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Letters to the Editor |
North Lakes Clinical, 6 High Wheatley, Ilkley LS29 8RX, United Kingdom
To the Editor:
I read with misgivings the recent communication by Fritz et al. (1) alleging that because under some circumstances an analog free thyroxine (FT4) immunoassay correlates total T4 and FT4 values, it does not measure FT4 but something akin to T4. I have shown repeatedly through mass action analyses (2)(3)(4) that many experiments, including the one reported by Fritz et al. (1) and others described in earlier papers(5)(6), cannot meaningfully address the working of any of these assays. Allegations of shortcomings cannot be substantiated if they depend on experiments using artificial T4 solutions in which serum T4-binding proteins are either lacking or are present at concentrations insufficient to prevent overlarge T4 abstraction by the assay antibody (conditions strictly invalid for FT4 measurements).
The amount of antibody that can be used in analog FT4 assays having a radioactive probe is limited (3). Constraints on the stability of molecules radiolabeled with carrier-free 125I atoms dictate the minimum usable amount of labeled probe for adequate tracer detection in the assay (3). To enable measurement, the minimum amount of stripping antibody must remove at least 1% of the total T4 from a normal serum sample (2)(3)(4). Although this amount far exceeds the actual number of FT4 molecules present in serum (approximately 0.02% of the total), the procedure remains valid because the serum-bound free equilibrium for FT4, bound T4 is negligibly disturbed, and the measured FT4 concentrations (obtained through calibration of standards) are not significantly affected (3)(4)(5). Sequestrations of more than approximately 3% invalidate any results. Because the FT4 concentration in ultrafiltrates or dialysates remains equivalent to only 0.02% of the total T4 (i.e., the ambient, undiluted FT4 concentration), any assay designed to sample 1% of T4 cannot detect this small amount, and total T4 assays similarly fail.
Results from varying serum (and therefore serum T4-binding protein) concentrations can also be explained quite differently. I understand that this early analog immunoassay retained a "TBG effect". That is, residual binding of the labeled analog to T4-binding globulin (TBG; and probably other proteins) was large enough to correlate FT4 values with TBG (partly but not completely mitigated by a T4 blocking agent). Thus, when serum amounts are altered by dilution or concentration, 2 connected events ensue. First, a more concentrated serum will have a higher concentration of TBG relative to antibody probe and act as a high-TBG serum, resulting in increased FT4. Conversely a diluted sample will mimic a low-TBG serum giving a low result. Second, intrinsic effects of dilution will occur. If in undiluted serum the assay samples 1%–2% of the total T4, in a 4-fold diluted serum, it will sample 4%–8%, an interval that lies well outside the window of validity of the assay, insofar as too much T4 is sequestered by the antibody probe to maintain equilibrium between FT4 and the T4 bound to the serum proteins. The assay essentially "collapses" into one more closely related to T4, and will do so in all cases in which too much T4 is sampled by the antibody.
Much of the work of Nelson et al. (5)(6) aimed at probing the working of analog FT4 assays is not applicable to the topic under discussion, but these investigators seem to suggest that in regard to these FT4 assays any experiment should be meaningful in any situation (whether physiologic or not). The cardinal property of direct 1-step assays (analog or labeled antibody) is that, although they are designed for all physiologic circumstances (i.e., they work with all sera regardless of serum T4-binding protein concentrations), they cannot be used with artificial solutions outside the assay’s limits of validity. This point has been argued elsewhere (2)(3)(4).
Another questionable implication (5)(6) is that analyses of the performance of this particular assay may apply generally to all such assays. On the contrary, FT4 assays are individual with regard to serum protein effects, assay ingredients, robustness to dilution, and percentage sampling by the antibody probe. General conclusions on the class of tests cannot be drawn by examining a single assay (3). Assays with residual analog binding to serum proteins cannot be easily compared with newer examples that lack this characteristic.
Finally, although ultrafiltration, equilibrium dialysis, and direct analog assays have the same aims, in regard to sampling and dilution these different assays represent opposite ends of a spectrum of valid methods. Experiments performed by one group of methods do not always apply to the other, because ultrafiltration and dialysis measure FT4 in the absence of serum proteins and bound T4, whereas analog assays accommodate their presence.
Footnotes
Editor’s Note: Dr. Fritz declined to reply for publication.
References
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