RENEB intercomparison exercises analyzing micronuclei (Cytokinesis-block Micronucleus Assay)

Figures & data

Table 1. Variations in the standard protocols of the CBMN assay between the participating labs in intercomparison 1 and 2.

Lab. No.	MN experience (years)	Medium	FCS (%)	Blood (%)	Culture time (h)	Time cyto B was added (h)	KCl + fixation methanol: acetic acid	Staining method
1	10	RPMI	10%	10%	72	24	KCl +4:1	Dapi
2	5	RPMI	10%	10%	70	23	KCl +4:1	Dapi
3	15	RPMI	10%	10%	72	24	KCl +5:1	Giemsa
4	>20	RPMI	10%	8%	72	24	KCl +4:1	Dapi
5	25	RPMI	10%	10%	70	23	KCl +4:1	Dapi, giemsa
6	16	RPMI	10%	10%	72	44	KCl +3:1	Giemsa
7	20	RPMI	25%	10%	72	44	3:1	Giemsa
8a	>15	Ham’s F10	20%	10%	72	44	KCl +3:1	Giemsa
9	15	RPMI	25%	10%	72	44	KCl +3:1	Giemsa
10	10	RPMI	10%	5%	72	44	KCl +3:1	Giemsa
11	20	RPMI	20%	10%	72	24	KCl +4:1	Giemsa
12	25	RPMI	10%	10%	70	23	KCl +4:1	Dapi
8b	2	RPMI	10%	5%	68	24	KCl +3:1	Dapi
13	8	RPMI	10%	6%	72	44	KCl +3:1	Dapi, giemsa
15	22	RPMI	10%	10%	70	23	KCl +4:1	Dapi
16	21	PB MAX	–	–	72	23	KCl +3:1	Giemsa

L8a is the lab participating in IC1, L8b is the lab participating in IC2.

Figure 1. Micronucleus calibration curves of the RENEB participating labs (L). Calibration curves are shown for automated (black lines), semi-automated (green lines) and manual (blue/purple lines) scoring methods. Labs 1, 2, 4, 5, 12 provided calibration curves for automated and semi-automated scoring. Labs 3, 6–11 provided calibration curves for manual micronucleus scoring. One calibration curve (L8) was set up for 200 kV X-rays, nine calibration curves were set up for Co-60 gamma-rays and two calibration curves (L1, L9) were set up for Cs-137 gamma-rays.

Table 2. Radiation characteristics and coefficients of the MN dose response curves of the RENEB labs.

Lab. No.	Radiation quality	Dose rate (Gy/min)	Irradiation temp (°C)	Scoring method	α (±SE)	β (±SE)	c (±SE)
1	Cs-137	0.48	37	Automated	0.061 (±0.011)	0.016 (±0.004)	0.027 (±0.002)
				Semi-auto	0.046 (±0.009)	0.032 (±0.004)	0.013 (±0.001)
2	60Co	0.4	37	Automated	0.057 (±0.011)	0.016 (±0.004)	0.011 (±0.004)
				Semi-auto	0.068 (±0.016)	0.015 (±0.006)	0.039 (±0.007)
3	60Co	0.26	37	Manual	0.016	0.056	0.007
4	60Co	0.8	37	Automated	0.059 (±0.011)	0.002 (±0.003)	0.010 (±0.005)
				Semi-auto	0.026 (±0.008)	0.012 (±0.003)	0.004 (±0.003)
5	60Co	1.27	37	Automated	0.029 (±0.012)	0.029 (±0.005)	0.038 (±0.005)
				Semi-auto	0.033 (±0.009)	0.032 (±0.004)	0.009 (±0.003)
6	60Co		37	Manual	0.063 (±0.011)	0.014 (±0.006)	0.008 (±0.003)
7	60Co	0.14	RT	Manual	0.024 (±0.002)	0.019 (±0.001)	0.012 (±0.001)
8	X-rays	1	RT	Manual	0.082 (±0.02)	0.009 (±0.003)	0.022 (±0.008)
	(200 kV, 4 mA)
9	Cs-137	0.63	RT	Manual	0.040 (±0.014)	0.017 (±0.004)	0.021 (±0.009)
10	60Co	<0.5	37	Manual	0.097 (±0.003)	0.014 (±0.010)	0.030 (±0.004)
11	60Co	1	37	Manual	0.049 (±0.004)	0.045 (±0.002)	0.020 (±0.001)
12	60Co	0.5	37	Automated	0.079 (±0.018)	0.007 (±0.001)	0.023 (±0.016)
				Semi-auto	0.061 (±0.008)	0.020 (±0.006)	0.002 (±0.005)

Table 3(a). Number of scored binucleate (BN) cells and observed micronucleus frequencies (micronuclei (MN)/1000 BN cells), stratified by scoring procedure for all labs participating in the first intercomparison. Mean MN frequencies ± SEM per scoring procedure are also presented.

