Optimisation of circulating biomarkers of cell death for routine clinical use.
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.


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.


CONCLUSIONS
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.
Greystoke
A
A
Department of Medical Oncology, Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester, UK.
Cummings
J
J
Ward
T
T
Simpson
K
K
Renehan
A
A
Butt
F
F
Moore
D
D
Gietema
J
J
Blackhall
F
F
Ranson
M
M
Hughes
A
A
Dive
C
C
D000818:Animals; D017209:Apoptosis; D015415:Biomarkers; D001793:Blood Preservation; D001800:Blood Specimen Collection / Q000379:methods; D018288:Carcinoma, Small Cell / Q000097:blood* / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D053538:Keratin-18 / Q000097:blood*; D008175:Lung Neoplasms / Q000097:blood* / Q000473:pathology; D010446:Peptide Fragments / Q000097:blood; D010949:Plasma; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D044967:Serum; D013048:Specimen Handling / Q000379:methods*; D013552:Swine / Q000097:blood
D015415:Biomarkers; D053538:Keratin-18; D010446:Peptide Fragments; D011933:Reagent Kits, Diagnostic; D020169:Caspases