Questionable High Free T4 Concentrations: When Confirming against an Alternative Method Is Not Enough 

free t4, immunoassay interferences, familial dysalbuminemic hyperthyroxinemia

Case Presentation

A 60-year old male was referred to the Endocrinology Department due to confusing results of thyroid tests during a routine medical check-up. The asymptomatic patient presented with increased free thyroxine (FT4) concentrations, and thyroid-stimulating hormone (TSH) and total thyroxine (T4) concentrations within the reference range (Table 1), with no tachycardia, distal tremor, visual impairment, or any other symptoms of thyrotoxicosis. He presented with a 10-kg weight gain over the previous few months (body mass index 27.7 kg/m²), which was attributed to changes in dietary and exercise habits due to the mobility restraints related to control of COVID-19 dissemination. Physical examination revealed neither goiter nor signs of orbitopathy, and there was no personal or family history of thyroid disorders. His usual medications were hydroxyzine, zopiclone, famotidine, and clonazepam. The patient’s clinical condition could not explain the high FT4 results with TSH within the reference range, which opened the possibility to consider unusual clinical situations.

Table 1

Analytical results in the different laboratories.

		Lab 1				Lab 2		Lab 3		Lab 4
		Elecsys COBAS (Roche)				Centaur (Siemens)		Centaur (Siemens)		Vitros (Ortho)
Test	Units	Reference range	2016	July 2021	July 2021 After HBTa	Reference range	May 2021	Reference range	July 2021	Reference range	July 2021
TSH	µU/mL	0.3–4.2	1.44	0.75	0.77	0.4-4.5	0.6	0.55–4.78	0.647
FT4b	pmol/L	12–22	29.57	32.3	35.08			11.4–22.6	36.7	10.0–28.2	10.0
FTIc						1.4–3.8	4.8
T4	nmol/L	66–181		148	182	63–135	167	58–140	171
FT3d	pmol/L	3.1–6.8	4.6		5.18	3.5–6.5	6.0	3.5–6.5	6.5
T3	nmol/L	1.3–3.1		1.55	1.54	1.2–2.8	1.9	0.9–2.7	1.7
Albumin	g/dL	3.2–5.2		4.95
SHBGe	nmol/L	20.6–76.7		30.3
ACEf	UI/L	13.3–63.9		5

Results in bold means “Out of reference range”.

^a

Heterophilic blocking tube.

^b

Free thyroxine index.

^c

Free triiodothyronine.

^d

Triiodothyronine.

^e

Sex hormone binding globulin.

^f

Angiotensin converting enzyme.

Initial results were confirmed in a new sample, so his endocrinologist consulted the biochemistry laboratory because of this lack of consistency with clinical presentation. Analytical records revealed similar results in 2016. Additionally, the patient provided analytical results from an outside laboratory, performed 2 months earlier using a different test method (Table 1, Lab 2), which showed TSH, total and free triiodothyronine within the reference range, but with both FT4 index and total T4 increased.

Albumin concentration, as a T4 carrier protein, was also evaluated and found within the reference range. Sex hormone-binding globulin and angiotensin-converting enzyme activities, whose expressions are upregulated by thyroid hormones, remained within the reference ranges (1, 2) (Table 1). No structural alteration was observed in pituitary gland by sella magnetic resonance imaging. All these analytical data and the absence of clinical symptoms suggested a potential interference in FT4 and total T4 measurement.

Discussion

Serial dilutions to corroborate linearity are commonly used to detect analytical interference in immunoassays. This, however, was not an option here since dilution affects the equilibrium between free and protein-bound T4.

FT4 is quantified in our laboratory by electrochemiluminescence using an Elecsys module (Cobas 8000 autoanalyzer, Roche Diagnostics) with a 1-step competitive configuration, including biotin-bound T4 as the marker and a ruthenium-labeled anti-T4 antibody (Table 2). Two possible types of interference—biotin or heterophilic antibodies—were considered (3). Endogenous increased biotin concentrations could compete with the biotin-coupled T4 for the streptavidin-coated particles, rendering falsely increased measured FT4 concentrations. However, the patient denied having taken biotin supplements.

