Radiation Protection Dosimetry (2014), Vol. 159, No. 1–4, pp. 105 –110 Advance Access publication 17 May 2014
doi:10.1093/rpd/ncu161
NEXT GENERATION PLATFORMS FOR HIGH-THROUGHPUT BIODOSIMETRY Mikhail Repin*, Helen C. Turner, Guy Garty and David J. Brenner Center for High-Throughput Minimally Invasive Radiation Biodosimetry, Columbia University Medical Center, New York, NY 10032, USA *Corresponding author: [email protected]
INTRODUCTION Established cytogenetic assays of human lymphocytes are recommended by the International Atomic Energy Agency (IAEA) for response to radiation emergencies(1). In particular, the dicentric assay, known for over 50 y(2), is considered the ‘gold standard’ in radiation biodosimetry(3, 4). Another well-known standardised cytogenetic technique for biological dosimetry is the cytokinesis-block micronucleus (CBMN) assay(1, 5). These two methods are regarded as the most suitable cytogenetic assays in the case of a mass-casualty radiation event due to their reliability and accuracy, when it will be necessary to analyse thousands of samples per day(6). However, a typical cytogenetic laboratory has a throughput less than a hundred of samples per day. Successful attempts to increase the throughput of cytogenetic assays by using 96-well plates, particularly for culturing and cell harvesting in the same plate, are known since the 1970s(7). The benefits from the introduction of high-throughput formats for cytogenetic assays are obvious: manipulating multiple samples simultaneously raises the throughput of the sample preparation as well as decreases the cost of the reagents required for analysis. Moreover, a number of companies have developed different robotic workstations for multiwell plates, including liquid-handling systems, microplate movers, microplate centrifuges, microplate incubators and microplate imaging systems. Nevertheless, high-throughput formats are not generally used for cytogenetic assays of human blood samples due to the lack of convenient approaches, and the conventional use of single vacutainers for blood collection and preparation of samples on microscope slides. In this paper, the general concept of the combined use of microplates and tube racks of standardised
formats at all stages of the biodosimetry assay protocols for increasing its throughput on the example of cytogenetic assays was described. ANSI/SLAS MICROPLATE STANDARDS There are currently a variety of microplates differing in the number of wells, volume and geometry, most of which conform to standards, administered by the American National Standards Institute (ANSI) and the Society for Laboratory Automation and Screening (SLAS). Most of manufactured biotechnological plates meet the Standards ANSI/SLAS 1-2004 through ANSI/SLAS 4-2004 that defines footprint, height, bottom outside flange and well positions. Thus, depending on the need, various combinations of standardised tube racks and plates from different manufactures can be used as platforms for bioassays. The standardisation of microplates allows the use of corresponding 8- or 12-channel manual pipettes or, more elaborate, liquid-handling systems with a 96-channel head for loading and aspirating liquid and for transferring samples between microplates in a high-throughput manner. Microtube racks for blood sample collection, cell culturing, fixation and storage For high-throughput biodosimetry assays, it would be preferable to collect the peripheral blood samples from fingers directly into high-throughput microplates using, for example, capillary-based syringes. However, loading blood samples directly into the wells of a microplate raises the obvious potential of human error (e.g. dispensing the samples into the wrong well or sample labelling). For this task the use
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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Here the general concept of the combined use of plates and tubes in racks compatible with the American National Standards Institute/the Society for Laboratory Automation and Screening microplate formats as the next generation platforms for increasing the throughput of biodosimetry assays was described. These platforms can be used at different stages of biodosimetry assays starting from blood collection into microtubes organised in standardised racks and ending with the cytogenetic analysis of samples in standardised multiwell and multichannel plates. Robotically friendly platforms can be used for different biodosimetry assays in minimally equipped laboratories and on cost-effective automated universal biotech systems.
M. REPIN ET AL.
of individual barcoded microtubes is preferable. These microtubes can then be organised in racks, having the same standards of microplate. Such systems are commercially available now (Figure 1A). Another advantage of using microtubes organised in standardised racks is that they allow for more robust sample tracking. Starting at the blood collection centres, samples can be tracked using the 2D barcodes pre-printed on the bottom of the tubes. To scan the bar code would simply require an inexpensive bar code reader to correlate data of individual with the sample. Similarly, at the biodosimetry laboratory, the information from the sample can be scanned from all tubes using a similar simple reader or a more elaborate 2D barcode plate reader which is also commercially available. Samples organised in standardised racks can be quickly transferred from tubes to the appropriate microplates. If the assay allows, the culturing and fixation of cells can be performed in the same tubes that were used for blood collection. This approach reduces the number of sample transfers, losses of cells and simplifies sample tracking operations. Microwell and microchannel plates for image analysis For imaging of cytogenetic preparations, 96-well plates with an optically clear film or glass bottom can be considered as having the most appropriate high-throughput format. An example of such a highcontent screening plate with square wells giving the maximum useful area is shown in Figure 1B. Although, the surface area of the bottom of each well is smaller by at least a factor of 24 compared with the 75` 25 mm standard microscope slide, the high cell density of the small sample size should be sufficient to generate statistics which are meaningful for triage assays. When the space of one well is not enough to obtain the required number of cells, for higher precision,
it is fairly straightforward to use several wells or plates for the duplicate sample, thus increasing the imaging area to score more cells. In principle, to carry out all steps of cytogenetic assay from blood collection to image analysis, it is enough to use only two platforms: (1) microtubes in rack and (2) 96-well imaging plate. However, other less common imaging plates compatible with ANSI/ SLAS formats can be considered for the development of the high-throughput biodosimetry assays. For example, four standard microscope slides can be fitted into a microplate footprint by using special adaptors (Figure 1C). An advantage of this set-up is that after preparation of the cytogenetic samples, the adaptor can be removed and the slides imaged individually using a standard microscope. This lowthroughput format can be extended further, by using slide adaptors with lattice to divide each slide into several zones, reaching the widely used 96-well plate format on four slides (for example, Microplate Hardware for four microscope slides from Arrayit Corporation, USA). Another possibility for the preparation of multiple samples on four slides in a ‘plateformat’ can be the use of expensive spotting devices which were developed for the preparation of microarrays on slides. Promising microfluidic technologies can also be used for cytogenetic assays(8). By adjusting their surface properties, microchannels can be made ‘self-loading’, so that specialised pumps are unnecessary for loading reagents. An example of microfluidic platforms designed in a standard microplate format is shown in Figure 1D. The microchannel positions in this plate are standardised and correspond to the position of 384-well plates, so that common laboratory 8-channel pipettes and standard 8-channel heads of automated systems can be used for loading samples and reagents. The surface of one microchannel is only 36 mm2 that is 50 times less the surface of a microscope slide.
