standardization of thyroid function tests · rms for free thyroid hormones: accomplished references...

Standardization of Thyroid Function Tests

Linda [email protected]

Saturday, Dec 3rd 201139th meeting

Location: Nycomed Belgium1080 Brussels

Belgian Thyroid Club -39th meeting - Dec 3rd 2011 22

Relatively high prevalence in adults Significant clinical consequences New perspectives resulting from researche.g.,1-3

Subclinical thyroid dysfunction linked to various adverse clinical outcomes (all-cause mortality; pregnancy)

1Singh S et al. Impact of subclinical thyroid disorders on coronary heartdisease, cardiovascular and all-cause mortality: a meta-analysis. Int J Cardiol 2008;125:41-48.2Haentjens P et al. Subclinical thyroid dysfunction and mortality: an estimate of relative and absolute excess all-cause mortality based on time-to-event data from cohort studies. Eur J Endocrinol 2008;159:329-41. 3Negro R et al. Thyroid antibody positivity in the first trimester of pregnancy is associated with negative pregnancy outcomes. J Clin Endocrinol Metab2011;96:E920-4.

Thyroid dysfunction: clinical importance


Clinical manifestations of thyroid disease vary in character and severity among patients

Symptoms often non-specific and slowly progressingAdequate laboratory assessment is needed

Strategies for laboratory testing and interpretation are welcome

Note: yearly volume of requests for testing of TSH and free thyroxine (FT4) alone is estimated to be in the order of 180 and 60 million US$, respectively#

#Weinzierl C, Beckman-Coulter, personal communication (2009).

Accuracy of clinical diagnosis limited


Guidelines – Ladenson PW et al. American Thyroid Associationguidelines for detection of thyroid dysfunction. ArchIntern Med 2000;160:1573-1575.– Gharib H et al. Subclinical thyroid dysfunction: a joint statement on management from the American Association of Clinical Endocrinologists, the American Thyroid Association, and the Endocrine Society. J Clin Endocrinol Metab 2005;90:581-585.– Bahn RS et al. Hyperthyroidism and other causes of thyrotoxicosis: management guidelines of the American Thyroid Association and American Association of Clinical Endocrinologists. Thyroid 2011;21:593-646.

Lab testing and interpretation strategies


Guidelines (continued)– Demers LM et al. 2002 Laboratory Medicine Practice Guidelines: laboratory support for the diagnosis and monitoring of thyroid disease. National Academy of Clinical Biochemistry (NACB) Washington (DC). http://www.aacc.org/members/nacb/archive/lmpg/thyroiddisease/pages/default.aspx.– Abalovich M et al. Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society clinical practice guideline. J ClinEndocrinol Metab 2007;92:S1-S47.– Stagnaro-Green A et al. Guidelines of the American Thyroid Association for the diagnosis and management of thyroid disease during pregnancy and postpartum. Thyroid 2011;21:1-45.



Leading journals– Dayan CM. Interpretation of thyroid function tests Lancet 2001;357(9256):619-624 [Review].– Stockigt J. Assessment of thyroid function: towards an integrated laboratory-clinical approach. Clin BiochemRev 2003;24:109-122.– Surks MI et al. Subclinical thyroid disease: scientific review and guidelines for diagnosis and management. JAMA 2004;291:228-238 [Review].



Not to forget:Hot topics currently under debate in scientific literatureAmong others: is screening for thyroid dysfunction in pregnancy worthwhile?e.g., 1,2

Do we need age-, gender- & ethnicity-specific TSH reference limits?e.g., 3

Should the TSH upper reference limit be lowered?e.g., 4,5

1Lazarus JH 2011 Screening for thyroid dysfunction in pregnancy: is it worthwhile? J Thyroid Res 2011, Article ID 397012 doi:10.4061/2011/397012. 2Negro R et al. Thyroid antibody positivity in the first trimester of pregnancy is associated with negative pregnancy outcomes. J Clin Endocrinol Metab 2011;96:E920-4.3Boucai L et al. An approach for development of age-, gender-, and ethnicity-specific thyrotropin reference limits. Thyroid 2011;21:5-11.4Brabant Get al. Is there a need to redefine the upper normal limit of TSH? Eur J Endocrinol 2006;154(5):633-7. Review.5Laurberg P et al. The TSH upper reference limit: where are we at? Nat Rev Endocrinol2011;7:232-239.



