Establishment of the upper reference limit for thyroid peroxidase autoantibodies according to the guidelines proposed by the National Academy of Clinical Biochemistry: comparison of five different automated methods

The estimation of the upper reference limit (URL) for autoantibodies against thyroid peroxidase (TPOAbs) is a controversial issue, because of an uncertainty associated with the criteria used to correctly define the reference population. In addition, the URL of TPOAbs is method-dependent and often arbitrarily established in current laboratory practice. The aim of this study was to determine the reference limits of TPOAbs in a male sample according to the National Academy of Clinical Biochemistry (NACB) guidelines, and to compare them with those obtained in a female group, for five third-generation commercial-automated immunoassay (IMA) platforms.

120 healthy males and 120 healthy females with NACB-required characteristics (younger than 30 years, TSH between 0.5 and 2.0 mIU/L, normal thyroid ultrasound, absence of thyroid disease and absence of other autoimmune diseases) were studied. Sera were analyzed for TPOAbs concentration using five IMA methods applied in automated analyzers: Immulite 2000 XPi (IMM); Maglumi 2000 Plus (MAG); Kryptor Compact Plus (KRY); Phadia 250 (PHA) and Liaison XL (LIA).

Differences in URLs could result from the different coating preparations of the TPO antigen (purified native or recombinant) on solid phase, which affect the proper exposure of the immunodominant epitopes recognized by the polyclonal antibodies present in serum of patients with autoimmune thyroid disease (AITD). Based on these findings, we suggest to overcome the proposal of the NACB guidelines which recommend to involve a single group of young male subjects, and propose, instead, to utilize two distinct groups: one of males and one of females. This new proposal removes the apparent contrast of an all-male reference group for a disease (such as AITD) that affects mainly females. However, in spite of the harmonization among methods provided by the use of an international standard preparation, the wide dispersion of quantitative results still observed in this study suggests the need for further efforts to better understand the cause of these discrepancies, focusing on TPO antigen preparations as the possible source of variability among different assays.