A comparison of free thyroxine concentration and the free thyroxine index as diagnostic tests of thyroid function

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1 Ann Clin Biochem 1981; 18: A comparison of free thyroxine concentration and the free thyroxine index as diagnostic tests of thyroid function J W TUTTLEBEE AND R BRD From the Endocrine Laboratory, Department of Chemical Pathology, Whittington Hospital, London N 19 SUMMARY Serum free thyroxine concentrations, measured by the mmo Phase kit, and free thyroxine index values were compared in 200 subjects classified according to age, sex, and clinical diagnosis. The free thyroxine concentration was as good as the free thyroxine index in hyperthyroid, hypothyroid, elderly, and acutely ill patients and a better diagnostic index of thyroid status in pregnancy and in oral contraception. t is generally accepted that non-protein bound or free thyroxine concentration in serum (FT4) is the most accurate biochemical index of thyroid status. Nwnerous methods for the estimation of Ff4 in serum have been described. The estimation of FT4 concentrations by equilibrium dialysis was first described by Sterling and Hegedus! in 1962 and later simplified by the aid of magnesium precipitation." Dialysis procedures have been used in combination with ion-exchange chromatography, 3 gas chromatography.f and radioimmunoassay.s" Other methods of measuring FT4 in serum include the uptake of radioactive thyroxine by Sephadex,? ultrafiltration," and polyacrylamide gel filtration." These methods are not readily adapted to a routine estimation of Ff4, and published data reveal considerable variation in the values obtained. Recently, a nwnber of commercial kits have become available for the routine assay of FT4. n this study we have used the Corning mmo Phase kit. lo Clark and Horn'! developed the 'free thyroxine index' (FT) which has been shown to correlate with the absolute FT4 concentration.p However, in pregnancy, contraceptive pill users, patients with severe non-thyroidal disease, and the elderly, the FT may not always reflect the FT4 concentration because ofthe abnormal levels of thyroxine-binding proteins encountered.p We have compared FT4 and Ff in groups of subjects classified according to age, sex, and clinical diagnosis to assess the possible advantages of FT4 as measured by the Corning mmo Phase kit. 88 Subjects and methods The following tests of thyroid function were used: serum thyroxine concentration (T4) by radioimmunoassayusing a polyethyleneglycol separation-s; serwn thyrotrophic hormone concentration (TSH) by radioimmunoassay using a double antibody separation'<; serum triiodothyronine concentration (T3) using the T3RA PEG kit (Radiochemical Centre, Amersham); triiodothyronine-uptake (T3 uptake) using the Thyopac-3 kit (Radiochemical Centre, Amersham). The FT was calculated as T4/T3-uptake. Ff4 was assayed using the mmo Phase kit (Corning Medical, Essex). This method utilises a novel approach, the basis being that the binding of thyroxine to immobilised antibody is proportional to the FT4. The assay uses two tubes, and each tube receives the same volume of serum and T4 tracer, but one of the tubes also receives a protein blocker which displaces all thyroxine from the thyroxinebinding proteins. After incubation with tracer, an equal quantity of immobilised antibody is added to each tube, binding is terminated by centrifugation, and the supernatant liquid is decanted. The counts in the two tubes are designated counts A and B. The concentration of thyroxine bound to immobilised antibody in tube A is functionally related to the FT4 concentration in the sample. The function first suggested was the ratio A/B.lo However, Ekins 16 pointed out that A/B depends not solely on the free hormone concentration in the sample but also on the concentration of T4-binding proteins and their

2 A comparison offree thyroxine concentration and the free thyroxine index 89 binding constants. He suggested another function, A.tT4, the product of the counts in tube A and the total thyroxine as given by the counts in tube B. nthis study we have used Ekin's modification, which is now recommended by Coming. The between-batch precision for all assays is shown in Table 1. The between-batch coefficient of variation (CV) for the Ff4 assay was 11' 9% for the low control and 14 2 % for the high control. Both controls were included in each of the 14 assays on which this work was based. The mean withinassay CV was 11 1 %, calculated from duplicate values in each run. Subjects were classified according to age, sex, therapy, and clinical diagnosis as follows: Hyperthyroid 20 patients (3 men and 17 women, age range years). Fifteen of the patients were clinically hyperthyroid, the remainder were being investigated for weight loss with tachycardia. All had T3 levels greater than 4 nmol/l. Hypothyroid 14 patients (1 man and 13 women, age range years). All the patients were clinically hypothyroid and had TSH levels greater than 20 mu/1. Pregnant 32 women (age range years) equally divided between the second and third trimesters of pregnancy. Table 1 Between-assay precision for the five thyroid function tests FT4 (pmol//) T4 ("mol/) FT Mea" TJ ("mol//) TSH (mu//) CV%Mea" CV%Mean CV%Mea" CV%Mean CV% S S Contraceptive pill users 26 women (age range years). These were clinically euthyroid and currently taking one of the forms of oestrogen-containing oral contraceptive. Acutely ill 45 patients (19 men and 26 women, age range years). All had severe non-thyroidal diseases (eg congestive cardiac failure, myocardial infarction, severe diabetes, carcinoma, renal failure). Elderly 21 subjects (10 men and 11women, age range years). These were attending an outpatient health clinic as part of a geriatric screening programme. Euthyroid 62 volunteer hospital staff (32 men and 30 women, age range years). Bloodsamples were obtained by venepuncture; the serum was separated by centrifugation at 2500 rpm for 10 minutes and stored at -20 C before assay. With the exception of T3 uptake, all assays were performed in duplicate. Results Table 2 shows the mean value, one SD, and 2 SD range for T4, T3-uptake, Ff and Ff4 in the seven groupsof subjects studiedand indicates thosegroups in which mean values are statistically significantly different from that of the euthyroid group at the 5% level. The individual values for free thyroxine concentration and free thyroxine index are indicated in Figs 1 and 2 respectively. The means for Ff4 and Ff in the seven groups of patients are combined in Table 3. The number of patients in each group whose values lie outside the euthyroid 2 SD range is shown, both as number of patients and also as the percentage of each group. Table 2 Mean and one standard deviation values, with two SD range, for serum T4, T3-uptake, Fr, and Fr4 in seven clinicalgroups A.ssay Hyperthyroid Hypothyroid Prelf"/J"t 0" A.cutely 1/1 Elderly euthyroid contraceptives (20) (4) (32) (26) (45) (20) (62) T4 Mean S S (nmol/l) SO ]S Range ~ S S T3-uptake Mean S S (%) SO Range 64 S FT Mean 2S 6 29 S l1s SO 67 8 S O S S 13 3 Range 116 (}"'387 2 (}...S9 S 4S S 4-16S S FT4 Mean 4S (pmol/) SO S Range 2S 9~S S S 2 S S-27 9 S (}"'26 4 Mean values not statistically significantly different from the euthyroid mean.

3 iii: 90 Tuttlebee and Bird Table 3 FT4 and FTl compared in the seven groups ofsubjects studied Free thyroxine index Free thyroxine concentration (pmolill Group No Mean ± SEM Abnormal Mean ± SEM Abnormal Hyperthyroid 20 2S 6± (100%) 4S 70±2 7S 20 (100%) Hypothyroid S± (100%) 8 90± (100%) Pregnant ±S 3 9 (28%) 19 oo±0 S4 3 (9%) Contraceptives 26 lls 4± (39%) 22 80± (15%) Acutely ill ±3 3 9 (20%) 17 70±0 7S 13 (29%) Elderly ±3 0 0 (0%) 21 60± (0%) Euthyroid ± (S%) 20 20± (5%) Hyp!'fthyroid Hypo1hyroid Pregnancy Oral conrrace'ptive Acutely ill Eld.,ly Euthyroid Fig. 1 Distribution of free thyroxine values in seven groups ofsubjects. Hypolhyro;d ~nancy Oral conrroc!'pcive" Aculpty ill Eld~rly EuthyroKJ Fig. 2 Distribution of free thyroxine index values in seven groups of subjects. Discussion 10 ' i.&.-...!.. ~ :...i : : t,-. "";''1- ~... '.~... &au. \..!!, 1-.6""-1 1, ~-~,, :... l J...~..J.. i ~ -..;...».~.., 1 J i, 100 i - ', l,() Fret" thyroxine (pmolll) ".... '" 200 JOO F'E'e thyroxin!' index. ::: The present study was undertaken to establish the value of routine assays of Ff4 using the Corning mmo Phase kit and to compare it with Ff in groups of subjects with different concentrations of serum thyroxine-binding proteins. The main T4-binding protein is thyroxine-binding globulin (TBG), which binds about 70% of the total > T4, the rest being bound to albumin, pre-albumin, and other T4-binding proteins. The concentration of TBG may be influenced by hormones, drugs, disease, and genetic factors.l" ncreased TBG concentration is encountered during pregnancy and in women taking oestrogen-containing contraceptives. The commonly used FT may be misleading in patients with abnormal concentrations of binding proteins although designed to compensate for such abnormalities.pr'" The value of measuring serum TBG concentration as a routine test of thyroid function has been reported by Burr,21 McDowell,22 and Leeureuil.P Burr and McDowell found that the derived index T4:TBG ratio correlated better with the thyroid state than the Ff. However, the calculation of the T4:TBG ratio makes no allowance for changes in the serum albumin and pre-albumin concentrations, which together bind about 30% of serum thyroxine. Lecureuil found that the T4:TBG ratio is inaccurate when the TBG concentrations are high or low and concluded that FT4 was better for differentiating between euthyroid subjects with normal, high, or low TBG concentrations and those with thyrotoxicosis and myxoedema. We found that, in hypothyroidism, hyperthyroidism, and in the elderly, Ff and Ff4 agreed equally well with thyroid status. Non-thyroidal illness is common in geriatric patients, and many clinically euthyroid patients with severe chronic non-thyroidal illness have abnormal thyroid function tests. n our series, this group was made up of nonhospitalised patients attending an outpatient geriatric clinic, and this may account for the fact that both the FT and Ff4 were within normal limits. Bayer and McDougal 24 estimated Ff4 by radioimmunoassay involving antibody-coated tubes used in the GammaCoat kit (Clinical Assays, Norfolk), and found this method to be superior to other thyroid function tests in patients with severe nonthyroidal illnesses. However, in our studywe obtained a similar distribution of values for both the FT and Ff4 in this group of patients. The relative concentrations of TBG, albumin, and pre-albumin will

4 A comparison offree thyroxine concentration and the free thyroxine index 91 depend on the nature of the underlying condition and could account for the wider spread of values obtained in this group compared with the euthyroid subjects. t might be expected that a derived index like FT would be less sensitive than FT4. Table 3 shows that out of 45 acutely ill patients FT was within the reference range in 36 and FT4 in 32. t is interesting to note that the total T4 concentration was within the reference range in 31 patients. There was agreement between the three tests in 30 patients. n the remaining 15, the total T4 was outside the reference range in nine, the FT4 in eight, and the FT in four. n the acutely ill patients we found the total T4 to provide a good screening test, and there appears to be no advantage in measuring 'free' thyroxine by whichever test in every patient. Five of the patients in which the total T4 was low had renal failure, and this could be accounted for by low T4-binding capacity. Neither FT4 nor FT had any outstanding advantages in the diagnosis of suspected hypothyroidism in acute illness, and both would need to be supplemented by the assay of serum thyrotrophin (TSH). Normal values in suspected hyperthyroidism would need to be supplemented by the assay of serum T3 to exclude the possibility of T3-thyrotoxicosis. Probably the most sensitive index of hyperthyroidism in this group of patients is the inhibition of TSH response to thyrotrophin releasing hormone (TRH). n pregnancy we found no significant difference in the mean values for FT4 and Ff in the second and third trimesters. However, Ff4 appeared to be better in correcting for raised T4-binding in both pregnancy and contraceptive pill user groups. The bettercorrelation offf4 with thyroid status may be accounted for by the fact that the mmo Phase method is based on the equilibrium of T4 only, and the calculation of the Ff requires a T3-uptake test. This test only indirectly reflects overall binding of T4 by thyroxine-binding proteins because prealbumin binds little or no T3. 8 No raised Ff4 values were found in pregnancy although four raised values were observed in the oral contraceptive group. The Ff4 value obtained by the mmo Phase method depends on assumptions made in the calculation, and the calculation itself has been the subject of discussion.t'' Further clinical evaluation will be necessary before the full value and limitation of this method are known. t has been shown that the GammaCoat (Clinical Assays, Norfolk) and the Lepetit (Uniscience Ltd, Cambs) kits for the estimation offt4 give comparable results.p On the basis of the present study it appears that the chief value of the FT4 assay would be in the diagnosis of thyrotoxicosis in pregnancy. t correlated better with the thyroid status than F in the oral contraceptive user group, but in other groups studied FT4 did not have any obvious advantage over the estimation of total