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Figure 1. Platforms for high-throughput biodosimetry: rack with 96 2D barcoded tubes with caps (Thermo Fisher Scientific, USA) (A); 96-square well plate with optically clear bottom (Ibidi GmbH, Germany) (B); slide-adaptor plate with four microscopic slides (Microfluidic ChipShop GmbH, Germany) (C) and 64 microfluidic channel plate (microfluidic ChipShop GmbH, Germany) (D).
NEXT GENERATION PLATFORMS FOR HIGH-THROUGHPUT BIODOSIMETRY
CYTOGENETIC PROCEDURES IN NEXT GENERATION PLATFORMS FOR HIGHTHROUGHPUT BIODOSIMETRY Procedures of current cytogenetic biodosimetry assays are based on relatively simple standard methods wellknown for a long time(9 – 11). To adapt these assays for next generation cytogenetic platforms, there is a need to address the reduced sample volumes and surface area. Blood sample collection
Cell culturing The CBMN and dicentric assays (as well as other cytogenetic assays) require culturing cells to division. This can all be done within the multiwell plate keeping in mind the need to select a plate with sufficient capacity according to the sample size. In the recent manual of the IAEA(1), emphasis was placed on the choice of culturing medium, its components (L-glutamine, serum, antibiotics, mitogens) and their testing before setting up the complete culturing medium. However, complete and tested cytogenetic media are commercially available and should be used whenever possible. Particularly, a ‘ready-to-use’ complete medium PB-MAX (Life Technologies, USA), gives satisfactory and reproducible results in the laboratory. Since in microtubes reduced culture volumes are used (up to 1 ml, compared with 5–10 ml using a
Cell harvesting and fixation At the end of the culturing, according to standard procedures, for both the dicentric and CBMN assays, lymphocytes must be treated with hypotonic solution and fixed. These steps involve multiple washes with fixative by centrifugation. The cell harvesting procedure in a microplate format is not complicated and similar to the traditional technique(7). Using traditional methods, 5–10 ml of blood cultures is transferred into 15-ml tubes and 10–30-min centrifugation times are used. With the use of tube plates/microplates, centrifugation time is cut to a few minutes, thus saving at least an hour from the total cell harvesting time. In a centrifuge, multiple 96-tube racks, 96-well plates and/or 384-well plates containing hundreds or even thousands of samples can be loaded and processed during one run. Few such runs give the throughput of sample processing far beyond the total current capacity of the established biodosimetry network with 4000 samples per day(17). Cell spreading and staining After harvesting and fixation of lymphocytes, cells can be prepared immediately for staining, stored at 2208C (for years) or shipped to other laboratories. For high-throughput preparation of cells for imaging and analysis, cell spreads can be prepared in screening multiwell and multichannel plates as well as on standard microscope slides. Cell spreads in multiwell plates can be made by dropping few microlitres of fixed cell suspension or by the use of a relatively straightforward but efficient cytospin technique for larger sample volumes. The
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Using the dicentric assay, 500– 1000 metaphase lymphocytes are typically necessary to provide a precise ionising radiation dose estimate. Samples of ,100 cells were historically regarded as inadequate for dose estimation(12); however, following the Chernobyl accident, it was found that 50 metaphase cells can give useful dose estimates using the dicentric assay(13). Nowadays, the IAEA considers 50 metaphase cells for the dicentric assay and 200 binucleated cells for CBMN assay as appropriate numbers for triage purposes(1). According to the last NATO dosimetry study the scored number of cells can be reduced from 50 to 20 using the dicentric analysis without loss of precision of triage dose estimates, at least for homogenous exposure scenarios(14). About 105 lymphocytes can be isolated from 30 ml of fingerstick-derived blood samples on average(15). Thus, for triage biodosimetry purpose, a sufficient number of lymphocytes for cytogenetic analysis can be collected from very small amounts of blood. Moreover, it is preferable to use fingerstick over venipuncture as it is minimally invasive, easier and faster(16). Here the authors propose to collect blood samples by fingerstick using a standard lancet and transfer samples using heparinised capillaries having a set volume (10 –100 ml) into individually barcoded microtubes organised in 96-tube racks (Figure 1A).
traditional assay), the cost per sample of using an offthe-shelf complete medium is not higher than that for a traditional assay with self-made medium. The pre-made medium has an added advantage that it undergoes strict quality control by the vendor, ensuring reliable and reproducible results. This is of critical importance when performing medical diagnostics on a large population. According to the recommendations of the IAEA(1), lymphocytes can be isolated before culturing; however, additional manipulations using lymphocyte separation medium and the transfer to culture plates are needed. Setting up the cultures is simplified in the case of using whole blood culturing. For whole blood samples, it is necessary only to add complete medium into tubes and put tube racks into a cell incubator. At that, it is unnecessary to close each tube, it is sufficient to put a lid on the rack. A compact cell incubator can fit several hundreds of plates, or thousands of samples. During culturing, concentrated solutions of demecolcine (Colcemid) or Cytochalasin B can be quickly added in a high-throughput manner to achieve the required final concentration.
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Imaging In principle, the imaging of cytogenetic samples prepared in high-throughput plates can be done on both, direct and inverted, microscope systems with an appropriate stage configuration adapted for the chosen format. Inverted microscope systems are preferable for usage with most of imaging plates due to their design. Samples in multiwell and multichannel plates can be scored manually; however, it is extremely labour intensive and incompatible with high throughput. Several companies offer high-content imaging systems for multiwell plates with automated hardware and specialised software (e.g. the IN CELL series, General Electric, USA). Commercial high-content imaging systems are expensive and are not available in most cytogenetic laboratories. More cost-effective, automated imaging systems for multiwell plates as well as for multichannel plates can be developed in house as it was done for microscope slides(15, 21).
the same approach of using the combination of tubes organised in standardised racks for blood collection and multiwell plates for screening can be used. Traditional protocols for proved biodosimetry assays can be used directly on new platforms in standard cytogenetic laboratories described above with corresponding volume adjustment; however, it is desirable to develop new protocols that will have higher throughput of biological sample preparation and will have smaller reagent consumption. In this connection, automation of assays using new robotic-friendly platforms described above and emerging cytogenetic techniques such as fluorescent in situ hybridization (FISH) in microchannels are of special interest. Automation For fast and efficient response to radiation events involving mass casualties, it has become imperative to automate cytogenetic dose assessment methods to increase throughput (1). Robotic systems for automation of cytogenetic specimen preparations are known for .20 y(24, 25) and focussed on by the IAEA in 2011(1). It is considered that such systems must have many specialised modules for different processes. For example, blood sampling module, lymphocyte isolation system, slide preparation device, a device for staining and the application of coverslips and so on(1). Each of these modules is expensive, in particular when requiring ‘automation-compatible’ devices which provide clearance for robotic loading of samples and allow control by centralised software. Currently, the direct automation of cytogenetic assays(24, 25) leads to expensive and inefficient systems with yields 1000 samples per week(26). An example of a higher throughput system is the Columbia University Rapid Automated Biodosimetry Tool (RABiT)(16). The RABiT isolates lymphocytes from blood samples and processes them in filter bottom multiwell plates. Application of the RABiT for highthroughput biodosimetry using the g-H2AX assay is described elsewhere(15). The task of the automation of cytogenetic assays on next generation platforms is simplified due to the use of the ANSI/SLAS microplate format from the beginning (blood samples arrive at cytogenetic centre already in 96 tube rack format) and availability of different corresponding automated microplate systems. Moreover, some pre-existing commercial robotic systems can be programmed for biodosimetry assays and used in the case of emergency. Care needs to be taken that the automated system is tested and pre-certified by the appropriate health authorities for its new intended use.
FUTURE DEVELOPMENTS New radiation-responsive biomarkers can be found and corresponding assays developed due to advances in proteomics(22) and genomics(23). For these assays,
Fluorescence in situ hybridization Fluorescent in situ hybridization improves the detection of different types of aberrations and can be used
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cytospin technique uses centrifugation to quickly sediment the cells onto the surface of glass slides or well bottoms to form a uniform monolayer(18). After few minutes of centrifugation, the fixative is removed from each well and cells remaining on the bottom surface are allowed to dry in air(11). The staining procedure can be completed by loading, for example, a low concentration DAPI solution in PBS buffer in a volume necessary to cover the entire surface of the well bottom. Low-throughput preparation of cell spreads on slides in plate adaptors can be done according to the standard protocol by dropping fixed cell suspension and waiting for complete evaporation of the fixative. Simple staining for scanning on a fluorescent microscope can be performed by dispensing small drops of DAPI with antifade and placing a coverslip on it. Alternatively, slides may be placed in an automatic stainer or stained manually. In the case of using special adaptors forming high-throughput format of 96 wells on four slides, the procedures of cell spreading and staining can be done similar to those procedures described above for multiwell plates. Preparation of cell spreads in microchannels is a more complicated task due to the action of liquid flow forces. In recent years, different methods for the preparation of cells for cytogenetic assays in microchannels including the preparation of metaphase cell have been developed(19, 20). Prepared samples can be stained in microchannels using only a few microlitres of dye, for example, DAPI solution.
NEXT GENERATION PLATFORMS FOR HIGH-THROUGHPUT BIODOSIMETRY
in all cytogenetic methods recommended by the IAEA for radiation dose estimations: translocation assay(1), dicentric assay(27), micronucleus assay(28) and PCC assay(29). Moreover, FISH can be used for the automation of the scoring process, because it facilitates the development of fast automated image systems(30, 31). The application of molecular cytogenetic methods for mass-casualty radiation events is complicated by the introduction of additional steps in the assay and is costly due to the expensive FISH probes. Nevertheless, FISH, in principle, can be performed in multiwell plates, but it is necessary to load at least 50 ml of probe to cover the surface of one well of standard 96-well plate without using coverslips that is comparable with the volume of 25 –30 ml of probe per microscope slide that is achieved by the use of coverslip. FISH miniaturization through the use of microfluidic technologies is a more promising approach in molecular cytogenetics due to the use of significantly reduced volumes of reagents corresponding to the dimensions of microchannels(8, 20). The preliminary experiments of this study have shown the possibility of using FISH technique on human lymphocytes in microchannels of plate compatible with the ANSI/SLAS microplate format (Figure 2).
FUNDING This work was supported by grant number U19 A1067773, the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, from the National Institute of Allergy and Infectious Disease, National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of National Institute of Allergy and Infectious Diseases or the National Institutes of Health.
REFERENCES
CONCLUSIONS The combined use of standardised microtube racks and multiwell plates represents a new path to increasing throughput of cytogenetic assays by an order of magnitude or more allowing their use in population screening following a radiological event. Moreover, new cytogenetic platforms having ANSI/SLAS microplate formats allow an automation of biodosimetry assays on universal biotech robots. 109
1. International Atomic Energy Agency. Cytogenetic dosimetry: applications in preparedness for and response to radiation emergencies. IAEA Emergency Preparedness and Response Series. IAEA (2011). 2. Bender, M. A. and Gooch, P. C. Types and rates of X-ray-induced chromosome aberrations in human blood irradiated in vitro. Proc. Natl Acad. Sci. USA 48, 522–532 (1962). 3. Pinto, M. M., Santos, N. F. and Amaral, A. Current status of biodosimetry based on standard cytogenetic methods. Radiat. Environ. Biophys. 49, 567–581 (2010). 4. Blakely, W. F., Salter, C. A. and Prasanna, P. G. Earlyresponse biological dosimetry—recommended countermeasure enhancements for mass-casualty radiological incidents and terrorism. Health Phys. 89, 494– 504 (2005). 5. Vral, A., Fenech, M. and Thierens, H. The micronucleus assay as a biological dosimeter of in vivo ionising radiation exposure. Mutagenesis 26, 11–17 (2011). 6. Rothkamm, K. et al. Comparison of established and emerging biodosimetry assays. Radiat. Res. 180, 111–119 (2013). 7. de Jong, B., Anders, G. J. and van der Meer, I. H. Chromosome preparations from microplate cultures of man, dog, rat, and mouse. Hum. Genet. 33, 295– 298 (1976).
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Figure 2. Metaphase spread of human lymphocyte with two dicentrics after exposure to 4 Gy of 137Cs gamma-rays prepared in a microchannel of the microfludic plate (shown in Figure 1D): DAPI-stained (A); FISH with centromere FAM-PNA probe (B) and FISH with telomere Cy3-PNA probe (C). Fifty microlitres of peripheral human blood was cultured and fixed in microtubes organised in rack (shown on Figure 1A) according to the IAEA protocol(1) for whole blood culture with five times reduced volumes and 2-min centrifugation time. Standard FISH was performed according to the manufacture’s recommendation (Panagene, Inc., Korea) with 7-ml volumes used for loading fixed cell suspension and reagents into microchannels of plate. Arrows show two dicentrics: ‘þ’—two centromere signals and ‘2’—with no telomere signal between centromeres.
M. REPIN ET AL. 21. Furukawa, A., Minamihisamatsu, M. and Hayata, I. Low-cost metaphase finder system. Health Phys. 98, 269– 275 (2010). 22. Ossetrova, N. I. and Blakely, W. F. Multiple bloodproteins approach for early-response exposure assessment using an in vivo murine radiation model. Int. J. Radiat. Biol. 85, 837– 850 (2009). 23. Tucker, J. D., Joiner, M. C., Thomas, R. A., Grever, W. E., Bakhmutsky, M. V., Chinkhota, C. N., Smolinski, J. M., Divine, G. W. and Auner, G. W. Accurate gene expression-based biodosimetry using a minimal set of human gene transcripts. Int. J. Radiat. Oncol. Biol. Phys. 88, 933–939 (2014). 24. Spurbeck, J. L., Carlson, R. O., Allen, J. E. and Dewald, G. W. Culturing and robotic harvesting of bone marrow, lymph nodes, peripheral blood, fibroblasts, and solid tumors with in situ techniques. Cancer Genet. Cytogenet. 32, 59– 66 (1988). 25. Hayata, I., Tabuchi, H., Furukawa, A., Okabe, N., Yamamoto, M. and Sato, K. Robot system for preparing lymphocyte chromosome. J. Radiat. Res. (Tokyo). 33(Suppl.), 231– 241 (1992). 26. Prasanna, P. G., Moroni, M. and Pellmar, T. C. Triage dose assessment for partial-body exposure: dicentric analysis. Health Phys. 98, 244–251 (2010). 27. Fomina, J., Darroudi, F., Boei, J. J. W. A. and Natarajan, A. T. Discrimination between complete and incomplete chromosome exchanges in X-irradiated human lymphocytes using FISH with pan-centromeric and chromosome specific DNA probes in combination with telomeric PNA probe. Int. J. Radiat. Biol. 76, 807–813 (2000). 28. Darroudi, F., Meijers, C. M., Hadjidekova, V. and Natarajan, A. T. Detection of aneugenic and clastogenic potential of X-rays, directly and indirectly acting chemicals in human hepatoma (Hep G2) and peripheral blood lymphocytes, using the micronucleus assay and fluorescent in situ hybridization with a DNA centromeric probe. Mutagenesis 11, 425 –433 (1996). 29. Brown, J. M., Evans, J. and Kovacs, M. S. The prediction of human tumor radiosensitivity in situ: an approach using chromosome aberrations detected by fluorescence in situ hybridization. Int. J. Radiat. Oncol. Biol. Phys. 24, 279– 286 (1992). 30. Lucas, J. N., Tenjin, T., Straume, T., Pinkel, D., Moore, D. II, Litt, M. and Gray, J. W. Rapid human chromosome aberration analysis using fluorescence in situ hybridization. Int. J. Radiat. Biol. 56, 35–44 (1989). 31. Schunck, C. and Mohammad, E. Automated analysis of FISH-stained HER2/neu samples with Metafer. Methods Mol. Biol. 724, 91– 103 (2011).
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8. Kwasny, D., Vedarethinam, I., Shah, P., Dimaki, M., Silahtaroglu, A., Tumer, Z. and Svendsen, W. E. Advanced microtechnologies for detection of chromosome abnormalities by fluorescent in situ hybridization. Biomed. Microdevices 14, 453– 460 (2012). 9. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M. and Hungerford, D. A. Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res. 20, 613– 616 (1960). 10. Hungerford, D. A. Leukocytes cultured from small inocula of whole blood and the preparation of metaphase chromosomes by treatment with hypotonic KCl. Stain Technol. 40, 333 –338 (1965). 11. Rothfels, K. H. and Siminovitch, L. An air-drying technique for flattening chromosomes in mammalian cells grown in vitro. Biotech. Histochem. 33, 73– 77 (1958). 12. Bender, M. A., Awa, A. A., Brooks, A. L., Evans, H. J., Groer, P. G., Littlefield, L. G., Pereira, C., Preston, R. J. and Wachholz, B. W. Current status of cytogenetic procedures to detect and quantify previous exposures to radiation. Mutat. Res. 196, 103 –159 (1988). 13. Pyatkin, E. K., Nugis, V. Yu. and Chirkov, A. A. Absorbed dose estimation according to the results of cytogenetic investigations of lymphocyte cultures of persons who suffered in the accident at Chernobyl atomic power station. Radiat. Med. 4, 52– 58 (1989). 14. Beinke, C. et al. NATO DOSIMETRY STUDY: Laboratory intercomparison of the dicentric chromosome analysis assay. Radiat. Res. 180, 129– 137 (2013). 15. Turner, H. C. et al. Adapting the g-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects. Radiat. Res. 175, 282– 290 (2010). 16. Garty, G., Karam, A. and Brenner, D. J. Infrastructure to support ultra high throughput biodosimetry screening after a radiological event. Int. J. Radiat. Biol. 87, 754– 765 (2011). 17. Maznyk, N. A., Wilkins, R. C., Carr, Z. and Lloyd, D. C. The capacity, capabilities and needs of the WHO BioDoseNet member laboratories. Radiat. Prot. Dosim. 151, 611–620 (2012). 18. Koh, C. M. Preparation of cells for microscopy using cytospin. Methods Enzymol. 533, 235– 240 (2013). 19. Zanardi, A., Bandiera, D., Bertolini, F., Corsini, C. A., Gregato, G., Milani, P., Barborini, E. and Carbone, R. Miniaturized FISH for screening of onco-hematological malignancies. Biotechniques 49, 497–504 (2010). 20. Vedarethinam, I., Shah, P., Dimaki, M., Tumer, Z., Tommerup, N. and Svendsen, W. E. Metaphase FISH on a chip: miniaturized microfluidic device for fluorescence in situ hybridization. Sensors (Basel) 10, 9831–9846 (2010).
doi:10.1093/rpd/ncu161
NEXT GENERATION PLATFORMS FOR HIGH-THROUGHPUT BIODOSIMETRY Mikhail Repin*, Helen C. Turner, Guy Garty and David J. Brenner Center for High-Throughput Minimally Invasive Radiation Biodosimetry, Columbia University Medical Center, New York, NY 10032, USA *Corresponding author: [email protected]
INTRODUCTION Established cytogenetic assays of human lymphocytes are recommended by the International Atomic Energy Agency (IAEA) for response to radiation emergencies(1). In particular, the dicentric assay, known for over 50 y(2), is considered the ‘gold standard’ in radiation biodosimetry(3, 4). Another well-known standardised cytogenetic technique for biological dosimetry is the cytokinesis-block micronucleus (CBMN) assay(1, 5). These two methods are regarded as the most suitable cytogenetic assays in the case of a mass-casualty radiation event due to their reliability and accuracy, when it will be necessary to analyse thousands of samples per day(6). However, a typical cytogenetic laboratory has a throughput less than a hundred of samples per day. Successful attempts to increase the throughput of cytogenetic assays by using 96-well plates, particularly for culturing and cell harvesting in the same plate, are known since the 1970s(7). The benefits from the introduction of high-throughput formats for cytogenetic assays are obvious: manipulating multiple samples simultaneously raises the throughput of the sample preparation as well as decreases the cost of the reagents required for analysis. Moreover, a number of companies have developed different robotic workstations for multiwell plates, including liquid-handling systems, microplate movers, microplate centrifuges, microplate incubators and microplate imaging systems. Nevertheless, high-throughput formats are not generally used for cytogenetic assays of human blood samples due to the lack of convenient approaches, and the conventional use of single vacutainers for blood collection and preparation of samples on microscope slides. In this paper, the general concept of the combined use of microplates and tube racks of standardised
formats at all stages of the biodosimetry assay protocols for increasing its throughput on the example of cytogenetic assays was described. ANSI/SLAS MICROPLATE STANDARDS There are currently a variety of microplates differing in the number of wells, volume and geometry, most of which conform to standards, administered by the American National Standards Institute (ANSI) and the Society for Laboratory Automation and Screening (SLAS). Most of manufactured biotechnological plates meet the Standards ANSI/SLAS 1-2004 through ANSI/SLAS 4-2004 that defines footprint, height, bottom outside flange and well positions. Thus, depending on the need, various combinations of standardised tube racks and plates from different manufactures can be used as platforms for bioassays. The standardisation of microplates allows the use of corresponding 8- or 12-channel manual pipettes or, more elaborate, liquid-handling systems with a 96-channel head for loading and aspirating liquid and for transferring samples between microplates in a high-throughput manner. Microtube racks for blood sample collection, cell culturing, fixation and storage For high-throughput biodosimetry assays, it would be preferable to collect the peripheral blood samples from fingers directly into high-throughput microplates using, for example, capillary-based syringes. However, loading blood samples directly into the wells of a microplate raises the obvious potential of human error (e.g. dispensing the samples into the wrong well or sample labelling). For this task the use
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://rpd.oxfordjournals.org/ at Carleton University on June 25, 2014
Here the general concept of the combined use of plates and tubes in racks compatible with the American National Standards Institute/the Society for Laboratory Automation and Screening microplate formats as the next generation platforms for increasing the throughput of biodosimetry assays was described. These platforms can be used at different stages of biodosimetry assays starting from blood collection into microtubes organised in standardised racks and ending with the cytogenetic analysis of samples in standardised multiwell and multichannel plates. Robotically friendly platforms can be used for different biodosimetry assays in minimally equipped laboratories and on cost-effective automated universal biotech systems.
M. REPIN ET AL.
of individual barcoded microtubes is preferable. These microtubes can then be organised in racks, having the same standards of microplate. Such systems are commercially available now (Figure 1A). Another advantage of using microtubes organised in standardised racks is that they allow for more robust sample tracking. Starting at the blood collection centres, samples can be tracked using the 2D barcodes pre-printed on the bottom of the tubes. To scan the bar code would simply require an inexpensive bar code reader to correlate data of individual with the sample. Similarly, at the biodosimetry laboratory, the information from the sample can be scanned from all tubes using a similar simple reader or a more elaborate 2D barcode plate reader which is also commercially available. Samples organised in standardised racks can be quickly transferred from tubes to the appropriate microplates. If the assay allows, the culturing and fixation of cells can be performed in the same tubes that were used for blood collection. This approach reduces the number of sample transfers, losses of cells and simplifies sample tracking operations. Microwell and microchannel plates for image analysis For imaging of cytogenetic preparations, 96-well plates with an optically clear film or glass bottom can be considered as having the most appropriate high-throughput format. An example of such a highcontent screening plate with square wells giving the maximum useful area is shown in Figure 1B. Although, the surface area of the bottom of each well is smaller by at least a factor of 24 compared with the 75` 25 mm standard microscope slide, the high cell density of the small sample size should be sufficient to generate statistics which are meaningful for triage assays. When the space of one well is not enough to obtain the required number of cells, for higher precision,
it is fairly straightforward to use several wells or plates for the duplicate sample, thus increasing the imaging area to score more cells. In principle, to carry out all steps of cytogenetic assay from blood collection to image analysis, it is enough to use only two platforms: (1) microtubes in rack and (2) 96-well imaging plate. However, other less common imaging plates compatible with ANSI/ SLAS formats can be considered for the development of the high-throughput biodosimetry assays. For example, four standard microscope slides can be fitted into a microplate footprint by using special adaptors (Figure 1C). An advantage of this set-up is that after preparation of the cytogenetic samples, the adaptor can be removed and the slides imaged individually using a standard microscope. This lowthroughput format can be extended further, by using slide adaptors with lattice to divide each slide into several zones, reaching the widely used 96-well plate format on four slides (for example, Microplate Hardware for four microscope slides from Arrayit Corporation, USA). Another possibility for the preparation of multiple samples on four slides in a ‘plateformat’ can be the use of expensive spotting devices which were developed for the preparation of microarrays on slides. Promising microfluidic technologies can also be used for cytogenetic assays(8). By adjusting their surface properties, microchannels can be made ‘self-loading’, so that specialised pumps are unnecessary for loading reagents. An example of microfluidic platforms designed in a standard microplate format is shown in Figure 1D. The microchannel positions in this plate are standardised and correspond to the position of 384-well plates, so that common laboratory 8-channel pipettes and standard 8-channel heads of automated systems can be used for loading samples and reagents. The surface of one microchannel is only 36 mm2 that is 50 times less the surface of a microscope slide.
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Figure 1. Platforms for high-throughput biodosimetry: rack with 96 2D barcoded tubes with caps (Thermo Fisher Scientific, USA) (A); 96-square well plate with optically clear bottom (Ibidi GmbH, Germany) (B); slide-adaptor plate with four microscopic slides (Microfluidic ChipShop GmbH, Germany) (C) and 64 microfluidic channel plate (microfluidic ChipShop GmbH, Germany) (D).
NEXT GENERATION PLATFORMS FOR HIGH-THROUGHPUT BIODOSIMETRY
CYTOGENETIC PROCEDURES IN NEXT GENERATION PLATFORMS FOR HIGHTHROUGHPUT BIODOSIMETRY Procedures of current cytogenetic biodosimetry assays are based on relatively simple standard methods wellknown for a long time(9 – 11). To adapt these assays for next generation cytogenetic platforms, there is a need to address the reduced sample volumes and surface area. Blood sample collection
Cell culturing The CBMN and dicentric assays (as well as other cytogenetic assays) require culturing cells to division. This can all be done within the multiwell plate keeping in mind the need to select a plate with sufficient capacity according to the sample size. In the recent manual of the IAEA(1), emphasis was placed on the choice of culturing medium, its components (L-glutamine, serum, antibiotics, mitogens) and their testing before setting up the complete culturing medium. However, complete and tested cytogenetic media are commercially available and should be used whenever possible. Particularly, a ‘ready-to-use’ complete medium PB-MAX (Life Technologies, USA), gives satisfactory and reproducible results in the laboratory. Since in microtubes reduced culture volumes are used (up to 1 ml, compared with 5–10 ml using a
Cell harvesting and fixation At the end of the culturing, according to standard procedures, for both the dicentric and CBMN assays, lymphocytes must be treated with hypotonic solution and fixed. These steps involve multiple washes with fixative by centrifugation. The cell harvesting procedure in a microplate format is not complicated and similar to the traditional technique(7). Using traditional methods, 5–10 ml of blood cultures is transferred into 15-ml tubes and 10–30-min centrifugation times are used. With the use of tube plates/microplates, centrifugation time is cut to a few minutes, thus saving at least an hour from the total cell harvesting time. In a centrifuge, multiple 96-tube racks, 96-well plates and/or 384-well plates containing hundreds or even thousands of samples can be loaded and processed during one run. Few such runs give the throughput of sample processing far beyond the total current capacity of the established biodosimetry network with 4000 samples per day(17). Cell spreading and staining After harvesting and fixation of lymphocytes, cells can be prepared immediately for staining, stored at 2208C (for years) or shipped to other laboratories. For high-throughput preparation of cells for imaging and analysis, cell spreads can be prepared in screening multiwell and multichannel plates as well as on standard microscope slides. Cell spreads in multiwell plates can be made by dropping few microlitres of fixed cell suspension or by the use of a relatively straightforward but efficient cytospin technique for larger sample volumes. The
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Using the dicentric assay, 500– 1000 metaphase lymphocytes are typically necessary to provide a precise ionising radiation dose estimate. Samples of ,100 cells were historically regarded as inadequate for dose estimation(12); however, following the Chernobyl accident, it was found that 50 metaphase cells can give useful dose estimates using the dicentric assay(13). Nowadays, the IAEA considers 50 metaphase cells for the dicentric assay and 200 binucleated cells for CBMN assay as appropriate numbers for triage purposes(1). According to the last NATO dosimetry study the scored number of cells can be reduced from 50 to 20 using the dicentric analysis without loss of precision of triage dose estimates, at least for homogenous exposure scenarios(14). About 105 lymphocytes can be isolated from 30 ml of fingerstick-derived blood samples on average(15). Thus, for triage biodosimetry purpose, a sufficient number of lymphocytes for cytogenetic analysis can be collected from very small amounts of blood. Moreover, it is preferable to use fingerstick over venipuncture as it is minimally invasive, easier and faster(16). Here the authors propose to collect blood samples by fingerstick using a standard lancet and transfer samples using heparinised capillaries having a set volume (10 –100 ml) into individually barcoded microtubes organised in 96-tube racks (Figure 1A).
traditional assay), the cost per sample of using an offthe-shelf complete medium is not higher than that for a traditional assay with self-made medium. The pre-made medium has an added advantage that it undergoes strict quality control by the vendor, ensuring reliable and reproducible results. This is of critical importance when performing medical diagnostics on a large population. According to the recommendations of the IAEA(1), lymphocytes can be isolated before culturing; however, additional manipulations using lymphocyte separation medium and the transfer to culture plates are needed. Setting up the cultures is simplified in the case of using whole blood culturing. For whole blood samples, it is necessary only to add complete medium into tubes and put tube racks into a cell incubator. At that, it is unnecessary to close each tube, it is sufficient to put a lid on the rack. A compact cell incubator can fit several hundreds of plates, or thousands of samples. During culturing, concentrated solutions of demecolcine (Colcemid) or Cytochalasin B can be quickly added in a high-throughput manner to achieve the required final concentration.
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Imaging In principle, the imaging of cytogenetic samples prepared in high-throughput plates can be done on both, direct and inverted, microscope systems with an appropriate stage configuration adapted for the chosen format. Inverted microscope systems are preferable for usage with most of imaging plates due to their design. Samples in multiwell and multichannel plates can be scored manually; however, it is extremely labour intensive and incompatible with high throughput. Several companies offer high-content imaging systems for multiwell plates with automated hardware and specialised software (e.g. the IN CELL series, General Electric, USA). Commercial high-content imaging systems are expensive and are not available in most cytogenetic laboratories. More cost-effective, automated imaging systems for multiwell plates as well as for multichannel plates can be developed in house as it was done for microscope slides(15, 21).
