Molecular Characterization of Testicular Germ Cell Tumors Using Tissue Microdissection.

Testicular germ cell tumors are among the most common malignancies seen in children and young adults. Genomic studies have identified characteristic molecular profiles in testicular cancer, which are associated with histologic subtypes and may predict clinical behavior including treatment responses. Emerging molecular technologies analyzing tumor genomics, transcriptomics, and proteomics may now guide precision management of testicular tumors. Laser-assisted microdissection methods such as laser capture microdissection efficiently isolate selected tumor cells from routine pathology specimens, avoiding contamination from nontarget cell populations. Laser capture microdissection in combination with next generation sequencing makes precise high throughput genetic evaluation effective and efficient. The use of laser capture microdissection (LCM) for molecular testing may translate into great benefits for the clinical management of patients with testicular cancers. This review discusses application protocols for laser-assisted microdissection to investigate testicular germ cell tumors.

Comparison of Hysteroscopic Cesarean Scar Defect Repair with 26 Fr Resectoscope and 16 Fr Mini-resectoscope: A Prospective Pilot Study.

STUDY OBJECTIVE: Several studies have been published on hysteroscopic treatment of cesarean scar defect using the 26 Fr resectoscope. This study compared the effects of the 26 Fr resectoscope with those of the 16 Fr mini-resectoscope in terms of efficacy, safety profile, and peri- and postoperative complications. DESIGN: A prospective cohort study. SETTING: Tertiary care university hospital (S. Orsola-Malpighi, Bologna, Italy). PATIENTS: Three hundred and nine women having symptoms and with a cesarean scar defect diagnosis were divided into 2 groups according to a temporal criterion: from March 2012 to March 2015, 155 consecutive women (control group) underwent isthmoplasty with the 26 Fr resectoscope (Karl Storz, Tuttlingen, Germany), whereas from April 2015 to March 2018, 154 consecutive women (study group) underwent isthmoplasty with the 16 Fr mini-resectoscope (Gubbini system, Tontarra Medizintechnik, Tuttlingen, Germany). INTERVENTIONS: One hundred and fifty-five women (control group) underwent isthmoplasty with the 26 Fr resectoscope, and 154 women (study group) underwent isthmoplasty with the 16 Fr mini-resectoscope. The so-called "channel-like" 360° endocervical resection technique was applied. MEASUREMENTS AND MAIN RESULTS: The isthmoplasty time with the 2 resectoscopes, excluding cervical dilatation, was similar (p = .25), whereas the overall surgical time was shorter in the case of the mini-resectoscope. The use of the 16 Fr mini-resectoscope was significantly associated with a reduced volume of distension medium used (p <.001) and a lower fluid absorption (p <.001). A significant increase (p = .01) in postoperative complications in the control group (9/155; 5.8%) compared with the study group (1/154; 0.7%) was also found. No significant reduction in discharge time was observed between the 2 groups (p = .13). Patient satisfaction immediately after surgery was significantly higher (p <.001) in the study group than in the control group. CONCLUSION: Isthmoplasty with a 16 Fr mini-resectoscope seems to be as effective as isthmoplasty with a 26 Fr resectoscope in reducing postmenstrual abnormal uterine bleeding and suprapubic pelvic pain. It is associated with a significant reduction in overall surgical time owing to the non-necessity of performing cervical dilatation. The 16 Fr mini-resectoscope facilitates surgery in small anatomical spaces such as the cervical canal and reduces the complication rate linked to blind maneuvers not respecting the uterine anatomy.

Microfluidic System for Rapid Isolation of Sperm From Microdissection TESE Specimens.

OBJECTIVES: To demonstrate a novel prototype microfluidic system for rapid isolation of sperm from real and simulated microdissection testicular sperm extraction samples. METHODS: The novel microfluidic system was tested using minced testicular biopsies from patients with nonobstructive azoospermia. The samples were split into 2 portions, conventional processing vs microfluidic. The embryologists were blinded to the processing protocol and searched the specimens for sperm after processing. We recorded the number of sperm found and the time to sperm identification and compared the sperm retrieval rates. RESULTS: When compared to conventional methods, samples processed through the microfluidic system were cleaner (decreased somatic cells/debris), with the average number of sperm identified per minute improving from 1.52 sperm per minute for the control and 13.5 sperm per minute with the device yielding an 8.88 fold improvement in the sperm found per minute for the device as compared to the control. Preliminary viability and morphology tests show a minimal impact on sperm processed through the microfluidic system. CONCLUSION: The presented microfluidic system can facilitate rapid and efficient isolation of sperm from microdissection testicular sperm extraction samples. A prospective clinical trial to verify these results is needed to confirm this preliminary data.

Comparative anatomical study on the role of zygomatic osteotomy in the extradural subtemporal approach to the clival region, when less is more.

PURPOSE: A great concern in performing the extradural subtemporal approach (ESTA) is the evaluation of the actual advantage provided by zygomatic osteotomy (ZO). Complications related to zygomatic dissection have been widely reported in the literature, making it of paramount importance to balance the actual need to perform it, against the risk of maneuver-related morbidity. Authors comparatively analyze the putative advantage provided by ZO in the ESTA in terms of anatomic exposure and surgical operability. Technical limits and potentials are critically revised and discussed. METHODS: A comparative microanatomical laboratory investigation was conducted. The operability score (OS) was applied for quantitative analysis of surgical operability. RESULTS: ZO was found to provide a weakly significant improvement in the surgical angle of attack (p value 0.01) (mean increase 3°). Maneuverability arch (MAC) increase related to ZO did not reach statistical significance (p value 0.09) (mean increase 2°). The variations provided by MAC increase on the conizing effect (CE) did not lead to an actual advantage in the real surgical scenario, modifying the vision area (VA) in terms of reduction of central vision area (CA) in favor of an increase of peripheral vision area (PA) only in the most caudal part of the surgical field. Ultimately, ZO did not influence the overall OS, scoring both ESTA-ZO+ and ESTA-ZO- 2 out of 3. CONCLUSION: In the ESTA, ZO does not provide an actual significant advantage in terms of surgical operability on clival and paraclival areas.

Metanephric organ culture.

Metanephric organ culture, or ex vivo embryonic kidney culture, was developed in the mid-twentieth century as a means to understand the development of the mammalian kidney and was used in early studies of polycystic kidney disease to explore mechanisms of renal cyst initiation by non-genetic factors. Following the identification of cystogenic genes, a resurgence of the use of metanephric organ culture occurred and has yielded insight into basic mechanisms of cystic dilation; facilitated identification of pathogenic pathways and potential therapeutic targets; and provided a system for evaluating therapeutic agents. This chapter provides detailed, step-by-step protocols with rationale and tips for the establishment, maintenance and treatment of metanephric organ cultures, and for performance of the most commonly employed secondary analyses of these cultures.

Investigating Mitophagy and Mitochondrial Morphology In Vivo Using mito-QC: A Comprehensive Guide.

Autophagy evolved as a mechanism to sustain cellular homeostasis during instances of nutrient deprivation. Mounting evidence has also clarified that under basal and stress conditions, selective autophagy pathways can target the destruction of specific organelles. Mitochondrial autophagy, or mitophagy, has emerged as a key quality control (QC) mechanism to sustain the integrity of eukaryotic mitochondrial networks. We recently reported the development of mito-QC, a novel reporter mouse model that enables the high-resolution study of mammalian mitophagy with precision, in fixed and live preparations. This model holds significant potential to transform our understanding of mammalian mitophagy pathways in vivo, in a variety of physiological contexts. We outline a detailed protocol for use of our recently described mito-QC mouse model, including tips and troubleshooting advice for those interested in monitoring mitophagy in vitro and in vivo.

A comparative study of surgical outcomes using a Colorado microdissection needle versus standard-size needle electrocautery in one-stage hypospadias repair using a transverse preputial island flap.

BACKGROUND: The purpose of this study was to compare the surgical outcomes of a Colorado microdissection needle (CMN) with that of a standard-size electrocautery needle in one-stage hypospadias repair using a transverse preputial island flap (TPIF). METHODS: The records of patients who received hypospadias repair from September 2012 to October 2013 were retrospectively reviewed. Patients were divided into a group that received repair using a CMN and those in which a standard-size electrocautery needle was used. Data collected and compared included age, types of hypospadias, duration of surgery, intraoperative blood loss, and postoperative edema and complications. RESULTS: There were 51 patients in the CMN group and 44 in the standard needle group, and the groups were similar with respect to age and type of hypospadias. The median surgery time for the CMN group was significantly shorter than that of the standard group (15.7 minutes vs. 20.6 minutes, respectively, P<0.001). At postoperative day 7 and day 30, the CMN group had significantly less patients with edema than the standard needle group (31.4% vs. 65.9%, P<0.01; and 37.3% vs. 79.5%, P<0.001, respectively). The overall complication rate has no significant difference between two groups. CONCLUSIONS: The use of CMN for tissue dissection and separation in hypospadias repair can facilitate foreskin degloving, shape the flap in a more efficient way, and help maintain adequate blood supply for the new urethra and its skin coverage.

The Use of Semitranslucent Rubber Pledgets During Microsurgical Dissection of Cerebellopontine Angle Tumors: Technical Note.

BACKGROUND AND IMPORTANCE: Dissection of cerebellopontine angle (CPA) tumors that abut or adhere to the brainstem or cranial nerves can be a challenging surgical endeavor. We describe the use of semitranslucent latex rubber pledgets in the tumor-brain interface as a method to improve visualization and protection of vital tissue during microsurgical dissection of CPA masses. The rubber pledgets are fashioned by cutting circular discs out of the cuff portion of talc-free, partially opaque latex gloves. These pledgets provide a semitranslucent, nonadherent membrane that can be placed between vital neural tissues and a tumor capsule to minimize trauma during dissection. The semitranslucent latex enables visualization of the underlying anatomical structures while also providing a protective surface onto which a suction device can be rested to facilitate clearance of the surgical field. CLINICAL PRESENTATION: A 56-yr-old woman with left ear tinnitus presented with a 3-cm CPA meningioma. During microsurgical dissection, rubber pledgets were used to preserve the interface between the brain stem, cranial nerves, and tumor capsule. The use of the rubber pledgets appeared to secure the interface between to tumor and the brain while at the same time protecting the cranial nerves, brainstem, and cerebellum. CONCLUSION: Semitranslucent rubber pledgets may facilitate microsurgical dissection of CPA tumors.

Comparison of Colorado Microdissection Needle Versus Scalpel Incision for Aesthetic Upper and Lower Eyelid Blepharoplasty.

PURPOSE: Traditionally, eyelid skin incisions with electro-cautery devices have been avoided due to the concerns of aesthetically unacceptable scar formation. The purpose of this study is to compare ecchymosis, cosmesis, and histologic tissue damage of incisions made with a scalpel or Colorado needle in patients undergoing upper and lower aesthetic blepharoplasty. To the best of authors' knowledge, no previous study has been performed before to compare these 2 modalities in aesthetic blepharoplasty surgery. METHODS: This is a multicenter, prospective, interventional, comparative case series. The study protocol was approved by Institutional Review Board in each institution. Patients underwent bilateral upper and/or transcutaneous lower blepharoplasty with 1 side randomly selected for skin incision with the scalpel, the other side with the Colorado needle. Ecchymosis was evaluated using a 10-point Likert scale and the wounds using a Hollander score. The margins of excised tissues were evaluated histologically. RESULTS: A total of 254 eyelids of 101 patients were included in the study. No significant difference was observed in ecchymosis on postoperative day 1 and 7 and scar cosmesis on day 30 and 180 between the 2 techniques. Histologically, necrosis was noted only with the Colorado needle sides (p = 0.001). No adverse events occurred on the Colorado needle side at any time after surgery. CONCLUSIONS: No clinical difference is noted between Colorado needle and scalpel incisions in terms of ecchymosis and scar cosmesis after aesthetic blepharoplasty.

Necessity of latency period in craniofacial distraction: Investigations with in vitro microdistractor and clinical outcomes.