Table 2

Competitive designs for FT4 measurement in the different autoanalyzers used in the study.

	Elecsys (Roche)	Centaur (Siemens)	Vitros (Ortho)
Design	One step	One step	One step
Support	Streptavidin-coated paramagnetic beads	Avidin-coated paramagnetic beads	Vessel coated with ligand
Capture	Biotin-bound T4	Biotinylated polyclonal rabbit anti-T4 antibody	Vessel coated with ligand
Detection	Ruthenium-bound polyclonal sheep anti-T4 antibody	Acridinium ester-labeled T4	HRPa-labeled sheep anti-T4 antibody

^a

Horseradish peroxidase.

To check potential interferences related to heterophilic antibodies, the sample was reanalyzed after pretreatment in heterophilic blocking tubes (Scantibodies Laboratory). The results remained unchanged with the exception of total T4, which was slightly increased, similar to the Lab 2 results (Table 1). Although unchanged FT4 concentrations after pretreatment do not completely rule out the presence of heterophilic antibodies, the probability of an interference of this type is reduced.

The sample was analyzed by a third laboratory, also using a Siemens Centaur analyzer (Table 2, Lab 3). Similar to the Roche assay, the Centaur assay uses a competitive format, differing mainly in the origin of anti-T4 antibody and the molecule that originates the luminescent signal. Both free and total T4 concentrations were increased (Table 1, Lab 3). This eliminated both antiruthenium and antisheep antibodies as the cause of the interference in the Elecsys method.

An increased FT4 concentration was more noticeable than that of total T4. A T4 binding alteration can be due to T4 displacement from its binding sites by drugs and endogenous molecules or by mutations in binding proteins. The pharmacological history of the patient failed to reveal any drug capable of causing such a displacement. Fatty acids, which can also displace hormones from their binding to proteins, were considered not interfering since triglyceride concentrations had been consistently within the reference range for years. Consequently, potential mutations of the albumin gene (ALB) were investigated by next-generation sequencing, with an R218H missense mutation being detected in heterozygosity. The R218H is the most common mutation in familial dysalbuminemic hyperthyroxinemia (FDH), a benign autosomal dominant condition where this amino acid change provokes a higher albumin affinity for thyroid hormones (4). This can increase the percentage of T4 transported by albumin by up to 30%, which results in increased total T4 concentrations with TSH values within the reference interval (5). Consequently, FDH could possibly explain the total T4 and TSH concentrations in this patient but not the increased FT4 concentrations. Free triiodothyronine concentrations can be increased in FDH caused by the less prevalent mutation (L66P) (3), not documented in this patient. This also agrees with the free triiodothyronine concentrations within the reference range in this patient.

The gold standard for free thyroid hormone measurement is equilibrium dialysis by which free and protein-bound hormones are physically separated (6). However, given that the process is complex and time-consuming, automated immunoassays are the most widely used techniques in clinical laboratories. These immunoassays do not involve physical separation, and antibodies sequester only a minimum part of free hormone to preserve the equilibrium between free and bound hormones. Nevertheless, they can be affected by issues specifically affecting thyroid hormone–binding proteins or general interferences common to immunoassays (3). The higher affinity with albumin in patients with FDH could thus affect FT4 concentrations measured using certain immunoassays, provoking substantial differences in their results depending on assay design (7).

In competitive assays, endogenous hormone and marked analogue compete for a limited quantity of antibody. Mutant albumin in 1-step competitive assays can bind more FT4 and labeled analogue than usual, due to its increased affinity, reducing the amount of analogue available to bind to the antibody. This leads to a weaker signal and higher measured results. In 2-step competitive designs, albumin is washed away prior to analogue addition, and therefore results should be less affected by this analytical interference. Our laboratory, and both Labs 2 and 3, used 1-step FT4 assays and obtained similar results, which was consistent with the concordance observed in patient’s results. Nevertheless, one group demonstrated that the Vitros (Ortho Diagnostics) 1-step competitive FT4 assay generated much lower concentrations of FT4 in patients with FDH who had the R218H mutation, suggesting that other factors may be involved in the differences (7).

