Interference In Immunoassays Research Articles

Evaluation of SARS-CoV-2 Serological Testing in Patients with Multiple Myeloma and Other Hematologic Malignancies on Monoclonal Antibody Therapies.

Background: Patients with hematological malignancies (HM), including multiple myeloma (MM), frequently suffer from immune deficiency-associated infectious complications because of both the disease and the treatment. Alarming results from China and the UK confirm the vulnerability of HM patients to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-driven coronavirus disease 2019 (COVID-19). Given that the immunoassay interference from the endogenous monoclonal immunoglobulin (M paraprotein) and treatment antibodies continually challenges the MM management, it is critical to evaluate the SARS-CoV-2 serology tests for suspected interference/cross-reactivity. Methods: We compared the degree of interference in three SARS-CoV-2 serology assay platforms in HM patients with and without COVID-19 and on various therapeutic monoclonal antibody (t-mAb) treatments. Further, we confirmed the cross-reactivity in pooled samples from normal and COVID-19 + samples spiked with respective antibodies in vitro. Results: None of the 93 HM patient samples with or without t-MAbs showed cross-reactivity on any of the three serology platforms tested. Conclusions: The tested three serologic assays for SARS-CoV-2 are specific and do not have cross-reactivity with M-components or t-MAbs indicating that they can be used safely in oncology practice and in research exploring the immunologic response to COVID-19 in patients with HM.

False hyperthyroidism caused by interference in immunoassays.

Immunoassays used to assess thyroid function are vulnerable to different types of interference that may affect clinical decision-making. We report the case of a 37-year-old woman who developed iatrogenic hypothyroidism after having received radioiode therapy who visited our hospital for her annual checkup. The patient was asymptomatic, without signs suggestive of thyroid disease. However, laboratory analysis proved otherwise: thyrotropin (TSH) 7.75mU/L, thyroxine (FT4) >7.7ng/dL. The inconsistency between her clinical symptoms and the biochemistry data raised the possibility of a methodological interference. A thorough evaluation of the main causes of interference was conducted in the laboratory to exclude the presence of interference in TSH and FT4. Finally, different interfering agents were identified, which affected free thyroid hormone and TSH determination.

The Role of Interferences in the Increasing Incidence of Vitamin D Deficiency.

Lipemia is one of the causes of interference in immunoassay and LC-MS/MS methods. Increased prevalence of vitamin D deficiency in the US, where obesity is gradually increasing, raises the suspicion that high levels of fat diet and blood lipid levels interfere with vitamin D measurement results. The focus of this study was to investigate the effect of blood lipid profiles on vitamin D results and prevent the matrix effect. In this study, 25OH vitamin D3 (25OHD3) levels of 100 samples consecutively accepted to biochemistry laboratory regardless of age and sex were measured by the LC-MS/MS method, and each sample was restudied after 1/10 dilution. After dilution restudy, two groups were obtained-group 1 (results deviating below 20%) and group 2 (results deviating above 20%)-and the difference between the groups was investigated. There were 79 patients in group 1 and 21 patients in group 2. In our study, lipid profiles (triglyceride, total cholesterol, HDL, LDL) from the same samples of consecutive vitamin D patients were studied. It was observed that the triglyceride, total cholesterol HDL, LDL, and 25OHD3 measurements of group 1 and group 2 were similar (p > 0.05). While the mean vitamin D value in the second group was 9.94 ± 7.85, the mean vitamin D value after dilution was measured as 39.23 ± 18.13 and was statistically significant. 25OHD3 concentrations of 21 patients out of 100 were found to be falsely low. Measurements were repeated to confirm the results. The matrix effect caused by exogenous and endogenous interferences in the blood could be a hidden factor increasing the prevalence of vitamin D deficiency by causing falsely low 25OHD3 values. Suspicious results should be remeasured by a dilution study.

Biotin immunoassay interference: A UK-based prevalence study.

Biotin interference in biotin-streptavidin-based immunoassays is increasingly reported due to individuals taking biotin-containing supplements and patients prescribed biotin. The reported prevalence of serum biotin above the lowest threshold (≥10 µg/L) for interference in Roche Diagnostics immunoassay tests is 0.8% in Australia and 7.4% in the USA. There are, however, no such data in UK populations. In a service evaluation, we therefore studied the prevalence of biotin interference in routine serum samples received in our laboratory. Biotin was measured in 524 anonymized surplus serum samples in which at least one immunoassay test had been requested. The median (95% confidence intervals) for serum biotin was 0.27 µg/L (0.07-0.93 µg/L). Serum biotin was <10 µg/L in all samples, <5 µg/L in 522 (99.6%) and <1 µg/L in 513 (98.1%) samples. In four samples, serum biotin was ≥2.5 µg/L (0.8%). These data indicate that the probability of biotin immunoassay interference in our patient population is extremely low, with the exception of assays reporting the lowest interference thresholds (e.g. Ortho Troponin I assay [threshold ≥2.5 µg/L]).