BACKGROUND: To determine the need for latency period in membranous bone distraction, we performed 1) in vitro comparison of preosteoblasts suspended in a 3D microdistraction model and 2) a clinical study comparing mandibular distraction cases with/without latency. METHODS: In the In Vitro study, Preosteoblasts polymerized in 3D-collagen gel were placed in a microdistractor and separated into three groups: 1) distraction with latency, 2) distraction without latency, and 3) static. After 2, 4, 6, and 8 days, cell proliferation, total protein levels, alkaline phosphatase activity, and osteogenic gene expression were assessed through RT-PCR. In the clinical study, patients underwent mandibular distraction in two groups: 1) latency and 2) no latency (n = 45). The rest of the distraction protocol was identical. Outcome was based on clinical examination, radiographs at six months, and 3D CT scans. RESULTS: In the In Vitro study, The distraction without latency group compared to the latency group had delays in: proliferation, total protein count, alkaline phosphatase activity, osteogenic gene expression in CBFA-1 (fourfold vs. eighteenfold), and in osteocalcin (twofold vs. sixfold). The distraction without latency group had higher apoptotic levels during the first four days compared to the latency group (68% vs. 14%). For the clinical study, similar perioperative complications (5% vs. 6%), X-ray mineralization (93% vs. 94%), bone volume, (8.6 vs. 9.1 cc) and bone density of central distraction zone (78% vs. 81%) were observed with or without latency. CONCLUSIONS: In vitro studies showed poorer results in cell survival, proliferation and osteogenic activity compared to distraction with latency; yet, clinically, there were no differences in distraction with latency versus without.

A patient with medulloblastoma in its early developmental stage.

Medulloblastoma is the most frequent malignant brain tumor of the posterior fossa in children and is considered an embryonal tumor. It has been suggested that medulloblastomas be categorized into 4 distinct molecular subgroups- WNT (DKK1), SHH (SFRP1), Group 3 (NPR3), or Group 4 (KCNA1)-since each subgroup is distinct and there is no overlap. The authors report on a 13-year-old boy with medulloblastoma. He presented with sudden-onset nausea and vomiting due to intratumoral hemorrhage. The medulloblastoma was thought to be in an early developmental stage because the tumor volume was extremely small. Immunohistochemical analysis showed that the tumor was mainly composed of DKK1- and NPR3-positive areas. The individual areas of the tumor stained only for DKK1 or NPR3, with no overlap-that is, DKK1 and NPR3 expression were mutually exclusive. Samples obtained by laser microdissection of individual areas and subjected to mass spectrometry confirmed that the expression patterns of proteins were different. Fluorescence in situ hybridization for chromosome 6 showed there were 2 distinct types of cells that exhibited monosomy or disomy of chromosome 6. These results demonstrated that distinct subtypes of medulloblastoma may be present within a single tumor, an observation that has not been previously reported. Our findings in this case indicate that early-stage medulloblastoma may include more than 1 distinct subtype and hint at factors involved in the origin and development of medulloblastomas.

The utility of endoscopic radical resection with microdissection electrodes for lingual thyroglossal duct cysts.

BACKGROUND: Lingual thyroglossal duct cysts (LTGDCs) are very rare and liable to be misdiagnosed as simple vallecular or mucus retention cysts. We recognized the importance of complete resection by means of the Sistrunk operation and applied the revised surgical technique to the treatment of LTGDCs. The aim of this study was to evaluate the results of surgical management of LTGDCs from the author's series and analyze its utility. PATIENTS AND METHODS: Twelve patients, 10 male and 2 female, who were diagnosed with LTGDCs between January 2007 and December 2012, underwent endoscopic radical resection with microdissection electrodes. All cases were evaluated by enhanced CT and flexible laryngoscope before surgery. We reviewed the collected data including presentation, CT findings, surgical techniques, postoperative complication, and recurrence. RESULTS: Most adult LTGDCs presented with foreign body sensation, while one infant presented acute upper airway obstruction. All cysts abutted on the hyoid bone and were located at the midline of the posterior tongue. Endoscopic radical resection with microdissection electrodes was possible by dissecting hyoid periosteum without significant morbidity. All patients excluding 1 infant were not intubated electively overnight and went home the following morning. All patients showed no evidence of recurrence during follow-up. CONCLUSIONS: We found that the diagnosis of LTGDCs must be based on the anatomic relationship with the hyoid bone by enhanced sagittal neck CT. Endoscopic radical resection with microdissection electrodes can be recommended for reducing recurrence and morbidity by dissecting the hyoid perichondrium in the treatment of LTGDCs.

