Cytokinesis Block Micronucleus (CBMN) Assay

Cytokinesis Block Micronucleus (CBMN) Assay
Lecture Module 7 Lecture: Cytokinesis Block Micronucleus (CBMN) Assay Purpose: This lecture reviews the role of the micronucleus assay for biological dosimetry. Learning objectives: Upon completion of this lecture, the participants will have insight in: The content and formation mechanisms of micronuclei Micronucleus dose response Micronucleus background frequencies Applications of the CBMN assay for biological dosimetry MN centromere assay for low dose detection MN automation for population triage Limitations of the MN assay Ongoing developments Practical issues: NDI, scoring criteria, protocols Duration: 2 hours References: INTERNATIONAL ATOMIC ENERGY AGENCY. Cytogenetic Dosimetry: Applications in Preparedness for and Response to Radiation Emergencies. A Manual. Technical Reports Series No. 405, IAEA, Vienna, 2011 Vral A. et al. The micronucleus assay as a biological dosimeter of in vivo ionising radiation exposure. MUTAGENESIS, 26(1), 11-17, 2011 Thierens H. & Vral A. The micronucleus assay in radiation accidents. ANNALI DELL ISTITUTO SUPERIORE DI SANITA, 45(3), , 2009 Baeyens A. et al. A semi-automated micronucleus-centromere assay to assess low-dose radiation exposure in human lymphocytes. IJRB, Early Online, pp 1-11, 2011 Fenech, M. Cytokinesis-block micronucleus cytome assay. NATURE PROTOCOLS, 2(5), , 2007

Contents What are micronculei and how are they formed
The CBMN assay for radiation dose assessment Micronucleus dose response Micronucleus background frequencies Applications of the CBMN assay for biological dosimetry Improvements of the CBMN assay 1) CBMN centromere assay 2) automated CBMN assay 3) CBMN cytome assay 4) semi-automated CBMN-centromere assay Limitations of the CBMN assay Ongoing developments Practical issues: NDI, Scoring criteria, protocols

What are micronuclei (MN) and how are they formed
Micronuclei are: small extranuclear fragments found in interphase cells Micronuclei represent: acentric chromosome fragments (MN-ac) or whole chromosomes (MN-wc) that are not incorporated in the daughter nuclei during cell division What are micronuclei (MN) and how are they formed? (slides 3-5): Micronuclei can be the result of small acentric chromosome fragments that are not incorporated into the daughter nuclei during cell division. They are enveloped by a nuclear membrane and appear as small nuclei - micronuclei – in the cytoplasm outside the main daughter nuclei. They arise during exposure to various clastogenic agents and are the result of non- or misrepaired DNA double strand breaks (dsb). MN can also contain whole chromosomes that lag behind at anaphase during nuclear division and so are not incorporated in the main nuclei. These MN-wc arise during exposure to aneugenic agents and represent the main fraction of spontaneously occurring MN. (Vral et al. Mutagenesis 2011) 3 3

Micronuclei arise during exposure to clastogenic agents (e.g.,ionizing radiation) (MN-ac) aneugenic agents (MN-wc) A small number of micronuclei (esp. MN-wc) appear spontaneously MN are not radiation specific! As MN can be produced by clastogenic as well as aneugenic agents, they are not radiation specific and as a consequence the CBMN assay is often used as a general toxicology test. 4 4

MN-ac are the result of : Non- or misrepaired DNA double strand breaks (DSB) MN-wc are the result of whole chromosomes that do not attach to the mitotic spindle 5 5

The cytokinesis block micronucleus (CBMN) assay
CBMN assay was developed by Fenech and Morley in 1985 In CBMN assay Cytochalasin B, a cytoplasmic division inhibitor, is added to cell culture to identify cells that underwent one division These cells are identified as binucleate (BN) cells CBMN assay is mostly applied to peripheral blood lymphocytes (PBL), but any cell that can divide can be used CBMN assay is often used as a general toxicology test In the CBMN assay the scoring of MN is restricted to once-divided cells, which are recognised by their binucleate (BN) appearance after inhibition of cytokinesis by cytochalasin B. Cytochalasin B prevents actine polymerisation to occur. This cytokinesis-block prevents confounding effects caused by variable cell division kinetics. 6 6