A closer look into the strategiesGuidelines sometimes quote absolute decision limits/cut-off values or reference intervals Literature sometimes reports on clinical trials in terms of absolute values without reference to the laboratory test used

Examples:… the panel defined the reference range of normal TSH concentration as 0.45 – 4.5 mIU/L… the panel recommends thyroid hormone therapy in individuals with elevated serum TSH concentrations whose FT4 concentration is below the reference range of 10.3 – 25.7 pmol/L… a TSH of 2.5 mIU/L has been accepted as the upper limit of normal TSH in the first trimester of pregnancy…


Measurement paradigmPrerequisite to work with absolute values

“Laboratory tests that claim the same measurandshould give equivalent results

within meaningful clinical constraints”


Measurement paradigm

Do current thyroidfunction tests giveequivalent results?


Working Group for Standardization of Thyroid Function Tests (WG-STFT) –

Chair: LM Thienpont (since 2005)

Mission statement:Develop reference measurement systems for thyroid

hormones, i.e., FT4 & FT3, TSH, TT4 & TT3”

Standardize the tests


Reference measurement system

Primary reference measurement procedure

Manufacturer’smaster procedure

End user’s routine measurement procedure

Primary calibrator

Manufacturer’sworking calibrator

Manufacturer’sproduct calibrator

Routine sample

MaterialCalibration

ProcedureValue assignment

Definition of measurand&unit

Result

Secondary reference measurement procedure

Trac

eabi

lity U

ncertainty


WG-STFT – Mission statementDevelopReference materialsReference measurement proceduresReference laboratories

Referencemeasurement

system

Implement (sustainable) standardization

Assess the current standardization status andquality of performanceMethod comparison with a panel of native sera

Investigate the feasibility of standardization

Before the mission statement “standardize”…


WG-STFT – Mission statementRMS for free thyroid hormones: accomplishedReferencesThienpont et al. Measurement of free thyroxine in laboratory medicine – Proposal of measurand definition. IFCC Working Group for Standardization of Thyroid Function Tests (WG-STFT). ClinChem Lab Med 2007;45:563-4.

Thienpont et al. Proposal of a candidate international conventional reference measurement procedure for free thyroxine in serum. IFCC Working Group for Standardization of Thyroid Function Tests(WG-STFT). Clin Chem Lab Med 2007;45:934-6.

Van Houcke SK, Van Uytfanghe K, Shimizu E, Tani W, Umemoto M, Thienpont LM. IFCC Working Group for Standardization of Thyroid Function Tests (WG-STFT). IFCC international conventional reference procedure for the measurement of free thyroxine in serum. Clin Chem Lab Med 2011;49:1275-81.


WG-STFT – Mission statementRMS for total thyroid hormones: accomplished ReferencesToussaint B, Klein CL, Wiergowski M. The certification of the mass fraction of thyroxine in a CRM intended for calibration. EUR 21872 EN (2006). ISBN 92-79-01032-8.

Ibidem. The certification of the mass fraction of triiodothyronine in a CRM intended for calibration. EUR 21893 EN (2006). ISBN 92-79-01067-0.

Thienpont LM, Van Uytfanghe K, Marriot J, Stokes P, Siekmann L, Kessler A, Bunk D, Tai S. Metrologic traceability of total thyroxinemeasurements in human serum: efforts to establish a network of reference measurement laboratories. Clin Chem 2005;51:161-8.

