (CLIA) resulted in slightly abnormal concentrations in serum (39.2 KIU/L) and very high concentrations in CSF (>280 KIU/L), thus prompting to proceed to serum dilutions to exclude a false negative result and a misdiagnosis. Different dilutions of serum resulted in nonlinear concentrations with endpoint result of 276,500 KIU/L at dilution 1:1000. CSF dilution was instead linear with endpoint result of 4050 KIU/L. In this case report we found that anti-GAD testing in CSF was essential to establish the clinical diagnosis and to suspect hook-effect in serum due to the excess of autoantibodies in this severe autoimmune condition.

Single monoclonal spike characterized as double monoclonal gammopathy in a patient with multiple myeloma: A rare finding.

Multiple myeloma (MM) is associated with the secretion of a unique monoclonal protein (M-protein) due to overproduction of immunoglobulin (Ig) by a clone of abnormally proliferating plasma cells. However, in 4% of the cases more than one M-protein can be found. This category of gammopathies is called "double monoclonal gammopathies." Here, we present a rare case of MM with double monoclonal gammopathy, where the presence of both M-proteins was observed in the single sharp peak on capillary zone electrophoresis (CZE). Further the interference of Hook effect is also discussed. Double monoclonal gammopathies need to be identified to increase diagnostic accuracy and reliability, and to get a better understanding of the disease pathogenesis and progression.

Double trouble: Unmasking two hook effects on Siemens Atellica® - Total PSA and total hCG assays.

The "hook effect" or "prozone phenomenon" occurs when the concentration of a particular analyte saturates the antibodies used in the test, resulting in falsely low or negative results despite the presence of high analyte concentrations. We report two recent cases of hook effect encountered with a widely used immunoassay analyzer, the Siemens Atellica® IM1600. The first case involves a patient with advanced metastatic prostate cancer whose total PSA (tPSA) concentration dropped dramatically from his last biological control. The second case concerns a pregnant woman whose total HCG (ThCG) levels were also subject to the hook effect and who was found to have a molar pregnancy. In both cases, a dilution step enabled to overcome this analytical concern and to obtain a correct result. In addition, a comparison of the sensitivity of different immunoassay analyzers to this phenomenon was carried out. To avoid this analytical error, an additional dilution step should automatically be performed when there is a clinical suspicion of elevated levels of tumor or hormone markers. Finally, the most affected manufacturers should adapt their assays, accordingly.

Diagnosis of hyperprolactinemia in women: A Position Statement from the Brazilian Federation of Gynecology and Obstetrics Associations (Febrasgo) and the Brazilian Society of Endocrinology and Metabolism (SBEM)

ABSTRACT Hyperprolactinemia is a frequent cause of menstrual irregularity, galactorrhea, hypogonadism, and infertility. The most common etiologies of hyperprolactinemia can be classified as physiological, pharmacological, and pathological. Among pathological conditions, it is essential to distinguish prolactinomas from other tumors and pituitary lesions presenting with hyperprolactinemia due to pituitary stalk disconnection. Proper investigation considering clinical data, laboratory tests, and, if necessary, imaging evaluation, is important to identify the correct cause of hyperprolactinemia and manage the patient properly. This position statement by the Brazilian Federation of Gynecology and Obstetrics Associations (Febrasgo) and Brazilian Society of Endocrinology and Metabolism (SBEM) addresses the recommendations for measurement of serum prolactin levels and the investigations of symptomatic and asymptomatic hyperprolactinemia and medication-induced hyperprolactinemia in women.

Experimental design for the development of a multiplex antigen lateral flow immunoassay detecting the Southern African Territory (SAT) serotypes of foot-and-mouth disease virus.

Antigenic lateral flow immunoassays (LFIAs) rely on the non-competitive sandwich format, including a detection (labelled) antibody and a capture antibody immobilised onto the analytical membrane. When the same antibody is used for the capture and the detection (single epitope immunoassay), the saturation of analyte epitopes by the probe compromises the capture and lowers the sensitivity. Hence, several factors, including the amount of the probe, the antibody-to-label ratio, and the contact time between the probe and the analyte before reaching the capture antibody, must be adjusted. We explored different designs of experiments (full-factorial, optimal, sub-optimal models) to optimise a multiplex sandwich-type LFIA for the diagnosis and serotyping of two Southern African Territory (SAT) serotypes of the foot-and-mouth disease virus, and to evaluate the reduction of the number of experiments in the development. Both assays employed single epitope sandwich, so most influencing variables on the sensitivity were studied and individuated. We upgraded a previous device increasing the sensitivity by a factor of two and reached the visual limit of detection of 103.7 and 104.0 (TCID/mL) for SAT 1 and SAT 2, respectively. The positioning of the capture region along the LFIA strip was the most influent variable to increase the detectability. Furthermore, we confirmed that the 13-optimal DoE was the most convenient approach for designing the device.

