A Mill Based Instrument and Software System for Dissecting Slide-mounted Tissue That Provides Digital Guidance and Documentation

Nils Adey; Dale Emery; Derek Bosh; Steven Callahan; John Schreiner; Yang Chen; Ann Greig; Katherine Geiersbach; Robert Parry

Disclosures

BMC Clin Pathol. 2013;13(29) 

In This Article

Discussion

Dissection of specific Areas Of Interest (AOIs) directly from slide-mounted tissue sections is commonly used to enrich for cell types of interest for further molecular analysis. Most clinical labs utilize manual dissection methods, typically of tumor samples, for cost and simplicity reasons. The only other viable choice for most clinical needs is Laser MicroDissection (LMD), which is extremely precise but also costly and often complicated. Because of this, a number of clinicians have expressed interest in a more automated, precise, and digitally documented approach to slide-mounted tissue dissection. This interest will likely grow as complex tests involving molecular techniques such as expression analysis, next generation sequencing, and proteomics are increasingly utilized. In cases where the downstream biochemistry is expensive, it may also become cost effective to improve the quality of the input sample. This interest lead to the development of the mesodissection platform described here. We use the term "meso" because the precision is between that of LMD and manual macrodissection methods. The current version mesodissection system provides better than 200 micron resolution and the joystick control allows users to obtain accuracy approaching +/- 60 microns from the intended region. In our experience, this is better than most users can obtain using a dissection microscope and scalpel. However, since the precision of both mesodissection and manual dissection are operator dependent, it is difficult to quantitate the degree of precision improvement. The mesodissection software allows a user (such as a pathologist) to make AOI decisions using an optical image, then annotate these AOIs on a digital image. Use of digital images can eliminate the need for hand annotated slides to be sent to the dissection lab thus minimizing logistical issues. In the laboratory, the serial tissue sections are digitally aligned, the AOIs electronically transferred to the live images, and used as a dissection guide. We find that the use of digital microscopic annotation typically results in more precision than hand annotation. Finally, the software generates a digital report of the entire dissection process.

Biochemical Considerations

The mesodissection system is compatible with both paraffinized and deparaffinized FFPE tissue sections mounted on standard glass slides. A variety of milling solutions have been tested and many more presumably can be used as long as they do not degrade the plastic xScisor and can hold the tissue fragments in suspension. For the latter reason mineral oil or SDS containing solutions were used to hold paraffinized fragments in suspension. When using mineral oil, the subsequent Proteinase K reaction can be performed by adding an aqueous solution to the recovered fragments and incubating on a heater-shaker. In these conditions, the tissue fragments migrate from the upper organic phase into the lower aqueous phase where the digestion occurs immediately. For expression analysis studies, dissection of still paraffinized tissue using organic milling solution is desirable as it minimizes exposure of the tissue to air and moisture prior to the Proteinase K step. A problem with the use of mineral oil is dissection visualization is somewhat impaired. Dissection visualization of paraffinized tissue sections is better using solutions containing SDS. SDS solutions can aspirate paraffinized tissue to a modest degree and SDS is compatible with Proteinase K. However, SDS must be removed prior to most subsequent biochemical reactions whereas post Proteinase K tissue lysates recovered from mineral oil can go directly into many downstream biochemistries.

Every type of transparent slide tested including both plain and charged glass was compatible with mesodissection as long as the tissue sections were reasonably well-adhered to the surface. Standard FFPE sections of most tissue types and thicknesses worked well, but occasional difficulty was encountered dissecting calcified tissue (the blade tends to ride over the top of the tissue). We found that frozen sectioned OCT embedded tissue was sometimes poorly adhered to the glass surface and when contacted by aqueous milling solution tended to dislodge rather than dissect. The problem tends to be worse with thicker sections (greater than 10 microns). In these cases, rinsing with an aqueous buffer to remove the OCT, dehydrating in alcohol, then dipping in molten paraffin and allowing to drain at an elevated temperature in order to remove the majority of molten paraffin greatly improved adhesion and provided good quality RNA dissection. Partial deparaffinization using AvanSci Bio's mineral oil/alcohol system allows for tissue staining and improved visualization.