		0 Gy		0.94 Gy		3.27 Gy		4.75 Gy PB
Scoring method	Laboratory	MN/1000 BN	BN	MN/1000 BN	BN	MN/1000 BN	BN	MN/1000 BN	BN
Automated	L1	54	2000	119	2999	480	2000	92	4000
	L2	17	3499	107	5069	469	2847	99	5130
	L4	112	4614	153	4903	439	3493	215	3988
	L5	53	3980	72	3794	206	3578	62	4373
	L12	23	3925	102	4011	439	1967	86	3996
Mean MN ± SEM		52 ± 17		111 ± 29		407 ± 113		111 ± 60
Mean MN ± SEM (without L4)		37 ± 10		100 ± 20		399 ± 129		85 ± 16
Semi-automated	L1	39	2000	99	2999	593	2000	107	4000
	L2	11	3499	85	5069	443	2847	94	5130
	L4	34	4614	71	4903	349	3493	89	3988
	L5	7	3980	45	3794	158	3578	19	4373
	L12	19	3924	99	4005	507	1945	79	3992
Mean MN ± SEM		22 ± 6		80 ± 10		410 ± 74		78 ± 15
Mean MN ± SEM (without L4)		19 ± 7		82 ± 13		425 ± 94		75 ± 19
Manual	L3	16	2000	32	2000	282	2000	72	2000
	L6	18	2000	112	2000	753	2000	187	2000
	L7	3	2000	78	2000	492	2000	136	2000
	L8	20	1000	142	1000	843	1000	376	1000
	L9	16	2000	55	2000	729	2000	77	2000
	L10	28	1500	113	1000	654	1003	206	1005
	L11	45	1298	54	1016	335	1001	135	1009
Mean MN ± SEM		21 ± 5		84 ± 15		584 ± 82		170 ± 39

PB: partial body (50%).

Table 3(b). Number of scored binucleate (BN) cells and observed micronucleus frequencies (micronuclei (MN)/1000 BN cells), stratified by scoring procedure for all labs participating in the second intercomparison. Mean MN frequencies ± SEM per scoring procedure are also presented.

		0.85 Gy		2.7 Gy
Scoring method	Laboratory	MN/1000BN	BN	MN/1000BN	BN
Automated	L1	86	4000	352	4000
	L8	45	4025	170	3036
	L4	62	3782	199	2623
	L5	58	4371	271	4386
	L12	104	3985	438	3987
	L2	60	8382	245	6351
	L13	63	1309	288	772
	L15	142	4000	439	2959
Mean MN ± SEM		78 ± 11		300 ± 36
Mean MN and BN ± SEM (without L13, L15)		69 ± 9		279 ± 41
Semi-automated	L1	82	4000	384	4000
	L8	56	4025	286	3036
	L4	52	3782	204	2623
	L5	49	4371	292	4386
	L12	83	3982	420	3974
	L2	56	8381	260	6350
	L13	82	1297	443	759
	L15	61	3967	272	2817
Mean MN ± SEM		65 ± 5		320 ± 30
Mean MN ± SEM (without L13, L15)		63 ± 6		308 ± 33
Manual	L3	88	2000	269	2000
	L5	59	1066	356	1043
	L6	106	2000	458	2000
	L7	24	2000	100	2000
	L9	87	1000	441	1000
	L10	139	2218	500	2160
	L11	91	2127	413	2184
	L13	97	2000	600	2000
	L15	165	2000	699	2000
	L16	142	2000	421	2000
Mean MN ± SEM		100 ± 13		426 ± 52
Mean MN ± SEM (without L13, L15, L16)		85 ± 14		362 ± 52
Total
Mean MN ± SEM		82 ± 7	3310 ± 371	355 ± 26
Mean MN ± SEM (without L13, L15, L16)		73 ± 6	3658 ± 461	319 ± 25

Table 4(a). Individual dose estimates reported by the laboratories participating in the first intercomparison exercise (laboratories: L1–L12), stratified by scoring procedure. Mean dose estimates ± SEM per scoring procedure are also presented.

