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Training Meeting HICARE in collaboration with IAEA
Hiroshima, Japan, June 11, 2013
The cytokinesis-block micronucleus (CBMN) assay
Tomisato Miura, Ph.D. Hirosaki university Graduate School of Health Sciences
BIODOSIMETRY IN THE 21st CENTURY
Miura T., Hirosaki University Graduate School of Health Sciences
Main Text
http://www-pub.iaea.org/MTCD/Publications/PDF/EPR-Biodosimetry%202011_web.pdf
Miura T., Hirosaki University Graduate School of Health Sciences
Contents
1. Overview of CBMN assay 2. General protocol 3. Application for Mass Casualty Events 4. Other Application
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay Sample accession for Cytogenetic Biodosimetry
IAEA, EPR-Biodosimetry 2011
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay Comparison of cytogenetic aberration assays
used for dose assessment
IAEA, EPR-Biodosimetry 2011
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay
Cell Cycle
IAEA, EPR-Biodosimetry 2011
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay
Cytokinesis
Molecular Biology of the Cell (© Garland Science 2008)
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay
Cytokinesis
Molecular Biology of the Cell (© Garland Science 2008)
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay Cytochalasin B inhibits actin polymerization
Atilla-Gokcumen GE, ACS Chem Biol (2010)
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay Micronuclei (MN) and nucleoplasmic
bridges (NPB) in the CBMN assay
Fenech M, Nat Protoc (2007)
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay The various possible fates of cultured
cytokinesis-blocked cells
Fenech M, Nat Protoc (2007)
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay Cells scored in the CBMN ‘‘cytome’’
assay
Fenech M, Nat Protoc (2007)
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay MN and NPB formation in cells undergoing
nuclear division
Fenech M, Nat Protoc (2007)
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay
Formation of NMs and NPBs
Thomas P, Mutagenesis (2003)
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay Inter-individual variation in the frequency of binucleated lymphocytes with micronuclei
Fenech M, Health Phys (2010)
10 healthy females aged 20–45 y 1.5 Gy (dose rate 5 Gy min-1)
Miura T., Hirosaki University Graduate School of Health Sciences
Overview of CBMN assay Effect of age and gender on MNC frequency in
peripheral lymphocytes
Bonassi S, Environ Mol Mutagen 2001
The spontaneous micronucleus yield increases systematically with age. Male: 0.35 MN/1000 BN cells/year
(nuclear power plant workers) 0.44 MN/1000 BN cells/year
(hospital workers) Baseline : 0.31 MN/1000/BN cells/year Female: 0.52 MN/1000/BN cells/year
IAEA, EPR-Biodosimetry 2011
The X-chromosome is almost completely responsible for gender difference
Miura T., Hirosaki University Graduate School of Health Sciences
Contents
1. Overview of CBMN Assay 2. General Protocol 3. Application for Mass Casualty Events 4. Other Application
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol Workflow in CBMN assay
Blood collection
PBMC
PBMC isolation
Blood culture
Hypotonic treatment/ Fixation
Slide preparation
Spreading
Cytocentrifugation
Cytochalasin B
PHA
Staining Scoring
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol Blood collection and shipment
A) Blood sampling should use the collection system, containing lithium or sodium heparin as an anticoagulant.
B) Blood should be collected (at least 5 ml), maintained at approximately 20oC (18-22oC) and sent to the CBMN laboratory as soon as possible within 72 hours after collection.
C) Precautions to ensure the integrity of the container and prevent leakage during shipment shall be observed.
D) Blood samples should be kept cool during shipping with a recommend time range of 6 to 22oC. A temperature recording device should be included to document that the temperature during shipment is controlled.;
E) The CBMN laboratory should be alerted of known infectious samples (HIV or hepatitis especially).
IAEA, EPR-Biodosimetry 2011
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol Isolation of peripheral blood lymphocytes
(mononuclear cells)
Density gradient centrifugation
BD Vacutainer® CPT™ Histopaque-1077, Lymphoprep, Ficoll, etc.
