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Technical Briefs |
1 Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium;2 Department of Chemical Endocrinology, University Medical Centre, Nijmegen, The Netherlands;
aaddress correspondence to this author at: Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium; fax 32-9-264-81-98; e-mail linda.thienpont{at}ugent.be
Abstract
Background: Serum-free thyroxine (FT4) testing is recommended for diagnosis or monitoring of thyroid dysfunction, particularly in cases of hormone binding abnormalities. However, the poor intermethod agreement among commercial FT4 assays suggests a need for standardization with a hierarchically higher measurement procedure. To that purpose, we applied equilibrium dialysis (ED) in combination with isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC-tandem MS).
Methods: After ED, we collected dialysate into tubes containing [13C6]-T4 for ID and [13C9]-T4 as carrier, purified the samples by solid-phase extraction, and analyzed them with LC/tandem MS. We evaluated the procedure’s analytical performance and tested its suitability for measurement of hypo-, eu-, and hyperthyroid serum FT4 concentrations. We conducted a pilot method comparison study with 3 commercial assays to investigate whether frozen sera could be used for the purpose of FT4 standardization.
Results: The within-run, between-run, and total CVs (inclusive ED) were 3.7%, 4.2%, and 5.6%, respectively (17.7 pmol/L; n = 20). The mean accuracy, estimated from recovery experiments with dialysate and dialysis buffer supplemented at 8.7, 18.7, and 33.5 pmol/L, and from analysis of certified sera gravimetrically diluted to 9.8, 19.2, and 34.8 pmol/L, was 98.0% to 102.8%. The procedure’s limit of detection and limit of quantification were 0.5 and 1.3 pmol/L, respectively. The method comparison demonstrated the suitability of the selected sera for standardization of FT4 assays and confirmed the lack of assay comparability.
Conclusions: We demonstrated that the described ED-ID-LC/tandem MS procedure and the selected type of sera qualify for standardization of FT4 measurements.
Serum-free thyroxine (FT4) testing is recommended for clinical evaluation of patients with suspected or diagnosed thyroid disease because the amount of T4 not bound to serum proteins is thought to closely reflect states of hypo-, eu- and hyperthyroidism, particularly in patients with hormone binding abnormalities(1)(2)(3). The lack of intermethod agreement among commercial FT4 assays, however, makes their suitability for clinical evaluation questionable [e.g., (4)(5)(6)(7)(8)(9)]. Therefore, recent guidelines on laboratory support emphasize the need for standardization of FT4 measurements(10)(11). The measurement procedure to be used for that purpose must include physical separation of free and protein-bound T4, either by equilibrium dialysis (ED) or ultrafiltration (UF). In addition, trueness-based quantification of T4 in dialysate or ultrafiltrate by isotope dilution-mass spectrometry (ID-MS) is a state-of-the-art procedure(10)(11)(12)(13). We selected and validated a procedure based on ED in combination with isotope dilution/tandem mass spectrometry (ID-tandem MS) and documented its suitability for measurement of hypo-, eu-, and hyperthyroid FT4 concentrations. We also applied it in a pilot split-sample comparison with 3 commercial test systems. To the best of our knowledge, only 1 alternative procedure, ID-MS after UF, is available(14).
In our performance of ED, we adhered to the conditions recommended in the C45-A consensus guideline(11). In brief, we first adapted the pH of the samples to 7.4 (at 37 °C) by adding 0.776 mol/L HEPES buffer(15). Then, we performed ED with a 20-cell dialyzer (Dianorm) incubated in a water bath for a minimum of 4 h at a mean (SD) temperature of 37 (0.2) °C. We used a 52.75 mmol/L HEPES dialysis buffer as described in Nelson et al.(16). The dialysis cells consisted of 2 halves of identical working volume (1.0 mL), between which a membrane of regenerated cellulose with a nominal cutoff of 5000 relative molecular mass (Mr) was fitted. Note that we validated the most critical parameters in ED, the pH of the buffer after dialysis, and the time necessary to reach equilibrium. A detailed description of the experimental design is available in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol52/issue9.
