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Thyroid Function Tests in Acutely Ill Patients. Comparison of Analogue Based Free Thyroid Hormone Assays with Free Thyroxine Index Russell R. Cooke & R. Pratt To cite this article: Russell R. Cooke & R. Pratt (1986) Thyroid Function Tests in Acutely Ill Patients. Comparison of Analogue Based Free Thyroid Hormone Assays with Free Thyroxine Index, Pathology, 18:1, 94-97 To link to this article: http://dx.doi.org/10.3109/00313028609090834
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Pathology (1986), 18, pp. 94-97
THYROID FUNCTION TESTS IN ACUTELY ILL PATIENTS. COMPARJSON OF ANALOGUE BASED FREE THYROID HORMONE ASSAYS WITH FREE THYROXINE INDEX RUSSELLR. COOKE AND R. PRATT Division of Chemical Pathology, Department of Laboratory Services, Wellington Hospifal
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Summary A prospective study of 100 acutely ill patients was carried out to assess the value of the free thyroxine (FT4) assay as a replacement screening procedure for the free thyroxine index (FTI). We found that the FT4 assay was significantly influenced by the albumin concentration, so that the number of follow-up tests required increased markedly. This was especially true at the low end of the FT4 range where the need for thyrotropin assays increased by 162%. The free triiodothyronine (FT3) assay was also shown to be albumin dependent. It is not useful to replace one set of difficulties due to protein binding with another, and overall it was concluded that it is not cost-effective to screen hospital patients for thyroid dysfunction using free hormone assays based on labelled analogue techniques. Key words: Thyroid function, free and bound hormone, albumin, screening, free thyroxine, free triiodothyronine
Accepted August 12, 1985
INTRODUCTI0N In recent years there has been an increasing trend towards screening patients for thyroid disorders with a direct FT4 measurement rather than the FTI. Most laboratories using FT4 methods use kits that depend on “labelled analogue” methods. It is claimed that this technique provides a direct measurement of FT4 because the thyroxine analogue used as a tracer binds only to a high affinity anti-T4 antibody and not to thyroxine binding globulin (TBG) or any other binding protein.’ As a single procedure for thyroid screening the FT4 could make a considerable saving in laboratory costs possible. However, the labelled analogue method has been criticized. Specimens from patients receiving heparin,2 from those with thyroxine a u t ~ a n t i b o d i e s ,and ~ with familial dysalbuminemic hyperthyr~xinema~ all give false results, though there are only a small number of patients in these categories. Possibly of greater importance is the observation4 that low FT4 results are seen in patients with severe non-thyroidal illness (euthyroid sick syndromeESS). It is well known that thyroid function tests are altered during severe illness,6 and it has been claimed that apart from any physiological disturbance, analogue tracer methods will give low results due t o the nature of the analogue. Amino et al.’ have demonstrated a dependence of some free hormone measurements on the serum albumin concentration. However, follow-up tests such as
thyrotropin (TSH) and triiodothyronine (T3) assays are available to clarify this situation. It is claimed by Vermaak et al.’ that a large increase in follow-up tests was required on the introduction of a FT4 method. This observation has been supported by some and disputed by others.”*” We therefore decided to take blood samples from 100 patients admitted to the medical, psychiatric and geriatric wards of a large teaching hospital and analyze the samples using both FTI techniques and FT4 methods. Thus we were able to compare the follow-up rates using the 2 methods of thyroid screening. T3, TSH and albumin measurements were also carried out on each sample.
PATIENTS AND METHODS Patien is All medical, geriatric and psychiatric patients admitted acutely over a 3 wk period were used in this study. Each patient had venous blood taken within 48 h of admission. A brief medical history was taken and an examination performed to exclude clinically obvious thyroid disease. One geriatric patient was subsequently proven to be hypothyroid and was excluded. Patients were subdivided into groups according to the nature of their illness (Table I ) . Thirty-two healthy laboratory workers (age range 17-58 yr) were used as a control group. All samples were centrifuged, aliquoted, and the serum was frozen at -20°C until analyzed.
Methods Total thyroxine was measured with the Abbott T4 RIA(PEG) kit (Abbott Laboratories, Diagnostic Division, North Chicago, 1L 60064, USA) and was multiplied with a T3 uptake result (Abbott Triobead) to give the FTI. In our laboratory the T3 uptake test is referred t o as the TBG binding ratio, or TBR. The reference interval for both the total T4 and FTI was 60-160 nmol/I. Criteria for doing follow-up tests were as follows:(1) I f FTI <76 then TSH assay (2) I f FTI >154 then T3 assay FT4 and FT3 were measured in all samples with Amerlex kits (Amersham International Ltd., Amersham, Bucks., UK). T3 was measured with an NML kit (Nuclear-Medical Laboratories, Irving, Texas 75061, USA) and TSH by an in-house double-antibody RIA. Albumin was measured by a Bromocresol purple dye binding method using a n Abbott VP bichromatic analyzer.” In a separate experiment we attempted to confirm the observation of others’ and extend it to FT3 measurements. Serum was obtained from 2 patients who had low albumin levels. The serum was aliquoted and various amounts of crystalline, lyophilized human serum albumin (Lot 108C-8085; Sigma Chemical Company, St Louis, MO 63178 USA) were weighed in. We measured the resultant FT4 and FT3 levels.
FT4 AND FTI IN ACUTELY ILL PATIENTS
Number of Patients in each Diagnostic Group
Diagnostic Group Obstructive airways disease Acute Psychiatric Gastrointestinal Bleeding Acute Geriatric Chest Pain Cardiac arrhythmias Stroke Heart failure Infections Others Normals
Number 12 10 4 10 20 4 5 6 12 16 32
TABLE2 Thyroid function and albumin results from euthyroid sick syndrome patients compared with results from normal subjects
Patients Mean (SEM) 99 109.1 (2.6) nmol/l 1.04 (0.01) 112.4 (2.7) 1.7 (0.04) nmol/l 1.44 mU/I 15.7 (0.5) pmol/l 4.82 (0.13) pmol/l 36.45 (0.53) g/l
Number T4 TBR FTI T3 TSH FT4 FT3 Albumin
Normals Mean (SEM) 32 11 I .8 (3.2) nmol/l 1.07 (0.02) 119.2 (3.3) 2.3 (0.06) nmol/l* 1.53 mU/I 15.5 (0.4) pmol/l 7.21 (0.17) pmol/l* 44.38 (0.50) g/l*
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RESULTS Significant differences (p <0.001) were observed between the patient group and the normal subjects for T3, FT3 and albumin concentrations. The T4, TBR, FTI, TSH and FT4 values were not significantly different (Table 2). Among the various patient groups the mean values for the parameters were similar. The only exception to this was the psychiatric group in which the mean T4 value was higher than other patient groups (p <0.001). We have not had sufficient experience with the FT4 assay to generate our own follow-up criteria. We therefore based our criteria on those from another hospital who had used the Amersham assay for 2 yr. These we modified slightly at the low end of the range, so that the percentage of the reference interval covered by the follow-up range was the same for both FT4 and FTI. The criteria used were: If FT4>20.0 then T3 assay If FT4< 12.0 then TSH assay The reference interval for FT4 was 10-23 pmol/l. When the FTI was used as the initial screening test, follow-up tests were required on 17 of the 99 patients (17.2%), of which 8 were a TSH assay and 9 a T3 assay (Table 3). When we used FT4 as the screening procedure 33 patients required follow-up tests (12 T3 and 21 TSH assays). The overall number of follow-up tests indicated by the FT4 assay results was 94% greater than those indicated by the FTI assay results. Nearly all of this increase was due to a larger number of patients with low FT4 values. As a group, the patients with FT4 values of less than 12 pmol/l had a mean albumin level that was significantly less (p <0.001) than the mean value for the remainder of the patients. The mean albumin values in those patients with an FTI of less than 75 showed no significant difference (p > 0.2) from the remainder. The number of results that fell outside the reference intervals was similar for each method: 7 for the FTI method and 12 for the FT4 method.
We then isolated a group of patients whose T3 value was less than 1.5 nmol/l, the lower end of our reference interval. These we called the serious euthyroid sick syndrome patients (SESS). These patients were mainly from the groups with infection, arrhythmia and heart failure. Of the SESS group, 6 were followed up after an FTI screen, and 14 with the FT4. In this group we again observed that using the FT4 as a screen doubled the number of follow-up assays that were required. Of the 25 patients with a low T3 value, 16 also had low albumin values, which confirmed the association between sickness, low T3 and low albumin values. We next considered those patients with an albumin level lower than the reference interval (male 38-48 g/l, female 34-46 gA). These numbered 38 and were significantly older (p <0.001) than the patients with normal albumin concentrations (Mean age 69.8 yr vs 48.5 yr). Twelve patients required follow-up tests after the FT4 assay, but when FTI was the screening test, only 3 patients required a follow-up assay. Thus in this group of hypoalbuminemic patients a 300% increase in TSH follow-ups occurred. We compared the 2 groups and found significant differences (p <0.01) in the mean values for T4, T3, FT4 and FT3, but not for the mean FTI values (Table 4). Ten patients with an albumin concentration of less than 30 g/l were investigated further. Follow-up TSH determinations were required in 7 of those screened with FT4 but only in 3 when FTI was the initial test. There were significant differences (p <0.05) for the variables age, FT4, FT3 and T3, but not T4 or FTI when we compared this low albumin group with all the other cases. The apparent dependency of the directly measured free hormone level on the albumin concentration was investigated further by performing correlation analysis on our patient results. Theoretically the FT4 concentration should not be dependent on the albumin concentration. TABLE 3 Number of follow-up assays required after initial thyroid tests
Initial Thyroid Test FTI FT4
T o T3 follow
Total Follow ups 17
Pathology (1986). 18, January
TAULE 4 comparison of Control and Hypoalbuminemic Patients. (Units are the same as in Table 2.)
Hypoalbuminemic patients Mean (SEM)
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Number Age T4 TBR FT I T3 TSH FT4 FT3
38 69.76 (1.91) 101.9 (4.3) 1.07 (0.02) 107.8 (4.37) 1.5 (0.1) 1.72 14.2 (0.6) 4. I 1 (0.2)
Control patients Mean (SEM) 93 48.55 (2.32)** 113.0 (2.4) 1.04 (0.01) 116.6 (2.52) 2.0 (0.04)** 1.36 16.2 (0.4)* 5.94 (0.15)**
However, we would expect the FT3 values to show strong correlation with the albumin values as both FT3 and albumin concentrations decline as severity of illness increases. If we correlate patients’ FT4 with their albumin concentrations, the correlation coefficient (R) is 0.41 (p <0.00005), whereas the correlation of FTI with albumin is 0.19 (p >0.01). If the FT4 measurement itself were not dependent, at least in part, on the albumin concentration, we would have expected the same R values. They are, however, very different. A partial correlation of FT4 with albumin, controlling for FTI, gives a correlation coefficient of 0.39 (p <0.001). This again emphasizes t he dependence of t he FT4 measurement on the albumin level. We tested the in vitro effects of albumin on the measured FT4 value by repeating an experiment of Amino et al.5 We extended it further to examine the effects of albumin on FT3 levels. The results of these experiments are summarized in Table 5. The apparent FT4 and FT3 concentrations rose as the albumin concentrations increased while the T4 and FTI remained essentially the same. As the albumin concentration changed to enter the reference range, the measured FT4 and FT3 values moved from the hypothyroid to the euthyroid range. Regressions on these data gave the following equations (with their associated Coefficients of Determination (CD)). Patient 1 FT4 = 0.22 [ALB] + 2.09 CD = 0.99 Patient 2 FT4 = 0.33 [ALB] + 2.98 CD = 0.99 Patient 1 FT3 = 0.07 [ALB] + 1.39 C D = 0.98 Patient 2 FT3 =0.07 [ALB] + 0.7 C D = 0.99 One can observe that the slopes for different patients were similar for each assay. T o eliminate the possibility TABLE5
“In Vitro” Variation of FT4 and FT3 with Albumin
16 27 34 40 45 14 23 32 36
2.5 3.2 3.5 4.2 4.4 1.6 2.3 3.0 3.2 3.4
49 55 52 56 56 69 72 75 74 75
52 62 58 64 62
8.5 9.7 10.9 12.2 7.3 10.7 13.6 14.9 16.0
87 94 95 93
that these results were caused by contamination of the albumin with thyroxine, we measured the apparent FT4 level in O.05M phosphate buffer containing 10 and 30 g/l of the albumin. N o FT4 was detectable.
DISCUSSION We have shown that the albumin concentration substantially affects FT4 and FT3 levels, both in vivo and in vitro, when labelled analogue techniques are used. This is of practical significance since follow-up assays are doubled if FT4 is used as the initial screening test. When comparable follow-up criteria for the two screening tests were employed, the FTI screen resulted in 8 follow-up TSH assays, while the FT4 caused 21, an increase of 162.5%. Patients who required a TSH after the FT4 screen, had mean albumin levels significantly different from the rest. This difference was not observed when the groups were separated according to the FTI, and analysis of the statistical dependency of this result showed a strong correlation with albumin concentration. This was true even when we controlled for the FTI result. The number of follow-up assays increased significantly when we adopted a FT4 screen, and the majority of the additional tests were associated with low albumin concentrations. This finding confirms the retrospective data of Vermaak et al.’ but disagrees with the findings of Mardell & Gamlen. None of these observations are conclusive evidence that the apparent FT4 levels are dependent on the albumin concentration but collectively they make a strong case that this is so when FT4 is measured by the labelled analogue method. We have confirmed this apparent albumin dependence and extended the results to the FT3 assay by in vitro studies. When albumin was added to serum and the resultant changes in thyroid function tests were measured, the FTI result remained constant while both of the free hormone measurements showed a linear dependence on the albumin levels. We assume that this albumin dependency resulted from the analogue binding to the albumin and thus affecting the amount of tracer available for participation in the immuno-reaction. It is recommended that the thyroid status of acutely ill patients be not routinely investigated, but this is not always possible. Thyroid dysfunction may be the cause of the presenting disorder and in such cases thyroid function tests are necessary. It is important that as few patients as possible be misdiagnosed as a result of the initial laboratory assessment: a laboratory test that compounds the problem rather than clarifying it should therefore be avoided. We have shown by prospective analysis of acutely ill patients that low albumin concentrations interfere with both FT4 and FT3 assays based on labelled analogues. This difficulty leads to a significant increase in the number of follow-up assays and therefore the cost. The problem appears linearly related to the albumin concentration and a correction factor could be derived (as with FTI or calcium), but this would defeat the purpose of free hormone assays. Any new test must have clear advantages over the test or tests it is seeking to supplant.
FT4 A N D FTI IN ACUTELY ILL PATIENTS
The free hormone assays based on labelled analogues fail to fulfil this basic criterion.
4. Stockigt JR, De Garis M, Csicsmann J et al. Limitations of a new free thyroxine assay (Amerlex Free T4). Clin Endocrinol 1981; 15: 313-8.
ACKNOWLEDGEMENTS To Dr H . C. Ford and Dr M. J. Crooke, Chemical Pathology, Wellington Hospital, for their helpful comments on the paper and to John Speed of Waikato Hospital for his sharing of two years experience of the FT4 assay with us.
5 . Amino N, Nishi K, Nakatani K et al. Effect of albumin
Address for correspondence: R.R.C., Division of Chemical Pathology,
Department of Laboratory Services, Wellington Hospital, Wellington, New Zealand
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SIR SAMUEL GARTH AT THE KIT KAT CLUB ...Garth immediately pulled out his list, which amounted to fifteen, and said: “It’s no great matter whether I see them to-night or not, for nine of them have such bad constitutions that all the physicians in the world can’t save them, and the other six have such good constitutions that all the physicians in the world can’t kill them.” John Timbs. Doctors and Patients. R. Bentley & Sons. 1873. Vol. 2. p.279.