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To develop a device for sectioning prostatectomy specimens that would facilitate comparison between histology and in vivo MRI.
A multi-bladed cutting device was developed, which consists of an adjustable box capable of accommodating a prostatectomy specimen up to 85 mm in size in the lateral direction, a “plunger” tool to press on the excised gland from the top to prevent it from rolling or sliding during sectioning, and a multi-bladed knife assembly capable of holding up to 21 blades at 4 mm intervals. The device was tested on a formalin fixed piece of meat and subsequently used to section a prostatectomy specimen. Histology sections were compared with T2-weighted MR images acquired in vivo prior to the prostatectomy procedure.
The prostatectomy specimen slices were very uniform in thickness with each face parallel to the other with no visible sawing marks on the sections by the blades after the cut. MRI and histology comparison showed good correspondence between the two images.
The developed device allows sectioning of prostatectomy specimens into parallel cuts at a specific orientation and fixed intervals. Such a device is useful in facilitating accurate correlation between histology and MRI data.
Non-invasive identification and localization of prostate cancer remains challenging. MRI is arguably the best non-invasive diagnostic method available, and various MRI techniques have been studied to improve accuracy of diagnosing prostate cancer (1–5). In order to determine the method’s success in detecting cancer, the MR images must be compared to the histological analysis of the excised prostate.
Methods have been developed to prepare the prostates removed by radical prostatectomy for histological analysis and subsequent correlation with MRI. The standard method involves a pathologist using a single-bladed histology knife to serially section the formalin-fixed specimen. It is difficult for even an experienced pathologist to consistently slice the specimen at the correct interval and to ensure that each cut is parallel to the plane corresponding to the MRI slices. Uneven section thickness may also result from the knife twisting during the cut or due to the compressive forces introducing shear strains on the specimen.
An alternate method was developed by Jackson et al. that used a multi-bladed knife and an adjustable base to hold and section the prostate specimen (6). This device was successful at sectioning prostate glands but was not ideal as it did not hold the prostate specimen or the blades tight enough to ensure perfectly parallel cuts. Since nothing held the prostate from the top, it was free to roll, slide or rotate under the knife. Short blades meant that more strokes were required to cut the gland, causing more surface damage. The purpose of this study was to develop a device that improved on the design used by Jackson et al. (6), which would enhance correlation between histology and MRI to evaluate the accuracy of a prostate cancer detection method. The device was required to create perfectly parallel and smooth slices at the same interval and orientation as the MRI images. The slices needed to be good quality with minimal distortion or damage for proper histological preparation and analysis.
A device (Figure 1) was developed to hold and section formalin-fixed cancerous prostate glands removed by prostatectomy. The prostate sections were then processed using whole mount histology protocol and digital images of the whole mount sections were compared to corresponding MRI images of the gland acquired in vivo. The device which is referred to as the “Multi-bladed Cutting Assembly” holds the prostate in the correct orientation for matching the histology and MRI sections, makes cuts at the same 4 mm interval as the MRI slices, performs perfectly parallel cuts without shearing and minimizes damage to the prostate specimen in the process.
In order to keep the orientation of the prostate gland fixed during the sectioning process, the device needs to apply a holding force from all sides. An adjustable box was designed and constructed that could accommodate a prostatectomy specimen of any size between 30 and 85 mm in the lateral direction (Figure 1. A). The application of slight pressure on the specimen from every direction succeeds in fixing it in place during the cutting process. Since the box is open from the top, a “plunger” tool was used to press on the excised gland from the top in between blades, preventing the gland from rolling or sliding and allowing the user to adjust pressure as needed during the cut.
The walls of the device, located laterally to the specimen, and the plunger, each include vertical slots spaced at 4 mm intervals that allow the blades of the knife to pass through the specimen. These slots (0.5 mm wide) are just slightly wider than the blades (0.25 mm), and slots on opposite walls are directly lined up with each other to ensure parallel cuts and to prevent any side to side movement of the blades. The specimen is held in place in the superior-inferior direction by two aluminum plates that fit through the slots in the lateral walls.
A separate multi-bladed knife is used in conjunction with the adjustable box. The knife assembly includes openings at 4 mm intervals for a maximum of twenty-one blades to be held (Figure 1. B). The blades of the knife fit through the vertical slots in the adjustable box and cut simultaneously through the sample. This multiple-blade design ensures that all cuts are made in a perfectly parallel fashion.
