[methods in molecular biology] genotoxicity assessment volume 1044 || micronucleus assay in human...

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Alok Dhawan and Mahima Bajpayee (eds.), Genotoxicity Assessment: Methods and Protocols, Methods in Molecular Biology, vol. 1044, DOI 10.1007/978-1-62703-529-3_10, © Springer Science+Business Media New York 2013

Chapter 10

Micronucleus Assay in Human Cells: Lymphocytes and Buccal Cells

Claudia Bolognesi and Michael Fenech

Abstract

The micronucleus (MN) assay, applied in different surrogate tissues, is one of the best validated cytogenetic techniques for evaluating chromosomal damage in humans. The cytokinesis-block micronucleus cytome assay (CBMNcyt) in peripheral blood lymphocytes is the most frequent method in biomonitoring human populations to evaluate exposure to genotoxic agents, micronutrient defi ciency, or excess and genetic instability. Furthermore recent scientifi c evidence suggests an association between an increased MN fre-quency in lymphocytes and risk of cancer and other age-related degenerative diseases. The micronucleus cytome assay applied in buccal exfoliated cells (BMNCyt) provides a complementary method for measuring DNA damage and cytotoxic effects in an easily accessible tissue not requiring in vitro culture. The protocol for CBMNcyt described here refers to the use of ex vivo whole blood involving 72 h of culture with the block of cytokinesis at 44 h. BMNCyt protocol reports the established method for sample processing, slide preparation, and scoring.

Key words Micronucleus , Chromosomal damage , DNA damage , Lymphocyte , Buccal cell , Cytome , CBMNcyt , BMCyt , Bud , Nucleoplasmic bridge

1 Introduction

The micronucleus cytome assay, applied in different surrogate tissues, is a comprehensive approach for evaluating genomic damage, cell death, and cytostasis in human populations. The cytokinesis- block micronucleus (CBMN) is the preferred assay to evaluate genomic damage in peripheral blood lymphocytes. The CBMN test, developed in the early 1980s [ 1 ] involving the block of cytokinesis with cytochalasin B (Cyt-B), allowed evaluation of the micronuclei frequency in once divided cells accumulated in the binucleated stage, overcoming the confounding effect associated with variations in in vitro cell division kinetics [ 2 ]. The CBMN assay more recently evolved in a comprehensive cytome assay (CBMNCyt) involving the cytological analysis of every scored cell for its viability, mitotic status, and chromosomal damage [ 3 ].

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This assay, applied simply as MN counting in binucleated cells or as comprehensive cytome mode, in isolated lymphocytes or whole-blood cultures, is widely used in human populations to assess the exposure to genotoxic agents, micronutrient defi ciency or excess [ 4 – 6 ], and genetic instability [ 7 , 8 ]. Recent studies report evidence that MN frequency in peripheral lymphocytes is associated with an increased risk of cancer and other age-related degenerative diseases [ 9 – 13 ] further justifying the potential application of CBMN assay in screening programs.

The buccal micronucleus cytome (BMNCyt) assay, as a mini-mally invasive application of the MN assay in exfoliated buccal mucosa cells, was successfully used in biomonitoring inhalation envi-ronmental and/or occupational exposure to genotoxic agents and in the evaluation of the impact of nutrition and lifestyle factors [ 14 ]. The complete potentiality of this assay is not known: important gaps yet subsist on the biological meaning of different cell types and nuclear alterations, other than micronuclei in buccal samples.

An international collaborative project on human micronucleus (HUMN) assays ( www.humn.org ) was launched in 1997, to better understand the signifi cance of this biomarker and to validate its use in human population studies. The fi rst phase of this project focused on CBMN in peripheral blood lymphocytes. The effects of meth-odological and demographic variables on MN frequency were defi ned through the analysis of a large database [ 15 – 17 ]. An inter-national slide scoring exercise using standardized scoring criteria was carried out and allowed to evaluate inter-laboratory and intra- laboratory variability affecting the analysis of data from multicenter studies [ 18 – 21 ]. Furthermore, the HUMN project also led to a prospective study showing that a high MN frequency in lympho-cytes predicts an increased risk of cancer [ 9 ].

