J. Endocrinol. Invest. 14: 1-9, 1991
Thyroid function evaluation by different commercially available free thyroid hormone measurement kits in term pregnant women and their newborns 1
E. Roti, E. Gardini2, R. Minelli, L. Bianconi, and M. Flisi Centro per 10 Studio, Prevenzione , Diagnosi e Cura delle Tireopatie Cattedra di Endocrinologia Universita di Parma, Via A. Gramsci 14, 43100 Parma, Italy
ABSTRACT. Evaluation of thyroid status by measurement of free thyroid hormone concentrations seems particularly helpful in conditions with altered serum binding proteins. In pregnancy, a condition of increased thyroxine binding globulin, serum free thyroxine (FT4) and free triiodothyronine (FT3) concentrations have been reported to be normal, increased or decreased. In the present study we have measured serum total and free thyroid hormone concentrations in pregnant women, their newborns and nonpregnant women. Serum FT 4 and FT3 concentrations have been measured with 10 different commercially available kits and the results obtained have been compared. Serum total thyroid hormone concentrations in pregnant women were significantly higher than in their newborns and in nonpregnant women. Maternal serum FT4 concentrations measured with the different kits were always significantly lower than values in nonpregnant women. Furthermore, with one kit, the mean maternal serum FT4 concentration was below the normal range and with many kits, a large number of maternal serum samples had serum FT4 concentrations below the normal range. With all kits, except two, neonatal serum FT4 concentrations were
higher than values in their respective mothers and, in general, lower than values in nonpregnant women. Serum FT3 concentrations in nonpregnant women were in the normal range, except with one kit, in which the mean serum FT3 concentration was below the normal range. Serum FT3 concentrations in newborns resulted markedly lower than in parturient and in non pregnant women. With almost all kits, serum FT3 values were below the normal range in many maternal samples. With one kit, maternal serum FT3 concentrations resulted higher than in nonpregnant women, whereas with the other kits serum FT3 concentrations were lower. Despite the variability of serum FT4 and FT3 concentrasions, serum TSH concentrations in pregnant women and their newborns, resulted in the normal range. These findings suggest that many kits for the measurement of serum free thyroid hormone concentrations do not seem adequate to evaluate the real thyroid status of pregnant women and newborns. Therefore, in these physiological conditions the measurement by commercial kits of serum FT4 and FT3 concentrations do not offer a real advantage in respect to serum total thyroid hormone determination.
INTRODUCTION Evaluation of thyroid status by measurement of free thyroid hormone concentrations seems to be particulary helpful in conditions with altered serum bind-
ing proteins such as thyroxine (T4) binding globulin (TBG) deficiency or excess , transthyretin (T 4 binding prealbumin) excess , variant albumin and prealbumin molecules with increased affinity for T4, variant TBG with decreased affinity for T4 and hypothyroninemias associated with non-thyroidal illness. In the above conditions, circulating free thyroid hormone concentrations should be in the normal range in spite of altered serum total iodothyronine concentrations (1-3). Increased serum TBG concentrations are always present in pregnancy. In pregnant women, the determination of free thyroid hormone concentrations has
l This study was supported in part by grant 88.00.628.04 of Consiglio Nazionale delle Ricerche, Roma by grant "Patologia del la Tiroide: Indagine dei Fattori Etiopatogenetici" of Ministero Pubblica Istruzione 40% , Roma, Italy. 2 Dr. Elian a Gardin i is a recipi ent of a Fellowship from Associazion e Volontaria Promozione Ricerca Tumori (A.VO.PRO.RI.T.), Parma, Italy. key-words:Thyroid hormones , pregnant wome n, newborns . Correspondence: Dr. Elio Roti , CattEid ra d i Endocrin ologia e Patologia Costi tuzionale , Via A. Gramsci 14, 43 100, Parma, Italy. Received June 14, 1990; acce pted Se ptember 18, 1990.