The CBMN assay for radiation dose assessment
Ionizing radiation is a strong clastogenic agent and thus a potent inducer of DSB and by consequence also of MN In CBMN assay for radiation dose assessment, MN are typically scored in 1000 binucleate PBL Radiation-induced MN in PBL contain mainly acentric chromosome fragments (MN-ac) resulting from non-repaired or misrepaired DNA dsb by non-homologous end joining (NHEJ) repair pathway As ionising radiation is a strong clastogenic agent, and thus a potent inducer of MN, the CBMN assay has proven to be a very reliable, thoroughly validated and standardized technique in the field of radiation biology to evaluate in vivo radiation exposure of occupational, medical or accidentally exposed individuals, and to assess individual in vitro radiosensitivity or cancer susceptibility. Radiation-induced chromosome aberrations such as micronuclei observed in PBL are mainly the result of unrepaired or misrepaired double strand breaks (dsb) by the non-homologous end joining (NHEJ) repair pathway. This repair pathway is active throughout the whole cell cycle and is especially important during Go and G1. It is an error prone repair pathway that seals the breaks without taking care of sequence homology. The main genes involved in this pathway are the heterodimer Ku70/Ku80 (also called XRCC6, XRCC5) and the catalytic subunit of DNA-PK called DNA-PKcs. Other important genes are XRCC4, Ligase 4 and Artemis, 7 7

Human Lymphocytes Advantages:
Easily obtained from the peripheral blood The majority of peripheral blood lymphocytes are in the G0 phase of the cell cycle Phytohaemagglutinin (PHA) converts these resting lymphocytes into dividing cells by which it is possible to visualize DNA lesions in metaphase chromosomes or interphase cells after division Radiation dose assessment with the CBMN assay is predominantly based on data obtained from human peripheral blood lymphocytes (PBL). PBL are easy to obtain by venipuncture and the cells represent a homogeneous population of mainly resting cells (i.e. cells in the Go phase) at the moment of in vivo irradiation. They can be stimulated by phytohaemagglutinin (PHA) to undergo in vitro mitosis. By this ‘in vitro’ stimulation DNA lesions can be visualised in metaphase chromosomes (e.g., the dicentric assay) or in the next interphase cells after division. Once divided cells can be recognised as binucleate cells after cytokinesis-block induced by adding cytochalasion B into the cultures. The lifetimes of lymphocytes are variable and the definition of lifespan can mean either that the cell dies or that it divides. The average time to death of a T cell is estimated to be around 20 years and this value applies to both naïve and memory cells. Remark: Resting lymphocytes collected in a blood sample are themselves the result of cell divisions occurring in vivo. One might therefore expect that some may already contain MN. Thus, it has been shown that scoring MN in mononucleate lymphocytes in conventional blood smears could be particularly useful for monitoring genetic damage in chronically exposed populations. Furthermore, scoring of MN in mononucleate cells could also be used as an interesting additional parameter in the CBMN assay (IAEA TR405 Manual).

The CBMN assay for radiation dose assessment
This figure shows the principle of the CBMN assay in PBL.

Micronuclei in binucleate lymphocytes
Photo A shows an overview of a MN slide after Giemsa staining for light microscopic analysis. It shows 3 BN cells, one monocucleate cell and one apoptotic cell. Photo B shows a few examples of BN cells: a BN cell without MN, one with 1 MN and one with 2 MN.

The MN dose response curve
Radiation-induced MN are strongly correlated with radiation dose and quality The in vitro MN dose-response calibration curves follow the same shape as described for the standard dicentric assay: for low LET radiation, MN follow a linear-quadratic dose response : y = c + αD + βD2 for high LET radiation a linear dependence y = c + αD is observed, with high LET radiation being more effective in generating MN at the same dose levels The dose response curves for MN; the linear and linear quadratic slopes; the effect of dose rate or fractionation; the dependence of radiation quality all follow the same principles as for the dicentric.

The MN dose response curve
An example of a typical MN dose response curve for low LET radiation (Co-60 -rays). The MN yields/1000 BN cells, together with the curve fitting through these points, are shown (bold line). The fitted curves for the upper and lower 95% confidence limits are shown as thin lines. As inter-individual differences in radiation-induced MN response exist, each data point in the dose response curve represents the mean value of data obtained from 10 different donors. MN dose response data can be fitted to the linear or linear quadratic models by the same software that has been developed for dicentrics. The most frequently used are the CABAS and Dose Estimate packages discussed elsewhere in this training course. MN frequency /1000 cells plotted against gamma ray dose 12 12

MN background frequencies
Drawback of the CBMN assay for applications in the low dose range: High and variable spontaneous frequency (2-36 MN/1000 BN cells)  unreliable for low dose assessment below ~ Gy X-or -rays A drawback of the CBMN assay compared to the dicentric assay is the interindividual variability of the spontaneous MN frequency. MN values ranging from 2 to 36 per 1000 BN cells have been reported. This background variability clearly poses limitations on using MN as a biological dosimeter for low doses, where pre-existing individual background frequencies are not known. Estimates have been made, suggesting that the CBMN assay in its basic form could only detect in vivo exposures in excess of Gy X- or gamma-rays. See the IAEA Manual for more discussion. 13 13

