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Technical Briefs |
Department of Laboratory Medicine, National University Hospital, Lower Kent Ridge Road, Singapore 119074
a author for correspondence: fax 65-7771757
The Vitros ECiTM analyzer (Ortho Clinical Diagnostics, Rochester, NY) uses a new enhanced chemiluminescence technology. The assay reagents and wells are supplied together in combined packs with calibration information stored on magnetic calibration cards with bar-coded calibrators.
We evaluated the following thyroid assays: thyrotropin (TSH), free thyroxine (fT4), free triiodothyronine (fT3), thyroxine (T4), and triiodothyronine (T3). Noteworthy is that the novel free thyroid hormone assay uses an alternative methodology to the frequently used one-step analog or two-step methodology. The conventional analog system uses a tracer-labeled T4 conjugate. The fT4 assay uses a peroxidase-labeled antibody to T4.
We assessed 872 consecutive subjects from a healthy adult population for TSH and fT4, and smaller cohorts of 120 and 146 subjects for the total hormone and fT3 reference intervals, respectively. The subjects conformed to accepted criteria for establishment of a reference population (1). In addition, patients on regular follow-up with established thyroid disorders were also used in the technical evaluation of the assays.
All procedures conformed to the Helsinki Declaration of 1975 and the 1996 revision.
Reference intervals were established using rank and percentile
confidence limits for euthyroid samples from a multiphasic screening
program collected from the local population. The subjects presented
with no evidence or clinical suspicion of thyroid abnormalities,
including family history of thyroid disorders, pituitary disorders, and
psychoses; were not on drugs known to interfere with thyroid gland
metabolism or hormone assays; and their results for hepatic and renal
function tests and complete blood counts were within the health-related
reference intervals (1). Table 1
shows the two-tailed 95th interpercentile reference intervals
obtained from groups of euthyroid subjects.
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We measured TSH in 11 pools of measurable hyperthyroid serum (repeated
over 5 consecutive days; n = 9). The TSH value at 20% CV
(functional sensitivity) was 0.0032 mIU/L. To provide an efficient
first-line test in screening for thyroid disorders in ambulatory
patients (2), TSH functional sensitivity is important
(3). As reported previously, a functional sensitivity of
0.0082 mIU/L for TSH was obtained by our laboratory on the DPC Immulite
(4). At 0.05–63 mIU/L we found within-day CVs 
3.5%
(n = 4) and a total CV 8.4% (n = 9).
The linearity of the TSH assay was determined with nine ratios of two serum samples with concentrations of 129 and 1.08 mIU/L. A linear correlation coefficient of 1.000 (n = 9) was obtained for TSH. Recoveries were 100–103%.
TSH results obtained from the Vitros ECi were compared with the DPC
Immulite and Abbott AxSym assays: ECi = 0.996 (Immulite) + 0.161
(n = 269; r = 0.993; Sy|x =
0.05; P <0.05); and ECi = 0.914 (AxSym) + 0.110
(n = 147; r = 0.999; Sy|x =
0.02; P <0.05). Bias plots show that the percent relative
difference of TSH between the Immulite or AxSym and the Vitros ECi is
spread evenly through the range 0.1–100 mIU/L (Fig. 1
). However, at values <0.1 mIU/L, a positive percent relative
difference was observed, indicating that the results obtained on the
Vitros ECi were lower than those obtained on either the Immulite or the
AxSym.
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The total imprecision of the free-hormone assays was measured using
pooled serum samples of five different concentrations covering the
dynamic range for fT4, fT3, T4, and T3.
The imprecision analysis was performed on nine occasions over 5
consecutive days. fT4 concentrations ranged from 5.1 to 77.4
pmol/L, with a within-day CV 3.2% and a total CV 3.1%.
fT3 concentrations ranged from 5.3 to 19.4 pmol/L, with a
within-day CV 5.0% and a total CV 4.0%. Total T4
concentrations ranged from 68 to 234 nmol/L, with a within-day CV
1.6% and a total CV 2.8%. Total T3
concentrations ranged from 2.0 to 6.3 nmol/L with a within-day CV
1.3% and a total CV 2.3%. In comparison, our current AxSym
analyzer had an interassay CV of 3.5–10% for fT4 at
concentrations of 4.0–77.2 pmol/L (5). Browning et al.
(6) recommend a total CV <8% for fT4 and <6% for
T4. These targets were achieved in this evaluation.
The linearity on dilution was tested for T4 and T3. This was performed by diluting different proportions of a patient sample pool from the upper and lower end of the dynamic range for each analyte to be evaluated. The "recovered" amount of analyte measured for T4 ranged from 88% to 99%, with a correlation coefficient of 0.99. The recovery for T3 ranged from 85% to 95%, with a correlation coefficient of 0.99.
