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Canadian Journal of Veterinary Research logoLink to Canadian Journal of Veterinary Research
. 2004 Apr;68(2):98–104.

Cortisol and free thyroxine determination by time-resolved fluorometry in canine serum

MD Parra 1, LJ Bernal 1, JJ Cerón 1,✉
PMCID: PMC1142152  PMID: 15188953

Abstract

Validation for canine serum of 2 commercially available time-resolved fluoroimmunoassays (TR-FIAs) designed for analysis of cortisol and free thyroxine (fT4) in human serum was carried out. Included was the study of interference by hemolysis, lipemia, and bilirubinemia. With the dissociation enhancement lanthanide fluoroimmunoassay kits, the intraassay coefficient of variation (CV) ranged from 6.4% to 8.7% for cortisol and from 5.3% to 9.8% for fT4; the interassay CVs ranged from 5.8% to 10.8% and from 3.9% to 14.1%, respectively. Accuracy was evaluated by comparing cortisol and fT4 results obtained with TR-FIA and those obtained with a validated enzyme-linked immunosorbent assay (ELISA) and an equilibrium dialysis (ED) assay, respectively. The regression equations obtained were y = 0.57x + 1.18 (r2 = 0.90) for cortisol and y = 0.87x + 0.82 (r2 = 0.93) for fT4. The limits of detection for cortisol and fT4 were 4.84 nmol/L and 2.68 pmol/L, respectively. The results of adrenocorticotropin-stimulation and dexamethasone-suppression tests were similar to those published previously; likewise, serial dilution of a canine serum sample with a high cortisol content demonstrated that the TR-FIA was immunologically specific. Serial dilution of a serum sample with a high fT4 concentration showed a methodologic bias, a dependence on serum binding capacity, which indicates that the results obtained with this method should be interpreted with caution. Finally, hemolysis and lipemia significantly interfered with cortisol and fT4 measurements, whereas bilirubinemia did not affect the results.

Introduction

Canine hyperadrenocorticism (Cushing’s syndrome) is caused by excessive production of cortisol by the adrenal cortex. Hypoadrenocorticism, which is relatively uncommon in dogs, results from deficient production of glucocorticoid, mineralocorticoid, or both. Definitive diagnosis of both disorders depends on cortisol measurement in serum samples in the course of several screening tests, such as the dexamethasone-suppression test or the adrenocorticotropin (ACTH)-stimulation test for the 1st disorder and the ACTH-stimulation test for the 2nd disorder (1).

Hypothyroidism is one of the most frequent endocrinopathies in dogs. Reliable biochemical tests are required for its diagnosis. Currently, combined measurements of the concentrations of serum thyroid-stimulating hormone (TSH) and total thyroxine (tT4) or free T4 (fT4) are recommended (2). However, determination of the fT4 concentration in canine serum samples has several advantages over traditional measurements of the tT4 concentration: overlap of fT4 concentrations between euthyroid and hypothyroid dogs is less frequent than overlap of tT4 concentrations (3), the fT4 concentration is not as likely as the tT4 concentration to be affected by nonthyroidal illness or drug therapy (4), and measurement of the fT4 concentration as a test for hypothyroidism has higher sensitivity (0.98), specificity (0.93), and accuracy (0.95) than measurement of the tT4 concentration, which has a sensitivity of 0.89, a specificity of 0.82, and an accuracy of 0.85 (5).

Time-resolved fluoroimmunoassay (TR-FIA) is a novel alternative method to the traditional tests. It has been successfully used to measure the concentrations of several human and animal hormones, including insulin (6), human chorionic gonadotropin (hCG) (7), androstenone (8), relaxin (9), and cortisol (10). One of the recent commercial applications of TR-FIA is dissociation enhancement lanthanide fluoroimmunoassay (DELFIA), which combines purified antigens or monoclonal antibodies labelled with lanthanide chelates that have a long fluorescence decay time (europium, samarium, or terbium), and a particular time-resolving fluorometer for nanosecond measurements (11). This technique offers the advantages of high sensitivity, stability of the reagents, lack of radiation, low background interference, and a wide test range (12,13).

The purpose of this study was to validate for canine serum 2 commercially available TR-FIAs for the determination of cortisol and fT4 concentrations in human serum: DELFIA cortisol and DELFIA fT4 kits (PerkinElmer Life Sciences, Wallac Oy, Turku, Finland). Additionally, we studied the effect of increasing concentrations of hemoglobin, lipids, and bilirubin on cortisol and fT4 measurements, since knowledge of these effects is critical for the correct interpretation of laboratory data (14).

Materials and methods

Methodology

In DELFIA, specific binding reagents (antibodies or antigens in TR-FIA) are labelled with nonfluorescent chelates, and fluorescence is developed after the binding reaction has taken place on a solid phase by the use of a dissociative fluorescence-enhancement solution (15).

In the cortisol assay tested in this study, there is a solid phase coated with streptavidin, which binds biotinylated antibodies. During a 1st incubation period on a plate shaker (Titramax 100, Heidolph Instruments, Schwabach, Germany), europium-labelled cortisol and the sample cortisol compete for a limited number of binding sites on cortisol-specific biotinylated monoclonal antibodies. Buffer and serum are then washed away with a DELFIA plate washer (Wallac), and an enhancement solution is added. Europium ions are dissociated from the labelled cortisol into the enhancement solution, where they form highly fluorescent chelates with components of the solution. The amount of fluorescence is inversely proportional to the quantity of cortisol in the test sample.

With the fT4 kit, anti-T4 monoclonal antibody is first reacted with a solid phase coated with anti-mouse IgG. Then the sample is added, and the fT4 reacts with the anti-T4 antibody. After this 2nd incubation, buffer and serum are washed away. In a 3rd incubation step, europium-labelled T4 is added; it occupies the remaining empty binding sites on the anti-T4 antibodies. As with the cortisol assay, after washing steps, an enhancement solution is added to dissociate the europium from the labelled T4. The amount of resulting fluorescence is inversely proportional to the amount of fT4 in the test sample.

The enhanced fluorescence was measured in a VICTOR 1420 multilabel counter (PerkinElmer, Wallac).

Animals

For the cortisol study, we used 10 adult male German Braco dogs, supplied by the Animal Center of Murcia University. The animals were determined to be healthy on the basis of physical and clinicopathological findings. For validation of the fT4 DELFIA kit, we used 20 random-source dogs of different breeds and sex; 12 were clinically healthy, 7 had hypothyroidism, and 1 had hyperthyroidism.

Blood samples were collected by venipuncture into Tapval tubes (Aquisel, Barcelona, Spain), allowed to clot at room temperature, and centrifuged at 3000 × g for 10 min. The serum samples obtained were stored at −20°C until analyzed.

Validation

Precision

For cortisol, intra-assay variation, expressed as the coefficient of variation (CV), was determined by analyzing in quadruplicate in the same assay 4 serum samples obtained after an ACTH-stimulation test and 4 samples with normal levels of cortisol. For fT4, intra-assay variation was assessed from differences between duplicate determinations on 18 canine samples: 6 with low, 6 with medium, and 6 with high fT4 content. For cortisol, interassay variation was assessed by analyzing the same 8 samples as used for assessing intra-assay variation on 5 d within 3 wk. For fT4, interassay variation was assessed by the use of 6 samples, 2 with low, 2 with medium, and 2 with high concentrations of hormone, on 5 d within 3 wk. Samples were kept frozen at −20°C between the assays.

Accuracy

For the DELFIA cortisol kit, the results for 10 samples of canine serum (1 with a low concentration, after administration of dexamethasone; 4 with basal concentrations, before administration of ACTH; and 5 with high concentrations, after administration of ACTH) were compared with those obtained with a previously validated ELISA (Enzymun-Test; Boehringer Mannheim, Ingelheim, Germany) (16). For the DELFIA fT4 kit, the results for 20 samples of canine serum were compared with those provided by a validated equilibrium dialysis (ED) assay (Nichols Institute Diagnostics, Bad Vilbel, Germany) (2).

Limit of detection

For both kits, the limit of detection, defined as the smallest concentration of analyte detectable by the method, was calculated by repeated analysis (n = 10) of the zero standard (cortisol- and fT4-free human serum) and expressed as the mean plus 2 standard deviations (s) (17).

Specificity

Immunologic specificity was evaluated by comparing parallelism of the standard curves obtained with the kits and the curves obtained from serial dilutions of canine samples with high concentrations of cortisol and fT4. A special diluent, DELFIA diluent I (Wallac), was used. Biologic specificity was tested only for cortisol, by the use of previously described dynamic tests (16), ACTH stimulation and low-dose dexamethasone suppression (LDDS), performed in 10 healthy dogs in different weeks to permit recovery to basal cortisol levels. Baseline blood samples were obtained. To avoid variation due to circadian rhythm, we conducted the tests first thing in the morning.

Effects of hemolysis, lipemia, and bilirubinemia

The effects of these interfering processes on cortisol and fT4 determinations were assessed following previously reported protocols for lipemia and bilirubinemia (14,18). Hemolysis was produced by freezing red cells at −20°C in order to not incorporate distilled water into the assay. For both assays, the hemolysate was added to pooled serum at final concentrations of 0.0, 0.25, 0.5, 1.0, 2.0, and 4.0 g/dL. A commercial emulsion of triglycerides (Lipofundina MCT/LCT 20%; Braun Medical, Barcelona, Spain) was added to homologous pooled serum samples at final concentrations of 0.0, 31.2, 62.5, 125, 250, 500, and 10 000 mg/dL. Bilirubin (Sigma-Aldrich, Spain) was dissolved in dimethylsulfoxide (DMSO) and the solution added to pooled serum samples at final concentrations of 0, 3.75, 7.5, 15, 30, and 60 mg/dL. For the hemoglobin and lipid series, DELFIA diluent I was used rather than DMSO.

Statistical analysis

The data were analyzed by standard methods. The significance level was set at 0.05. Analysis of variance was used. Precision data were reported as mean ± s. Simple regression analysis was used to compare results obtained by TR-FIA with those obtained by ELISA and equilibrium dialysis (ED).

Results

Intra- and interassay CVs for the cortisol and fT4 DELFIAs appear in Table I. With respect to intra-assay variation, samples with a normal concentration of cortisol had a CV of 6.4%, whereas samples with a high concentration of cortisol had a CV of 8.7%. For fT4 the CVs ranged from 5.3% for samples with a low concentration of this hormone to 9.8% for samples with a medium concentration. With respect to interassay variation, samples with a medium concentration of cortisol had a CV of 5.8%, whereas samples with a high concentration had a CV of 10.8%. Samples with a high fT4 concentration had the lowest CV (3.9%), and samples with a medium concentration had the highest CV (14.1%).

Table I.

Intra-assay and interassay precision of cortisol and free thyroxine (fT4) concentrations in canine serum as determined by time-resolved fluoroimmunoassay (TR-FIA)

Coefficient of Variation (%)
Hormone and sample Concentration, mean ± s Intra-assay Interassay
Cortisol, nmol/L (n = 4)
 Before ACTH given 31.7 ± 2.0 6.4 5.8
 After ACTH given 158.2 ± 13.8 8.7 10.8
fT4, pmol/L (n = 6)
 Low concentration 7.6 ± 0.4 5.3 7.0
 Medium concentration 22.6 ± 2.6 9.8 14.1
 High concentration 98.1 ± 8.5 7.5 3.9
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s — standard deviation; ACTH — adrenocorticotropin

On comparing the results of TR-FIA for cortisol and fT4 with those obtained by ELISA and ED, the following regression equations were obtained: for cortisol, y = 0.57x + 1.18, where y = TR-FIA result (nmol/L) and x = ELISA result (nmol/L) (Figure 1); and for fT4, y = 0.87x + 0.82, where y = TR-FIA result (pmol/L) and x = ED result (pmol/L) (Figure 2). Measurements obtained with TR-FIA were highly correlated with those provided by ELISA (r2 = 0.90) and ED (r2 = 0.93).

Figure 1.