E. Roti, E. Gardini, R.Minelli, et al.
from the umbilical cord at birth, respectively. Parturient women were 29.2± 1.2 (mean±SE) years old with a mean gestational age of 39.8±0.2 weeks. All the newborns, 9 females and 14 males, were normal with a mean weight of 3,332±94 g. A regional neonatal thyroid screening program based upon T4 and TSH determinations in blood collected on filter paper on day 4 revealed no thyroid abnormalities. Blood samples were also obtained from 14 healthy, 30.6± 1.2 year old non pregnant women without a history of thyroid disease. Pregnant and nonpregnant women were not receiving any medication. Serum samples were kept at 20C until hormone analyses.
led to variable results. Employing equilibrium dialysis, serum free thyroxine (FT 4) and serum triiodothyronine (FT3) concentrations have been found to be increased (4-7), decreased (8-11) or normal (12-17). In spite of these findings, pregnant women in the absence of thyroid disease are euthyroid (18). To help resolve these discrepancies, we have evaluated 10 different commercially available kits to measure FT4 and FT3 concentrations in 23 pregnant women, their newborns and 14 nonpregnant women. MATERIALS AND METHODS Subjects
Thyroid hormone determinations Total T4 (TT4) and total T3 (TT3) were measured by radioimmunoassay with materials obtained from the Division Baxter Diagnostics-Clinical Assays (Baxter SA, Maurepas, France). Serum thyrotropin (TSH)
Blood samples were obtained from 23 healthy, term pregnant women without a history of thyroid disease and from their newborns. Maternaf and neonatal blood specimens were obtained from an antecubital vein during parturition and
Table 1 - Kits employed for the measurement of serum FT4 and FT3 concentrations in nonpregnant women, parturients and their newborns. Commercial
Supplier
Normal range
Method
Name
FT4 (pmol/I) FT3(pmol/l)
FT4
FT3
single step
9.4-25.0
3.3-8.2
5.4
6.0
FT 4/FT3 Solid Phase Becton Dickinson Component System Italia s.p.a (Milan, Italy)
single step
90-21.9
4.3-8.3
6.3
3.4
RIA-coat FT4/FT3
Byk-Gulden Italia s.p.a. (Cormano, Italy)
single step
90-283
3.4-7.7
9.3
8.9
Coat-A-Count FT4/FT3
Diagnostic Product Corporation (Los Angeles, California)
single step
10.3-25.7
2.2-6.8
5.4
5.9
Magic 1 Step FT4/FT3
Ciba-Corning Kontron Italia s.p.a. (Milan, Italy)
single step
11.6-32.2
2.9-7.7
14.3
11.9
Seria FT4/FT3
Ares-Serono Diagnostici s.p.a. (Milan, Italy)
single step
100-237
2.8-6.9
5.7
4.8
RIA-gnost FT4/FT3
Behringwerke AG (Marburg, W Germany)
two steps
90-24.5
3.5-6.0
6.3
3.0
Gamma Coat 125-I-FT4/FT3
Division Baxter Diagnostics/Clinical Assay (Maurepas, France)
two steps
9.4-25.9
1.9-6.8
5.6
13.4
Liso-phase FT 4 kit
Sclavo s.p.a. (Milan, Italy)
physical separation of bound and free hormone
8.6-18.0
3.0
Amerlex-MAB FT4 Kit
Amersham International pic (Amersham, England)
125-1 monoclonal Ab 11.0-24.0 against T4; magnetic separation of free and antibody bound T4
4.1
Amerlex-M FT 4/FT3 RIA Kit
Amersham International pic (Amersham, England)
Intraassay CV %
2
Free thyroid hormones in term pregnant women
concentrations were measured by a sensitive immunoradiometric (IRMA) method with reagents supplied by Ares-Serono (Milan, Italy). The normal range for serum thyroid hormone and TSH concentrations were: TT4, 58-148 nmol/l; TT3, 1.2-3.1 nmol/l ; TSH, 0.4-3.8 mUll. An estimate of thyroid hormone binding (THBR) was assessed by a competitive binding assay method (Division Baxter Diagnostics-Clinical Assays). Free T4 index (FT41) and free T3 index (FT31) were calculated by the product of n 4 x THBR and TT3 x THBR, respectively. Serum free T4 (FT4) concentrations were measured by 10 commercially available kits as reported in Table 1. The Amerlex-M free T4 RIA, Free T4 solid phase component system, RIA-coat FT 4, Coat-A-Count Free T4, Magic 1 Step FT 4 and Seria Free T4 kit, employ the so called "single step" method free hormone determination in which a 125 1 -T4 analogue, theoretically modified to prevent binding with serum proteins. The kit supplied by Ares-Serono utilizes the 125 1 -T4 analogue and an anti-T4 monoclonal antibody. The Riagnost FT4 and Gamma Coat 1251-FT4 kits utilize the so called "two step" assay for free thyroid hormone determinations. The unknown serum is added to the anti T4 antibody coated tube. Free T4 reacts with the binding sites of the antibody. The serum is then removed and 1251-T4 is added. The tracer binds to the unoccupied T4 binding sites on the antibody. With the Liso-phase FT 4 kit the separation of free and bound T4 fractions is obtained by Sephadex LH-20 column chromatography followed by direct RIA of free hormone. The Amerlex-MAB FT4 Kit employs a high specific activity 1251-labelled monoclonal antibody raised against T4 and uses a magnetic separation of free and antibody bound T4. Due to limitations of serum volume, serum free T3 (FT3) concentrations were measured with only eight commercially available kits. We have employed the following "single step" labeled analog methods:
Amerlex-M Free T3 RIA Kit, Free T3 Solid Phase Component System , RIA Coat FT3, Coat A Count Free T3, Magic 1 Step-FT3 and Seria Free T3. The RIA-gnost FT3 and the Gamma Coat 125 1_ T3 Radioimmunassay Kits, both "two step" methods were also employed (Table 1). The normal ranges, indicated by the manufacturers, for serum FT 4 and FT3 concentrations are reported in Table 1. For each determination, all serum samples were evaluated in the same assay, carefully following the procedures recommended by the manufacturers. Statistical analyses were conducted by paired and unpaired Student's t tests and by linear correlation, as appropriate. All the results are reported as mean±SE. RESULTS Total thyroid hormone concentrations; free thyroid hormone indexes and TSH concentrations
The intraassay coefficients of variation were: TT 4, 4.3%; TT3, 9.5% ; TSH 2.8%; and THBR, 1.9%. Serum n 4, n3, and TSH concentrations and THBR, FT41, and FT3 I values in nonpregnant women , in parturient women and their newborns are reported in Table 2. Serum n 4 concentrations in parturient women were significantly higher (unpaired Student's ttest, p
Thyroid function evaluation by different commercially available free thyroid hormone measurement kits in term pregnant women and their newborns 1
E. Roti, E. Gardini2, R. Minelli, L. Bianconi, and M. Flisi Centro per 10 Studio, Prevenzione , Diagnosi e Cura delle Tireopatie Cattedra di Endocrinologia Universita di Parma, Via A. Gramsci 14, 43100 Parma, Italy
ABSTRACT. Evaluation of thyroid status by measurement of free thyroid hormone concentrations seems particularly helpful in conditions with altered serum binding proteins. In pregnancy, a condition of increased thyroxine binding globulin, serum free thyroxine (FT4) and free triiodothyronine (FT3) concentrations have been reported to be normal, increased or decreased. In the present study we have measured serum total and free thyroid hormone concentrations in pregnant women, their newborns and nonpregnant women. Serum FT 4 and FT3 concentrations have been measured with 10 different commercially available kits and the results obtained have been compared. Serum total thyroid hormone concentrations in pregnant women were significantly higher than in their newborns and in nonpregnant women. Maternal serum FT4 concentrations measured with the different kits were always significantly lower than values in nonpregnant women. Furthermore, with one kit, the mean maternal serum FT4 concentration was below the normal range and with many kits, a large number of maternal serum samples had serum FT4 concentrations below the normal range. With all kits, except two, neonatal serum FT4 concentrations were
higher than values in their respective mothers and, in general, lower than values in nonpregnant women. Serum FT3 concentrations in nonpregnant women were in the normal range, except with one kit, in which the mean serum FT3 concentration was below the normal range. Serum FT3 concentrations in newborns resulted markedly lower than in parturient and in non pregnant women. With almost all kits, serum FT3 values were below the normal range in many maternal samples. With one kit, maternal serum FT3 concentrations resulted higher than in nonpregnant women, whereas with the other kits serum FT3 concentrations were lower. Despite the variability of serum FT4 and FT3 concentrasions, serum TSH concentrations in pregnant women and their newborns, resulted in the normal range. These findings suggest that many kits for the measurement of serum free thyroid hormone concentrations do not seem adequate to evaluate the real thyroid status of pregnant women and newborns. Therefore, in these physiological conditions the measurement by commercial kits of serum FT4 and FT3 concentrations do not offer a real advantage in respect to serum total thyroid hormone determination.