The Hook Effect: A Case Study of a Giant Invasive Prolactinoma With Falsely Low Serum Prolactin.

Prolactinomas are benign pituitary tumors also known as prolactin-secreting adenomas (PSA). These tumors cause excessive secretion of prolactin (hyperprolactinemia), a hormone responsible for lactation. Diagnosing hyperprolactinemia relies on measuring prolactin levels in the blood, and elevated serum levels of prolactin are typically indicative of prolactinoma. The hook effect occurs in immunological tests such as the prolactin level test. When the amount of prolactin present in the sample is too high and exceeds the binding capacity of the antibodies being used, the test result may indicate falsely low levels of prolactin, which is the hook effect. The present study describes the case of a male patient who presented with neck pain and difficulty swallowing. MRI revealed a giant (>40mm) extradural tumor affecting the clivus, anterior fossa, pterygopalatine, and bilateral infratemporal fossae as well as the petrous apex and bilateral cavernous sinuses. Endocrinological investigation yielded no specific abnormalities. An occipitocervical fixation (arthrodesis) was proposed with simultaneous extended endoscopic endonasal resection. Surgery succeeded in resecting a portion of the clival tumor and the anterior fossa. Measurement of prolactin levels several weeks post-surgery found them to be extremely high, confirming the hook effect.

Development of molecular and antigenic-based rapid tests for the identification of African swine fever virus in different tissues.

African swine fever (ASF) is a severe haemorrhagic infectious disease affecting suids, thus representing a great economic concern. Considering the importance of the early diagnosis, rapid point of care testing (POCT) for ASF is highly demanded. In this work, we developed two strategies for the rapid onsite diagnosis of ASF, based on Lateral Flow Immunoassay (LFIA) and Recombinase Polymerase Amplification (RPA) techniques. The LFIA was a sandwich-type immunoassay exploiting a monoclonal antibody directed towards the p30 protein of the virus (Mab). The Mab was anchored onto the LFIA membrane to capture the ASFV and was also labelled with gold nanoparticles for staining the antibody-p30 complex. However, the use of the same antibody for capturing and as detector ligand showed a significant competitive effect for antigen binding, so required an experimental design to minimize reciprocal interference and maximize the response. The RPA assay, employing primers to the capsid protein p72 gene and an exonuclease III probe, was performed at 39 °C. The limit of detection of the method was assessed using a plasmid encoding the target gene and resulted in 5 copy/µL. The new LFIA and RPA were applied for ASFV detection in the animal tissues usually analysed by conventional assays (i.e., real-time PCR), such as kidney, spleen, and lymph nodes. A simple and universal virus extraction protocol was applied for sample preparation, followed by DNA extraction and purification for the RPA. The LFIA only required the addition of 3% H2O2 to limit matrix interference and prevent false positive results. The two rapid methods (25 min and 15 min were needed to complete the analysis for RPA and LFIA, respectively) showed high diagnostic specificity (100%) and sensitivity (93% and 87% for LFIA and RPA, respectively) for samples with high viral load (Ct < 27). False negative results were observed for samples with low viral load (Ct > 28) and/or also containing specific antibodies to ASFV, which decreased antigen availability and were indicative of a chronic, poorly transmissible infection. The simple and rapid sample preparation and the diagnostic performance of the LFIA suggested its large practical applicability for POC diagnosis of ASF.

ELISA Essentials: Surfaces, Antibodies, Enzymes, and Substrates.

The enzyme-linked immunosorbent assay is a powerful analytical tool for the assessment of the kind and quantity of specific analytes found within a biological sample. It is based upon the exceptional specificity of antibody recognition of its cognate antigen and the power of enzyme-mediated signal amplification for sensitivity. However, development of the assay is not without challenges. Here, we describe the essential components and features necessary to successfully prepare and carry out the ELISA format.

Interference in ELISA.

ELISA is a well-established technique used worldwide to quantify analytes present in a diverse milieu of biological samplings. It is especially important to clinicians who rely on the accuracy and precision of the test to administer patient care. Those results are to be held with great scrutiny since the assay is subject to error caused by interfering substances found in the sample matrix. In this chapter, we examine the nature of such interferences and discuss approaches to identify and offer remedies to remove the interference and validate the assay.

A Mechanistic Pharmacodynamic Modeling Framework for the Assessment and Optimization of Proteolysis Targeting Chimeras (PROTACs).

