Despite the benefits of using immunoassays in clinical laboratories, including high specificity and sensitivity and short turnaround time, various types of interference can lead to inaccurate results that cloud clinical decision-making. To minimize the effect of assay interferences, manufacturers have adopted measures such as package insert warnings and blocking agents to reduce nonspecific binding.
Immunoassays of thyroid function have improved considerably over the years. For example, third-generation thyroid-stimulating hormone (TSH) assays have a functional sensitivity of 0.01 mIU/L compared to 1.0 mIU/L with first-generation assays. However, interferences remain a significant problem in thyroid function testing, and although the clinical laboratory is responsible for correctly reporting these, this “task proves difficult because the interferences may be unique to an individual and change over time, inducing false positive or false negative results,” according to a review published in Endocrine Reviews.
The researchers focused on describing interferences that commonly affect immunoassays of TSH, free thyroxine (T4), and free triiodothyronine (T3). Findings were based on a review of >100 studies published between 1981 and 2017.
- MacroTSH: “Unlike TSH, which is a small bioactive hormone of 28 kDa easily filtered by the kidney, [macroTSH] is a large molecule of at least 150kDa that likely accumulates in the circulation, resulting in falsely increased TSH levels,” wrote the researchers. It is mostly composed of immunoglobulin G-bound TSH and is likely inactive.
- Biotin and anti-streptavidin antibodies: In immunoassay platforms based on biotin-streptavidin interaction, inaccurate measures of thyroid function may occur in patients receiving biotin supplements or in the presence of anti-streptavidin antibodies. A misdiagnosis of Graves’ disease, for example, can occur from interference by anti-streptavidin antibodies.
- Anti-ruthenium antibodies: These antibodies may lead to false results in systems that use ruthenium labels. Screening for their presence is important to limit unnecessary additional tests and inappropriate drug prescriptions.
- Thyroid hormone autoantibodies, primarily against T4 and T3: “[Immunoglobulin G] isotypes with a polyclonal autoreactive response” and a prevalence of up to 40% in patients with autoimmune disorders. “In the absence of [thyroid hormone autoantibodies], the labeled tracer and free hormones in the sample compete for binding sites on the capture antibody,” as explained in the paper. “In the presence of anti-T4 and anti-T3 [thyroid hormone autoantibodies], however, autoantibodies may bind to both the measured analyte and labeled tracer, thereby skewing the true concentration of thyroid hormones.”
- Heterophilic antibodies: These can cause false results by interfering with various sites within the assay process and should be clearly indicated in patient files because of potentially prolonged effects.
Clinical effect of false results
The review also revealed the clinical effect of interference in thyroid immunoassays, based on 150 patient cases described in the literature reviewed. The authors observed a negative clinical effect in approximately 50% of cases, most commonly being the unnecessary prescription of L-thyroxine (37%), superfluous use of thyroid-releasing hormone stimulation tests (16%) and thyroid scans (15%), and the unnecessary prescription of antithyroid drugs (12%).
In some cases, the interference was discovered years after the inappropriate treatment was initiated, and patients experienced adverse treatment-related effects. The paper further notes the possibility that assay interference may cause falsely normal results in patients who actually have thyroid dysfunction. However, it is less likely that these cases will be detected.
“Discrepancies between assay values and previous results obtained with the same test, as well as other biochemical parameters or clinical setting are paramount in the suspicion and detecting process of an interference,” the researchers stated. Familiarity with the patient’s clinical history is also important, as “certain patients are more prone to developing an interference, be it because of recent immunization, transfusion, autoimmune disease, monoclonal therapy, or contact with pets.”
These interferences can often be identified through techniques including assay method comparison, dilution, and blocking reagents studies. The combined use of these methods is sufficient to identify antibody interference in 90% of suspected cases. Polyethylene glycol precipitation is an additional technique that may be used.
Endocrinology Advisor interviewed Christian Nasr, MD, medical director of the Thyroid Center at the Cleveland Clinic, Ohio, for further discussion about thyroid interference on immunoassays.
Endocrinology Advisor: What are the potential effects of interference on immunoassays?
Dr Nasr: There are many potential negative repercussions of assay interference that may affect patients and healthcare systems through inaccurate diagnosis, unnecessary testing, and/or inappropriate management. The clinician may be fooled by such an abnormal test, which may lead to further diagnostic testing — biochemical, immunological, or imaging — that would be otherwise unnecessary. Such testing may add to the confusion and make the clinician consider unlikely diagnoses. Such unnecessary cost may accrue exponentially, and may lead to erroneous and potentially dangerous treatment decisions.
Endocrinology Advisor: How can physicians minimize these issues in clinical practice?
Dr Nasr: First, the clinician needs to be educated about these possible scenarios. Such education could take place in different venues, including medical society meetings, local conferences, lab communications, and staff meetings, for example. The lab director should be available and ready to discuss possible causes of assay interference. Next, the laboratory will need to know which reference lab is able to do further testing on the samples and communicate with the ordering provider.
Endocrinology Advisor: What are additional recommendations for clinicians?
Dr Nasr: Providers should simply use their clinical acumen and always suspect possible assay interference when the test results do not correlate with the clinical situation. The most challenging scenario is when there is a true lab abnormality along with the assay interference. The same cautious approach should be followed in this case. Providers should always communicate with available experts including their lab personnel.
Favresse J, Burlacu MC, Maiter D, Gruson D. Interferences with thyroid function immunoassays: clinical implications and detection algorithm. Endocr Rev. 2018;39(5):830-850.