- Email: [email protected]
Analytical and clinical validation of the new ultrasensitive Roche Thyroglobulin II assay
Analytical and clinical validation of the new ultrasensitive Roche thyroglobulin II assay Marcelo C. Batista, Carlos E.S. Ferreira, Paulo R.S. Ferreira, Adriana C.L. Faulhaber, Andr´e L.O. Silva, Crist´ov˜ao L.P. Mangueira PII: DOI: Reference:
S0009-9120(16)30467-2 doi:10.1016/j.clinbiochem.2016.11.019 CLB 9423
To appear in:
Clinical Biochemistry
Received date: Accepted date:
22 October 2016 18 November 2016
Please cite this article as: Batista Marcelo C., Ferreira Carlos E.S., Ferreira Paulo R.S., Faulhaber Adriana C.L., Silva Andr´e L.O., Mangueira Crist´ov˜ ao L.P., Analytical and clinical validation of the new ultrasensitive Roche thyroglobulin II assay, Clinical Biochemistry (2016), doi:10.1016/j.clinbiochem.2016.11.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Analytical and clinical validation of the new ultrasensitive Roche Thyroglobulin II assay
T
Author names and affiliations: Marcelo C. Batistaa, Carlos E. S. Ferreiraa,
IP
Paulo R. S. Ferreiraa, Adriana C. L. Faulhabera, André L. O. Silvaa, Cristóvão L.
a
SC R
P. Mangueiraa Clinical Lab, Hospital Israelita Albert Einstein
Bloco E – 2⁰ andar
MA
São Paulo – SP CEP 05652-900
NU
Av. Albert Einstein, 627
Brazil
[email protected]
D
E-mail:
TE
[email protected]
CE P
[email protected] [email protected] [email protected]
AC
[email protected]
Corresponding author: Marcelo C. Batista Hospital Israelita Albert Einstein Av. Albert Einstein, 627 Bloco E - 2⁰ andar – Laboratório Clínico São Paulo – SP CEP 05652-900 Brazil Phone: 55 11 2151-5555
ACCEPTED MANUSCRIPT Fax: 55 11 2151-0408 Cell phone: 55 11 99140-0282
IP
Running head: New Roche Thyroglobulin II assay
T
E-mail: [email protected]
SC R
Keywords: thyroglobulin; immunoassay; electrochemiluminescence; thyroid cancer
NU
Abbreviations CV: coefficient of variation
DTC: differentiated thyroid cancer
LOQ: limit of quantitation
Tg: thyroglobulin
TSH: thyroid-stimulating hormone
TE
D
MA
CE P
Funding: This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
AC
This work was presented in part as a poster on July 29, 2014, at the 2014 AACC Annual Meeting in Chicago, USA.
ACCEPTED MANUSCRIPT Manuscript Thyroglobulin (Tg) is a serum marker of differentiated thyroid carcinoma
T
(DTC) and thus should be accurately measured down to levels of 0.10 μg/L. In
IP
the past decade, a few second generation immunometric assays have been
SC R
developed with such sensitivity [1]. In this study, we evaluated the analytical and clinical performance of the new ultrasensitive Roche Tg-II assay
NU
(Mannheim, Germany).
Serum samples with anti-Tg < 20 IU/mL (Roche assay) were selected
MA
from our routine workload. Thyroglobulin was measured with Tg-II assay in Roche E170 Modular and Access Tg assay in Access 2 or Unicel DxI 800 (2
according
to
Beckman-Coulter,
Brea,
USA).
Both
are
TE
results,
D
equipments using the same principles/reagents and yielding nearly identical
electrochemiluminometric or enzymatic chemiluminometric, sandwich type
CE P
assays calibrated against the same reference material BRC 457 (European Commission). Access has a functional sensitivity < 0.10 μg/L [1] and is
AC
considered by many the gold standard Tg immunoassay. Tg-II limit of quantitation (LOQ, lowest concentration with CV < 20%) was < 0.08 µg/L (CV=16.8%), as determined by running 2 low Tg serum pools for 3 months using 2 different reagent and calibrator lots. Total imprecision of 3 serum samples and 2 Roche controls was < 5.5% at 0.28-77 µg/L. Linearity was confirmed at 0.21-493 µg/L. Tg-II levels correlated well (r=0.99), but were on average 27% higher (mean bias 3.73 µg/L) compared to Access in 226 samples. This difference is slightly above Tg total error (21.9%) according to biologic
variation
database
(www.westgard.com/quality-requirements.htm,
access 10/22/2016) and is most likely due to heterogeneity in antibodies used in
ACCEPTED MANUSCRIPT the assays [1], which may have varying specificity for multiple Tg epitopes (for more details on analytical validation, see Supplementary Material).