The ID-MS procedure used for quantification of T4 in dialysate was derived from our reference measurement procedure for total T4(17)(18)(19). In short, a candidate T4 reference material was used for calibration and [13C6]-T4 for ID. The dialysate was collected into a polypropylene tube containing an amount of [13C6]-T4 equivalent to the endogenous T4 content and a 10-fold higher amount of [13C9]-T4 as carrier. From then on, all volumetric steps that contributed to the accuracy were gravimetrically controlled. The dialysate (1 mL) was processed by Oasis MAX solid-phase extraction and subsequently analyzed with an electrospray API 4000TM double stage MS (Applied Biosystems) coupled to an Agilent 1100 Series HPLC, equipped with a Hypersil BDS C18 column (150 x 2.1 mm, 5 µm). Gradient elution was performed with a mixture of acetonitrile/water/formic acid. T4 and [13C6]-T4 were monitored in the positive multiple reaction mode (ion transitions at m/z 778 to 732 and 784 to 738, respectively). Calibration was done by direct analysis of mixtures of equivalent amounts of T4/[13C6]-T4 (
80 fmole of each), also containing the carrier. For methodologic details and representative ion chromatograms, refer to the online Data Supplement and its Fig. 1
.
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We performed a method comparison study to investigate whether the selected types of samples were suitable for standardization of FT4 immunoassays. The method comparison included the Dimension RxL (Dade Behring), Elecsys 2010 (Roche), and Vitros Eci (Ortho) and used 6 sera purchased from Solomon Park Research Institute. The samples were obtained from single blood donations by apparently healthy donors and were prepared according to the CLSI C37-A protocol, but without filtration(20). Samples were shipped on dry ice.
For the validation of the performance characteristics of the ED-ID-LC/tandem MS procedure, we used the following materials: (a) 1 serum pool from blood donations by apparently euthyroid donors for imprecision, inclusive of ED; (b) HEPES buffer (52.75 mmol/L) and pooled serum dialysate containing endogenous T4 (mean, 16.5 pmol/L; 95% confidence interval (CI), 16.3–16.7 pmol/L; n = 10), both supplemented with T4 concentrations at 8.7, 18.7, and 33.5 pmol/L, respectively, for accuracy, trueness, and imprecision of ID-MS measurement; (c) 2 lyophilized sera certified for total T4 and gravimetrically diluted with HEPES buffer (52.75 mmol/L) to concentrations of 9.8, 19.2, and 34.8 pmol/L, respectively, for accuracy, trueness, and imprecision; (d) 10 aliquots sampled from a T4/[13C6]-T4 mixture, 5 measured directly by ID-MS, the other 5 measured after processing to determine the difference between direct and processed calibrators; (e) samples selected from the method comparison study for interference, ion suppression, limit of detection (LOD), and limit of quantification (LOQ); (f) 10 aliquots from a serum dialysate pool; [13C6]-T4 was added to 5 aliquots before processing and to the other 5 after processing, to determine absolute recovery. For demonstration of the suitability of the ED-ID-LC/tandem MS procedure for analysis of eu-, hypo-, and hyperthyroid FT4 concentrations, we used sera from the Laboratory for Hormonology of the Department of Clinical Chemistry of Ghent University Hospital (Ghent, Belgium) and handled them according to the local Ethics Committee guidelines. For details on the experimental design of the method comparison and validation protocol, refer to the online Data Supplement.
Statistical data analysis was done with CBstat and according to the EP5-A2 protocol(21) (see below).
Because both ED and UF are qualified methods for the separation of free- from protein-bound T4, we initially tested them in parallel (for a description of the UF conditions see the online Data Supplement). These experiments revealed several technical drawbacks of UF: centrifugation with thermostatic control at 37 °C is cumbersome and requires preheating, results depend on the membrane cutoff, the viscosity of undiluted serum makes it necessary to perform UF centrifugation for at least 45 min or to pool ultrafiltrates to obtain a UF yield consistent with the LOQ of ID-MS, and device or batch-dependent protein leakage may occur. In contrast, the selected ED design allows dialysis of undiluted serum under physiologic conditions of temperature and pH within 4 h and has sufficient sensitivity for subsequent ID-MS measurement (see below).