INTRODUCTION Evaluation of thyroid status by measurement of free thyroid hormone concentrations seems to be particulary helpful in conditions with altered serum bind-
ing proteins such as thyroxine (T4) binding globulin (TBG) deficiency or excess , transthyretin (T 4 binding prealbumin) excess , variant albumin and prealbumin molecules with increased affinity for T4, variant TBG with decreased affinity for T4 and hypothyroninemias associated with non-thyroidal illness. In the above conditions, circulating free thyroid hormone concentrations should be in the normal range in spite of altered serum total iodothyronine concentrations (1-3). Increased serum TBG concentrations are always present in pregnancy. In pregnant women, the determination of free thyroid hormone concentrations has
l This study was supported in part by grant 88.00.628.04 of Consiglio Nazionale delle Ricerche, Roma by grant "Patologia del la Tiroide: Indagine dei Fattori Etiopatogenetici" of Ministero Pubblica Istruzione 40% , Roma, Italy. 2 Dr. Elian a Gardin i is a recipi ent of a Fellowship from Associazion e Volontaria Promozione Ricerca Tumori (A.VO.PRO.RI.T.), Parma, Italy. key-words:Thyroid hormones , pregnant wome n, newborns . Correspondence: Dr. Elio Roti , CattEid ra d i Endocrin ologia e Patologia Costi tuzionale , Via A. Gramsci 14, 43 100, Parma, Italy. Received June 14, 1990; acce pted Se ptember 18, 1990.
E. Roti, E. Gardini, R.Minelli, et al.
from the umbilical cord at birth, respectively. Parturient women were 29.2± 1.2 (mean±SE) years old with a mean gestational age of 39.8±0.2 weeks. All the newborns, 9 females and 14 males, were normal with a mean weight of 3,332±94 g. A regional neonatal thyroid screening program based upon T4 and TSH determinations in blood collected on filter paper on day 4 revealed no thyroid abnormalities. Blood samples were also obtained from 14 healthy, 30.6± 1.2 year old non pregnant women without a history of thyroid disease. Pregnant and nonpregnant women were not receiving any medication. Serum samples were kept at 20C until hormone analyses.
led to variable results. Employing equilibrium dialysis, serum free thyroxine (FT 4) and serum triiodothyronine (FT3) concentrations have been found to be increased (4-7), decreased (8-11) or normal (12-17). In spite of these findings, pregnant women in the absence of thyroid disease are euthyroid (18). To help resolve these discrepancies, we have evaluated 10 different commercially available kits to measure FT4 and FT3 concentrations in 23 pregnant women, their newborns and 14 nonpregnant women. MATERIALS AND METHODS Subjects
Thyroid hormone determinations Total T4 (TT4) and total T3 (TT3) were measured by radioimmunoassay with materials obtained from the Division Baxter Diagnostics-Clinical Assays (Baxter SA, Maurepas, France). Serum thyrotropin (TSH)
Blood samples were obtained from 23 healthy, term pregnant women without a history of thyroid disease and from their newborns. Maternaf and neonatal blood specimens were obtained from an antecubital vein during parturition and
Table 1 - Kits employed for the measurement of serum FT4 and FT3 concentrations in nonpregnant women, parturients and their newborns. Commercial
Supplier
Normal range
Method
Name
FT4 (pmol/I) FT3(pmol/l)
FT4
FT3
single step
9.4-25.0
3.3-8.2
5.4
6.0
FT 4/FT3 Solid Phase Becton Dickinson Component System Italia s.p.a (Milan, Italy)
single step
90-21.9
4.3-8.3
6.3
3.4
RIA-coat FT4/FT3
Byk-Gulden Italia s.p.a. (Cormano, Italy)
single step
90-283
3.4-7.7
9.3
8.9
Coat-A-Count FT4/FT3
Diagnostic Product Corporation (Los Angeles, California)
single step
10.3-25.7
2.2-6.8
5.4
5.9
Magic 1 Step FT4/FT3
Ciba-Corning Kontron Italia s.p.a. (Milan, Italy)
single step
11.6-32.2
2.9-7.7
14.3
11.9
Seria FT4/FT3