the same approach of using the combination of tubes organised in standardised racks for blood collection and multiwell plates for screening can be used. Traditional protocols for proved biodosimetry assays can be used directly on new platforms in standard cytogenetic laboratories described above with corresponding volume adjustment; however, it is desirable to develop new protocols that will have higher throughput of biological sample preparation and will have smaller reagent consumption. In this connection, automation of assays using new robotic-friendly platforms described above and emerging cytogenetic techniques such as fluorescent in situ hybridization (FISH) in microchannels are of special interest. Automation For fast and efficient response to radiation events involving mass casualties, it has become imperative to automate cytogenetic dose assessment methods to increase throughput (1). Robotic systems for automation of cytogenetic specimen preparations are known for .20 y(24, 25) and focussed on by the IAEA in 2011(1). It is considered that such systems must have many specialised modules for different processes. For example, blood sampling module, lymphocyte isolation system, slide preparation device, a device for staining and the application of coverslips and so on(1). Each of these modules is expensive, in particular when requiring ‘automation-compatible’ devices which provide clearance for robotic loading of samples and allow control by centralised software. Currently, the direct automation of cytogenetic assays(24, 25) leads to expensive and inefficient systems with yields 1000 samples per week(26). An example of a higher throughput system is the Columbia University Rapid Automated Biodosimetry Tool (RABiT)(16). The RABiT isolates lymphocytes from blood samples and processes them in filter bottom multiwell plates. Application of the RABiT for highthroughput biodosimetry using the g-H2AX assay is described elsewhere(15). The task of the automation of cytogenetic assays on next generation platforms is simplified due to the use of the ANSI/SLAS microplate format from the beginning (blood samples arrive at cytogenetic centre already in 96 tube rack format) and availability of different corresponding automated microplate systems. Moreover, some pre-existing commercial robotic systems can be programmed for biodosimetry assays and used in the case of emergency. Care needs to be taken that the automated system is tested and pre-certified by the appropriate health authorities for its new intended use.
FUTURE DEVELOPMENTS New radiation-responsive biomarkers can be found and corresponding assays developed due to advances in proteomics(22) and genomics(23). For these assays,
Fluorescence in situ hybridization Fluorescent in situ hybridization improves the detection of different types of aberrations and can be used
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cytospin technique uses centrifugation to quickly sediment the cells onto the surface of glass slides or well bottoms to form a uniform monolayer(18). After few minutes of centrifugation, the fixative is removed from each well and cells remaining on the bottom surface are allowed to dry in air(11). The staining procedure can be completed by loading, for example, a low concentration DAPI solution in PBS buffer in a volume necessary to cover the entire surface of the well bottom. Low-throughput preparation of cell spreads on slides in plate adaptors can be done according to the standard protocol by dropping fixed cell suspension and waiting for complete evaporation of the fixative. Simple staining for scanning on a fluorescent microscope can be performed by dispensing small drops of DAPI with antifade and placing a coverslip on it. Alternatively, slides may be placed in an automatic stainer or stained manually. In the case of using special adaptors forming high-throughput format of 96 wells on four slides, the procedures of cell spreading and staining can be done similar to those procedures described above for multiwell plates. Preparation of cell spreads in microchannels is a more complicated task due to the action of liquid flow forces. In recent years, different methods for the preparation of cells for cytogenetic assays in microchannels including the preparation of metaphase cell have been developed(19, 20). Prepared samples can be stained in microchannels using only a few microlitres of dye, for example, DAPI solution.
NEXT GENERATION PLATFORMS FOR HIGH-THROUGHPUT BIODOSIMETRY
in all cytogenetic methods recommended by the IAEA for radiation dose estimations: translocation assay(1), dicentric assay(27), micronucleus assay(28) and PCC assay(29). Moreover, FISH can be used for the automation of the scoring process, because it facilitates the development of fast automated image systems(30, 31). The application of molecular cytogenetic methods for mass-casualty radiation events is complicated by the introduction of additional steps in the assay and is costly due to the expensive FISH probes. Nevertheless, FISH, in principle, can be performed in multiwell plates, but it is necessary to load at least 50 ml of probe to cover the surface of one well of standard 96-well plate without using coverslips that is comparable with the volume of 25 –30 ml of probe per microscope slide that is achieved by the use of coverslip. FISH miniaturization through the use of microfluidic technologies is a more promising approach in molecular cytogenetics due to the use of significantly reduced volumes of reagents corresponding to the dimensions of microchannels(8, 20). The preliminary experiments of this study have shown the possibility of using FISH technique on human lymphocytes in microchannels of plate compatible with the ANSI/SLAS microplate format (Figure 2).
FUNDING This work was supported by grant number U19 A1067773, the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, from the National Institute of Allergy and Infectious Disease, National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of National Institute of Allergy and Infectious Diseases or the National Institutes of Health.
REFERENCES
CONCLUSIONS The combined use of standardised microtube racks and multiwell plates represents a new path to increasing throughput of cytogenetic assays by an order of magnitude or more allowing their use in population screening following a radiological event. Moreover, new cytogenetic platforms having ANSI/SLAS microplate formats allow an automation of biodosimetry assays on universal biotech robots. 109
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Figure 2. Metaphase spread of human lymphocyte with two dicentrics after exposure to 4 Gy of 137Cs gamma-rays prepared in a microchannel of the microfludic plate (shown in Figure 1D): DAPI-stained (A); FISH with centromere FAM-PNA probe (B) and FISH with telomere Cy3-PNA probe (C). Fifty microlitres of peripheral human blood was cultured and fixed in microtubes organised in rack (shown on Figure 1A) according to the IAEA protocol(1) for whole blood culture with five times reduced volumes and 2-min centrifugation time. Standard FISH was performed according to the manufacture’s recommendation (Panagene, Inc., Korea) with 7-ml volumes used for loading fixed cell suspension and reagents into microchannels of plate. Arrows show two dicentrics: ‘þ’—two centromere signals and ‘2’—with no telomere signal between centromeres.
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