A long-awaited letter

[diogenes]

In 2015 I gave a talk to a TUK forum on the poor quality control and performance of free T4 and free T3 assays then used. The situation hasn't changed in the meantime, but I got a letter accepted in a journal on this very subject: It will appear very soon but since it will be behind a paywall I'm giving it in full here (sorry for the length):

Global FT4 immunoassay standardization.

Response to: Kratzsch J et al. Global FT4 immunoassay standardization: an expert opinion review

John E. Midgley

[e-mail]

North Lakes Clinical, Ilkley LS29 8PT,

UK Corresponding author: John E. Midgley, North Lakes Clinical, Ilkley LS29 8PT,

To the Editor,

Even after many years of use, harmonisation of free thyroid hormone assays still appears to be a distant prospect [1]. Forty years after the invention and production of the one-step tests for free thyroxine (FT4) and free triiodothyronine (FT3), in which I had a role as an inventor [2], rationalisation and harmonising of such assays (including the two-step alternatives from different manufacturers) has still not been resolved. Why is this so?

I would like to briefly comment on three intertwined obstacles that must be overcome to achieve a successful outcome.

A major barrier to progress has been the various later attempts at exploring the basic nature of these assays. All of them misapprehend how the assays work, being either theoretically bogus [3], or practically invalid either from a general mistaken examination by disassembly of the assay components [3–7]. Illegitimate claims result both from such an approach, while in some studies the wrong conditions were used to compare so called “gold standard” methods with only a few examples of working assays [8]. There can be technical difficulties when performing “gold standard” methods such as equilibrium dialysis and especially tandem mass spectrometry/ultrafiltration of undiluted sera where, despite its apparent validity, potential problems with membrane integrity under pressure, and other extrusion artefacts can seriously affect the results for free hormone measurement [6, 8]. The technical pressure on equilibrium dialysis, on the other hand, is somewhat less demanding, readily demonstrating its validity through its robust response to dilution of the serum sample, by maintaining constancy of measurement of free hormone concentrations [9]. However, in many studies using these techniques, general conclusions were derived from only examining a small selection of available tests, thereby tarring all with the same brush. This is neither justified nor acceptable.

In a more general sense, the first problem for assay rationalisation is the general failure of assay designers to properly understand the complexity of devising and testing these assays for routine use. In the initial phase and trialling of the first such tests, to thoroughly examine the assays’ performance, the development used sera from many classes of individuals This was done not only in the frequently encountered circumstances, such as differentiation of hypo-, eu- and hyperthyroid groups, those on T4 therapy, and in the three trimesters of pregnancy, but additionally, individuals with variations in thyroxine binding protein concentrations (TBG) from zero to four times the mean concentration of the reference range, analbuminemics, familial dysalbuminemic hyperthyroxinemics (FDH), sera with high concentrations of T4 and T3-containing autoantibodies, and various states and forms of nonthyroidal illness (NTI) [10]. The tests were additionally supported by comparison with equilibrium dialysis. Correlations between TBG, transthyretin and albumin with FT4 or FT3 were also examined in healthy persons. By such comprehensive testing protocols, appropriate findings in the rare groups consolidated the assays’ accuracy and precision in the more common cases. Rarely, if at all, has any other test since been validated using a similar rigorous examination before submission for licence to market the method. Rather methods were merely copied and justified in commonly encountered patient groups without applying a deeper understanding of the care needed to produce a comprehensively acceptable performance in more challenging situations. The exhaustive protocol of testing used in the first assays uncovered hitherto unknown problems, which were largely corrected in later developments [10]. Such duty of care was no longer maintained by recent relaxed protocols. Crucially, awareness of the especial difficulties of producing a valid FT3 test appears to be lacking. This requires an order of magnitude of greater understanding of the tests even than for FT4. The apparent success of such imperfect assays in general cases does not validate their performance in rare sera. Modification costs may contribute to an unwillingness to refine existing assays, because the cost of improvement may outweigh the perceived additional financial return

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The second failure has been by the regulators who determine the criteria and give permission for the tests to be marketable. The necessary basic validations appropriate for a free analyte assay are far more searching than required for the total hormone tests. Not only should precision and specificity be examined, but also the special requirements for assay validity. These include (1) robustness of the assay results to progressive serum dilution, (2) acceptable performance in rare categories, and (3) extraction of analyte by the test’s antibody sufficiently small as to negligibly affect FT4 or FT3 measurement in all categories. Regulatory requirements are instead rooted in the requirements for validation of total hormone assays, which though necessary, are here fundamentally inadequate as a test of validity.

