6Medications that distortwith particular referencthyroxine

Jim R. Stockigt, MD, FRACP, FRCa,b,* ee

rat

stralia

* Corresponding author. Monash University, Melbourne, Australia.E-mail address: jrs@netspace.net.au (Stockigt).

Contents lists available at ScienceDirect

Best Practice & Research ClinicalEndocrinology & Metabolism

journal homepage: www.elsevier .com/locate/beem

Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767sample dilutionaspirinfurosemidecarbamazepinenon-steroidal anti-inammatory drugsnon-esteried fatty acidsheparinserum total T4

improves sensitivity and specicity of thyroid diagnosis, but theseassays are not impeccable. Estimation of serum free T4 conve-niently accommodates variations in the concentration ofthyroxine-binding globulin (TBG), but no current technique reli-ably reects the in vivo free T4 concentration in numerous othersituations. The effect of circulating competitors that increase T4and T3 in vivo, in particular, many medications, is under-estimatedby current free hormone estimates that involve sample dilution.Non-esteried fatty acids generated during sample storage andincubation can spuriously increase the measured free T4 estimate,especially after in vivo treatment with heparin. These artefacts areunlikely to be overcome by current assay strategies. Total serumT4, corrected for alterations in TBG concentration, gives a morerobust estimate of thyroxine concentration than current methodsof free hormone estimation and should now be reintroduced as thegold standard.

2009 Elsevier Ltd. All rights reserved.Keywords:free T4 interferenceEndocrinologist , Chen-FHead of Immunoassay LaboaMonash University, Melbourne, Australiab Epworth and Alfred Hospitals, Melbourne, AucDorevitch Pathology, Melbourne, Australia1521-690X/$ see front matter 2009 Elsevier Ldoi:10.1016/j.beem.2009.06.004in vitro tests of thyroid function,e to estimates of serum free

PA, Professor of Medicine, ConsultantLim, MAACB, MAgrSci, PhD,ory c

The combination of serum thyroid-stimulating hormone (TSH)with measurement of circulating thyroid hormones greatlytd. All rights reserved.

laboratory parameters, it is highly desirable for results to be interpretable in relation to consensus

reference intervals that do not vary depending on the choice of method. In this article, we considerwhether current assays satisfy these objectives and outline situations in which the results may bepotentially misleading, with particular attention to the in vitro effects of medications. Drugs that alterthe secretion of TSH or thyroid hormones in vivo are excluded, as are the syndromes of thyroidhormone resistance1 and situations in which immunoreactivity of TSH may not accurately reect thebiologic activity of this glycoprotein.2,3

The major part of this review will consider how free T4 and T3 estimates are inuenced by serumconstituents that cause discrepancies between the in vivo hormone level and the apparentconcentration in the assay tube. These effects reect the difculty of estimating free hormoneconcentrations in the presence of proteins that bind numerous other ligands in addition to T4 and T3.Free hormone estimates may be spuriously high if components that displace T4 and T3 from proteinbinding, such as non-esteried fatty acids (NEFA), are generated during sample storage or incubation,a particular problem in heparin-treated patients. Conversely, the measured free hormone concen-tration may be falsely low in diluted samples that contain medications that compete for proteinbinding of T4 or T3.

Assays for TSH can be subject to interference by heterophilic antibodies, which may also distort themeasurement of other analytes. Assays for T4 and T3, either total or free, are vulnerable to interferencefrom circulating auto-antibodies, which produce divergent artefacts in the assay, depending on thetechnique that is used to separate bound from free hormone (see below).

It is important to re-emphasise that all current assays for thyroid hormones and TSH involve thecomparison of known and unknown samples, based on the assumption that the assay signal is solelya function of the concentration of analyte in the sample at the moment of collection. If any of thefollowing three conditions is breached, the assay is likely to give a spurious result.

a. Competition for antibody binding between the assay tracer and free hormone is identical insamples and standards.

b. Any constituent, generated during sample storage and incubation, that inuences the concen-tration of analyte or the assay signal, is similar in samples and standards.

c. Dilution-dependent dissociation of bound hormone is similar in samples and standards.

