Study design, setting, participants, and methods
The development of the mesodissection system followed an ISO 9001 compliant phase review process. The breadboard prototype instrument was developed at AvanSci Bio using a modified commercial milling machine and the consumables were constructed from common hardware components and pipet tips. The laboratory prototype instrument and software were developed at AvanSci Bio, ARUP Laboratories, and the Scientific Computing and Imaging (SCI) Institute at the University of Utah, using a combination of custom and off the shelf components. Production consumables were manufactured from custom injection molded components. The imaging software was written in C++ and interfaces with various hardware drivers. The initial laboratory prototype was presented in the exhibit hall of the Association of Molecular Pathology 2011 Annual Meeting to obtain potential user feedback. This feedback was used to refine the design of the current instrument.
Milling instrument and xScisor disposable
A new platform that uses milling technology to dissect tissue from slide-mounted tissue sections was developed (See Figures 1 and 2 for a detailed description). In the basic mill design, an object is attached to a table (stage) capable of controlled X and Y axis movement such that it can be driven against a fixed rotating cutting bit thereby shaping the object. However, as milling is typically not used for the purpose of collecting fragments, a plastic mill bit termed the xScisor was developed that simultaneously dispenses liquid, cuts tissue, and aspirates the tissue fragments from the surface of the glass slide (Figure 3). Low cost manufacturing methods were developed so the xScisor could be disposable to prevent sample cross-contamination. Because tissue is relatively soft compared to glass, a spring pressure controlled system was designed such that the xScisor blade rests on the slide surface with sufficient downward force to cut through the tissue but glides across the glass slide. Milling tissue from glass slides also provided the opportunity to place a digital microscope below the slide in order to view the process, direct the dissection, and generate digital documentation.
Software and workflow
A software package was developed that was modeled after a typical manual slide-mounted tissue dissection workflow found in many molecular pathology labs (discussed above, see Figure 4 for a more detailed description). This software provides an interface to digitally indicate AOIs and save dissection reference images. Alternatively, the dissection reference image can be generated by digital scanning technology if it is saved in either .jpg, .png. or .tif file format. In the laboratory, the reference image was imported from the database, aligned to the magnified live view from a serial tissue section, the AOI transferred to overlay the live image and guide the dissection, and finally a digital report in PDF format was generated.
Tissue samples used in this study
All human FFPE tissue sections used for the studies described here were derived from left over/unused normal regions of placenta, liver, bowel, kidney, and skin specimens from the Anatomic Pathology Gross Room at the University of Utah (Department of Pathology, School of Medicine). As these tissue sections were anonymized, they are exempt from IRB approval. All human tissues were processed using standard FFPE methods, and the resulting tissue blocks and the mouse-human fusion tissue blocks were sectioned at 5 micron thickness. The formalin perfused mouse liver and kidney tissues that were used to make the mouse-human fusion blocks were surplus tissues obtained from studies of the mouse olfactory system done at the University of Utah (Department of Physiology). All the mouse olfactory tissue sections were from the same source, but had been OCT embedded, and then frozen sectioned at 10 to 60 microns thickness. These olfactory tissue sections were surplus from the intended studies. For the studies described here, the OCT was replaced with paraffin as described below.
Resolution and accuracy
Both resolution and accuracy were quantitated using Microsoft PowerPoint by visual examination of the post-dissection digital images including the scale bars generated by the mesodissection software. The accuracy values were determined by attempting to fill the difference between the intended and actual boundary with a line of relative width of 50, 100, or 250 microns (Figure 5A and B). Standard deviation was calculated using Microsoft Excel.
