Transthyretin and Retinol binding protein 4: analytical evaluation of the immunonephelometric method|Transtiretina e Proteina legante il retinolo 4: una valutazione analitica del metodo immunonefelometrico

Introduction: transthyretin cardiac amyloidosis (ATTR-CA) is a progressive disease in which circulating levels of TTR contribute to risk stratification of disease progression and represent a biomarker of response to therapy. Aim of this study is to evaluate the analytical characteristics of the nephelometric method used to measure TTR and Retinol binding protein 4 (RBP4). Methods: paired serum (S) and plasma lithium heparin (Li-ep) samples from healthy subjects and patients with ATTR-CA were collected. Reproducibility analysis, based on the CSLI EP05-A3 protocol, was conducted using S and Li-ep pools where TTR and RBP4 were measured on the automated system Atellica NEPH 630 (Siemens Healthineers). S and Li-ep pools were analysed also by a native electrophoresis on 4-20% polyacrylamide gel associated with western-blot for TTR and RBP4. Results: no differences were observed in the measurement of TTR and RBP4 in Li-ep and S. The nephelometric method had a similar analytical performance in both matrices (CV% repeatability Li-ep and S: TTR 1.92 and 2.52; RBP4 2.25 and 1.49; CV% within lab-precision: TTR 2.40 and 2.90; RBP4 2.30 and 2.14). Electrophoresis highlighted the presence of high molecular weight forms, TTR tetramers bound to 1 or 2 RBP4 and TTR trimers and dimers in both matrices. Conclusion: measurement of circulating TTR and RBP4 can be performed on both Li-ep and S. The evaluation of the nephelometric assay showed suitable performances to use it in the clinical context of ATTR-CA. Electrophoretic investigations revealed the presence of several TTR circulating forms, thus further studies are necessary to understand which one is quantified by nephelometry.