Cells shall be cultured at 37 ± 0.5oC either as whole blood, enriched lymphocyte suspension (buffy coat) or isolated lymphocytes
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol
Blood culture
PBMC
Cytochalasin B (3.0 ~ 6.0 ug/ml)
PHA
37 ± 0.5 oC 0 h 44 h 72 h
Harvest
Fenech M, Nat Protoc (2007)
Duplicate cultures should be performed from each blood sample per individual.
Experimental variation: results for A + C versus B + D Scorer bias: results for A + B versus C + D
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol
Harvest and slide preparation
IAEA, EPR-Biodosimetry 2011
Air drying Cytocentrifugation
• Swelling 0.075 M KCl for 10-15 minutes • Fixation 5:1 methanol:acetic acid * Stockable at – 20 oC freezer after fixation
Overtreatment with hypotonic solution may induce loss of cytoplasm and result in poor satiability of cytoplasm.
Drying, fixing and staining of cells and slide preparation
Harvesting of cells using cytocentrifugation
Appropriate safety protection including gloves must be worn.
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol
Staining and Microscopy
Cells should be stained appropriately so that nuclei and micronuclei can be clearly visualized.
Giemsa Acridine Orange DAPI
Bright field microscopy Fluorescence microscopy
Observation of cells at a magnification of at least 400x is required for scoring of cells and micronuclei. For optimal scoring, however, a higher magnification (eg. 1000x) is recommended.
DIFF Quick
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol
Criteria for Scoring
(a) The cells must be binucleated (BN). (b) The two nuclei in a BN cell shall have intact nuclear membranes and be
situated within the same cytoplasmic boundary. (c) The two nuclei in a BN cell shall be approximately equal in size, staining
pattern and staining intensity. (d) The two nuclei within a BN cell may be unconnected or may be attached
by one or more fine nucleoplasmic bridges, which are no wider than 1/4th of the nuclear diameter.
(e) The two main nuclei in a BN cell may touch but ideally should not overlap each other. A cell with two overlapping nuclei can be scored only if the nuclear boundaries of either nucleus are distinguishable.
(f) The cytoplasmic boundary or membrane of a BN cell shall be intact and clearly distinguishable from the cytoplasmic boundaries of adjacent cells.
The cytokinesis blocked cells that may be scored for MN frequency should have the following characteristics:
IAEA, EPR-Biodosimetry 2011
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol
Criteria for scoring micronuclei
(a) The diameter of MN in human lymphocytes usually varies between 1/16th and 1/3rd of the mean diameter of the main nuclei, which corresponds to 1/256th and 1/9th of the area of one of the main nuclei in a BN cell, respectively.
(b) MN are non-refractile and can therefore be readily distinguished from artefacts such as staining particles.
(c) MN are not linked or connected to the main nuclei. (d) MN may touch but not overlap the main nuclei and the
micronuclear boundary should be distinguishable from the nuclear boundary.
(e) MN usually have the same staining.
MN are morphologically identical to but smaller than the main nuclei. They also shall have the following characteristics:
IAEA, EPR-Biodosimetry 2011
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol
Scoring
IAEA, EPR-Biodosimetry 2011
< 0.5 Gy: at least 2000 binucleated cells > 0.5 Gy: at least 1000 binucleated cells should be scored
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol The background micronucleus frequency
The mean MN value for the appropriate age and gender shall be subtracted from the observed MN frequency in lymphocytes after the radiation exposure event to deduce the likely induced MN frequency value.
Estimation of the radiation exposure dose from the standard curve
1 to 25 year 26 to 50 year 51 to 75 year
at least 3 males 3 females
3 age groups Base-line values
Both genders Observed MN
frequency
Induced MN
frequency value
IAEA, EPR-Biodosimetry 2011
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol
Dose Response Curve
IAEA, EPR-Biodosimetry 2011
At least 8 doses should be used in the range up to 5 Gy
Miura T., Hirosaki University Graduate School of Health Sciences
General Protocol
Nuclear division index (NDI) • Mono-, bi- and multi-nucleated cells are viable cells with an intact cytoplasm
and normal nucleus morphology. • They may or may not contain one or more MN or nuclear buds (NBUD) and
in the case of bi- and multi-nucleated cells they may or may not contain one or more NPB.
* Necrotic and apoptotic cells should not be included amongst the viable cells scored.
