Need for Revision of Diagnostic Limits for Medullary Thyroid Carcinoma with a New Immunochemiluminometric Calcitonin Assay

To the Editor:

Calcitonin is the most sensitive tumor marker for both diagnosis and postoperative surveillance of medullary thyroid carcinoma (MTC).1 Healthy control individuals virtually always have a serum calcitonin concentration <10 ng/L, and patients with higher basal calcitonin concentrations are recommended to undergo a pentagastrin-stimulation test (PGT) to exclude the presence of MTC. Stimulated peak serum calcitonin concentrations >100 ng/L are >90% specific for a diagnosis of C-cell disease, either C-cell hyperplasia or MTC (1). These diagnostic limits were established on the basis of data from studies that used the manual Cisbio immunoradiometric calcitonin assay and were subsequently validated for the Nichols calcitonin assay(2).

Many centers now use the Liaison assay (Diasorin) as an alternative to the discontinued Nichols assay. Bieglmayer et al. have shown that the Liaison calcitonin assay is positively biased compared with the Nichols assay (3). The manufacturer’s product insert for the Liaison assay currently recommends a basal calcitonin concentration of >10 ng/L as the diagnostic limit for further investigation for MTC, despite the fact that the upper reference limit for males (18.6 ng/L) exceeds this limit. A recent study published in Clinical Chemistry proposed revision of the 10-ng/L limit for measuring basal calcitonin with the Liaison assay(4).

The Liaison assay is a 1-step direct, 2-site immunochemiluminometric assay that uses specific affinity-purified mouse monoclonal antibody–coated magnetic microparticles and an alternative affinity-purified mouse monoclonal antibody conjugated to an isoluminol derivative. The functional sensitivity (defined as the concentration with a CV ≤20%) reported by the manufacturer is 8 ng/L; however, when calculated in our laboratory with 90 paired samples, we obtained a functional sensitivity of 4 ng/L.

We measured basal serum calcitonin and stimulated calcitonin with the Liaison assay in 83 healthy volunteers [43 females (age range, 18–59 years; mean, 32 years) and 40 males (range, 22–72 years; mean, 39 years)] who were recruited to the study. The study participants provided informed consent. Nonfasting 5-mL blood samples were collected into plain serum Vacutainer® tubes (Becton Dickinson). Ten of the 43 women (age range, 27–36 years; mean, 32 years) and 12 of the 40 men (range, 25–48 years; mean, 33 years) underwent a PGT, which was performed in the nonfasting state. A 5-mL blood sample was collected at 0 min and at 1, 2, 3, 5, and 10 min after a single intravenous bolus of pentagastrin (0.5 μg/kg). In these volunteers, the 0-min sample was used to determine the basal calcitonin reference intervals. All blood samples were allowed to clot and were centrifuged within 20 min of collection. Serum aliquots were stored at −20 °C and assayed in batches.

A rank-based nonparametric method was used to calculate reference intervals. The fifth-percentile values were lower than the functional sensitivity of the assay (4 ng/L) for both males and females. The 95th percentile (and hence the upper limit of the reference interval for basal calcitonin) was 21.9 ng/L (median, 9.8 ng/L) for males and 11.1 ng/L (median, 5.7 ng/L) for females (Fig. 1 ). Basal calcitonin values for 0-min samples obtained via a cannula for the PGT were similar to those of the remaining basal calcitonin samples. The median peak stimulated calcitonin concentration was 28.2 ng/L in males (range, 19.5–110.0 ng/L) and 11.0 ng/L in females (range, <4 ng/L to 38.6 ng/L).

Figure 1.

Basal serum calcitonin concentrations in males (n = 40) and females (n = 43).

Indicated are the current diagnostic limit of 10 ng/L (solid line) and the manufacturer’s upper reference limits for males (dashed line) and females (dotted line).

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In our study, the upper limit of the reference interval in healthy males was similar to that recommended by the manufacturers of the Liaison calcitonin assay; however, the upper limit for healthy females is twice the manufacturer’s recommended value of 5.5 ng/L. Thirteen percent of healthy male volunteers and 54% of healthy female volunteers had basal calcitonin concentrations that exceeded the manufacturer’s recommended upper reference limit. When a basal calcitonin diagnostic limit of 10 ng/L was used to indicate the need for further assessment with the PGT (1), 48% of healthy males and 19% of healthy females had a basal calcitonin value greater than this limit. After pentagastrin stimulation, 1 individual had a peak stimulated calcitonin value >100 ng/L (basal, 16.6 ng/L; peak, 110 ng/L).

Our data show an upper reference limit for males that is almost twice that for females. This result is supported by postmortem studies demonstrating that men have twice the number of C cells than females (5). Therefore, the validity of applying a single diagnostic limit for basal and stimulated calcitonin for both males and females remains questionable.

The information gained from this study is insufficient to make any definite recommendation regarding changes to the currently used cutoff limits for stimulated calcitonin concentrations, and further studies are needed. This study offers strong evidence, however, to suggest that the current diagnostic limits of >10 ng/L for basal concentrations are too low when the Liaison assay is used. Extrapolating these cutoffs obtained with the Cisbio assay to the Liaison assay has the potential to lead to unnecessary thyroid biopsies. Thus, our data support calls for a revision of the diagnostic limits for basal serum calcitonin with the Liaison assay (4).

Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors’ Disclosures of Potential Conflicts of Interest:Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: NIHR Biomedical Research Centre funding scheme. R. Ramachandran, IPSEN Research Foundation; W.S. Dhillo, NIHR Clinician Scientist Award and Wellcome Trust Value in People Award; N.M. Martin, HEFCE Clinical Senior Lecturer Award.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank the Sir John McMichael Centre, Imperial College Healthcare NHS Trust, for allowing us to use their facilities.

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