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References analyzer. These assays utilize an anti-T4 monoclonal
1. Slocum RH, Cumings JG. Amino acid analysis of physiological antibody and a T4-analog fluorescein-labeled tracer for
samples. In: Hommes FA, ed. Techniques in diagnostic human
binding and signal detection, respectively. We report
biochemical genetics. New York: Wiley Liss, 199 1:87-126.
2. Rosenbium R. Stability of glutamine vitro. Proc Soc Exp Biol
in here correlations T
of the Cobas#{174}-FPotal T4 and T-
Med 1965;119:763-5. Uptake assays with two commercially available assays
3. Dickinson JC, Rosenblum H, Hamilton PB. Ion exchange of T4 and T-uptake, TDx#{174} (Abbott Laboratories, Abbott
chromatography of the free amino acids in the plasma of the Park, IL) and Emit#{174} (Syva Co., Palo Alto, CA). Both of
newborn infant. Pediatrics 1965;36:2-13.
4. De Wolfe MS, Baskurt 5, Cochrane WA. Automatic amino acid these assays, like the Cobas-FP methods, are homoge-
analysis of blood serum and plasma. Clin Biochem 1967;1:75-81. neous assays adapted to run on small, automated
5. PerryTL, Hansen S. Technical pitfalls leading to errors in the clinical analyzers. Further, we used the Cobas-FP
quantitation of plasma amino acids. Clin Chim Acts 1969;25:
Total T4 and T-Uptake assay results to calculate a free
53-8.
6. Armstrong MD, Stave U. A study of plasma free amino acid thyroxine index (VF4I; an estimate of free T4 concen-
levels. I. Study of factors affecting validity of amino acid analyses. tration) for comparisons with direct RLA measure-
Metabolism 1973;22:549-60. ments of free T4 (with a two-step RIA from Incstar,
7 Olek KV, Uhlhaas 5, Wardenbach P, Yaniaguchi M. Zuverhas-
Stillwater, MN). The principles of the procedures and
sigkait von Aminosaure-Bestimmungen aus menschlichem Se-
rum bei unterschiedlichen Lagerungsbedingungen. J Clin Chem further details for all assays are described in the
Clin Biochem 1979;17:599-604. respective manufacturers’ package inserts. Perfor-
8. Ukida M, Schafer K, Bode JC. Effects of storage at -20#{176}Con mance of the assays for use in the present comparison
the concentration of amino acids in plasma. J Clin Chem Clin
Biochem 1981;19:1193-5. study was done according to the manufacturers’ in-
9. Schaefer A, Piquard F, Haberey P. Plasma amino acids anal- structions.
ysis: effect of delayed samples preparation and of storage. Cliii Clinical specimens (n = 99) for ‘F4 and T-uptake
Chim Acta 1987;164:163-9. method comparisons were patients’ sera submitted for
10. Jacob R, Barrett E. Chromatographic analysis of glutamine
in plasma. J Chromatogr 1982;229:188-92. thyroid testing to a large hospital laboratory; the
11. Khan K, Blaak E, Elia M. Quantifying intermediary metab- present tests were performed after all routine analyses
olites in whole blood after a simple deproteinization step with were complete. Specimens were chosen to encompass a
sulfosalicylic acid. Clin Chem 1991;37:728-33. range of normal and abnormal (high or low) test results
12. Parvy P, Bardet J, Rabier D, Gasquet M, Kamoun P. hntra-
and interlaboratory quality control for assay of amino acids in for T4 and T-uptake on the basis of the values mea-
biological fluids: 14 years of the French experience. Clin Chem sured by the hospital’s routine methods (commercial
1993;39:1831-6. 1251 RIA methods). No diagnostic information (i.e.,
13. Saito Y, Carro-Corona 0, Pepinsky R. X-ray examination of pregnancy states or thyroid function) from these tests
molecular configuration of asparagine in crystalline 1.-asparagine
monohydrate. Science 1954;121:435-6. of record was used for selecting or categorizing the
samples in the method comparisons reported here.
Each specimen was subsequently assayed in singlet for
T4 and T-uptake by the Emit, TDx, and Cobas-FP
Two Fluorescence Polarization Immunoassays for systems. TDx and Cobas-FP T-uptake results were
Total Thyroxine and T-Uptake Quantification, Eoan M. reported in T-uptake units; each Emit result was
Cadoff,’ Charles M. Cheng,2 Holly G. Jerome,2 Laura D. converted to a thyroid-hormone-binding ratio (THBR),
Klein,2 and Stuart M. Palmer2’3 (1 UMDNJ-Robert according to the manufacturer’s instructions. FT4I was
Wood Johnson Med. School, New Brunswick, NJ; calculated as total T4 concentration divided by T-
2Phe Diagnostic Systems, 1080 US Highway 202, uptake for TDx and Cobas-FP and as total T4 times
Somerville, NJ 08876-377 1; author for correspondence: THBR for Emit-again, according to the manufactur-
fax 908-253-7891) ers’ instructions.
For the between-methods comparison, we used direct
Homeostasis of thyroid function and the physiologi- linear regression analysis for the T4 methods and for
cal role played by thyroxine (T4; 3,5,3’,5’-tetraiodothy- the TDx and Cobas-FP T-uptake assays. In the regres-
ronine) have been well described (1, 2). Furthermore, sion analysis of Emit T-uptake vs the other two meth-
quantification of the available binding sites on proteins ods, we used the inverse of THBR because of the
specific for ‘F4 (termed T-uptake) is useful in assessing difference between the Emit and FP systems: PP sys-
the relative T4-protein equilibria existing in hypo-, tems allow for direct measurement of thyroid-hormone-
hyper-, and euthyroid individuals (3). Because the binding sites through the binding of a labeled T4 analog
concentration of unbound or free thyroid hormone 14 to unoccupied sites on T4-binding globulin (TBG) or
correlates with the metabolic state of an individual, other serum proteins; the higher assay signal produced
assessment of total T4 and T-uptake capacity, com- reflects the greater number of available binding sites in
bined with an estimate of free T4, may help in diag- the FP assays. The Emit T-uptake signal is a measure
noses of suspected thyroid disorders or in general of the residual T4 that remains unbound after the
thyroid-function screenings (4). addition of a constant, saturating amount of the thy-
Roche Diagnostic Systems (Somerville, NJ) has re- roid hormone, and therefore is an indirect measure of
cently developed two fluorescence polarization (FP) the unoccupied sites; in this assay, there is an inverse
assays for quantifring total T4 and T-uptake in serum relationship between assay signal and T-uptake capac-
or plasma samples with the Cobas Fara Il#{174} centrifugal ity. The manufacturer’s instructions call for multiply-
466 CLINICAL CHEMISTRY, Vol. 41, No. 3, 1995
ing the total T4 by THBR to calculate the Emit VF41, The results for samples from these limited popula-
thus, the Emit THBR has an inverse proportionality to tions of patients indicate that the Cobas-FP results
the T-uptake measured by FP. agree closely with those of other commercially avail-
Specimens (n = 103) for the free T4 comparison were able immunoassays for T4 and T-uptake, and that the
patients’ sera submitted to a large reference laboratory calculated F’r4I by Cobas-FP has potential use as a
for thyroid testing; and again, they were assayed after surrogate for direct free T4 measurement. The Co-
all other testing was complete. These samples were bas-FP assays for total T4 and ‘F-uptake are easy to use
chosen also to encompass a range of normal or abnor- because the Cobas Fara II analyzer carries out all
mal results for T4 or ‘F-uptake methods of the reference pipetting, mixing, reading, and data reduction auto-
laboratory [Ciba Corning (Medfleld, MA) Magic#{174} 1251 matically; moreover, this analyzer can be programmed
RIA methods] and no patients’ diagnoses information to generate FF4I values for each sample by requesting
was available. These specimens were subsequently both assays on a single sample cup. The system has a
tested in duplicate for free T4 with the two-step RIA throughput of =85 test results per hour and needs
and for total T4 and T-uptake with the Cobas-FP calibration only once a month.
reagents. The Cobas-FP-calculated F4I was compared
with the free T4 RIA results by direct linear regression This work was supported by a grant to the University of
Medicine and Dentistry of New Jersey at the Robert Wood
analysis. Johnson University Hospital from Roche Diagnostic Systems,
Results of the method comparison regression analy- Inc., a member of the Roche Group.
ses are summarized in Table 1. The TDx, Emit, and
References
Cobas-FP total T4 assay results for this patient popu-
1. DeGroot U, Niepomniszcze H. Biosynthesis of thyroid hor-
lation all agree closely, as indicated by the high regres- mone: basic and clinical aspects. Metabolism 1977;26:665-718.
sion coefficients and the slopes close to unity. Close 2. Schirnmel M, Utiger RD. Thyroidal and peripheral production
agreement was also observed among the three T- of thyroid hormones. Ann Intern Med 1977;87:760-8.
uptake methods, although the two FP assay systems 3. Fernandez-Ulloa M, Mason HR. Thyroid. In: Kaplan LA, Pesce
show a closer correlation with each other than with AJ, eds. Clinical chemistry. Theory, practice and correlation, 2nd
ed. St. Louis: CV Mosby, 1989:620-38.