All materials used to construct the multi-bladed cutting assembly were purchased from McMaster-Carr Supply (Cleveland, Ohio) except when noted.
The base of the cutting assembly acts as the main body from which the rest of the device is assembled (Figure 1. A). The main part of the base is made from 3/4″ nylon with a width of 4″ (102 mm) and an approximate length of 4.2″ (107 mm). A custom made tool was used to cut blade slots lengthwise in the nylon. Wall slots running perpendicularly to the blade slots at 3/8″ (9.5 mm) intervals 11 mm deep were milled into the base with a 4 mm diameter end mill. 4×10 mm aluminum bars act as slats that fill empty slots beneath the prostate sample to help support it during cutting. Aluminum pieces 1/8″ thick were screwed into each side of the nylon sheet to close each end of the wall slots and to act as feet for the device.
The walls of the device hold the prostate by adjusting to the smallest separation distance allowed by the device without seriously deforming the prostate. The two lateral walls are made from 1/4″ aluminum cut out using an Omax Water Jet Cutting Machine (Omax Corporation, Kent, Washington). The outline of the walls and the vertical slots were cut using the water jet, and the final width was finished to 4″. The bottom 10 mm of each wall was thinned from 1/4″ (6 mm) to 4 mm using a mill. The thin bottom allows the walls to be inserted into the slots in the base of the device. The superior-inferior plates are made from 0.02″ aluminum and the locations of these plates can be adjusted at 4 mm intervals to conform to the superior-inferior size of the specimen (Figure 2).
The slit walls of the plunger are made from 1/2″ polypropylene plastic cut using a water jet cutter and milled to a 45° angle on the tips. The two walls were then hot glued to a flexible 1/16″ piece of polypropylene bent in three places on a break press. Polypropylene was chosen for its low cost, durability and chemical resistance. The two slit walls touch at the tips when at rest, while when squeezed at the top, they open up to fit a wide range of prostate sizes. The plunger holds the prostate in place by wedging its slit walls between the lateral walls of the holding box and the prostate specimen (Figure 3).
The multi-bladed knife holds the required number of blades for each specimen in tension to prevent them from deflecting during the cutting process (Figure 1. B). The No. 260 Type (L) Feather Trimming Blades (Fisher Scientific, Ottawa, Ontario) are spaced at 4 mm intervals to match the slots in the lateral walls. Each blade has one hole at both ends which are used to affix the blade to the knife frame. One end of each blade is held in an assembly of three layers of 1/4″ aluminum cut on the water jet and joined together by screws. A 1/8″ diameter aluminum rod is slid between two of the layers and through one of the two holes in each blade. Arms made of 3/8″ thick aluminum milled 18 mm high and 245 mm long extend parallel to the blades and hold another 1/4″ aluminum plate that screw tightens to hold a 1/8″ nylon rod through the second hole in each blade and pull the blades tight. The tensioning plate is attached and tightened after the blades have been inserted through the slots in the lateral walls. The blades are inserted from the side of the lateral walls and the slots in the plunger are used to guide the blades through.
A rack was also designed to hold the blades so that they could be cleaned simultaneously in solution. Cleaning of the blades allow them to be reused for subsequent specimens. The blade rack is made from 1/4″ aluminum legs attached by 1/8″ nylon rod. Two 1/8″ diameter aluminum rods extend horizontally to hold the dirty blades with 3/8″ nylon spacers used to keep blades from sticking to each other (Figure 1. C).
During the design process, various prototypes were tested using formalin fixed meat. Beef was decided on as it was easy to obtain and had a similar consistency to a prostatectomy specimen. Beef steak was purchased at a local grocery store and cut using a utility knife to the same shape and size as a typical prostatectomy specimen. The beef was then injected with and submerged in formalin for over 24 hours. After removal from the solution, the beef phantom had very similar texture to a formalin fixed prostate gland.
Once the specimen has been prepared by the pathologist, with inking of the resection margins and removal of the seminal vesicles and vas deferens, and the apical and bladder neck resection margins as thin tissue dough-nuts, the set up of the multi-bladed cutting assembly can be started. The first part of setting up the cutting assembly requires placing the prostate specimen on the base where the spacing of the lateral walls can be determined. The base slats are arranged so that the walls will fit in the correct slots and the lateral walls are then inserted into the base on each side of the specimen. Once held in place, the specimen is then rotated manually to the orientation that will produce the proper slice direction. The superior-inferior plates are then slid through the lateral walls so that they hold the prostate tightly, as shown in Figure 2.
