Free Thyroxine (FT4) and Free Triiodothyronine (FT3) Estimate Tests

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Index Methods: FT4I and FT3I

Index methods are free hormone estimate tests that require two separate measurements.[146] One test is a total hormone measurement (TT4 or TT3) the other is an assessment of the thyroid hormone binding protein concentration using either an immunoassay for TBG or a T4 or T3 "uptake" test called a Thyroid Hormone Binding Ratio (THBR). Alternatively, indexes may be calculated from a TT4 measurement paired with an estimate of the free T4 fraction determined by isotopic dialysis. In this case, the quality and purity of the tracer employed critically impacts the accuracy of the index.[149,153,154]

Calculation of a FT4I using TBG only improves diagnostic accuracy compared with TT4 when the TT4 abnormality results from an abnormal concentration of TBG. In addition, the TT4/TBG index approach is not fully TBG independent, nor does it correct for non TBG-related binding protein abnormalities or for TBG molecules which have abnormal affinity.[141,155,156,157,158] Thus, despite the theoretical advantages of using a direct TBG measurement, TT4/TBG indexes are rarely used because TBG binding capacity can be altered independent of changes in the concentration of TBG protein, especially in patients with NTI.[99] In addition, TBG binding reflects 60 - 75% of the binding capacity thus relying on TBG alone excludes hormone binding effects due to transthyretin and albumin abnormalities.

"Uptake" tests have been used to estimate protein binding of thyroid hormones since the 1950s. Two different types of "uptake tests" have been used. "Classical" uptake tests add a trace amount of radiolabeled T3 or T4 to the specimen and allow the labeled hormone to distribute across the thyroid hormone binding proteins in exactly the same way as endogenous hormone.[146,154] Since only a trace amount of labeled T3 or T4 is used, the original equilibrium is barely disturbed. The distribution of the tracer is dependent upon the saturation of the binding proteins. Addition of a secondary binder or adsorbent (anion exchange resin, talc, polyurethane sponge, charcoal, or antibody-coated bead, etc.) results in a redistribution of the T3 or T4 tracer into a new equilibrium, that now includes the binder. The tracer counts sequestered by the adsorbent are dependent on the saturation of the binding proteins: the higher the saturation of the binding proteins, the greater the amount of tracer in the adsorbent. The amount of added tracer taken up onto the absorbant results in an indirect measure of TBG. When the TBG concentration is low, TBG binding sites are highly saturated with T4 so that a smaller amount of added T3 tracer binding will bind to TBG and more will be being taken up by the adsorbent. Conversely, when the TBG concentration is high, TBG saturation with T4 is low, more tracer binds unoccupied TBG binding sites and less becomes bound to the adsorbent. Unfortunately the relationship between THBR and TBG concentration is non-linear, such that index testing usually does not correct TT4 abnormalities resulting from grossly abnormal TBG concentrations.[158]

It has been recommended that a normal serum sample standard be used to normalize the response of the assays and allow the reporting of the result as a ratio to normal i.e. a "Thyroid Hormone Binding Ratio (THBR)".[154] "Classic" uptake assays used T3 tracer because the lower T3-TBG binding affinity relative to T4-TBG resulted in a higher isotopic uptake by adsorbant and thus shorter counting times. However, since the validity of using a T3-uptake test to correct a TT4 value is questionable, some current non-isotopic assays use a "T4-uptake". Many manufacturers still use the "classical" approach to produce T3 uptake assays in which the mean normal percent uptake can vary from 25% to 40% (bound counts/total counts). Traditionally, the free thyroxine index, sometimes called a "T7" is derived from the product of a T3-uptake test and a TT4 measurement, often expressed as a % uptake (adsorbent bound counts divided by total counts).

Guideline 12. Thyroid Hormone Binding Ratio (THBR) or "Uptake" Tests

  • "Uptake" tests should be called "Thyroid Hormone Binding Ratio" tests, abbreviated THBR and include an indication of which hormone is used, i.e. THBR (T4) or THBR (T3).

  • A T4 signal is preferred over T3 for THBR measurements, to better reflect T4 binding protein abnormalities.

  • THBR values should be reported as a ratio with normal serum, the latter having an assigned value of 1.00.

  • THBR calculations should be based on the ratio between absorbent counts divided by the total minus absorbent counts, rather than the ratio between absorbent counts and total counts.

  • The THBR result should be reported in addition to the total hormone and free hormone index value.

  • THBR tests should not be used as an independent measurement of thyroid status, but should be interpreted in association with a TT4 and/or TT3 measurement and used to produce free hormone estimates (FT4 or FT3 indexes).

"Classic" T3-uptake or THBR tests are typically influenced by the endogenous T4 concentration of the specimen. This limitation can be circumvented by using a very large excess of a non-isotopically labeled T4 tracer with an affinity for thyroid binding proteins comparable to that of T4. Current THBR tests usually produce normal FT4I and FT3I values when TBG abnormalities are mild (i.e. pregnancy). However, some of these tests may produce inappropriately abnormal index values when patients have grossly abnormal binding proteins (congenital TBG high or low, familial dysalbuminemic hyperthyroxinemia (FDH), thyroid hormone autoantibodies or NTI) and in the presence of some medications that influence thyroid hormone protein binding [Section-3 B3(c)vi].

The first free hormone tests developed in the 1960s were indexes, calculated from the product of the free hormone fraction from a dialysate multiplied by the TT4 measurement (made by PBI and later RIA).[159,160] The free fraction index approach was later extended to measure the rate of transfer of isotopically-labeled hormone across a membrane separating two chambers containing the same undiluted specimen. The free hormone indexes calculated with isotopic free fractions are not completely independent of TBG concentration and furthermore are influenced by radiochemical purity, the buffer matrix and the dilution factor employed.[161,162]

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