References Slocum RH Cumings JG Amino acid analysis of by ericaburns


									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
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
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,
                                                                                          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.
                                                                               hepatocytes.    Though the production of IL-6 and CRP has

                                                                                                CLINICAL   CHEMISTRY,      Vol. 41, No. 3, 1995    467

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