Fresh Unfixed Tissue Research Articles

Microdissection of the supraoptic nucleus and posterior pituitary gland from fresh unfixed tissue

The rat supraoptic nucleus (SON) contains magnocellular neurons that project long axons that terminate in the posterior pituitary gland. To perform molecular characterization of these regions, such as transcriptome and methylome profiling, it is necessary to obtain large quantities of high-quality RNA and DNA. Prior methods to isolate molecular material from these small regions required fixing or freezing and laser microdissection of whole tissue, which can compromise recovery and integrity. We have established a straight-forward method of dissecting out the SON and posterior pituitary gland from fresh, unfixed tissue that allows for the isolation of RNA or DNA without compromising nucleic acid integrity. Furthermore, this method can be used as a framework for the microdissection of any region of the brain to isolate any sensitive material. In this manuscript, we describe step-by-step instructions from the macro scale dissection, to brain sectioning, and finally the microdissection of the appropriate tissue.•Transcardial perfusion without fixative prevents the shortcomings of nucleic acid cross-linking.•A fast method and the maintenance of tissue in ice-cold HBSS during dissection and sectioning prevents nucleic acid degradation.•A vibratome is used for the sectioning of fresh brain tissue without freezing or gelatin embedding (i.e. cryostat or microtome).

Real-Time Evaluation of Thyroid Cytology Using New Digital Microscopy Allows for Sample Adequacy Assessment, Morphological Classification, and Supports Molecular Analysis.

Thyroid cytological examination, a key tool in preoperative thyroid nodule evaluation, is specific and accurate; some drawbacks are due to inadequate or indeterminate cytological reports and there is a need for an innovative approach overcoming the limits of traditional cytological diagnostics. Fluorescence laser confocal microscopes (FCM) is a new optical technique for allowing immediate digital imaging of fresh unfixed tissues and real-time assessment of sample adequacy and diagnostic evaluation for small biopsies and cytological samples. Currently, there are no data about the use of FCMs in the field of thyroid nodular pathology. The aims of this study were to test FCM technology for evaluating the adequacy of FNA samples at the time of the procedure and to assess the level of concordance between FCM cytological evaluations, paired conventional cytology, and final surgical histology. The secondary aim was to define the integrity of nucleic acids after FCM evaluation through NGS molecular analysis. Sample adequacy was correctly stated. Comparing FCM evaluation with the final histology, all cases resulting in malignant or suspicious for malignancy at FCM, were confirmed to be carcinomas (PPV 100%). In conclusion, we describe a successful application of FCM in thyroid preoperative cytological evaluation, with advantages in immediate adequacy assessment and diagnostic information, while preserving cellular specimens for permanent morphology and molecular analysis, thus improving timely and accurate patient management.

Histoarchitectural and Histoenzymatic Studies on Gizzard of Guinea Fowl (Numida meleagris)

Background: The gizzard is a muscular stomach of the Gastrointestinal tract of bird that holds sparing crystals which aids as the mechanical multure component for food material to be ingested by birds. Its specialty is to grinding of ingested food material after secretion of HCL (Hydrochloric Acid) and pepsinogen enzymes in the proventriculus. Methods: The twelve samples of gizzard of guinea fowl were fixed in 10% NBF (Neutral buffered formalin) and Bouin’s fluid for histological while chilled acetone and chilled ethanol for histochemical studies. The fresh unfixed tissues were used for the cryostat section for the demonstration of different enzymes.Result: The tubular glands were main feature of mucosal layer but sometime acinar glands also found as the replacement of tubular glands with lymphoid aggregation. The horizontal koilin was also noticed between long mucosal folds. Tunica submucosa was discovered in squeezed manner in comparison to the mucosa. The tunica muscularis has shown inner circular and outer longitudinal as the thick layer. It was intermingling with collagen and elastic fibers. Histochemically the gizzard was examined for glycogen, alkaline phosphatase, acid phosphatase and succinic dehydrogenase.

Rapid pathology of lumpectomy margins with open-top light-sheet (OTLS) microscopy.

Open-top light-sheet microscopy is a technique that can potentially enable rapid ex vivo inspection of large tissue surfaces and volumes. Here, we have optimized an open-top light-sheet (OTLS) microscope and image-processing workflow for the comprehensive examination of surgical margin surfaces, and have also developed a novel fluorescent analog of H&E staining that is robust for staining fresh unfixed tissues. Our tissue-staining method can be achieved within 2.5 minutes followed by OTLS microscopy of lumpectomy surfaces at a rate of up to 1.5 cm2/minute. An image atlas is presented to show that OTLS image quality surpasses that of intraoperative frozen sectioning and can approximate that of gold-standard H&E histology of formalin-fixed paraffin-embedded (FFPE) tissues. Qualitative evidence indicates that these intraoperative methods do not interfere with downstream post-operative H&E histology and immunohistochemistry. These results should facilitate the translation of OTLS microscopy for intraoperative guidance of lumpectomy and other surgical oncology procedures.

Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining.

BackgroundFor decades, the Vibratome served as a standard laboratory resource for sectioning fresh and fixed tissues. In skilled hands, high quality and consistent fresh unfixed tissue sections can be produced using a Vibratome but the sectioning procedure is extremely time consuming. In this study, we conducted a systematic comparison between the Vibratome and a new approach to section fresh unfixed tissues using a Compresstome. We used a Vibratome and a Compresstome to cut fresh unfixed lymphoid and genital non-human primate tissues then used in situ tetramer staining to label virus-specific CD8 T cells and immunofluorescent counter-staining to label B and T cells. We compared the Vibratome and Compresstome in five different sectioning parameters: speed of cutting, chilling capability, specimen stabilization, size of section, and section/staining quality.ResultsOverall, the Compresstome and Vibratome both produced high quality sections from unfixed spleen, lymph node, vagina, cervix, and uterus, and subsequent immunofluorescent staining was equivalent. The Compresstome however, offered distinct advantages; producing sections approximately 5 times faster than the Vibratome, cutting tissue sections more easily, and allowing production of larger sections.ConclusionsA Compresstome can be used to generate fresh unfixed primate lymph node, spleen, vagina, cervix and uterus sections, and is superior to a Vibratome in cutting these fresh tissues.

Role of Intraoperative Pathology Consultation in Skeletal Tumors and Tumor-Like Lesions

Early and accurate detection of bone tumors and their staging are important since some of them are highly malignant. Intraoperative pathological consultation in bone tumors and tumor-like conditions is quite complex; however, it allows improvement in prognosis and limb salvage. Present study was conducted on 52 patients who underwent surgical procedure after clinical and radiological diagnosis of bone tumors/tumor-like conditions. Fresh unfixed tissue was quickly inspected grossly, followed by preparation of imprint smears and frozen section which were evaluated by two pathologists separately and compared subsequently with reports of paraffin-embedded sections. Clinical reasons for intraoperative consultation were to make diagnosis in 65.4% of cases and to determine resection margin status in 21.1% while in 13.5% of cases, it was for both indications. Diagnostic yield of imprint smears was 87.8% (13 malignant, 22 benign, and 1 tumor-like) and of frozen section was 90.2% (16 malignant, 19 benign, and 2 nonneoplastic) while paraffin sections could diagnose specific tumors in 95.1% (18 malignant, 18 benign, and 3 nonneoplastic). Although frozen section had better sensitivity (88.2%), it had less specificity (94.7%) as compared to imprint smears (76.5% and 100%, resp.). Imprint cytology and frozen section together provide a quick, safe, and reliable intraoperative provisional tissue diagnosis in skeletal tumors and tumor-like conditions.

Analyzing agreement patterns of intraoperative central nervous system lesion reporting according to type and grade

Background:Neuropathology centers are expected to offer a prompt and accurate intraoperative diagnosis regarding tumor/lesion type and grade on fresh unfixed tissue. Level of diagnostic accuracy according to type and grade and also, the experience at a new center has not been reported before.Aims:The aim of this study is to review the agreement patterns according to tumor/lesion type and grade between intraoperative and final histopathologic diagnosis in central nervous system (CNS) lesion samples received by a newly established neuropathology center at a tertiary care neuropsychiatric hospital.Materials and Methods:Agreement between intraoperative and final histopathologic diagnosis was classified as: (I) Grade in agreement but type not in agreement; (II) grade not in agreement but type in agreement; (III) grade and type both not in agreement; (IV) grade and type both in agreement.Statistical Analysis:Confidence interval (CI) of agreements was calculated for various categories of neoplastic as well as non-neoplastic lesions. CI was also calculated for groups where n × p and n × (1 − p) were more than 5, i.e., fulfilled the requirement of the central limit theorem.Results:On retrospective analysis of 333 cases, 284 (85.3%) cases were categorized as neoplastic while 49 (14.7%) cases were categorized as non-neoplastic. Among the neoplastic lesions agreement was seen in 237 (83.5%) cases while 47 (16.5%) cases showed disagreement. Similarly in non-neoplastic category; 46 (93.9%) cases showed agreement while 3 (6.15%) cases showed disagreement. Of the non-neoplastic lesions, one case fell into the agreement category I, 2 in category III and 46 in IV. Among neoplastic lesions, there were 21 cases in agreement category I, 17 in II, 9 in III and 237 in IV. On analyzing the accuracy of intraoperative reporting according to tumor type, the break up was: - Astrocytic: 2 (I), 16 (II), 2 (III), 86 (IV); oligodendroglial: 8 (I), 1 (II); ependymal: 2 (III), 6 (IV); embryonal: 23 (IV); cranial and spinal nerve tumors: 2 (II), 21 (IV); choroid plexus tumors: 4 (IV); meningeal tumors: 3 (I), 1 (III), 49 (IV); metastatic tumors: 3 (I), 17 (IV); cysts (tumor-like conditions): 14 (IV); neuronal and mixed neuronal glial tumors: 1 (III); malignant lymphoma: 1 (III); sellar tumors: 17 (IV); and mixed gliomas: 5 (I).Conclusions:This study identifies problem areas of CNS intraoperative reporting, in a new center, with reference to tumor typing and grading. It may forewarn upcoming centers of neuropathology about the potential problem areas of intraoperative reporting.

