Abstract
Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0-100 » ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored.
Original language | English |
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Pages (from-to) | 720-728 |
Number of pages | 9 |
Journal | Clinical Chemistry and Laboratory Medicine |
Volume | 62 |
Issue number | 4 |
Early online date | 30 Oct 2023 |
DOIs | |
Publication status | Published - 25 Mar 2024 |
Bibliographical note
© 2023 Walter de Gruyter GmbH, Berlin/Boston.Funding
Research funding : The study was supported by The Netherlands Organization for Scientific Research (NWO) via LIFT grant 731.017.405. Roche Diagnostics partially sponsored the CYFRA 21-1 reagents used in the study. The funding organizations did not play a role in the design of the study, choice of enrolled patients, review and interpretation of data or preparation of approval of manuscripts.
Funders | Funder number |
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Nederlandse Organisatie voor Wetenschappelijk Onderzoek | 731.017.405 |
Keywords
- cytokeratin 19 fragment 21-1
- immunoaffinity (IA)
- liquid chromatography-tandem mass spectrometry (LC-MS/MS)
- lung cancer diagnostics
- protein precipitation
- tryptic digestion
- Chromatography, Liquid/methods
- Antigens, Neoplasm
- Humans
- Tandem Mass Spectrometry/methods
- Lung Neoplasms/diagnosis
- Biomarkers, Tumor
- Carcinoma, Non-Small-Cell Lung/diagnosis
- Keratin-19
- Liquid Chromatography-Mass Spectrometry