DIUTHAME enables matrix-free mass spectrometry imaging of frozen tissue sections.

RATIONALE: A recently developed matrix-free laser desorption/ionization method, DIUTHAME (desorption ionization using through-hole alumina membrane), was examined for the feasibility of mass spectrometry imaging (MSI) applied to frozen tissue sections. The permeation behavior of DIUTHAME is potentially useful for MSI as positional information may not be distorted during the extraction of analytes from a sample. METHODS: The through-hole porous alumina membranes used in the DIUTHAME chips were fabricated by wet anodization, were 5 µm thick, and had the desired values of 200 nm through-hole diameter and 50% open aperture ratio. Mouse brain frozen tissue sections on indium tin oxide (ITO)-coated slides were covered using the DIUTHAME chips and were subjected to MSI experiments in commercial time-of-flight mass spectrometers equipped with solid-state UV lasers after thawing and drying without matrix application. RESULT: Mass spectra and mass images were successfully obtained from the frozen tissue sections using DIUTHAME as the ionization method. The mass spectra contained rich peaks in the phospholipid mass range free from the chemical background owing to there being no matrix-derived peaks in that range. DIUTHAME-MSI delivered high-quality mass images that reflected the anatomy of the brain tissue. CONCLUSIONS: Analytes can be extracted from frozen tissue by capillary action of the through-holes in DIUTHAME and moisture contained in the tissue without distorting positional information of the analytes. The sample preparation for frozen tissue sections in DIUTHAME-MSI is simple, requiring no specialized skills or dedicated apparatus for matrix application. DIUTHAME can facilitate MSI at a low mass, as there is no interference from matrix-derived peaks, and should provide high-quality, reproducible mass images more easily than MALDI-MSI.

Mind the gap: Micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae.

Cryo-focused ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-lamella workflow is a low-throughput technique and can easily be hindered by technical issues like the bending of the lamellae during the final cryo-FIB-milling steps. The severity of lamella bending seems to correlate with crinkling of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin lamella, leading to its bending and breakage. To protect the lamellae from such forces, we milled "micro-expansion joints" alongside the lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of micro-expansion joints drastically decreases bending of lamellae milled from eukaryotic cells grown and frozen on EM grids. Furthermore, we show that this adaptation does not create additional instabilities that could impede subsequent parts of the cryo-lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them a straightforward solution against cryo-lamella bending to increase the throughput of in situ structural biology studies.

Cryoembedder, automatic processor/stainer, liquid nitrogen freezing, and manual staining for frozen section examination: A comparative study.

Frozen section examination (FSE) reshaped surgical pathology and is characterized by a high accuracy. Nonetheless pathologists can experience artefacts that can compromise or defer the diagnosis. We compared a commercial system, composed by a cryoembedder and a processor/stainer, to our FSE protocol. Feasibility of diagnosis as well as overall architecture, cytology, and staining, were scored under the following conditions: Traditional (liquid nitrogen freezing and manual staining), Only-Presto (liquid nitrogen freezing and commercial processor/stainer), Only-PrestoCHILL (cryoembedder and manual staining), and PrestoSystem (cryoembedder and processor/stainer). Scores were compared across the different experimental conditions. PrestoSystem had significantly higher scores than Traditional, Only-Presto or Only-PrestoCHILL in all categories (Wilcoxon test; all P-value <.001); similarly, also Only-Presto and Only-PrestoChill had significantly higher scores than Traditional in all categories. Only-PrestoCHILL had significantly higher scores than Only-Presto in Cytology and Architecture. In conclusion the control of pre-analytical variables provided reproducible results, of a higher quality.

Development of Functional Requirements for Ex Vivo Pathology Applications of In Vivo Microscopy Systems: A Proposal From the In Vivo Microscopy Committee of the College of American Pathologists.

CONTEXT.­: In vivo microscopy (IVM) allows direct, real-time visualization of tissue histology in living patients without the need for tissue removal, processing, or staining. The IVM technologies in clinical use include confocal microscopy and optical coherence tomography. These technologies also show promise for use with pathology specimens (ex vivo microscopy [EVM]). However, few systems designed for EVM are commercially available, at least in part because of the lack of defined minimal functional requirements (FRs). OBJECTIVE.­: To develop minimal FRs for likely high-volume pathology applications of EVM. DESIGN.­: The IVM Committee of the College of American Pathologists identified potential EVM pathology applications based on the published literature. A subcommittee of IVM and EVM early adopters and experts then defined FRs for the most likely EVM applications. RESULTS.­: Potential EVM applications include assessment of margins, adequacy of needle biopsies and aspirates for diagnosis, and transplant tissues; selection of tissue for molecular studies or biorepository; and guidance in block selection from gross specimens. The first 3 applications were selected for development of FRs. The FRs were identified based on existing laboratory practices and guidelines and input from experts in the field and included device footprint and portability, specimen preparation, imaging time, field of view or resolution, morphologic diagnostic capability, yield, accuracy, ease of use, safety, and cost. CONCLUSIONS.­: Consensus was achieved on FRs that would accommodate the selected EVM applications. Publication and dissemination of those FRs will provide guidance to engineers, researchers, and vendors on how to optimally adapt IVM technologies for EVM for widespread adoption by pathologists.