More recently a new project (HUMNxl project) was launched to validate the MN assay in exfoliated buccal cells following the same steps experienced for the CBMN assay in human peripheral blood lymphocytes [ 22 , 23 ].

Detailed standardized protocols for CBMNCyt and BMCyt assays were established by the HUMN consortium including sampling, slide preparation, and staining and scoring criteria by taking into account the available procedures, confounding factors, and staining artifacts [ 24 , 25 ].

A special issue, recently published in Mutagenesis (January 2011), provides an overview of the potential applications of the MN test in different surrogate tissues in different fi elds and of the latest advances in understanding the molecular mechanisms under-lying the MN induction and kinetics.

The aim of this chapter is to provide guidelines for the applica-tion of micronucleus cytome protocols in peripheral lymphocytes and exfoliated buccal cells in biomonitoring of human populations.

The experimental protocol for CBMNCyt assay described below refers to the use of ex vivo whole blood culture. The method


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applied to isolated lymphocyte culture was described in detail in a previous paper [ 24 ]. The assay involves the block of cytokinesis with cytochalasin at 44 h and cell harvesting at 72 h of culture. A comprehensive approach is described involving the evaluation of preexisting MN (frequency of MN in mononucleated cells), other different nuclear anomalies (nuclear buds and nucleoplas-mic bridges), and cytotoxicity biomarkers (necrotic and apop-totic cells).

The protocol for BMCyt assay reports the established method for sample processing, slide preparation, and scoring. The proce-dure involves repeated washing with a buffer solution to remove bacteria and cell debris and a fi ltration process to obtain single-cell suspension. Criteria for scoring micronuclei and other nuclear anomalies in buccal cells are also described.

2 Materials

1. Biological safety cabinet (Class II biosafety cabinet to provide personnel, environment, and product protection).

2. CO 2 cell culture incubator. 3. Centrifuge: Benchtop centrifuge speed 0–3,000 rpm operating

at −5 to 25 °C. 4. Cytocentrifuge is recommended for slide preparation, but not

mandatory. Cytocentrifuge cups are supplied with the instru-ment and need to be assembled following the instructions.

5. Microscope with excellent optics for bright-fi eld and fl uores-cence examination of stained slides at 400× and 1,000× magnifi cation.

1. Test tubes for culturing cells. 2. Conical 15 mL polypropylene test tubes. 3. Microscope slides pre-cleaned/ready to use (76 × 26 mm and

1 mm thick) wiped with alcohol and allowed to dry. The slides are stored at −20 °C before use.

4. Coverslips—22 × 50 mm. 5. Filter or Whatman fi lter paper. 6. Multichannel cell counter.

1. Culture medium: RPMI 1640 medium with 2 mM glutamine and 25 mM Hepes sterile liquid. Store at 4 °C. Use at 37 °C when preparing whole blood culture.

2. Fetal bovine serum (FBS): Heat-inactivated, sterile. Store frozen at −20 °C. Thaw in a 37 °C water bath before adding to the culture medium. Once thawed, FBS will remain stable at 4 °C for 3–4 weeks ( see Note 1 ).

2.1 Laboratory Equipment

2.2 Materials for CBMN Cyt Protocol

2.3 Reagents for CBMN Cyt Protocol

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3. Antibiotic/antimycotic solution: 10,000 U/mL penicillin, 10 mg/mL streptomycin, and 25 μg/mL amphotericin.

4. Phytohemagglutinin (PHA) M form: 1 mg/mL, liquid. PHA should be aliquoted into a volume convenient for use in sterile tubes. The aliquots may be stored at −20 °C for up to 6 months. If lyophilized powder form is available reconstitute to prepare 1 mg/mL solution.

5. Lymphocyte culture medium: RPMI-1640 medium, 10 % FBS, 1 % antibiotic/antimycotic solution. Mix 500 mL of RPMI-1640 medium, 50.6 mL FBS, and 6.14 mL of antibiotic/antimycotic solution. Prepare the culture medium in sterile culture-grade glass or plastic bottles. Prepare aliquots of cul-ture tubes containing 4.7 mL of the culture medium. Culture medium can be stored for 1 week at 4 °C before use. 75 μg of PHA is added to each test tube immediately before preparing the cultures.

6. Dimethyl sulfoxide (DMSO). 7. Cyt-B: 300 μg/mL Cyt-B prepared in DMSO and isotonic

saline. Stored at −20 °C ( see Note 2 ). 8. Hypotonic solution: 0.075 M Potassium chloride (KCl).

Prepare 500 mL of solution by dissolving 2.8 g of KCl in 500 mL of distilled water. Do not store. Use only freshly pre-pared solution.

9. 100 % Methanol. 10. Prefi xing solution: 3:5 ratio of methanol:glacial acetic acid.

Mix and put at −20 °C before use. This procedure should be performed in a well-ventilated fume hood with appropriate safety precaution.

11. Fixing solution: 5:1 ratio of methanol:glacial acetic acid. Prepare an adequate volume of fi xing solution. The fi xative should be freshly prepared each time and used at 4 °C. This procedure should be performed in a well-ventilated fume hood with appropriate safety precaution.

12. Sorensen buffer: 9.07 g/L of potassium dihydrogen phosphate (KH 2 PO 4 ), 11.87 g/L of disodium hydrogen phosphate dehy-drate (Na 2 HPO 4 ), pH 6.8. To obtain 100 mL of Sorensen buf-fer solution (pH 6.8), mix 53.4 mL of KH 2 PO 4 with 46.6 mL of Na 2 HPO 4 . Utilize this fi nal solution to prepare Giemsa staining solution.

13. Staining solution: 2 % (v/v) Giemsa in Sorensen buffer. Filter 50 mL of Giemsa’s azur-eosin- methylene blue solution with fi lter paper. Protect from light. Prepare 200 mL of Giemsa staining solution (2 % v/v) by adding 192 mL of distilled water with 4 mL of Sorensen buffer at room temperature.

14. Eukitt or DePex mounting medium.


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1. Small-headed toothbrushes. 2. 30–50 mL polystyrene containers or test tubes. 3. Centrifuge tubes. 4. Swinnex fi lter holders. 5. 100 μm Nylon net fi lters. 6. 10 mL Syringes. 7. Filtercards. 8. Microscope slides superfrosted (76 × 26 mm 1 mm thick)

wiped with alcohol and allowed to dry. 9. 22 × 50 mm Coverslips. 10. Eukitt or DePex mounting medium.

1. Buccal cell buffer: 1.6 g of Tris–HCl, 38.0 g of ethylenediami-netetraacetic acid (EDTA) tetra sodium salt, and 1.2 g of NaCl. Weigh and dissolve in 600 mL of Milli-Q water. Make up the volume to 1,000 mL. Adjust pH to 7.0 using 5 M HCl and autoclave at 121 °C for 30 min. The buffer will last for up to 3 months when stored at room temperature.

2. Saccomanno’s fi xative. 3. Fixing solution: 3:1 (v/v) ethanol:glacial acetic acid. Prepare

an adequate volume of fi xing solution. The fi xative should be freshly prepared each time and used at 4 °C. This procedure should be performed in a well-ventilated fume hood with appropriate safety precaution.

4. DMSO. 5. Light green cytoplasmic stain: Dissolve 1 g of light green in

450 mL of Milli-Q water. When dissolved, make up to 500 mL and fi lter through Whatman No. 1 fi lter paper. Store in the dark at room temperature where it should remain active for 3 years.

6. Ethanol. 7. 5 M HCl. 8. Schiff ’s reagent.

3 Methods

1. Collect fresh blood (2 mL is enough to prepare duplicate cultures for baseline and in vitro-challenged lymphocytes) by venepuncture into vacutainer blood tubes with sodium or lithium heparin anticoagulant. All the procedures of cell culturing and treatment must be performed in a Class II biosafety cabinet.

2. Cultures are prepared within 24 h of phlebotomy ( see Note 3 ).

2.4 Materials for BMN Cyt Protocol

2.5 Reagents for BMN Cyt Protocol

3.1 CBMN Cyt Protocol

3.1.1 Sampling and Whole Blood Cell Culture


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3. 0.3 mL heparinized blood is added to 4.7 mL of complete medium RPMI-1640 supplemented with 10 % FBS, antibiot-ics, and antimycotics in round-bottom culture test tubes. Set up duplicate cultures per subject and/or treatment.

4. Add 75 μL of PHA solution to each test tube. 5. Incubate the test tubes at 37 °C with lids loose in a humidifi ed

atmosphere containing 5 % CO 2 . 6. At 44 h, add 100 μL of the 300 μg/mL Cyt-B solution. 7. Incubate the test tubes at 37 °C. 8. At 28 h after the addition of Cyt-B, harvest the cells for slide

preparation.

1. Transfer the content of each culture to centrifuge tubes. 2. Centrifuge the cell suspension for 10 min at 146 × g . 3. Discard the supernatant and replace with 5 mL of 0.075 M

KCl hypotonic solution. 4. Mix the cells gently into suspension and keep the test tubes at

room temperature for 5 min to allow red blood cell lysis to occur ( see Note 4 ).

5. Add 400 μL of prefi xing solution to each test tube and gently mix ( see Note 5 ).

6. Centrifuge the cell suspension for 10 min at 146 × g . 7. Discard the supernatant and replace with 5 mL of cold methanol

(−20 °C). 8. Gently mix the cell suspension. Samples in methanol can be

stored for months ( see Note 6 ). 9. Centrifuge the cell suspensions in methanol for 10 min at

228 × g . 10. Discard the supernatant and replace with 5 mL of fi xing solution. 11. Gently mix the cell suspension and centrifuge for 10 min at

228 × g . 12. Repeat steps 9 and 10 twice. 13. Discard the supernatant and fi nally resuspend the pellet in

500 μL of fi xing solution.

1. Drop the cell suspension directly onto clean iced slides. 2. Dry the slides in air for at least 20 min. 3. Immerse the microscope slides for 5 min at room temperature

in staining dishes containing 2 % Giemsa solution. 4. Rinse the slides for 5 min in deionized water. 5. Place the slides to be coverslipped on tissue paper. 6. Put two large drops of Eukitt or DePex (use a plastic dropper)

on each coverslip.

3.1.2 Harvesting of Cells

3.1.3 Slide Preparation and Staining


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7. Invert the slide and place on the coverslip. Allow the DePex to spread. Turn the slide over so that the coverslip is on top, and press the coverslip gently to expel any excess DePex and air bubbles.

8. Place the slides on a tray and leave overnight in the fume hood to dry.

9. Store the slides in the slide boxes at room temperature.

1. Slides should be coded before scoring. Slide scoring should be performed by a person not aware of the experiment conditions using transmitted light microscopy at 400× magnifi cation ( see Note 7 ).

2. As a fi rst step, 500 cells are scored for viability and mitotic status. Frequencies of viable (with intact cytoplasm and normal nuclear morphology) mono-, bi-, and pluri-nucleated cells are evaluated to calculate the nuclear division index:

i e NDI N Mono Bi Multi. . [ ( ) ( ) ( )] /= + +2 3 500

Necrotic and apoptotic cells are identifi ed and enumer-ated, but not included among the viable cells scored. The pho-tomicrographs of the different cell types considered in the CBMN Cyt are shown in Figs. 1 , 2 , 3 , 4 , and 5 . Necrotic cells are identifi ed by the presence of numerous vacuoles in the cytoplasm and sometimes in nuclei and damaged cytoplasmic membranes (Fig. 4 ). Apoptotic cells are identifi ed by the pres-ence of chromatin condensation and in the late stages by nuclear fragmentation in intact cytoplasm (Fig. 5 ).

3. Only binucleated cells are scored for DNA damage biomarkers which include micronuclei (MNi), buds, and nucleoplasmic bridges (NPBs). A minimum of 1,000 binucleated cells for each culture needs to be scored ( see Note 8 ).

3.1.4 Slide Scoring

Fig. 1 Mononucleated cell


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Fig. 3 Multinucleated cell

Fig. 4 Necrotic cell

Fig. 2 Binucleated cell


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(a) Binucleated cells have two nuclei equal in size, staining pattern and intensity sometimes touching, but not over-lapping, each other (Fig. 2 ).

