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Clinical Chemistry 47: 1353-1363, 2001;
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(Clinical Chemistry. 2001;47:1353-1363.)
© 2001 American Association for Clinical Chemistry, Inc.

Reviews

Direct and Indirect Free Thyroxine Assay Methods: Theory and Practice

John E.M. Midgleya

aNorth Lakes Clinical, 6 High Wheatley, Ilkley, West Yorkshire LS29 8RX, United Kingdom. E-mail je.midgley{at}virgin.net.co.uk.


   Abstract
Top
 Abstract
The Theory Underlying Free...
 Early Approaches to FT4...
 Direct FT4 Immunoassays
 Summary
 References
 
Background: For the diagnosis of thyroid disease, measurement of "free hormone" is generally accepted as an appropriate measure. However, valid assays measuring the free fraction of thyroxine (FT4) ideally must perform without bias, despite large variations in the concentrations and affinities of serum T4-binding proteins in the population. Several approaches have been taken to overcome such bias, and these have created considerable controversy in the field over the past decade.

Approach: This review, from both a historical and an analytical standpoint, charts the progress made over more than 30 years in improvements to the performance of assays in common use for the measurement of FT4 in serum or plasma. It reexamines the theory behind early approaches to such assays [for example, the free thyroxine index (FTI) method], that preceded more accurate, two-step immunoassays or one-step analog techniques. It evaluates the continuous refinements to the latter assays that by now have largely supplanted the FTI approach and where the deficiencies that so exercised clinical chemists in the past have been virtually eliminated in the leading assays.

Content: The basic Mass Action theory underpinning all such methods is discussed by assessing how far each particular approach obeys the criteria the theory imposes. In this, it is not the intention of the review to dissect individual commercial or academic assays, but rather to give guidance where appropriate as to how any assay said to measure FT4 can be conveniently evaluated by those intending to use it. Examples are given where inappropriate tests may wrongly imply assay invalidity by misinterpreting how FT4 assays work.

Summary: Detailed knowledge of the underlying theory is essential when devising tests for direct FT4 assays, to ensure that such tests do not overstep the practical limits of assay validity.


   The Theory Underlying Free Thyroxine Assays
Top
 Abstract
 The Theory Underlying Free...
Early Approaches to FT4...
 Direct FT4 Immunoassays
 Summary
 References
 
general background
For the diagnosis of thyroid disease, the "free hormone" concept (1) is generally accepted [with exceptions (2)(3)(4)] as an appropriate measure. This implies that the supply of thyroxine (T4) 1 or triiodothyronine (T3) into cells is governed by their unbound (free) concentrations in serum rather than the protein-bound fractions. Valid assays measuring the free fraction of T4 (FT4) ideally must perform without bias, despite large variations (both absolute and relative) in the concentrations (and affinities) of serum T4-binding proteins found in the population.

No assays measure the actual unbound molecules of T4 in serum. Regardless of the probe used, be it a dialysis membrane, a nonspecific binder, or an antibody, unbound T4 is abstracted or dissociated in excess of the original unbound hormone in the sample. This does not matter as long as the amount of hormone sampled is so small a fraction of the total (bound and unbound) that the equilibrium between bound and free forms in serum is minimally disturbed.

For a euthyroid subject with serum binding protein concentrations within the appropriate reference intervals, ~0.01–0.02% of the total serum T4 is present as FT4. The Mass Action relationship at equilibrium between the two fractions of bound and free hormone for a single protein is:

where Kequil is the equilibrium binding constant of the protein for T4.

This equation transforms to:

Consequently, the measured FT4 concentration will remain essentially unaltered, provided that the ratio of occupied/unoccupied sites holds virtually constant. Hence, measurements of FT4 are valid only with minimum dissociation of bound T4 from the serum proteins. From the known equilibrium constants for the three serum binding proteins, thyroxine-binding globulin (TBG), transthyretin, and albumin (5)(6), a maximum of 5% of the protein-bound T4 in serum can be dissociated into the free form, but preferably much less than this (7).

Any valid assay must encompass the whole physiologic range of serum binding protein concentrations. This can be defined as the ideal "window of validity" for any FT4 assay. Within this window, no serum presented to the assay is affected (with regard to disturbance of the bound-free equilibrium for T4) so as to be significantly influenced by either the T4 sampling process or by procedures that might otherwise affect the equilibrium. Minimization of disturbance is especially difficult for sera with very low binding capacities for T4 (e.g., with low concentrations of TBG and/or albumin). When operating outside their windows, assays no longer measure FT4. Various methods adhere to the above requirement to different extents, and even within any given common technology, individual assays may differ detectably in their compliance. Hence, uniquely in immunoassay technology, the mode of operation and window of validity of any given FT4 assay (or its equivalent) are individually defined, so that subtle differences arise in the assay’s performance when compared with any other. Several factors cause this. These do not merely concern arbitrary judgments by commercial manufacturers, or perhaps lack of awareness, as to how extensively the wide variations in serum in the human population should be examined during development of their assays. They also include differences in concentration, specificity, and affinity for T4 of any antibodies used; the extent of serum dilution by assay ingredients; the choice of assay reagents such as buffers to mimic physiologic conditions; the choice and concentration of analog or competitive binding reagent; and the time and temperature of the assay incubation process (7)(8)(9)(10)(11)(12)(13)(14).