The third culpable entity is the lenient reaction and tolerant attitude of the medical discipline to this unsatisfactory state of affairs. It is surprising that in the intercourse between producer and customer there has been no active demand by the latter for improving accuracy, precision and robustness in the various assays offered. It is usual that if offerings vary so much in a product, the customer will demand to have a more consistent performance in all. In the current environment, some financial constraints are likely to unduly influence the selection of assay groups over performance. The problem is accentuated by the present system of multi-assay offerings in a dedicated system. If within a given platform, methods for FT4 and FT3 are inadequate, it becomes very difficult to measure these separately by a different system. Such variation in test results causes unnecessary revalidation work if tests are subject to a change in product.

In conclusion, for significant progress in rationalisation of tests to occur, regulators themselves needed to strengthen the requirements for valid working assays. This must include robustness to progressive serum dilution and independence from transport protein differences. Only stricter licensing rules will force producers to deliver on the promise of rationalisation and harmonisation of contemporary FT4 and FT3 assays, which is well within their capability and the public interest.

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1) Kratzsch J, Baumann NA, Ceriotti F, Lu ZX, Schott M, van Herwaarden AE, et al. Global FT4 immunoassay standardization: an expert opinion review. Clin Chem Lab Med 2020. doi.org/10.1515/cclm-2020-1696.

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2.Midgley JEM, Wilkins TA. New methods of free thyroid hormone assay. In: Bizollon Ch A, editor Physiological peptides and new trends in radioimmunoassay. Elsevier/North Holland; 1981. p. 215–34.

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3.Ekins RP. Measurement of free hormones in blood. Endocr Rev 1990;11:5–46. doi.org/10.1210/edrv-11-1-5.

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4.Nelson JC, Nayak S, Wilcox RB. Variable underestimates of serum free thyroxine (T4) immunoassays of free T4 concentrations in simple solutions. J Clin Endocrinol Metab1994;79:1373–5. doi.org/10.1210/jc.79.5.1373.

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5.Midgley JEM, Christofides ND. Legitimate and illegitimate tests of free-analyte assay function. Clin Chem 2009;55:439–41. doi.org/10.1373/clinchem.20....

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6.Midgley JEM. “All that glisters is not gold”: ultrafiltration and free thyroxine measurement; with apologies to W Shakespeare. Clin Biochem 2010;44:151–3. doi.org/10.1016/j.clinbioch....

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7.Midgley JEM. Spurious conclusions on analog free thyroxine assay performance. Clin Chem 2007;53:1714. doi.org/10.1373/clinchem.20....

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8.Midgley JEM, Christofides ND. Inaccuracies in free thyroid hormone measurement by ultrafiltration and tandem mass spectrometry. Clin Chem 2009;55:2228–9. doi.org/10.1373/clinchem.20....

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9.Thienpont LM, Faix JD, Beastall G. Standardisation of FT4 and harmonization of TSH measurements – a request for input from endocrinologists, and other physicians. Endocrine2015;62:855–6. doi.org/10.1507/endocrj.ej1....

•10.Christofides ND, Sheehan CP. Enhanced chemiluminescence labeled-antibody immunoassay (Amerlite-MAB) for free thyroxine: development and technical validation. Clin Chem 1995;41:17–23. doi.org/10.1093/clinchem/41....

[linda96]

Thank you Diogenes. I am interpreting this as the TH assay kits are not being used correctly? That the assay kits are being used without proper validation in different assay machines, not correlating the results to compare accurately with the same kits used in other assay machines?

[Musicmonkey]

I am struggling to understand the significance of this as a lay person. Does it mean that the way blood tests for T3 and T4 are being carried out is not accurate?