Generalized mathematical model for immunoassay interference

As part of a landmark review of the antigen excess, Jacobs et al. (Autoimmunity Reviews 2015;14:160-167) contrast how immunoassay "interference" by non-target biomolecules can cause spurious readings for clinical diagnostic tests. The purpose of the present Letter is to complement and analytically extend this description by Jacobs et al. by briefly presenting a generalized mathematical model of immunoassay interference that is derived from a simple mechanistic system of differential equations. Derivation includes expressions for the location of the peak concentration of bound reporter as well as that maximum concentration of target biomolecule at which immunoassay interference drives concentration of bound reporter to zero. This parsimonious, mechanistic, and generalized model may prove foundational to analytic study of immunoassay interference. More comprehensive models for specific classes of immunoassays have and can be developed.

Immunoassay interference on thyroid functions tests during treatment with Nivolumab

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Biotin interference in cardiac troponin immunoassay – where the wild things are?

Biotin interference in cardiac troponin immunoassay – where the wild things are?

Pre-existing canine anti-IgG antibodies: implications for immunotherapy, immunogenicity testing and immunoassay analysis

One of the most enigmatic features of humoral immunity is the prevalent presence of circulating autoantibodies against IgG. These autoantibodies consist of several subsets, including rheumatoid factors, anti-Fab/anti-F(ab′)2-autoantibodies, and anti-idiotypic antibodies. Anti-IgG autoantibodies can impair the safety and efficacy of therapeutic antibodies and interfere with immunogenicity tests in clinical trials. They can also cross-react with allospecific IgG, presenting as heterophilic antibodies that interfere with diagnostic immunoassays. Owing to these factors, recent years have seen a resurgent interest in anti-IgG autoantibodies, but their underlying clinical significance, as well as biological roles and origins, remain opaque. Increased knowledge about canine anti-IgG autoantibodies could facilitate the development of canine immunotherapies and help in understanding and counteracting immunoassay interference. This study investigated the clinical significance and interconnection of heterophilic antibodies, anti-Fab, and anti-F(ab′)2-autoantibodies in dogs. We performed a 2-year prospective follow-up of dogs with heterophilic antibodies and analyzed serum for anti-Fab and anti-F(ab′)2-autoantibodies. Canine heterophilic antibodies can persist for at least 2 years in serum. A widespread occurrence of anti-Fab and anti-F(ab′)2-autoantibodies was found, with reactivity to cryptic epitopes in the IgG hinge region and sporadic cross-reactivity with mouse IgG. Canine anti-Fab and anti-F(ab′)2-autoantibodies are thus potential sources of clinical immunogenicity and immunoassay interference.

Prevalence of detectable biotin in The Netherlands in relation to risk on immunoassay interference

Despite the increasing awareness about biotin interference with immunoassays, so far, only two studies have quantified the prevalence of elevated biotin in patient populations. In a US study, over 7% had biotin concentrations exceeding 10 ng/mL, whereas in an Australian study only 0.8% of ED samples contained biotin exceeding 10 ng/mL. At present, representative data for the European population are lacking. In this study, we investigated biotin prevalence in The Netherlands in a representative cohort of routine laboratory requests in our laboratory using an LC-MS/MS assay for quantification of biotin in human plasma. In our study, we found 0.2% of samples exceeding 10 ng/mL of biotin, a finding more or less in line with the Australian data. Even though the biotin prevalence appears to be low, with concomitant low to moderate biotin concentrations, it is by no means a rare phenomenon. Laboratories like ours are likely to experience biotin positive samples on a daily basis with variable impact on patient care depending on the analytical bias from the immunoassay platform used. Our simple and robust LC–MS/MS assay for quantification of biotin in human samples may contribute to better understanding of the systemic concentrations seen after moderate- and high-dose biotin supplementation and the extent of immunoassay interference.