(b) MNi have the same staining pattern and intensity as the main nuclei. The diameter of MNi ranges from 1/16 to 1/3 of one of the main nuclei. MNi are not linked or connected to the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary (Fig. 6 ).

(c) Buds are very similar to MNi with the exception that they are connected to one of the main nuclei. Sometimes the buds appear as a small protrusion of the main nuclei (Fig. 7 ).

Fig. 5 Apoptotic cell

Fig. 6 Binucleate cell with one micronucleus


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(d) NPBs are continuous DNA-containing structures linking the two nuclei. The width of an NPB may vary but it should not exceed 1/4 of the diameter of the nuclei (Fig. 8 ).

1. Before starting the cell collection subjects are requested to rinse the mouth two times with 100 mL of water to remove cell debris.

2. Samples are collected by using two small-headed toothbrushes (one for each cheek), which are gently but fi rmly rotated ten times against the inside of the cheek wall in a circular motion to collect buccal cells.

3. Buccal cell samples are collected into two 30 mL containers labeled LC (left cheek) and RC (right cheek), each containing 20 mL of Saccomanno’s fi xative ( see Note 9 ).

3.2 BMNCyt Protocol

3.2.1 Buccal Cell Collection

Fig. 7 Binucleated cell with one micronucleus and a bud

Fig. 8 Binucleated cell with one micronucleus and a nucleoplasmic bridge (NPB)


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4. Buccal cell suspension fi xed in Saccomanno’s solution can be stored at 4 °C for months.

1. Transfer the cell suspension from the containers into two cen-trifuge tubes and centrifuge for 10 min at 580 × g .

2. Aspirate the supernatant leaving approximately 2 mL of cell suspension. Briefl y vortex the cells. Add 8 mL of buccal cell buffer at room temperature and resuspend the cells.

3. Centrifuge the cells for 10 min at 580 × g . 4. Aspirate the supernatant leaving approximately 1 mL of cell

suspension and add 10 mL of buccal cell buffer. 5. Centrifuge the cell suspension for 10 min at 580 × g . 6. Aspirate off the supernatant leaving approximately 1 mL of cell

suspension and add 5 mL of buccal cell buffer. Briefl y vortex the cells ( see Note 10 ).

7. Homogenize the cell suspension for 2–3 min using a handheld tissue homogenizer at medium intensity ( see Note 11 ).

8. To increase the number of clearly separated cells, pass the cell suspension 5–6 times into a syringe using an 18 G needle.

9. Pool the cell suspension from the left and right cheek tubes into a 20 mL syringe, pass the cells through a 100 μm nylon fi lter held in a swinnex holder, and collect the fi ltered cells in a 15 mL centrifuge tubes ( see Note 12 ).

10. Centrifuge the cell suspension for 10 min at 580 × g . 11. Remove the supernatant and resuspend the cells in 1 mL of

buccal cell buffer. 12. Dilute 100 μL of cell suspension into 900 μL of buccal cell

buffer and count the cells using a counting chamber. 13. To further aid in the cellular disaggregation, add 50 μL of

DMSO per mL of cell suspension. 14. Fix the cells using the required volume of ethanol:glacial acetic

acid 3:1 to give a concentration of 80,000 cells per mL. 15. Using a Pasteur pipette drop 100–150 μL of cell suspension

onto a pre-cleaned/ready-to- use microscope slide.

1. Put the fi xed slides (including a spare control slide) for 1 min each in Coplin jars with 50 % and then 20 % ethanol sequen-tially, and then wash with Milli-Q water for 2 min in Coplin jars ( see Note 13 ).

2. Immerse the slides in a Coplin jar of 5 M HCl, for 30 min, and then rinse in running tap water for 3 min (since the cells are now fi xed they will not be lost while rinsing). Include a nega-tive control with each batch to check for effi cacy of 5 M HCl treatment by placing a sample slide in Milli-Q water for 30 min instead of in 5 M HCl ( see Note 14 ).

