Interpretation of heterophile-blocking tube results in thyroid function testing using analytical change limits: An exploratory study of clinically discordant cases

INTRODUCTION: This study aimed to evaluate an analytical framework for interpreting heterophile-blocking tube (HBT) results in thyroid function tests (TFTs) by comparing HBT-associated changes in thyroid-stimulating hormone (TSH), free thyroxine (fT4), and free triiodothyronine (fT3) with analyte-specific analytical change limits (ACLs), documenting transitions across laboratory reference intervals, and comparing changes across the three analytes measured in the same treated aliquot.
METHODS: This single-center, prospective, exploratory observational study enrolled 41 adults whose TFT results were discordant with their clinical presentations. Untreated and HBT-treated aliquots of the same serum specimen were analyzed in the same run. Signed percentage changes were compared with ACLs of ±2.77 × the analytical coefficient of variation (CVa) (12.47% for TSH, 15.84% for fT3, and 14.40% for fT4), and paired concentrations were compared using the Wilcoxon signed-rank test with Holm adjustment. Changes in the three analytes from the same treated aliquot were compared, and the sensitivity of the classification to the imprecision estimate was examined.
RESULTS: Median signed changes were -0.45% for TSH, -6.47% for fT3, and -2.11% for fT4; only fT3 showed a significant paired reduction (adjusted p=0.0038). The ACL was exceeded in 8/41 (19.5%) fT3, 1/39 (2.6%) TSH, and 2/41 (4.9%) fT4 results. The rates of TSH and fT4 exceedance were close to those expected from analytical variation alone (p=0.86 and p=0.61, respectively), whereas fT3 exceeded the limit approximately four times more often (p=0.0008). fT3 changes showed no significant correlation with concurrent changes in TSH or fT4, clustered in specimens with elevated pre-HBT fT3 levels, and remained more frequent at every assumed CVa ranging from 1.8% to 8%. Threshold-supported normal-ization occurred in four fT3 results and in none of the TSH or fT4 results.
DISCUSSION AND CONCLUSION: An imprecision-based ACL combined with reference-interval transition provides a transparent criterion for HBT interpretation. HBT-responsive changes occurred predominantly in fT3, whereas TSH and fT4 in the same aliquots were largely unaffected, arguing against a nonspecific pretreatment effect. A change exceeding the ACL is compatible with, but does not by itself prove, heterophile antibody interference and should be interpreted together with the com-plete thyroid profile, the clinical context, and, where necessary, independent confirmatory testing.

Keywords: Endocrinology, heterophile antibodies, immunoassay, thyroid function tests