Characterization of Microscopic Multicellular Foci in Grossly Normal Renal Parenchyma of Von Hippel-Lindau Kidney.

Background and Objectives: This study aims to describe the earliest renal lesions in patients with von Hippel-Lindau (VHL) disease, especially the multicellular microscopic pathologic events, to get information into the genesis of renal neoplasms in this condition. Materials and Methods: Multicellular events were identified, and 3dimensional reconstruction was performed in grossly normal kidney parenchyma from VHL disease patients by using H&E-stained slides previously prepared. Results: The lesions were measured and the volume of clusters was calculated. Immunohistochemistry was performed for downstream HIF-target protein carbonic anhydrase 9 (CAIX) as well as CD34 for assessment of angiogenesis. We divided lesions into four types according to lesion height/size. The number of lesions was markedly decreased from lesion 1 (smallest) to lesion 2, then from lesions 2 to 3, and again from lesion 3 to 4. Distribution was highly consistent in the four cases, and the same decrement pattern was seen in all blocks studied. The volumes of clusters were measured and divided into three categories according to their volume. The most frequent pathologic event in VHL kidneys was category 1 (smallest volume), then category 2, and then category 3. Conclusion: We demonstrate that tracking histologic and morphologic changes in 3 dimensions of multicellular microscopic pathologic events enabled us to confirm a protracted sequence of events from smaller to larger cellular amplification events in VHL kidney.

Myelin phagocytosis by astrocytes after myelin damage promotes lesion pathology.

Astrocytes are key players in the pathology of multiple sclerosis and can assume beneficial and detrimental roles during lesion development. The triggers and timing of the different astroglial responses in acute lesions remain unclear. Astrocytes in acute multiple sclerosis lesions have been shown previously to contain myelin debris, although its significance has not been examined. We hypothesized that myelin phagocytosis by astrocytes is an early event during lesion formation and leads to astroglial immune responses. We examined multiple sclerosis lesions and other central nervous system pathologies with prominent myelin injury, namely, progressive multifocal leukoencephalopathy, metachromatic leukodystrophy and subacute infarct. In all conditions, we found that myelin debris was present in most astrocytes at sites of acute myelin breakdown, indicating that astroglial myelin phagocytosis is an early and prominent feature. Functionally, myelin debris was taken up by astrocytes through receptor-mediated endocytosis and resulted in astroglial NF-κB activation and secretion of chemokines. These in vitro results in rats were validated in human disease where myelin-positive hypertrophic astrocytes showed increased nuclear localization of NF-κB and elevated chemokine expression compared to myelin-negative, reactive astrocytes. Thus, our data suggest that myelin uptake is an early response of astrocytes in diseases with prominent myelin injury that results in recruitment of immune cells. This first line response of astrocytes to myelin injury may exert beneficial or detrimental effects on the lesion pathology, depending on the inflammatory context. Modulating this response might be of therapeutic relevance in multiple sclerosis and other demyelinating conditions.

Spatio-temporal transcriptome of the human brain.

Brain development and function depend on the precise regulation of gene expression. However, our understanding of the complexity and dynamics of the transcriptome of the human brain is incomplete. Here we report the generation and analysis of exon-level transcriptome and associated genotyping data, representing males and females of different ethnicities, from multiple brain regions and neocortical areas of developing and adult post-mortem human brains. We found that 86 per cent of the genes analysed were expressed, and that 90 per cent of these were differentially regulated at the whole-transcript or exon level across brain regions and/or time. The majority of these spatio-temporal differences were detected before birth, with subsequent increases in the similarity among regional transcriptomes. The transcriptome is organized into distinct co-expression networks, and shows sex-biased gene expression and exon usage. We also profiled trajectories of genes associated with neurobiological categories and diseases, and identified associations between single nucleotide polymorphisms and gene expression. This study provides a comprehensive data set on the human brain transcriptome and insights into the transcriptional foundations of human neurodevelopment.

Pembrolizumab for patients with melanoma or non-small-cell lung cancer and untreated brain metastases: early analysis of a non-randomised, open-label, phase 2 trial.