We serially diluted patient samples with a protein-free matrix (normal saline 1:2 to 1:16) and assayed for fT4 and fT3. Samples from late pregnancy and critically ill subjects were included. Mean fT4 results were, for samples from euthyroid subjects, 103%, 107%, 109%, and 99% of the undiluted sample; mean fT3 results were, for samples from euthyroid subjects, 100%, 102%, 98%, and 101%; for samples from late-pregnancy subjects, 95%, 95%, 100%, and 109%; for samples from critically ill subjects, 89%, 90%, 92%, and 87%, with three of the groups within ± 10%, as required by Ekins (7). On the Vitros ECi analyzer, the fT4 assay uses a patented assay design, including an anti-T4 antibody labeled with horseradish peroxidase, that has been optimized to minimize the effect of binding protein alterations. This fT4 and fT3 assay withstood up to a 1:16 sample dilution (maintaining constancy in the ratio of free hormone to binding proteins) in a protein-free matrix. Albumin is added to the free hormone assay reagent by a number of manufacturers to buffer the effects of increased nonesterified fatty acids that develop in serum in vitro. In our hands, in pregnant subjects, where the equilibrium is altered and the binding capacity is increased, slightly higher fT4 was observed on the Vitros ECi for values >10 pmol/L; values <10 pmol/L were slightly lower than the AxSym results. Our evaluation data suggested that in critically ill subjects with an altered equilibrium and a low binding capacity, the albumin falsely lower the fT4 concentration reported by the AxSym. In this context, there is no albumin added to the ingredients of the free hormone assays of Vitros ECi. There is also minimal sample:reagent dilution (1:5), a factor that minimizes the underestimation of fT4 when T4 binding inhibitors are present (8). These findings in the free hormone assay currently are being investigated further.
The fT4, fT3, T4, and T3 results compared with the Abbott AxSym: ECi fT4 = 1.143 (AxSym) - 3.009 (n = 338; r = 0.978; Sy|x = 0.12; P <0.05); ECi fT3 = 1.161 (AxSym) + 1.723 (n = 115; r = 0.932; Sy|x = 0.07; P <0.05); ECi T4 = 0.87 (AxSym) + 1.327 (n = 112; r = 0.983; Sy|x = 0.55; P <0.05); ECi T3 = 1.045 (AxSym) + 0.174 (n = 109; r = 0.961; Sy|x = 0.02; P <0.05).
The bias plots of the free and total hormones are shown in Fig. 1
.
T3 and fT3 results on the Vitros ECi were significantly
higher (fT3 ranging from 20% to 150% higher; T3 up to
50% higher at lower concentrations) than the AxSym. T4 results
reported on the Vitros ECi are 10–20% lower than AxSym results. fT4
values <10 pmol/L on the AxSym appear to be higher. The lower values
on the Vitros ECi may be considered more valid because there is no
albumin in the assay reagent to buffer the effects of nonesterified
fatty acids (7).
In conclusion, the technical performance, ease of operation, and rapid turnaround time makes the Vitros ECi a consideration for routine use in thyroid evaluation.
Acknowledgments
This work was supported by Ortho Clinical Diagnostics, Singapore, who kindly supplied all the test kits used in the analyses. We thank the staff of the Department of Laboratory Medicine, National University Hospital, Singapore, for technical assistance.
References
The following articles in journals at HighWire Press have cited this article:
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  M. L. Campbell-Yeo, A. C. Allen, K. S. Joseph, J. M. Ledwidge, K. Caddell, V. M. Allen, and K. C. Dooley Effect of Domperidone on the Composition of Preterm Human Breast Milk Pediatrics, January 1, 2010; 125(1): e107 - e114. [Abstract] [Full Text] [PDF]
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 M. P Rayman, A. J Thompson, B. Bekaert, J. Catterick, R. Galassini, E. Hall, M. Warren-Perry, and G. J Beckett Randomized controlled trial of the effect of selenium supplementation on thyroid function in the elderly in the United Kingdom Am. J. Clinical Nutrition, February 1, 2008; 87(2): 370 - 378. [Abstract] [Full Text] [PDF]
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 M. L. Rawlins and W. L. Roberts Performance Characteristics of Six Third-Generation Assays for Thyroid-Stimulating Hormone Clin. Chem., December 1, 2004; 50(12): 2338 - 2344. [Abstract] [Full Text] [PDF]
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R. Sapin and M. d'Herbomez Free Thyroxine Measured by Equilibrium Dialysis and Nine Immunoassays in Sera with Various Serum Thyroxine-binding Capacities Clin. Chem., September 1, 2003; 49(9): 1531 - 1535. [Full Text] [PDF]
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A. Price, C. Burgin, I. Catch, and M. Cruise Functional Sensitivity and Recovery of Thyroid-stimulating Hormone Clin. Chem., November 1, 2001; 47(11): 2067 - 2067. [Full Text] [PDF]
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R. Sapin, J.-L. Schlienger, F. Gasser, E. Noel, B. Lioure, F. Grunenberger, B. Goichot, and D. Grucker Intermethod Discordant Free Thyroxine Measurements in Bone Marrow-transplanted Patients Clin. Chem., March 1, 2000; 46(3): 418 - 422. [Full Text] [PDF]
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H. A. Hendriks, W. Kortlandt, and W. M. Verweij Standardized Comparison of Processing Capacity and Efficiency of Five New-Generation Immunoassay Analyzers Clin. Chem., January 1, 2000; 46(1): 105 - 111. [Abstract] [Full Text] [PDF]
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