To begin, use multi or biparametric MRI to locate the tumor and its position in the prostate gland before surgery. Next, prepare the patient with abdominal access, insufflation, and chemo port placement. Set the pneumoperitoneum to 12 millimeters.
Make an incision on the peritoneum to release the bladder and dissect the perirectal space. Now, open the pelvic fascia on either side and perform a complete dissection of the anterior and lateral aspects of the prostate and bladder. Open the bladder neck and identify the proximal urethra.
Section the proximal urethra to gain access to the posterior aspects of the prostate and bladder. Next, dissect the seminal vesicles and the rectum space, followed by the lateral aspects of the prostate. Dissect the apex.
Then section the distal urethra. Then complete the vesico-urethral anastomosis. Extract the prostate, then suture the incision shut.
Place the freshly collected prostate in a dry container. To process the prostate for sample collection, first select the targeted zones based on the MRI information. With a disposable automatic needle, retrieve cylindrical samples from the selected areas.
Then identify the outer zone of the cylinder and clamp it with a pair of tissue forceps. Place the cylinder on a slide and mark the outer zone with tissue ink. Place the collected cylinders horizontally on a sample holder disc filled with cryostat embedding medium and transfer them to a microtome cooling plate for cutting.
Next, attach the sample holder disc to the specimen chuck. Use the OCT compound to embed the tissue sample before sectioning. Use a cryostat microtome to cut the sample at 25 degrees Celsius.
Collect thin cylinder-shaped tissue sections onto treated microscope slides. Fix the slides in 96%ethanol, then hydrate them in distilled water. Stain the sections in Harris hematoxylin for one minute.
Now treat the slides with 30%ammonium hydroxide to turn the hematoxylin blue. Then stain the washed slides with eosin for 30 seconds. Then dehydrate the slides with increasing titrations of alcohol, followed by clearing with 100%xylene.
Mount the slides with GPX and place a cover slip on the stained tissue. Use a light microscope to analyze the stained sample, discriminating areas with the carcinoma and determining the Gleason score of the tumor. Mark precise tumor zones on the slides.
Then with the surgical blade, macro-microdissect the sample areas marked on the slide within the OCT disc. When the embedding tissue begins to defrost, lift the tissue with a pair of forceps and place them into correctly labeled cryotubes. This technique was used to obtain tumor material from 61%of the cases studied.
Out of the 16 prostates were tumor acquisition was unsuccessful, 10 had less than 10%tumor load and none exceeded 20%An ROC curve analysis showed an area under the curve of 0.843, indicating that this method can deliver satisfactory tumor acquisition results. Histopathological correlation of the radical prostatectomy specimen versus the processed cylinder biopsy was performed using hematoxylin and eosin staining. Different histological patterns were found along the cylinder, such as Gleason pattern 3, which showed small well-formed glands lined by cancerous cells.
Irregularly-shaped glands with enlarged atypical cells were observed in the Gleason pattern 4 sections. The samples with prostatic intraepithelial neoplasia showed crowded cells within ductal spaces.
