Fixation in Form-Acetic allows hyaluronic acid detection in mouse ovaries

Lay summary To visualise tissues to determine the presence of disease or simply to understand anatomy, it is important to preserve fresh tissue. Fixatives are chemical solutions that preserve tissues to enable microscopic evaluation. However, some fixatives introduce artefact such as shrinkage of cells. Recently, a new fixative, Form-Acetic, was developed that is superior for preserving the structure of ovary tissue and allows investigation of ovary composition. One component of the ovary is hyaluronic acid (HA), which plays a crucial role in normal ovary function and fertility. Importantly, HA is sensitive to different fixative solutions. Therefore, it is meaningful to verify whether Form-Acetic is suitable for detecting HA. In this study, adult mouse ovaries were fixed in Form-Acetic and HA was detected. All HA-containing structures in the ovary were clearly distinguished which proves that the novel fixative allows the detection of HA.

Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity

Maintaining organ homeostasis requires complex functional synergy between distinct cell types, a snapshot of which is glimpsed through the simultaneously broad and granular analysis provided by single-cell atlases. Knowledge of the transcriptional programs underpinning the complex and specialized functions of human kidney cell populations at homeostasis is limited by difficulty accessing healthy, fresh tissue. Here, we present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 high-quality cells to map healthy kidney at high resolution. Our sex-balanced dataset revealed sex-based differences in gene expression within proximal tubular cells, specifically, increased anti-oxidant metallothionein genes in females and the predominance of aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed between male and female proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. Within the immune niche, we identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions and validated findings by flow cytometry. We observed significant heterogeneity in resident myeloid populations and identified an MRC1+ LYVE1+ FOLR2+ C1QC+ population as the predominant myeloid population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, revealing novel insights into the complexity of renal parenchymal cells and kidney-resident immune populations.

Liver Iron Concentration Assessed by SQUID Biosusceptometry Compared to Heat-dried Liver Biopsy: A Blinded Study.

Objective Biomagnetic liver susceptometry (BLS) is a noninvasive method to quantify liver iron concentration (LIC). Here we report our findings from a prospective study which validates in vivo LIC from a SQUID biosusceptometer by in vitro LIC in fresh tissue and paraffin-embedded biopsies from patients at risk for iron overload.Materials and Methods LIC was measured by BLS and biopsy. LIC by biopsy were measured in 40 dry weight fresh tissue and paraffin-embedded liver biopsy samples. LIC from biopsies and total iron scores from histology were compared to biosusceptometry. In addition, the wet-to-dry weight ratio was determined.Results Liver iron concentrations measured by BLS and in 40 fresh tissue biopsies were related by a factor of 6.0 ± 0.2 (r2 = 0.88). Similar results were obtained from comparisons with deparaffinized biopsies (6.6±0.3, r2=0.87) and histology (6.7±1.3, r2=0.47). In contrast, a mean wet-to-dry weight ratio of 4.1 ± 0.7 was achieved from biopsies immediately weighed after the biopsy procedure.ConclusionLIC derived from two independent measures, the historical biopsy gold standard and biosusceptometry, were highly correlated. When comparing biosusceptometry with wet weight biopsies, the liver tissue sample size is critical.

Intraoperative discrimination of native meningioma and dura mater by Raman spectroscopy

Meningiomas are among the most frequent tumors of the central nervous system. For a total resection, shown to decrease recurrences, it is paramount to reliably discriminate tumor tissue from normal dura mater intraoperatively. Raman spectroscopy (RS) is a non-destructive, label-free method for vibrational analysis of biochemical molecules. On the microscopic level, RS was already used to differentiate meningioma from dura mater. In this study we test its suitability for intraoperative macroscopic meningioma diagnostics. RS is applied to surgical specimen of intracranial meningiomas. The main purpose is the differentiation of tumor from normal dura mater, in order to potentially accelerate the diagnostic workflow. The collected meningioma and dura mater samples (n = 223 tissue samples from a total of 59 patients) are analyzed under untreated conditions using a new partially robotized RS acquisition system. Spectra (n = 1273) are combined with the according histopathological analysis for each sample. Based on this, a classifier is trained via machine learning. Our trained classifier separates meningioma and dura mater with a sensitivity of 96.06 $$\pm $$ ± 0.03% and a specificity of 95.44 $$\pm $$ ± 0.02% for internal fivefold cross validation and 100% and 93.97% if validated with an external test set. RS is an efficient method to discriminate meningioma from healthy dura mater in fresh tissue samples without additional processing or histopathological imaging. It is a quick and reliable complementary diagnostic tool to the neuropathological workflow and has potential for guided surgery. RS offers a safe way to examine unfixed surgical specimens in a perioperative setting.

A method for rapid flow-cytometric isolation of endothelial nuclei and RNA from archived frozen brain tissue

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.

The PASS Course: Practical Anatomy and Surgical Skills Course as a Pragmatic Primer for Senior Medical Students Pursuing Surgical Residency

The senior year of undergraduate medical education has been scrutinized for lacking emphasis from educators and value for students. Surgical residency program directors and medical students have reported different sets of perceived weaknesses as surgical trainees enter residency. With this in mind, we developed a novel rotation for senior medical students pursuing surgical residency. The rotation incorporates practical didactics, robust skill and simulation training, and an enriching anatomy experience that entails dissections and operations on embalmed and fresh tissue cadavers. To our knowledge, this is the first reported formal training experience for medical students that involves working with fresh tissue cadavers, which have been described as effective models for live human tissue in the operating room. We describe our multifaceted curriculum in detail, discuss its organization, and elaborate on its potential value. We also provide detailed explanations of the curriculum components so that other surgical educators may consider adopting them.

