Total Thyroxine (TT4) and Total Triiodothyronine (TT3) Methods

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Methods for Measuring Total Thyroid Hormones

Serum total T4 and total T3 methods (TT4 and TT3) have evolved through a variety of technologies over the past four decades. The PBI tests of the 1950s that estimated the TT4 concentration as "protein-bound iodide" were replaced in the 1960s first by competitive protein binding methods and later in the 1970s by radioimmunoassay (RIA) methods. Currently, serum TT4 and TT3 concentrations are measured by competitive immunoassay methods that are now mostly non-isotopic and use enzymes, fluorescence or chemiluminescence molecules as signals.[135] Total hormone assays necessitate the inclusion of an inhibitor (displacing or blocking agent) such as 8-anilino-l-napthalene-sulphonic acid (ANS), or salicylate to release the hormone from binding proteins.[136] The displacement of hormone binding from serum proteins by such agents, together with the large sample dilution employed in modern assays, facilitates the binding of hormone to the antibody reagent. The ten-fold lower TT3 concentration, as compared with TT4 in blood, presents technical sensitivity and precision challenges, despite the use of a higher specimen volume.[137] Although reliable high-range TT3 measurement is critical for diagnosing hyperthyroidism, reliable normal-range measurement is also important for adjusting antithyroid drug dosage and detecting hyperthyroidism in sick hospitalized patients, in whom a paradoxically normal T3 value may indicate hyperthyroidism.

Despite the availability of highly purified preparations of crystalline L-thyroxine and L-triiodothyronine (i.e. from the United States Pharmacopoeia (16201 Twinbrook Parkway, Rockville, MD 20852) no TT4 or TT3 reference methods have yet been established.[138,139] Further, the hygroscopic nature of the crystalline preparations can affect the accuracy of gravimetric weighing.[140] Secondly, the diluents used to reconstitute L-T4 and L-T3 preparations for use as calibrators are either modified protein matrices or human serum pools that have been stripped of hormone by various means. In either case, the protein composition of the matrix used for the calibrators is not identical to patient sera. This can result in the protein binding inhibitor reagent (e.g. ANS) releasing different quantities of hormone from calibrator matrix proteins than from the TBG in patient specimens. This may impact the diagnostic accuracy of testing when the binding proteins are abnormal, such as in NTI.

Guideline 9. For Manufacturers Developing TT4 and TT3 Methods

Method biases should be reduced by:

  • The development of L-T4 and L-T3 reference preparations and establishing international reference methods.

  • Ensuring that instruments are not sensitive to differences between human serum and the calibrator matrix.

  • Ensuring that during the test process, the amount of thyroid hormone released from serum binding proteins is the same as that released in the presence of the calibrator diluent.