Given complexity of this magnitude and the failure of many producers to fully grasp its implications, it is unsurprising that, even after 20 years of development, individual FT4 methods still vary considerably in performance (i.e., their windows of validity fall short of the ideal). However, regardless of these differences, routine laboratories can perform useful investigations to test the validity of each assay method. This review, in choosing the most commonly used methods for the measurement of FT4 in serum, aims to reevaluate their validity and to suggest ways in which the limits of this validity can be simply tested. Individual named assays will not be examined in detail. There are too many to be evaluated in a limited space, and the continual appearance and replacement of commercial assays would render any review rapidly obsolete.

The following analysis of methods for measuring FT4 includes an examination of the T3-uptake test [and thus the indirect free thyroxine index (FTI) approach], and explains why shortcomings in performance can occur (9)(15)(16)(17)(18). Direct one- and two-step immunoassays are then considered, comparing these in concept and performance with assays ranging from the FTI to the so-called "gold-standard" methods of equilibrium dialysis and ultrafiltration.

The direct one-step assays have recently been criticized as both failing to correctly measure FT4 concentrations in protein-free and other artificial solutions and to obey classic tests for recovery of analyte (19)(20)(21)(22)(23)(24). Many of these experiments have, however, been conducted outside the window of validity of the assays. For example, if the antibody in valid FT4 assays can be allowed to bind 0.01–1% of the total hormone in a serum sample, recovery estimations, in the classic sense, have no meaning. It is not surprising, therefore, that studies of artificial solutions containing serum T4-binding proteins and T4 showed apparent correlations of FT4 with binding protein concentrations (19)(20)(21)(22)(23)(24). These tests were also performed under conditions in which the equilibrium between bound and free hormone was sufficiently perturbed to disobey the essential criterion for a valid assay. Such experiments, in which (unlike the case with equilibrium dialysis or ultrafiltration) the serum binding proteins, their T4 load, and the antibody probe interact in solution, teach the need for caution in devising suitable tests for assay validity.

important factors affecting ft4 measurements
As emphasized earlier, the primary rule for valid measurement of FT4 in any assay is that, by the assay process, serum protein-bound T4 is minimally displaced into the free phase, with insignificant disturbance of the endogenous bound-free equilibrium for T4 in the sample (7)(9). The permissible amount of hormone so displaced can be many times that of the concentration of endogenous FT4 (7)(9). Disturbances of the equilibrium can take several forms. T4 can be sequestrated, through the free fraction, onto the binding sites of a probing antibody. Simultaneously, the bound hormone is further partially dissociated simply by dilution of the serum when assay ingredients for FT4 measurement are added. In gold-standard methods, such as equilibrium dialysis, dilution is the key function to control, because the serum is first dialyzed before the separated dialysate is assayed for T4 by a sensitive total-hormone assay (25)(26)(27). Conversely, in direct one- or two-step immunoassays for FT4, both dilution of serum and simultaneous sequestration of T4 by the antibody probe are equally relevant. These influences are, in the latter case, approximately additive. If the antibody sequestrates 1% of the total T4 from a sample containing 0.02% free in the original serum, this is equivalent to a dilution of 50-fold. If the addition of assay ingredients to the sample dilutes it by 10-fold, then the overall effect is approximately equivalent to a 60-fold dilution. If, however, the original serum sample was first diluted 2-fold (e.g., to conduct a dilution test), then the fixed amount of antibody will remove ~2% of the total hormone from the serum proteins and this, combined with the 10-fold dilution of the assay process itself, will approximate to a 110-fold dilution. Equivalent effects occur in a serum naturally containing one-half the normal binding capacity for T4 (i.e., sera with lower endogenous binding protein concentrations should act in a given assay as if a serum with concentrations within the reference intervals had been diluted appropriately).

In some FT4 immunoassays, one of the assay ingredients may be albumin, which was initially added to modulate the residual binding of analog to the serum T4-binding proteins (7) but was subsequently used to mitigate the effects of substances such as nonesterified fatty acids (NEFAs) on T4 binding to the serum binding proteins. However, exogenous albumin may also distort (lower) true serum FT4 values by its own sequestration of T4 from the serum equilibrium system. (A detailed discussion will be presented later in the text.)

For the earlier T3-uptake test used in determining FTI, two simultaneous influences are at work. One is the potential of the competing binder (often nonspecific), used to bind the excess T3 not bound to the serum binding proteins, to also bind T4 (18). The other is the potential for the high concentration of the T3 label, necessary to saturate the unoccupied binding sites, to dissociate some of the bound T4 from its bound sites on the serum proteins by direct competition. From Mass Action equations, relatively little of the bound T4 need be dissociated before there is unacceptable distortion of the FTI result away from a valid FT4 measurement.

Finally, factors either endogenous in the sample in vivo or developed in vitro during assay incubation may interfere with T4 binding in serum, again displacing enough bound T4 to seriously affect (increase) the FT4 or FTI measurement. This is especially noticeable when certain drugs or other substances are present that compete with T4 for some of its serum protein binding sites, or where similarly competing NEFAs are produced in vitro in heparinized patients (28)(29)(30)(31)(32)(33).

All of these factors must be evaluated and their effects recognized in the development of valid assays for FT4. Failure to do so, especially with regard to antibody sequestration of T4 and the extent of intrinsic serum dilution by an assay, can compromise the validity of the measurements, so that FT4 values may correlate with one or more of the serum binding protein concentrations. The same phenomenon will occur with the T3-uptake test if the amount of label and the effect of the competing binder unduly disturb the natural bound-free equilibrium of the serum proteins.


   Early Approaches to FT4 Measurement
Top
 Abstract
 The Theory Underlying Free...
 Early Approaches to FT4...
Direct FT4 Immunoassays
 Summary
 References
 
the fti
The FTI approach (34)(35) provided the first convenient estimation of FT4 in serum in the routine laboratory. This method combines two measurements: total T4 by immunoassay and T3 uptake. From the basic Mass Action equation described earlier, total T4 closely approximates the serum protein-bound T4 (occupied sites), and T3 uptake represents the concentration of unoccupied sites. FTI, as an index of FT4, is therefore expressed as the product of the two tests/100. The values obtained do not, of course, represent actual FT4 concentrations (34).

The total T4 measurement of the FTI is perfectly valid. However, the commonly held interpretation of the way in which the T3-uptake test works (34) is probably incorrect. This holds that the T3 added merely occupies the vacant binding sites on the serum T4-binding proteins, the excess spilling over for collection by a competing binding agent. Scrutiny of the Law of Mass Action casts doubt on this simple interpretation.

Serum contains two classes of T4-binding proteins: TBG, as a low-concentration, high-affinity protein; and transthyretin and albumin as high-concentration, low-affinity binders. For a normal euthyroid serum, the TBG concentration is ~2000-fold lower than the combined concentrations of transthyretin and albumin, but TBG binds ~60% of the total T4 present (5)(6). Whereas most of the TBG binding sites are occupied, <1% of the transthyretin and albumin sites bind T4. When excess T3 is added in the T3-uptake test, enough must be added to both occupy high- and low-affinity binding proteins and provide sufficient excess for sequestration by the added binding probe. This creates a dilemma. If enough T3 must be added to fill the large excess of high-concentration, low-affinity binding sites (virtually all of which are unoccupied), then this concentration will be high enough to disturb T4 binding to TBG. The affinity of TBG for T3 is only ~10% that for T4 (36)(37)(38), but the quantity of T3 necessary for the assay will outweigh this smaller affinity, allowing T3 to significantly displace T4 from the TBG binding sites. The effect of the fixed amount of added T3 on the T4 displacement phenomenon will be proportionately smaller for high-TBG sera and proportionately greater for low-TBG sera. The T3-uptake test (through the FTI) thus inevitably displays a TBG dependency (14)(15)(16)(18). Thus, because the added labeled T3 is preferentially bound to the large excess of unoccupied transthyretin and albumin sites, the T3-uptake test should also produce falsely low FTI results in nonthyroidal illness (NTI) where the concentrations of such proteins are often reduced (39)(40)(41)(42).

The nonspecific binders used in many T3-uptake assays (18) can also create additional problems by directly sequestrating some T4 as well as T3. With such a variety of effects (14)(16)(17)(18), it is not surprising that, for example, the imperfect normalization of, e.g., high-TBG late-pregnancy sera into the mid- or high-normal range for FTI should have been accepted historically as the correct placement, when more accurate measurements lacking TBG distortions place such sera lower in the reference interval (43)(44)(45).

The FTI approach is therefore one in which the rules for valid FT4 assays are not met. It produces a hybrid result, with a very narrow window of validity, between a total T4 measurement and a true FT4 measurement. The positioning of a given FTI in the scale between these two extremes depends on the conditions defining the T3-uptake test (45). These include the concentration of T3 used relative to the volume of serum tested, the degree of dilution of serum in the assay conditions, and the amount and specificity of the competing binder used to sequestrate the excess T3. Assays using more specific secondary binders that do not incidentally sequestrate T4 from serum should be superior to those that use nonspecific binders.