MON-477 Anti-Streptavidin Interference in Multiple Hormones Immunoassays: Case Report

Automated immunoassays are the most commonly current methods used in clinicial laboratory to hormone tests, due short turnaround time and high specificity and sensibility. However immunoassays are not free from interferences. Several immunoassays use biotin-streptavidin interaction to anchor antigen-antibody complexes in solid phase and so are susceptible to biotine intake or anti-streptavidin antibody. In sample with these interferents, the results in competitive assays are falsely high and in non-competitive assays are falsely low. OBJECTIVE: To report a case of multiple hormones immunoassay interference by an anti-streptavidin antibody. MATERIAL AND METHODS: A 11-years-old boy with child obesity, and no other symptoms, had discordant laboratory results with markedly elevated free T4, total T4 and total T3 with normal TSH. Repeated measurements in the same sample and in a new collected sample confirmed the initial results and revealed low PTH and high TRAb without hypocalcemia or hyperthiroidism clinical pictures. All the parameters were measured on the automated Cobas e602, Roche plataform.The patient and your family denied biotin ingestion, so anti-streptavidin interference could explain the multiple interference.The serum was pre-incubated with streptavidin microparticles during an hour and centrifuged for 10min (3000 rpm) to removed the analytical interference. RESULTS: The results of post-incubation with microparticles were all in the normal range. Three control samples were not affected by the incubation. Since luminescence is inversely proportional to analyte concentration in competitive assays, low signals lead to falsely high levels (like in free T4, total T4, Total T3 and TRAb in our patient). The reverse occurs with immunometric assays, in which low signals result in falsely reduced values (like in PTH in our patient). CONCLUSION: In literature, streptavidin antibodies seem to be a rare occurrence. In samples with biotin or anti-streptavidin suspection, pre-incubation with streptavidin microparticles is a simple and effective procedure to remove this interference.

Macroprolactinemia Detection by Magnetically Assisted Polyethylene Glycol Precipitation: Potential for Automation.

Macroprolactin is an immunoglobulin-prolactin complex that is not bioactive in vivo but the prolactin component remains immunoreactive. The complex is a universal source of interference in prolactin immunoassays and commonly results in misdiagnosis of hyperprolactinemia with consequent clinical mismanagement of patients. Removal of macroprolactin by precipitation with polyethylene glycol (PEG) is an effective technique for identifying such patients but unfortunately not universally employed due to the manual nature of the procedure. We developed a modified PEG precipitation technique using magnetic nanoparticles that we termed Magnetically Assisted PEG Precipitation (MAPP). This procedure was verified against an established PEG precipitation procedure. The MAPP procedure we developed was robust, reproducible, and affords the potential for automation of macroprolactin screening in clinical laboratories. Comparisons of prolactin levels obtained following MAPP in sera from patients with either true hyperprolactinemia or macroprolactinemia generated results comparable to that of conventional PEG precipitation. The MAPP technique yields results comparable to those of traditional PEG precipitation. Elimination of the need for centrifugation affords the possibility of automation and hence more widespread adoption of routine PEG screening by clinical laboratories.

Interference From High-Dose Biotin Intake in Immunoassays for Potentially Time-Critical Analytes by Roche.

Immunoassays using the interaction between streptavidin and biotin are used for clinical chemical analytes on platforms by many different manufacturers. The design can be susceptible to interference from high-dose biotin intake in patients, which remains an often-overlooked confounder despite recently increased awareness. To evaluate an easily implementable method of in vitro biotin depletion for the removal of biotin interference in immunoassays for potentially time-critical analytes. A biotin stock solution was made and de-identified patient samples were spiked to reach a biotin concentration of 1.126 × 106 pg/mL, the maximum reported biotin concentration 1 to 2 hours after a single oral dose of 300 mg biotin. Then, the resulting interference in Elecsys immunoassays for cortisol, cyclosporine A, tacrolimus, digitoxin, thyroid-stimulating hormone, free triiodothyronine, free thyroxine, C-peptide, insulin, N-terminal pro-B-type natriuretic peptide, troponin T high sensitive, human immunodeficiency virus, procalcitonin, β human chorionic gonadotropin, toxoplasma immunoglobulin M, and toxoplasma immunoglobulin G was evaluated before and after biotin depletion using streptavidin particles. All tested immunoassays, with the exception of toxoplasma immunoglobulin M and toxoplasma immunoglobulin G, suffered from significant biotin interference. The depletion protocol removed assay interference due to biotin and produced results that were close or identical to initial prespike measurements. Despite an increase in turnaround times, biotin adsorption is a feasible countermeasure for biotin interference in Elecsys immunoassays. Until test kits with an increased resistance to the interference from high-dose biotin intake are distributed, the evaluated protocol can provide results properly reflecting the patient's clinical condition.