The impact of misleading individual assay results can be minimised by considering serum T4 andTSH together, taking account of their characteristic feedback relationship and the assumptions andlimitations that underpin this diagnostic relationship.4 While there is no single artefact that concur-rently distorts the results for serum free T4 and TSH to simulate a false diagnosis, the combination ofa low free T4 estimate with suppressed serum TSH is a challenging and ambiguous nding that may beattributable to severe illness, or to the in vivo and in vitro effects of medications. While true centralhypothyroidism can occur as a neuroendocrine effect of critical illness5, a similar combination of resultscan be induced by medications, for example, when high dose furosemide is given together withdopamine or glucocorticoids. The latter two each suppress serum TSH4, while the former displaces T4from its binding proteins6 and also leads to spuriously low estimates of free T4 due to a dilution-dependent in vitro artefact (see below). Whenever the cause of an apparent thyroid abnormality isunclear, it is crucial to take account of medications.6

Effects of sample storage

It is widely accepted that thyroid hormones and TSH are stable when serum is stored at 4 C for upto 1 week.7 Stability on T4 and TSH of lter paper spots used for neonatal screening when stored at 4 C

8Introduction

The aim of in vitro testing of thyroid function is to accurately reect the in vivo activity of keyanalytes such as thyroid-stimulating hormone (TSH), thyroxine and triiodothyronine. For these key

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767754has been conrmed for at least 1 year.

Whether binding proteins remain stable over long periods is less certain, although there is noevidence to the contrary. The storage artefact that is most likely to inuence free hormone estimates isthe generation of NEFA from serum triglyceride, particularly in heparin-treated patients (see below).

Sample carry-over

When serum is sampled on automated assay platforms, sample carry-over has the potential todistort results. This issue is particularly relevant for an analyte such as TSH, where the concentrationmay vary at least 1000-fold between severe primary hypothyroidism and hyperthyroidism. The limitedinformation that is available is reassuring. For example, it was estimated that carry-over in TSH assayswas

antibody interference, assay of total hormone in suspect and control samples after ethanol extraction isdenitive.18

Anomalous protein binding of assay tracer

Labelled analogues of T4, designed to minimise their protein binding in serum, have been used astracers in free T4 assays for over 25 years. That objective is readily achieved for thyroxine-bindingglobulin (TBG), but not for albumin.20 When the labelled analogue is protein-bound to a greater extentin the unknown than in the standard serum, less tracer is available to compete for the assay antibody,giving a falsely high free T4 estimate, as in familial dysalbuminemic hyperthyroxinemia20 and withiodothyronine-binding immunoglobulins.19 Conversely, if serum albumin is subnormal, or its bindingsites are occupied by other ligands21, free T4 estimates by analogue tracer methods tend to be low,becausemore tracer is available in the sample than in the standard. The early analogue free T4methodswere so highly dependent on albumin that the free T4 estimate was virtually zero in euthyroid subjectswith hereditary analbuminaemia.20 In renal failure, analogue assays give low serum free T4 values byup to 40% in pre-dialysis samples21 (see below).

The term T4 or T3 analogue is used in two different senses. The term analogue tracer assay is bestconned to methods where a labelled, chemically modied T4 molecule and antibody are incubatedwith the serum sample; serum free T4 and the labelled analogue compete for a limited number ofantibody sites. In a different sense, labelled analogues may be used to quantify unoccupied solid-phasebinding sites after the serum component has been removed. Two-step assays for free T4, in which

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767756a tracer is added after the serum component has been removed, are generally free of this artefact.