Ares-Serono Diagnostici s.p.a. (Milan, Italy)
single step
100-237
2.8-6.9
5.7
4.8
RIA-gnost FT4/FT3
Behringwerke AG (Marburg, W Germany)
two steps
90-24.5
3.5-6.0
6.3
3.0
Gamma Coat 125-I-FT4/FT3
Division Baxter Diagnostics/Clinical Assay (Maurepas, France)
two steps
9.4-25.9
1.9-6.8
5.6
13.4
Liso-phase FT 4 kit
Sclavo s.p.a. (Milan, Italy)
physical separation of bound and free hormone
8.6-18.0
3.0
Amerlex-MAB FT4 Kit
Amersham International pic (Amersham, England)
125-1 monoclonal Ab 11.0-24.0 against T4; magnetic separation of free and antibody bound T4
4.1
Amerlex-M FT 4/FT3 RIA Kit
Amersham International pic (Amersham, England)
Intraassay CV %
2
Free thyroid hormones in term pregnant women
concentrations were measured by a sensitive immunoradiometric (IRMA) method with reagents supplied by Ares-Serono (Milan, Italy). The normal range for serum thyroid hormone and TSH concentrations were: TT4, 58-148 nmol/l; TT3, 1.2-3.1 nmol/l ; TSH, 0.4-3.8 mUll. An estimate of thyroid hormone binding (THBR) was assessed by a competitive binding assay method (Division Baxter Diagnostics-Clinical Assays). Free T4 index (FT41) and free T3 index (FT31) were calculated by the product of n 4 x THBR and TT3 x THBR, respectively. Serum free T4 (FT4) concentrations were measured by 10 commercially available kits as reported in Table 1. The Amerlex-M free T4 RIA, Free T4 solid phase component system, RIA-coat FT 4, Coat-A-Count Free T4, Magic 1 Step FT 4 and Seria Free T4 kit, employ the so called "single step" method free hormone determination in which a 125 1 -T4 analogue, theoretically modified to prevent binding with serum proteins. The kit supplied by Ares-Serono utilizes the 125 1 -T4 analogue and an anti-T4 monoclonal antibody. The Riagnost FT4 and Gamma Coat 1251-FT4 kits utilize the so called "two step" assay for free thyroid hormone determinations. The unknown serum is added to the anti T4 antibody coated tube. Free T4 reacts with the binding sites of the antibody. The serum is then removed and 1251-T4 is added. The tracer binds to the unoccupied T4 binding sites on the antibody. With the Liso-phase FT 4 kit the separation of free and bound T4 fractions is obtained by Sephadex LH-20 column chromatography followed by direct RIA of free hormone. The Amerlex-MAB FT4 Kit employs a high specific activity 1251-labelled monoclonal antibody raised against T4 and uses a magnetic separation of free and antibody bound T4. Due to limitations of serum volume, serum free T3 (FT3) concentrations were measured with only eight commercially available kits. We have employed the following "single step" labeled analog methods:
Amerlex-M Free T3 RIA Kit, Free T3 Solid Phase Component System , RIA Coat FT3, Coat A Count Free T3, Magic 1 Step-FT3 and Seria Free T3. The RIA-gnost FT3 and the Gamma Coat 125 1_ T3 Radioimmunassay Kits, both "two step" methods were also employed (Table 1). The normal ranges, indicated by the manufacturers, for serum FT 4 and FT3 concentrations are reported in Table 1. For each determination, all serum samples were evaluated in the same assay, carefully following the procedures recommended by the manufacturers. Statistical analyses were conducted by paired and unpaired Student's t tests and by linear correlation, as appropriate. All the results are reported as mean±SE. RESULTS Total thyroid hormone concentrations; free thyroid hormone indexes and TSH concentrations
The intraassay coefficients of variation were: TT 4, 4.3%; TT3, 9.5% ; TSH 2.8%; and THBR, 1.9%. Serum n 4, n3, and TSH concentrations and THBR, FT41, and FT3 I values in nonpregnant women , in parturient women and their newborns are reported in Table 2. Serum n 4 concentrations in parturient women were significantly higher (unpaired Student's ttest, p