T
To evaluate the clinical performance of Tg-II in distinguishing DTC
IP
patients with and without active disease, we tested 2 different cutoffs for
SC R
suppressed Tg drawn under thyroxine replacement (0.10 and 0.27 µg/L) and also for endogenous/exogenous TSH-stimulated Tg (1.00 and 2.00 µg/L) [2,3]. Access Tg below these limits was considered an indication of disease-free
NU
status. Suppressed Access Tg was < 0.10 or 0.27 µg/L in 42 and 51 samples,
MA
respectively (Figure 1A); in the former group, Tg-II was < 0.10 µg/L in all but one specimen (0.10 µg/L), whereas in the latter group Tg-II was < 0.27 µg/L in all 51 samples. Stimulated Access Tg was < 1.00 or 2.00 µg/L in 18 and 27
TE
D
samples, respectively (Figure 1B); in the former group, Tg-II was < 1.00 µg/L in all 18 samples, whereas in the latter group Tg-II was < 2.00 µg/L in all but one
CE P
specimen (Access Tg 2.90 µg/L and Tg-II 1.28 µg/L). In summary, our results are consistent with 2 recent reports [4,5] and
AC
suggest Tg-II is a highly sensitive and reproducible assay with an excellent agreement
(96-100%)
with
Access
in
distinguishing
absent
and
persistent/recurrent DTC. Nonetheless, because of a positive Tg-II bias, labs planning to switch to this assay should ideally establish a new baseline for each patient by running a few samples in the old and new assay [1].
ACCEPTED MANUSCRIPT References 1. Giovanella L, Feldt-Rasmussen U, Verburg FA, Grebe SK, Plebani M, Clark
T
PM. Thyroglobulin measurement by highly sensitive assays: focus on
IP
laboratory challenges. Clin Chem Lab Med 2014;53:1301-14.
SC R
2. Giovanella L, Treglia G, Sadeghi R, Trimboli P, Ceriani L, Verburg FA. Unstimulated highly sensitive thyroglobulin in follow-up of differentiated
NU
thyroid cancer patients: a meta-analysis. J Clin Endocrinol Metab 2014;99:440-7.
MA
3. Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Nikiforov YE, Pacini F, Randolph GW,, Sawka AM, Schlumberger M, Schuff KG, Sherman
D
SI, Sosa JA, Steward DL, Tuttle RM, Wartofsky L. 2015 American Thyroid
TE
Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association
CE P
Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid 2016;26:1-133.
AC
4. Netzel BC, Grebe SKG, Leon BGC, Castro MR, Clark PM, Hoofnagle NA, Spencer CA, Turcu AF, Algericas-Schimnich A. Thyroglobulin (Tg) testing revisited: Tg assays, TgAb assays, and correlation of results with clinical outcomes. J Clin Endocrinol Metab 2015;100:E1074-E1083. 5. Rotteveel-de Groot DM, Ross HA, Janssen MJR , Netea-Maier RT, Oosting JD , Sweep FCGJ , van Herwaarden AE. Evaluation of the highly sensitive Roche thyroglobulin II assay and establishment of a reference limit for thyroglobulin-negative patient samples. Pract Lab Med 2016;5:6-13.
ACCEPTED MANUSCRIPT Legends Figure 1. Serum Tg levels measured with Beckman-Coulter Access and Roche
T
Tg-II assay in patients with differentiated thyroid carcinoma; 71 samples were
IP
drawn under levothyroxine replacement (suppressed Tg, Fig. 2A) and 27 after
SC R
TSH stimulation (stimulated Tg, Fig. 2B). The Y-axis is displayed on logarithm scale to enhance visualization of low Tg levels, with values < 0.10 µg/L plotted
NU
as 0.04-0.09 µg/L. The highest suppressed Tg level is shown off scale, with
AC
CE P
TE
D
MA
Access/Tg-II values displayed in parentheses.