One of these factors seems to be the buffer chloride content. Chloride concentrations have been reported to increase FT4 measurement by equilibrium dialysis, especially in patients with FDH, due to the inhibition of T4 binding to albumin (8). Another study analyzed FT4 concentrations by symmetric dialysis with different chloride conditions mimicking those used in some autoanalyzers, in sera from patients with FDH, and in a serum pool from healthy controls (9). Chloride concentrations minimally affected FT4 measurement in the control group. However, varying the chloride concentration provoked important changes in the serum of patients with FDH, which present a much higher T4 percentage bound to albumin. In the absence of chloride, symmetric dialysis rendered results similar to those of Vitros analyzer, known to use no chloride, whereas with high chloride concentration, FT4 concentrations were much higher, similar to those obtained by other analyzers using high chloride buffers. However, buffer influence must not be limited to chloride content, since other components such as pH or detergent composition and concentration may also alter T4 binding to albumin, or other parameters involved in T4 measurement. Similarly, the differences between analyzers in terms of nature of the exogenous ligand and whether it is immobilized may also contribute to explain the differences observed in FT4 concentrations. In this sense, it is important that manufacturers detail the composition of reagents due to their potential influence in the results and their consequent interpretation.

The IFCC established the separation of FT4 by equilibrium dialysis or ultrafiltration followed by detection by mass spectrometry as a reference measurement procedure (6). However, methods using equilibrium dialysis/ultrafiltration followed by mass spectrometry–based measurement are not interchangeable. In addition, it should be considered that these procedures are not routinely available in clinical laboratories.

In light of the previous discussion, we requested the evaluation of the patient’s FT4 concentrations by another laboratory (Lab 4), employing a Vitros analyzer. As expected, FT4 concentrations were in the lower part of reference range, which more closely agreed with the clinical condition of the patient and his TSH status (Table 1).

An antithyroid treatment may be inappropriately indicated in patients with FDH based on an increased FT4 result (10). However, the decision to initiate antithyroid treatment should incorporate TSH results and clinical symptoms. The patient was informed about these results indicating that no treatment was necessary to correct his FT4 concentrations.

To conclude, it is important to carefully investigate incongruent FT4 concentrations in relation to TSH and/or the clinical conditions, ruling out potential sources of interference including testing for FDH. An alternative methodology should be carefully examined to corroborate the results to avoid a similar analytical problem.

Questions to Consider

1. What can interfere with thyroid hormone immunoassays?

2. Which are the strategies to rule out their presence?

3. How can mutations in the ALB gene affect thyroid hormone concentrations?

Points to Remember

• In FDH, mutations in the ALB gene lead to increased total T4 concentrations due to the increased affinity of albumin for thyroid hormones.

• Although there is no physiological increase in FT4 concentrations, falsely increased concentrations can be observed, depending on the immunoassay used.

• When looking for an alternative method to confirm analytical results, methodologies should be examined in detail, since they may share potential sources of interference.

• Communication between analyst and clinician can help to unravel the causes behind incongruent results.

Nonstandard Abbreviations

T4, thyroxine; FT4, free thyroxine; TSH, thyrotropin; FDH, familial dysalbuminemic hyperthyroxinemia.

Human Genes

ALB, albumin.

Author Contributions

All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.

Employment or Leadership

Á. González is a member of the Spanish Society for Laboratory Medicine. E. Alegre, Scientific Advisory Committee of Jose Luis Castaño Foundation from the Spanish Society for Laboratory Medicine.

Consultant or Advisory Role

None declared.

Stock Ownership

None declared.

Honoraria

None declared.

Research Funding

None declared.

Expert Testimony

None declared.

Patents

None declared.

Other Remuneration

Á. González and E. Alegre received funding to attend Euromedlab 2021 from Roche Diagnostics.

Author notes

Authors’ Disclosures or Potential Conflicts of Interest:Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: