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

Table 4 Extended 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	2545	13.96	28.08	12727	20.57	25.67	2146	13.34	4.59	39391	6.94	7.38	560	14.32	25.77
AFP	500-600000	pg/mL	12038	23.54	20.99	56960	19.11	10.29	10754	21.43	3.25	493993	12.38	18.42	1489	33.13	63.64
Total PSA	50-60000	pg/mL	1421	34.29	31.87	6172	17.44	13.78	1052	18.72	3.67	8246	16.03	12.46	68	130.09	151.66
CA 15-3	0.5-600	U/mL	12	30.11	18.58	62	29.25	12.98	11	34.01	2.93	33	23.72	17.26	26	30.49	27.59
CA 19-9	2-3000	U/mL	64	22.71	17.91	329	11.98	19.1	54	19.2	2.71	36	15.18	26.88	10	16.18	36.06
CA 125	2-4000	U/mL	74	14.42	22.04	370	12.5	11.07	68	17.29	2.42	317	7.72	12.62	6.13	18.13	37.03
β-HCG	0.2-400	mU/mL	7.85	20.62	20.74	42	16.95	16.23	7	26.88	2.69	1.2	29.09	52.2	0.55	17.3	74.36
CYFRA 21-1	500-900000	pg/mL	24985	69.07	36.07	152731	81.41	52.97	16601	53.61	2.5	8627	50.45	55.77	3506	31.82	30.8
HE4	2000-3000000	pg/mL	55766	22.11	21.46	295050	19.92	10.63	55043	22.45	2.1	2288	19.7	68.01	2590	22.73	54.77
Prolactin	500-600000	pg/mL	11952	23.76	21.49	60823	13.52	9.33	10863	14.81	2.05	12191	17.16	20.41	10602	29.43	28.46
Leptin	500-600000	pg/mL	13001	29.19	15.47	64533	19.61	8.82	11020	23.3	1.88	14622	16.75	18.61	9145	31.06	27.03
OPN	2000-3000000	pg/mL	57576	58.11	28.1	240688	24.08	13.88	42845	25.18	5.71	75488	41.77	25.01	10700	62.83	47.91
HGF	100-120000	pg/mL	2546	15.19	20.7	13471	19.05	12.85	2171	22.65	2.04	894	7.18	27.83	436	29.6	49.81
MIF	100-120000	pg/mL	2263	46.52	45.1	12332	20.48	18.05	2220	23.01	2.93	876	62.88	48.48	517	45.1	47.35
sFas	100-150000	pg/mL	2533	27.15	18.62	13212	20.06	12.63	2775	27.38	1.77	4005	29.39	21.39	2472	39.44	28.26
sFasL	50-60000	pg/mL	1245	19.09	20.91	6023	21.37	6.63	1093	22.37	1.98	< MR	< MR	< MR	< MR	< MR	< MR
TRAIL	10-12000	pg/mL	253	20.17	21.78	1269	17.13	10.13	220	21.55	2.05	72	11.04	32.6	98	26.7	35.57
VEGF	50-60000	pg/mL	1146	46.21	23	6155	45.49	8.97	1100	43.93	3.26	< MR	< MR	< MR	< MR	< MR	< MR
IL-6	2-3000	pg/mL	54	17.46	26.94	268	17.39	23.92	54	24.51	5.54	21	14.19	41.05	< MR	< MR	< MR
IL-8	5-6000	pg/mL	124	75.72	21.22	668	45.79	22.2	111	30.7	3.13	26	36.91	38.62	6.70	90.89	40.54
TNFα	5-6000	pg/mL	117	18.6	23.55	583	16.6	11.16	111	19.41	2.52	16	15.5	28.1	7.85	22.35	49.24
TGFα	10-12000	pg/mL	238	18.16	21.43	1210	19.49	7.74	221	22.29	2.56	27	17.64	45.12	18	19.22	48.85
FGF2	50-50000	pg/mL	1104	25.12	23.52	5708	17.67	9.9	1068	19.05	3.55	68	9.43	49.91	80	15.21	32.41
SCF	20-30000	pg/mL	603	12.44	19.52	2951	16.66	11.26	549	17.88	2.01	79	31.9	40.75	59	22.35	32.17

Inter-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.