Factors influencing the MN background levels: age, gender, diet, exposure to environmental mutagens age: systematic increase with age gender: systematically higher in females vs. males  males: MN/1000BN/year  females: MN/1000BN/year The two most important factors influencing MN background frequency, besides dietary factors and exposure to a wide range of environmental chemicals - clastogens and aneugens, are age and gender. Large scale biomonitoring studies have shown that the spontaneous micronucleus yield increases systematically with age. 14 14

What do spontaneous MN contain? Analysis of MN content was performed using a FISH pan-centromeric probe  the majority of spontaneous MN contain a centromere (> 70%)  whole chromosome  the age increase is due to an increase in centromere-positive MN *often the X-chromosome is involved  explains the observed gender difference Analysis of the MN for the presence of centromeres, by using a pan-centromeric FISH probe, shows that the majority of spontaneous MN contain a centromere (> 70%). Furthermore, the age increase of the spontaneous MN frequencies can be attributed almost totally to centromere-positive MN, reflecting an increased chromosome loss with age. The X-chromosome is almost completely responsible for this spontaneously occurring chromosome loss, hence the higher female background. For instance in a study performed on hospital workers by Thierens et al. (Mutagenesis, 2000) total MN yields of 16.4/1000 for males (mean age = y) and 23.5/1000 for females (mean age = 41.8 y) were found. The number of centromere-negative MN in this study was not significantly different between males (6.7MN) and females (7.7MN). 15 15

This figures shows the principle of the combined micronucleus-centromere assay. The red dots represent the centromeres visualised by a FISH pan-centromeric probe staining. MN containing acentric fragments are centromere-negative, while MN containing whole chromosomes are centromere-positive. 16 16

Centromere negative positive The photos show BN lymphocytes with a centromere-negative and a centromere-positive micronucleus. The centromeres are stained with a FISH pan-centromeric probe (spectrum orange) and the nuclei and micronuclei are counterstained blue with DAPI. The cells are visualised by fluorescence microscopy. 17 17

Applications of the CBMN assay for biological dosimetry (1)
Chernobyl nuclear power plant and Semipalatinsk nuclear test site studies CBMN assay was applied to assess protracted exposure, due to the incorporation of long-lived radionuclides Conclusions: MN frequencies measured in a large number of residents living in the vicinity of the nuclear sites were increased and significantly associated with the estimated internal absorbed dose The CBMN assay has been used to measure radiation exposure in radiation accidents and in large-scale biomonitoring studies. In some accident studies, the CBMN assay was applied to assess protracted exposure, due to the incorporation of long-lived radionuclides by residents in the vicinity of the Chernobyl nuclear power plant and of the Semipalatinsk nuclear test site. MN frequencies measured in a large number of residents in both studies were increased and significantly associated with the estimated internal absorbed dose. Note: Chernobyl nuclear power plant: 80 individuals who were located between 100–200 km from Chernobyl at the time of the accident in 1986 were tested for their MN frequency in BN lymphocytes between 1989 and 1991 after migration to the USA. In this study whole body counts for 134Cs and 137Cs were performed, so that the MN frequency could be related to body dose. The Semipalatinsk nuclear test site: this area has been highly contaminated with radioactive fallout during 40 years of continual weapons testing ( ). Individuals living near the site have been exposed to both internal and external radiation. In order to assess the effects of prolonged radiation, MN analysis was performed in people living in different contaminated villages and one control village. 18 18

For accidents involving few people, only a limited number of MN studies are available: (1) The Istanbul accident with an unshielded former radiotherapy Co source (10 scrap metal workers) (2) Accident (Europe) with a 50 kV contact radiotherapy X ray device (one hospital worker) Blood samples taken between 1 and 6 months after the accident Conclusions: MN-derived dose estimates were in striking agreement with dose values obtained from dicentric studies For radiation accidents involving only few patients, the dicentric assay has generally been used to assess radiation damage soon after the accident, and only a limited number of studies on micronucleus-based dosimetry are available. In the study describing the Istanbul accident where ten scrap metal workers were irradiated by an unshielded former radiotherapy 60Co source and in the study reporting the accident of a hospital worker exposed to a 50 kV contact radiotherapy X-ray device during maintenance, several cytogenetic endpoints were scored. (See IAEA Manual for citations). In both radiation accidents, blood was sampled at different time points, varying between 1 month and 6 months, after the accident took place. In the Istanbul accident, lymphocytes, sampled 1 month after the exposure, were assayed for MN as well as for dicentrics and translocations. MN derived dose estimates gave values in the range 0.7–2.7 Gy, in excellent agreement with doses obtained from dicentrics and FISH. For the hospital worker, a dose estimate of 0.73 Gy was obtained with the CBMN assay in good agreement with a value of 0.62 Gy by dicentrics. 19 19