The blades of the multi-bladed knife assembly are loaded so that they match the open slots whose plane passes through the specimen. Once inserted through the lateral walls of the cutting assembly, the blades are inserted into the tension plate and tightened. It is important that the blades are sufficiently taut to avoid lateral deflection or twisting while cutting.
The final step before cutting is to insert the plunger between the walls and wedge it against the prostate. This is demonstrated in Figure 3 where the inferior plate has been removed for better visualization. Once the prostate is fully immobilized with the plunger, the knife can be lowered into the prostate specimen and sectioning can be completed.
The average superior-inferior length of each prostate specimen is around 44 mm requiring an average number of 10 blades per procedure.
The processing and paraffin embedding of the prostatectomy specimen was carried out in a routine fashion, except oversized baskets and blocks were used to accommodate the intact sections of the prostate gland. Whole mount sections were cut using Lieka RM2245 whole body rotatory microtome, and submitted as intact transverse sections mounted on oversize glass slides for hematoxylin and eosin (H&E) staining. The oversized slides were scanned at 2.5x and 20x magnification using digital image analysis system from Bacus Lab (Olympus Global). Digital images of the H&E stained sections were subsequently registered to the corresponding T2-weighted MRI images. The registration was carried out with a software procedure developed in house using b-splines algorithm. The software carries out non-rigid body registration using the boundary of the entire prostate gland manually outlined on MRI and histology images.
The multi-bladed cutting assembly was used on a prostate specimen removed by radical prostatectomy and prepared by the study pathologist. The adjusted assembly was successful at holding the prostate tightly so that it could be correctly aligned with the cutting slots and trusted to stay in position. With the plunger finally set in place, the prostate could not move out of position.
The act of cutting the prostate with the cutting assembly required a moderate force to pull the blades through the tissue in one long downward cutting motion. A sawing motion to push down through the prostate was needed, as more than one stroke was required to cut completely through the prostate gland. Once the cut was finished with the cutting assembly, there was a thin layer of tissue remaining at the bottom of each cut which held the slices together. The pathologist completed the cut at each slice with a scalpel once the specimen was removed from the box and laid out on the table (Figure 4).
The specimen slices looked very uniform in thickness with each face parallel to the other. There were no visible sawing marks on the sections.
Figure 5 shows a representative in vivo T2-weighted image (top left) and the corresponding whole mount H&E section (top right) from a prostatectomy specimen cut with the developed cutting assembly. The correspondence between the MRI image and the histology section is clearly apparent. The prostate gland was manually segmented on the T2-weighted image (center left) and the H&E section was registered to it (center right). The accuracy of the registration is confirmed by the checkerboard test (bottom).
The multi-bladed cutting assembly design can make more accurate, consistent and better quality cuts than the method of cutting off one slice at a time. By making all cuts at the same time, slices are ensured to be parallel and of uniform thickness. The way in which the cutting assembly holds specimens allows greater control over the orientation of the slices than manual one slice at a time cutting. Tight slot tolerances allow cuts to be made back and forth without any sawing marks left on the specimen’s sections, producing a better surface for microtome slices to be taken from.
Parallel and uniform cuts generated by the developed cutting assembly facilitate more accurate correlation between MRI and histology data, which is evident from the very good correspondence between MRI and histology sections shown in Figure 5. Anatomical details of the prostate gland can be easily identified on both MRI and histology slices, which confirms the very close locations of both sections within the prostate gland. As a result, the two images can be reliably registered to each other, which is confirmed by the checkerboard overlay seen at the bottom of Figure 5.
The average time required to set-up and slice a prostate is 10–15 minutes with another twenty minutes required to clean the device after use. The slicing device could be used with other small organs that need to be sectioned at the same slice thickness.
The main limitation of the current device is that it produces sections with a fixed slice thickness. Although it may be desirable to build a device with an adjustable slice thickness, that was no the objective of the present design, since the MRI slice thickness was decided on during the initial MRI protocol development.
In conclusion, we presented a device that allows reliable sectioning of prostatectomy specimens with parallel cuts at a specific orientation and fixed intervals. Such a device is useful in facilitating accurate correlation between histology and MRI necessary in testing accuracy of an MRI method in prostate cancer diagnosis.
Contract Grant Sponsor: Canadian Institutes for Health Research