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.

Efficacy of the second micro-testicular sperm extraction after failed first micro-testicular sperm extraction in men with nonobstructive azoospermia.

OBJECTIVE: To evaluate the efficacy of the second micro-testicular sperm extraction (TESE)in men with nonobstructive azoospermia in whom the first micro-TESE failed. DESIGN: Retrospective. SETTING: Private clinic. PATIENT(S): One hundred twenty-five men with nonobstructive azoospermia with failed previous micro-TESE. The patients were divided into 2 groups according to their surgical sperm retrieval status during the second micro-TESE. If sperm could not be found, these patients were classified as Group 1, and, if sperm was found, the patients were classified as Group 2. The 2 groups were compared for clinical parameters and pathologic findings. INTERVENTION(S): Micro-TESE. MAIN OUTCOME MEASURE(S): Surgical sperm retrieval status. RESULT(S): Sperm was recovered successfully in 23 of 125 (18.4%) men with the second micro-TESE. Testicular volume was significantly lower in Group 2 (8.2 ± 5.4 mL) than Group 1 (11.3 ± 5.3 mL). Seven of 14 (50%) patients with Klinefelter's Syndrome had sperm recovery with repeat micro-TESE. The sperm retrieval rate was significantly higher in the Leydig cell hyperplasia and tubular sclerosis groups than in the Sertoli cell only and maturation arrest groups (54.5%, 10.1%, and 18.6%, respectively). CONCLUSION(S): On the basis of our results, 18.4% of men with failed first micro-TESE had a probability of sperm retrieval with the second micro-TESE. Patients with successful sperm recovery had smaller testicular volumes than those with a failed second attempt. Severe testicular atrophy was not a contraindication for the second micro-TESE in such patients.

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.

Methods to Visualize Auxin and Cytokinin Signaling Activity in the Shoot Apical Meristem.

Visualizing the distribution of hormone signaling activity such as auxin and cytokinins is of key importance for understanding regulation of plant development and physiology. Live imaging and genetically encoded hormone biosensors and reporters allow monitoring the spatial and temporal distribution of these phytohormones. Here, we describe how to cultivate live shoot apical meristems after dissection for observation under the confocal microscope for up to 4 days. The shoot apical meristems are maintained on an appropriate medium allowing them to grow and initiate new organs at a frequency similar to plants grown on soil. Meristems expressing hormone biosensors and reporters allows following hormone signaling activity distribution at high spatiotemporal resolution without chemical fixation, an approach that that can also be applied to follow the dynamics of expression in vivo of any fluorescent marker.

Studying Na+ and K+ channels in aldosterone-sensitive distal nephrons.

Aldosterone-sensitive distal nephron (ASDN) including the distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct (CD) plays an important role in the regulation of hormone-dependent Na+ reabsorption and dietary K+-intake dependent K+ excretion. The major Na+ transporters in the ASDN are thiazide-sensitive Na-Cl cotransporter (NCC), epithelial Na+ channel (ENaC), pendrin/Na+-dependent Cl--bicarbonate exchanger (NDCBE). Whereas major K+ channels in the ASDN are Kir4.1 and Kir5.1 in the basolateral membrane; and Kir1.1 (ROMK) and Ca2+ activated big conductance K+ channel (BK) in the apical membrane. Although a variety of in vitro cell lines of the ASDN is available and these cell models have been employed for studying Na+ and K+ channels, the biophysical properties and the regulation of Na+ and K+ channels in vitro cell models may not be able to recapitulate those in vivo conditions. Thus, the studies performed in the native ASDN are essential for providing highly physiological relevant information and for understanding the Na+ and K+ transport in the ASDN. Here we provide a detailed methodology describing how to perform the electrophysiological measurement in the native DCT, CNT and cortical collecting duct (CCD).

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.

Site-specific gene expression analysis using an automated tissue micro-dissection punching system.

Site-specific gene expression analyses are important for understanding tissue functions. Despite rapid developments in DNA-related technologies, the site-specific analysis of whole genome expression for a tissue remains challenging. Thus, a new tool is required for capturing multiple tissue micro-dissections or single cells while retaining the positional information. Here, we describe the development of such a system, which can pick up micro-dissections by punching a tissue repeatedly in a very short period, e.g., 5 s/sampling cycle. A photo of the punched tissue provides information on the dissected positions, allowing site-specific gene expression analysis. We demonstrate the site-specific analysis of a frozen tissue slice of mouse brain by analyzing many micro-dissections produced from the tissue at a 300-µm pitch. The site-specific analysis provided new insights into the gene expression profiles in a tissue and on tissue functions. The analysis of site-specific whole genome expression may therefore, open new avenues in life science.

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.

A multicentre study of the precision and accuracy of the TruSlice and TruSlice Digital histological dissection devices.

BACKGROUND: Five key factors enabling a good surgical grossing technique include a flat uniformly perpendicular specimen cutting face, appropriate immobilisation of the tissue specimen during grossing, good visualisation of the cutting tissue face, sharp cutting knives and the grossing knife action. TruSlice and TruSlice Digital are new innovative tools based on a guillotine configuration. The TruSlice has plastic inserts whilst the TruSlice Digital has an electronic micrometre attached: both features enable these dissection factors to be controlled. The devices were assessed in five hospitals in the UK. MATERIAL AND METHODS: A total of 267 fixed tissue samples from 23 tissue types were analysed, principally the breast (n = 32) skin (30), rectum (28), colon (27) and cervix (17). Precision and accuracy were evaluated by measuring the defined thickness, and the consistency of achieving the defined thickness of tissue samples taken respectively. Both parameters were expressed as a total percentage of compliance for the cohort of samples accessed. RESULTS: Overall, the mean (standard deviation) score for precision was 81 (11) % whilst the accuracy score was 82 (11) % (both p < 0.05, chi-squared test), although this varied with type of tissue. Accuracy and precision were strongly correlated (rp = 0.83, p < 0.001). CONCLUSION: The TruSlice Digital devices offer an assured precision and accuracy performance which is reproducible across an assortment of tissue types. The use of a micrometre to set tissue slice thickness is innovative and should comply with laboratory accreditation requirements, alleviating concerns of how to tackle issues such as the 'measurement of uncertainty' at the grossing bench.