Evaluation of a method allowing preservation of fresh lymph nodes for flow cytometric immunophenotyping

Flow cytometric immunophenotyping (FCI) of lymph nodes (LN) requires fresh unfixed tissue, with analysis being carried out within few hours post surgery. This study evaluated a novel method for fresh LN preservation, in order to allow histomorphology-based FCI. This study was carried out prospectively on 30 LN with suspected involvement by haematolymphoid neoplasms (HLN). FCI was performed on each fresh and post cryopreserved LN cell suspension. Percentage positivities (PP) and mean fluorescent intensities (MFI) were calculated on both preparations for a combination of T and B-lymphoid antigens together with viability. The cryopreservation method applied in this study did not affect significantly PP and had minor impact on MFI of the tested antigens. Overall, there was minimal decrease in PP and MFI on the cryopreserved cells when compared with fresh cells, for most antigens with only a mild increase in apoptotic cells. However, these changes were not diagnostically significant, since both reactive processes and HLN present within analyzed LN could be identified and differentiated. Viability was more than 75% for all cryopreserved LN composed of haematolymphoid cells. The method presented in this study confers the possibility of storing fresh LN biopsies for later FCI, thus allowing a morphology-based immunophenotypic approach. This would allow a more sensitive, specific, and cost-effective management of LN specimens, whilst maintaining the important benefits provided by FCI.

Rapid intraoperative diagnosis of tumors of the eye and orbit by squash and imprint cytology

Purpose To evaluate the diagnostic accuracy of intraoperative squash and imprint cytologic techniques in ocular and orbital lesions. Design Observational case series. Methods Forty-five consecutive ocular and orbital biopsy specimens subjected to squash and imprint cytologic techniques for rapid intraoperative diagnosis were included in this study. The indications for which the surgeon requested intraoperative diagnosis included: (1) infiltrative lesions where a preoperative tissue diagnosis was not available, (2) discrepancy between preoperative and intraoperative clinical diagnosis, and (3) unusual clinical presentations with an uncertain diagnosis. Fresh unfixed tissue was used for making squash preparations and imprint smears. Alcohol-fixed smears were stained with hematoxylin and eosin, whereas air-dried smears were stained with the Giemsa stain. The residual tissue was subjected to routine processing for histopathologic examination. Main outcome measure Correlation of intraoperative cytologic diagnosis with the final histologic diagnosis was the primary outcome measure. The correlation was considered to have complete concordance if the diagnosis matched with respect to nature of the lesion (benign vs. malignant) as well as with the further classification of the benign or the malignant lesion. Partial concordance was considered when there was agreement with respect to the nature of the lesion, but there was a mismatch in the further classification of the benign or the malignant lesion. Results Biopsies were obtained from 45 patients whose ages ranged from 1 to 79 years (median, 21 years). The sites of biopsy included eyelids (n = 4), conjunctiva (n = 6), intraocular tissue (n = 3), and orbit (n = 32). Adequate cellularity was obtained in all cases by squash and imprint techniques. A cytologic diagnosis could be made within 5 to 8 minutes after receiving the sample. Of 44 cases in which a cytologic diagnosis could be made, 21 (48%) were malignant and 23 (52%) were benign. Cytologic analysis was inconclusive in 1 case. The cytologic–histologic correlation showed complete concordance in 41 of 45 cases (91%) and partial concordance in 3 cases. Sensitivity and specificity of the squash and imprint technique in detecting malignant lesions was 100%. Conclusions Assuming the availability of a skilled cytologist, ophthalmic pathologist, or both, squash and imprint preparations are useful for rapid and reliable intraoperative diagnosis of ocular and orbital lesions.