Blunt microdissection for endovascular treatment of infrainguinal chronic total occlusions.

PURPOSE: To present a systematic safety evaluation of the CrossBoss blunt microdissection catheter for crossing peripheral chronic total occlusions (CTOs). METHODS: Between July 2010 and July 2011, 15 patients (all men; mean age 60.7±9.1 years) underwent endovascular treatment of 17 infrainguinal CTOs that were resistant to guidewire passage, so the blunt microdissection catheter was employed to recanalize the artery. Fourteen lesions were de novo and 3 were in-stent restenoses. Sixteen lesions were in the superficial femoral artery; 8 of 17 CTOs were TASC II type D. Extensive calcification was present in 12 lesions. Mean lesion length was 182.9±66.2 mm (range 57-296). RESULTS: Procedural success was 100% and successful crossing without the use of a re-entry device (technical success) was achieved in 15 cases. Twelve lesions were stented. Average fluoroscopy time was 36.5±21.2 minutes (143.8±76.9 Gy*cm (2) radiaton dose area product), during which a mean 172.1±62.2 mL of iodinated contrast were used. Two patients had access site hematomas that were treated conservatively, and there was no perforation, distal embolization, amputation, or need for urgent revascularization. During the mean follow-up of 11.4±0.1 months, 1 patient died, and none required an amputation or surgical revascularization. There was a significant improvement in ankle-brachial index (0.6±0.1 to 0.8±0.2, p=0.001) and symptoms as assessed by Rutherford class at 1 year. Four of 17 limbs required secondary revascularization procedures within 1 year. CONCLUSION: The CrossBoss blunt microdissection catheter facilitated successful crossing of CTOs in patients with infrainguinal lesions following unsuccessful guidewire crossing, with an acceptably low rate of periprocedural complications and significant improvement in symptoms.

The "bespoke" upper eyelid blepharoplasty and brow rejuvenation.

Blepharoplasty of the upper eyelids is one of the most commonly performed procedures in aesthetic plastic surgery. In this article, we describe our approach to the patient with aging of the periorbita. At all times, the approach is tailored to the individual's needs, trying to achieve a natural result that will not in any way affect the function of the eyelid. Our current approach and techniques for upper eyelid blepharoplasty and brow lifting are described.

Laser-based microdissection of single cells from tissue sections and PCR analysis of rearranged immunoglobulin genes from isolated normal and malignant human B cells.

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene family-specific primers recognizing nearly all V gene segments together with primers for the J gene segments. By sequence analysis of V genes from distinct cells, the clonal relationship of the B lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D, and J gene usage. Moreover, the presence and pattern of somatic Ig V gene mutations give valuable insight into the stage of differentiation of the B cells.

Infratentorial supracerebellar resection of a pineal tumor using a high definition video exoscope (VITOM®).

A telescope based high definition (HD) video system (VITOM®, Karl Storz GmBH & Co., Tuttlingen, Germany) has recently been proposed as an alternative to the operating microscope for microneurosurgery. It remains unclear which clinical situations will benefit from its advantages. In light of the uncomfortable surgeon position and fatigue often associated with pineal region surgery, we used the VITOM® HD exoscope system to perform an infratentorial supracerebellar resection of a pineal tumor. The VITOM® dramatically improved surgeon comfort and ease of operating by permitting the surgeon to stand upright and in a comfortable position and avoid the need to extend the arm or assume an awkward. position commonly encountered when using the microscope for these approaches. The marked improvement in surgeon comfort afforded by the VITOM® exoscope indicates that this system may have significant advantages over traditional microscope based surgery for tumors of the pineal region approached using an infratentorial supracerebellar approach.