Competitor inuences on measured serum free T4 and T3 concentrations

In contrast to the binding proteins that carry steroid hormones, which show little interaction withother ligands, binding sites for circulating thyroid hormones show broad interaction with NEFAs andnumerous common medications22,23 (Table 1). Displacement of T4 and T3 from binding proteins candistort diagnostic tests and may inuence hormone delivery and clearance. Despite the fact thatimportant drug competitors for T4 binding to TBG have afnities between three orders of magnitude(furosemide) and almost seven orders of magnitude (aspirin) less than that of T4 itself22,23, they maycause signicant displacement of T4. Afnity for the specic binding site is only one of three major

Table 1T4 displacement in vitro by drug competitors at relevant therapeutic concentrations; undiluted serum, 37 C.

Medication aAfnity for TBG(relative to T4 103)

bFree Fractionpercent

cTherapeutic Concentrationumol/l

cT4% displacement(undiluted serum)

Fenclofenac 1.5 0.5 270 90Aspirin 0.03 11 1800 62Meclofenamic acid 8 0.2 60 39Diunisal 0.07 0.7 320 37Mefenamic acid 2.7 0.6 80 31Phenylbutazone 0.04 0.9 320 31Flufenamic acid 0.6 0.2 70 10Diclofenac 3.9 0.5 10 7Carbamazepine 40 44d

Phenytoin 0.3 5.8 80 1345d

Furosemidee 11 1.8 330 530e

Iopanoic acidf 0.1 1.2 200 8Heparin

factors that inuence competitor potency. Total therapeutic concentrations of important drugcompetitors range from almost 2000 mM for aspirin and 330 mM for furosemide.22,23 In addition toafnity and total concentration, the potency of a competitor will be inuenced by its own free fraction,a variable that is highly dependent on assay dilution (see below). Relative displacement of T4 and T3 isgenerally equivalent23, as predicted for two ligands that share the same specic binding sites. Poten-tially important interactions of drug competitors with the T4 binding sites on transthyretin caninuence free T4 values when TBG values are very low, or serum T4 is extremely high.24

Principles of competition

When there is competition between several ligands for a shared specic binding site, competition isa function of their relative free concentrations in that milieu. Assessment of the potency of substancesthat compete for thyroid hormone binding in serum is complicated by the fact that these competitorsare themselves 9099% bound to albumin in undiluted serum. These albumin sites are shared withother ligands; occupancy of those sites becomes an additional key factor in determining competition.

Dilution effects

For highly bound ligands such as T4, it is technically easier to perform free hormone estimates withdiluted serum. In the absence of competitors, such measurements give useful comparisons betweensamples with high, normal and low free hormone concentrations. However, it is a problem inherent inall assays for free T4 and T3, that the free concentrations of hormone and binding competitors do notchange in parallel with progressive sample dilution.23 In reality, it is extremely difcult to establishsystems in which the relative concentrations of free hormone, competitor(s) and unoccupied bindingsites remain in the relationship that applies in vivo.

Mass action dictates that there is dissociation of a bound ligandwith progressive sample dilution, sothat free concentration is at rst well maintained, but then decreases signicantly as the reservoir ofbound ligand becomes depleted.23,25 Because important drug competitors have a much smallerreservoir of bound ligand, their free concentration declines with progressive dilution before the free T4concentration alters.23,25 For a hormone such as T4, with a free fraction of about 1:4000, progressivedissociationwill sustain the free T4 concentration at 1:100 dilution, while a drug that has a free fractionof 1:50 will show a marked decrease in free concentration after only 1:10 dilution. Thus, the effect ofa competitor to increase free T4 can be under-estimated, the error being greatest in assays with thehighest sample dilution. Use of undiluted or minimally diluted serum in ultraltration or equilibriumdialysis systems minimises this artefact.26,27 It should be noted that when undiluted serum is dialysedor ltered against a volume of buffer, that volume should be included in the dilution factor. Asemphasised by Ekins28, the position of the dialysis membrane, within a constant total volume, isirrelevant in determining either the nal free ligand concentration or the effects of dialysablecompetitors.

Co-dilution and pre-dilution

Data on competitor effects are confusing unless details such as dilution and albumin concentrationare clearly dened. The terms pre-dilution and co-dilution are useful in considering potential arte-facts.29 Pre-dilution occurs when the concentration of binding proteins is progressively decreasedbefore test concentrations of competitor are added (Fig. 1). The lower the concentration of albumin, thehigher the occupancy of available binding sites, thus leading to a disproportionate increase in the freecompetitor concentration; this effect magnies competitor potency.30 Thus, when potential compet-itors are added to pre-diluted serum, their effect is prone to over-estimation. For example, in the case ofa highly albumin-bound NEFA, the T4-displacing effect of 5 mM oleic acid in undiluted serum could bematched almost exactly by 0.5 mM oleic acid in serum diluted 1:1031 (Fig. 2).

The reverse artefact is seen with co-dilution, which occurs when whole serum, containing

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767 757a competitor or test additive, is serially diluted so that total concentrations of binding proteins,

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767758Fig. 1. When serum is pre-diluted, followed by addition of a particular concentration of competitor, the effect of the competitor willbe over-estimated as albumin occupancy increases. If a competitor is added to serum, followed by simultaneous co-dilution ofbinding proteins, hormone and competitors, its effect on T4 binding will be progressively be under-estimated with dilution.hormone and competitors diminish in parallel. If a competitor is less highly protein bound than thehormone itself, its potency will be progressively under-estimated with sample dilution.

A co-dilution effect was the clue that led to the recognition of furosemide as an important inhibitorof T4 binding in serum.32 When T4 binding was studied in serial dilution in critically ill

Fig. 2. Effect of oleic acid increments on T4 free fraction with progressive serum dilution (equilibrium dialysis, 37 C, Tris chloridebuffer, pH 7.4). The effect of 5 mmol/l oleic acid in undiluted serum is reproduced by 0.5 mmol/l oleic acid in serum diluted 1:10.(Reference 31).

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767 759hypothyroxinaemic patients, those who had received high-dose furosemide showed amarked increasein free T4 fraction that was obscured with sample dilution (Fig. 3).

The importance of a dilution artefact was demonstrated in comparing the T4-displacing effect offurosemide in three commercial free T4 assays; the effect was most obvious in the method with least

Fig. 3. The furosemide-induced increase in free T4 fraction is progressively lost with sample dilution. (Equilibrium dialysis, 37 C,1 ml serum compartment, 20 ml total volume). Serum dilution of 1:50 is equivalent to 1:1000 dilution of free ligand. (Reference 32).sample dilution33 (Fig. 4). Similarly, therapeutic concentrations of phenytoin and carbamazepineincreased the free concentration of T4 by 4050% using ultraltration of serum that had not beendiluted, while no increase in T4 free fraction was seen (i.e., the free hormone estimate was spuriouslylow) using a commercial single-step free T4 assay after 1:5 serum dilution.27 During continuing drugtherapy, total T4 was lowered by 2550%; measured free T4 concentrations were normal in theultraltration assay and spuriously low in the assay that used diluted serum.27

Fig. 4. Effect of increasing concentrations of serum furosemide on apparent free T4 concentration, by three commercial methodsthat vary in sample dilution. The T4-displacing effect of the competitor is progressively obscured with increasing sample dilution.(Redrawn from ref 33, Hawkins RC. Clin Chem 1998; 44:2550).

0.6 mmol/l. It is well known that NEFA can inhibit serum binding of T4, but the relevance of this

concentrations >3 mmol/l will increase free T4 by displacement from TBG30,36, but these concen-

trations are uncommon in vivo. However, in heparin-treated patients, serum NEFA may increase tothese levels as a result of heparin-induced mobilisation of lipoprotein lipase from endothelium invivo that leads to increased NEFA in vitro during sample storage or incubation36 (Fig. 5). (Samplescollected with heparin are unaffected, as heparin itself has negligible direct effect on T4 binding.) Asa result of this artefact, serum NEFA concentrations at the time of assay can be much higher thanthey were in vivo.37 As shown in Table 2, both incubation for 22 h at 37 C and 7 days storage ofserum at 4 C increases serum concentrations of NEFA to levels that will inhibit T4 binding in serumtaken after full anticoagulant doses of heparin; a similar effect occurs without heparin if serumtriglyceride is markedly increased. This phenomenon could account for some reports of apparentincreases in free T4 and free T3 fraction in critically ill subjects. Because of the incubation at 37 Cthat is involved in equilibrium dialysis and ultraltration methods, it is an exaggeration to regardthese techniques as impeccable gold standard techniques of free T4 estimation, particularly insubjects who have received heparin. Where heparin has been given, assays of total T4 and T3 arelikely to be more informative than free T4 and T3 estimates, unless the serum is treated withadditives to avoid in vitro generation of NEFA.37