Figure 1

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Correlation between the results of time-resolved fluoroimmunoassay (TR-FIA) and enzyme-linked immunosorbent assay (ELISA) in the measurement of cortisol concentrations in serum samples from 10 dogs.

Figure 2.

Figure 2

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Correlation between the results of TR-FIA and equilibrium dialysis (ED) in the measurement of free thyroxine (fT4) concentrations in serum samples from 20 dogs.

The sensitivity, or limit of detection, calculated as twice the s from the mean response for the zero control samples, was 4.84 nmol/L for cortisol and 2.68 pmol/L for fT4.

Results obtained after diluting a sample with a high content of cortisol and fT4 are shown in Table II and Figures 3 and 4. Dilution caused a decrease in cortisol concentration proportional to the dilution factor (except for the highest dilution), and the curve constructed by using serial dilutions of canine serum was parallel to that obtained with the human standards. Likewise, fT4 showed a strong decrease in concentration when samples were diluted, and the 2 curves, the standard curve and that obtained from serial dilutions of a serum sample, were parallel.

Table II.

Cortisol and fT4 concentrations in serially diluted serum samples from a dog as determined by TR-FIA

Concentration
Hormone Dilution Uncorrected Corrected
Cortisol (nmol/L) Undiluted 419.3 419.3
1:2 179.8 359.6
1:4 90.6 362.4
1:8 52.7 422.1
1:16 41.0 656.0
fT4 (pmol/L) Undiluted 70.6 70.6
1:2 27.9 55.8
1:4 13.3 53.2
1:8 9.7 77.6
1:16 8.7 139.2
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Figure 3.

Figure 3

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Parallelism between dilution curves for canine serum (▪) and human standards (⋄) in the cortisol assay. cps — counts per second.

Figure 4.

Figure 4

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Parallelism between dilution curves for canine serum (▪) and human standards (⋄) in the free thyroxine (fT4) assay.

Data obtained after ACTH stimulation and LDDS are presented in Table III. The ACTH administration produced a significant increase (P = 0.0015) from basal cortisol levels, whereas LDDS caused a significant decrease (P = 0.021). The highest value after ACTH administration was 326.72 nmol/L and the lowest after LDDS 4.84 nmol/L.

Table III.

Serum cortisol concentrations (nmol/L) for 10 dogs at baseline and after adrenocorticotropin (ACTH) stimulation and low-dose dexamethasone suppression (LDDS) as determined by time-resolved fluoroimmunoassays (TR-FIA)

After test
Measure At baseline ACTH LDDS
Mean 41.36 207.98a 10.45b
SD 20.67 92.91 7.99
Minimum 15.89 102.71 4.84
Maximum 75.01 326.72 22.42
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a

Significantly higher than baseline value (P = 0.0015)

b

Significantly lower than baseline value (P = 0.021)

The effect of the interfering substances can be appreciated in the corresponding interferographs, where data points represent the mean of duplicate determinations, x-axes increasing concentrations of hemoglobin (Figure 5), triglycerides (Figure 6), or bilirubin (Figure 7), and y-axes the percentage change in cortisol or fT4 concentration for a given concentration of the added substance. The addition of fresh hemolysate to serum samples significantly increased the cortisol concentration (P = 0.022), but only with the highest hemoglobin concentrations (2 and 4 g/dL). On the other hand, the same hemolysate produced a generalized decrease in fT4 concentration, although the differences were not significant (P = 0.29). The addition of triglycerides, independent of the amount, significantly decreased the cortisol concentration (P = 0.014); however, increasing triglyceride amounts caused a significant and continuous increase in fT4 concentration (P = 3.49 × 10−6, r2 = 0.92). Different bilirubin concentrations did not affect the concentration of cortisol (P = 0.64) or fT4 (P = 0.95).

Figure 5.

Figure 5

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Effect of added hemoglobin on serum cortisol (▪) and free thyroxine (fT4) (♦) determinations.

Figure 6.

Figure 6

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Effect of added triglycerides on serum cortisol (▪) and free thyroxine (fT4) (♦) determinations.

Figure 7.

Figure 7

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Effect of added bilirubin on serum cortisol (▪) and free thyroxine (fT4) (♦) determinations.

Discussion

The lack of species-specific commercial kits for cortisol and fT4 measurements in canine samples led us to use, for this study, kits developed and validated for human samples. However, use of human assays or human reference laboratories for assaying animal specimens is unwise unless the assays have been validated adequately (19). Hence, 4 standard criteria for validating immunoassays were assessed with the TR-FIAs; namely, precision, accuracy, sensitivity, and specificity (20). Additionally, effects of hemolysis, lipemia, and bilirubinemia were studied.

The precision study of the cortisol assay provided intra- and interassay CVs of 6.4% to 8.7% and 5.8% to 10.8%, respectively. These values are higher than those obtained with an automated ELISA (16) and similar to those obtained with a manual ELISA (21) and a radioimmunoassay (RIA) (22) but lower than those obtained with a chemiluminescent assay (20).

The intra-assay CVs for fT4 ranged between 5.3% and 9.8%. These values are higher than those described with an ELISA (23). However, a different ELISA (24), an assay based on chemiluminescence (25), and a direct ED assay (26) provided CVs similar to ours. Our interassay CVs for fT4 (3.9% to 14.1%) were higher than those reported by some authors (2,23), although values of 13% (24) and 15.3% (27) have also been reported.

Pipetting and dispensing of reagents were done manually in our study, in contrast to other studies (2,17,20,23,24), in which automated autoanalyzers automatically performed all the assay steps. This fact could have influenced the CVs. Further studies would be desirable to determine if the CVs could be improved with the use of an automated DELFIA Dispense Unit or another automated system for pipetting samples and reagents instead of a conventional multipipette.

The accuracy of DELFIA correlated well with the accuracy of a previously validated ELISA in measuring cortisol concentrations and with the accuracy of an ED assay in measuring fT4 concentrations, as reflected in the r2 values of 0.90 and 0.93, respectively. Thus, for dogs, TR-FIA seems an accurate technique for determining the serum concentration of these types of compounds, as it is for human cortisol (27) and bovine cortisol (11), as well as for many other hormones (7,8).

The limit of detection for the DELFIA cortisol assay was very similar to that obtained with 2 RIAs (28,29) and lower than the limit of detection with chemiluminescence (20). Only ELISA (16) provided a lower detection limit (2.76 nmol/L). The limit of detection for the DELFIA fT4 assay was very similar to that with an ELISA (24) and an ED (26) and lower than that obtained with several RIAs (4,30,31). The assays allow the detection of cortisol and fT4 concentrations as low as 4.84 nmol/L and 2.68 pmol/L, respectively, well beyond the reported normal concentrations in canine serum (16,31). Therefore, TR-FIA can be considered a highly sensitive method that may allow the detection of the small cortisol and fT4 concentrations needed for the diagnosis of Cushing’s syndrome, hypoadrenocorticism, and hypothyroidism in dogs.

With regard to specificity of the assays, the DELFIA cortisol kit showed a decrease in cortisol concentration when the degree of dilution of the sample increased. Furthermore, the curve obtained after diluting the canine sample paralleled that obtained with the human standards; therefore, the assay can be considered immunologically specific. However, we recommend avoiding the dilution of samples since the diluent might interfere with the assay, as may have happened with the highest dilution. This process would not be necessary in routine practice since the assay range supplied by the kit (0 to 1600 nmol/L) is much wider than cortisol reference ranges in healthy dogs and after ACTH stimulation. This assay range is also wider than that provided by ELISA, which has an upper standard of 1399 nmol/L (16). On the other hand, our results from ACTH stimulation and LDDS indicate that the TR-FIA can detect significant changes (increases and decreases, respectively) in cortisol concentrations after administration of the test substance, agreeing with previous reports (1,31) and demonstrating the biologic specificity of the assay.

The DELFIA fT4 kit showed a marked decrease in fT4 concentration when the degree of dilution of the sample increased; this fact could indicate the presence of a methodologic bias, a dependence on serum binding capacity (sBC), since theoretically dilution of serum should not reduce the fT4 concentration (32). The appearance of an sBC-dependent bias in a method can be related to 3 factors: excessive sample dilution; T4 sequestration by anti-T4 antibodies included in the assay reagents; and the presence in the assay reagents of albumin, which binds T4 and increases T4 sequestration (33). Any of these factors might have been present in our study, but the last one is especially likely to have been responsible for an sBC-dependent bias since the T4-Eu tracer stock solution provided with the kit and the buffer used in sample dilution (DELFIA diluent I) contain albumin.

In the veterinary literature, only 1 ELISA (23) has been reported to show a lower concentration of fT4 when samples were diluted, but this fact was not referred to as an sBC-dependent bias. Since a very good correlation existed between the results with TR-FIA and ED in our study, apparently TR-FIA could be used to measure canine fT4 with the precaution of not diluting the samples. However, some methods with a high sBC-dependent bias have been reported to yield falsely low fT4 values in severely ill human patients with nonthyroidal conditions. On the other hand, some tests with this bias have shown results that have good agreement with those of ED (considered the reference or “gold-standard” method) in these patients (33). Therefore, the clinical implications and significance of this bias have not been clearly defined, and additional clinical studies should be done.

Concerning the interfering substances, the manufacturer of the DELFIA kits indicates that, for human serum samples, hemoglobin at a concentration of 5 g/L or less and bilirubin at a concentration of 30 mg/dL or less do not interfere with the assays, but triglycerides at a concentration above 500 mg/dL may cause variation in cortisol and fT4 concentrations. We observed that high hemoglobin concentrations (2 and 4 g/dL) significantly affected the cortisol determination with the DELFIA system; however, lower hemoglobin concentrations did not affect the assay. An ELISA showed that nonsignificant changes could be detected after the addition of a hemolysate at concentrations of 0.5 g/dL or less (higher concentrations were not tested) (18), which agrees with our results. The hemolysate produced a decrease in fT4 levels unrelated to the amount of hemoglobin added, as in the previous study (18), although the decreases were not significant. Unknown chemical interactions may have occurred between red cell components and assay reactants, causing the absence of correlation between percentage change and hemoglobin concentration. For this study we rejected a conventional method of lysing saline-washed red cells in distilled water since distilled water interfered with the assays, producing falsely high cortisol measurements (cortisol dilutions with uncorrected concentrations: 1:1, 687 nmol/L; 1:2, 513 nmol/L; 1:4, 546 nmol/L) and fT4 measurements. Therefore, hemolysis was produced by freezing red cells at −20°C.

Lipemia was demonstrated to negatively bias cortisol concentrations. Possible causes are the lipid-soluble nature of cortisol and inaccurate pipetting of lipemic samples because of their higher viscosity (29). Previous reports regarding the effect of lipemia on cortisol determination have contradictory results. An RIA showed that the addition of a 100% fat emulsion reduced the mean cortisol concentration in heparinized plasma by 12% (29), whereas an ELISA indicated a slight positive bias for cortisol levels with triglyceride addition (18). Our interferograph for fT4 exhibited an increase in concentrations in proportion to the triglyceride levels that could be anticipated by linear regression analysis, as previously described with an ELISA (18). Probably triglycerides prevent the binding between labelled cortisol and antibodies, causing low fluorescence and consequently high cortisol measurements.

Overall, the different results achieved depending on the species or method make it necessary to assess the effect of the interfering substances in each case, and therefore the data presented here will be valid only for the specified reagents and the VICTOR 1420 multilabel counter.

In conclusion, the DELFIA human cortisol kit has been demonstrated to be precise, accurate, sensitive, and specific for determination of the cortisol concentration in canine serum samples, and it could be used in veterinary clinical pathology laboratories as an alternative to ELISA. Bilirubinemic samples can be assayed without significant interference, but the results with lipemic and very hemolytic samples should be interpreted with caution since lipemic samples will underestimate the cortisol concentration and samples with a high hemoglobin content will overestimate it.

The DELFIA human fT4 kit has also shown good precision and accuracy and high sensitivity with canine serum samples, but the sBC-dependent bias indicates that results should be interpreted with caution until further studies can clarify this subject. Hemolysis could produce slight underestimation of fT4 concentrations, and increases due to lipemia can be predicted by linear regression analysis.

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