Application for biomonitoring Several studies have been performed on large populations of nuclear power plant workers and hospital staff  a dose dependent increase of MN and 0.03 MN per 1000 BN cells/mSv was found in 2 different studies performed on nuclear power plant workers (receiving accumulated doses ranging from 10 to 248 mSv) Application of CBMN-centromere assay in first study resulted in increase of MN per 1000 BN cells/mSv, with dose-dependent increase completely due to centromere-negative micronuclei  CBMN assay, especially CBMN-centromere assay allows accumulated doses exceeding 50 mSv to be detected, at population level Large scale biomonitoring studies on radiation workers, like nuclear power plant workers and hospital staff, showed that the CBMN assay, and especially the CBMN assay combined with FISH staining for centromeres can detect radiation-induced chromosomal damage at the population level for accumulated doses received occupationally exceeding 50 mSv. These biomonitoring studies, examining rather large populations (ranging between 70 and 270 subjects) of radiation workers occupationally exposed to accumulated doses ranging from 10 to 248 mSv, showed a clear dependence of micronucleus formation on the accumulated dose. In a study performed by Thierens et al. on nuclear power plant workers, a linear regression of age-corrected individual micronucleus frequencies showed an increase of MN per 1000 BN cells/mSv. Application of the CBMN-centromere assay in a second study performed by Thierens et al. on nuclear power plant workers resulted in a comparable, dose-dependent increase of MN, i.e MN per 1000 BN cells/mSv. This second study further demonstrated that dose-dependence is completely due to centromere-negative micronuclei, thus pointing to the clastogenic effect of ionizing radiation. In a biomonitoring study of Vaglenov et al., an increase of 0.03 MN per 1000 BN cells/mSv was also found in nuclear power plant workers. (See IAEA Manual for citations) 20 20

Improvements of the CBMN assay (1)
Development of the CBMN-centromere assay  to increase the low dose sensitivity Development of the CBMN cytome assay  to score more endpoints related to radiation exposure Development of the CBMN assay for automated scoring  to allow rapid analysis of large sample sizes (triage) Development of a combined CBMN-centromere assay for automated scoring  to combine high speed MN analysis with a more accurate assessment in the low dose range The many applications of the CBMN assay in radiation biodosimetry highlight its important role in assessing radiation exposure but also its shortcomings. By tackling its strong as well as its weak points, important developments have been made. The ultimate goals of these developments were (i) to further increase the sensitivity of the CBMN assay for low-dose assessment, (ii) to make the assay more specific for radiation by scoring nucleoplasmic bridges in BN cells and (iii) to adapt the assay for rapid analysis of a large number of samples. A last improvement was the development of a micronucleus-centromere assay for automated analysis which combines high speed MN analysis with a more accurate assessment in the low dose range. 21 21

The CBMN-centromere assay allows discrimination between centromere-positive MN (MNCM+)(whole chromosomes) and centromere-negative MN (MNCM-)(acentric chromosome fragments) several studies showed that the majority of spontaneous MN are CM+ (70% and more), while most radiation-induced MN are CM- the number of MNCM+ shows only a very small increase with dose manual scoring of MNCM- lowers detection limit to Gy As it has been shown that most of the radiation-induced micronuclei originate primarily from acentric fragments while spontaneous MN generally contain whole chromosomes the application of the CBMN-centromere assay, which uses a pan-centromeric probe to discriminate between centromere-positive and -negative MN, substantially increases the sensitivity of the CBMN assay in the low dose range. The majority of spontaneous micronuclei are centromere-positive (73%) while most radiation induced MN are centromere-negative. The number of centromere- positive MN showed only a very small increase with dose (5.3/Gy/1000 BN cells). By manual scoring of centromere-negative micronuclei in 2000 binucleated cells a detection limit, at the 95% confidence limit, of 0.1 Gy was achieved. 22 22

Scoring of nucleoplasmic bridges (NPBs) in the CBMN cytome assay In CBMN cytome assay NPBs, joining two nuclei in BN cell, can be scored NPBs originate from dicentric chromosomes Another improvement of the CBMN assay is the CBMN cytome assay where a spectrum of chromosomal damage can be assessed including breakage, asymmetric chromosome rearrangement, chromosome loss and non-disjunction as well as necrosis, apoptosis and cytostasis. This method is also specifically used to measure nucleoplasmic bridges (NPB), a surrogate biomarker of dicentric chromosomes which result from either telomere end-fusions or mis- repair of DNA double strand breaks. NPB appear as a strand joining the two nuclei in a BN cell. A strong correlation has been observed between NPB, and dicentric chromosomes and this suggests that NPB could be used for biodosimetry. This application still needs more research. 23 23

Advantage of scoring NPBs for biological dosimetry NPB background frequency is lower than for MN and is not affected by gender NPBs provide a direct method of measuring asymmetrical chromosome rearrangement (dicentrics, rings) in once divided cells NPBs are more radiation specific then MN NPBs are increased in a dose-related manner and correlate well with dicentric and ring chromosome frequencies analyzed in metaphase cells analysis of NPBs is quicker compared to dicentric analysis as in the CBMN assay a large number of BN cells are accumulated As NPB develop from dicentric chromosomes that span across between the two daughter nuclei, one is in fact looking at dicentrics in another way –in simpler images than metaphases. The advantages of dicentrics over MN i.e., greater specificity to radiation and no gender effect therefore also apply to NPB. NPB however have the same advantages as MN compared with dicentrics in more rapid and less labour-intensive scoring. This opens up real prospects of NPB making a significant contribution to the range of biodosimetry techniques that are available for deployment. Furthermore the MN/NPB ratio may provide a “fingerprint” of specific genotoxic exposure. 24 24

Automated scoring for the CBMN assay among all cytogenetic methods, CBMN assay allows most easy and rapid scoring of radiation damage to further increase the throughput of this technique, automated scoring procedures have been developed automated MN scoring is very attractive for: 1) population triage in case of large scale radiation accidents 2) for large-scale assessment of genetic damage in radiation workers receiving a high radiation burden (biomonitoring) automated MN scoring results in a more reproducible analysis Compared to the dicentric assay, which is the standard technique for biological dosimetry, the easy and rapid scoring of MN makes this method very attractive for (1) large scale assessment of genetic damage in radiation workers receiving a high radiation burden and for (2) population triage in case of large scale radiation accidents. Automation of the MN scoring further results in a more reproducible analysis of MN frequencies due to withdrawal of the subjective MN identification by technical staff. 25 25

Automated scoring for the CBMN assay different image analysis systems for automated MN scoring are available: - MN software module integrated in the metaphase finder system MSearch of Metasystems 1) identification of 2 adjacent similar nuclei (e.g. DAPI stained) 2) MN scoring in a circular region defined around the 2 nuclei of the BN cell - MN analysis system running on the PathFinder Cellscan capture station 1) identification of the cytoplasm of Giemsa-stained cells 2) detection of the number of nuclei in the cell, allowing identification of BN cells 3) automated scoring of MN in the BN cell Several algorithms for automated image analysis of the CBMN assay were already developed in the 1990s. These have been much improved so that currently there are two approaches. One automatically identifies by morphological criteria BN cells by the occurrence of two adjacent similarly DAPI stained nuclei. In a second step, MN are detected automatically in a circular area defined around the 2 nuclei of the BN cell (see gallery of BN cells with MN in next slides). A further evaluation of the detected yield of MN by a scorer is not necessary. Another system uses a capture station and two MN analysis workstations. This system identifies firstly the cytoplasm of Giemsa stained cells, then detects the number of nuclei in the cell thus allowing identification of BN cells and in a third step it scores the MN. 26 26

Automated scoring for the CBMN assay The Metasystems instrument in use with the MNScore module for automated scoring of micronuclei 27 27

Automated scoring for the CBMN assay Example of BN cells with and without MN captured by the MN module for automated MN scoring of Metasystems on 1 slide automated scoring of MN in about 2000 BN cells can be performed in less than 8 minutes Example of BN cells captured for automated MN scoring (MNScore) On one slide automated scoring of MN in about 2000 BN cells can be performed in less than 8 minutes. Illustrations produced in the lab of Prof. Vral, Ghent University, Belgium Gallery of captured BN cells with MN 28

The CBMN assay for automated scoring the figure shows a dose response curve obtained for automated MN scoring (by MNScore of Metasystems), based on MN data of 10 individuals Results dose of 1 Gy can be detected with accuracy of 0.2 Gy 95% CI of 0 Gy and 1 Gy doses do not overlap accurate dose estimations are also achieved at higher doses of 2 and 3 Gy The fully automated MN scores obtained in this study were highly correlated with the manual MN scores (r2 = 0.917) and demonstrated that a visual validation was not needed. The reference dose response curve obtained for automated MN scoring, based on MN data of 10 individuals, showed that the uncertainty on a dose determination of 1 Gy amounts to 0.2 Gy. The 95 % confidence intervals of the 0 Gy and 1 Gy doses did not overlap. Accurate dose estimations were also achieved at the higher doses of 2 and 3 Gy. Therefore, the MN scoring system is able to distinguish exposures with doses of 1, 2 or 3 Gy, at very high speed: on one slide 2000 BN cells can be scored for micronuclei in less than 8 minutes. Taken from Willems et al., IJRB 86(1),2-11, 2010 29