T4 and F supplemented by the assay of serum T3 or TSH to confirm the presence or absence of hyperthyroidism or hypothyroidism respectively. The procedures adopted will depend on the individual laboratory and the facilities available. n a laboratory using an automated method and preparing their own reagents, the cheapest test is total T4 concentration. This test can be supplemented with FT in selected cases. The estimation of FT using Thyopac 3 can be carried out in 2 hours and costs about 1 per test. n a laboratory using a nonautomated test method for total T4, the mmo Phase kit has the advantage that total T4 and FT4 can be calculated for each test if required; but, in our experience, the precision was less satisfactory than that of F. The cost per test is comparable to that of FT, and a test can be carried out in 4 hours. Both methods are technically straightforward. References 1 Sterling K, Hegedus A. Measurement offree thyroxine concentration in human serum. J Clin nvest 1962; 41: Sterling K, Brenner MA. Free thyroxine in human serum: simplified measurement with the aid of magnesium precipitation. J Clin nvest 1966; 45: 155--{i3. Bird R, Abiodum MO. Measurement of serum percentage free thyroxine by equilibrium dialysis and ion-exchange chromatography. Clin Chim Acta 1973; 48: Petersen BA, Giese RW, Larsen PR, Karger BL. Measurement of free thyroid hormones in serum by dialysis and gas chromatography. Clin Chem 1977; 23: Ekins RP, Ellis SM. The radioimmunoassay of free thyroid hormones in serum. Proceedingsofthe Seventh nternational Thyroid Conference, Boston, 1975: Jiang N, Tue K. Determination of free thyroxine in serum by radioimmunoassay. Clin Chem 1977; 23: Levinson SS, Rieder SV. Parameters affecting a rapid method in which Sephedex is used to determine the percentage of free thyroxine in serum. Clin Chem 1974; 20: Thorson SC, Wilkins GE, Schaffrin M, Morrison RT, Mcntosh HW. Estimation of serum free thyroxine concentration by ultafiltration. J Lab Clin Med 1972; 80: McDonald LJ, Robin N, Siegel L. Free thyroxine in serum as estimated by polyacrylamide gel filtration, Clin Chem 1978; 24: 652--{i. 10 Lidgard GP. Free T4: a new advanced RA assay. Med Lab World 1978: 5:

5 92 11 Clark F, Hom DB. Assessment of thyroid function by the combined use of the serum protein bound iodine and resin uptake of J181-T3. J Clin Endocrinol Metab 1965; 25: Wellby ML, O'Halloran MW, Marshall J. A comparison of effective thyroxine ratio, free thyroxine index and free thyroxine concentration in correcting for thyroxine binding abnormalities in serum. J Clin Endocrinol Metab 1974; 3: Felicetta J, Green WL. Value of free thyroxine index. N Engl J Med 1980; 302: & Nye L, Hassan M, Willmott E, Landon J. ntroduction of a rapid, simple radioimmunoassay and quality control scheme for thyroxine. J Clin Pathol 1976; 29: Hall R, Amos J, Ormston BJ. Radioimmunoassay of human serum thyrotrophin. Br Med J 1971; 1: Ekins R. Commercial radioimmunoassay for free thyroxine. Lancet, 1979; 1: Oppenheimer JR. Role of plasma proteins in the binding, distribution and metabolism of thyroid hormones. N Engl J Med 1968; 278: Premachandra BN, Gossain VV, Perlstein 1M. ncreased free thyroxine in a euthyroid patient with thyroxine binding globulin deficiency. J Clin Endocrinol Metab 1976; 42: Sheridan P, Newton KE, Payne RD. nterpretation of serum total thyroxine concentrations in patients Tuttlebee and Bird with abnormal thyroxine binding proteins. Br Med J 1978; 1: 477. '0 Roosdorp N, Joustra M. A numerical comparison of the use of T3-uptake values and of thyroxine binding globulin levels for the estimation of free thyroxine in serum. Clin Chim Acta 1979; 98: Burr WA, Ramsden DB, Evans SE, Hogan T, Hoffenberg R. Concentration of thyroxine binding globulin: value of direct assay. Br Med J 1977; 1: McDowell DR. An evaluation of serum thyroxine binding globulin as a routine test of thyroid function. Ann Clin Biochem 1979; 16: Lecureuil M, Cronzat-Reynes G, Bernard J, Choffel C. Correlation of free thyroxine index and thyroxine: thyroxine binding globulin ratio, with the free thyroxine concentration as measured by the thyroxine and thyroxine binding globulin radioimmunoassays. Clin Chim Acta 1978; 87: Bayer MF, McDougall R. Radioimmunoassay of free thyroxine in serum: comparison with clinical findings and results of conventional thyroid function tests. Clin Chem 1980; 26: Tuttlebee JW. Assay of free thyroxine in serum: a preliminary comparison of three commercial kits. Med Lab Sci 1981; 38: Acceptedfor publication 29 January 1981