the cytokinesis-block micronucleus (cbmn) assay€¦ · miura t., hirosaki university graduate...

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

http://www-pub.iaea.org/MTCD/Publications/PDF/EPR-Biodosimetry 2011_web.pdf




Contents

1. Overview of CBMN assay 2. General protocol 3. Application for Mass Casualty Events 4. Other Application


Overview of CBMN assay Sample accession for Cytogenetic Biodosimetry

IAEA, EPR-Biodosimetry 2011


Overview of CBMN assay Comparison of cytogenetic aberration assays

used for dose assessment



Overview of CBMN assay

Cell Cycle




Cytokinesis

Molecular Biology of the Cell (© Garland Science 2008)


Overview of CBMN assay Cytochalasin B inhibits actin polymerization

Atilla-Gokcumen GE, ACS Chem Biol (2010)


Overview of CBMN assay Micronuclei (MN) and nucleoplasmic

bridges (NPB) in the CBMN assay

Fenech M, Nat Protoc (2007)


Overview of CBMN assay The various possible fates of cultured

cytokinesis-blocked cells



Overview of CBMN assay Cells scored in the CBMN ‘‘cytome’’

assay



Overview of CBMN assay MN and NPB formation in cells undergoing

nuclear division




Formation of NMs and NPBs

Thomas P, Mutagenesis (2003)


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)


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


The X-chromosome is almost completely responsible for gender difference


Contents

1. Overview of CBMN Assay 2. General Protocol 3. Application for Mass Casualty Events 4. Other Application


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


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).



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


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


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


General Protocol

Harvest and slide preparation


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.


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


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:



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:



General Protocol

Scoring


< 0.5 Gy: at least 2000 binucleated cells > 0.5 Gy: at least 1000 binucleated cells should be scored


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



General Protocol

Dose Response Curve


At least 8 doses should be used in the range up to 5 Gy


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.


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.


Contents



Application for Mass Casualty Events

Option 1: Triage scoring Option 2: Automation


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


Option 1: Triage scoring

Triage model in CBMN assay

McNamee JP, Radiat Prot Dosimetry (2009) 200 BNC/15 min


Option 2: Automation Commercially available automated system

Zeiss/MetaSystems


Option 2: Automation The RABiT Rapid Automated Biodosimetry Tool


Center for High-Throughput Minimally-Invasive Radiation Biodosimetry, Colombia University, U.S.A.


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.


Option 2: Automation High throughput cytogenetic laboratory and automation


The Armed Forces Radiobiology Research Institute (AFRRI), U.S.A.


Contents



Other Application

Assessment for Medical Exposure



Other Application

Assessment for Medical Exposure

Senthamizhchelvan S, Phys Med (2009)


Other Application Assessment for Environmental Toxicology

Bolognesi C & Fenech M, Nat Protoc (2012)

Mussel


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


Hirosaki University Graduate School of Health Sciences

Tomisato Miura, Ph.D. E-mail: [email protected]

the cytokinesis-block micronucleus (cbmn) assay€¦ · miura t., hirosaki university graduate...

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