The field of targeted protein degradation is growing exponentially. Yet, there is an unmet need for pharmacokinetic/pharmacodynamic models that provide mechanistic insights, while also being practically useful in a drug discovery setting. Therefore, we have developed a comprehensive modeling framework which can be applied to experimental data from routine projects to: (1) assess PROTACs based on accurate degradation metrics, (2) guide compound optimization of the most critical parameters, and (3) link degradation to downstream pharmacodynamic effects. The presented framework contains a number of first-time features: (1) a mechanistic model to fit the hook effect in the PROTAC concentration-degradation profile, (2) quantification of the role of target occupancy in the PROTAC mechanism of action and (3) deconvolution of the effects of target degradation and target inhibition by PROTACs on the overall pharmacodynamic response. To illustrate applicability and to build confidence, we have employed these three models to analyze exemplary data on various compounds from different projects and targets. The presented framework allows researchers to tailor their experimental work and to arrive at a better understanding of their results, ultimately leading to more successful PROTAC discovery. While the focus here lies on in vitro pharmacology experiments, key implications for in vivo studies are also discussed.

Development of carbon nanoparticles-based soluble solid-phase immune sensor for the quantitative diagnosis of inflammation.

Quantitative immunodiagnosis is one of the most commonly used methods for in vitro diagnostics. Various bioanalytical methods have been developed to quantitatively diagnose immune analytes; however, they require blood dilution pretreatment, reaction mixing, complicated experimental steps, and can cause diagnostic errors due to the hook effect. To address this issue, we introduced a simple immunoassay based on carbon nanoparticles (CNPs). The assay was designed to have high flexibility for use in various in vitro diagnostic devices by constructing a soluble solid-phase immune sensor with high solubility using antibody-conjugated CNPs and polymer materials. Excellent performance was achieved using a free-antibody system with dual calibration. To verify the performance of this method with high reliability, canine C-reactive protein was selected as the immune analyte. Interestingly, our method efficiently mitigated the hook effect with outstanding performance in a one-step reaction without blood dilution or reaction mixing. The detection range of the target can be effectively controlled using free antibodies. Therefore, our CNP-based immunodiagnosis method may advance the commercialization of point-of-care immune biosensors.

Corrigendum: Common pitfalls in the interpretation of endocrine tests.

[This corrects the article DOI: 10.3389/fendo.2021.727628.].

A Case of a Negative Urine Pregnancy Test in a Multiple Gestation Pregnancy.

Urine pregnancy tests (UPTs) are a highly reliable method of detecting pregnancy, with reported 100% sensitivity and 99.2% specificity. This test relies on the detection of ß-human chorionic gonadotropin (ß-hCG) molecules in the urine through a two-site sandwich immunoassay. Although a nearly perfect test, it is common knowledge that this test can be falsely negative if performed too early in the pregnancy when urinary ß-hCG concentrations fall below detectable levels. Less commonly known is that the test may provide a false-negative result when urinary ß-hCG concentrations are extremely elevated, such as gestational trophoblastic disease or multiple gestations. Here, we present a case of a patient with a prior positive urine pregnancy test who presents with symptoms consistent with early pregnancy. Repeat testing resulted in a negative urine pregnancy test. Additional workup revealed significantly elevated serum quantitative ß-hCG and bedside ultrasound revealed multiple gestation intrauterine pregnancy. The patient ultimately delivered triplets by repeated caesarean section. It is important for physicians to understand and recognize the limitations of the urine pregnancy test in order to best facilitate care for patients who may have a false-negative pregnancy test result, as there are significant risks of improper patient management with a multiple gestation pregnancy or gestational trophoblastic disease.

Hook effect in gestational trophoblastic disease: An emergency department case presentation.

Gestational trophoblastic disease is a process that affects ≈1 of 1000 pregnancies. If left untreated, this can progress to potentially life-threatening complications with malignancy such as choriocarcinoma.  The emergency physician must be aware of the presentation and complications of this disease process, including the difficulties in diagnosis.  In this case presentation, the authors discuss the presentation and diagnostic process of a patient in the emergency department as well as the phenomenon known as the hook effect, which may complicate the decision-making process.

An improved method for rapid identification of hook effect samples in HBsAg quantitative assay.

Quantitative hepatitis B surface antigen assay is widely used for diagnosis of hepatitis B virus infection; however, specimens with high levels of the antigen can cause false-negative results (Hook effect), which needs to be resolved. The hook effect samples and non-hook effect samples were detected on the LiCA® 500 instrument using three methods, viz., 1, 2, and 3. Method 1, the currently used procedure, was performed in two steps with a total reaction time of 25 min in a final volume of 250 µL: first incubation was with two reagents for 15 min and then with one other reagent for 10 min. In Method 2, all three reagents were added in one step with a final volume of 250 µL, and the total reaction time was still 25 min. In Method 3, the improved method, all three reagents were added in one step while the final volume was only 130 µL and the total reaction time was only 1 min. Signal values of the non-hook effect samples obtained using Method 2 were significantly lower than those with Method 1, showing competitive inhibition. The hook effect samples tested with Method 2 approximated those obtained using Method 1. Method 3 took 1 min and differentiated hook effect samples successfully, similar to the results with Method 2 which took 25 min. Changing the timing of one reagent addition and incubation time in Method 3 provided a rapid and effective method for the identification of hook effect. The results were more clearly distinguishable due to the phenomenon of competitive inhibition. Method 3 can be considered an improvement on the chemiluminescence platform.

Correlation between Results of Semi-Quantitative and Quantitative Tests for Hepatitis B Virus Surface Antigen among Patients Achieving Viral Suppression with Antiviral Treatment.

Background: Hepatitis B virus (HBV) infection remains a threat to global public health. Serum hepatitis B surface antigen (HBsAg) has been used in screening for HBV infection. Quantitative HBsAg assays are useful for monitoring the natural history of HBV infection and its response to therapy. The aim of this study was to determine the relationship between quantitative (qHBsAg; IU/mL) and semi-quantitative (sqHBsAg; signal-to-cutoff ratio [S/Co]) HBsAg titers in patients with chronic hepatitis B (CHB). Methods: We retrospectively included 284 samples with HBV DNA < 20 IU/mL from patients who had simultaneous qHBsAg (using electrochemiluminescence assay) and sqHBsAg tests. Patients were grouped according to their serum HBV-envelope antigen (HBeAg) status (HBeAg-negative, n = 239 and HBeAg-positive, n = 45). The Spearman test was used to analyze the correlation between the quantitative and semi-quantitative assays. Results: There was a significant linear correlation between sqHBsAg and qHBsAg in the HBeAg-negative patients (qHBsAg [IU/mL] = 0.0094 × sqHBsAg [S/Co]1.323; adjusted R2 = 0.8445; p < 0.001). There was a substantial hook effect in the assays from the HBeAg-positive patients, so we performed a stratified analysis according to qHBsAg <1000 IU/mL or ≥1000 IU/mL and found a significant positive linear correlation between sqHBsAg S/Co and qHBsAg (qHBsAg [IU/mL] = 0.072 × sqHBsAg [S/Co]1.331; adjusted R2 = 0.7878; p < 0.001) in HBeAg-positive patients with qHBsAg titers of <1000 IU/mL and a significant negative correlation in HBeAg-positive patients with qHBsAg titers of ≥1000 IU/mL (qHBsAg [IU/mL] = 8.987 × 1014 × sqHBsAg [S/Co]−3.175; adjusted R2 = 0.6350; p < 0.001). Conclusions: There was a highly linear, positive correlation between qHBsAg and sqHBsAg in HBeAg-negative CHB patients. The hook effect led to a negative correlation in HBeAg-positive CHB patients with qHBsAg titers ≥1000 IU/mL.

Prolactin immunoassay: does the high-dose hook effect still exist?

PURPOSE: Measurement of prolactin in clinical laboratories is an important component in the management of patients with pituitary adenoma. Prolactin measurement is known to be sensitive to the high-dose hook effect, in the presence of extremely high prolactin concentrations. This interference is referred to in most recent articles discussing prolactin assays and the management of prolactin-secreting pituitary adenomas. The objective of our study was to evaluate if the high-dose hook effect remains relevant in current practice, when using currently available assays. METHODS: Serum from a patient with a giant macroprolactinoma was assayed using all of the available prolactin assays in France in 2020, using native serum and after dilution. Technical inserts from assays were reviewed to assess the information on analytical principles, numbers of steps, and any reference to high dose hook effect. RESULTS: Fourteen assay kits were studied by 16 laboratories; all were two-site immunometric assays, mostly using one step (11/14). Results obtained after dilution varied from 17,900 µg/L to 86,900 µg/L depending on the assay used. One tested assay was sensitive to the high-dose hook effect leading to a falsely lower prolactin concentration when measuring native serum (150 µg/L compared to 17,900 µg/L after dilution). CONCLUSION: The high-dose hook effect still exists in a very small minority of prolactin assays. The evolution of assay methods may lead to new assays that remain sensitive to this effect in the future. We therefore advise that the hook effect should still be mentioned in prolactin assay recommendations.