3.2.2 Buccal Cell Harvesting and Slide Preparation

3.2.3 Slide Staining


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3. Drain the slides and place them in Coplin jar containing Schiff ’s reagent for 60 min in the dark at room temperature. Rinse the slides in running water for 5 min and then rinse again in Milli-Q.

4. Counterstain the slides in 0.2 % light green for 20–30 s and rinse well in Milli-Q water.

5. Immediately place the slides face down on Whatman No. 1 fi lter paper to blot away any residual moisture.

6. Place the slides on a slide tray and allow the slides to dry for about 20 min.

7. Examine the cells at ×100 and ×400 magnifi cation to assess the effi ciency of staining and the density of the cells.

8. Place the slides to be coverslipped on tissue paper and follow steps 6 – 9 of Subheading 3.1.3 of the CBMN Cyt protocol.

1. Slides should be coded and scored blindly using transmitted light and under fl uorescence microscopy at 1,000× magnifi cation.

2. MN and other nuclear anomalies are identifi ed under transmit-ted light and confi rmed under fl uorescence ( see Note 15 ).

3. As a fi rst step 1,000 cells are scored for viability. The photomi-crographs of the different buccal cell types considered in the BMN Cyt are reported in Fig. 9 . The frequency of basal cells, differentiated cells, and cells with anomalies associated with cell death is evaluated. (a) Basal cells are oval or roundish in shape with a large

nucleus:cytoplasm ratio relative to differentiated cell. They are typically smaller in size than differentiated cells (Fig. 9a ).

(b) Differentiated cells are angular and fl at in shape with a smaller nucleus:cytoplasm ratio compared with the basal cell. They are larger than basal cells (Fig. 9b ).

(c) Buccal cells with condensed chromatin are differentiated cells with areas of tightly packed chromatin in the nucleus usually in a striated parallel pattern (Fig. 9c ).

(d) Karyorrhectic cells are characterized by extensive nuclear chromatin aggregation with a loss of nuclear integrity indicative of nuclear fragmentation (Fig. 9d ).

(e) Pyknotic cells have a small shrunken nucleus (diameter ranges from 1/3 to 2/3 of that of a normal nucleus) that is intensely and uniformly stained (Fig. 9e ).

(f) Karyolytic cells have nuclei that are completely depleted of DNA which appear as Feulgen-negative ghostlike images (Fig. 9f ).

3.2.4 Slide Scoring for Buccal MN


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Fig. 9 Various cells evaluated in the buccal micronucleus cytome assay. ( a ) Basal cell; ( b ) differentiated cell; ( c ) buccal cell with condensed chromatin; ( d ) karyorrhectic cell; ( e ) pyknotic cell; ( f ) karyolytic cell


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4. Only differentiated cells are scored for nuclear alterations: MN and buds. A minimum of 2,000 differentiated cells for treatment are suggested to be scored. (a) MNi are round or oval Feulgen-positive bodies not linked

or connected with the main nucleus with the same texture and stain intensity as the main nucleus but with a size that is 1/3–1/16 of the nucleus (Fig. 10a–c ).

(b) Buds appear as small nuclear Feulgen-positive bodies con-nected with the main nucleus or as small protrusions of the nuclei. Buds usually have 1/3–1/16 diameter of the main nucleus but in some cases could be more than 1/2 the main nucleus (broken eggs; Fig. 11a–c ).

4 Notes

1. Avoid repeated refreezing and thawing of the FBS. 2. This material is toxic and a possible teratogen. It must always

be purchased in sealed vials. The preparation of this reagent must be carried out in a cytoguard cabinet and the following personal protection must be used: Tyvek gown, P2 dust mask, double- nitrile gloves, and safety glasses. Preparation : Take the 50 mg vial of Cyt-B from −20 °C and allow it to reach room

Fig. 10 Micronucleus as observed in the buccal micronucleus cytome assay. ( a ) and ( b ) Mononucleated buccal cell with MN; ( c ) binucleated buccal cell with MN

Fig. 11 Buds as observed in the buccal micronucleus cytome assay. ( a ) Mononucleated cell with nuclear bud; ( b ) and ( c ) cells with “broken egg” buds