The routine laboratory can probe the relationship of a given FTI with TBG concentrations in serum (which should be carried out only with a panel of euthyroid sera having a wide range of TBG concentrations from otherwise healthy subjects, and excluding either pregnant or nonthyroidally ill subjects). It can also place the FTI measurements along the arbitrary scale from total T4 to FT4 (46). One need only measure total T4, FTI, and FT4 by selected methods on a panel of ~150–200 routine (not severely ill or pregnant) subjects (9), including some hypo- and hyperthyroid patients, to encompass virtually the whole range of expected values for each marker. It is assumed that each assay method has approximately the same imprecision. Any pair of the three markers is now correlated. If the FTI is acting as a hybrid assay, imperfectly correcting for the natural variation in serum binding protein concentrations in the panel, then it will correlate more closely with total T4 than will the FT4 assay (9). The extent to which the correlations differ is a rough measure of how effectively the FTI approach corrects for binding-protein differences. If changing from FTI to an FT4 assay is contemplated, then several FT4 assays could be used as comparators. In general, given equivalent imprecision, the smaller the correlation coefficient with total T4, the more likely is it that the assay in question is correcting completely for serum binding protein variations.

In addition, a valid FT4 assay should show better discrimination than FTI at the euthyroid-hyperthyroid borderline of the reference interval (9)(46). Performance at the hypothyroid borderline may not be so clearly altered (9)(46).

thyroxine (t4) uptake (t-uptake) assays
In this method, labeled T4 (or a suitable analog) is added in large excess to serum (47). The excess hormone (or analog) fills the hitherto unoccupied protein T4-binding sites and spills over into a free fraction, which can be measured. Just as for T3-uptake methods, T4-uptake techniques (47) may suffer from similar distortions arising from the very different concentrations and binding affinities of TBG on the one hand vs transthyretin and albumin on the other (see above). Before addition of additional hormone, TBG sites are already occupied by endogenous T4 to a large extent, whereas transthyretin and albumin sites are not. Much of the added hormone is thus bound to transthyretin and albumin. This means that the distribution of added hormone is more sensitive to differences, both relative and absolute, in the concentrations of these binding proteins. A decrease in such concentrations relative to TBG, especially in NTI, may strongly affect the T4-uptake value, giving, in turn, low index values (48). These problems, as with T3 uptake, again produce a hybrid result, with a truncated window of validity, in which serum T4-binding protein effects distort the values. The only advantage over the T3-uptake test is that when labeled T4 is used, the same hormone is being used to assess unoccupied sites, with the same affinities as the hormone already occupying the serum binding protein sites. However, if a labeled T4 analog is used instead (T-uptake), its affinity for TBG (relative to T4) may be attenuated more than for transthyretin and albumin. This will exaggerate the influence of transthyretin and albumin even further and may lead to expected falsely low results in NTI, where concentrations of these proteins usually are low.


   Direct FT4 Immunoassays
Top
 Abstract
 The Theory Underlying Free...
 Early Approaches to FT4...
 Direct FT4 Immunoassays
Summary
 References
 
two-step immunoassays
In the period 1978–1980, challenges arose to the hegemony of FTI, which fell increasingly under suspicion of inadequate performance. These included a variety of so-called kinetic FT4 assays (49)(50), followed by a two-step approach (51). The former assays had only a relatively brief existence and are now obsolete. However, the two-step approach to FT4 was basically valid (51) and was a major advance over what was then available commercially. In this test, serum is first incubated with a small quantity of immobilized T4-specific antibody, which takes up a very small quantity of T4 from the sample, minimally disturbing the original serum bound-free equilibrium. After equilibrium is reached, the serum and antibody are separated (51). The immobilized antibody containing bound T4 is then washed repeatedly to remove traces of serum. It is then incubated with a labeled probe (e.g., a suitable T4 derivative or, initially, 125I-labeled T4), and the occupancy of antibody binding sites is measured as for a typical immunoassay.

The two-step assay no doubt is independent, by definition, of the influence of the serum proteins and their bound T4. There are, however, potential problems that must be addressed. The first is the need to remove rigorously all traces of serum proteins before the second incubation is begun. Even minor amounts of binding proteins could sequestrate the labeled probe because of their high affinity. Modern two-step assays avoid this problem by use of T4 conjugated with macromolecules (e.g., enzymes). Such conjugates usually have negligible affinity for serum protein binding sites. A second problem is the possibility of "back displacement" of T4 bound in the first incubation by competition with labeled probe in the second, and by the resulting nonequilibrium conditions for T4 binding (i.e., the phase of serum and its remaining T4 has been washed away). Again, macromolecular T4 conjugates can minimize the first effect because usually the conjugate will bind much less avidly to the antibody than does T4. The use of T3 conjugates may reduce competition even further. The potential for reequilibration of antibody-bound T4 remains however, and suitably avid antibodies should be used to minimize this effect. Short (nonequilibrium) second incubations can also help.

one-step analog immunoassays
To avoid the inconvenience of using two separate processes, with their potential for greater overall imprecision, Dr. T.A. Wilkins and I invented and developed the one-step analog technique for the measurement of FT4 (8)(9)(52) and FT3 (53). In these assays, the labeled T4 (T3) then used as a competing species for antibody binding was replaced by a chemically modified T4 (T3) analog. This had the property of binding avidly to a T4 (T3)-specific antibody, but much less avidly than the native hormone to the serum binding proteins (7)(8)(9)(54). The aim of the system was to improve the convenience of two-step approaches while maintaining validity.

Accordingly, one-step approaches made redundant the initial separation of serum binding proteins required by the two-step approach (51) and superficially resembled the corresponding total-hormone assays. Early analog methods, although they performed adequately in correcting for the physiologically encountered differences in TBG and transthyretin concentrations in serum, were correlated with serum albumin concentrations (7)(55)(56)(57)(58). Although the assays were at the forefront compared with anything else then commercially available, they were criticized for imperfect performance (55)(56)(57)(58)(59)(60). Vehement controversy between us and others (2)(3)(4)(7)(59)(60)(61)(62)(63) as to the exact working and validity of such assays regrettably generated far more heat than light and led to hesitation in some countries (especially the US) in adopting the methodology. The critics, however, paid little attention to the fact that the alternative FTI methods, the most frequently used assays at the time, had greater shortcomings in the same and other areas. As an example of this critical misinterpretation, the correlation of late-pregnancy sera that produced low-normal analog FT4 values with serum albumin concentrations was alleged to demonstrate the invalidity of these assays (55). It is now known that this correlation is merely a physiologic one and not an assay shortcoming (45). Other studies alleged, wrongly, that the assays were merely measuring the "albumin-bound" fraction of T4, and not FT4 (57). Practical experience in the development of the modern one-step assays lacking interferences also showed that the stringent criteria laid down by theorists (2)(3)(4)(59)(60) for the permitted amounts and affinities of antibodies in valid FT4 assays could be considerably relaxed and still give acceptable dose-response values (64)(65).

At first (8)(9), the enforced use of radioactive labeling probes limited analog immunoassay design. Assay development was constrained by the maximum specific activity of the label compatible with the analog’s chemical stability. Detection of adequate amounts of signal for convenient analysis required the antibody to sequestrate slightly >1% of the total hormone in euthyroid sera with reference binding protein concentrations (7)(8)(9), still well within the window of validity for the assay (7)(8)(9). Albumin was also added to modulate the biasing effects of variable serum albumin concentrations (7).

Since then, improvements in the performance of analog FT4 and FT3 assays (64)(65) have eliminated interactions of the labeled probe with any of the common serum binding proteins. Newer nonradioactive labels, with higher detection sensitivity, use smaller amounts of antibody, consequently extracting a lower percentage of hormone (64)(65). This gives three advantages. The first advantage is that when measuring sera with abnormal T4-binding capacities, intrinsic bias is minimized. The second advantage (65) is that, when they lack added albumin, assays are more robust to progressive serum dilution (a useful technique, although sometimes overinterpreted as a test for assay validity and absence of bias). The third advantage is that FT4 results no longer significantly correlate with serum binding protein concentrations, even albumin (64)(65)(66).

Comparison of the performance of the newer and original assays confirmed earlier forecasts (7) that the need for improvement lay in the abolition of biasing effects attributable to albumin. This comprises the hypoalbuminemia of severe NTI (still a controversial area in regard to the value of thyroid function tests) and the rare conditions of analbuminemia, familial dysalbuminemic hyperthyroxinemia, and the presence in serum of strongly binding T4 or T3 autoantibodies. The first three have been addressed with good analog one-step assays (67)(68), and all, including the vast majority of sera containing autoantibodies, have been addressed successfully in the newer immunometric assays, where the antibody rather than the analog is labeled (64)(65).

With modern one-step assays, therefore, comparing new methods with earlier ones is more difficult than comparing an FTI with any FT4 method. This is because, as stated above, the possible improvements in diagnostic performance become increasingly marginal as the learning curve in development progresses. For the routine laboratory, the most relevant test of the validity of any FT4 assay is to correlate, in euthyroid sera with wide variations in binding protein concentrations, the FT4 value with each of the proteins TBG, transthyretin, and albumin. Once again, subjects receiving drugs such as dilantin or aspirin, and nonthyroidally ill and pregnant subjects should not be included in the correlations because these may have altered physiology dependent on their condition. Specificity and sensitivity measurements are also recommended for discrimination of hypo- and hyperthyroidism from the reference interval.

