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Letters to the Editor |
1 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, Departments of2 Pathology and 3 Medicine, University of Utah, Salt Lake City, UT
aAddress correspondence to this author at: ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, Fax 801-584-5207, E-mail tanya.sandrock{at}aruplab.com
To the Editor:
In Graves disease, autoantibodies activate thyroid-stimulating hormone (TSH)1 receptor, leading to hyperthyroidism caused by increases in intracellular cAMP and thyroid hormone. We developed a nonradioactive assay to measure thyroid-stimulating immunoglobulins (TSIs).
Fasting serum samples were collected and deidentified for the reference interval study, which was approved by the University of Utah ethical review board (IRB #7740). Chinese hamster ovary–hTSHR-25–adherent cells (passage <30) (Leonard Kohn, Interthyr Research Foundation) (1) were propagated in Ham F12-media supplemented with 5% serum and were trypsinized and dispensed into a 96-well plate at a density of 16 000 cells/well 48 h before use. The medium was removed from the cells, and 100 µL of Hanks balanced salt solution was added to each well. To predilute serum samples, 70 µL of serum was dispensed into 530 µL hypotonic Hanks Balanced Salt Solution in a 2-mL deep 96-well block (2). We then added 85 µL of diluted serum to each well of the cell culture plate. The final volume/well was 185 µL. All samples were tested in triplicate. Cells were incubated at 5% CO2, 37 °C for 90 min.
After stimulation with sera, cAMP in the supernatant was measured using a cAMP-RIA reagent set (Amersham Biosciences). Lysis of the remaining monolayer and detection of intracellular cAMP concentrations were performed with the DiscoverRx HitHunter cAMP XS enzyme fragment complementation (EFC-cAMP) assay according to the manufacturer’s instructions. For a 96-well plate, we used 25 µL of cAMP XS antibody/lysis, 25 µL of enzyme donor–cAMP conjugate, and 50 µL of enzyme acceptor/chemiluminescence substrate. The cAMP calibrator was diluted in Hanks Balanced Salt Solution/3-isobutyl-1-methylxanthine mixture as described above, and 15 µL of the diluted calibrator was placed in a separate 96-well cell culture plate and treated identically to the cells. After substrate addition, the plate was incubated for 18 h at room temperature. We then transferred 60 µL to 1 white 384-well plate (Thermolabsystems) and read it on a luminometer (Thermolabsystems). All dispensing steps were performed using the Tecan Freedom Evo 200. Calibrators were plotted using a 4-parameter best-fit analysis. TSH concentrations were measured on either the Immulite or Roche Modular E170. We performed data analyses by use of the online EP evaluator (David G. Rhoads; http://www.dgrhoads.com) and CBstat (Kristian Linnet). Indeterminate was defined as 110%–129% of the TSI reference interval value; a positive result was set at 
130% and was consistent with Graves disease.
Results for the same stimulated cells processed with the RIA-cAMP and the EFC-cAMP methods correlated well [R = 0.96 (P < 0.001), n = 49, slope = 1.06, and intercept = –18.27 (Fig. 1
)]. Two samples positive by RIA-cAMP were negative and indeterminate by EFC-cAMP [mean (SD) RIA-cAMP and EFC-cAMP values 156% (12.66%), 95% (2.9%); and 140% (6.0%), 119% (1.7%), respectively], and 1 sample was indeterminate by RIA-cAMP and positive by EFC-cAMP ([117% (8.6%), 143% (11.2%)]. An additional 376 samples assayed with the RIA-cAMP method were processed using the EFC-cAMP method. The correlation for all 425 samples was R = 0.92, slope 1.048, intercept = –2.2 by Deming regression. Use of the EFC-cAMP assay according to the traditional method may lead to false-positive results at TSH concentrations 10 mU/L. Such occurrences should not be a problem, however, because patients with Graves disease with hyperthyroidism should have TSH concentrations below the reference interval (<0.4 mU/L) (data not shown).
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Results for 5 of 6 Graves patient samples provided by Kronus from patients clinically diagnosed with Graves disease were positive (TSH concentrations 370%, 382%, 371%, 262%, and 316%) and 1 was indeterminate (115% of the reference interval TSI value). For correlation, 3 samples were assayed both in our laboratory and a second reference laboratory: the results were 100%, 106%; 446%, 278%; and 149%, 180% of the TSI reference interval value; respectively. Results for 1 sample positive for EFC-cAMP were negative twice by RIA-cAMP (85%, 106%) and positive by TRAb (68% inhibition of TSH binding). Intraassay precision was tested by running 5 samples 3 to 4 times in triplicate, with an observed CV of 10%. During a 20-day period, with a mean sample result of 429%, the interassay CV was 27%. The interassay CV for a second control was 9.6%. Two cAMP controls, tested independent of the Chinese hamster ovary cells for 140 runs, had a CV of 15%.
Data regarding assay specificity (including the results for testing samples from patients with Hashimotos disease) and sensitivity for the cell line were previously published (1). Pooled normal-reference serum run 11 times in triplicate yielded an SD of 5.5% of the reference interval value. In addition, 46 serum samples from healthy individuals were tested using the EFC-cAMP method (mean 94.4%, median 94.5%, and SD 6.8%). Results for the normal samples were 80% to 106% of the control (data not shown).
This EFC-cAMP assay is a nonradioactive surrogate for RIA-cAMP for studying antibody-mediated increases in cAMP concentrations in response to activation of the G-coupled TSH receptor.
Acknowledgments
Grant/Funding Support: This work was supported by the ARUP Institute for Clinical and Experimental Pathology®
Financial Disclosures: None declared.
Acknowledgments: We thank Leonard Kohn for providing the Chinese hamster ovary cell line expressing the TSH receptor (Leonard Kohn, The Interthyr Research Foundation), Linda Seiler for excellent technical expertise, Hayden Jeffreys for samples from Graves patients (Kronus), and Lois Hybl and Tom Martins for critical reading of the manuscript.
Footnotes
1 Nonstandard abbreviations: TSH, thyroid-stimulating hormone; TSI, thyroid-stimulating immunoglobulin; EFC-cAMP, cAMP XS enzyme fragment complementation. 
References
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109%; indeterminate is 110%–129%; a positive result is