The heparin-induced artefact is accentuated if serum triglyceride concentrations are increased, ifphenomenon is complex, for three reasons. First, the competitor potency of NEFA is markedly over-estimated if test concentrations are added to pre-diluted serum31 (see above, Fig. 2). Second, there isthe possibility that the concentration of NEFA present in the assay tube may be greater than the in vivoconcentration, due to generation of NEFA from triglyceride during sample storage and incubation, anartefact is especially important in samples taken during heparin treatment (see below). Third, theT4-displacing effect of NEFA in vivo may not occur from direct interaction with TBG, but by cascadeeffects as a result of the displacement of other ligands from the albumin sites that normally limit theirfree concentration (see below).

The heparin artefact

Heparin treatment in vivo can lead to an unusual in vitro artefact that gives spuriously highestimates of circulating free T4. In the presence of a normal serum albumin concentration, NEFAEstimation of competitor potency in serum under simulated in vivo conditions

The potency of competitors for T4 binding under in vivo conditions can be assessed by equilibriumdialysis of undiluted serum, adding the test substances in amounts that achieve appropriate nalconcentrations of competitor in the serum compartment at equilibrium.31 Addition of each testsubstance was based on its serum binding, measured by a non-isotopic spectrophotometric method inwhich transit of drug from buffer to serum compartment is proportional to serum binding.34 Inundiluted serum at 37 C, in vitroT4 displacement>20% occurs with numerousmedications at relevantconcentrations in the serum compartment at equilibrium (Table 1). Notably, the hierarchy of potency ofvarious competitors at their relevant therapeutic concentrations in whole serum is strikingly differentfrom their afnity for TBG in isolation.29,31 The effect of continuing therapy in vivowill be inuenced bythe kinetics of the competitor (see below). Measurement of the T4 free fraction in undiluted serum islaborious, because even minimal free iodine contamination of the iodinated T4 tracer leads to majorover-estimates of the free thyroxine fraction. This artefact can be avoided by magnesium chlorideprecipitation of authentic tracer in each dialysate in the presence of excess unlabelled hormone ascarrier.35

Competition by non-esteried fatty acids (NEFA)

A diversity of NEFA, each intensely albumin-bound at multiple sites, normally circulates atconcentrations up to 0.5 mmol/l, comparable to the normal serum albumin concentration of about

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767760serum albumin concentration is low, and particularly by prolonged incubation at 37 C (Table 2). Under

propyl-2-furanpropanoic acid (CMPF) , a naturally occurring furanoid acid that accumulates in renal

Fig. 5. Effect of heparin in vitro to increase the apparent concentration of serum free T4. Heparin acts in vivo (left) to liberate

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767 761failure, have the potential to exert indirect or cascade effects on T4 binding in serum. At concentra-tions that caused only minimal direct inhibition of T4 binding in undiluted normal serum, oleic acidand CMPF each augmented the T4-displacing effect of several drug competitors for T4 binding inthese conditions, doses of heparin as low as 10 units may be responsible for in vitro increases in serum-free T4.38 Low-molecular-weight heparin preparations appear to have a similar effect.39

Interaction between competitors

Increasing concentrations of any substance that shares albumin-binding sites with a competitorcould increase the free concentration of that competitor. Both oleic acid40 and 3-carboxy-4-methyl-5-

41

lipoprotein lipase from vascular endothelium. Lipase acts in vitro (right) to increase the concentration of non-esteried fatty acids(NEFA).undiluted serum40,41 (Fig. 6). By such a mechanism, free hormone concentrations can be inuenced bysubstances that have little direct interaction with hormone-binding sites.