Emit. The decreased correlation coefficients in the
4. Alexander NM. Thyroid-function tests [Editorial]. Clin Chem
Cobas-FP vs Emit and TDx vs Emit regressions may be 1984;30:827-8.
due to differences between direct and indirect ap- 5. Cheng C, Fischberg E, Palmer S. The effect of abnormally low
proaches to the measurement of thyroid-hormone-bind- and high T4 values on the correlation between T-uptake FP and
ing sites. The agreement between FP methods suggests T-3 uptake RIA assays [Abstract]. Clin Chem 1993;39:1166.
that similar binding phenomena are being measured
by both. The FF41 calculated from the Cobas-FP assays
showed a strong correlation with the two-step RIA free
T4 results for the specimens tested (r = 0.978). This Interleukln-6 and C-Reactive Protein During Pediatric
suggests that the FT4I calculation for the Cobas meth- Cardiopulmonary Bypass, Deiphine Behr,1 Main
ods reasonably approximates the measured free T4 Hernuann,”2’5 Philippe Pouard,3 Isabelle Spizzi,’
values over the wide range ofT4 and T-uptake concen- Francine Leca,4 Pascal Vouhe,4 and Ohuanesse
trations of the samples in this study. Further studies Ekindjian”2 [i Lab. Central de Biochijn., H#{244}pital
are in progress to confirm the clinical accuracy of the Laennec, 42 rue de S#{232}vres, Paris, France; 2 Lab. de
Cobas-FP FT4I results in other situations observed Biochim. Cell. de l’Inflam., Tour D4, Univ. Paris XI, 5
rue J-B Clement, 92296 Ch#{226}tenay Malabry cedex,
previously to cause interferences in other methods (5).
France; H#{244}pital
Dept. d’Anesth#{233}sie-R#{233}animation,
Laennec; Service de Chirurgie Cardio-Vasculaire,
Table 1. Method comparison results for T4, T-uptake,
H#{244}pital
Laennec; author for correspondence: (fax 01-42
and FT4I determinatlons.
34 19 87)]
Methods: y, x Slope y-intercept R SES SEEC
T4,nmol/L (n = 99; specimen range: 13.8-254.6 nmol/L by TDx) Patients undergoing cardiopulmonary bypass (CPB) are
Cobas-FP, TDx 1.09 4.73 0.961 0.032 12.44 liable to develop a postperfusion syndrome characterized
Cobas-FP, Emit 0.99 16.42 0.973 0.024 10.45 by a whole-body inflammatory reaction (1). The biological
Emit, TDx 1.09 -11.06 0.982 0.022 8.42 activities of cytokines suggest intense participation of
T-uptake” (n = 99; specimen range: 0.33-1.81 units by TDx) these inflammatory events during CPB. Cytokines are
Cobas-FP, Thx 0.89 0.11 0.990 0.013 0.031 synthesized by many cell types and possess endocrine,
Cobas-FP, Emit 0.56 0.31 0.833 0.038 0.121 paracrine, or autocrine activities. Their local synthesis
TDx, Emit 0.63 0.23 0.833 0.042 0.135 and action in endothelium are particularly important (2).
Free T4index, pmoIIL (n = 103; specimen range: 0.13-100.9 Secretion of interleukin-lp (IL-lp) and tumor necrosis
pmol/L by RIA) factor-a (TNF-a) can be induced by the presence of endo-
Cobas FT4I, 3.97 38.70 0.978 0.086 17.84 toxins, gram-negative bacterial products, and complement
free T4 RIA anaphylatoxins C3a/C5a (3, 4). Factors triggering IL-6
Linear regression analyses of assays performed on samples tested in synthesis include IL-1/3, TNF-a, and gram-negative bac-
singlet (duplicate for AlA). terial products (5). IL-113 and TNF-a induce the release of
I) In % T-uptake for Emit, units for FP methods. acute-phase proteins such as C-reactive protein (CRP) by
SES, standard error of the slope; SEE,standard error of the y-estimate.
#{176}
hepatocytes. Though the production of IL-6 and CRP has
CLINICAL CHEMISTRY, Vol. 41, No. 3, 1995 467
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