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Clinical assessment of a simultaneous radioimmunoassay (RIA) for free thyroxine (FT4) and thyrotropin (TSH) in the diagnosis of thyroid disorders
CSCC/CAMB POSTER ABSTRACTS
30
A SIMPLE, S E N S I T I V E FLUOROMETRIC A S S A Y FOR THE D E T E R M I N A T I O N OF A T P IN P L A T E L E T S
Seudeal, N a r a c e D. and Thibert, Rojer J., Dept. of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4 ATP measurements have been used to check for RBC viability and to determine microbial numbers. Recently, decreased platelet ATP levels have shown a 100% correlation with a muscle biopsy test for the disease, malignant hyperthermia (Solomons, C. C. et al. (1980) New Engl. J. Med. 303, 642). A method is proposed to determine ATP levels in platelets using a coupled enzymatic system. 3-Phosphoglycerate (3-PGA) is converted to 1,3-di-PGA in the presence of phosphoglycerokinase. The 1,3-di-PGA is then converted to glyceraldehyde 3-phosphate (GAP) in the presence of GAP dehydrogenase. This sequence of reactions produces I mol of NAD ÷ and constitutes the assay for ATP as provided by a commercially available kit. This assay, however, is not sensitive enough to measure ATP levels in platelets. An increased sensitivity has been obtained by converting GAP to dihydroxyacetone phosphate (DHAP) in the presence of trioseisomerase and DHAP to glycerol 3-phosphate (GP) in the presence of GP dehydrogenase. The entire sequence of reactions produces 2 tools of NAD +. The NAD + is reacted with acetone in the presence of NaOH to form a highly fluorescent condensation product. The formation of this product is achieved by boiling for 2 min. The excitation and emission wavelengths for maximum fluorescence of this product are 365 and 460 nm, respectively. A pH optimum of 2.0-6.0 is obtained for the formation of this product and once formed it is stable for at least 24 hr. However, upon irradiation (365 nm), the product starts to decompose after 4 rain, thus, readings must be taken within the 4 min after excitation. A plot of fluorescence versus concentration of ATP shows a linear relationship between 0 - 4 nmols ATP with a correlation coefficient of 0.9997. Native fluorescence from interfering NADH is removed with HC1 prior to the condensation reaction. Platelets are isolated by a centrifugation technique and dissolved in guanidine. HC1 (8.0 M). This obviates the necessity of a tedious ultra-filtration step in the preparation of the platelets for analysis.
31
A N ENZYME-COUPLED COLORIMETRIC A S S A Y FOR S E R U M C H O L I N E S T E R A S E : A P P L I C A T I O N TO THE C O B A S - F A R A T M CENTRIFUGAL A N A L Y Z E R
Moses GC, Ross ML, Henderson AR. Dept. of Clin Biochem, University Hospital (University of Western Ontario), PO Box 5339, London, Ont, N6A 5A5 A simple enzyme-coupled colorimetric assay for serum cholinesterase (EC 3.1.1.8) activity has been adapted to the Cobas Fara T M bench-top centrifugal Analyzer. The procedure is based on that described by Abernethy HM et al. (Clin Chem 1986; 32: 194-7). In the assay choline produced from hydrolysis of benzylcholine during a 10-rain incubation at 25°C is determined by a coupled reaction scheme consisting of choline oxidase, peroxidase, phenol and 4-amino-antipyrene and a cholinesterase inhibitor, physostigmine, to prevent further benzoylcholine hydrolysis during the colour development. The assay requires 5 ~1 of a 21-fold diluted serum in a total reactions volume of 260 ~l. Analytically the proposed method performs satisfactorily and shows good correlation with the manual visible (Abernethy et al.) and hte classical 240 n m (UV) kinetic procedures; regression equations and correlation 292
coefficients are as follows: Fara visible (U/mL) = 1.0084 manual visible - 0.109 r = 0.839, n = 15 Fara visible (U/mL) = 0.7070 kinetic 240 n m to 0.0477 r = 0.913, n = 32 Cholinesterase activity measurements, standardized with choline iodide, were linear in the range of 80-3100 U/L patient sera and a commercially available h u m a n serum butyryl cholinesterase (Sigma Diagnostics). The proposed procedure is suitable for routine determination of total serum cholinesterase activity in serum, but it can also be used to predict succinyl choline sensitivity and/or detect genetic variants using appropriate substrates.
