Thyroglobulin Measurement Giovanella

Marburg Summer School on Thyroid Cancer Management 2017
Thyroglobulin measurement
Prof. Dr. med. Luca Giovanella MD PhD

Clinic for Nuclear Medicine and Competence Centre for Thyroid Diseases
Oncology Institute of Southern Switzerland - Bellinzona (Switzerland)
Department of Clinical Chemistry and Laboratory Medicine
Ente Ospedaliero Cantonale (EOC) - Bellinzona (Switzerland)
Luca Giovanella MD PhD Thyroglobulin measurement
Disclosures
Advisory Board
Roche, BRAHMS TermoFisher, Bayer, Genzyme
Research grants
Roche, BRAHMS TermoFisher, Bayer, Genzyme,
Siemens, AstraZeneca
Speaker fees
Roche, BRAHMS TermoFisher, Siemens, Genzyme
Thyroglobulin: a thyroid-specific biomarker
8q24
chaperonin
• Folding
• Homo-dimerization
• Glycosilation
660 kDa
Marker of the “presence/absence” of thyroid cells ( DTC cells)

Tg 144’090 μg/L
Agenda
Tg measurement in DTC management
 Laboratory
- Assay methods
- Analytical problems
 Clinical practice
- Interpretation criteria
- Emerging problems (i.e. non-ablated patients)

Analytical methods
▪ Circulating thyroglobulin measurement
Immunoassays
RIA standard curve
Radioimmunoassay (RIA)
Immunometric assays (IMAs)
IMA standard curve

Analytical sensitivity
 Functional Sensitivity (FS)

 Limit of Quantification (LOQ)
▪ MEASURABLE / REPORTABLE
▪ DETECTABLE BUT NOT MEASURABLE

 Limit of Detection (LOD)
▪ UNDETECTABLE
 Limit of Blank (LOB)
▪ BACKGROUND
1. LOB: Highest measurement result that is likely to be observed for a blank sample [= meanblank+1.645 (SDblank)].
2. LOD: Lowest amount of analyte in a sample that can be detected, although perhaps not quantified as
an exact value [= LoB+1.645 (SD low concentration sample)].
3. LOQ: Lowest amount of analyte in a sample that can be quantitatively determined with stated
acceptable precision and trueness, under stated experimental conditions (IFCC)
4. FS: Lowest amount of analyte that can be quantitatively determined with an inter-assay coefficient-
of-variation < 20% (NACB)
High sensitivity: clinical relevance

positive
FS ~ 1 µg/L
negative
negative
FS ~ 0.1-0.2 µg/L rhTSH
rhTSH
Tgbasal < 0.1-0.2 g/L = Tgstimulated < 1-2 g/L
Spencer et al. Thyroid 2010 , Giovanella et al. JCEM 2014

Analytical problems
▪ Standardization
Molecular etherogeneity
(i.e. splicing of Tg-mRNA, glycosilation and iodination).
 Certified Reference Material (BCR® 457)
▪ Inter-assays biases remain >10%-15%.
▪ Use the same assay during the patient’s follow-up.
▪ Using different assays may disrupt serial monitoring.
▪ If change unavoidable  patient’s rebaseline needed
▪ Inter-assays variability ▪ Inter-labs variability

Analytical problems
▪ Standardization
Analytical problems
▪ Interferences
▪ Limited agreement between different assays
 Calibration against the 1st IR Preparation 65/93.
 Rebaseline if change in TgAb assay is unavoidable
▪ TgAb cutoff
Access Centaur Immulite 2000
IU/mL Elecsys Roche
Beckmann Siemens Siemens
FS 10 1.8 20 10
Literature 22 4 44 20
▪TgAb immunoassay:
MCO 115 60 60 40
Positive
Tg discarded
Negative
Tg validated
Interferences Conventional recovery
Tg autoantibodies (TgAb) Added Recovery Tg
Tg recovery
Clinically relevant Tg range
No interference
1 40 ug/L
RR recovery
(4%CV at 40 ug/L)
Interference Mini-recovery
Added Recovery Tg
Tg2 – Tg1
TgRR = x 100
[aTg]
Clinically relevant Tg range
Tg1 = Tgmeasured Tg (without recovery Tg)
Tg2 = Tg1 plus recovery Tg
aTg: recovery Tg concentration
1 2.5 ug/L
TgRR = Tg recovery rate
RR variance
(4%CV at 2.5 ug/L)
Tg-mrec reflex – preliminary data

Giovanella L et al, Clin Chem Lab Med 2013; 51: 449-453.
Verburg FA et al, Horm Metab Res 2012; 44: 555-557.
Interferences
PositiveTg autoantibodies (TgAb)
MS/MS - liquid chromatography

PROS
 Highly specific
 Not interferred by Abs
CONS
 Suboptimal FS at 0.5 ug-Tg/L).
Undetectable Tg in ~40% of TgAb-positive pts.
with proved DTC recurrence
 Sample preparation ~ 20 hours
 Technically demanding
Proteins span 10-orders of magnitude (i.e. 1
peptide from Tg / 40 millions peptides from Albumin)
 Hardware and technical expertise

TgAb as a surrogate tumor marker
TgAb2 -
TgAb2 +
▪ Preablation undetectable Tg and positive TgAb1