BACKGROUND: Immunotherapy targeting the PD-1 axis has activity in several tumour types. We aimed to establish the activity and safety of the PD-1 inhibitor pembrolizumab in patients with untreated brain metastases from melanoma or non-small-cell lung cancer (NSCLC). METHODS: In this non-randomised, open-label, phase 2 trial, we enrolled patients aged 18 years or older with melanoma or NSCLC with untreated brain metastases from the Yale Cancer Center. Patients had at least one untreated or progressive brain metastasis between 5 and 20 mm in diameter without associated neurological symptoms or the need for corticosteroids. Patients with NSCLC had tumour tissue positive for PD-L1 expression; this was not required for patients with melanoma. Patients were given 10 mg/kg pembrolizumab every 2 weeks until progression. The primary endpoint was brain metastasis response assessed in all treated patients. The trial is ongoing and here we present an early analysis. The study is registered with ClinicalTrials.gov, number NCT02085070. FINDINGS: Between March 31, 2014, and May 31, 2015, we screened 52 patients with untreated or progressive brain metastases (18 with melanoma, 34 with NSCLC), and enrolled 36 (18 with melanoma, 18 with NSCLC). A brain metastasis response was achieved in four (22%; 95% CI 7-48) of 18 patients with melanoma and six (33%; 14-59) of 18 patients with NSCLC. Responses were durable, with all but one patient with NSCLC who responded showing an ongoing response at the time of data analysis on June 30, 2015. Treatment-related serious and grade 3-4 adverse events were grade 3 elevated aminotransferases (n=1 [6%]) in the melanoma cohort, and grade 3 colitis (n=1 [6%]), grade 3 pneumonitis (n=1 [6%]), grade 3 fatigue (n=1 [6%]), grade 4 hyperkalemia (n=1 [6%]), and grade 2 acute kidney injury (n=1 [6%]) in the NSCLC cohort. Clinically significant neurological adverse events included transient grade 3 cognitive dysfunction and grade 1-2 seizures (n=3 [17%]) in the melanoma cohort. INTERPRETATION: Pembrolizumab shows activity in brain metastases in patients with melanoma or NSCLC with an acceptable safety profile, which suggests that there might be a role for systemic immunotherapy in patients with untreated or progressive brain metastases. FUNDING: Merck and the Yale Cancer Center.

Prion-Protein-interacting Amyloid-ß Oligomers of High Molecular Weight Are Tightly Correlated with Memory Impairment in Multiple Alzheimer Mouse Models.

Alzheimer disease (AD) is characterized by amyloid-ß accumulation, with soluble oligomers (Aßo) being the most synaptotoxic. However, the multivalent and unstable nature of Aßo limits molecular characterization and hinders research reproducibility. Here, we characterized multiple Aßo forms throughout the life span of various AD mice and in post-mortem human brain. Aßo exists in several populations, where prion protein (PrP(C))-interacting Aßo is a high molecular weight Aß assembly present in multiple mice and humans with AD. Levels of PrP(C)-interacting Aßo match closely with mouse memory and are equal or superior to other Aß measures in predicting behavioral impairment. However, Aßo metrics vary considerably between mouse strains. Deleting PrP(C) expression in mice with relatively low PrP(C)-interacting Aßo (Tg2576) results in partial rescue of cognitive performance as opposed to complete recovery in animals with a high percentage of PrP(C)-interacting Aßo (APP/PSEN1). These findings highlight the relative contributions and interplay of Aßo forms in AD.

Varied autopsy findings in five treated patients with Gaucher disease and parkinsonism include the absence of Gaucher cells.

Enzyme replacement therapy is standard of care for patients with Gaucher disease, as it significantly improves skeletal, visceral, and hematological symptoms. Few pathological studies have documented the extent of pathological findings in treated patients. Autopsy findings in five treated patients, who ultimately developed parkinsonism, ranged from the complete absence of Gaucher pathology to extensive involvement of multiple tissues, without correlation to age, genotype, spleen status, or dose/duration of therapy. Additional autopsies may elucidate modifiers and biomarkers contributing to disease burden and response to therapy.

A 10-minute prototype assay for tissue degradation monitoring in clinical specimens.