		Estimated doses (Gy)
		True doses (Gy)
Scoring method	Laboratory	0 Gy	0.94 Gy	3.27 Gy	4.75 Gy PB
Automated	L1	0.39	1.14	3.71	4.59
	L2	0.00	0.82	3.55	5.01
	L4	1.65	2.29	6.27	13.60
	L5	0.39	0.71	1.98	2.28
	L12	0.00	0.93	3.88	7.29
	Mean ± SEM	0.49 ± 0.30	1.18 ± 0.29	3.88 ± 0.69	6.55 ± 1.93
	Mean ± SEM (without L4)	0.20 ± 0.11	0.90 ± 0.09	3.28 ± 0.44	4.79 ± 1.03
Semi-automated	L1	0.43	1.07	3.59	6.19
	L2	0.00	1.01	3.71	7.04
	L4	0.83	1.50	4.33	7.29
	L5	0.00	0.66	1.70	4.81
	L12	0.26	1.16	3.75	7.63
	Mean ± SEM	0.30 ± 0.15	1.08 ± 0.14	3.42 ± 0.45	6.59 ± 0.51
	Mean ± SEM (without L4)	0.17 ± 0.11	0.97 ± 0.11	3.19 ± 0.50	6.42 ± 0.61
Manual	L3	0.16	0.41	2.79	5.86
	L6	0.00	1.29	5.39	7.89
	L7	0.00	1.32	4.40	5.62
	L8	0.10	1.09	3.83	5.90
	L9	0.00	0.67	5.39	5.24
	L10	0.00	1.00	4.00	5.60
	L11	0.38	0.47	2.10	3.23
	Mean ± SEM	0.09 ± 0.05	0.89 ± 0.14	3.99 ± 0.47	5.62 ± 0.52
Mean TOTAL ± SEM		0.27 ± 0.10	1.03 ± 1.11	3.79 ± 0.29	6.18 ± 0.59
Mean TOTAL ± SEM (without L4)		0.14 ± 0.04	0.92 ± 0.07	3.59 ± 0.27	5.61 ± 0.39

Uncertainty interval for triage dose estimation.

0.5 Gy for 0 Gy and 0.94 Gy.

20% of true dose for the higher doses: 0.65 Gy for 3.27 Gy and 0.95 Gy for 4.75 Gy.

grey: dose estimates lying outside the uncertainty interval.

PB: partial body (50%).

Table 4(b). Individual dose estimates reported by the laboratories participating in the second intercomparison exercise (laboratories: L1–L16), stratified by scoring procedure. Mean dose estimates ± SEM per scoring procedure are also presented.

		Estimated dosesTrue doses (Gy)
Scoring method	Laboratory	0.85	2.7
Automated	L1	0.87	2.72
	L8	0.27	2.82
	L4	0.46	2.39
	L5	0.48	2.73
	L12	1.15	3.42
	L2	0.29	2.09
	Mean ± SEM (Gy)	0.58 ± 0.14	2.69 ± 0.18
Semi-automated	L1	1.00	2.55
	L8	0.51	2.86
	L4	0.95	3.03
	L5	0.85	2.79
	L12	1.15	2.79
	L2	0.66	2.55
	L13	0.66	2.05
	L15	1.17	3.65
	Mean ± SEM (Gy)	0.87 ± 0.09	2.78 ± 0.16
Mean ± SEM (Gy) (without L13, L15)		0.85 ± 0.1	2.76 ± 0.08
Manual	L3	1.10	2.69
	L5	0.83	3.09
	L6	0.92	2.40
	L7	0.38	1.59
	L9	1.13	3.94
	L10	0.99	3.29
	L11	0.76	2.37
	L13	0.70	2.35
	L15	1.34	3.53
	L16	2.03	4.10
	Mean ± SEM (Gy)	1.02 ± 0.14	2.94 ± 0.25
Mean ± SEM (Gy) (without L13, L15, L16)		0.87 ± 0.10	2.77 ± 0.29
Mean TOTAL ± SEM (Gy)		0.86 ± 0.08	2.82 ± 0.12
Mean TOTAL ± SEM (Gy) (without L 13, L 15, L 16)		0.78 ± 0.07	2.74 ± 0.12

Uncertainty interval for triage dose estimation.

0.50 Gy for 0.85 Gy.

0.54 Gy for 2.7 Gy (20% of the true dose).

Grey: values lying outside the uncertainty interval.

Figure 2. Variance/mean (σ²/μ) values, representing the overdispersion of the MN frequency distribution with respect to the Poisson distribution, are shown for the sham-irradiated control (0 Gy), the total body simulated (0.94 Gy, 3.27 Gy) and partial body (PB) simulated (4.75 Gy) exposures of the first intercomparison. Per dose point, the σ²/μ values are grouped per scoring method: automated scoring (black symbols), semi-automated scoring (green symbols), manual scoring (blue symbols). Values of σ²/μ ≥ 1.5 (dotted line) were taken as being indicative for partial body exposure.

Figure 3. z-scores obtained in the first (a) and second (b) intercomparison exercise for the comparison of individual labs’ dose estimates with the true dose. The z-scores of all the participating labs are given for the different dose points and scoring methods [automated (black symbols), semi-automated (green symbols) or manual (blue symbols)]. Dose estimates are classified as satisfactory (| z | ≤ 2), questionable (2 < | z | < 3), and unsatisfactory (| z | ≥ 3).

Figure 4. Comparison of the dose estimates obtained by scoring 1, 2 or 4 slides in automated (a), semi-automated (b) or manual mode (c). Dose estimations for the 0.85 Gy and the 2.70 Gy dose points are given for each lab. The dotted lines represent the uncertainty intervals.