The RABiT: a rapid automated biodosimetry tool for radiological triage. II. Technological developments

Abstract

Purpose: Over the past five years the Center for Minimally Invasive Radiation Biodosimetry at Columbia University has developed the Rapid Automated Biodosimetry Tool (RABiT), a completely automated, ultra-high throughput biodosimetry workstation. This paper describes recent upgrades and reliability testing of the RABiT.

Materials and methods: The RABiT analyses fingerstick-derived blood samples to estimate past radiation exposure or to identify individuals exposed above or below a cut-off dose. Through automated robotics, lymphocytes are extracted from fingerstick blood samples into filter-bottomed multi-well plates. Depending on the time since exposure, the RABiT scores either micronuclei or phosphorylation of the histone H2AX, in an automated robotic system, using filter-bottomed multi-well plates. Following lymphocyte culturing, fixation and staining, the filter bottoms are removed from the multi-well plates and sealed prior to automated high-speed imaging. Image analysis is performed online using dedicated image processing hardware. Both the sealed filters and the images are archived.

Results: We have developed a new robotic system for lymphocyte processing, making use of an upgraded laser power and parallel processing of four capillaries at once. This system has allowed acceleration of lymphocyte isolation, the main bottleneck of the RABiT operation, from 12 to 2 sec/sample. Reliability tests have been performed on all robotic subsystems.

Conclusions: Parallel handling of multiple samples through the use of dedicated, purpose-built, robotics and high speed imaging allows analysis of up to 30,000 samples per day.

Figure 1

a), b) characteristics of the Histone H2AX phosphorilation (γ-H2AX) assay and the Cytokinesis Block Micronucleus assay (CBMN) assay. c) Concept of implementation of the two assays in the RABiT (in this case assuming a 36 hour useful life span of the γ-H2AX assay).

Figure 2

Photo of the RABiT, showing the various subsections. The Lymphocyte Harvest station is shown in close up in figure 5 below. The imaging system is shown in Figure 8 below.

Figure 3

Hardware structure of the RABiT. The top tier (Main Computer, MC) controls the overall operation of the RABiT. Two additional computers control the cell harvesting system (CH, dealing with the robotics and CHP dealing with the capillary imaging and barcode reading). An additional dedicated computer controls the imaging system (IS). The middle tier includes systems with dedicated controllers: The SCARA robot, the Osprey UV laser (UV), The Liconic LTX220 incubator, the Eppendorf 5810R centrifuge and the Caliper Sciclone ALH300. The bottom tier consists of the low-level peripherals: The lymphocyte harvest station is controlled by two computers. The CHP computer controls the two barcode readers (BR) one for reading the plate barcodes and one for reading the capillary barcodes and a CCD camera for imaging the reed blood cell band. The CH computer controls a dispensing valve (DV) a rotating drive motor (RDM), an XYZ stage and a rotation stage (Φ) The Transfer to Substrate System (TTS) (Chen et al. 2010b), controlled directly by MC, consists of a feed plate slide (FPS), for loading a plate, a Peel Clamp (PC) and Peel Clamp Slide (PCS) for removing the under drain from the multiwell plate, a Tape Roller (TR) and Barcode Stamp (BS). Finally, the Imaging system consists of an 3 axis gantry stage (XYZ) and tape spooler (TS) for coarse sample motion, a Piezoelectric Z stage (PZS) for fine focusing and a galvanometric scan head (SH) for fine motions within the sample. A barcode reader (BR) is provided for sample identification. Each image is acquired using an Image intensifier (II) and a CMOS camera (CMOS). Sample illumination is provided from a mercury lamp (Hg) and an ultraviolet light emitting diode (LED). Images are stored on a 15TB hard disk array (HDA) and then to a T120 tape library.

Figure 4

Sample collection kit developed for the RABiT. Shown are the capillary holder (full in insert) with a prototype padded shipping container and ice pack, required phlebotomy supplies and the data collection card with an attached barcoded capillary.

Figure 5

The newly designed lymphocyte harvesting station. Showing the incoming bucket and plate queue, the XYZ stage, with the capillary-filled bucket and plate currently being processed. One capillary is in each of the stations: A – Lymphocyte dispense, capillary picking and discarding; B – capillary imaging, C – barcode reading and D – cutting.

Figure 6

Design drawing of the capillary gripper for a) the original RABiT (Salerno et al. 2007) and b) the modified design.

Figure 7

Close-up of the laser cutting station. The Rotating Drive Motor (RDM) is used for spinning the capillary to ensure uniform cutting.

Figure 8

Photos of the RABiT imaging system. a) Top view showing the illumination light path as well as the three imaging light paths, separated by dichroic mirrors. Each imaging path passes through a 4x relay lens and an image intensifier (II) to a CMOS camera. The nucleus is imaged in blue (Hoechst 33342), through a 455nm emission filter while the cytoplasm or γ-H2AX foci are imaged in orange, through a 565nm emission filter. The beads used for focusing are imaged through an additional cylindrical lens and 625nm emission filter. b) Front view, showing the sample, XYZ stage, Barcode reader(BR), Piezo Z stage (PZS) scan head (SH) and objective (10x). c) Close up view of the illumination path with the dichroic used for merging light from the UV light emitting diode (LED) exposed.

Figure 9

Image of a 2 mm × 2 mm region of a filter containing stained lymphocytes, stitched from 100 individual images. The edge of the 9 mm diameter filter is marked by a dashed line.

Figure 10

Force required to extract capillaries from the centrifuge bucket. The error bars correspond to the standard error ±√[p x (1-p)/N] , where p is the is the value of each bar in the histogram and N=41 is the number of trials.

Figure 11

Images of 10 μm beads through a cylindrical lens. The Z-values correspond to the position of the objective lens, mounted on a piezo-actuator.

Figure 12

Measured aspect ratio of 10 μm fluorescent beads as a function of objective lens position (line). The values shown correspond to the vertical extent of a bead image divided by its horizontal extent. As a comparison, the symbols show the apparent diameter of a lymphocyte nucleus, imaged simultaneously, on a second camera. Beyond ±5 μm or so, the image of the nucleus becomes too fuzzy to extract a diameter.

Figure 13

Settling time of the scan head as a function of deflection distance along the two axes.

Figure 14

Settling time for the piezoelectric Z stage (PZS) following a 100 μm motion of the objective lens. The upper curve corresponds to the requested position whilst the lower curve corresponds to the actual position. 1 vertical division corresponds to 50 μm. 1 horizontal division corresponds to 10 msec.

Figure 15

Images of irradiated lymphocytes (0 Gy, 2 Gy and 5 Gy), processed using the RABiT γ-H2AX assay protocol, as described in (Turner et al. 2011), and imaged using the RABiT imaging system. The outlines around each nucleus were generated from the nuclear image (not shown) by the RABiT software. Fluorescence is integrated within these boundaries. The right hand half of the 0 Gy panel shows an image with the gain increased in post processing to show that the non-irradiated lymphocytes to not have any fluorescence above background The other panels have not had any post processing other than background subtraction performed in the RABiT and reduction from 12 bits to 8 bits.