We recently identified alpha II spectrin as a Tissue Degradation Indicator (TDI) and demonstrated that intrinsic spectrin-breakdown levels reliably reveal tissue degradation status in biospecimens. With the present study, we introduce an in vitro biological assay to mimic the endogenous spectrin-breakdown process and serve as degradation monitor (DM). By initiating the DM at the time of specimen collection and by attaching the DM to respective specimens, specimen degradation can be assessed by DM readout without specimen consumption. Using a protease inhibitory assay and protease-targeted immunoassays, we identified calpain as the protease responsible for degradation-induced spectrin breakdown. To recapitulate spectrin degradation in vitro, we developed several enzymatic assays in test tubes by incubating recombinant spectrins and synthetic Fluorescence Resonance Energy Transfer (FRET)-based spectrin peptides with purified human and porcine calpains. The in vitro assays reliably performed in different environments for a limited time due to loss of calpain activity. To maintain longer calpain activity, we introduced cultured cells as calpain providers into the in vitro assays. Under a variety of degradative conditions, including 4°C, 13°C, 23°C, 29°C, 37°C, freezing, and freeze-thaw steps, we compared the use of this prototype DM to the intrinsic spectrin cleavage assay (ISCA) in specimen degradation assessment using animal models. A strong correlation (r=0.9895) was detected between the DM-revealed degradation and the ISCA-revealed degradation. Notably, the DM-based degradation assessment takes only 10min and does not jeopardize the tissue itself, whereas the ISCA-based degradation assessment needs to sacrifice tissues and takes several hours to accomplish. Our data suggests the application of an in vitro degradation monitor for fast, real time, and non-invasive assessment of specimen degradation. This observation could lead to a transformative product dedicated to biospecimen quality control. This study also addresses critical, yet unmet needs for developing a universal standard for specimen degradation measurement.

Radiologic and histologic consequences of radiosurgery for brain tumors.

Progressively enlarging encephalopathic changes are now well-documented effects of gamma knife radiosurgery (GKRS) occurring ~3-30 months after treatment of both benign and malignant brain lesions. These changes can be variably associated with inflammatory demyelination and necrosis and/or recurrent tumor. While radiographic differentiation between encephalopathic changes and recurrent tumor is of high clinical relevance, confident interpretation of post-radiosurgery imaging changes can be challenging or even impossible in some cases. Gadolinium-enhanced MRI of these lesions reveals variable amounts of enhancing and non-enhancing components within these lesions that have not been clearly correlated with structural-pathologic change. The goal of this study is to characterize the histopathological changes associated with enhancing versus non-enhancing regions of GKRS-treated lesions. MRI images of patients with progressive, etiologically ambiguous brain lesions following GKRS were reviewed prior to explorative neurosurgery. Chosen for this study were lesions in which distinct areas of enhancement and non-enhancement of at least 5 mm in size could be identified (n = 16). Distinctly enhancing and non-enhancing areas were separately biopsied and histologically evaluated. Only cases with uniform histological results are presented in this study. Enhancing and non-enhancing areas in post GKRS lesions represent separate pathological changes. Radiographically enhancing areas correlate either with recurrent tumor growth or inflammatory demyelinating changes. Lack of radiographic enhancement correlates with coagulative necrosis if the sample is taken from the center of the lesion, or with reactive astrocytosis if the sample is taken from the periphery. Separate biopsy of enhancing and non-enhancing regions of post-GKRS encephalopathy was able to confirm that the pathologies in these areas are distinct. These findings allow for better-informed correlation of histological and radiological changes and a better understanding of post-treatment tissue pathology.

Significance of histology in determining management of lesions regrowing after radiosurgery.

Brain metastases treated with stereotactic radiosurgery may show delayed enlargement on post-treatment imaging that is of ambiguous etiology. Histopathologic interpretation of brain specimens is often challenging due to the presence of significant radiation effects admixed with irradiated residual tumor of indeterminate viability. The purpose of this study was to assess the impact of histologic findings on clinical outcomes following resection of these lesions. Between 2004 and 2010, 690 patients with brain metastases were enrolled in a prospective gamma knife data repository, and lesions requiring excision were identified. Tissue specimens were divided into four groups based on the ratio of treatment related inflammatory changes (TRIC) to tumor cells, and subsequently patient outcomes were assessed. Of 2,583 metastases treated, 36 were excised due to symptomatic enlargement. Only TRIC, without residual evidence of tumor, was seen in 36 % (13/36) of specimens. Resection of these lesions resulted in 100 % local control in follow-up. Of the remaining 23 lesions that contained any viable-appearing tumor within the resected specimen, 8 recurred after resection. Lesions that enlarged in the first 6 months were more likely to contain higher amounts of residual tumor cells. Patients with even <2 % tumors cells on excision had significantly worse local control (75 vs. 100 %, p = 0.024) and survival (HR 0.27, p = 0.029) compared with those patients with exclusively TRIC. In summary, our findings underscore the importance of surgically obtaining tissue in a method that facilitates complete lesional interpretive histology in order to accurately guide ongoing patient management.

VHL-deficient vasculogenesis in hemangioblastoma.

Hemangioblasts are capable of differentiation into vascular structures and blood. Patients with von Hippel-Lindau (VHL) disease develop hemangioblastomas which are composed of VHL-deficient tumor cells with protracted hemangioblastic differentiation potential. In a subset of these tumors, hemangioblastic differentiation is characterized by different stages of red blood cell formation. It has remained controversial, however, whether VHL-deficient hemangioblastic cells are similarly capable of differentiating into vascular cells and functioning vascular structures in vivo. By histologic, immunohistologic and microdissection-based genetic analysis of 60 VHL disease-associated hemangioblastomas, we re-examined the controversial question whether VHL-deficient neoplastic hemangioblastic cells are capable of vascular differentiation (vasculogenesis). In most tumors (n=47), there was no evidence of either vasculogenesis or hematopoiesis; tumor cells were either scattered between reactive angiogenetic vascular structures or arranged in solid clusters. A subset of tumors (n=13), however, revealed vaculogenetic structures that were composed of cuboidal or flat cells and frequently contained red blood cell precursors or mature red blood cells. Microdissection-based deletion analysis of epithelial cells confirmed them to be VHL-deficient tumor cells. Immunohistochemistry for CD31 was consistently negative in these structures, and no evidence could be obtained for connectivity with reactive vasculature. We demonstrate that hemangioblastic differentiation capacity of VHL-deficient hemangioblastic cells includes not only erythropoiesis, but also differentiation into primitive vasculogenetic structures. Tumor cells, however, do not appear to have the potential of terminal differentiation into mature and functional vascular structures.

Neuronal and glioma-derived stem cell factor induces angiogenesis within the brain.

Stem cell factor (SCF) is overexpressed by neurons following brain injury as well as by glioma cells; however, its role in gliomagenesis remains unclear. Here, we demonstrate that SCF directly activates brain microvascular endothelial cells (ECs) in vitro and induces a potent angiogenic response in vivo. Primary human gliomas express SCF in a grade-dependent manner and induce normal neurons to express SCF in brain regions infiltrated by glioma cells, areas that colocalize with prominent angiogenesis. Downregulation of SCF inhibits tumor-mediated angiogenesis and glioma growth in vivo, whereas overexpression of SCF is associated with shorter survival in patients with malignant gliomas. Thus, the SCF/c-Kit pathway plays an important role in tumor- and normal host cell-induced angiogenesis within the brain.

Pediatric Prostatic Alveolar Rhabdomyosarcoma Presenting with Metastatic Spinal Cord Compression in the Thoracic Spine: A Case Report and Review of the Literature.

Rhabdomyosarcoma (RMS) is a pediatric malignancy with a variable prognosis depending on tumor stage and genotype. There has been a significant improvement in survival rates over the past decades. However, aggressive variants can metastasize to locations that are difficult to treat. We report a case of prostatic alveolar rhabdomyosarcoma with metastases to the bone marrow and thoracic spine in a child. The patient was treated with a multimodal approach that included surgical resection of the epidural mass; the administration of vincristine, dactinomycin, and cyclophosphamide; and radiotherapy. Unfortunately, after six months, the patient showed disease progression and was started on secondary-line treatment. This case illustrates the difficulties in managing end-stage metastatic rhabdomyosarcoma and is the first report of prostatic rhabdomyosarcoma presenting with spinal cord compression in a child.

Intrinsic indicators for specimen degradation.