Kinetics of the competitor

The kinetics of the competitor itself will determine how it inuences hormone binding in vivo.Competitors of short half-life such as furosemide or salicylatewill show uctuating effects on hormonebinding so that free hormone estimates may vary depending on the time between dosage andsampling.42,43 By contrast, competitors of long half-life, such as phenytoin or carbamazepine27, will

Table 2Effect of heparin, time and temperature on in vitro generation of non-esteried fatty acids (NEFA).

Sample storage Normal subjects Serum NEFA mmol/l Diabetic (Serum Tg >5 mmol/l)

Heparin 10 IU Heparin 25,000 U/day

Thawed from 80 C 0.24 0.03 0.30 0.14 0.77 0.11 0.53 0.234 C, 7 days 0.41 0.04* 0.36 0.15 2.90 1.10*22 C, 4 h 0.17 0.04 1.30 0.67* 1.10 0.33*22 C, 20 h 0.23 0.05 3.29 1.33* 1.31 0.48*37 C, 4 h 0.19 0.05 2.88 1.17* 1.03 0.16*37 C, 22 h 0.35 0.05* 0.48 0.21* 3.49 1.18* 2.23 1.10*Serum was frozen at 80 C after sampling; mean SD; n> 4; *p< 0.05.

result in a new steady state with a lowered total hormone concentration with an increased freefraction, resulting in a normal free hormone estimate.27

Potential binding competitors in critical illness

It has been suggested that free T4 and T3 results could be inuenced in critical illness by circulatingcompetitors for T4 and T3 binding.44 Concentrations of NEFA above 2 mmol/l can inhibit T4 binding,and samples taken during heparin treatment can achieve these levels during storage and incubation37

(see above). Pre-dilution effects may have inuenced the earlier studies that suggested a possible directeffect of oleic acid on T4 binding, tested by addition of oleic acid to diluted serum.45 Conversely,

Fig. 6. Addition of 0.3 mmol/l CMPF, which alone produces minimal inhibition of T4 binding, augments T4 displacement by fourmedications that interact with T4 binding to TBG (undiluted serum, equilibrium dialysis, 37 C). Drugs were added to producerelevant therapeutic concentrations in the serum compartment at equilibrium: furosemide 0.91, fenclofenac 4.7, diunisal 20, aspirin300 umol/l. (Reference 41).

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767762a serum dilution factor of 1:64 (dialysate and diaysand) could have attenuated any potential effect ina study that showed no evidence of a T4-binding inhibitor in sera from a large group of critically illpatients46, that is, there may have been a substantial co-dilution effect. So far, there is a lack of studieswith undiluted sera from critically ill, heparin-free subjects whose drug therapy is fully documented.Sample storage and incubation times would need to be minimised to avoid in vitro generation ofNEFA.37 A further possibility that might inuence tissue delivery of thyroid hormone is the potentialeffect of TBG degradation at the site of sepsis or inammation.47,48

Between-method differences

It is cause for concern that estimates of free T4 show large between-method differences in threesituations where thyroid function is difcult to assess on clinical grounds alone: critical illness, renalinsufciency and late pregnancy. By contrast, total T4 measurements in these situations givepredominantly normal results (see below).

Critical illness

Marked method-dependent differences in apparent free T4 concentration have been demonstratedin some studies of critical illness. Sapin et al.49 used six commercial free T4 kits to study 20 previouslyeuthyroid subjects in a clearly dened example of critical illness, 7 days after bone marrow trans-plantation, during multiple drug therapy, including heparin and glucocorticoids (Fig. 7). Methods thatinvolved sample incubation at 37 C gave high free T4 values in 2040% of their study subjects, while

Fig. 7. Free T4 estimated by six different methods in 20 previously euthyroid bone marrow recipients on the seventh day aftertransplantation. Free T4 estimates are increased or decreased, depending on the type of assay. Serum total T4 remained normal in 19of the 20 subjects, while serum TSH was subnormal in 11, independent of the method used. Therapy included heparin andglucocorticoids at the time of sampling. Mid-reference ranges for each method have been normalized to 100%. (Re-drawn from ref49, Sapin R et al, Clin Chem 2000; 46:41820).

