Overview

• Thyroxine (total thyroxine/tT4/T4) is the main secretory product of thyroid gland, as both free and protein bound forms.
• Over 99.9% of thyroxine is reversibly bound to carrier proteins.
• The minimal free unbound fraction (free T4/fT4/fT4eq/fT4d) is metabolically active and also converted to tri-iodothyronine (T3) for cellular effects.
• Diagnostic assays for thyroxine measure both protein-bound (inactive) and free forms.
• Free T4 assays are designed to measure only the free unbound component but have varied accuracy, in part when using cross reacting human assays for veterinary species.
• The addition of equilibrium dialysis to the testing (pre-incubation) improves the accuracy of free T4 measurement (fT4eq/fT4d) in veterinary species.

Sampling

Tests

Methodologies

• fT4eq/fT4d Equilibrium dialysis (“modified”):
  • Free T4 isolated from serum by passive diffusion through a semipermeable membrane, which allows only free T4 molecules to pass into the dialysate, but not larger plasma proteins and protein bound T4.
  • This diffusion is passive, taking hours and is typically set up and left overnight to run, the dialysate then tested the following day.
  • Control material should be tested to ensure the filtration system did not fail.
  • A slightly different “standard” dialysis technique using radioactive tracer T4 is also available and has similar performance.
  • Radioimmunoassay (RIA/IRMA) on the dialysate. 
• fT4: ‘Analogue’ competitive EIAs (eg CLIA) or RIAs:
  • Tubes or magnetized particles with coated T4 antibody, their final radioactivity inversely proportional to the sample’s free T4.
  • Have been designed to measure fT4 but in veterinary medicine (cf. human) appear to be less accurate, typically underestimating canine values.

Availability

• Some commercial laboratories use analogue assays (fT4).
• Fewer specialist laboratories use equilibrium dialysis (fTeeq/fT4d).

Validity

Sensitivity

• Typically high sensitivity for equilibrium dialysis (>90% typically). 
• Varies with analogue non dialysis method, eg in hypothyroid dogs (TgAA positive dogs with low fT4eq/fT4d) 62% human assay, 75% veterinary assay but often no better, or worse than, comparative routine T4 assays.
• As a singular test, fT4eq is no better than combined [T4] with [TSH].

Specificity

• Good specificity (>90%) for equilibrium dialysis (fT4eq/fT4d), less so direct analogue platforms (fT4).
• As a singular test, fT4eq/fT4d is no better than combined [T4] with [TSH].

Technique (intrinsic) limitations

• fT4:
  • Anti T4 antibodies Thyroid: T4AA assay affect only these analogue tests for free T4, the membrane in equilibrium dialysis preventing such antibodies entering the tested diasylate. 
• fT4eq/fT4d:
  • Samples are often run in duplicate to improve quality control, with repeat testing if the duplicate values are significantly different.
  • The semi-permeable membrane can fail, so the test has to be repeated (eg significantly lipemic or hemolyzed samples). 
• fT4eq/fT4d more expensive and time consuming, analogue fT4 assays more expensive (cf. standard T4 measurement).

Technician (extrinsic) limitations

• Equilibrium dialysis involves several practical steps in which technician factors may be important, eg filtration set up, sample loading.

Result Data

Further Reading

Publications

Refereed papers

• Recent references from VetMedResource and PubMed.
• Randolph J F et al (2015) Free Thyroxine Concentrations by Equilibrium Dialysis and Chemiluminescent Immunoassays in 13 Hypothyroid Dogs Positive for Thyroglobulin Antibody. JVIM 3, 877-881 PubMed Full Article.
• Behrend E N, Kempannien R J & Young D W (1998) Effect of storage conditions on cortisol, total thyroxine and free thyroxine concentrations in serum and plasma of dogs. JAVMA 212, 1564-1568.
• Larsson M G (1988) Determination of free thyroxine and cholesterol as a new screening test for hypothyroidism. JAAHA 24, 209-217.
• Peterson M E, Melian C & Nicholls R (1997) Measurement of serum total thyroxine, triiodothyronine, free thyroxine and thyrotropin concentrations for diagnosis of hypothyroidism in dogs. JAVMA 211, 1396-1402.

Other sources of information

• Specific commercial laboratory offering free T4 in regard to their methodology and reference data populations etc.