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temperature. Sterilize the top of the rubber seal with ethanol, but do not remove the seal. Vent the vial seal with a sterile needle and using a syringe add 2.5 mL of sterile DMSO and mix gently (Cyt-B should dissolve readily in DMSO). Remove the 2.5 mL from the vial and eject into a sterile 50 mL Falcon tube. Again add 2.5 mL of sterile DMSO into the vial and remove the contents into the 50 mL Falcon tube. Pipette 20 mL of sterile DMSO into the tube to reach a fi nal volume of 25 mL Cyt-B solution (2 mg/mL) and mix gently. Transfer into a plas-tic sterile bottle, and add 141.7 mL of 0.9 % isotonic saline to reach a fi nal volume of 166.7 mL. This gives a fi nal concentra-tion of 300 μg/mL Cyt-B solution. Mix and dispense adequate volumes into sterile 2 mL cryogenic capped vials to make mul-tiple aliquots and store at −20 °C for up to 12 months.

3. Blood samples can be stored before culturing up to 24 h between 15 and 22 °C without any signifi cant effect on cell survival, mitogen stimulation, and MN frequency [ 26 ]. The time and storage temperature are critical for obtaining a suffi cient number of in vitro-dividing lymphocytes.

4. Hypotonic treatment is a critical step for slide preparation using whole blood cultures. Mild treatment is suggested to avoid the loss of necrotic and apoptotic cells and cells with NPBs.

5. The use of prefi x provides a mild cell fi xation avoiding the formation of cell clumps.

6. This step is introduced to stop or interrupt the process of cell preparation, when large number of samples needs to be processed simultaneously.

7. Detailed scoring criteria established by the HUMN project ( http://www.humn.org ) were described in previous papers [ 19 , 24 ]. For a more comprehensive photo gallery refer to Fenech [ 19 ] and Fenech et al. [ 24 ]. Visual scoring, even though a time- consuming process requiring specifi c expertise, is still the preferred and only method of scoring slides for the complete set of biomarkers in micronucleus cytome assays. The automated scoring of MN is now possible allowing a high throughput for this specifi c biomarker and also can reduce the variability due to the subjective evaluation. Several automated image analysis systems were developed and they are at an advanced stage of validation [ 27 – 30 ].

8. In exceptional circumstances, such as folate defi ciency, addi-tional nuclear anomalies may be formed defi ned as “fused” nuclei (FUS), “circular” nuclei (CIR), and “horse- shoe” nuclei (HS) and could be included as additional CIN biomarkers in the lymphocyte CBMN-Cyt assay [ 31 ].

9. The method for buccal cell collection should be kept constant in order to obtain samples with a homogeneous distribution of the different cell types. It was observed that repeated vigorous


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brushing on the same area can increase the percent of basal cells [ 25 ]. The use of Saccomanno’s fi xative allows preserving the cell suspensions at 4 °C for months before processing.

10. The washing procedure with buffer solution helps to remove bacteria and cell debris, which could confound the scoring.

11. The homogenization step allows single-cell suspension to be obtained.

12. The cell sampling is performed on the inside of both cheeks to maximize cell collection and to obtain a homogeneous cell sus-pension, avoiding unknown biases that may be caused by sam-pling one cheek only. After the fi rst steps of cell processing the cell suspensions from both cheeks are pooled together and then fi ltered to discard cell clumps.

13. The sequential treatment with 50 and 20 % ethanol and then wash with Milli-Q water hydrate the cells before treatment with 5 M HCl.

14. Schiff ’s reagent is a solution that combines chemically with aldehydes to form a bright red product. The treatment of slides with 5 M HCl converts some of the deoxyribose in DNA to aldehydes which can be then detected by Schiff ’s reagent.

15. The staining technique is a critical step in BMCyt. The Feulgen–light green procedure is recommended to score the cell types in bright fi eld and reconfi rm the nuclear alterations using far-red fl uorescence [ 25 ]. The use of staining procedures that are not specifi c for DNA are discouraged because they yield false-positive results [ 32 ]. Detailed scoring criteria were described in previous papers [ 25 ]. For a more comprehensive photo gallery refer to Thomas et al. (2009) [ 25 ].

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