▪ TgAb2 = postablation (6-12 months) TgAb
Kim W G et al. JCEM 2008;93:4683-

4689
Decreased TgAb
Unchanged TgAb
Increased gAb
Interferences
Heterophilic antibodies
Screening
▪ removal of interfering antibodies with Hab-blocking
▪ HAb prevalence: 0.4-1%
reagent, normal mouse serum, immobilized protein A
▪ Falsely increased Tg (rarely decreased)
column or polyethylene glycol.
Giovanella L et al, Clin Chem Lab Med 2009; 47: 952-954. ▪ test repetition with an alternative assay
Verburg FA et al, Horm Metab Res 2010; 42: 736-739.
▪ measurement of serial dilutions of suspected samples
Interferences
Hook effect
▪ assay antibodies sequestered by excess antigen,

▪ formation of sandwich-complexes becomes less likely
▪ very rare in modern IMAs

▪ serial dilutions = Tg increased
Interpretation criteria
(i.e. response assessment)

Early follow-up
Assay-specific cutoffs
Luca Giovanella MD PhD DTC Risk Stratification
Serum thyroglobulin in non-ablated patients
What is the role of serum Tg measurement in patients who have not undergone RAI
remnant ablation?
RECOMMENDATION 64
Periodic serum Tg measurements on thyroid hormone therapy should be considered
during follow-up of patients with DTC who have undergone less than total thyroidectomy
and in patients who have had a total thyroidectomy but not RAI ablation.
While specific cutoff levels of Tg that optimally distinguish normal residual thyroid
tissue from persistent thyroid cancer are unknown, rising Tg values over time are
suspicious for growing thyroid tissue or cancer.
ATA 2015
Serum thyroglobulin in non-ablated patients
RECOMMENDATION 50
A) Postoperative disease status (i.e., the presence or absence of persistent disease) should
be considered in deciding whether additional treatment (e.g., RAI, surgery, or other
treatment) may be needed. (Strong recommendation, Low-quality evidence)
B) Postoperative serum Tg (on thyroid hormone therapy or after TSH stimulation) can
help in assessing the persistence of disease or thyroid remnant and predicting potential
future disease recurrence. The Tg should reach its nadir by 3–4 weeks postoperatively in
most patients. (Strong recommendation, Moderate-quality evidence)
C) The optimal cutoff value for postoperative serum Tg or state in which it is measured
(on thyroid hormone therapy or after TSH stimulation) to guide decision-making
regarding RAI administration is not known. (No recommendation, Insufficient evidence)
Serum thyroglobulin in non-ablated patiens
 Postoperative serum thyroglobulin monitoring for patients who have functioning thyroid
remnants that typically give rise to serum basal Tg2GIMA concentrations in the 0.05–0.5 μg/l
or less range when TSH is suppressed.
Spencer C et al. Curr Opinion Endocrinol Diabetes Obes. 2014
 Tg will become a significantly less-useful marker in this scenario, and will either have to
be replaced by tumor-specific markers (e.g., molecular markers for tumor-specific mutations)
or more sophisticated Tg reference intervals, mathematically normalized to TSH level and
residual thyroid tissue tailored to individual patients, will have to be established.
Grebe SKG. Expert Rev Endocrinol Metab 2010
Tg in DTC patients: summary
 High-sensitive Tg
 Negative TgAb/if positive monitor TgAb trend
 ROC curve-derived cutoff
 Not useful after lobectomy
 No interpretation criteria after thyroidectomy alone

Serum Tg: pre-therapy measurement
 PSA pre-therapy mandatory

 CEA pre-therapy mandatory
 Tg pre-therapy not recommended
TM[C]
t

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Marburg Summer School on Thyroid Cancer Management 2017

Prof. Dr. med. Luca Giovanella MD PhD

Thyroglobulin: a thyroid-specific biomarker

Marker of the “presence/absence” of thyroid cells ( DTC cells)

Tg measurement in DTC management

- Emerging problems (i.e. non-ablated patients)

▪ Circulating thyroglobulin measurement

Immunometric assays (IMAs)

IMA standard curve

 Functional Sensitivity (FS)

▪ DETECTABLE BUT NOT MEASURABLE

High sensitivity: clinical relevance

FS ~ 0.1-0.2 µg/L rhTSH

Tgbasal < 0.1-0.2 g/L = Tgstimulated < 1-2 g/L

Spencer et al. Thyroid 2010 , Giovanella et al. JCEM 2014

 Certified Reference Material (BCR® 457)

▪ Inter-assays biases remain >10%-15%.

▪ Use the same assay during the patient’s follow-up.

▪ Using different assays may disrupt serial monitoring.

▪ If change unavoidable  patient’s rebaseline needed

▪ Inter-assays variability ▪ Inter-labs variability

 Calibration against the 1st IR Preparation 65/93.

 Rebaseline if change in TgAb assay is unavoidable

Interferences Conventional recovery

Tg autoantibodies (TgAb) Added Recovery Tg

Tg-mrec reflex – preliminary data

MS/MS - liquid chromatography

 Not interferred by Abs

 Sample preparation ~ 20 hours

 Hardware and technical expertise

TgAb as a surrogate tumor marker

▪ Preablation undetectable Tg and positive TgAb1

Kim W G et al. JCEM 2008;93:4683-

▪ assay antibodies sequestered by excess antigen,

▪ very rare in modern IMAs

(i.e. response assessment)

Serum thyroglobulin in non-ablated patients

Serum thyroglobulin in non-ablated patients

Serum thyroglobulin in non-ablated patiens

Tg in DTC patients: summary

 Negative TgAb/if positive monitor TgAb trend

 ROC curve-derived cutoff

 Not useful after lobectomy

 No interpretation criteria after thyroidectomy alone

Serum Tg: pre-therapy measurement

 PSA pre-therapy mandatory

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