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767 763analogue tracer methods that are inuenced by tracer binding to albumin, gave subnormal estimates offree T4 in 2030%. By contrast, total T4 was normal in 19 of these 20 presumably euthyroid subjects.Serum TSH was 10-fold above normal.When organic acids that are retained in renal failure were added to normal serum, the apparent free T4concentration diminished using an analogue tracer method, increased in an equilibrium dialysis methodand was unaltered with a labelled antibody method.21 All three methods showed an apparent rise infree T4 after heparin treatment for haemodialysis, consistent with an effect of NEFA generated duringsample incubation.30,36 The effects on apparent free T3 were similar, or more marked.21

Pregnancy

The demonstration that maternal hypothyroxinaemia in the rst trimester can have an adverseeffect on later psychomotor development has focussed attention on precise assessment of thyroidfunction in pregnancy; current uncertainty in the interpretation of free T4 estimates in pregnancy is

unhelpful, especially as serum TSH shows trimester-dependent variations. While it is clear that normalpregnancy is associated with a marked increase of about 40% in total T4 concentration, there is noconsensus as to whether free T4 is maintained at normal levels51, or whether the mean free hormoneconcentration decreases slightly, with values still predominantly within the normal range.52 Inter-pretation is complex because, particularly in late pregnancy, there are marked discrepancies between

Fig. 8. Comparison of three types of serum free T4 estimate, pre and post maintenance haemodialysis, in 11 uremic subjects.Horizontal bars indicate the median, the boxes the range from 25 to 75%. Estimates of free T4 by an analog tracer method were lowerthan by equilibrium dialysis. Apparent free T4 by all three methods increased after heparinization for hemodialysis. Concentrationsof three organic acids, indoxyl sulphate (IS), indole acetic acid (IAA) and hippuric acid (HA) were diminished by hemodialysis, butremained >10-fold above normal. The effect on apparent free T4 of adding these organic acids to normal serum, varied widelybetween methods (see text). (Re-drawn from ref 21, Iitaka et al. Clin Endocrinol 1998; 48: 73946).J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767764various methods of estimating free T4, with strong negative bias in some methods.53,54 In a compar-ative study of free T4 by seven commercial methods in 23 euthyroid women at term, Roti et al. foundthat albumin-dependent methods showmarked negative bias, with up to 50% subnormal values, whileother methods gave values above their non-pregnant reference interval.53

A recent study that conrmed marked negative bias in free T4 estimates during pregnancy hasquestioned the wisdom of continuing to rely on free thyroxine estimates during pregnancy.54 Incontrast to two-kit free T4 methods, total serum T4 and its derivative, the free thyroxine index, showedthe anticipated inverse relationship with serum TSH, with historically consistent results in numerousreports.54 Thus, because of consistency between methods, total T4 measurement may be superior tofree T4 estimation as a guide to therapy during pregnancy, provided that the reference values takeaccount of the normal oestrogen-induced increase in TBG. If free T4 estimates continue to be used inpregnancy, clinicians will need to interpret results in relation to reference intervals that are bothtrimester and method specic.

The reasons for wide method-dependent discrepancies in free T4 values in pregnancy remainunclear. Albumin is lower and serum triglyceride and NEFA concentrations increase in late preg-nancy55, phenomena that can inuence estimates of free T4. The possibility of a pregnancy-relatedcirculating inhibitor of thyroid hormone binding that is undetected in diluted samples may meritfurther study.

