Diagnostic Dilemma: A Case of Undetectable TSH

Introduction: Under steady-state conditions, measurement of TSH is accepted as the best assessment of thyroid function. The widely used TSH chemiluminometric assays have very low limits of detection and can help distinguish between the various causes of subnormal TSH. However, when evaluating a patient with abnormal thyroid tests but without thyroid symptoms, an appraisal of the test should be considered. Clinical Case: A 63-year-old South Asian man was referred to endocrinology for evaluation of a non-detectable TSH (<0.01 µIU/mL) that was reproduced on repeat testing, both using Siemens ADVIA Centaur TSH3-UL immunoassay. The patient was clinically euthyroid and denied taking biotin supplements. Testing of thyroid hormone showed normal values for free T3, total T3, free T4, and total T4. Additional labs included normal studies for free thyroxine by equilibrium dialysis, thyroid stimulating immunoglobulin, and heterophilic antibodies. Thyroid uptake and scan showed uniform uptake of 5.1% and 15.1% at 2-hours and 24-hours, respectively, with no dominant nodules. Hypothalamic-pituitary hormonal testing and MRI pituitary were both normal as well. When TSH testing was repeated on a separate platform, Roche’s eCLIA immunoassay, detectable values were obtained (TSH 6.48 µIU/mL). Conclusions: Testing of serum TSH by commercially available immunoassays is based on the sandwich method in which one antibody binds to the β-subunit of TSH and the other to the α-β interface. Most assays use monoclonal antibody pairs to achieve high selectivity. Immunoassay tests are prone to interferences, particularly by way of altering the measurable concentration of the analyte or by altering antibody binding (1). In this case, the presence of detectable TSH depended on the platform by which it was measured. This finding suggests a TSH-β variant with impaired immunoreactivity but functionally normal bioactivity. Such a mutation has been previously reported to occur five times more frequently among South Asian individuals than the general population (2). Genetic testing was offered to the patient to confirm this suspicion but was declined. It is incumbent on the clinician to reconcile a test result that is discordant with the clinical presentation. Having a fundamental understanding of the principles of the testing platform can assist in identifying potential sources of error. Failing to recognize a possible interference can lead to unnecessary healthcare expenditures, misdiagnosis and inappropriate management, potentially at a cost to the patient’s wellbeing. When faced with an undetectable TSH with otherwise normal thyroid hormones and unremarkable clinical picture, it is best to repeat the TSH test using a different available platform. References: (1)Favresse J et al. Endocr Rev. 2018;39(5):830-850(2)Pappa T et al. Thyroid. 2015 Aug;25(8):869-76

Prevalence of heterophilic antibodies in serum samples from horses in an equine hospital, and elimination of interference using chicken IgY

Background Heterophilic antibodies in serum and plasma can interfere with mammalian antibodies in immunoassays and result in false test results, usually false positive. Although studies screening for heterophilic antibodies as well as elimination studies have been conducted in dogs and cats, knowledge of the presence of heterophilic antibodies in other species in veterinary medicine is limited. In this study, a 2-site sandwich-type interference assay that detects anti-mouse antibodies was used to detect heterophilic antibodies in a population of horses treated in an animal hospital. Results A total of 194 serum samples from 127 individual horses were analyzed. There were 11/127 (8.7%) interference-positive horses, and these were analyzed in an assay exchanging the capture mouse IgG with chicken IgY. The positive samples were negative in the chicken IgY assay, indicating elimination of a possible interference, with the chicken-based assay. Four interference-positive samples were from geldings, and anti-Müllerian hormone (AMH) was analyzed from these samples. AMH concentrations were negative in these samples as expected in geldings, indicating that the heterophilic antibodies did not cause interference in the AMH assay. Conclusion The present study shows that there are heterophilic antibodies in horse serum samples like in samples from humans, dogs, and cats. The use of chicken-based reagents, such as chicken IgY, which do not cross-react with mammalian IgG, eliminates the effects of interfering antibodies in the samples. Equine heterophilic antibodies do not necessarily cause interference in commercial immunoassays.

