Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay

Table 3 Inter-assay imprecision

			QC 1			QC 2			St 5			Pool 1			Pool 2
Biomarker	Measuring	Unit	Conc	FI	Conc	Conc	FI	Conc	Conc	FI	Conc	Conc	FI	Conc	Conc	FI	Conc
	range		Mean	Mean CV%	Mean CV%	Mean	Mean CV%	Mean CV%	Mean	Mean CV%	Mean CV%	Mean	Mean CV%	Mean CV%	Mean	Mean CV%	Mean CV%
CEA	100-120000	pg/mL	2505	10.45	3.92	10933	8.88	7.62	2150	11.53	4.15	38897	4.53	6.58	466	15.14	14.43
AFP	500-600000	pg/mL	11980	13.92	5.32	54865	12.72	5.09	10703	12.14	3.43	455084	8.59	20.68	1598	34	66.04
Total PSA	50-60000	pg/mL	1230	15.82	5.11	5880	11.51	11.21	1057	8.61	0.32	7762	7.88	4.59	< MR	< MR	< MR
CA 15-3	0.5-600	U/mL	12	18.57	6.38	57	21.29	2.85	11	20.52	1.79	29	16.48	8.48	24	27.78	21.2
CA 19-9	2-3000	U/mL	61	20.3	4.79	292	12.19	4.58	54	11.44	2.19	33	17.49	7.29	10	19.24	23.19
CA 125	2-4000	U/mL	71	13.39	3.23	347	11.19	3.9	68	11.73	1.91	302	6.26	8.27	5.53	22.25	36.3
β-HCG	0.2-400	mU/mL	8	17.8	4.39	39	16.3	1.48	7	15.48	2.41	0.65	4.49	61.53	< MR	< MR	< MR
CYFRA 21-1	500-900000	pg/mL	29454	16.78	27.37	155151	15.64	40.96	16614	42.71	1.14	9724	38.02	50	3506	21.25	30.8
HE4	2000-3000000	pg/mL	55648	27.52	5.48	291745	23.45	3.46	55123	23.76	2.74	2276	14.67	63.85	< MR	< MR	< MR
Prolactin	500-600000	pg/mL	11868	26.35	4.16	60195	15.98	2.69	10922	14.69	1.97	12057	21.22	10.08	10765	35.99	20.91
Leptin	500-600000	pg/mL	13056	23.59	2.44	62955	14.97	3.92	10971	15.63	1.81	13430	18.98	8.79	8468	36.1	23.77
OPN	2000-3000000	pg/mL	53895	45.75	4.01	236975	29.04	4.62	43171	31.41	2.38	73954	46.72	8.5	10870	85.54	22.7
HGF	100-120000	pg/mL	2452	13.41	12.19	12766	16.96	8.28	2154	20.58	1.7	785	7.96	11.57	283	10.19	20.14
MIF	100-120000	pg/mL	2230	19.69	7.89	11061	14.32	4.89	2209	12.31	1.68	574	8.59	13.68	513	63.56	62.9
sFas	100-150000	pg/mL	2486	27.52	6.36	12705	21.86	6.83	2779	26.23	1.37	3548	30.07	7.78	2217	51.45	22.99
sFasL	50-60000	pg/mL	1219	16.69	4.68	5898	20.4	2.92	1091	17.72	1.62	< MR	< MR	< MR	< MR	< MR	< MR
TRAIL	10-12000	pg/mL	252	13.53	4.72	1233	13.51	4.09	221	15.17	1.85	65	10.05	9.26	92	27.2	23.98
VEGF	50-60000	pg/mL	1127	25.4	4.27	6110	24.51	8.87	1115	18.99	3.76	< MR	< MR	< MR	< MR	< MR	< MR
IL-6	2-3000	pg/mL	49	14.49	9.34	226	22.21	17.12	53	18.81	4	16	12.3	14.64	< MR	< MR	< MR
IL-8	5-6000	pg/mL	122	8.85	5.21	605	11.32	3.73	110	9.31	1.98	33	9.31	8.37	6.71	25.35	40.54
TNFα	5-6000	pg/mL	114	21.44	5.52	562	16.31	6.81	110	16.78	2.28	13	19.54	8.14	6.41	26.96	40.75
TGFα	10-12000	pg/mL	237	16.15	6.26	1180	13.07	3.33	221	15.5	1.97	24	16.87	23.88	18	24.27	32.78
FGF2	50-50000	pg/mL	1101	11.17	8.98	5571	13.86	5.71	1066	12.75	3.89	55	11.36	68.81	79	19.8	40.04
SCF	20-30000	pg/mL	599	15.96	10.08	2813	13.38	4.75	550	14.69	1.93	58	11.5	10.22	50	24.27	22.5

Inter-assay imprecision results for all 24 tested markers based on FI as well as observed concentration (Obs Conc) results of assays 1-5 for synthetic quality controls QC 1, QC 2, standard 5 as well as for physiological serum pools 1 and 2. AFP: Alpha-fetoprotein; CA 125: Cancer antigen 125; CEA: Carcinoembryonic antigen; CV: Coefficient of variation; CYFRA 21-1: Cytokeratine 19-fragment; FGF2: Fibroblast growth factor-2; FI: Fluorescent intensity; HE4: Human epididymis protein 4; HGF: Hepatocyte growth factor; IL-6: Interleukin-6; MIF: Macrophage migration inhibitory factor; MR: Measuring range; Conc: Concentration; OPN: Osteopontin; QC 1: Quality control 1; SCF: Stem cell factor; sFas: Soluble Fas; sFasL: Soluble Fas-ligand; TGFα: Transforming growth factor-α; TNFα: Tumor necrosis factor-α; total PSA: Total prostate-specific antigen; TRAIL: Tumor necrosis factor related apoptosis-inducing ligand; VEGF: Vascular endothelial growth factor; β-HCG: β-human chorionic gonadotropin.