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Clinical Chemistry 44: 1002-1007, 1998;
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(Clinical Chemistry. 1998;44:1002-1007.)
© 1998 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

Misleading serum free thyroxine results during low molecular weight heparin treatment

Heather P. Stevenson1, G. Pooler, R. Archbold2, Paul Johnston3, Ian S. Young2, and Brian Sheridan2,a

1 Directorate of Biochemical Medicine, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK.
Departments of
2 Clinical Biochemistry and
3 Cardiology, Royal Hospitals Trust, Belfast BT12 6BA, UK.
a Author for correspondence. Fax 44-1232-312014.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Measured free thyroxine concentrations in serum increase markedly after intravenous heparin administration, but the effect of heparin administered subcutaneously has not been adequately documented. We found in vitro increases of up to 63% in measured FT4 after a single dose of subcutaneous heparin (enoxaparin, 2000 units) in nine healthy volunteers, and the magnitude of these increases was correlated with initial serum triglyceride concentrations (r = 0.93, P <0.005) and in vitro free fatty acid release (r = 0.88, P <0.005). In 10 cardiac inpatients receiving repeated doses of enoxaparin (2000 units twice daily), measured FT4 increased by up to 171% in specimens taken 2–6 h after injection. When specimens were obtained 10 h after injection, the effect appeared to be minimized, with in vitro increases of <40%, but such increases may still be sufficient to cause interpretative errors. If FT4 estimation is absolutely necessary in patients receiving enoxaparin, specimens should be taken >=10 h postdose and analyzed within 24 h.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interpretation of thyroid function test results for inpatients is complicated by the frequent occurrence of nonthyroidal illness and treatment with drugs such as heparin and salicylates. In particular, the finding of increased free thyroxine (FT4)1 in conjunction with thyroid-stimulating hormone (TSH) concentrations within the reference interval may lead to unnecessary investigation or even inappropriate treatment if causes such as drug interference are not considered. Specimens drawn from patients receiving intravenous heparin treatment can show marked increases in FT4 concentrations after even a brief preanalytical delay, whereas storage of blood from healthy volunteers for up to 7 days at room temperature before analysis has been shown not to affect the measured FT4 concentration (1). The mechanism of this phenomenon is in vivo release of lipoprotein lipase and hepatic lipase from the vascular endothelium (2)(3). The lipolytic activity of these enzymes in vivo and/or in vitro leads to a rise in the serum concentration of free fatty acids, which displace thyroid hormones from their binding proteins.

Low molecular weight (LMW) heparins are derivatives obtained by fractionation or depolymerization of heparin. They have greater bioavailability and longer half-lives than unfractionated (UNF) heparin and may lead to fewer bleeding complications. Enoxaparin (Clexane) is frequently used at doses of 2000 units (20 mg) once or twice daily in the management of patients with a high risk of deep vein thrombosis or pulmonary embolism.

The artifactual increase in measured serum FT4 concentrations caused by intravenous UNF heparin has been recognized for some time (4)(5) and has been demonstrated using a variety of analytical techniques (equilibrium dialysis, ultracentrifugation, and direct immunoassay). However, the effect of subcutaneous LMW heparin on thyroid function tests has not been extensively studied. This effect will depend on the time the sample is taken relative to the injection time, the triglyceride concentration in the serum, and the length of time between venesection and analysis during which in vitro lipolysis occurs.

Reports on lipolysis after LMW-heparin administration have given conflicting results, which may be caused by the use of different heparin preparations. Persson et al. (2) demonstrated a maximal release of lipoprotein lipase one hour after subcutaneous LMW heparin injection (Kabivitrum AB preparation, 5000 units) with comparable or greater enzyme release than with subcutaneous UNF heparin (5000 units). Harenberg et al. (6) suggested that, although subcutaneous LMW heparin 21–23 led to greater lipase release than intravenous UNF heparin, the release of free fatty acids was similar. However, Myrmel et al. (7) compared the effects of LMW heparin (Fragmin, 5000 units) and UNF heparin (5000 units) on lipolysis in hip replacement patients and concluded that the former had substantially lower lipolytic effect.

