Automatic versus manual lymphocyte fixation: impact on dose estimation using the cytokinesis-block micronucleus assay

The lymphocyte cytokinesis-block micronucleus (CBMN) assay is a biodosemeter for the exposure to ionizing radiation. We examined the feasibility to implement a fully automated cell harvesting system for binucleate lymphocyte (BN) fixation. We compared fully automated versus manual BN fixation and evaluated its relevance on the accuracy of dose estimates using the CBMN. First, dose-response curves based on X-ray irradiated blood samples of ten healthy donors (0-4 Gy, dose rate 1.0 Gy/min) were established. BN was either prepared manually or fully automatically using the Hanabi cell harvester system PII. Slides were finally scored following an automatic or semi-automatic approach using the Metafer4 platform. The variance was calculated per dose and separately for each of the four fixation and scoring combinations. Thereafter, a serial of 16 blood samples of unknown exposure doses (0-3.9 Gy X-ray) was analyzed. Employing the four fixation and scoring combinations, we compared the number of dose estimates lying outside the ±0.5 Gy interval and the mean absolute difference (MAD) and examined sensitivity, specificity and accuracy of doses merged into binary dose categories of clinical significance. Irrespective of the fixation procedure, we observed at doses ≤1.0 Gy about 2-4 times higher median variances for the automated scoring procedure over the semi-automated approach (p ≤ 0.03). The lowest median variance was observed for automatic fixation + semi-automated scoring (135) which was even 2 times lower relative to manual fixation + semi-automated scoring (276, p = 0.04). These differences became negligible after doses >1.0 Gy. For the automatic fixation procedure, we also observed a tendency toward borderline significant higher numbers of dose estimates falling into the ±0.5 Gy interval (25 %, p = 0.08) and lower MAD values (50 %, p = 0.09), which was predominantly caused by the accuracy of dose assessment >1.0 Gy. Regarding the discrimination of binary dose categories of clinical significance, we observed a good agreement of both fixation procedures. The implementation of the automatic cell harvesting system considerably reduces the workload and results in dose estimates with a tendency of being slightly more accurate as they are after a manual fixation.