Heat pretreatment increases resolution in DNA flow cytometry of paraffin-embedded tumor tissue.

Flow cytometry of single-cell suspensions prepared by enzymatic digestion from formalin-fixed, paraffin-embedded tissue suffers from several major drawbacks. The most important factors that influence the results are the high and unpredictable coefficients of variation (CVs) of the G0/G1 peak in the DNA histogram and reduction of propidium iodide (PI) intercalation with DNA, resulting from protein cross-linking by formalin. In this study we introduce a heating step (2 h incubation in citrate solution at 80 degrees C) prior to a brief pepsin digestion of tissue sections in the protocol for DNA content analysis of formalin-fixed and paraffin-embedded tissue. This new method is compared with established methods for the preparation of cell suspensions from frozen and paraffin-embedded tissues with respect to cell yield, DNA histogram resolution, DNA dye saturation kinetics, cell cycle parameters, and antigen retrieval in various epithelial and nonepithelial tissues. The recovery of single cells from the paraffin sections was doubled by the heat treatment step, while the limited time of proteolysis resulted in decreased cell debris. Furthermore, an increased fraction of cells became cytokeratin-positive, while these immunocytochemically stained cells also exhibited a higher mean fluorescence intensity. The DNA histograms prepared from cell suspensions obtained according to this new protocol showed a significantly improved resolution, leading to a better identification of peridiploid cell populations. Heat pretreatment of paraffin-embedded archival tissue sections showed PI saturation kinetics similar to, or even better than, those of fresh unfixed tissues, independent of duration of fixation. This new method, making use of routinely available antigen retrieval principles, thus allows high-resolution DNA analysis of routinely fixed and paraffin-embedded tissue samples. Using external reference cells, inter- and intralaboratory standardization of DNA histograms can be achieved.

Orbital lymphomas and Castleman's disease

A series of 100 consecutive orbitotomies for tumour was assessed. Lymphoid infiltrations contributed 27% and malignant non-Hodgkin lymphoma was the commonest primary malignant orbital tumour in this series. The clinical and histological features of the first histologically proven case of primary orbital Castleman's disease are reported, as are the results of a study of 27 patients with pure orbital lymphoid infiltrations. The natural history of conjunctival lymphomas differs significantly from that of pure orbital lymphomas and for this reason no conjunctival lymphomas were considered in this series (which therefore contrasts with some previous reports). Histological examination with fresh unfixed tissue available for immunohistochemical staining enhances the diagnostic accuracy of these varied underlying diseases. More than one biopsy may be required before the diagnosis is established. These are significant factors given the differing lines of management for each disorder.

Methods for histochemical demonstration of vascular structures at the muscle-bone interface from cryostate sections of demineralized tissue

In tissue decalcified with MgNa2EDTA at a neutral pH activity for ATPase can used be for demonstration of the vascular structures at the muscle-bone interface. The GOMORI method for alkaline phosphatase is only of value, when fresh unfixed tissue is to be examined. The azo-dye method for alkaline phosphatase failed to give satisfactory results, and so did the alpha-amylase PAS method. 5'-nucleotidase activity is present in both capillaries and in cells lining the surfaces of bones, while larger blood vessels are poorly stained.

Nachweis und analyse von aldehydfuchsin-positiven faserstrukturen im bindegewebe der menschlichen eileiterschleimhaut. I. Voruntersuchungen und darstellung des morphologischen substrats

Investigating the biomorphosis of the human uterine tube, it was possible to detect aldehyde fuchsin positive fibers in the lamina propria mucosae. These fibers can be regarded as sulphur containing scleroproteins relating to the histochemical findings of oxidation, thiosulphation, methylation, sensitivity to pepsin and papain, respectively. For the visualization of the morphological substratum it was necessary to treat the fixed or fresh unfixed tissue chemically by oxidation and thiosulfation before the slices were stained with aldehyde fuchsin. Among several oxidantia examined in preliminary trial tests, peracetic acid, performic acid, potassium permanganate, bromine, ammonium persulphate (mixing with sulphuric acid), periodic acid (t = 24 h) effected the aldehyde fuchsin staining of the fibers. It is presumably that the effective oxidantia are able to destroy protein bound disulphides localizing in the connective tissue fibers. Aldehyde fuchsin was prepared as recommended by MOWRY (1975, paraldehyde fuchsin) and by BOCK and OCKENFELS (1970, crotonaldehyde fuchsin). Besides, solutions of paraldehyde fuchsin according to MOWRY's modification prepared with basic fuchsin as well as pararosanilin (C. I. 42 500) was analyzed by spectrophotometry and by thin layer chromatography. The dye analysis has shown that the dye consists of various components and the composition of the dye bath changes with increasing age of the solution. By the way, the freshly prepared solutions were used between 3 and 18 days after preparation. Generally, the peracetic acid - aldehyde-fuchsin stain describing by FULLMER and Lillie (1958 a) was suitable for the reproducible demonstration of the aldehyde fuchsin fibers in oviducts from several stages of life. In neonatal uterine tubes the fibers spread over the mucosa as a multidimensional network, and in senile ones they are localized zonally near the epithelium. It was possible to visualize the aldehyde fuchsin positive fibers also in other organs and tissue formations, e.g. in perichondria of the tracheobronchial tree, in the lung, small intestine, and uterus.

