Related Experiment Video
Updated: Jul 7, 2026
Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications
Published on: February 28, 2014
Optimisation of circulating biomarkers of cell death for routine clinical use
A Greystoke1, J Cummings, T Ward
1Department of Medical Oncology, Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester, UK.
Background:
M30 and M65 enzyme-linked immunosorbent assays detect circulating cytokeratin 18 fragments released during caspase-dependent or total cell death, respectively, and have potential as biomarkers in epithelial cancers. While these assays have been validated, their robustness for routine clinical use is unknown.
Patients And Methods:
M30 and M65 were measured in matched serum and plasma samples from 31 lung cancer patients and 18 controls.
Results:
Time allowable between sample acquisition and processing is critical for assays in clinical use. A 4-h delay in processing at room temperature increased M30 (P < 0.0001), an effect minimised by incubation on ice. M30 and M65 in serum were resistant to processing variations including delays. Serum and plasma measurements correlated well although M30 but not M65 was lower in serum (P < 0.0005). Less variation between duplicate assays was observed in serum. Prolonged storage (-80 degrees C) led to increased M30 (12%, 6 months; 34%, 1 year). Sample dilution in the supplied assay diluent proved non-linear, whereas dilution in donor serum or porcine plasma restored linearity up to a ratio of 1 : 6.
Conclusion:
We present recommendations that improve the reliability of these assays for clinical use and recommend serum as the preferred matrix with data more resistant to variations in collection.
Insights
The M30 and M65 assays for cytokeratin 18 fragments show promise for epithelial cancer biomarkers. Recommendations are provided to enhance assay reliability for clinical use, favoring serum samples for greater stability.
Area of Science:
- Biochemistry
- Oncology
- Clinical Diagnostics
Background:
- M30 and M65 enzyme-linked immunosorbent assays detect cytokeratin 18 fragments, indicating caspase-dependent or total cell death.
- These assays show potential as biomarkers in epithelial cancers, but their clinical robustness is unconfirmed.
Purpose of the Study:
- To evaluate the robustness of M30 and M65 assays for routine clinical use.
- To identify optimal sample handling and processing conditions for reliable biomarker detection.
Main Methods:
- M30 and M65 levels were measured in serum and plasma from lung cancer patients and controls.
- Investigated the impact of sample processing delays, storage conditions, and dilution methods on assay results.
Main Results:
- Processing delays at room temperature increased M30, an effect mitigated by cold incubation.
- Serum M30 and M65 levels were resistant to processing variations, unlike plasma.
- Serum showed less assay variation and M30 was lower than in plasma; prolonged storage increased M30.
- Dilution linearity was restored using donor serum or porcine plasma.
Conclusions:
- Recommendations are provided to improve the reliability of M30 and M65 assays for clinical application.
- Serum is recommended as the preferred sample matrix due to its resistance to collection variations.