Development of a combined CBMN-centromere assay for automated scoring to enhance sensitivity, reliability and processing time of MN assay by combining automated MN assay with FISH pan-centromere scoring This will allow :  systematic biomonitoring of radiation workers exposed to low doses  more accurate assessment of exposure in second phase of mass radiation casualty event - after early triage - when the time constraint will be less strict The disadvantage of the standard CBMN assay is the high and variable spontaneous MN frequency so that only exposures in excess of Gy can be detected. The scoring of MN and recognition of centromeres in MN to allow detection of low dose exposures requires more time and effort compared to the standard CBMN assay. Therefore development of a combined automated MN- centromere scoring procedure would enable a higher sensitivity with a reduced scoring time. It is envisaged that such a combined CBMN-centromere assay for automated scoring in a case of mass radiation casualties will allow a more accurate assessment of exposures in second phase analysis following early triage when time constraints are less strict. 30 30

Development of a combined CBMN-centromere assay for automated scoring In this figure, the data for MNtotal, MNCM+ and MNCM- are graphically represented for doses up to 0.5 Gy. It shows that the increase in the total MN number with increasing dose is almost completely due to the increase in MNCM- , while the amount of MNCM+ remains rather constant. Only for higher doses of 1 and 2 Gy (not shown here) a slight increase in MNCM+ was observed. When calculating the percentage of MNCM-, it was shown in this paper that the spontaneous MN are mainly centromere-positive (range %, mean %) while the % of centromere-negative MN quickly increases with dose (% MNCM- = 32% for a dose of Gy and increasing to 94 % for a dose of 2 Gy). This figure shows the average of the total number of MN (MNtotal), MNCM+ and MNCM- for 10 donors as a function of dose (up to 0.5Gy). The error bars are SEM. (modified from Baeyens et al., IJRB, 2011) 31 31

Development of a combined CBMN-centromere assay for automated scoring Conclusion Semi-automated CBMN-centromere assay combines high speed MN analysis with a more accurate assessment in the low dose range, down to 0.05 Gy which makes it of special interest for large-scale radiation applications 32 32

Limitations of the CBMN assay (1)
Major limitations of CBMN assay are related to: retrospective dosimetry accidents involving partial body irradiation The major limitations of the CBMN assay are related to retrospective dosimetry and accidents involving partial body irradiation. 33 33

Retrospective dosimetry In some accident studies MN data were compared with data obtained by the FISH translocation test, which is the endpoint of choice for retrospective dosimetry Istanbul accident (10 scrap metal workers) and accident of hospital worker with a radiotherapy X ray device (slide 19) blood samples were taken at different time points after the accident (1month -1year) In the radiation accident studies already described before (Istanbul and a hospital worker), where blood samples were taken some months after the accident, micronucleus and dicentric data were also compared with FISH translocation data. Translocations, representing stable chromosome aberrations, are the most reliable endpoint for retrospective dosimetry. 34 34

Retrospective dosimetry Results and conclusions  dose estimates obtained by scoring MN (comparable to dicentrics) are lower (20 to 30%) than dose estimates obtained by scoring translocations  correction of the MN values for the delayed blood sampling in the hospital worker (MN disappear with a half-life of 342 days) result in a dose estimate comparable to that obtained with FISH translocations  underestimation of radiation doses because MN (and dicentrics) are unstable chromosome aberrations which have a limited in vivo persistence In the Istanbul accident, the dose estimates obtained by MN and dicentric scoring were about 20 to 30% lower than the dose estimates obtained by translocation scoring. For the hospital worker, exposed to a 50 kV contact radiotherapy X-ray device, a blood sample was also taken 1 year later and the different endpoints were scored again. The data showed that MN disappear with a half-life of 342 days (for dicentrics a half-life of 377 days was observed). These results are in agreement with the decline in MN yields down to about 60 % observed after 1 year post-treatment in radiotherapy patients. Correction of the MN values for the delayed blood sampling in the hospital worker resulted in a dose estimate that was in good agreement with the dose estimate obtained by scoring FISH translocations. Underestimation of radiation doses in situations of delayed blood sampling is related to the fact that MN as well as dicentrics represent unstable chromosome aberrations which have a limited in vivo persistence, especially after high dose irradiation. For this reason, these cytogenetic assays are less suitable for old or long-term exposures. 35 35

