Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The micronucleus (MN) assay is a well-established test for quantifying DNA damage. However, scoring the assay using conventional techniques such as manual microscopy or feature-based image analysis is laborious and challenging. This paper describes the methodology to develop an artificial intelligence model to score the MN assay using imaging flow cytometry data.

Abstract

The micronucleus (MN) assay is used worldwide by regulatory bodies to evaluate chemicals for genetic toxicity. The assay can be performed in two ways: by scoring MN in once-divided, cytokinesis-blocked binucleated cells or fully divided mononucleated cells. Historically, light microscopy has been the gold standard method to score the assay, but it is laborious and subjective. Flow cytometry has been used in recent years to score the assay, but is limited by the inability to visually confirm key aspects of cellular imagery. Imaging flow cytometry (IFC) combines high-throughput image capture and automated image analysis, and has been successfully applied to rapidly acquire imagery of and score all key events in the MN assay. Recently, it has been demonstrated that artificial intelligence (AI) methods based on convolutional neural networks can be used to score MN assay data acquired by IFC. This paper describes all steps to use AI software to create a deep learning model to score all key events and to apply this model to automatically score additional data. Results from the AI deep learning model compare well to manual microscopy, therefore enabling fully automated scoring of the MN assay by combining IFC and AI.

Introduction

The micronucleus (MN) assay is fundamental in genetic toxicology to evaluate DNA damage in the development of cosmetics, pharmaceuticals, and chemicals for human use1,2,3,4. Micronuclei are formed from whole chromosomes or chromosome fragments that do not incorporate into the nucleus following division and condense into small, circular bodies separate from the nucleus. Thus, MN can be used as an endpoint to quantify DNA damage in genotoxicity testing1.

The preferred method for quantifying M....

Protocol

1. Data acquisition using imaging flow cytometry

NOTE: Refer to Rodrigues et al.16 with the following modifications, noting that the acquisition regions using IFC may need to be modified for optimal image capture:

  1. For the non-Cyt-B method, perform a cell count using a commercially available cell counter following the manufacturer's instructions (see Table of Materials) on each culture immediately before culture and immed.......

Representative Results

Figure 1 shows the workflow for using the AI software to create a model for the MN assay. The user loads the desired .daf files into the AI software, then assigns objects to the ground truth model classes using the AI-assisted cluster (Figure 2) and predict (Figure 3) tagging algorithms. Once all ground truth model classes have been populated with sufficient objects, the model can be trained using the RF or CNN algorithms. Foll.......

Discussion

The work presented here describes the use of deep learning algorithms to automate the scoring of the MN assay. Several recent publications have shown that intuitive, interactive tools allow the creation of deep learning models to analyze image data without the need for in-depth computational knowledge18,19. The protocol described in this work using a user interface-driven software package has been designed to work well with very large data files and permit the cr.......

Acknowledgements

None.

....

Materials

NameCompanyCatalog NumberComments
15 mL centrifuge tubeFalcon352096
Cleanser - Coulter Clenz Beckman Coulter8546931Fill container with 200 mL of Cleanser.  https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=8546931#2/10//0/25/
1/0/asc/2/8546931///0/1//0/
ColchicineMilliporeSigma64-86-8
Corning bottle-top vacuum filter MilliporeSigmaCLS4307690.22 µm filter, 500 mL bottle
Cytochalasin BMilliporeSigma14930-96-25 mg bottle
Debubbler - 70% IsopropanolMilliporeSigma1.3704Fill container with 200 mL of Debubbler.  http://www.emdmillipore.com/US/en/product/2-Propanol-70%25-%28V%2FV%29-0.1-%C2%B5m-filtred,MDA_CHEM-137040?ReferrerURL=https%3A%2F%2Fwww.google.com%2F
Dimethyl Sulfoxide (DMSO)MilliporeSigma67-68-5
Dulbecco's Phosphate Buffered Saline 1XEMD MilliporeBSS-1006-BPBS Ca++MG++ Free 
Fetal Bovine SerumHyCloneSH30071.03
Formaldehyde, 10%, methanol free, Ultra PurePolysciences, Inc.04018This is what is used for the 4% and 1% Formalin. CAUTION: Formalin/Formaldehyde toxic by inhalation and if swallowed.  Irritating to the eyes, respiratory systems and skin.  May cause sensitization by inhalation or skin contact. Risk of serious damage to eyes.  Potential cancer hazard.  http://www.polysciences.com/default/catalog-products/life-sciences/histology-microscopy/fixatives/formaldehydes/formaldehyde-10-methanol-free-pure/
Guava Muse Cell AnalyzerLuminex0500-3115A standard configuration Guava Muse Cell Analyzer was used.
Hoechst 33342Thermo FisherH357010 mg/mL solution
MannitolMilliporeSigma69-65-8
MEM Non-Essential Amino Acids 100XHyCloneSH30238.01
MIFC - ImageStreamX Mark IILuminex, a DiaSorin company100220A 2 camera ImageStreamX Mark II eqiped with the 405 nm, 488 nm, and 642 nm lasers was used.
MIFC analysis software - IDEASLuminex, a DiaSorin company100220"Image analysis sofware"
The companion software to the MIFC (ImageStreamX MKII)
MIFC software - INSPIRELuminex, a DiaSorin company100220"Image acquisition software"
This is the software that runs the MIFC (ImageStreamX MKII)
Amnis AI softwareLuminex, a DiaSorin company100221"AI software"
This is the software that permits the creation of artificial intelligence models to analyze data
Mitomycin CMilliporeSigma50-07-7
NEAA Mixture 100xLonza BioWhittaker13-114E
Penicllin/Streptomycin/Glutamine solution 100XGibco15070063
Potassium Chloride (KCl)MilliporeSigmaP9541
Rinse - Ultrapure water or deionized waterNANAUse any ultrapure water or deionized water.  Fill container with 900 mL of Rinse.
RNaseMilliporeSigma9001-99-4
RPMI-1640 Medium 1xHyCloneSH30027.01
Sheath - PBSMilliporeSigmaBSS-1006-BThis is the same as Dulbecco's Phosphate Buffered Saline 1x  Ca++MG++ free.  Fill container with 900 mL of Sheath.
Sterile waterHyCloneSH30529.01
Sterilizer - 0.4%–0.7% HypochloriteVWRJT9416-1This is assentually 10% Clorox bleach that can be made by deluting Clorox bleach with water.  Fill container with 200 mL of Sterilzer.
T25 flaskFalcon353109
T75 flaskFalcon353136
TK6 cellsMilliporeSigma95111735

References

Explore More Articles

Micronucleus AssayImaging Flow CytometryArtificial IntelligenceAI ModelConvolutional Neural NetworkCNNMononucleatedBinucleatedPolynucleatedIrregular MorphologyNon apoptotic PopulationBright FieldDNATagging ToolImage Tagging

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved