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

Table 1 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	2505	7.24	8.87	10933	2.32	3.39	2150	1.29		38897	2.29	3.98	466	6.77	7.78
AFP	500-600000	pg/mL	11980	3.81	4.23	54865	2.4	3.29	10703	2.88		455084	3.46	12.87	1598	4.99	11.99
Total PSA	50-60000	pg/mL	1230	5.97	7.9	5880	2.06	3.41	1057	1.92		7762	2.46	4.16	< MR	< MR	< MR
CA 15-3	0.5-600	U/mL	12	9.1	8.43	57	4.06	4.5	11	3.05		29	6.48	6.35	24	3.87	3.7
CA 19-9	2-3000	U/mL	61	9.54	7.35	292	5.27	7.69	54	3.2		33	3.35	2.66	10	1.65	1.75
CA 125	2-4000	U/mL	71	6.77	6.68	347	4.14	4.4	68	2.27		302	4.15	4.36	5.53	6.23	11.25
β-HCG	0.2-400	mU/mL	8	8.86	5.54	39	5.56	4.25	7	1.54		0.65	6.37	36.09	< MR	< MR	< MR
CYFRA 21-1	500-900000	pg/mL	29454	7.92	6.98	155151	3.7	3.18	16614	1.52		9724	4.22	4.77	3506	8.07	13.12
HE4	2000-3000000	pg/mL	55648	8.73	6.98	291745	5.17	5.23	55123	4.1		2276	8.16	9.58	< MR	< MR	< MR
Prolactin	500-600000	pg/mL	11868	5.69	4.73	60195	3.04	3.55	10922	2.61		12057	4.37	3.69	10765	3.08	2.52
Leptin	500-600000	pg/mL	13056	8.97	5.05	62955	2.75	2.38	10971	1.94		13430	7.17	4.04	8468	2.78	1.48
OPN	2000-3000000	pg/mL	53895	8.94	7.32	236975	3.72	6.02	43171	0.91		73954	4.46	4.01	10870	2.75	2.25
HGF	100-120000	pg/mL	2452	9.76	8.64	12766	4.06	4.28	2154	2.86		785	3.11	2.86	283	5.65	7.44
MIF	100-120000	pg/mL	2230	7.83	5.97	11061	4.5	4.45	2209	3.06		574	1.89	1.68	513	14.31	15.66
sFas	100-150000	pg/mL	2486	6.3	5.53	12705	3.19	2.77	2779	2.4		3548	4.52	3.89	2217	1.67	1.47
sFasL	50-60000	pg/mL	1219	5.65	5.71	5898	2.17	2.28	1091	2.86		< MR	< MR	< MR	< MR	< MR	< MR
TRAIL	10-12000	pg/mL	252	7.23	7.41	1233	2.16	2.26	221	2.49		65	3.4	3.62	92	3.32	3.5
VEGF	50-60000	pg/mL	1127	13.04	8.8	6110	3.97	2.78	1115	4.41		< MR	< MR	< MR	< MR	< MR	< MR
IL-6	2-3000	pg/mL	49	9.2	9.27	226	4.33	4.21	53	3.43		16	5.66	7.02	< MR	< MR	< MR
IL-8	5-6000	pg/mL	122	5.53	5.67	605	2.19	2.34	110	1.71		33	5.85	6.7	6.71	4.49	9.43
TNFα	5-6000	pg/mL	114	7.77	7.97	562	2.88	3.17	110	2.25		13	4.56	5.66	6.41	3.12	6.68
TGFα	10-12000	pg/mL	237	6.36	6.29	1180	3.02	3.11	221	1.23		24	5.17	8.75	18	4.49	11.59
FGF2	50-50000	pg/mL	1101	13.38	9.41	5571	2.61	2.85	1066	3.08		55	8.71	18.11	79	9.57	14.52
SCF	20-30000	pg/mL	599	8.41	7.97	2813	3.02	2.88	550	1.17		58	6.99	10.97	50	8.32	13.4

Intra-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.