Accidents involving partial body irradiation in practice, most accidents involve partial body exposures (PBE)  problem with PBE: underestimation of dose because of presence of undamaged lymphocytes present outside the irradiation field For dicentric assay mathematical methods exist to calculate PBE The contaminated Poisson method (Dolphin 1970) based on the finding that after whole body (WB) irradiation dicentrics are Poisson distributed The Qdr method (Sasaki and Miyata 1968) For CBMN assay as MN are already slightly overdispersed after WB irradiation the applicability of a MN frequency distribution analysis with respect to a PBE is questionable and still needs further investigation A second limitation of the CBMN assay and also of the dicentric assay, is related to the fact that most accidents involve partial body exposures. In these situations, undamaged PBL, that reside outside of the irradiation field, will dilute the aberration yield, leading to an underestimation of the dose delivered to the exposed tissue. For the dicentric assay however mathematical methods exist to calculate partial body exposure: 1) the contaminated Poisson method (Dolphin 1970) which is based on the fact that after whole body irradiation dicentrics are Poisson distributed and 2) the Qdr method (Sasaki and Miyata 1968). These calculation methods work well but are only useful when a significant part of the body has received a relatively high dose. Both methods are fully described in the IAEA Manual As the distribution of MN is anyhow slightly overdispersed, the suitability of a MN frequency distribution analysis with respect to a partial body irradiation is questionable and still needs further investigation. 36 36

The CBMN assay: practical issues
Scoring criteria for the CBMN assay Nuclear division index (NDI) Protocols 37 37

Scoring criteria for the CBMN assay (1)
Criteria for manual scoring of BN cells Cytokinesis-blocked cells should have following characteristics: (a) cells should be binucleated (BN) (b) two nuclei in a BN cell should be situated within same cytoplasm (c) two nuclei in a BN cell should be approximately equal in size, staining pattern and intensity (d) two nuclei within BN cell may be unconnected or may be attached by one or more fine NPB (e) two main nuclei in BN cell may touch but ideally should not overlap each other (f) BN cells should not overlap each other 38 38

Criteria for manual scoring of MN MN are morphologically identical to but smaller than the main nuclei. They also have the following characteristics: diameter of MN in human lymphocytes should be smaller then 1/3rd of the mean diameter of the main nuclei MN are non-refractile and can therefore be readily distinguished from artefacts such as staining particles MN are not linked or connected to the main nuclei MN may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable MN usually have the same staining intensity as the main nuclei but occasionally staining may be more or less intense These are the criteria that are universally adopted and have been in operation for many years. 39 39

Criteria for scoring NPB in the CBMN-cytome assay Description is given in: - The IAEA Manual and - Cytokinesis-block micronucleus cytome assay. M. Fenech, Nature Protocols 2(5), , 2007 40 40

Nuclear division index (NDI)
In the CBMN assay, the relative frequencies of cells with 1, 2, 3, etc nuclei (NDI) can be used to define cell cycle progression and how this is affected by radiation exposure Calculation of the NDI NDI= M1 + M2 + M3 + M4 / N - 500 cells are scored - M1 to M4 represent the number of cells with one to four nuclei and N is the total number of viable cells scored More details about the calculation of the NDI and its uncertainty are described in the IAEA Manual When scoring CB lymphocyte preparations one observes cells with 1, 2, 3, etc., main nuclei. The relative frequencies of the cells may be used to define cell cycle progression of the lymphocytes after mitogenic stimulation and how this has been affected by the exposure. This is referred to as the NDI. Cells to be scored for NDI should have the following characteristics: mono-, bi and multi-nucleated cells are viable cells with an intact cytoplasm and normal nucleus morphology containing one, two, three or more nuclei respectively. They may or may not contain one or more MN or nuclear buds (NBUDs) and in the case of bi- and multi-nucleated cells they may or may not contain one or more NPBs. Necrotic and apoptotic cells should not be included amongst the viable cells scored. For the calculation of the NDI 500 viable cells are scored to determine the frequency of cells with 1, 2, 3 or 4 nuclei. M1 to M4 in the formula represent the number of cells with one to four nuclei and N is the total number of viable cells scored. The published methods for calculating NDI do not consider its uncertainty. A method for deriving the uncertainty has not been published. It is however described in the IAEA Manual and due to its complexity, a fully worked example is shown in an Annex of the Manual. 41 41

