Clinical Lab Handbook

Container/Tube: SST

Alternate Specimen: Red Top, Lithium Heparin-Gel

Specimen Volume: 5 mL of serum

Collection Instructions: Allow specimen to clot for 30 minutes. Spin specimen in centrifuge.

Chemiluminescence Immunoassay

A competitive immunoassay technique is used, which depends on a competition between T4 present in the sample with a horseradish peroxidase (HRP)-labeled T4 conjugate for a limited number of binding sites on a sheep anti‑T4 antibody presented in the liquid phase. Binding protein effects are eliminated by use of an appropriate buffer containing displacement agent. The antigen-antibody complex is captured by a donkey anti‑sheep second antibody coated on the wells. Unbound materials are removed by washing.

The bound HRP conjugate is measured by a luminescent reaction. A reagent containing luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer agent, is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent (a substituted acetanilide) increases the level of light produced and prolongs its emission. The light signals are read by the system. The amount of HRP conjugate bound is indirectly proportional to the concentration of T4 present.

Thyroxine accounts for at least 90% of circulating protein-bound iodine. While >99.9% of T4 is protein-bound, primarily to thyroxine-binding globulin (TBG), it is the free fraction that is biologically active. In most patients the total T4 level is a good indicator of thyroid status, however it can sometimes be inadequate, and diagnostic efficiency may be improved by use of a total T4 test in conjunction with other tests. T4 levels may be altered in conditions affecting the capacity of the thyroid hormone binding proteins, e.g. pregnancy, when a test for triiodothyronine (T3) or T4 uptake may be used with the total T4 result to calculate the free T4 index (FT4I) to estimate the level of metabolically active free T4.

5.5-11.0 µg/dL

Monday through Sunday

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