S0009-9120(16)30467-2 doi:10.1016/j.clinbiochem.2016.11.019 CLB 9423
To appear in:
Clinical Biochemistry
Received date: Accepted date:
22 October 2016 18 November 2016
Please cite this article as: Batista Marcelo C., Ferreira Carlos E.S., Ferreira Paulo R.S., Faulhaber Adriana C.L., Silva Andr´e L.O., Mangueira Crist´ov˜ ao L.P., Analytical and clinical validation of the new ultrasensitive Roche thyroglobulin II assay, Clinical Biochemistry (2016), doi:10.1016/j.clinbiochem.2016.11.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Analytical and clinical validation of the new ultrasensitive Roche Thyroglobulin II assay
T
Author names and affiliations: Marcelo C. Batistaa, Carlos E. S. Ferreiraa,
IP
Paulo R. S. Ferreiraa, Adriana C. L. Faulhabera, André L. O. Silvaa, Cristóvão L.
a
SC R
P. Mangueiraa Clinical Lab, Hospital Israelita Albert Einstein
Bloco E – 2⁰ andar
MA
São Paulo – SP CEP 05652-900
NU
Av. Albert Einstein, 627
Brazil
[email protected]
D
E-mail:
TE
[email protected]
CE P
[email protected] [email protected] [email protected]
AC
[email protected]
Corresponding author: Marcelo C. Batista Hospital Israelita Albert Einstein Av. Albert Einstein, 627 Bloco E - 2⁰ andar – Laboratório Clínico São Paulo – SP CEP 05652-900 Brazil Phone: 55 11 2151-5555
ACCEPTED MANUSCRIPT Fax: 55 11 2151-0408 Cell phone: 55 11 99140-0282
IP
Running head: New Roche Thyroglobulin II assay
T
E-mail: [email protected]
SC R
Keywords: thyroglobulin; immunoassay; electrochemiluminescence; thyroid cancer
NU
Abbreviations CV: coefficient of variation
DTC: differentiated thyroid cancer
LOQ: limit of quantitation
Tg: thyroglobulin
TSH: thyroid-stimulating hormone
TE
D
MA
CE P
Funding: This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
AC
This work was presented in part as a poster on July 29, 2014, at the 2014 AACC Annual Meeting in Chicago, USA.
ACCEPTED MANUSCRIPT Manuscript Thyroglobulin (Tg) is a serum marker of differentiated thyroid carcinoma
T
(DTC) and thus should be accurately measured down to levels of 0.10 μg/L. In
IP
the past decade, a few second generation immunometric assays have been
SC R
developed with such sensitivity [1]. In this study, we evaluated the analytical and clinical performance of the new ultrasensitive Roche Tg-II assay
NU
(Mannheim, Germany).
Serum samples with anti-Tg < 20 IU/mL (Roche assay) were selected
MA
from our routine workload. Thyroglobulin was measured with Tg-II assay in Roche E170 Modular and Access Tg assay in Access 2 or Unicel DxI 800 (2
according
to
Beckman-Coulter,
Brea,
USA).
Both
are
TE
results,
D
equipments using the same principles/reagents and yielding nearly identical
electrochemiluminometric or enzymatic chemiluminometric, sandwich type
CE P
assays calibrated against the same reference material BRC 457 (European Commission). Access has a functional sensitivity < 0.10 μg/L [1] and is
AC
considered by many the gold standard Tg immunoassay. Tg-II limit of quantitation (LOQ, lowest concentration with CV < 20%) was < 0.08 µg/L (CV=16.8%), as determined by running 2 low Tg serum pools for 3 months using 2 different reagent and calibrator lots. Total imprecision of 3 serum samples and 2 Roche controls was < 5.5% at 0.28-77 µg/L. Linearity was confirmed at 0.21-493 µg/L. Tg-II levels correlated well (r=0.99), but were on average 27% higher (mean bias 3.73 µg/L) compared to Access in 226 samples. This difference is slightly above Tg total error (21.9%) according to biologic
variation
database
(www.westgard.com/quality-requirements.htm,
access 10/22/2016) and is most likely due to heterogeneity in antibodies used in
ACCEPTED MANUSCRIPT the assays [1], which may have varying specificity for multiple Tg epitopes (for more details on analytical validation, see Supplementary Material).
