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

Table 2 Extended intra-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	2545	8.32	11.34	12727	7.61	12.23	2146	4.11		39391	2.1	3.63	560	8.09	5.67
AFP	500-600000	pg/mL	12038	6.09	6.9	56960	3.08	4.23	10754	4.45		493993	2.39	8.73	1489	6.87	11.8
Total PSA	50-60000	pg/mL	1421	5.76	7.78	6172	3.75	6.27	1052	4.1		8246	1.59	2.69	68	9.76	7.02
CA 15-3	0.5-600	U/mL	12	9	8.7	62	4.18	4.96	11	4.32		33	5.31	5.23	26	9.19	8.66
CA 19-9	2-3000	U/mL	64	10.63	8.63	329	4.83	7.1	54	5.05		36	2.9	2.39	10	4.87	6.17
CA 125	2-4000	U/mL	74	7.02	7.03	370	3.61	3.78	68	2.84		317	3.59	3.64	6.13	4.74	8.54
β-HCG	0.2-400	mU/mL	7.85	10.62	7.02	42	4.89	3.67	7	5.08		1.2	6.76	18.28	0.55	8.17	31.39
CYFRA 21-1	500-900000	pg/mL	24985	7.62	8.22	152731	12.13	4.28	16601	3.32		8627	4.34	5.73	3506	8.33	13.12
HE4	2000-3000000	pg/mL	55766	11.42	9.39	295050	4.28	4.45	55043	1.9		2288	6.47	7.67	2590	11.67	8.86
Prolactin	500-600000	pg/mL	11952	7.86	6.96	60823	3.43	4.13	10863	4.49		12191	3.31	2.83	10602	6.1	4.71
Leptin	500-600000	pg/mL	13001	12.79	7.06	64533	2.79	2.4	11020	5.73		14622	5.67	3.18	9145	6.58	3.4
OPN	2000-3000000	pg/mL	57576	9.61	8.58	240688	4.93	9.29	42845	3.77		75488	3.61	3.35	10700	5.59	4.85
HGF	100-120000	pg/mL	2546	9.92	8.68	13471	3.23	3.3	2171	2.51		894	2.82	2.53	436	7.6	8.8
MIF	100-120000	pg/mL	2263	10.84	10.06	12332	5.12	5.99	2220	5.19		876	2.06	1.86	517	11.4	17.25
sFas	100-150000	pg/mL	2533	7.15	6.72	13212	3.91	3.47	2775	3.94		4005	3.54	3.07	2472	4.88	4.34
sFasL	50-60000	pg/mL	1245	6.35	6.49	6023	2.9	3.15	1093	5.13		< MR	< MR	< MR	< MR	< MR	< MR
TRAIL	10-12000	pg/mL	253	8.52	8.64	1269	2.65	2.72	220	3.73		72	4.22	4.71	98	6.19	6.44
VEGF	50-60000	pg/mL	1146	16.04	11.12	6155	4.61	3.56	1100	2.88		< MR	< MR	< MR	<MR	< MR	< MR
IL-6	2-3000	pg/mL	54	11.72	12.01	268	6.35	6.11	54	1.79		21	6.25	7.44	< MR	< MR	< MR
IL-8	5-6000	pg/mL	124	7.8	9.02	668	6.56	9.49	111	3.94		26	5.05	9.13	6.70	6.22	9.43
TNFα	5-6000	pg/mL	117	10.43	10.35	583	3.68	3.8	111	4.86		16	5.27	9.14	7.85	5.41	4.61
TGFα	10-12000	pg/mL	238	8.86	8.94	1210	3.78	4.05	221	4.91		27	3.99	9.09	18	5.32	9.69
FGF2	50-50000	pg/mL	1104	20.18	14.17	5708	2.97	3.07	1068	2.04		68	9.63	13.35	80	7.72	7.97
SCF	20-30000	pg/mL	603	8.04	7.66	2951	4.39	4.32	549	3.96		79	6.49	8.93	59	8.5	15.12

Intra-assay imprecision results for all 24 tested markers based on FI as well as observed concentration (Obs Conc) results of all assays 1-10 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.