Influence of sample preparation optimization on the accuracy of dose assessment of an automatic non-fluorescent MN scoring system

Abstract

Purpose

Automatizing the scoring of the cytokinesis-blocked micronucleus assay spares a lot of valuable time. The dose-effect relationship can be applied reliably for dose estimation if the quality of the slides is the same from the perspective of the used image processing algorithm. This aspect brings in additional requirements against the quality of the slides compared to the conventional visual scoring.

Materials and methods

An add-in software was created to the non-fluorescent RS-MN automatic MN scoring system which is capable of measuring quantitatively the degree of typical anomalies. The image processing is less reliable when the presence of these anomalies is more frequent. The behavior of the designed sample quality parameters (SQPs) was tested on in vitro irradiated peripheral blood samples (0, 1, and 2 Gy) obtained from a healthy donor and also on samples from patients undergoing low dose-rate brachytherapy.

Results

We examined 20 different SQPs and identified two that are independent and correlate significantly with the error of the fully automatic MN frequency. One is related to the size of the cells and the other reflects the homogeneity of the environment. An equation was established which presents a connection between the error of the auto MN frequency and the SQPs. By adding a fourth cleaning step to the conventional sample preparation and changing the pre-dripping temperature of the slide, the SQP can be modified, and consequently, the sample quality can be improved. The gain in accuracy is 54 ± 10 MN per 1000 binucleated cells, which corresponds to the effects of 0.5 Gy. Around the lowest limit of detection (<0.5 Gy), it means a 50–100% drop in the error of dose, which is significant. With sample quality harmonization, the positive predictive value was raised to 80–93% depending on the dose.

Conclusions

With the technique described in this paper, the suitability for automated scoring of a micronucleus slide can be tested quantitatively and objectively. A method is presented with which in some cases the uncertainty of the assessed doses due to variance in sample quality can be decreased or if it is not possible its bias can be predicted. The proposed protocol leads to more reliable estimation of dose. The SQPs are designed in a way that they have the potential to be adapted to similar systems.

Keywords:

• Automatic micronucleus assay (MN)
• biodosimetry
• sample preparation optimization
• quantified slide quality

Acknowledgments

The study was possible thanks to the help of the entire R&D team at Radosys Ltd., who made the prototype of the RS-MN microscope available and supported the research and development of the software.

Disclosure of interest

The authors report no conflict of interest.

Additional information

Funding

This research was partially supported by the Hungarian National Research, Development and Innovation Fund (Grant VKSZ 14-1-2015-0021) and by the Hungarian Thematic Excellence Programme (Grant TKP2020-NKA-26).

Notes on contributors

Tímea Hülber

Tímea Hülber, M.Sc., is a Ph.D. student at the Institute of Nuclear Techniques at Budapest University of Technology and Economics and works as an R&D physicist at Radosys Ltd. She is specialized in ionizing radiation measurements, including biodosimetry and solid-state nuclear track detectors.

Zsuzsa S. Kocsis

Zsuzsa S. Kocsis, Ph.D., works as a radiobiologist in the National Institute of Oncology, Center of Radiotherapy, Department of Radiobiology and Diagnostic Onco-Cytogenetics.

Judit Németh

Judit Németh, M.Sc., is a biochemical engineer working as an R&D engineer at Radosys Ltd.

Enikõ Kis

Enikő Kis, M.Sc., is a Ph.D. student at the National University of Public Service. She works as a radiation biologist at the Department of Radiobiology and Radiohygiene of the National Public Health Center in Budapest, Hungary

Francesco d’Errico

Francesco d’Erico, Ph.D., is a Professor at the School of Engineering, University of Pisa, Pisa, Italy and at Yale University, School of Medicine New Haven, CT, USA specialized in radioprotection physics.

Géza Sáfrány

Géza Sáfrány, M.D., Ph.D., D.Sc., is a medical doctor specialized in radiation biology and radiohygiene. He is the head of the Department of Radiobiology and Radiohygiene of the National Public Health Center in Budapest, Hungary.

Csilla Pesznyák

Csilla Pesznyák, M.Sc., Ph.D., is a medical physicist specialized in radiotherapy, including dosimetry, radiation biology, radiation protection, and education organization. She is an associate professor at the Institute of Nuclear Techniques at Budapest University of Technology and Economics.