IAEA, EPR-Biodosimetry 2011
M1 M2 M3 M4
NDI may be used to define cell cycle progression of the lymphocytes after mitogenic stimulation. The index is in itself not sufficiently robust for direct application as a biodosimeter. Nevertheless the assay is frequently employed as a useful research tool for understanding the cell cycling kinetics of the cultures.
Miura T., Hirosaki University Graduate School of Health Sciences
Contents
1. Overview of CBMN Assay 2. General Protocol 3. Application for Mass Casualty Events 4. Other Application
Miura T., Hirosaki University Graduate School of Health Sciences
Application for Mass Casualty Events
Option 1: Triage scoring Option 2: Automation
Miura T., Hirosaki University Graduate School of Health Sciences
Application for Mass Casualty Events
Option 1: Triage scoring
DIC assay Dose estimation Triage dose assessment 1,000 metaphases
or 100 DICs
- Triage scoring
- QuickScan
50 metaphases or 30 DICs
50 metaphases or 30 DICs
w/o Chr# counting
CBMN assay
Miura T., Hirosaki University Graduate School of Health Sciences
Option 1: Triage scoring
Triage model in CBMN assay
McNamee JP, Radiat Prot Dosimetry (2009) 200 BNC/15 min
Miura T., Hirosaki University Graduate School of Health Sciences
Option 2: Automation Commercially available automated system
Zeiss/MetaSystems
Miura T., Hirosaki University Graduate School of Health Sciences
Option 2: Automation The RABiT Rapid Automated Biodosimetry Tool
IAEA, EPR-Biodosimetry 2011
Center for High-Throughput Minimally-Invasive Radiation Biodosimetry, Colombia University, U.S.A.
Miura T., Hirosaki University Graduate School of Health Sciences
Option 2: Automation The RABiT Rapid Automated Biodosimetry Tool
Garty G, Health Phys (2010)
Center for High-Throughput Minimally-Invasive Radiation Biodosimetry, Colombia University, U.S.A.
Miura T., Hirosaki University Graduate School of Health Sciences
Option 2: Automation High throughput cytogenetic laboratory and automation
IAEA, EPR-Biodosimetry 2011
The Armed Forces Radiobiology Research Institute (AFRRI), U.S.A.
Miura T., Hirosaki University Graduate School of Health Sciences
Contents
1. Overview of CBMN Assay 2. General Protocol 3. Application for Mass Casualty Events 4. Other Application
Miura T., Hirosaki University Graduate School of Health Sciences
Other Application
Assessment for Medical Exposure
IAEA, EPR-Biodosimetry 2011
Miura T., Hirosaki University Graduate School of Health Sciences
Other Application
Assessment for Medical Exposure
Senthamizhchelvan S, Phys Med (2009)
Miura T., Hirosaki University Graduate School of Health Sciences
Other Application Assessment for Environmental Toxicology
Bolognesi C & Fenech M, Nat Protoc (2012)
Mussel
Miura T., Hirosaki University Graduate School of Health Sciences
References
• IAEA. Cytogenetic Dosimetry: Applications in Preparedness for and Response to Radiation Emergencies. EPR-Biodosimetry, 2011
• Molecular Biology of the Cell (© Garland Science 2008) • Atilla-Gokcumen GE, et al. ACS Chem Biol, 5(1):79-90, 2010 • Fenech M. Nat Protoc, 2(5)1084-104, 2007 • Thomas P, et al. Mutagenesis, 18(2):187-94, 2003 • Fenech M. Health Phys, 98(2):234-43, 2010 • Bonassi S, et al. Environ Mol Mutagen, 37(1):31-45, 2001 • McNamee JP, et al. Radiat Prot Dosimetry, 135(4):232-42, 2009 • Garty G, et al. Health Phys, 98(2):209-17, 2010 • Senthamizhchelvan S, et al. Phys Med, 25(2):82-7, 2009 • Bolognesi C, Fenech M. Nat Protoc, 17;7(6):1125-37, 2012
Miura T., Hirosaki University Graduate School of Health Sciences
Hirosaki University Graduate School of Health Sciences
Tomisato Miura, Ph.D. E-mail: [email protected]