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

Abstract

Aim of the studyThe 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.Methods120 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).ResultsA statistically significant difference (p < 0.05) between medians in male and female groups was observed for PHA (2.6 and 3.1 IU/mL, respectively) but not for the other four methods. Scatter plots of TPOAbs values revealed a wide dispersion with very different coefficients of variation between the five methods, varying from 48.6 % for KRY in females to 126.3 % for MAG in females. The URLs differed in males and females according to the method: 28.7 and 29.0 IU/mL for IMM, 24.6 and 25.4 IU/mL for MAG, 6.4 and 6.9 IU/mL for KRY, 8.3 and 10.0 IU/mL for PHA and 14.2 and 17.9 IU/mL for LIA, respectively. Such URLs were lower than those stated by the manufacturers except for LIA in females. The difference between URLs ranged from a minimum of 11.3 % (LIA in males) to a maximum of 66.8 % (PHA in males).ConclusionsDifferences 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.

Definition of the upper reference limit for thyroglobulin antibodies according to the National Academy of Clinical Biochemistry guidelines: comparison of eleven different automated methods

“…The first relevant result of the present study was the demonstration of differences between TgAb URLs claimed in the package insert (m-URL) and those obtained in the male reference sample (e-URL): with the exception of ARC and MAG method, e-URLs were lower than those proposed by the manufacturers, the difference ranging from 2.33 to 88.85%. These results were similar to those described in two previous studies dealing with the definition of TPOAb reference limits, determined by several current IMA platforms [ 30 , 31 ]. In our opinion, these discrepancies could be related to the lack of strict criteria in the selection of the subjects for the reference group.…”

“…These results were similar to those described in two previous studies dealing with the definition of TPOAb reference limits, determined by several current IMA platforms [30, 31]. In our opinion, these discrepancies could be related to the lack of strict criteria in the selection of the subjects for the reference group.…”

Subclinical hypothyroidism and antithyroid autoantibodies in women with subfertility or recurrent pregnancy loss

“…There are a range of anti‐TPOAb assays available, with differing detection limits and thresholds for test positivity predetermined by the manufacturer. Most laboratories use one of five third‐generation commercial automated immunoassay (IMA) platforms and the cut‐off values for the upper limit of normal generally fall in the range of 10–50 iu/ml 12 . These variations are an accepted part of normal UK practice.…”

Cut-offs for thyroid peroxidase and thyroglobulin antibodies in early pregnancy

“…Currently used methods for measurement of these autoantibodies are mainly automatic immunoassays, the methods are not standardized, and assay principles are not completely similar. Thus, results obtained with one method may not be comparable with results of another method (21,22). Consequently, cut-offs are not interchangeable between the available methods emphasizing the need for method-specific cut-offs.…”