Dilution tests on sera are valuable, but require restraint. The useful limits of dilution experiments can be calculated from the affinity constants and concentrations of each of the serum T4-binding proteins, for which the binding potential (BP) is given as the product of the two parameters ({Sigma}K1,2,3P1,2,3). For a normal euthyroid serum, BP is ~6000 (4000 from TBG, 1300 from transthyretin, and 700 from albumin). Sera such as those in NTI could have a BP value ~15–20% of this (i.e., ~900–1200). Therefore, dilution tests on a normal serum need only be pursued out to a dilution (over and above the dilution of serum intrinsic in the assay itself) of no more than five- to sixfold. If the assay in question is sufficiently (not necessarily perfectly) robust out to this dilution factor, then it is likely that sera with BP values of 900–1200 are being measured without undue bias. Sera with BP values lower than this are relatively rare. Dilution of sera with low BP values is unnecessary except as an academic exercise to probe the limits of validity of the assay. The methodology is then being stretched into nonphysiologic regions that may exceed its window of validity, and false indications of assay failure may ensue. It may well be that an assay is designed to encompass, without undue bias, the BP values for the vast majority of sera encountered, but can rapidly fail outside this range.

Comparison of results with those from a gold-standard method such as equilibrium dialysis or ultrafiltration is also valuable, if such facilities are available. However, careful choices of sera should be made, and nonthyroidally ill subjects should not generally be included, nor should serum from patients known to have taken drugs that can affect the binding of T4 to the serum binding proteins (see below).

immunometric one-step analog methods
More recently, further advances have been made in one-step FT4 analog technology, with two key innovations (64)(65). The first innovation involved transferring the detection label from the analog itself to a specific T4-binding monoclonal antibody and immobilizing the analog as a complex insoluble matrix. In the second innovation, a cross-reacting hormone (T3) was used rather than T4 itself for binding to antibody as the basis of insoluble matrix formation (insoluble T3-matrix) (64)(65). This invention confronted the outstanding problem of how to measure FT4 in sera containing avid autoantibodies against T4 or T3, as well as finally preventing the interference of any other serum binding protein in the assay (64)(65)(66). In this assay, the affinity of the insoluble T3-matrix for the antibody is manyfold less than for FT4. Thus, so that the relative rates of binding of the insoluble T3-matrix and FT4 to the labeled antibody are roughly equivalent, the concentration of this matrix must be correspondingly manyfold higher than FT4 (64)(65). Hence, occupation of the immobilized T3-matrix by a small quantity of the antibody probe is negligible. This allows the gross excess of unoccupied insoluble T3-matrix binding sites to bind large amounts of extraneous autoantibodies without significantly affecting the binding ability of the labeled antibody probe (64)(65)(69)(70). The method has been shown to accommodate the vast majority of such sera within the correct range for their underlying functional state (69)(70), unlike labeled-analog methods, which are still often susceptible to interference (71).

In addition, it was realized that the basic mode of action of direct FT4 immunoassays, using avid antibodies, could be expressed as a simple competition for binding of FT4 and analog (or insoluble analog-matrix) until an apparent equilibrium is reached (64)(65). The small, extremely slow dissociation of antibody-bound analyte and analog thus greatly simplified the original complex arguments (7) that sought to explain the assay system (64)(65), permitting the amounts and affinities of antibody and analog to be more variable than the original, more rigid theory allowed (2)(3)(4)(7)(59)(60).

albumin in the assay ingredients in one-step assays
Initially, extra albumin was provided in the assay ingredients of the first one-step assays to ameliorate the biasing effects of residual binding of the analog tracer to endogenous albumin in the serum sample (7). This inevitably inserted an additional bias. The smaller the binding protein concentrations in any given serum, the greater was the influence of the added albumin on the assay through its own T4 sequestration. However, direct experiments, using added albumin, showed that (a) the binding properties of added albumin were variable and (b) the bias was important only when serum albumin concentrations (but not those of TBG or transthyretin) differed significantly from the norm (7)(58). Accordingly, in these assays, FT4 measurements correlated only with serum albumin concentrations (7)(66). Even then, the effects were not marked until albumin concentrations had fallen well below the reference interval for that protein [e.g., in conditions ranging from severe NTI to analbuminemia (7)(58)]. Observed bias within the reference interval for albumin concentrations did not have significant consequences for diagnostic discrimination (66).

