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Introduction

Micronucleus assays are designed to detect

and quantify damages in the genetic

material. DNA damage is induced by

various substances and treatments,

including chemicals, environmental

pollution, and pharmaceuticals. As marker

of DNA damages lagging chromosome

fragments are quantified in micronucleus

assays. When dividing, cells transform

some of the fragments to micronuclei,

which are then visible in the microscope.

Their number reflects the amount of

damage the agent has caused in the DNA.

The Mammalian Erythrocyte Micronucleus

Assay (often also referred to as In-Vivo

Micronucleus Assay) is commonly used in

tests of chemical agents for their impact on

DNA. It involves animals (usually rodents)

treated with the testing agent. In contrast to

in-vitro assays results also reflect influences

of metabolic processes in the live animal.

Target cells are the red blood cells

(erythrocytes) taken from bone marrow or

peripheral blood. Hereby immature

erythrocytes (Polychromatic E.; PCE) are

analyzed, as the mature erythrocytes

(Normochromatic E.; NCE) deplete DNA

and RNA and do not show micronuclei

anymore. PCE and NCE are distinguished

by color (after applying a May-Gruenwald-

Giemsa stain), and the ratio between both

populations bears information about cell

cycle delays due to toxic side effects of the

testing agent.

Details of the mammalian erythrocyte

micronucleus assay are described by the

OECD Guideline #474 from 1997. With

Metafer MetaCyte evaluation of the assay

can be fully automated. Automation

requires cellulose column purified samples

according to the method described first by

Romagna and Staniforth (Mutation Res.

213, 1989).

Fig. 1: Metafer gallery of erythrocytes (PCE are selectively shown). The number of micronuclei is displayed

in the right gallery image corner. The number in the left corner stands for the cell sub-population.

Histograms below show the sub-populations (left) and the micronuclei distribution (right).

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Mammalian Erythrocyte Micronucleus Assay

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Functionality

Scoring automation of the Mammalian Erythrocyte Micronucleus Assay requires samples

prepared from column purified cell solutions (see Romagna and Staniforth, Mutation Res. 213,

1989). It is also recommended that slides are prepared with the help of cyto-centrifugation, so

that erythrocytes are flattened and, thus, easier to analyze for the presence of micronuclei.

With Metafer MetaCyte and the standard classifier for the test a sample is typically scanned in

2 - 4 min. The following functionality is available:

1. Imaging: Slides are automatically scanned (40x magnification), and cells are acquired as

color images (if a monochrome camera is used, an RGB LED light source is required to

generate color images).

2. Color Value Analysis: The first 1,000 cells (amount is adjustable) are used for a color

value analysis. Since color values are highly dependent on slide preparation, it is

required to analyze the absolute values based on a representative number of cells. The

limit between the two subpopulations is the defined by sectioning the color value

histogram into 10% sections, and then determining the least populated section

(excluding the first and the last sections, as those are generally low populated). Cells

within this section are automatically considered as ambiguous, and excluded from the

analysis. As a result a histogram of 4 cell populations is generated: (i) the first 1,000 cells

used for color value determination, (ii) ambiguous cells, (iii) PCE, and (iv) NCE.

3. Micronucleus Analysis: Micronuclei are counted automatically in PCE and NCE.

Micronucleus counts are shown in the gallery images of each cell.

4. Stop Condition: After the analysis of a definable number of PCE the scan is

automatically stopped.

5. Reporting: Data are summarized in an adjustable report showing (i) the number of PCE,

(ii) the number of NCE, (iii) the ratio of PCE and NCE, (iv) the number and ratio of PCE

with micronuclei, and (v) the number and ratio of NCE with micronuclei. Additionally the

number of undefined cells and the search parameters are given.

Automation of the Mammalian Erythrocyte Micronucleus Assay is compliant to OECD guideline

#474, and can be used in GLP environments if the optional Metafer security module is installed.

Search parameters can be adjusted, and reports are customizable. MetaSystems recommends

to perform validation experiments prior to implementation for studies.

Contact MetaSystems Worldwide

Headquarters: MetaSystems Germany tel +49 6205 39610 info@metasystems.de

Asia and Australia: MetaSystems Asia tel +852 2587 8333 info@metasystems-asia.com

USA and Canada: MetaSystems Group tel +1 617 924 9950 info@metasystems.org

India MetaSystems India tel +91 9535 778801 info@metasystems-india.com

Web: www.metasystems-international.com

MetaSystems White Paper

Mammalian Erythrocyte Micronucleus Assay