Efficiency
Serially cut tissue sections were either deparaffinized with AvanSci Bio’s mineral oil/alcohol system, or left paraffinized, then dissected by hand or using the mesodissection system. In order to dissect the same AOIs using both methods, the post manual dissected tissue sections were digitally outlined and used as guides for mesodissection. The resulting tissue samples were recovered in one of the following liquids: TE (10 mM Tris, 1 mM EDTA, pH 8.5), TE + 0.1% SDS, or light mineral oil, then subject to Proteinase K digestion by adding 5 μg of Proteinase K to the samples in TE and TE + SDS, or by adding 70 μl of TE with 5 μg proteinase K to the samples in mineral oil. Incubation was performed on the Thor (AvanSci Bio) programmable heater-shaker: 65°C 30 min 1500 rpm; 95°C 10 min 1500 rpm; 25°C 1 min 450 rpm. The samples were then centrifuged and assayed for genomic DNA concentration using PicoGreen according to the manufactures’ protocol (Life Technologies) as a measure of total sample recovered.
Purity
To make the mouse-human fusion blocks, the formalin treated tissue samples described above were held in close proximity while casting in paraffin. The resulting 5 micron slide-mounted tissue sections were deparaffinized and dissected as indicated. The recovered tissue samples were Proteinase K digested as described above, then subject to both dual amplicon multiplex endpoint PCR with Taq Platinum using the recommended conditions (Life Technologies) and 2% agarose gel electrophoresis, or single amplicon qPCR using the Power SYBR Green master mix and the recommended conditions (Life Technologies). The PCR primer pair (amplicons) sequences (directed to either the mouse or human Cox1 mitochondrial genes) are: AGGGGACCCAATTCTCTACCA + CTCCGTGTAGGGTTGCAAGT (mouse) and TTCGGCGCATGAGCTGGAGTCC + AGTTGCCAAAGCCTCCGATT (human).
RNA isolation, reverse transcription, and quantitative PCR
The formalin perfused, OCT embedded, frozen sectioned, 10 to 60 microns thick mouse olfactory tissue sections that were obtained for this study were relatively poorly adhered to the slides surface and tended to be dislodged rather than cut by the spinning xScisor blade. Therefore, the tissue sections were gently rinsed in 0.1X PBS to remove the OCT, dipped in molten paraffin for 5 minutes, then the excess paraffin allowed to drain by standing the slide on edge at 70°C for 5 minutes. These tissue sections were milled using 70 μl per sample of light mineral oil, then 70 μl of TE, pH 8.5 containing 5 μg of protease K was added and the tubes mixed. Protease K digestion was carried out using the Thor heater-shaker at 60°C 30 min 1500 rpm; 82°C 15 min 1500 rpm; 25°C 1 min 450 rpm. The overlaying mineral oil was removed using Wicking Strips (AvanSci Bio), then 90 μl PKD buffer (Qiagen) was added to achieve 160 μl total volume. Next, 16 μl of DNase booster buffer and 10 μl of DNase I (Qiagen) were added to each tube, the tubes incubated at room temperature for 15 minutes and the RNA isolated as described in the RNeasy FFPE kit (Qiagen). The manual dissected material was processed using the limonene deparaffinization protocol described in the RNeasy FFPE kit. cDNA was generated using the Applied BioSystems High Capacity Reverse Transcription kit and Quantitative PCR was performed using Applied BioSystems Power SYBR Green reagent as described in the manufacturer’s protocol. The primers pairs used in the qPCR were GATGACTGAGTACCTGAACCG + CAGAGACAGCCAGGAGAAATC (mouse Bcl-2 cDNA) and GCCCTCCGTATCTTACTTCAAG + GCGGTCCAGGTAGTTCATG (mouse Cyclin D1 cDNA).
FISH
Slide-mounted tissue sections were deparaffinized using d-limonene or AvanSci Bio’s mineral oil/alcohol system, then mesodissected using TE plus 0.1% Tween-20 as the milling solution. Recovered tissue fragments were centrifuged for 2 minutes, the majority of the supernatant discarded, the fragments resuspended, and 2 μl aliquots were spotted onto Fisher Scientific Capillary Gap plus slides (130 micron) and baked at 65°C for 2 hours. These slides were then subject to tissue FISH processing using the Kreatech recommended FFPE Tissue FISH protocol.