Perfusion fixation in brain banking: a systematic review.

BACKGROUND: Perfusing fixatives through the cerebrovascular system is the gold standard approach in animals to prepare brain tissue for spatial biomolecular profiling, circuit tracing, and ultrastructural studies such as connectomics. Translating these discoveries to humans requires examination of postmortem autopsy brain tissue. Yet banked brain tissue is routinely prepared using immersion fixation, which is a significant barrier to optimal preservation of tissue architecture. The challenges involved in adopting perfusion fixation in brain banks and the extent to which it improves histology quality are not well defined. METHODOLOGY: We searched four databases to identify studies that have performed perfusion fixation in human brain tissue and screened the references of the eligible studies to identify further studies. From the included studies, we extracted data about the methods that they used, as well as any data comparing perfusion fixation to immersion fixation. The protocol was preregistered at the Open Science Framework: https://osf.io/cv3ys/ . RESULTS: We screened 4489 abstracts, 214 full-text publications, and identified 35 studies that met our inclusion criteria, which collectively reported on the perfusion fixation of 558 human brains. We identified a wide variety of approaches to perfusion fixation, including perfusion fixation of the brain in situ and ex situ, perfusion fixation through different sets of blood vessels, and perfusion fixation with different washout solutions, fixatives, perfusion pressures, and postfixation tissue processing methods. Through a qualitative synthesis of data comparing the outcomes of perfusion and immersion fixation, we found moderate confidence evidence showing that perfusion fixation results in equal or greater subjective histology quality compared to immersion fixation of relatively large volumes of brain tissue, in an equal or shorter amount of time. CONCLUSIONS: This manuscript serves as a resource for investigators interested in building upon the methods and results of previous research in designing their own perfusion fixation studies in human brains or other large animal brains. We also suggest several future research directions, such as comparing the in situ and ex situ approaches to perfusion fixation, studying the efficacy of different washout solutions, and elucidating the types of brain donors in which perfusion fixation is likely to result in higher fixation quality than immersion fixation.

From fix to fit into the autoptic human brains.

Formalin-fixed, paraffinembedded (FFPE) human brain tissues are very often stored in formalin for long time. Formalin fixation reduces immunostaining, and the DNA/RNA extraction from FFPE brain tissue becomes suboptimal. At present, there are different protocols of fixation and several procedures and kits to extract DNA/RNA from paraffin embedding tissue, but a gold standard protocol remains distant. In this study, we analyzed four types of fixation systems and compared histo and immuno-staining. Based on our results, we propose a modified method of combined fixation in formalin and formic acid for the autoptic adult brain to obtain easy, fast, safe and efficient immunolabelling of long-stored FFPE tissue. In particular, we have achieved an improved preservation of cellular morphology and obtained success in postmortem immunostaining for NeuN. This nuclear antigen is an important marker for mapping neurons, for example, to evaluate the histopathology of temporal lobe epilepsy or to draw the topography of cardiorespiratory brainstem nuclei in sudden infant death syndrome (SIDS). However, NeuN staining is frequently faint or lost in postmortem human brain tissues. In addition, we attained Fluoro Jade C staining, a marker of neurodegeneration, and immunofluorescent staining for stem cell antigens in the postnatal human brain, utilizing custom fit fixation procedures.

The State of Cell Blocks and Ancillary Testing: Past, Present, and Future.

Cell blocks are an integral part of cytology, but their utility is recognized probably more now than ever before, largely owing to the significant role they play in ancillary testing, particularly molecular diagnostics. Modifications to improve the cell block method initially introduced more than a century ago have been made over the years. Though their value is acknowledged and they are widely used across laboratories, cell block preparations are not standardized and results of ancillary testing performed on them are inconsistent. This article reviews the state of cell blocks-summarizes the more common, currently available and used methods and their corresponding advantages and shortcomings, outlines the role of alternative techniques (eg, smears), and proposes methods to optimize results.

The evolution of pre-analytic factors in Anatomic Pathology.

Anatomic Pathology has continuously evolved since launch by Virchow in Berlin. The era from 1990 to 2010 saw the rise of immunohistochemistry and its application for diagnosis, prognosis, and prediction of response to therapy. Currently the next wave of evolution is ongoing; molecular pathology, with emphasis on alterations to DNA, and expression of mRNA as biomarkers. The interrogation of biomolecules by specific probes is more demanding on specimens than the traditional application of histologic stains to tissue. This issue is juxtaposed to the fact that the majority of specimens are purely evaluated by histomorphology, for which current specimen practices are adequate. The capacity to identify a priori which cassette of tissue is appropriate for molecular analysis is difficult, if not impossible, the goal is to improve the quality of all pathology specimens in an economically viable model to enable advanced assay, when applicable.

How technology is reshaping the practice of nongynecologic cytology: frontiers of cytology symposium.

To pay tribute to the Founders of Acta Cytologica, this Golden Anniversary symposium on nongynecologic cytology revives the written symposium style of the 1950s. Participants from countries throughout the world were asked how new technologies are currently applied in their laboratories and whether future advances and challenges can be predicted. The specific questions and the participants' answers follow.

Retention of fluorescent probes during aldehyde-free anhydrous freeze-substitution.

Fluorescent probes are widely used for microscopy of live-cell processes, but few such probes can also be used with classically fixed or otherwise immobilized material, and none has been used without aldehyde fixation, which can introduce artefacts of structure and probe localization. Here we show that the fluorescence patterns in fungal hyphae loaded with chloromethyl aminocoumarin (CMAC), and then anhydrously freeze-substituted, without any aldehyde fixation, are similar to those seen in living hyphae. Probe loss into the mounting medium (Spurr's resin) with CMAC and five other probes tested indicated that some unwanted solubilization of probe occurred during embedding, but nevertheless vacuoles could be imaged by their retention of probe.