32
CLINICAL A S S E S S M E N T OF A SIMULTANEOUS RADIOIMMUNOASSAY (RIA) FOR FREE THYROXINE (FT+) A N D T H Y R O T R O P I N (TSH) IN THE D I A G N O S I S OF THYROID D I S O R D E R S
J. Kalra, P. S. Raina, K. L. Massey, P. K. Chattopadhyay and V. A. Laxdal, Department of Pathology, University Hospital and University of Saskatchewan, Saskatoon, Saskatchewan S7N 0X0 The immunoradiometric assays (IRMA) for serum thyrotropin (TSH) are being used as the initial screening test for evaluating thyroid status. However, this test has limitations in differentiating subclinical hypothyroidism from overt hypothyroidism and in some of those euthyroid patients who may have subnormal values for TSH. We have evaluated the analytical and clinical value of "SimulTRAC" FT4157Co]/TSH [~I] RIA method (Becton Dickinson Immunodiagnostics, Orangeburg, New York) which measures serum levels of FTt and TSH simultaneously in a single tube. Dual standards containing FT, and TSH or patient samples are incubated with dual antibody at 37°C for 2.5 hours. The dual tracer containing FTt [57Co] and TSH [~5I] is then added and the tubes are again incubated at 37°C for 1.5 hours. Bound and unbound TSH and FT4 are separated by precipitation with a second antibody. The precipitate is separated by centrifugation and counted in a gamma counter which discriminates between the disintegration produced by [1~5I]and [~7Co] tracers. The reference interval for euthyroid subjects (N = 53) by this method for serum TSH and FT4 were 0.7 to 7 . 2 m U / L and 11.8 to 27.5pmol/L respectively and were within the reference range listed by the manufacturer. The sensitivity for the reliable detection of serum TSH and FT4 were 0.18 mU/L and 0+8 pmol/L respectively. The intra and inter-assay coefficient of variation (CV) were less than 12% at different serum concentrations and the analytical recovery were 92 to 104%. Our study included 63 patients who were divided into different groups on the basis of their clinical findings. I n the hyperthyroid group (n = 18) all the patients were correctly diagnosed except three, in whom FT4 was within normal range whereas the TSH value was subnormal. In the hypothyroid group (n = 23) 18 patients were correctly diagnosed as overt hypothyroid and 5 as subclinically hypothyroid. All the patients who were clinically euthyroid (n = 11) or had nonthyroidal illness (n -- 11) were also correctly diagnosed. Our observations indicate that the SimulTRAC F T J T S H assay results correlated well with the clinical status of patients. This assay may be useful as a single test as it also provides improved diagnostic accuracy between patients with overt hypothyroidism and subclinical hypothyroidism. CLINICAL BIOCHEMISTRY, VOLUME 20, AUGUST 1987
30
A SIMPLE, S E N S I T I V E FLUOROMETRIC A S S A Y FOR THE D E T E R M I N A T I O N OF A T P IN P L A T E L E T S
Seudeal, N a r a c e D. and Thibert, Rojer J., Dept. of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4 ATP measurements have been used to check for RBC viability and to determine microbial numbers. Recently, decreased platelet ATP levels have shown a 100% correlation with a muscle biopsy test for the disease, malignant hyperthermia (Solomons, C. C. et al. (1980) New Engl. J. Med. 303, 642). A method is proposed to determine ATP levels in platelets using a coupled enzymatic system. 3-Phosphoglycerate (3-PGA) is converted to 1,3-di-PGA in the presence of phosphoglycerokinase. The 1,3-di-PGA is then converted to glyceraldehyde 3-phosphate (GAP) in the presence of GAP dehydrogenase. This sequence of reactions produces I mol of NAD ÷ and constitutes the assay for ATP as provided by a commercially available kit. This assay, however, is not sensitive enough to measure ATP levels in platelets. An increased sensitivity has been obtained by converting GAP to dihydroxyacetone phosphate (DHAP) in the presence of trioseisomerase and DHAP to glycerol 3-phosphate (GP) in the presence of GP dehydrogenase. The entire sequence of reactions produces 2 tools of NAD +. The NAD + is reacted with acetone in the presence of NaOH to form a highly fluorescent condensation product. The formation of this product is achieved by boiling for 2 min. The excitation and emission wavelengths for maximum fluorescence of this product are 365 and 460 nm, respectively. A pH optimum of 2.0-6.0 is obtained for the formation of this product and once formed it is stable for at least 24 hr. However, upon irradiation (365 nm), the product starts to decompose after 4 rain, thus, readings must be taken within the 4 min after excitation. A plot of fluorescence versus concentration of ATP shows a linear relationship between 0 - 4 nmols ATP with a correlation coefficient of 0.9997. Native fluorescence from interfering NADH is removed with HC1 prior to the condensation reaction. Platelets are isolated by a centrifugation technique and dissolved in guanidine. HC1 (8.0 M). This obviates the necessity of a tedious ultra-filtration step in the preparation of the platelets for analysis.
