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Technical Briefs |
1
The Immunoassay Laboratory, Department of Clinical Biochemistry, Barts and The London NHS Trust, 51-53 Bartholomew Close, London EC1A 7BE, United Kingdom
a author for correspondence: fax 44-207-796-4676, e-mail
L.A.Perry{at}mds.qmw.ac.uk
Serial serum measurements of thyroglobulin (TGN) are used in the management of differentiated thyroid carcinoma as a complement to total body scans in monitoring the presence of residual or ectopic functioning thyroid tissue following surgery or ablative radiotherapy (1).
To minimize the direct labor and reagent costs and because the shelf-life is short, manual radiometric assays (CIS method) are performed infrequently. By contrast, the clinical demand is for more frequent assays to complement radioiodine scanning and minimize the time patients spend in a hypothyroid state while on the protocol for monitoring thyroid cancer recurrence.
The aim of our study was to compare a manual radiometric method with a fully automated chemiluminescence immunoassay that might allow more frequent performance of the test without producing unacceptable personnel costs.
Blood samples were collected from 41 consecutive patients undergoing follow-up measurements of TGN and thyroid hormone after thyroid ablation or surgery. Blood was collected in tubes without additives or separator gel. Serum samples were stored at 4 °C until assayed the same day or stored at -20 °C until the next assay.
Serum TGN was measured by both the CIS ELSA-HTG (High Wycombe, Bucks,
United Kingdom) and the DPC Immulite (Llanberis, Gwynedd, Wales)
methods. The CIS method is a manual antibody-coated tube IRMA with a
working range of 
1.5–500 µg/L (lowest reporting concentration
varies from batch to batch) and a shelf-life of 5 weeks. The CIS-quoted
reference range, derived from 115 presumably healthy subjects, is 5–25
µg/L (81%). Ten percent of subjects had TGN <5 µg/L, and 9% of
subjects had TGN >25 µg/L. The DPC Immulite method is a
chemiluminescent enzyme immunometric assay (random access) with a
working range of 0.5–300 µg/L and a shelf-life of 6 months. The
assay has an adjustment check every 2 weeks. The Immulite range,
derived from 55 healthy laboratory volunteers, is 0.5–55 µg/L
(95%).
Three internal quality-control (QC) pools were run in each DPC Immulite
assay. The low QC pool was from a single donor, whereas the medium and
high QC pools were samples from a single patient diluted appropriately
in normal donor serum to give concentrations of 10 and 100 µg/L.
Two internal QC pools were run in each CIS ELSA-HTG assay. The low QC
was a manufacturer-supplied QC that came with the reagent set,
whereas the high QC was a sample from a single patient diluted in
normal male donor serum to give a result of 50 µg/L.
Agreement between the two assays was assessed by the method of Bland
and Altman (2).
Between-assay imprecision assessed with internal QC pools was better for the Immulite assay [CVs of 9.2%, 8.2%, and 6.6% at 1.7 (n = 13), 10.0 (n = 13), and 117 µg/L (n = 20), respectively] than for the CIS method [CVs of 14% and 9.5% at 9.0 and 55 µg/L, respectively (n = 11 and 19, respectively)]. Data are not based on equal numbers of samples in each group because of the limited availability and changeover of in-house QC pool material.
We measured 41 patient samples by both methods: 32 had measurable results by both methods; 3 results were below the working range of both assays; 4 results were measurable by Immulite (1.8, 5.1, 7.3, and 9.2 µg/L) but below the CIS working range (<1.4 µg/L); 1 result was above the working range of both assays; and 1 result was above the Immulite working range but measurable by the CIS method (445 µg/L).
Immulite serum TGN concentrations strongly associate with CIS:
Immulite = [1.04 (95% confidence interval, 0.95–1.13) x
DPC] + 0.87 (-5.4 to 7.1); r = 0.974; P
<0.0001; n = 31. One patient result (CIS result, 85.5 µg/L;
Immulite result, 283 µg/L) was excluded from the correlation as being
a reproducible but significant outlier. Fig. 1
is a Bland–Altman difference plot demonstrating absence of
detectable dose-related bias.
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Some results (Table 1
) differed significantly in clinical implications. Such a
difference was also reported by others comparing the Immulite assay
with the BRAHMS LUMItest TGN assay (3) in 59 patients with
thyroid carcinoma; TGN was detectable in 8 cases with the Immulite but
not the BRAHMS assay. False-positive Immulite results could lead to
unnecessary ablative therapy for patients, but true positives could
indicate earlier identification of tumor recurrence. This difference
could not be attributed to calibration differences because both assays
use the same reference material and overall there was good agreement
between the results.
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TGN autoantibody interference is documented to be a problem in TGN assays: RIAs can over- and underestimate TGN concentrations, whereas dual monoclonal immunometric assays only underestimate them (4). The Immulite assay uses a polyclonal capture antibody and a monoclonal signal antibody, whereas the CIS assay uses monoclonal capture and signal antibodies. Therefore, we asked whether the CIS assay underestimating TGN concentration because of TGN autoantibody interference.
TGN antibody status was checked in four of seven patients from whom sufficient sample was available; only one of the four was positive by an agglutination method (anti-TGN = 1/402, and anti-thyroid peroxidase = 1/1602). Thus, TGN autoantibody interference seems unlikely to account for the discrepant results.
In the seven patients for whom repeat analysis confirmed the clinical discordance, the Immulite result was always higher. In two patients (patients D and G), there were no supporting clinical or radiological details, whereas another two patients (patients E and F) had not had recent imaging. The remaining three patients (patients, A, B, and C) had negative diagnostic 131I whole body scans, with serum TGN undetectable by the CIS assay but detectable by the Immulite assay. A possible explanation for the discrepancy between the measurable TGN by Immulite and the negative 131I scan is the type of isotope used for the diagnostic scan. The use of 123I rather than 131I for diagnostic scanning offers increased sensitivity in detection of tumor recurrence (Prof. A. Grossman, Department of Endocrinology, St. Bartholomew’s Hospital, London EC1A 7BE, United Kingdom, personal communication). Of the three patients with a negative 131I scan, one (patient C) had 7 days earlier had a positive 123I scan, which suggested early recurrence and was consistent with the serum TGN result by Immulite (7.3 µg/L; result by CIS <1.4 µg/L). The other two patients were not scanned with 123I.
An alternative explanation is that the measurable results by Immulite are falsely positive. Caution should be exercised until there is more evidence that the increased TGN in patients with negative scans ultimately represents true positivity for disease recurrence.
Finally, another possibility is that the Immulite assay is detecting different molecular forms of TGN that the tumor may be secreting that are not recognized by other assays or at least not with the same affinity if recognized.
In conclusion, the Immulite TGN assay is more sensitive and demonstrated improved precision when compared with the CIS method. The Immulite assay is more easily able to meet the clinical demand requirements in terms of turnaround time. Although there was good agreement overall between the two methods, the TGN results in some individuals differed more than twofold and/or were clinically discordant. In such situations, it is necessary to run and report results from both assays in parallel for several measurements in each patient, taking into account clinical and radioisotopic scan data before changing assays.
Acknowledgments
We are grateful to DPC for providing two reagent sets free of charge for this study.
References
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