Interference in thyroid function immunoassays: clinical consequences

Thyroid function tests are prone to analytical interference, which can cause misleading results when performed on automated immunoassay analyzers. We present a case of a 68-years old woman diagnosed with primary hypothyroidism and chronically treated with levothyroxine. Her status has been followed-up in several different institutions and before readmission to our institute, she was diagnosed as T3 toxicosis according to the lab results of suppressed TSH, normal FT4 and highly elevated FT3 values. Due to lack of toxic symptoms, our clinician suspected FT3 test interference, which was confirmed in our lab by performing the test on a different immunoassay platform. In conclusion, every discrepancy between clinical presentation and laboratory test results has to be inspected by close communication between clinicians and laboratory specialists. Our goal was to raise the awareness within the healthcare community about the interference in immunoassays affecting different kit manufacturers and analytical platforms in order to avoid erroneous diagnosis and mistreatment of patients. Key words: immunoassay, interference, free triiodothyronine, mistreatment

Understanding and managing biotin interference in immunoassays

Understanding and managing biotin interference in immunoassays

Assessment of Risk for Interference by Circulating Biotin in Samples Received for High Sensitivity Troponin-T, Thyrotropin, and for Prostate Specific Antigen Testing by Immunoassays.

Biotin interference in streptavidin-based immunoassays is known and may lead to erroneous results and thus to diagnostic error. The recent increase in reports of biotin interference in immunoassay-based testing has been attributed to increased intake of biotin supplements by the public and to the high dose biotin therapy in patients with neurological and inherited disorders. Circulating biotin levels greater than 20 ng/mL are reported to exhibit interference in high sensitivity troponin T (hs-TnT), thyroid stimulating hormone (TSH), and in prostate specific antigen (PSA) among other assays when using our Cobas® 6000 immunoassay analyzer (Roche Diagnostics, IN, USA). This study aims to examine the risk for biotin interference among our patient population. Serum and plasma leftover samples from 183 different patients were collected following completion of hs-TnT (53 samples), TSH (45 samples), and PSA (85 samples) testing. Aliquots were stored frozen at -20°C until analysis. Biotin concentrations in these samples were measured using an ELISA (ALPCO, Salem, NH, USA) according to the manufacture's protocol. Samples with biotin levels of 20 ng/mL or greater were considered as high-risk samples (HRS) for biotin interference. The overall concentrations of biotin in our patients' samples ranged from 0.02 ng/mL to 11.38 ng/mL (median 0.42 ng/mL). The median and (range) biotin concentrations in hs-TnT, TSH, and PSA samples were 0.27 ng/mL (0.02 - 6.86 ng/mL), 0.39 ng/mL (0.08 - 11.38 ng/mL), and 0.47 ng/mL (0.09 - 7.73 ng/mL), respectively. Although there was no significant difference between biotin levels in samples for TSH or PSA measurement (p = 0.85), biotin in samples for PSA and for hs-TnT and in samples for TSH and hs-TnT were significantly different (p = 0.049 and 0.089), respectively. None of the samples had biotin levels greater than or equal to 20 ng/mL. Using representative samples with requests for hs-TnT, TSH, and PSA testing, where reliable performance for the selected assays at their lowest measurement range is required for clinical intervention, among our study population the risk was considered minimal as their circulating biotin levels were less than 20 ng/mL. However, educating clinicians and laboratory users regarding the potential of biotin interference is always recommended.

Chemistry of Biotin-Streptavidin and the Growing Concern of an Emerging Biotin Interference in Clinical Immunoassays.

Overconsumption of biotin (5–100 mg daily) as a supplement by the general population poses a significant problem for clinical immunoassays (IAs) based on biotin–streptavidin (SA) interactions. This affinity pair has been exploited in immunoassays because of its avidity, sensitivity, specificity, and stability. The elevated biotin level in plasma varies from patient to patient, and its severe interference cannot easily be predicted and quantified. Thus, immunoassay manufacturers must investigate the biotin interference in the developed immunoassays to satisfy the threshold of 3510 ng/mL (14 367 nM), as stipulated by the FDA. There is no concrete solution to circumvent the biotin interference without extra costs and technical difficulties, albeit different strategies have been attempted. They include the IA format with biotinylated reagents prebound to streptavidin, the removal of biotin from the specimen, sample treatment, and biotin interference-free assays. The general public has been instructed to stop taking biotin supplements for 48 h or even weeks before the test, depending on the specific test, dose, and frequency of biotin uptake. As lab-based techniques cannot accommodate an enormous number of public samples, a rapid analytical procedure for biotin is urgently needed to quantify for its interference in immunoassays.

Immunoassay interferences and their impact on patient care

Immunoassay interferences and their impact on patient care

Understanding and managing interferences in clinical laboratory assays: the role of laboratory professionals.