Falsely markedly elevated 25-hydroxyvitamin D in patients with monoclonal gammopathies

ObjectivesMonoclonal immunoglobulins can cause interference in many laboratory analyses. During a 4 month period we observed seven patients with monoclonal disease and falsely extremely elevated 25-hydroxyvitamin D (25(OH)D) results above 160 ng/mL (>400 nmol/L) measured using an immunoassay from Abbott Diagnostics. Based on these findings, we studied the occurrence of falsely elevated 25(OH)D in samples with paraproteins and investigated possible mechanisms of the observed interference.Methods25(OH)D was analyzed using the Architect i2000 platform from Abbott Diagnostics and a higher order method, liquid chromatography-mass spectrometry (LC-MS/MS), in serum samples from 50 patients with known monoclonal disease. Patients with falsely elevated 25(OH)D were included in further studies to elucidate the cause of interference. Spuriously elevated results were in addition analyzed on two alternative platforms (Siemens and Roche).ResultsFalsely elevated 25(OH)D levels were present in eight patients on the Abbott analyzer and one on the Siemens platform. Results from Roche were comparable with LC-MS/MS. Additional investigations excluded elevated concentrations of rheumatoid factor and heterophilic antibodies as the cause of interference in the Abbott assay.ConclusionsLaboratories should be aware of the risk of falsely elevated 25(OH)D in samples run on the Architect analyzer from patients with monoclonal disease. Highly elevated vitamin D results should be diluted and if the dilution is non-linear, rerun by a different method, preferably LC-MS/MS. In patients with spuriously elevated 25(OH)D without known monoclonal disease, the laboratory should consider requesting protein electrophoresis to exclude paraprotein interference.

Falsely Elevated Thyroid-Stimulating Hormone Results due to Interference by M-Component of IgG-Lambda Type

Heterophilic antibodies but also M-components can interfere with laboratory tests causing erroneous results. We report the case of a 75-year-old man with myeloma and a monoclonal immunoglobulin component (M-component) that caused elevated thyroid-stimulating hormone (TSH) results. The M-component was of the IgG-lambda type. Thyroid markers were analyzed repeatedly, and there was a clear association between IgG concentrations and TSH values (R2 = 0.724). The highest TSH value was 75 mIU/L. Polyethylene glycol (PEG) precipitation of intact immunoglobulins was used to investigate if there was an antibody-related interference problem. The PEG treatment normalized the TSH value, showing that the cause of the elevated TSH result was due to interference caused by the M-component. In conclusion, it is important to remember that both heterophilic antibodies and M-components may cause erroneous results.

FRI0579 RHEUMATOID FACTOR IS ASSOCIATED WITH FALSELY ELEVATED RESULTS IN COMMERCIAL IMMUNOASSAYS: DATA FROM AN EARLY ARTHRITIS COHORT