Variable degrees of molecular degradation occur in human surgical specimens before clinical examination and severely affect analytical results. We therefore initiated an investigation to identify protein markers for tissue degradation assessment. We exposed 4 cell lines and 64 surgical/autopsy specimens to defined periods of time at room temperature before procurement (experimental cold ischemic time (CIT)-dependent tissue degradation model). Using two-dimensional fluorescence difference gel electrophoresis in conjunction with mass spectrometry, we performed comparative proteomic analyses on cells at different CIT exposures and identified proteins with CIT-dependent changes. The results were validated by testing clinical specimens with western blot analysis. We identified 26 proteins that underwent dynamic changes (characterized by continuous quantitative changes, isoelectric changes, and/or proteolytic cleavages) in our degradation model. These changes are strongly associated with the length of CIT. We demonstrate these proteins to represent universal tissue degradation indicators (TDIs) in clinical specimens. We also devised and implemented a unique degradation measure by calculating the quantitative ratio between TDIs' intact forms and their respective degradation-modified products. For the first time, we have identified protein TDIs for quantitative measurement of specimen degradation. Implementing these indicators may yield a potentially transformative platform dedicated to quality control in clinical specimen analyses.

High-throughput secretomic analysis of single cells to assess functional cellular heterogeneity.

Secreted proteins dictate a range of cellular functions in human health and disease. Because of the high degree of cellular heterogeneity and, more importantly, polyfunctionality of individual cells, there is an unmet need to simultaneously measure an array of proteins from single cells and to rapidly assay a large number of single cells (more than 1000) in parallel. We describe a simple bioanalytical assay platform consisting of a large array of subnanoliter microchambers integrated with high-density antibody barcode microarrays for highly multiplexed protein detection from over a thousand single cells in parallel. This platform has been tested for both cell lines and complex biological samples such as primary cells from patients. We observed distinct heterogeneity among the single cell secretomic signatures that, for the first time, can be directly correlated to the cells' physical behavior such as migration. Compared to the state-of-the-art protein secretion assay such as ELISpot and emerging microtechnology-enabled assays, our approach offers both high throughput and high multiplicity. It also has a number of clinician-friendly features such as ease of operation, low sample consumption, and standardized data analysis, representing a potentially transformative tool for informative monitoring of cellular function and immunity in patients.

Nervous system involvement in von Hippel-Lindau disease: pathology and mechanisms.

Patients with von Hippel-Lindau disease carry a germline mutation of the Von Hippel-Lindau (VHL) tumor-suppressor gene. We discuss the molecular consequences of loss of VHL gene function and their impact on the nervous system. Dysfunction of the VHL protein causes accumulation and activation of hypoxia inducible factor (HIF) which can be demonstrated in earliest stages of tumorigenesis and is followed by expression of VEGF, erythropoietin, nitric oxide synthase and glucose transporter 1 in VHL-deficient tumor cells. HIF-independent functions of VHL, epigenetic inactivation of VHL, pVHL proteostasis, and links between loss of VHL function and developmental arrest are also described. A most intriguing feature in VHL disease is the occurrence of primary hemangioblastic tumors in the nervous system, the origin of which has not yet been entirely clarified, and current hypotheses are discussed. Endolymphatic sac tumors may extend into the brain, but originally arise from proliferation of endolymphatic duct/sac epithelium; the exact nature of the proliferating epithelial cell, however, also has remained unclear, as well as the question why tumors almost consistently develop in the intraosseous portion of the endolymphatic sac/duct only. The epitheloid clear cell morphology of both advanced hemangioblastoma and renal clear cell carcinoma can make the differential diagnosis challenging, recent developments in immunohistochemical differentiation are discussed. Finally, metastasis to brain may not only be caused by renal carcinoma, but may derive from VHL disease-associated pheochromocytoma/paraganglioma, or pancreatic neuroendocrine tumor.

Evolution of tumefactive lesions in multiple sclerosis: a 12-year study with serial imaging in a single patient.

We describe the acute presentation and the long-term evolution of recurrent tumefactive lesions (TLs) in a patient with relapsing-remitting multiple sclerosis. Five TLs occurred on three different occasions over a period of 12 years and these were followed by 73 serial magnetic resonance images (MRI). TL evolution was described by means of magnetization transfer imaging (MTI) and cerebrospinal fluid tissue specific imaging (TSI) over the follow-up period. During the study period, the patient had three clinical relapses with only minimal disability progression. MTI demonstrated that only the peripheral portion of each TL reverted to pre-lesional MT ratios within six months' post-enhancement. Recurring TLs may present a similar pattern of evolution that may be associated with a long-term favourable clinical outcome.

Novel mutations in the CLCN1 gene of myotonia congenita: 2 case reports.