In 1987, Wilson et al. (8) reported an increase in measured serum FT4 concentrations 2 h after the subcutaneous injection of LMW heparin (CY222) into healthy volunteers but suggested that the effect may not occur at prophylactic dosages. In 1996, Juame et al. (9) reported a case of artifactual FT4 increase, by equilibrium dialysis measurement, in a patient receiving intralipid and subcutaneous heparin (5000 units). In a study on healthy volunteers, these authors found increased lipolysis up to 2 h after subjects received subcutaneous heparin when triglyceride was added to serum as a substrate. This phenomenon does not appear to have been further investigated with respect to the time-course of the effect or its dependence on analytical methods.

The aim of this study was to use current laboratory methods to examine the effect of subcutaneous LMW- heparin injection on lipolysis and thyroid function tests, with or without delay in analysis, for healthy volunteers and hospital inpatients.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Blood specimens from nine healthy, nonfasting male volunteers (ages 33–54 years) and 19 cardiac inpatients (13 male, 6 female, ages 42–87 years) were collected, using a tourniquet, into Greiner tubes containing serum separator gel. The blood samples were allowed to clot for 30–45 min, and the serum was stored in aliquots for analysis with minimal delay or stored at -20 °C, 4 °C, or room temperature before analysis.

Ethical approval was granted by the Research Ethics Committee, The Queen's University of Belfast. Enoxaparin (Clexane, Rhône-Poulenc Rorer) was administered to healthy volunteers by subcutaneous injection of 2000 units, and blood specimens were obtained immediately before the injection and at timed intervals later. Specimens from cardiac inpatients were obtained with informed consent unless drawn for routine laboratory tests.

Serum albumin and triglyceride concentrations were measured on a Vitros 750 instrument (J & J Clinical Diagnostics). Free fatty acid concentrations were estimated by an enzymatic method with a working range of 0.01–2.00 mmol/L (Randox Laboratories) on a centrifugal analyzer (Cobas FARA, Roche Diagnostic Systems) in undiluted or diluted serum (1:3 or 1:5) that had been stored at room temperature for 0–48 h and then frozen at -20 °C until analysis. The reference intervals for fasting serum are as follows: albumin, 30–45 g/L; triglycerides, 0.68–1.97 mmol/L; and free fatty acids, 0.1–0.9 mmol/L. FT4 and TSH concentrations were estimated by fluoroimmunoassay on an AutoDELFIA instrument (Wallac Oy) with sample pipetting completed ~3 h after venipuncture or after 24 or 48 h storage at room temperature. The reference intervals for serum FT4 and TSH are 7.6–19.7 pmol/L and 0.45–4.5 x 10-3 IU/L, respectively.

Statistical analyses included a paired t-test and the Pearson correlation for gaussian results, or Wilcoxon matched-pairs signed rank and Spearman's rank tests for nongaussian results. Results are expressed as mean ± 1 SE (range). The between-batch imprecision for each analysis was as follows: albumin, CV = 1.0% at 28.0 g/L; triglycerides, CV = 1.1% at 1.36 mmol/L; free fatty acids, CV = 2.5% at 1.11 mmol/L; FT4 CV <=3.5% at 10.9–26.3 pmol/L; and TSH CV <=2.0% at 0.54–20.1 x 10-3 IU/L.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
effect of delay in analysis of serum from healthy volunteers
The triglyceride concentrations in serum obtained from nonfasting volunteers before heparin injection ranged from 0.56 to 4.09 mmol/L. Free fatty acid concentrations increased during storage for 2 days at room temperature, and the increase was correlated with the initial triglyceride concentration (r = 0.93, P = 0.0004, n = 9) (Table 1 ). The free fatty acid concentrations, which were all <0.91 mmol/L, did not substantially alter measured FT4 or TSH concentrations relative to between-batch and intraindividual variability (10)(11).


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  		Table 1. In vitro lipolysis and thyroid function test results before and after a single subcutaneous heparin injection in nine healthy volunteers.

effect of a single subcutaneous enoxaparin injection in healthy volunteers
Specimens obtained 3 h after enoxaparin injection and analyzed with minimal delay (<9 h), had increased free fatty acid concentrations but no increase in FT4 or TSH relative to preheparin specimens (Table 1Up ). When analysis of the specimens obtained 3 h postheparin was delayed up to 48 h with storage at room temperature, free fatty acid concentrations increased further, and FT4 concentrations increased by 2–63% relative to results obtained without delay (mean increase, 2.6 ± 0.8 pmol/L). The percentage increase in serum FT4 concentration after a 48-h delay was correlated with the triglyceride concentration (r = 0.93, P = 0.0003, n = 9) and with the free fatty acid concentration measured after 48 h (r = 0.88, P = 0.0018, n = 9).

In serum samples obtained 24 and 48 h postheparin, the free fatty acid concentrations measured before and after a delay in analysis were similar to those seen in preheparin specimens, and only small changes in FT4 concentrations were observed.

There was no substantial change, relative to between-batch and intraindividual variability, in serum TSH concentrations after heparin injection in healthy volunteers, either with or without delay in analysis.

effect of multiple doses of enoxaparin in cardiac inpatients
Storing sera taken from cardiac inpatients 2–6 h postheparin at room temperature for 48 h caused marked increases in the free fatty acid and FT4 concentrations in 4 of 10 patients (Fig. 1 and Table 2 ). At 2 h postheparin, the percentage in vitro increase in FT4 (range 10–161%) was correlated with serum triglyceride concentration (1.08–3.34 mmol/L, rs = 0.79, P = 0.006, n = 10), free fatty acid concentration (1.43–4.90 mmol/L, rs = 0.82, P = 0.004, n = 10), and the free fatty acid/albumin molar ratio (2.2–7.4, rs = 0.73, P = 0.015, n = 10; Fig. 2 ).



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  		Figure 1. Free fatty acid release during 48 h storage at room temperature, in normal serum from healthy volunteers (n = 9), and in serum taken from cardiac inpatients (n = 10) after enoxaparin administration (2000 units twice daily) (mean ± SE).


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  		Table 2. Effect of enoxaparin on FT4 concentrations measured after 48-h storage of specimens at room temperature.



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  		Figure 2. Thyroxine displacement from thyroxine-binding proteins in serum (stored at room temperature for 48 h) from 19 postheparin cardiac inpatients as a function of final free fatty acid concentration and the free fatty acid/albumin ratio.

The percentage increase in FT4 was calculated from results obtained by analysis of each serum sample with minimal delay and after 48 h storage at room temperature.

When single serum samples obtained 10 h after the last enoxaparin injection in 18 patients were analyzed initially after <18 h storage at 4 °C and then after 48 h at room temperature, a small in vitro increase in FT4 was observed, with a mean change of 8.1 ± 12.9% (range -5 to 40%, P = 0.028; Table 3 ). The release of free fatty acids during the 24- or 48-h incubation at room temperature was significantly greater (P = 0.005) in 10-h postheparin samples than that seen in preheparin volunteer serum. In 3 of the 18 patients studied at 10 h postheparin, the FT4 results obtained after a 48-h delay (21.0, 26.2, and 27.2 pmol/L) were above the upper reference limit (19.7 pmol/L), whereas results obtained after minimal delay were 19.0, 19.8, and 19.4 pmol/L, respectively. All of these patients had serum TSH concentrations within the reference interval, and routine delay in analysis may have given an anomalous pattern of results.


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  		Table 3. Serum free fatty acids and thyroid function test results in specimens taken from hospital inpatients 10 h after the last enoxaparin injection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Laboratory investigation of thyroid function in acutely ill patients is, unfortunately, prone to difficulty as a result of preanalytical factors such as "nonthyroidal illness" and the presence in serum of drugs that may displace thyroxine from its binding proteins, alter conversion of thyroxine to triiodothyronine, stimulate thyroxine degradation, alter TSH secretion, or cause in vitro lipolysis (12). From a recent audit in this hospital, it was established that ~0.3% of specimens received by the Regional Endocrine Laboratory were from inpatients receiving subcutaneous LMW heparin, and of these specimens, only 35% were analyzed on the same working day as venipuncture, with the remainder being subject to a preanalytical delay of 24 h or more. Specimens are transported from several hospitals to the laboratory at ambient temperature, and in vitro lipolysis may occur before they arrive in the laboratory.