[diogenes Remembering in reply to Musicmonkey]

My answer is a bit technical. You can swap between different tests with different ranges and correct for difference , so long the two tests parallel each other all the way - that is at each point over the whole range you can use the same fractional correction. Unfortunately this is not always true so in those cases you mention, no the test(s) are not all accurate over all the range. It won't matter if the distortions are nowhere near diagnostic sensitive areas but will otherwise. This is why some tests are better than others at diagnosing. And why when changing test methods a new reference range has to be developed by the lab.

[Musicmonkey in reply to diogenes]

I see, thank you. So our doctors really need to be aware of this and ensure that labs are making these adjustments where necessary.

[diogenes Remembering in reply to Musicmonkey]

It is absolutely necessary.

[Nat107 in reply to diogenes]

Hi Diogenes Is that why everyone who has thyroid testing blood tests have different ranges as per depending on where you live and which labs they are sent to. It is strange their can be so many different ranges for each blood test a person has depending on this criteria of each lab instead of all being bog standard same ranges across the country at least.

I would just like to mention if that’s ok, in regards to GP care and non testing of T3, most GP’s are just not prepared to do this test, maybe due to cost of both the test & if say needed treatment of T3, When Both my husband and myself queried this as we had reason to with my GP & nurses who were constantly pushing to put both myself & my husband on statins because our cholesterol was 5.5 & 6, which didn’t think was that extremely high, I mentioned it could be related to the fact we both had hypothyroidism for many years and it is in fact related to low T3 levels. They were not happy I even brought the subject up so eventually I asked them to test our blood for T3 levels, and we would take it from there. they eventually brought us into surgery for blood tests and a talking too, thinking they were going to help us at last we were happy, but later found they had not even tested our T3 but TPO antibodies instead, but strangely after this, we didn’t hear a dickie bird regarding our test or statins, found it really strange as the phone call conversation we had which both of us received was very pushy indeed and was near on trying to scare us into accepting statins as we were now prone to many serious health symptoms because of our cholesterol levels, strangely the phone calls stopped and we heard no more after requesting our T3 be checked first to see if it was relating to our ‘high cholesterol’!

So it seems to us it is not really about wanting to make sure we are all round functioning ok by testing all vital levels but just sticking to testing our Tsh & T4. To save money and make more by dishing out statins.

Thank you for your information post Diogenes and trying to help, your information is always so interesting to read

Have a great day x

[diogenes Remembering in reply to Nat107]

What you say about ranges and how they are used is completely understood. But understand one thing! Decisions made from tests especially at the borders of any test where it could be "something or nothing" are purely biochemically based. But medicine is not a science; it is at base an art between the revisitation of the patient's history and the doctor who has to work on that basis. Numbers are only hints as to what to do next if anything; they are not set in stone as regards diagnosis. They have to be viewed within the interplay between doctor and patient acting preferably as equals in the drama.

[Nat107 in reply to diogenes]

Totally agree

[tattybogle in reply to diogenes]

well said diogenes

:)

[DippyDame in reply to diogenes]

If only medics understood this...

[Littlebee]

Thanks Diogenes this gives me an insight into the complexities in testing and how important it is that someone who thoroughly understands these tests and their manufacture is listened to and the learning is acted upon.

[LindaC]

Thank you diogenes 🎶😇 Congratulations, too.

[helvella Administrator Thyroid UK]

With apologies to Buffy Sainte-Marie (and others):

It is five point two, and it's six point four

He tests with Siemens and with Bayers

It is all of thirty-one, and it's only seventeen

It's been a danger for a thousand years

[Annib1]

Thanks Diogenes. It is a minefield. I wrote twice to practice manager and to GP with copies to both. I wanted to find out what they were willing to test for so I could test privately for the rest. After no response I telephoned the manager and she said she had discussed with senior clinicians in the practice and decided to leave it to the GP. I rang the GP to say I wanted fT4 and fT3 as well as TSH for my next blood tests as I was about to consult a private endo and wanted a baseline for levo only before he changed me to something else so she agreed to write this request on the lab forms. While waiting for test results I was changed to NDT but the NHS lab results came back as TSH only. So I do not have a baseline. Who is in charge here? The GPS or the labs or the CCGs? The guidelines all make a fuss about GP and patient cooperating in care plans and this makes a mockery. Rant over!