Background:Immunoassays are used to measure a range of analytes in clinical laboratories. Rheumatoid factor (RF) and other patient antibodies, such as heterophilic antibodies, can bind animal antibodies used in immunoassays and cause erroneous results, which may lead to misdiagnosis and incorrect treatment of patients.1Objectives:To assess RF reactivity to animal antibodies and to test if selected commercial immunoassays are vulnerable to interference from RF-positive sera.Methods:Samples from 124 patients with RF-positive rheumatoid arthritis (RA) included in the Norwegian Very Early Arthritis Clinic (NOR-VEAC)-cohort2were included in the study. Samples from patients with seronegative RA (n=51) and psoriatic arthritis (n=15) from the same cohort were included as controls. Reactivity to mouse IgG1, mouse IgG2a, rabbit IgG, bovine IgG, sheep/goat IgG and human IgG was analysed using in-house interference assays detecting antibodies able to cross-link the animal or human antibodies. RF-positive sera with strong reactivity to mouse IgG1 were selected for testing in three commercial immunoassays previously shown to be susceptible to interference from heterophilic antibodies; Abbott Architect Total β-hCG assay, BioRad 27-plex cytokine assays and Roche Elecsys Soluble Transferrin Receptor (sTfR).3Samples were tested before and after addition of blocking aggregated murine IgG1 (interference protection). Interference was defined as a discrepancy between the unblocked and blocked samples likely to influence clinical interpretation of the results and exceeding the reported assay imprecision with a considerable margin.Results:We found considerably stronger reactivity toward animal antibodies, particularly mouse IgG1 and rabbit IgG, in sera from RF-positive RA-patients compared to the control group (Fig. 1a-b). In the Abbott β-hCG assay, interference was shown in 6 out of the 30 tested sera (Fig. 2a). In the 27-plex cytokine assays, interference was demonstrated in 7 out of 10 tested sera (for 3-14 analytes). Furthermore, 17 out of the 27 cytokine assays were found to be susceptible to interference (Fig. 2b). Interference was shown in 2 out of 33 samples in the sTfR assay. In unblocked samples, sTfR values were 8.1 and 8.2 mg/L, vs. 4.2 mg/L and 6.0 mg/L in blocked samples, respectively. Additionally, 3 sera had >25% relative difference, but the results were within the reference range.Figure. 1(a-b)Figure. 2(a-b)Conclusion:Reactivity to animal antibodies used in immunoassays is common in sera from RF-positive RA patients and are associated with falsely elevated results in commercial immunoassays. In our cohort, interference was demonstrated in a considerable proportion of samples in the Abbott hCG and 27-plex cytokine assays. Physicians as well as researchers, laboratories and assay manufacturers must be alert to the risk of falsely elevated test results in RF positive RA patients, particularly when results are unexpected or discordant with clinical findings. False test results may interfere with research results, and also lead to potentially harmful diagnostic and therapeutic interventions if unrecognised.References:[1] Bolstad N, et al. Heterophilic antibody interference in immunometric assays.Best Pract Res Clin Endocrinol Metab2013;27(5):647-61.[2] Norli ES, et al. Diagnostic spectrum and 2-year outcome in a cohort of patients with very early arthritis.RMD Open2017;3(2):e000573.[3] Bolstad N, et al. Heterophilic antibody interference in commercial immunoassays; a screening study using paired native and pre-blocked sera.Clinical Chem Lab Med2011;49(12):2001-6.Disclosure of Interests:Johanna Elin Gehin Speakers bureau: Roche, Rolf Anton Klaasen: None declared, Ellen Sauar Norli: None declared, Silje Watterdal Syversen Speakers bureau: Roche, Thermo Fisher, Guro Løvik Goll Consultant of: Novartis, Pfizer, Speakers bureau: Abbvie, Biogen, Boehringer Ingelheim, Orion Pharma, Eli Lilly, Novartis, Pfizer, MSD, Roche, UCB, David J Warren: None declared, Tore K. Kvien Grant/research support from: Received grants from Abbvie, Hospira/Pfizer, MSD and Roche (not relevant for this abstract)., Consultant of: Have received personal fees from Abbvie, Biogen, BMS, Celltrion, Eli Lily, Hospira/Pfizer, MSD, Novartis, Orion Pharma, Roche, Sandoz, UCB, Sanofi and Mylan (not relevant for this abstract)., Paid instructor for: Have received personal fees from Abbvie, Biogen, BMS, Celltrion, Eli Lily, Hospira/Pfizer, MSD, Novartis, Orion Pharma, Roche, Sandoz, UCB, Sanofi and Mylan (not relevant for this abstract)., Speakers bureau: Have received personal fees from Abbvie, Biogen, BMS, Celltrion, Eli Lily, Hospira/Pfizer, MSD, Novartis, Orion Pharma, Roche, Sandoz, UCB, Sanofi and Mylan (not relevant for this abstract)., Kjell Johannes Nustad: None declared, Maria D Mjaavatten Speakers bureau: Pfizer, Abbott, Nils Bolstad Consultant of: Pfizer, Janssen, Speakers bureau: Orion Pharma, Napp Pharmaceuticals, Takeda, Roche, Novartis

Non-coronarogenic causes of increased cardiac troponins in clinical practice

Cardiospecific isoforms of troponins are the most sensitive and specific biomarkers for the diagnosis of myocardial infarction. However, though elevated troponin levels indicate myocardial damage, they do not determine the cause and mechanism of the damage. With the new highly sensitive methods, very minor damages of the heart muscle can be detected. Myocardial damage can occur in many non-coronarogenic diseases. In this review, we discuss the mechanisms of elevation, the diagnostic value of cardiac troponins in the renal failure, tachyarrhythmias, endocarditis, myocarditis, pericarditis, sepsis, neurogenic pathologies (stroke), pulmonary embolism. In addition, we pay attention to the main reasons for a false-positive increase of the concentration of cardiac troponins: heterophilic antibodies, rheumatoid factor, alkaline phosphatase, cross-reactions with skeletal muscle troponins.

Persistent free-floating pelvic trophoblastic cysts following an interstitial ectopic pregnancy

Persistent trophoblast after ectopic pregnancy has been demonstrated at the surgical site or as peritoneal implants. A 37-year-old woman (G5P2) experienced persistently low levels of beta-human chorionic gonadotropin (hCG) after surgical treatment for an interstitial pregnancy. Evaluation for persistent trophoblast, gestational trophoblastic neoplasm and heterophilic antibodies was negative. After 15 months without resolution, she elected for hysterectomy. We found four smooth, freely floating avascular cysts intraoperatively; pathological evaluation identified the cysts as trophoblastic tissue. Serum beta-hCG resolved postoperatively and remained negative at 1 year. Our case demonstrates the novel finding of trophoblastic tissue existing as free-floating cysts in the peritoneal cavity. With appropriate suspicion, these cysts can be identified on radiologic investigation and removed laparoscopically.