ECTS2013 Poster Presentations Calciotropic and phosphotropic hormones and mineral metabolism (33 abstracts)
Mass spectrometric immunoassay for intact parathyroid hormone: correlation with immunoassay and application to clinical samples
L Couchman 1 , D Taylor 1 , B Krystans 1, , M Lopez 1, , A Prakash 1, , D Sarracino 1, , A Garces 1, , M Vogelsang 1, , S Peterman 1, , G Vadali 1, , S Robinson 1, & C Moniz 1
1Kings College Hospital, Clinical Biochemistry, London, UK; 2ThermoFisher Scientific, BRIMS, Boston, Massachusetts, USA; 3ThermoFisher, Scientific, Hemel Hempstead, UK.
Introduction: Parathyroid hormone (PTH) measurement is of use in i) differential diagnosis of hypercalcaemia and ii) patients with renal impairment at risk of bone disease. PTH immunoassays are complicated by cross-reactivity with truncated (inactive) variant forms, which accumulate in patients with renal impairment. PTH assay variability is a critical governance issue in renal medicine, suggesting an MS-based reference method is required.
Aim: To develop a mass spectrometric immunoassay (MSIA) for intact PTH quantification, and to compare results with a PTH immunoassay.
Methods: Plasma PTH was immunocaptured onto MSIA pipette tips pre-bound with anti-PTH antibody, using an automated liquid handler (Versette). Captured analytes were eluted from the tips, digested and specific tryptic peptides analysed by LC–MS/MS, using labelled peptides and recombinant PTH standards for assay calibration. Samples (n=357) analysed by immunoassay (Advia Centaur) then re-analysed by MSIA for comparison.
Results: The MSIA assay demonstrated excellent linearity (R2=0.90–0.99), sensitivity (LOQ, 16 pg/ml), specificity and precision (CV <10%). Significant findings were i) poor correlation (R2=0.67) between the immunoassay and the surrogate N-terminal tryptic peptide (aa 1–13) for intact PTH, ii) better correlation (R2=0.86) of ‘mean PTH’ by MSIA (i.e. the mean concentration of all tryptic peptides, including variant forms), iii) identification of novel variant forms in samples from patients with renal disease, and iv) the commonly-cited variant form PTH 7–84 was not detected.
Conclusions: This approach allows rapid, automated immunoenrichment achieving high sensitivity and selectivity. MSIA allows the simultaneous monitoring of intact and variant PTH forms. Correlation of the PTH MSIA assay using only the N-terminal tryptic peptide (aa 1–13) with immunoassay demonstrated that the immunoassay overestimates the amount of active PTH. This difference was greater in patients with renal impairment, in whom PTH concentrations direct clinical decisions.
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