We used the mesodissection system to generate samples for two common applications, RT-qPCR and FISH. The results show that mesodissected samples are compatible with RNA expression analysis using RT-qPCR. The recovered sample-mineral oil mixture can be added directly to a Proteinase K reaction without an extraction and centrifugation step. Elimination of this step likely improved the RNA yield compared to a commonly used limonene deparaffinization protocol. We demonstrate that fragments of tissue generated by mesodissection can be re-adhered to slides and used for FISH analysis. This application could be used to increase FISH throughput as well as minimize hybridization probe and precious sample usage. A collaborator has used the mesodissection system to obtain samples for protein analysis by mass spectrometry and reports the system is comparable in quality and yield to other methods of tissue sample retrieval (Dr. David Krizman, Oncoplex Diagnostics). We also showed that the mesodissection system is capable of dissection directly from tissue blocks. Dissection directly from tissue blocks produces a lot of sample since the cut depth approaches 100 microns without the need to generate and dissect individual slide-mounted tissue sections. A possible downside is that the deeper the dissection, the less information one has about the material being recovered. Our further development of this capability is dependent on user interest, as it will require engineering of clamps mounted on the X-Y stage to properly position the tissue blocks and improvements to the dissection visualization methods.

Factors Improving Dissection Performance

In addition to the factors discussed above, the following list of milling variables can improve mesodissection performance and should be considered when operating the instrument: 1) The cutting effectiveness, diameter of the cut area, and wear rate of the blade is a function of the downward force of the mill head, controlled using the adjustable position spring stop. Once properly set, it is usually not necessary to readjust the spring pressure. However, sometimes it is necessary to increase pressure when using larger blades, particularly when cutting paraffinized tissue, and in this situation, going over the same area multiple times can be helpful. 2) Coordinated movement of the digital AOI with the live image of the tissue section is dependent on precise calibration of the instrument's slide stage with the 2iD software. 3) The aspiration rate can affect recovery efficiency; in general, increase the aspiration rate to the point where most of the dissection solution is used for a given sample, then centrifuge to pellet the recovered sample and discard excess dissection solution. 4) When doing point dissections, it is necessary to depress the pulse button for a couple seconds in order to aspirate the dissected tissue, otherwise aspiration occurs only with transverse stage movement. 5) To obtain clean cut edges, the leading edge of the blade should be rotating into the leading edge of dissection. For example, because the blade rotates counter-clockwise, dissection around the outside edge of an AOI should proceed in a counter-clockwise direction. 6) When dissecting deparaffinized tissue with TE or water, the dissected tissue occasionally forms clumps, which can be flung outside of the aspiration area by the rotating xScisor rather than being aspirated. Addition of a small amount of detergent (i.e. 0.1% Tween-20) to the milling solution can greatly reduce the formation of tissue clumps and improve aspiration. 7) The design of the xScisor results in simultaneous dispense and aspirate functions because both functions are controlled by the same movement of the plunger. The aspiration volume is greater than the dispense volume by the volume of the xScisor plunger. The effect is the column of collected liquid is regularly broken by small air bubbles. If a small amount of milling liquid is pre-aspirated, these air bubbles can be used as boluses to physically separate dissected areas or place an air bolus between the plunger and the recovered liquid, which can be used to increase recovery efficiency. The disadvantage of pre-aspiration is a reduction of total aspiration capacity.

Next Generation Instrument

While the current mesodissection instrument provides a number of advantages related to the digital workflow, it is joystick driven and thus typically does not result in a time savings compared to hand dissection of larger AOI's. A number of laboratories have expressed interest in automation and therefore, we are developing the next generation instrument with a fully automated stage. The AOIs will be transferred manually as this step requires user verification. However, once transferred, the software will automatically design a milling path for each AOI and perform the dissection at a significantly faster rate. This additional capability not only frees user from dissecting using the joystick, but minimizes any impact of impaired dissection visualization when using unstained and still paraffin embedded tissues.

Comments

3090D553-9492-4563-8681-AD288FA52ACE

processing....