Thienpont LM, Van Uytfanghe K, Marriott J, Stokes P, Siekmann L,Kessler A, Bunk D, Tai S. Feasibility study of the use of frozenhuman sera in split-sample comparison of immunoassays with candidate reference measurement procedures for total thyroxineand total triiodothyronine measurements. Clin Chem 2005;51:2303-11.


WG-STFT – Mission statementRMS for TSH: accomplishedReferencesStöckl D, …Thienpont LM et al. Current Stage of Standardization of Measurements of Specific Polypeptides and Proteins Discussed in Light of Steps Needed Towards a Comprehensive Measurement System. Discussion paper from the Members of the EQA Working Group B on Target Values in External Quality Assessment. Eur J ClinChem Clin Biochem 1997;35:719-32.

Thienpont LM. Accuracy in Clinical Chemistry - Who Will Kiss Sleeping Beauty Awake? Clin Chem Lab Med 2008;46:1220-2.

Thienpont LM, Van Houcke SK. Traceability to a common standard for protein measurements by immunoassay for in-vitro diagnostic purposes. Clin Chim Acta 2010;411:2058-61.

Miller WG … Thienpont LM et al. Roadmap for Harmonization of clinical laboratory measurement procedures. Clin Chem2011;57:1108-17.


WG-STFT – Mission statementAssessment of the current standardization status and quality of performance: accomplishedReferencesThienpont LM et al.; for the IFCC Working Group on Standardization of Thyroid Function Tests. Report of the IFCC Working Group forStandardization of Thyroid Function Tests.Part 1: Thyroid-Stimulating Hormone. Clin Chem 2010; 56:902-11.Part 2: Free Thyroxine and Free Triiodothyronine. Clin Chem 2010;56:912-20.Part 3: Total Thyroxine and Total Triiodothyronine. Clin Chem 2010;56:921-9.

Thienpont LM et al. Standardization activities in the field of thyroid function tests: a status report. Clin Chem Lab Med 2010;48:1577-83.


Measurement paradigm

Do current thyroidfunction tests giveequivalent results?


Standardization statusFree thyroxine (FT4)IFCC WG-STFT – Project phase I (2008)

y = 0.5066x + 1.2198 (D)

y = 0.9111x + 1.2935 (E)

0

10

20

30

0 10 20 30FT4 ID-LC/MS (pmol/L)

Mea

ns, a

ll (p

mol

/L) .

-80-60-40-20

020406080


Diff

eren

ce, m

eans

, all

(%) . F #3: +101%

Mean MS

17.08

10.008

1012

14

16

1820

22

A B C D E F G H I J K L M N P Q R

Procedure

FT4,

mea

ns (p

mol

/L) .

. 15 tests

Means varied from 10 to 17 pmol/L

Except 2 tests, all measured FT4 much lower than the RMP (up to 42%)

Hyperthyroid FT4 conc. with 1 test, still eu- with another

Standardization is needed


Standardization statusFree triiodothyronine (FT3)IFCC WG-STFT – Project phase I (2008)

14 tests

Means varied from 3.7 to 6.5 pmol/L

Most tests werenegatively biased vsthe RMP (up to 30%)

1 test had a positive bias of 22%

Standardization is neededFT3 ED ID-MS (pmol/L)

FT3

Mea

ns R

outin

e (p

mol

/L)

FT3

Rou

tine

(pm

ol/L

)

FT3

Diff

eren

ce R

outin

e (%

)

FT3 ED ID-MS (pmol/L)

y = 0.70x + 0.01 (A)

y = 1.14x + 0.4 (O)

1

3

5

7

9

1 2 3 4 5 6 7-60

-40

-20

0

20

40

60

1 2 3 4 5 6 7

Mean MS

3.7

6.5

3

4

5

6

7

A B C D E F G H I J K M N O


Standardization statusTSHIFCC WG-STFT – Project phase I (2008)

1.60

2.27

1.4

1.6

1.8

2.0

2.2

2.4

A B C E F G H I J K L M N O P RProcedure

TSH

, med

ians

(mIU

/L) ..

y = 1.1556x - 0.0702 (C)

y = 0.8045x + 0.0213 (K)

0

20

40

60

80

0 20 40 60 80TSH All median (mIU/L)

Mea

ns, a

ll (m

IU/L

) .