On the basis of these observations, we opted for ED. In experiments performed with a model solution, however, we found a discrepancy between ED and UF. This observation indicates the need for a recommended or standard procedure rather than a trueness-based separation technique, as previously observed by Holm et al.(13)(22). Therefore, we relied for ED on the C45-A guideline and the study by Nelson et al.(11)(16). For a detailed discussion of the rationale for selecting ED, we refer to the online Data Supplement.
The performance characteristics of the ED-ID-LC/tandem MS procedure are summarized in Table 1
. The within-run, between-run (according to EP5-A2), and total CVs of the procedure, inclusive of ED, were 3.7%, 4.2%, and 5.6%, respectively (FT4 concentration: 17.7 pmol/L). The total CV was confirmed from the data for repeated analysis of sera from hypo-, eu-, and hyperthyroid patients (see Table 1 in the online Data Supplement). The maximum CVs for ID-MS measurement of T4 extracted from dialysate, dialysis buffer, and gravimetrically diluted certified sera were 2.8 (within-run), 2.3 (between-run), and 3.0% (total CV), respectively. The mean accuracy assessed from recovery studies was 98.2% to 102.8% (dialysate), 98.4% to 99.0% (dialysis buffer), and 98.0% to 100.0% (gravimetrically diluted certified sera). Statistical analysis of the data obtained for the 2 series of calibration mixtures did not reveal substantial differences (P = 0.96 in the 2-sided F-test and P = 0.17 in the 2-sided t-test with equal variances). There was no indication of ion suppression (identical isotope ratios within the experimental error) or of interference by other than T4/[13C6]-T4 analytes during measurement. The LOD and LOQ were estimated at 0.5 pmol/L [signal-to-noise (S/N) 3] and 1.3 pmol/L (S/N 6), respectively. The absolute recovery of solid-phase extraction was 85.0% (95% confidence interval, 82.3–87.7%). With this LOD and recovery, euthyroid FT4 concentrations were typically measured at an S/N ratio of 75–100. The Deming regression data of the method comparison (Fig. 1
) are consistent with previous reports(9) and confirm the need for standardization: the test assays provide discrepant results (the mean FT4 concentrations of the sera varied from 10 pmol/L to 14.5 pmol/L) and deviate from ED-ID-LC/tandem MS to a different extent (average deviations: –3%, –18%, and –33%).
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In conclusion, the presented study showed that our measurement procedure, based on ED and trueness-based ID-LC/tandem MS measurement of dialysate, qualifies as a method for standardization of FT4 measurements. At the same time, our results demonstrate that samples processed according to the C37-A protocol are suitable for use in standardization. These results provide a basis for worldwide standardization of FT4 measurements under the auspices of the International Federation of Clinical Chemistry and Laboratory Medicine(23). An inaugural meeting of the group is foreseen at the 2006 AACC Annual Meeting(24).
Acknowledgments
We gratefully acknowledge the financial support of the European Commission through project G6RD-CT-2001-00587. The authors also thank W. Lambert (Laboratory for Toxicology, Ghent University) for the measurement facilities given (GOA 12051204; Bijzonder Onderzoeks Fonds, University of Ghent).
References
The following articles in journals at HighWire Press have cited this article:
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  B. Yue, A. L. Rockwood, T. Sandrock, S. L. La'ulu, M. M. Kushnir, and A. W. Meikle Free Thyroid Hormones in Serum by Direct Equilibrium Dialysis and Online Solid-Phase Extraction-Liquid Chromatography/Tandem Mass Spectrometry Clin. Chem., April 1, 2008; 54(4): 642 - 651. [Abstract] [Full Text] [PDF]
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