INTRODUCTION: Myotonia Congenita is an inherited myotonia that is due to a mutation in the skeletal muscle chloride channel CLCN1. These mutations lead to reduced sarcolemmal chloride conductance, causing delayed muscle relaxation that is evident as clinical and electrical myotonia. METHODS: We report the clinical presentations of two individuals with Myotonia Congenita (MC). RESULTS: Patient 1 has been diagnosed with the recessive form of MC, known as the Becker variant, and Patient 2 has been diagnosed with the dominant form of MC, known as the Thomsen variant. In both patients, the diagnosis was made based on the clinical presentation, EMG and CLCN1 gene sequencing. Patient 1 also had a muscle biopsy. CONCLUSIONS: Genetic testing in both patients reveals previously unidentified mutations in the CLCN1 gene specific to Myotonia Congenita. We report the salient clinical features of each patient and discuss the effects and common types of CLCN1 mutations and review the literature.

Expression of Hemangioblast Proteins in von Hippel-Lindau Disease Related Tumors.

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome that targets a highly selective subset of organs causing specific types of tumors. The biological basis for this principle of organ selectivity and tumor specificity is not well understood. VHL-associated hemangioblastomas share similar molecular and morphological features with embryonic blood and vascular precursor cells. Therefore, we suggest that VHL hemangioblastomas are derived from developmentally arrested hemangioblastic lineage keeping their potential of further differentiation. Due to these common features, it is of major interest to investigate whether VHL-associated tumors other than hemangioblastoma also share these pathways and molecular features. The expression of hemangioblast proteins has not yet been assessed in other VHL-related tumors. To gain a better understanding of VHL tumorigenesis, the expression of hemangioblastic proteins in different VHL-associated tumors was investigated. The expression of embryonic hemangioblast proteins Brachyury and TAL1 (T-cell acute lymphocytic leukemia protein 1) was assessed by immunohistochemistry staining on 75 VHL-related tumors of 51 patients: 47 hemangioblastomas, 13 clear cell renal cell carcinomas, 8 pheochromocytomas, 5 pancreatic neuroendocrine tumors, and 2 extra-adrenal paragangliomas. Brachyury and TAL1 expression was, respectively, observed in 26% and 93% of cerebellar hemangioblastomas, 55% and 95% of spinal hemangioblastomas, 23% and 92% of clear cell renal cell carcinomas, 38% and 88% of pheochromocytomas, 60% and 100% of pancreatic neuroendocrine tumors, and 50% and 100% of paragangliomas. We concluded that the expression of hemangioblast proteins in different VHL-associated tumors indicates a common embryological origin of these lesions. This may also explain the specific topographic distribution of VHL-associated tumors.

Qualitative Comparison of Cryostat- versus Snap-Frozen Neurosurgical Intraoperative Consultations.

Background. Frozen sections (FS) are common in neurosurgery to address varied clinical concerns. Artifacts in central nervous system (CNS) FS can be severe and affect or hinder interpretation. We performed a case-control study using a semiquantitative scale: the Histologic Preservation Score (HPS), and a quantitative scale: the Ice Crystal Vacuolization Score (ICVS), to compare the histologic quality yielded by snap- versus cryostat freezing techniques. Material and Methods. All specimens were sectioned in 2 halves, one half was used for FS and the other for permanent evaluation. HPS assigns a distortion score to the FS sample using the non-frozen half as the comparator: 1 = minimal, 2 = slight, 3 = moderate, 4 & 5 = severe. The ICVS is the average size in µm of the 5 largest vacuoles/0.05 mm2, evaluated on digitized slides. Results. 86 CNS-FS were collected: 22 snap- and 64 cryostat-FS. Significant differences in HPS: 2.28 versus 2.84 (p <0.05) and ICVS 7.47 versus 14.56 (p < 0.001) were obtained for snap- versus cryostat-FS, respectively. HPS and ICVS showed a strong correlation: R2 = 0.63, p < 0.0001. Histologic distortion was worse for neuroglial than mesenchymal tissue by both methods; however, a significant difference was only observed in cryostat-FS: HPS: 3.23 versus 2.33, p < 0.001; ICVS: 16.86 µm versus 10.26 µm, p < 0.001. Conclusion. Snap-FS yields better histologic quality than cryostat-FS for CNS-FS, and the difference is more pronounced in neuroglial samples. HPS and ICVS correlate strongly, indicating that the histologic quality is inversely proportional to water-crystallization. These results may apply to other areas of surgical pathology.