-120-100-80-60-40-20

020406080

100120

0.00 0.10 10.00

TSH All median log(mIU/L)

Diff

eren

ce, m

eans

, all

(%) . R #39: +159%

16 testsMedians of 11 tests differed <10% from the surrogate RMP (overall median) Using the respective regression equations and a TSH RI from 0.4 to 4 mIU/L, the most discrepant tests would give values ranging from 0.34 to 3.24 mIU/L, and 0.39 to 4.55 mIU/L

Lowering the upper reference limit is impossiblewithout standardization


Standardization statusTotal thyroxine (TT4)IFCC WG-STFT – Project phase I (2008)

101.76

74.73

65707580859095

100105110

A B C E F G H K L M PProcedure

TT4,

mea

ns (n

mol

/L) ..

y = 1.251x - 6.8799 (H)

0

50

100

150

200

250

300

0 50 100 150 200 250 300TT4 ID-LC/MS (nmol/L)

Mea

ns, a

ll (n

mol

/L) .

-60

-40

-20

0

20

40

60

0 50 100 150 200 250TT4 ID-LC/MS (nmol/L)

Diff

eren

ce, m

eans

, all

(%) .

y = 0.785x + 7.88(M: w ithout high)

11 tests

Means varied from 75 to 102 nmol/L

Overall good agreement with the RMP: 7 tests gave means that differed less than 10%

Only 4 tests need standardization


Standardization statusTotal triiodothyronine (TT3)IFCC WG-STFT – Project phase I (2008)

TT3 ID-MS (nmol/L)

TT3

Mea

ns R

outin

e (n

mol

/L)

TT3

Rou

tine

(nm

ol/L

)

TT3

Diff

eren

ce R

outin

e (%

)

TT3 ID-MS (nmol/L)

y = 1.17x - 0.23 (C)

y = 1.31x + 0.01 (K)

0.5

1.0

1.5

2.0

2.5

3.0

0.5 1.0 1.5 2.0-40

-20

0

20

40

60

80

0.5 1.0 1.5 2.0

Mean MS1.45

1.89

1.21.31.41.51.61.71.81.92.02.1

A B C E F H I J K L N M

12 tests

Means varied from 1.45 to 1.89 pmol/L

Nearly all tests were positively biased vsthe RMP: 7 deviated >10%; 2 of them even >20% (up to 32%)

Standardization is needed


Quality of performanceAssessment of quality of performance Against quality specifications, e.g., TE limits.After recalculation of results with regression equation, e.g., FT4 best and worst quality of performanceIFCC WG-STFT – Project phase I (2008)

#8: 0.4

-6

-4

-2

0

2

4

6


E-D

iff R

ecal

-R1/

R1

(pm

ol/L

) ..

#8: -1.0

-6

-4

-2

0

2

4

6


G-D

iff R

ecal

-R1/

R1

(pm

ol/L

) ..


Quality of performanceAssessment of quality of performance IFCC WG-STFT – Project phase I (2008)

TSHK B R

r2 = 0.998 r2 = 0.992 r2 = 0.972

TS H Mean‐All (mIU/L )

TSH Diff Rec

al Rep

licate (m

IU/L)

TSH Diff Rec

al Rep

licate (m

IU/L)

TSH Diff Rec

al Rep

licate (m

IU/L)

T S H Mean‐All (mIU/L ) T S H Mean‐All (mIU/L )