Implications for free and total hormone assays

It is uncertain whether the limitations of current free T4 methodology, as outlined above, are likelyto be overcome by further technical innovations. Whether free T4 is estimated by two-step methodsthat separate a fraction of the free T4 pool from the binding proteins before the T4 assay is performed,

or by one-stepmethods designed to give a signal that is proportional to the free T4 concentration in thepresence of binding proteins, the frequency of non-specic abnormalities has become a major disad-vantage of these techniques. One-step methods generally become invalid when the sample andstandard differ in their binding of assay tracer20,25, but two-step methods are generally free of thisartefact. However, both types of method are vulnerable to artefacts that result from generation of NEFAduring sample storage and incubation and both fail to reect the in vivo effect of binding inhibitors,which is obscured by sample dilution. Especially in samples from heparin-treated patients, generationof NEFA during sample storage and incubation can be sufcient to displace T4 directly or to augmentthe effect of other competitors by cascade effects. The heparinNEFA effect may remain an insuperablepre-analytical problem, unless a simple, non-toxic additive can be developed that will block heparin-induced lipase from the moment of sample collection.

The problems documented here have not yet been addressed in a recently developed free T4 andfree T3 methods56, regarded as a major advance in the routine assessment of free thyroid hormones.57

The method uses on-line solid phase extraction liquid chromatography/tandem mass spectrometryto measure the free T4 and T3 in protein-free dialysate after 20 h dialysis of serum against an equalvolume of buffer at pH 7.2 (effective dilution 1:1), using a 96-well dialysis plate. Assay standards areprepared in protein-free buffer. Detection limit, internal quality controls and assay precision methodsappeared to be satisfactory, but this technique gave consistently higher values than establishedmethods. On that basis,method-specic reference intervals would need to be developed. Assessment ofpotential complexities related to heparin-induced lipolysis, pregnancy or critical illness has not beenreported.

There has been a general trend to regard equilibrium dialysis methods, however cumbersome, asthe gold standard for free T4 estimation. The artefacts summarised here suggest that this view is no

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767 765longer tenable and that total T4 measurement is the more robust parameter. Notably, denitive studiesof the pituitarythyroid axis in critical illness58,59 have relied on total rather than free T4 as thepreferred index of thyroid hormone secretion.

Whatever techniques are developed to achieve precise laboratory assessment, diagnostic accu-racy cannot be assured without collaboration across the laboratoryclinical interface. Numerousdiagnostic errors can occur if laboratory results are classied without regard for the clinicalcontext.60

Clinical points

Free T4 estimates are unreliable in several common diagnostically difcult situations. Because free T4 reference intervals vary widely between methods, interpretation requiresmethod-specic ranges.

Total serum T4 shows much less variation between methods. When corrected for variationsin TBG and interpreted in relation to TSH, total T4 is the more reliable parameter in situationsof diagnostic difculty.

The medication history is crucial whenever tests of thyroid function are anomalous, or whenthe sick euthyroid syndrome is considered.

Research agenda

Why do many free T4 estimates show strong negative bias in late pregnancy? Is thisphenomenon due to a circulating binding inhibitor that is obscured by sample dilution?

Can an effective non-toxic additive be developed that will block the in vitro action of heparin-induced lipase that causes spurious NEFA-induced elevation of free T4?

Can technology for rapid ultra-ltration of undiluted serum be developed for routine use?

J.R. Stockigt, C.-F. Lim / Best Practice & Research Clinical Endocrinology & Metabolism 23 (2009) 753767766References

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Medications that distort in vitro tests of thyroid function, with particular reference to estimates of serum free thyroxineIntroductionEffects of sample storageSample carry-overHeterophilic antibody interferenceEndogenous T4- and T3-binding antibodiesAnomalous protein binding of assay tracerCompetitor influences on measured serum free T4 and T3 concentrationsPrinciples of competitionDilution effectsCo-dilution and pre-dilutionEstimation of competitor potency in serum under simulated in vivo conditionsCompetition by non-esterified fatty acids (NEFA)The heparin artefactInteraction between competitorsKinetics of the competitorPotential binding competitors in critical illness

Between-method differencesCritical illnessRenal failurePregnancy

Implications for free and total hormone assays

Research agendaReferences