T
To evaluate the clinical performance of Tg-II in distinguishing DTC
IP
patients with and without active disease, we tested 2 different cutoffs for
SC R
suppressed Tg drawn under thyroxine replacement (0.10 and 0.27 µg/L) and also for endogenous/exogenous TSH-stimulated Tg (1.00 and 2.00 µg/L) [2,3]. Access Tg below these limits was considered an indication of disease-free
NU
status. Suppressed Access Tg was < 0.10 or 0.27 µg/L in 42 and 51 samples,
MA
respectively (Figure 1A); in the former group, Tg-II was < 0.10 µg/L in all but one specimen (0.10 µg/L), whereas in the latter group Tg-II was < 0.27 µg/L in all 51 samples. Stimulated Access Tg was < 1.00 or 2.00 µg/L in 18 and 27
TE
D
samples, respectively (Figure 1B); in the former group, Tg-II was < 1.00 µg/L in all 18 samples, whereas in the latter group Tg-II was < 2.00 µg/L in all but one
CE P
specimen (Access Tg 2.90 µg/L and Tg-II 1.28 µg/L). In summary, our results are consistent with 2 recent reports [4,5] and
AC
suggest Tg-II is a highly sensitive and reproducible assay with an excellent agreement
(96-100%)
with
Access
in
distinguishing
absent
and
persistent/recurrent DTC. Nonetheless, because of a positive Tg-II bias, labs planning to switch to this assay should ideally establish a new baseline for each patient by running a few samples in the old and new assay [1].
ACCEPTED MANUSCRIPT References 1. Giovanella L, Feldt-Rasmussen U, Verburg FA, Grebe SK, Plebani M, Clark
T
PM. Thyroglobulin measurement by highly sensitive assays: focus on
IP
laboratory challenges. Clin Chem Lab Med 2014;53:1301-14.
SC R
2. Giovanella L, Treglia G, Sadeghi R, Trimboli P, Ceriani L, Verburg FA. Unstimulated highly sensitive thyroglobulin in follow-up of differentiated
NU
thyroid cancer patients: a meta-analysis. J Clin Endocrinol Metab 2014;99:440-7.
MA
3. Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Nikiforov YE, Pacini F, Randolph GW,, Sawka AM, Schlumberger M, Schuff KG, Sherman
D
SI, Sosa JA, Steward DL, Tuttle RM, Wartofsky L. 2015 American Thyroid
TE
Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association
CE P
Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid 2016;26:1-133.
AC
4. Netzel BC, Grebe SKG, Leon BGC, Castro MR, Clark PM, Hoofnagle NA, Spencer CA, Turcu AF, Algericas-Schimnich A. Thyroglobulin (Tg) testing revisited: Tg assays, TgAb assays, and correlation of results with clinical outcomes. J Clin Endocrinol Metab 2015;100:E1074-E1083. 5. Rotteveel-de Groot DM, Ross HA, Janssen MJR , Netea-Maier RT, Oosting JD , Sweep FCGJ , van Herwaarden AE. Evaluation of the highly sensitive Roche thyroglobulin II assay and establishment of a reference limit for thyroglobulin-negative patient samples. Pract Lab Med 2016;5:6-13.
ACCEPTED MANUSCRIPT Legends Figure 1. Serum Tg levels measured with Beckman-Coulter Access and Roche
T
Tg-II assay in patients with differentiated thyroid carcinoma; 71 samples were
IP
drawn under levothyroxine replacement (suppressed Tg, Fig. 2A) and 27 after
SC R
TSH stimulation (stimulated Tg, Fig. 2B). The Y-axis is displayed on logarithm scale to enhance visualization of low Tg levels, with values < 0.10 µg/L plotted
NU
as 0.04-0.09 µg/L. The highest suppressed Tg level is shown off scale, with
AC
CE P
TE
D
MA
Access/Tg-II values displayed in parentheses.