Reproducibility of single-pass donor DSAEK tissue preparation with the MORIA single-use microkeratome.

PURPOSE: To assess the reproducibility of single-pass cutting for preparation of ultra-thin (≤120µm) donor cornea grafts in Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: All consecutive patients of DSAEK performed using the MORIA One Use microkeratome (MORIA, Antony, France) in between June 2014 and August 2015. Patient and donor central corneal thickness (CTT), microkeratome head, remaining stromal bed thickness (RSBT), number of cut and graft thickness at 1 month and 6 months postoperatively were recorded in this single-center prospective study. RESULTS: The mean preoperative donor graft CCT was 569.0±45.1µm and the mean donor endothelial graft CCT immediately after the cut was 116.5±28.7µm. At 1 month postoperatively, the mean CCT was 102.8±35.9µm. At 6 months, the mean CCT was 89.4±26.2µm. In all eyes, the mean CCT decreased from the post-cut (116.5±28.7µm) to the last visit (89.4±26.2µm) (P<0.01) due to in vivo deturgesence of the graft. We obtained 77.5% ultra-thin DSAEK immediately after the cut, 77.5% at 1 month and 89.8% after 6 months. CONCLUSION: Single-pass technique with the MORIA One Use microkeratome offers safe and reproducible DSAEK tissue preparation and allows achieving ultra-thin DSAEK in 89.8% of cases.

Development of new and accurate measurement devices (TruSlice and TruSlice Digital) for use in histological dissection: an attempt to improve specimen dissection precision.

Histological dissection of human tissue has relied on conventional procedures, which have largely remained unchanged for decades. Practices to determine measurement parameters employed in these procedures have largely relied on the use of rulers and weighing scales. It is well documented in the scientific literature that both fixation and processing of tissue can significantly affect the viability of the of tissue sections both for tinctorial and immunocytochemical investigations. Both of these factors can be compounded in their negative effects by inappropriate sampling of tissue at histological cut up. There are five key factors to ensure good surgical grossing technique, flat uniformly perpendicular specimen cutting face, appropriate immobilisation of the tissue specimen during grossing, good visualisation of the cutting tissue face, sharp cutting knives and the grossing knife action. Meeting these factors implies the devices are fit for purpose. Here we describe an innovative approach to designing cut up devices to improve accuracy and precision, which take these five key requirements into consideration. The devices showed accuracy and precision, enabling tissue slices to be produced in a uniformly perpendicular fashion to within 2 mm in thickness and to enable consistency and reproducibility of performance across a series of tissue types. The application of a digital rule on one of these devices ensures accuracy and also enables quality control issues to be clearly assessed. As cellular pathology laboratories conform to ever increasing standards of compliance and performance in practice, the advent of assured precision and accuracy at cut up is awaited. Recommendations from accreditation bodies such as the United Kingdom Accreditation Service (UKAS) continue to push for improvements in this area of histological investigation. These newly designed devices may give the answers to these requirements and provide the impetus for a new generation of innovative equipment for histological dissection.

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.

Position-Specific Gene Expression Analysis Using a Microgram Dissection Method Combined with On-Bead cDNA Library Construction.

Gene expression analysis is a key technology that is used to understand living systems. Multicellular organisms, including plants, are composed of various tissues and cell types, each of which exhibits a unique gene expression pattern. However, because of their rigid cell walls, plant cells are difficult to isolate from the whole plant. Although laser dissection has been used to circumvent this problem, the plant sample needs to be fixed beforehand, which presents several problems. In the present study, we developed an alternative method to conduct highly reliable gene expression profiling. First, we assembled a dissection apparatus that used a narrow, sharpened needle to dissect out a microsample of fresh plant tissue (0.1-0.2 mm on each side) automatically from a target site within a short time frame. Then, we optimized a protocol to synthesize a high-quality cDNA library on magnetic beads using a single microsample. The cDNA library was amplified and subjected to high-throughput sequencing. In this way, a stable and reliable system was developed to conduct gene expression profiling in small regions of a plant. The system was used to analyze the gene expression patterns at successive 50 µm intervals in the shoot apex of a 4-day-old Arabidopsis seedling. Clustering analysis of the data demonstrated that two small, adjacent domains, the shoot apical meristem and the leaf primordia, were clearly distinguishable. This system should be broadly applicable in the investigation of the spatial organization of gene expression in various contexts.

A Smart Haptic Hand-Held Device for Neurosurgical Microdissection.

Microneurosurgery requires dexterity, precision and delicate force application in order to be carried out safely and effectively. Neurosurgeons must apply sufficient force in order to carry out microsurgical procedures effectively but not excessive force such that iatrogenic injury occurs. This paper presents a smart hand-held microsurgical instrument that indicates to the surgeon when a force-threshold has been exceeded by providing vibrotactile feedback. Many existing haptic-feedback systems, particularly master-slave robotic platforms, are large, highly complex, and costly. By comparison, the proposed device is compact, fail-safe and low cost. Two psychophysical user studies were carried out to assess the proposed vibrotactile force-threshold feedback system. A cadaveric pilot study was carried out to evaluate the device in a microdissection task. In all the studies performed, the haptic dissector device has shown to be effective in providing real-time feedback in terms of force application during microsurgical tasks.

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.