-3

-2

-1

0

1

2

3

0 1 2 3 4 5 6 7-3

-2

-1

0

1

2

3

0 1 2 3 4 5 6 7-3

-2

-1

0

1

2

3

0 1 2 3 4 5 6 7


Free T4 in pregnancy Comparison (trimester specific) of FT4 values by ED ID-LC/tandem MS and 3 immunoassays in non-pregnant controls (n = 26) and pregnant (n = 107)

0

5

10

15

20

25

Control 1st 2nd 3rd

Con

cent

ratio

n (p

mol

/L)

FT4 ED-ID/tandem MS

0

5

10

15

20

25

Control 1st 2nd 3rd

Con

cent

ratio

n (p

mol

/L)

FT4 Cobas

0

4

8

12

16

20

Control 1st 2nd 3rd

Con

cent

ratio

n (p

mol

/L)

FT4 ARCHITECT

0

5

10

15

20

Control 1st 2nd 3rd

Con

cent

ratio

n (p

mol

/L)

FT4 Immulite

Δ9% Δ29% Δ9% Δ25%

Δ10% Δ25%

~ ~

~~ ~Δ14%


Free T4 in pregnancy

0

5

10

15

20

25

Control 1st 2nd 3rd

Con

cent

ratio

n (p

mol

/L)

FT4 ED-ID/tandem MS

0

5

10

15

20

25

Control 1st 2nd 3rd

Con

cent

ratio

n (p

mol

/L)

FT4 Cobas

0

4

8

12

16

20

Control 1st 2nd 3rd

Con

cent

ratio

n (p

mol

/L)

FT4 ARCHITECT

0

5

10

15

20

Control 1st 2nd 3rd

Con

cent

ratio

n (p

mol

/L)

FT4 Immulite

Δ9% Δ29% Δ9% Δ25%

Δ10% Δ25%

~ ~

~~ ~Δ14%

Anckaert E, Poppe K, …Thienpont LM et al. FT4 immunoassays may display a pattern during pregnancy similar to the equilibrium dialysis ID–LC/tandem MS candidate reference measurement procedure in spite of susceptibility towards binding protein alterations. Clin Chim Acta 2010;411:1348-53.


First conclusionCurrent thyroid function tests need standardization

Diagnosis/follow-up against test specific reference intervals/clinical decision thresholds is still recommended

Standardization efforts must be accompanied by assessment of the quality of thyroid tests

Communicate in a transparent way with clinicians on the influence of analytical quality on a test result

Infer adequate quality specifications preferably in consultation with clinicians

Improve the quality of some thyroid function tests


First conclusionTesting of FT4 in pregnancy

Immunoassays are sensitive to binding proteinalterations, but to a grossly different extent

Some show the “true” changes of “FT4” duringpregnancy (as observed with the referencemeasurement procedure ED ID-LC/tandem MS)

FT4 in pregnancy can be tested with commercial tests, but results have to be interpreted test specific; the same test should be used for follow-up


Feasibility of standardizationPhase II - Proof of concept – FT4Objective: demonstrate consistency in time of methodcomparisons against the reference measurementprocedure (or surrogate –) (= sustainability)

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

A B C D E F G H I J K L M

Rat

io A

ssay

-mea

n/M

S-m

ean

.

Phase 2 Phase 1


Feasibility of standardizationPhase II - Proof of concept – TSH

0.7

0.8

0.9

1.0

1.1

1.2

1.3

D O M F B H J A K G C L N E

Ratio

Ass

ay-m

ean/

Trim

med

-mea

n . Phase 2 Phase 1


Feasibility of standardizationStandardization = recalibration on the basis of a method comparisonOutcome of standardization – Phase I & IIFT4 – Situation before and after recalibration

-60

-30

0

30

10 20 30FT4 ED ID-MS (pmol/L)

Diff

eren

ce, m

eans

, all

(%) .

#8

-60

-30

0

30

10 20 30FT4 ED ID-MS (pmol/L)

Diff

eren

ce, m

eans

-rec

al, a

ll (%

) .