31
A N ENZYME-COUPLED COLORIMETRIC A S S A Y FOR S E R U M C H O L I N E S T E R A S E : A P P L I C A T I O N TO THE C O B A S - F A R A T M CENTRIFUGAL A N A L Y Z E R
Moses GC, Ross ML, Henderson AR. Dept. of Clin Biochem, University Hospital (University of Western Ontario), PO Box 5339, London, Ont, N6A 5A5 A simple enzyme-coupled colorimetric assay for serum cholinesterase (EC 3.1.1.8) activity has been adapted to the Cobas Fara T M bench-top centrifugal Analyzer. The procedure is based on that described by Abernethy HM et al. (Clin Chem 1986; 32: 194-7). In the assay choline produced from hydrolysis of benzylcholine during a 10-rain incubation at 25°C is determined by a coupled reaction scheme consisting of choline oxidase, peroxidase, phenol and 4-amino-antipyrene and a cholinesterase inhibitor, physostigmine, to prevent further benzoylcholine hydrolysis during the colour development. The assay requires 5 ~1 of a 21-fold diluted serum in a total reactions volume of 260 ~l. Analytically the proposed method performs satisfactorily and shows good correlation with the manual visible (Abernethy et al.) and hte classical 240 n m (UV) kinetic procedures; regression equations and correlation 292
coefficients are as follows: Fara visible (U/mL) = 1.0084 manual visible - 0.109 r = 0.839, n = 15 Fara visible (U/mL) = 0.7070 kinetic 240 n m to 0.0477 r = 0.913, n = 32 Cholinesterase activity measurements, standardized with choline iodide, were linear in the range of 80-3100 U/L patient sera and a commercially available h u m a n serum butyryl cholinesterase (Sigma Diagnostics). The proposed procedure is suitable for routine determination of total serum cholinesterase activity in serum, but it can also be used to predict succinyl choline sensitivity and/or detect genetic variants using appropriate substrates.
32
CLINICAL A S S E S S M E N T OF A SIMULTANEOUS RADIOIMMUNOASSAY (RIA) FOR FREE THYROXINE (FT+) A N D T H Y R O T R O P I N (TSH) IN THE D I A G N O S I S OF THYROID D I S O R D E R S
J. Kalra, P. S. Raina, K. L. Massey, P. K. Chattopadhyay and V. A. Laxdal, Department of Pathology, University Hospital and University of Saskatchewan, Saskatoon, Saskatchewan S7N 0X0 The immunoradiometric assays (IRMA) for serum thyrotropin (TSH) are being used as the initial screening test for evaluating thyroid status. However, this test has limitations in differentiating subclinical hypothyroidism from overt hypothyroidism and in some of those euthyroid patients who may have subnormal values for TSH. We have evaluated the analytical and clinical value of "SimulTRAC" FT4157Co]/TSH [~I] RIA method (Becton Dickinson Immunodiagnostics, Orangeburg, New York) which measures serum levels of FTt and TSH simultaneously in a single tube. Dual standards containing FT, and TSH or patient samples are incubated with dual antibody at 37°C for 2.5 hours. The dual tracer containing FTt [57Co] and TSH [~5I] is then added and the tubes are again incubated at 37°C for 1.5 hours. Bound and unbound TSH and FT4 are separated by precipitation with a second antibody. The precipitate is separated by centrifugation and counted in a gamma counter which discriminates between the disintegration produced by [1~5I]and [~7Co] tracers. The reference interval for euthyroid subjects (N = 53) by this method for serum TSH and FT4 were 0.7 to 7 . 2 m U / L and 11.8 to 27.5pmol/L respectively and were within the reference range listed by the manufacturer. The sensitivity for the reliable detection of serum TSH and FT4 were 0.18 mU/L and 0+8 pmol/L respectively. The intra and inter-assay coefficient of variation (CV) were less than 12% at different serum concentrations and the analytical recovery were 92 to 104%. Our study included 63 patients who were divided into different groups on the basis of their clinical findings. I n the hyperthyroid group (n = 18) all the patients were correctly diagnosed except three, in whom FT4 was within normal range whereas the TSH value was subnormal. In the hypothyroid group (n = 23) 18 patients were correctly diagnosed as overt hypothyroid and 5 as subclinically hypothyroid. All the patients who were clinically euthyroid (n = 11) or had nonthyroidal illness (n -- 11) were also correctly diagnosed. Our observations indicate that the SimulTRAC F T J T S H assay results correlated well with the clinical status of patients. This assay may be useful as a single test as it also provides improved diagnostic accuracy between patients with overt hypothyroidism and subclinical hypothyroidism. CLINICAL BIOCHEMISTRY, VOLUME 20, AUGUST 1987