Association between Fusobacterium nucleatum and patient prognosis in metastatic colon cancer

Recent evidence suggests that Fusobacterium nucleatum (Fn) is associated with the development and progression of colorectal cancer. We aimed to delineate the clinical implications of Fn in metastatic colon cancer. We performed quantitative polymerase chain reaction (qPCR) using DNA samples from synchronous metastatic colon cancer patients with either formalin-fixed paraffin-embedded (FFPE) archival primary site tumor samples or fresh colon tissues. Progression-free survival (PFS)1 and PFS2 were defined as PFS of first- and second-line palliative settings. qPCR for Fn was successfully performed using 112 samples (FFPE, n = 61; fresh tissue, n = 51). Forty-one and 68 patients had right-sided and left-sided colon cancer, respectively. Patients with Fn enriched right-sided colon cancers had shorter PFS1 (9.7 vs. 11.2 months) than the other subgroups (HR 3.54, 95% confidence interval [CI] 1.05–11.99; P = 0.04). Fn positive right-sided colon was also associated with shorter PFS2 (3.7 vs. 6.7 months; HR 2.34, 95% CI 0.69–7.91; P = 0.04). In the univariate analysis, PFS1 was affected by differentiation and Fn positive right-sided colon cancer. The multivariate analysis showed that differentiation (HR 2.68, 95% CI 1.40–5.14, P = 0.01) and Fn positive right-sided colon (HR 0.40, 95% CI 0.18–0.88, P = 0.02) were associated with PFS1. Fn enrichment in right sided colon was not associated with overall survival (OS). Fn enrichment has significantly worse prognosis in terms of PFS1 and PFS2 in patients with right-sided metastatic colon cancers.

PSII-B-24 Use of the Elnady technique of tissue preservation to preserve specimens for use in undergraduate animal science courses

The use of tissue specimens for undergraduate instruction is a very valuable tool. However, fresh tissue specimens are not always available and many common preservation techniques can result in discoloration, offensive odors, and/or dangerous chemical residues. The Elnady Technique was developed as a means to produce tissue specimens that “are realistic, durable, have no offensive odor, and are dry, soft and flexible” (Elnady, F.A. 2016 The Elnady Technique: An innovative, new method for tissue preservation. Altex. 33:237–242. doi:10.14573/altex.1511091). Briefly for soft tissue, specimens were preserved by fixing in formalin for one week. The tissue specimen was then dehydrated with a series of acetone baths changed weekly. Once the tissue was fully dehydrated, the specimen was impregnated in glycerin by full immersion in a glycerin bath for one to two weeks. Excess glycerin was then removed by draining followed by immersion in cornstarch for one to two weeks. Cornstarch residue was removed with a soft brush, and the specimen was stored in a plastic bag. Multiple specimens (including female reproductive tract of the cat, goat, horse, and sow; digestive tract of barred owl, cat, chicken, and dog; one day old lamb stomach; goat rumen, reticulum, omasum, and abomasum; deer testicle; and sheep heart and kidney) have been preserved and used in various animal science course laboratories (75 laboratory sections and over 1000 students at Berry College). Some of the specimens have been in use for five years and are still in usable condition. The Elnady Technique has proven to be a useful means of preserving tissue samples used in undergraduate animal science courses.

Intraoperative Assessment of IDH Mutation Status and Tumor Invasioni in Glioma

Introduction/Objective Maximizing surgical resection in gliomas, while avoiding compromising non-infiltrated tissue, is associated with survival benefit. Current methodologies are suboptimal in providing rapid, intraoperative molecular characterization of tissue. We address this unmet need by using desorption electrospray ionization mass spectrometry (DESI-MS) for the intraoperative molecular assessment of gliomas. Methods/Case Report This prospective study uses intraoperative DESI-MS analysis of fresh tissue to evaluate IDH mutations via 2-hydroxyglutarate intensity and TCP via measurement of N-acetylaspartic acid (NAA) intensity and characteristic lipid profiles in less than three minutes. Blinded review of the tissue smears by a neuropathologist is used to validate IDH mutation status and TCP estimates. Results (if a Case Study enter NA) Presently, 529 biopsies from 85 enrolled patients have been collected and analyzed at two institutions. TCP assessment based on NAA intensity in 203 biopsies at the first institution yielded sensitivity, specificity, and accuracy values of 91, 76, and 83%, whereas TCP estimates via characteristic lipid profiles yielded 76, 85, and 81%, respectively. Assessment of IDH mutation status of 71 core biopsies yielded sensitivity, specificity, and accuracy values of 89, 100, and 94%. Ongoing validation of the methodology is being performed at a second institution, where we have collected 282 biopsies from 36 patients. IDH mutation assessment of the first 15 patients indicate 100% sensitivity, specificity, and accuracy. Conclusion This study represents the first and largest study using DESI-MS for the intraoperative evaluation of IDH status and TCP measurement in gliomas. Prospectively, we propose to modify our DESI-MS system to allow estimation of IDH mutation status and TCP in surgical cavities without the need for a biopsy by placing a surgical material along the margin and transferring material from the blot to a microscope slide prior to DESI-MS analysis. We envision molecular analysis by DESI-MS as a complementary technique to histopathology capable of providing additional clinical information in near real-time.