Feasibility of standardization

FT4 – Between test CV before (□) and after (Δ) recalibration

0

5

10

15

20

10 15 20 25 30FT4 ED-ID-MS (pmol/L)

Bet

wee

n as

say

CV

(%)


Feasibility of standardizationD

iffer

ence

Mea

ns (%

)

Diff

eren

ce M

eans

(%)

Trimmed Mean (mIU/L) Trimmed Mean (mIU/L)

-40-30-20-10

010203040

0 2 4 6 8 10-40-30-20-10

010203040

0 2 4 6 8 10

Outcome of standardizationPhase I & II TSH – Situation before and after recalibration


Feasibility of standardization

TSH – Between test CV before (□) and after (Δ) recalibration

0

5

10

15

20

0 2 4 6 8 10TSH Trimmed mean (mIU/L)

Bet

wee

n as

say

CV

(%)


Second conclusionStandardization is feasible

Technical means are available

Disseminate on activities/plans of the WG

Implement (sustainable) standardization


TransferabilityWay forward


Way forwardTransformation of WG-STFT into CommitteeObjective: involve a broader forum of stakeholders in preparation of the implementation phase

Stakeholders(IVD manufacturers)Physicians and their patients Laboratory directorsProfessional societiesPharmaceutical companies Regulatory agencies

Additional vehiclesMedical journals


Way forwardUp to now, feasibility of standardization shown from method comparison with samples from “apparently healthy” individuals

BUT

What about the performance of the tests on clinical samples?

Clinical samples, repository panels are needed!

Submit commercial tests to a method comparison with samples from different patient populations (hypo-, eu- and hyperthyroid patients) – Project Phase III (2012)


Phase III studyEstablish a strong physician–laboratory interfaceJanuary 2010 – Call to clinicians for:- Interest in joining the WG activities/discussions - Help with procurement of clinical samples/repository panels

Several positive replies (Europe, Japan, USA), BUT: all restricted to interest in joining the WG activities

Hindrances with regard to procurement of clinicalsamples- Approval by local ethical committees- Needed sample volume (15 mL serum/~ 30 mL blood)

Route not further pursued


Phase III studyClinical samples: FT4 n = 90: TSH n = 10015 mL of serum per sample

Commercial source for samples

Contact person: Dr. Jim BoushellAddress: 10 Commerce Way, Norton, MA 02766, USA


Phase III study


Phase III studyCriteria for enrolment of individualsPreferably, not under treatment.If treated, information on the type of treatment and when it has been started should be captured.

Exclusion of patients diagnosed with a severe non-thyroidal illness (NTI) (state of dysregulation with abnormal levels of T3, T4, FT3 and/or FT4 although the thyroid gland does not appear to be dysfunctional).

In practice, exclusion of critically ill patients suffering from CKD, liver cirrhosis, sepsis, trauma, advanced (active) malignancy, prolonged fasting/starvation, heart failure, MI, psychiatric disorder.

Agreement on a fair cost price per donation (450 US$)


Phase III study: status update (30/11/11)

Population Details Target n Enrollment n

TSH A1 <<<conc. 10 2A2: 0.01- 0.1 mIU/L* 10 1A3: 0.1-0.3 mIU/L 10 9B: 0.3-3.0 mIU/L Eu – 30 30C1: 3.0-50 mIU/L 20 20C2: >50 mIU/L 20 2

FT4 D: > 2.2 ng/dL Hyper – 30 5

E: 0.8-2.2 ng/dL Eu – 30 29F: 0.2-0.8 ng/dL Hypo – 30 9

Hypo –

Hyper-thyroid

*Test specific


Call of the Chair of WG-STFTWould you be willing to support us by

active involvement in patient (and sample) recruitment?


[email protected]. +32 9 264 81 04

IfIf yesyes, contact me!, contact me!

standardization of thyroid function tests · rms for free thyroid hormones: accomplished references...

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