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

doi:10.5662/wjm.v4.i4.219

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World J Methodol. Dec 26, 2014; 4(4): 219-231
Published online Dec 26, 2014. doi: 10.5662/wjm.v4.i4.219

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.

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.

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.

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.

Citation: Hermann N, Dreßen K, Schildberg FA, Jakobs C, Holdenrieder S. Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay. World J Methodol 2014; 4(4): 219-231
URL: https://www.wjgnet.com/2222-0682/full/v4/i4/219.htm
DOI: https://dx.doi.org/10.5662/wjm.v4.i4.219