Protocols (1) Standard Cytokinesis-block Micronucleus protocol
a heparinized blood sample is taken and complete culture medium (RPMI+ supplements + 10% FCS ) is prepared The protocol is fully described in an annex of the IAEA Manual. In the next sequence of slides the main steps are illustrated. The blood sample is collected using lithium heparin anticoagulant. Culture medium is composed of RPMI-1640 medium supplemented with 10 to 15% heat inactivated fetal calf serum, L-glutamine and antibiotics. 42 42

0.5 ml of blood is added to complete culture medium Typically 0.5 mL of whole blood is added to 4.5 mL of complete culture medium 43 43

Phytohaemagglutinin (PHA) is added as the mitogen 100 μL of Phytohaemagglutinin (e.g. PHA-M, Sigma, 25 mg/25 mL H2O) is added to the culture to give a final concentration of 20 μg/mL. 44 44

Blood culture is incubated at 37°C and with 5%CO2 The blood is cultured in tissue culture flasks at 37°C, 5% CO2 in air in a humidified atmosphere. 45 45

Protocols (5) Standard Cytokinesis-blocked Micronucleus protocol
after 24 hours incubation cytochalasin B is added to block cytokinesis 20 μL cytochalasin-B (Cyt-B) is added to the culture, at 24 h post PHA stimulation, to give a final concentration of 6 μg/mL. This is the optimum concentration for accumulating BN cells in whole blood cultures. As Cyt-B is difficult to dissolve in aqueous solution a Cyt-B stock solution should be prepared in dimethylsulphoxide (5 mg Cyt-B in 3.3 mL DMSO) and aliquoted and stored at -20ｰC until required. 46 46

Cells are harvested 3 days after culture start (between 68-72h): Harvest protocol: Blood cultures are centrifuged The culture is terminated between h post PHA stimulation. The chosen harvest time should maximize the number of BN cells and minimize the number of mononucleated and multinucleated cells. The cell suspension is transferred from its culture flask to a centrifuge tube and spun gently at 180 g for 10 min. 47 47

Harvest protocol: Cells are hypotonically treated with cold KCl (0.075 M) KCl The supernatant culture medium is removed and the ells are hypotonically treated with 7 mL of cold (4°C) 0.075M KCl to lyse red blood cells. Immediately after, the cell suspension is centrifuged at 180 g for 10 min. 48 48

Harvest protocol: After centrifugation cells are fixed with fixative solution (10 methanol / 1 acetic acid / 11 Ringer salt solution). For the automated scoring 4/1/5 ratio is used. The supernatant is removed and replaced with 7 mL freshly made fixative consisting of methanol: acetic acid (10:1) diluted 1:1 with Ringer’s solution (4.5 g NaCl, 0.21g KCl, 0.12 g CaCl2 in 500 mL H2O). For the automated scoring a 4/1/5 ratio is used. The fixative should be added whilst agitating the cells to prevent clumps forming. The cells are then centrifuged again at 180 g for 10 min. 49 49

Harvest protocol: After centrifugation cells are washed another 2 or 3 times with a fix solution containing 10 methanol / 1 acetic acid (for automation: 4/1) The cells are washed with two or three further changes of freshly prepared fixative consisting of methanol:acetic acid (10:1) (4:1 for automated scoring), this time without Ringer’s solution, until the cell suspension is clear. 50 50

Harvest protocol: After the last fixation step, the supernatant is removed and cells suspended in about 1 ml of fixative are dropped on clean slides, air dried and stained with Giemsa (for manual scoring) or DAPI (for automated analysis). After removing the supernatant to 1 mL or less above the cell pellet (depending on pellet size), the cells are resuspended gently, and the suspension (~ 40 l) is dropped onto clean glass slides and allowed to air dry. For light microscope analysis cells can be stained in 2-6% Giemsa (e.g. Giemsa’s Azur-Eosin-Methylene blue solution, Merck) in HEPES buffer (0.03M ; pH 6.5) during min in the dark, followed by a quick rinse in distilled H2O and air dried. For fluorescence microscopy cells can be stained in acridine orange (10 μg/mL in phosphate buffered saline pH 6.9) for 2-3 sec. For automated MN analysis (with the MN module of Metasystems) cells are stained with DAPI-Vectashield (antifade mounting solution containing DAPI). DAPI-Vectashield is dropped on the slide and a coverslip is added. 51 51

Giemsa stained slide DAPI stained slide The photos show views (x 40 objective) of BN cells stained with Giemsa for light microscopy or with DAPI for fluorescence microscopy. Illustrations produced in the laboratory of Prof. Vral, Ghent University, Belgium 52 52

Cytokinesis Block Micronucleus (CBMN) Assay

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Cytokinesis Block Micronucleus (CBMN) Assay

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