Over the next 20 years, continual improvements in the properties of the analogs eventually led to the abolition of their binding to serum proteins (64)(65). Accordingly, there was no need to add albumin expressly to counteract bias from residual binding effects. However, another problem manifested itself, which extra albumin in the assay ingredients helped to ameliorate. In patients receiving heparin injections, blood lipoprotein lipase is activated (72)(73). When blood is withdrawn from such patients, NEFAs are produced in vitro, often to very high concentrations (74)(75)(76)(77)(78). The NEFAs in turn are capable of displacing substantial quantities of bound T4 from the serum protein T4-binding sites and can increase the measured FT4 considerably, often grossly out of the reference interval (30)(31)(74)(75)(76)(77)(78). Albumin in the assay ingredients opposed this artificial increase in FT4 by "mopping up" the NEFAs produced, thereby restoring most values to normality (30)(31). Thus, assays containing albumin can be used with heparinized subjects (with caution).

In severe NTI, there are additional problems. Initially, in the days of FTI supremacy, it was believed that without overt primary thyroid dysfunction, such patients were essentially euthyroid, although there were considerable decreases in the concentration of total T4 into the hypothyroid range, not wholly corrected by FTI (40)(41)(42)(79)(80).

Gold-standard methods for FT4 measurement variously gave results below, within, and above the euthyroid reference interval (27)(41)(42)(81)(82)(83)(84)(85). Simultaneously, thyrotropin (TSH) concentrations could also be low, normal, or occasionally above normal (86)(87)(88)(89), although not necessarily in harmony with the FT4 values. It was postulated that in NTI, the serum contained nondialyzable substances that could bind competitively to the T4-binding proteins, displacing bound T4 into the free phase (90)(91)(92). This explanation rationalized how the gold-standard FT4 measurements could be so out of line with total T4 values, even when the serum binding protein concentrations (especially transthyretin and albumin) were also severely decreased. Nevertheless, FT4 estimates straddling the reference interval also create problems in discriminating true primary hypo- and hyperthyroidism in subjects with NTI from subjects with no apparent thyroid dysfunction.

It was, no doubt, hoped that when new, more convenient assays were available, these anomalies would disappear. However, over the past 20 years, the exact status of the NTI subject with regard to FT4 in serum has remained as controversial as ever and certainly cannot be conceived as "steady-state" with regard to thyroid function indicators (93). In NTI, overlaying any general level of thyroid function, there seem to be continuous (independent) ebbs and surges in the production and retention of substances either directly or indirectly related to FT4 as the disease progresses (81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91)(92). These can include serum binding proteins (94); interfering substances, including administered drugs (32)(33); TSH (90)(91)(92); and probably production of T4 and its conversion to T3 (95)(96). The true thyroidal state of the subject can thus be obscured by relatively short-lived changes in measured indicators, which may, at random, show up in single samples taken from a panel of NTI patients. This highlights the danger of diagnosing NTI patients by single samples, making it difficult to compare assay performance if the panel of sera inevitably contains examples of these random events. Indeed, because of the tendency of FT4 and TSH concentrations independently to enter and leave the euthyroid reference interval, it is recommended that both markers be measured in NTI and that careful monitoring is done to rule out effects that might alter the concentration of either marker, e.g., administration of albumin-binding drugs (66)(97). Only in this way can we assess the underlying integrated level of thyroid function and discount transient anomalies. The difficulties in diagnosing hypothyroidism accompanying NTI have been vividly described (98).

Most studies comparing FT4 assay performance in NTI have not taken this complexity fully into account and are generally poorly designed. Assays with biases attributable to added albumin have often been criticized for giving below-normal values when TSH is presumably within the reference interval (31)(32)(96). However, because gold-standard assays lacking added albumin can equally give diagnostically meaningless above-normal values, misplaced confidence in their validity has often led to support for assays that show the same effect (94). Here there is a logical inconsistency. If albumin-containing FT4 assays can read too low and TSH is, in a random NTI sample, transiently high, a misdiagnosis of hypothyroidism could occur. Conversely, if albumin-free assays give above-normal values when TSH is transiently low, a misdiagnosis of hyperthyroidism could equally be made. Neither methodology thus can be completely trusted in this situation. Additionally, albumin-containing assays may mitigate the transient effects on FT4 of albumin-binding drugs in serum and depict the underlying thyroidal function more exactly.

Finally, the potential effects of assay incubation on the concentration of interfering substances in serum should not be underestimated. In an "albumin-free" assay using a serum whose T4-binding sites are already saturated with other molecules, the FT4 measurement is exquisitely sensitive to any further production of interference in vitro. Published studies show that FT4 values are increased when NEFA concentrations are >2.5 mmol/L (78)(99)(100)(101). The rate of further increase of FT4 on increases in NEFAs is ~7–10 pmol/L per mmol/L additional NEFAs (7)(30)(78). Therefore, an increase in NEFAs (or the combination of some other interferent + NEFAs) of 0.2 mmol/L will cause an (in vitro-dependent) increase in measured FT4 of ~1.5–2 pmol/L. Albumin-containing assays can at least partially mitigate this effect (30). The amount of exogenous albumin in the assay buffers that can be used to minimize interference in FT4 assays depends very much on its properties of T4 binding as well as its concentration. It is well known that commercial sources of albumin can vary markedly in their T4-binding characteristics (58). Such sources are properly termed preparations rather than well-defined purified compounds because denaturation of albumin can occur to various degrees during production. However, a calculation can be made of how large a BP for added albumin will distort the FT4 values of most sera to an acceptably minimal extent relative to the assay calibrators. This would approximate, at most, to 10–25% of that found endogenously in a serum with binding protein concentrations within the reference intervals. Thus, in an assay using a 10-µL sample and 100 µL of reagent, the bovine serum albumin content of the reagent should be <1.2 g/L.

Hence, FT4 values are not grossly affected until endogenous albumin concentrations are less than half-normal and TBG concentrations are also substantially diminished. Albumin preparations with weaker T4-binding affinities can be used in greater quantities because the smaller affinity offsets the effects of higher concentration in producing a suitable BP factor. Supplemental data on this topic, in the form of an Appendix, are available in the on-line version of this journal (http://www.clinchem.org/content/vol47/issue8/).

Selecting assays that either do or do not contain albumin in their buffers appears to be a trade-off of intrinsic bias in FT4 values downward in NTI (and in all other situations where the binding capacity is reduced), caused by the added albumin, against increased sensitivity of an albumin-free assay to extraneous in vivo and in vitro effects. Depending on their experience with discrimination of thyroidal disease in NTI subjects, assay users must decide for themselves which to use.

A carefully controlled study on large numbers of subjects, with a defined and graded severity of systemic illness, is urgently needed, with full knowledge of medication taken and with multiple sampling, in which outcomes are known and where substantial numbers of NTI patients with primary hypo- and hyperthyroidism are also included. Such a study, using an albumin-containing and an albumin-free assay for comparison, together with TSH measurements, would more usefully evaluate the sensitivity and specificity of each assay in diagnosing thyroid disease in these conditions. In this way, one could offset the misdiagnosis frequency from the inevitable biases in the albumin-containing assay against the misdiagnosis frequency in albumin-free assays.

It is unfortunate that albumin is the only useful (and inexpensive) protein able to mitigate the effects of interfering substances on FT4 values in heparinized, drug-affected, or nonthyroidally ill patients. However, chemical modification of albumin may greatly attenuate or destroy its binding of T4 without destroying its ability to bind other substances, such as NEFAs (102). The binding sites for NEFAs and other substances are manyfold more than for T4. Judicious protein modification might produce a substance still able to quench interference without causing significant bias through extra T4 binding. This might yield an assay giving superior discrimination in NTI through nullification of the influence of interfering substances while not otherwise suffering from bias from albumin addition.


   Summary
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 Abstract
 The Theory Underlying Free...
 Early Approaches to FT4...
 Direct FT4 Immunoassays
 Summary
References
 
The performance of convenient assays for FT4 has improved greatly since the first attempts (FTI) in 1965. Index methods were accepted until it became clear that they were still strongly influenced by TBG. In the meantime, certain "truths" had come to be accepted by many workers, not the least that in late pregnancy, FT4 results should lie in the middle of or in the upper regions of the euthyroid reference intervals. Better assays, correcting more completely for TBG concentrations, forced a sometimes painful period of readjustment in this and other areas, which has taken 20 years or more to reconcile in some regions of the world. By now, the better assays for FT4 have eliminated the biasing effects that arose in the early assays from residual binding of labels to serum albumin. The most recent improvements have also largely addressed the remaining problem of T4 autoantibodies in serum. The main problem outstanding in using FT4 assays is that of diagnosis in NTI. This is a problem that lies more in the vagaries of patient physiology than in any hope of a final, simple, and definitive answer in an ideal assay. The scope for further improvement in FT4 assay methodology is very limited. Most of the residual problems (albumin dependency, dilution performance) have been addressed, and a final decision can be taken by diagnosticians, not merely whether to terminate their use of FTI, with all its shortcomings, but which direct FT4 assay to use, based on simple comparisons of each assay’s diagnostic properties with those of other assays.


   Footnotes
 
1 Nonstandard abbreviations: T4, thyroxine; T3, triiodothyronine; FT4, free T4; TBG, thyroxine-binding globulin; FTI, free thyroxine index; NEFA, nonesterified fatty acid; NTI, nonthyroidal illness; BP, binding potential; and TSH, thyrotropin.


   References
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 Abstract
 The Theory Underlying Free...
 Early Approaches to FT4...
 Direct FT4 Immunoassays
 Summary
 References
 

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