A single subcutaneous dose of LMW heparin in healthy volunteers was shown to increase the release of free fatty acids in specimens taken 3 h after the dose. This increase in in vitro lipolysis coincides with the peak anticoagulant activity of LMW heparin (13), and both actions appear to become undetectable after 24 h. This lipolysis was found to be associated with increases in the serum FT4 concentration measured after a 24- or 48-h delay. Free fatty acid concentrations as low as 1.2 mmol/L were found to be associated with increases of >10% in FT4 concentrations, whereas Christofides and Sheehan (14) suggested that nonesterified fatty acid concentrations >3 mmol/L are required to displace thyroxine from binding proteins, given normal albumin concentrations. Because the extent of interference in FT4 measurement by free fatty acids may be method-dependent, this difference may imply a greater susceptibility of the AutoDELFIA assay to the free fatty acid concentration than the Amerlite-MAB assay used by Christofides and Sheehan.

The decrease in serum TSH that was documented by Faber et al. (15) in specimens obtained 15 min after UNF- heparin injection (5000 units) and Wilson et al. (8) at 2 h after LMW-heparin injection (27 500 anti-Xa units), was not observed in this study on healthy volunteers. This is in keeping with the suggestion that thyroxine displacement does not occur in vivo.

To establish the best approach to testing thyroid function in patients receiving enoxaparin, serum from patients on twice-daily doses was analyzed after a 48-h incubation at room temperature. A marked increase of measured FT4 was observed 2–6 h postdose, and substantial increases in the FT4 concentration also occurred in samples taken 10 h after the last dose, although the magnitude of these changes was large enough to lead to possible misinterpretation in only 3 of 18 patients.

The increase in FT4 concentrations seen after heparin injection in cardiac inpatients showed an important relationship with the serum free fatty acid/albumin molar ratio as suggested by Liewendahl (16). The reason some patients show greater in vitro thyroxine displacement from binding proteins than others appears to be largely related to the triglyceride concentration in the blood sample, although it may also depend on lipase activities or other factors. It would appear from our data that clinically significant artifacts in FT4 results are unlikely when the specimen is found to have triglyceride concentrations less than the upper reference limit for fasting individuals. Mendel et al. (17), in a study into the effect of UNF heparin, similarly did not find a substantial increase of measured FT4 in subjects with plasma triglycerides <1.69 mmol/L.

The finding of increased lipolysis in all specimens taken 2–12 h after enoxaparin injection in patients receiving twice-daily doses of 2000 units suggests that measurement of FT4 should be avoided where possible in such patients. However, if FT4 estimation is necessary, the increasingly fast turnaround times of automated FT4 assays and specimen refrigeration may allow minimization of the artifact if specimens are taken at an appropriate time. The findings in this study suggest that specimens for FT4 estimation should be taken at least 10 h after the last enoxaparin injection, and arrangements should be made to prevent a delay of >24 h before analysis of specimens stored at 4 °C to minimize the risk of misinterpretation of results because of preanalytical lipolysis.


   Footnotes
 
1 Nonstandard abbreviations: FT4, free thyroxine; TSH, thyroid-stimulating hormone; LMW, low molecular weight; and UNF, unfractionated.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Diver MJ, Jones PJ, Hughes JG, Hipkin LJ, West CR. Effect of preanalytical conditions on measurements of free thyroxine and total triiodothyronine [Letter]. Clin Chem 1996;42:1124.[Free Full Text]
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  8. Wilson R, Gordon D, Johnstone RV, Johnstone P, Beastall GH, Thomson JA. The parallel effects of a very low molecular weight heparin (CY222) on Anti Xa activity and thyroid hormone levels in blood. Scott Med J 1987;32:76-78. [Web of Science][Medline] [Order article via Infotrieve]
  9. Juame JC, Mendel CM, Frost PH, Greenspan FS, Laughton CW. Extremely low doses of heparin release lipase activity into the plasma and can thereby cause artifactual elevations in the serum free thyroxine concentrations as measured by equilibrium dialysis. Thyroid 1996;6:79-83. [Web of Science][Medline] [Order article via Infotrieve]
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