The recently raised concerns regarding biotin interference in immunoassays have increased the awareness of laboratory professionals and clinicians of the evidence that the analytical phase is still vulnerable to errors, particularly as analytical interferences may lead to erroneous results and risks for patient safety. The issue of interference in laboratory testing, which is not new, continues to be a challenge deserving the concern and interest of laboratory professionals and clinicians. Analytical interferences should be subdivided into two types on the basis of the possibility of their detection before the analytical process. The first (type 1) is represented by lipemia, hemolysis and icterus, and the second (type 2), by unusual constituents that are not undetectable before analysis, and may affect the matrix of serum/plasma of individual subjects. Type 2 cannot be identified with current techniques when performing the pre-analytical phase. Therefore, in addition to a more careful evaluation and validation of the method to be used in clinical practice, the awareness of laboratory professionals should be raised as to the importance of evaluating the quality of biological samples before analysis and to adopt algorithms and approaches in the attempt to reduce problems related to erroneous results due to specific or non-specific interferences.

Characterization of canine anti-mouse antibodies highlights that multiple strategies are needed to combat immunoassay interference

Immunoassays are widely used for detection and quantification of analytes in biological samples, but are vulnerable to analytical errors caused by interfering sample substances. Of particular interest are endogenous anti-animal antibodies that may bind to the immunoassay antibodies and cause erroneous test results. This phenomenon is a hazard to patient safety in both human and veterinary medicine. Here, we demonstrate that anti-mouse antibodies in dogs bind selectively to different regions of the murine IgG molecule, cross-react with IgG from different species, and consist of all major antibody classes present in canine serum (IgA, IgG and IgM). The antibody characteristics varied among individuals and their prevalence differed between two dog breeds. The selective binding to different IgG regions suggests that the antibodies might not originate from immunization through exposure to mice or other species. These findings show that canine anti-mouse antibodies are highly heterogeneous in nature and therefore require a combination of strategies to be counteracted.

Biotin Measurement by Targeted LC-MS/MS: Method Development and Application to the Evaluation of Immunoassay Interference in Thyroid Function Tests

Abstract Introduction While biotin is widely used as a health and beauty supplement, patients rarely report the use of biotin supplements to clinicians. This can negatively impact patient care since biotin can falsely elevate or lower results in immunoassays that use biotinylated components. On the Beckman DxI platform, total T3 (T3), free T3 (FT3), and free T4 (FT4) assays are falsely elevated by excess biotin, while TSH and total T4 (T4) are unaffected. The goals of this study were to develop a method to measure serum biotin, to determine biotin concentrations in samples submitted for thyroid function testing, and to correlate biotin concentrations with immunoassay result patterns suggestive of biotin interference. Methods We developed and validated a targeted LC-MS/MS method to measure serum biotin (AMR: 5.4-500 mcg/L). Two collections of patient samples were tested using automated Beckman DxI assays for TSH (RR: 0.4-5.0 μIU/mL), T3 (RR: 73-178 ng/dL), FT3 (RR: 2.4-4.1 pg/mL), T4 (RR: 4.8-10.8 mcg/dL), and FT4 (RR: 0.6-1.2 ng/dL). The first collection included consecutive samples submitted for TSH and FT4 testing. To increase the probability of identifying samples with biotin interference, a middleware preselection algorithm was used to identify samples with a results pattern suggestive of biotin interference (normal to high TSH with elevated T3, FT3, and/or FT4). Results Two of the 94 samples consecutively submitted for thyroid function tests demonstrated biotin levels &gt;AMR. TSH and FT4 were within normal ranges for one, while the other had normal TSH and elevated FT4 (biotin: 26 mcg/L). The preselection algorithm targeting normal to high TSH with elevated T3, FT3, and/or FT4 results flagged 83 samples, 21.7% of which had measurable biotin (5.71-194.3 mcg/L). Immunoassay results for TSH, T3, FT3, T4, and FT4 were obtained where there was sufficient volume for testing. Of these 38 samples, all samples with biotin &gt;24 mcg/L had elevated results for 2(+) affected assays. Conclusions In this study, a targeted LC-MS/MS method to measure biotin in serum samples was developed and used to estimate the distribution of biotin concentrations in samples submitted for thyroid function testing. Comparison of immunoassay result patterns with biotin concentrations suggested an interference threshold around 24 mcg/L. The evaluation of consecutively submitted samples showed that biotin in excess of this interference threshold in patients receiving thyroid function tests on the Beckman DxI platform is rare. However, combining the preselection algorithm with quantitative biotin measurement by LC-MS/MS demonstrated that biotin interference does occur in these patient samples and should be evaluated when there are confounding results or result patterns suggestive of interference. In addition, the biotin LC-MS/MS method described here has additional clinical utility in the evaluation of suspected interference in any immunoassay that utilizes biotinylated components.