Standardization of thyroid function tests is important to ensure comparable results across laboratories. For tests of total T3 and T4, a reference measurement system exists using primary calibrators and isotope dilution mass spectrometry. For free T3 and T4, a conventional reference measurement procedure was established using equilibrium dialysis and liquid chromatography tandem mass spectrometry. For TSH, the measurand was defined as intact total TSH accounting for glycosylation, and an "All Procedure Trimmed Mean" statistical approach using multiple commercial assays on patient samples was adopted as a surrogate reference measurement procedure.

1. Standardization of
Thyroid Function Tests
Ola H. Elgaddar
MD, PhD, MBA, CPHQ - Lecturer of Chemical Pathology
Medical Research Institute - Alexandria University
Ola.elgaddar@alexu.edu.eg

2. Clinical laboratory tests performed
by different laboratories have
comparable results

3. 1.Imprecision +
2.systematic differences in method results, which
can arise from inherent differences in method
design +++ (Very obvious in immunoassays)

4. Is this a problem??
Immunoassays are critical to many clinical
decisions, and many parameters are present
in several guidelines…..... Assuming that
there is no difference between test results
assayed in different settings!

5. UK NEQAS [Edinburgh], Department ofLaboratoryMedicine,Royal Infirmary ofEdinburgh,Edinburgh EH164SA,UK

6. Harmonization of Measurements
Any process that enables the establishment
of equivalence of reported values produced
by different measurement procedures for the
same measurand, (i.e. the quantity
intended to be measured)

7. Harmonization is achieved via:
1) Method #1 = Standardization:
The conventional approach which depends
on the application of a well-understood
reference measurement procedure (RMP)
and commutable reference materials to
establish the calibration for each available
routine measurement procedure.

8. Commutability
The ability of a reference material to have
inter-assay properties comparable to the
properties demonstrated by authentic
clinical samples when measured by more
than one measurement procedure

9. The core component of standardization is
the “establishment of metrological
traceability” which requires the availability of
an ISO conform reference measurement
system (RMS)

10. Components of ISO conform RMS:
ØDefined measurand,
ØUnits for expression of measurement
results (ideally the Système International
d’Unités or SI),
ØDifferent hierarchical levels of materials
and measurement procedures.

11. Metrological Traceability

12. Accordingly, the provided lab results in the
lower part of the hierarchy (routine IVD
methods), will provide measurements
traceable to the top of the hierarchy, within
stated uncertainty constraints.

13. Joint Committee for Traceability in Laboratory Medicine (JCTLM)

14. Harmonization is achieved via:
2) Method #2:
Considered only when RMPs are not
available and is based on application of
sound statistical methodology to produce
arbitrary equivalence of otherwise disparate
results generated by the various non-
standardized routine measurement
procedures (i.e: surrogate RMPs)

15. ØThyroid function tests
(TFTs) are among the most
commonly used tests in
both primary and secondary
healthcare settings
ØMany factors affect the
interpretation, and hence
the clinical decision to be
taken based on the TFTs
results.

16. ØThese factors could be
related to the physiology of
the hypothalamic – pituitary
– thyroid axis, and hence
the distribution of TH in the
the tissue.
ØSome physiological / pathological /
pharmacological cofounders could be present as
well affecting TH results….
ØAnd finally, the difference in TH results could be
attributed to the lab itself due to a standardization
problem

19. Till the end of this presentation, all data
is obtained either from the C-STFT
official website, or from their progress
reports released periodically!

20. Aim of the project
The C-STFT aims at global
standardization of thyroid function testing
and works towards this with the
international in vitro diagnostic (IVD)
industry.

21. Aim of the project
The overall expected benefit:
ØProduce equivalent laboratory data so
that common reference intervals and/or
clinical practice decision limits can be
used;
ØInclude laboratory data in electronic
patient records;

22. Aim of the project
The overall expected benefit:
ØDevelop evidence-based clinical
practice guidelines for application of
consistent standards of medical care;
ØCombine laboratory data across studies to
translate research into patient care &
disease prevention activities.

23. Total T3 & T4

24. Standardization of TT4 and TT3
The mission was easy as there is an existing
SI- traceable reference measurement
system that could be recommended by the
C-STFT to the IVD industry for
implementation in accordance with the ISO
17511.

25. Such RMS is composed of:
qPrimary calibrators (IRMM 468 & 469)
qReference methods (isotope dilution-mass
spectrometry, ID/MS)
qReference laboratories offering
measurement services
The committee refers to the database of the
Joint Committee for Traceability in
Laboratory Medicine (JCTLM)

28. Free T3 & T4

29. Standardization of FT4 & FT3
When the C-STFT started its activities in
2005, no reference measurement system
was available for free thyroid hormones
(FT4, FT3). Therefore, the C-STFT started
from scratch with defining the measurand,
and proposing a reference measurement
procedure (RMP)

30. Definition of the measurand
According to the International Union of Pure and
Applied Chemistry (IUPAC)/IFCC format, the
component was ‘‘T4 that is not bound to proteins’’
(present in the water fraction of plasma or serum),
the kind-of-quantity to measure the ‘‘amount-of-
substance concentration’’ and the system ‘‘plasma
or serum at physiological conditions (pH 7.40,
temperature 37.0°C)’’. The preferred unit for
expression of measurement results was pmol/L.

31. The conventional Reference
Measurement Procedure (cRMP)
qIdeally, metrological traceability of assays should
be established with a trueness-based RMP as
key element
qSince free thyroid hormones have to include a
physical step for separation of the free from
bound fraction (by equilibrium dialysis (ED) or
ultrafiltration (UF), it is by no means possible to
unequivocally demonstrate that the T4
concentration in dialysate or ultrafiltrate is
identical to the true free hormone concentration
in the original sample

32. The conventional Reference
Measurement Procedure (cRMP)
qTherefore, the C-STFT decided to propose
an international “conventional” RMP
(cRMP)
qIt should be based on ED and be combined
with direct determination of the T4
concentration in the dialysate with a
trueness-based RMP utilizing ID-liquid
chromatography/ tandem MS (ID-
LC/tandem MS) as measurement principle.

33. The conventional Reference
Measurement Procedure (cRMP)
This implied that according to this proposal
the measurand was operationally defined as
“T4 in the dialysate from ED of serum
prepared under defined conditions”. The
convention did not apply to the ID-LC/tandem
MS part of the cRMP, provided it is calibrated
with the certified T4 primary calibrator IRMM
468, already available in the total T4
reference measurement system

35. No CRM present when
you search for FT4

37. TSH

38. Harmonization of TSH
qTSH is a glycoprotein circulating in the
bloodstream as mixture of components
comprising disease-specific glycoforms.
qCurrent TSH immunoassays all are
traceable to the same WHO IRP TSH
80/558, and express their measurements in
mIU/L.

39. Harmonization of TSH
qThe WHO IRP TSH 80/558 is from human
cadaver pituitary and may comprise
glycosylation forms that differ from those
typically present in serum TSH. Therefore,
the above issues of non commutability
applies, and hence, IRP-traceable TSH
measurements do not give equivalent or
interchangeable results.

40. Before defining the TSH measurand
TSH measurement is mixture analysis. This
implies that each component in the mixture
must contribute significantly to the
diagnostic application of the assay used for
mixture analysis, and that the latter should
measure the components in the mixture in
an “equimolar” way, but to the diagnostic
relevant extent.

41. Before defining the TSH measurand
qThis means that the important changes in overall
concentration of the mixture in health and
disease should outweigh the minor changes in
composition of the mixture in individual samples.
qThis requirement of equimolar measurement is
of utmost relevance for TSH, since glycoforms
are different in euthyroidism versus subclinical
and overt hypothyroidism, and because there is
evidence that immunoassays may be sensitive
to the differences

42. Definition of TSH measurand
The C-STFT defined the measurand as
“TSH, intact, total, glycosylation
encountered in diagnostic applications which
should be specified”.
This definition requires in first instance that
TSH assays examined for harmonization
deliver a measure for “total TSH”. It also
implies that harmonization should be based
on the glycoforms present in native samples.

43. The “All Procedure Trimmed Mean
APTM” as surrogate RMP for TSH
Ø Current TSH assays are expressed in IUs
(concentrations in mIU/L). However, it is to
expect that one day it will be possible to
establish the relation between the IU and mass
units (SI) for TSH.
Ø From this point of view, the reference
measurement system for TSH and its realization
should be viewed as continuum from discovery
of the analyte to final translation into the SI, with
different needs at different stages of the
continuum

44. The “All Procedure Trimmed Mean
APTM” as surrogate RMP for TSH
Ø Therefore, the C-STFT decided to preserve the
traceability of TSH assays to the WHO IRP in
their proposed harmonization process, and
recommends the “All Procedure Trimmed Mean”
(APTM, expressed in IU) as surrogate RMP. It is
proposed to statistically derive the APTM from
data of a dedicated method comparison study
with native human samples, in which as many
TSH assays as possible participate

45. The “All Procedure Trimmed Mean
APTM” as surrogate RMP for TSH
ØAccording to this proposal, the APTM
targeted panel becomes the key tool in the
harmonization process.
ØIt has the big advantage that it is composed
of samples that contain the typical
glycoforms of TSH in the blood circulation,
thus are as commutable as possible.

46. The “All Procedure Trimmed Mean
APTM” as surrogate RMP for TSH
ØAfter being assigned with the APTM
(expressed in mIU/L) derived from the
measurement values of the WHO traceable
assays that participated in the method
comparison study, the panel is intended
to serve as a new measurement
standard, in other words, at that point in
the harmonization process, the IU of the
WHO IRP is transferred to the panel.

47. The “All Procedure Trimmed Mean
APTM” as surrogate RMP for TSH
Note that for the process to be successful,
proof of sufficient correlation of the
concerned assays to the APTM is a
prerequisite. Also the new standard should
be sustainable. This requires that the IU is
transferred from the first panel to the follow-
up panels via analogous method comparison
studies.