Laser capture microdissection applications in breast cancer proteomics.

Breast cancer tissues are characterized by cellular heterogeneity, representing a mixture of, e.g., healthy epithelial ducts, invasive or in situ tumor cells, surrounding stroma, infiltrating immune cells, blood vessels, and capillaries. As a consequence, protein extracts from whole tissue lysates also represent a variety of cell types present in the tissues under examination. This, however, seriously hampers the analysis of tumor cell-specific signals, which is of interest when performing biomarker discovery-type of studies. Therefore, laser capture microdissection is a perfect tool to isolate a relatively pure population of cells of interest, such as tumor cells. In this chapter, we describe the use of the PALM MicroBeam system for laser microdissection and pressure catapulting. Protocols are provided for sectioning, staining, microdissection, sample preparation, and mass spectrometric analysis of snap frozen breast cancer tissue.

Laser-capture microdissection and transcriptional profiling in archival FFPE tissue in prostate cancer.

Prognostic markers can improve prediction of the behaviour of a cancer at the point of diagnosis. A key value of any prognostic marker is at the point of tumour diagnosis. In the context of prostate cancer, this implies profiling in the diagnostic formalin-fixed, paraffin-embedded (FFPE) transrectal ultrasound-guided (TRUS) needle biopsy. TRUS needle biopsies commonly contain both stromal and epithelial cells, and malignant glands are found as isolated foci within this tissue. Using the entire biopsy for genetic analysis inevitably results in a significant contamination of malignant cells with benign tissue. This combination of minimal tumour yields and tissue heterogeneity have so far prohibited prognostic transcript and microarray molecular studies in needle biopsies. Laser-capture microdissection (LCM) allows enriched cell populations to be accurately isolated from heterogeneous tissue, hence facilitating analysis of different components from a single tissue sample. Here, we describe its use in isolating tumour cells in archival FFPE prostate needle biopsies and subsequent application for RNA extraction and quantitative real-time PCR (QPCR).

Laser-capture microdissection for Factor VIII-expressing endothelial cells in cancer tissues.

The immunostaining based Laser-capture microdissection (LCM) method called immune-LCM allows us to quantify the mRNA. Immune-LCM has recently been introduced to enhance identification of cells carrying a particular protein from frozen tissue samples. We have recently performed the immune-LCM of formaldehyde-fixated, paraffin-embedded tissues immunostained with a monoclonal antibody Factor VIII. This method could be useful for quantitative gene expression analysis in blood vessels from tumors of patients that have been treated with antiangiogenic drugs, allowing for validation of the effect of drug on the expected targets. Such capability might be exceedingly useful for the evaluation of the bioactivity of new drugs. This method is also useful to compare gene expression patterns in tumor cells versus endothelial cells during tumor progression or tumor angiogenesis.

Mixed epithelial and stromal tumors of the kidney: evidence for a single cell of origin with capacity for epithelial and stromal differentiation.

Mixed epithelial and stromal tumors are uncommon biphasic tumors of the kidney, with cystic and solid areas composed of a morphologically diverse stroma, including ovarian-like stroma and an epithelial component with considerable heterogeneity. Little is known about the histogenesis and clonal origins of these tumors. A total of 21 mixed epithelial and stromal tumors of the kidney from female patients who underwent radical or partial nephrectomies were examined. The epithelial and stromal components, and also adjacent non-neoplastic renal parenchymal tissues were separately laser microdissected from sections prepared from formalin-fixed and paraffin-embedded tissues for X chromosome inactivation analysis. Nineteen of the 21 tumors were informative. Seven of these informative cases showed random X chromosome inactivation pattern in both epithelial and stromal components of mixed epithelial and stromal tumors of the kidney. Nonrandom inactivation of the X chromosome was found in 12 of 19 informative tumors. The same pattern of nonrandom inactivation of the X chromosome was seen in both epithelial and stromal components in all 12 of the tumors with nonrandom X chromosome inactivation. Our data support the theory that the stroma and epithelium arise from a common cell of origin.