Sedimentation analysis of ribonucleic acid extracted from isolated Mauthner nerve fibre components.

Abstract— Segments of spinal cords from goldfish or from carp were incubated in vitro in the presence of RNA precursors for varying periods of time. Mauthner nerve fibres were isolated from the fresh unfixed tissue, or, for the separate analyses of axon and myelin sheath, from the fixed spinal cord.The myelinated Mauthner fibre isolated from the incubated spinal cord showed RNA synthesis. A considerable part of the material sedimented at 4S, but part of the nucleic acid was recovered at higher sedimentation values, up to 30S. Newly synthesized RNA was extracted from the isolated myelin sheath as well as from the axon. Isolated myelinated Mauthner fibres were also incubated in vitro with RNA precursors. In this case incorporation occurred exclusively in material sedimenting at 4S or lower. The turnover rate for RNA from the fibre was of a higher order than that of the bulk of RNA from the spinal cord. The findings of RNA synthesis in these tissue components lacking nuclei could possibly be explained as owing to mitochondria.Studies by electron microscopy demonstrated the extent of purity of the isolated components and it was found that contamination was so small as to make it unlikely that the RNA investigated originated in contaminating tissue.

Thermoelectric Apparatus for the Preparation of Fresh-Frozen Tissue Sections

THE application of the Peltier effect in the preparation of frozen histological sections has been described by Ioffe1and Brown and Dilly2, who cooled the microtome stage by means of thermoelements constructed of semi-conductor materials. Sections of fixed material can be obtained in this way; but it is not possible to cut sections of fresh unfixed tissue by merely cooling the microtome stage. For this purpose it is necessary to cool not only the stage, but also the knife, and although this can be done with solid carbon dioxide the method is not very satisfactory.

Some improvements in the preparation of fresh frozen sections.

For the histochemical demonstration of sensitive enzymes it is necessary to use fresh unfixed tissue sections. With the following procedure one can constantly obtain such sections 10-20μ thick with relative ease. Schanze's sliding microtome is employed. The microtome knife is deeply cooled by placing blocks of dry ice on its surface, and is provided with a device for preventing the sections from rolling up. The microtome is operated in an ordinary refrigerator maintained at a temperature of 0-3°C. For this purpose, the door of the refrigerator is replaced by a wooden door provided with a glass window, gloved arm holes, and a small door.

Thin sections from unfixed tissues for histochemical staining.

Sections were cut from fresh unfixed tissues by means of a microtome provided with an apparatus for the simultaneous cooling of the knife and freezing stage. These sections were of uniform thickness and were found to be very suitable for histochemical staining. Such sections were immersed while still frozen in the fluid which contained the necessary chemicals for a specific technic. After remaining in the fluid for an appropriate time, the sections were put on slides and dried in warm air. The remaining steps were carried out on the slides. Several histochemical procedures (phosphatase, esterase, glycogen) were found to give good results when this technic was used.

Method and results of a study of the distribution of iodine between cells and colloid of thyroid glands

Method.—Use was made of the fact that from frozen sections, as prepared for histological study of fresh unfixed tissues, the colloid of the thyroid gland completely disappears out of the acini when the sections are floated in physiological salt solution. The sections were picked out by means of a needle, washed in another solution of salt solution, dried at 105° C., weighed and analysed for iodine by the technique of Kendall. The iodine in the colloid portion was sometimes determined by evaporation of the salt solution containing the colloid, and at other times differentially by analysis of the whole dried gland. Results.—In a series of about thirty experiments on dogs'thyroids the thyroid iodine was found in the majority of instances to be wholly in the colloid as the cell portion was free from iodine. In a smaller percentage of cases the cell mass gave only qualitative tests for iodine, which at the present stage of this investigation might be considered due to unopened small acini.