Immunostaining of Skeletal Tissues.

Immunohistochemistry (IHC) is a routinely used technique in clinical diagnosis of pathological conditions and in basic and translational research. It combines anatomical, immunological, and biochemical methods and relies on the specific binding of an antibody to an antigen. Using the technique with mineralized tissues is more challenging than with soft tissues. Demineralizing the samples allows for embedding in paraffin wax, and also facilitates cryosectioning. This chapter describes methods for IHC on formaldehyde-fixed, demineralized, paraffin-embedded, or frozen sections to detect antigens in skeletal tissues.

Laser Capture Microdissection on Frozen Sections for Extraction of High-Quality Nucleic Acids.

Many cancers harbor a large fraction of nonmalignant stromal cells intermixed with neoplastic tumor cells. While single-cell transcriptional profiling methods have begun to address the need to distinguish biological programs in different cell types, such methods do not enable the analysis of spatial information available through histopathological examination. Laser capture microdissection offers a means to separate cellular samples based on morphological criteria. We present here an optimized method to retrieve intact RNA from laser capture microdissected tissue samples, using pancreatic ductal adenocarcinoma as an example, in order to separately profile tumor epithelial and stromal compartments. This method may also be applied to nonmalignant tissues to isolate cellular samples from any morphologically identifiable structure.

What is the accuracy of frozen section in the diagnosis of mucinous ovarian tumours? A 9-year review of performance in a Greek tertiary referral centre.

PURPOSE: This retrospective study aimed to evaluate the diagnostic accuracy of the intra-operative frozen sections (FS) of mucinous ovarian tumours (mOT). METHODS: Between 2007 and 2015, a total of 105 mucinous ovarian samples were collected during laparotomy. The intra-operative FS accuracy was evaluated and potential factors correlated with increased inaccuracy assessed using both univariate and multivariate analysis. RESULTS: The overall FS accuracy was 82.6%, while diagnostic discrepancy observed in 18/105 cases, including under-diagnosis in 14 and over-diagnosis in four cases. Amongst six cases diagnosed as benign with FS, five were upgraded to low malignant potential and one to malignant in the final formalin fixed, paraffin embedded section (FFPES). Amongst the 37 low malignant potential (LMP) cases, two were finally diagnosed as benign and eight as malignant. Amongst malignant tumours the diagnostic agreement occurred in 21/23 cases, while solely two cases were over-diagnosed. The false FS interpretation resulted in inadequate surgical management in 8/105 (7.6%) cases. Misdiagnosis had a statistically significant association with tumour size greater than 13 cm. The ratio of tumour size per number of frozen sections (TSFSR) less than 8 found to be an independent predictor of inaccuracy [OR 2.46, 95% confidence interval (CI) 1.74-3.46, P < 0.001]. CONCLUSIONS: The accuracy rate of FS in our study was 82.6%. Frozen section had low accuracy amongst LMP tumours adversely influencing the adequate surgical management. This discordance seems to reflect adverse predictors such as the LMP heterogeneity, maximal tumour diameter and low TSFSR.

Sustainable Development of Pathology in Sub-Saharan Africa: An Example From Ghana.

CONTEXT: - Pathology services are poorly developed in Sub-Saharan Africa. Komfo Anokye Teaching Hospital in Kumasi, Ghana, asked for help from the pathology department of the University Hospital of North Norway, Tromsø. OBJECTIVE: - To reestablish surgical pathology and cytology in an African pathology department in which these functions had ceased completely, and to develop the department into a self-supporting unit of good international standard and with the capacity to train new pathologists. DESIGN: - Medical technologists from Kumasi were trained in histotechnology in Norway, they were returned to Kumasi, and they produced histologic slides that were temporarily sent to Norway for diagnosis. Two Ghanaian doctors received pathology training for 4 years in Norway. Mutual visits by pathologists and technologists from the 2 hospitals were arranged for the introduction of immunohistochemistry and cytology. Pathologists from Norway visited Kumasi for 1 month each year during 2007-2010. Microscopes and immunohistochemistry equipment were provided from Norway. Other laboratory equipment and a new building were provided by the Ghanaian hospital. RESULTS: - The Ghanaian hospital had a surgical pathology service from the first project year. At 11 years after the start of the project, the services included autopsy, surgical pathology, cytopathology, frozen sections, and limited use of immunohistochemistry, and the department had 10 residents at different levels of training. CONCLUSIONS: - A Ghanaian pathology department that performed autopsies only was developed into a self-supported department with surgical pathology, cytology, immunohistochemistry, and frozen section service, with an active residency program and the capacity for further development that is independent from assistance abroad.

A new laboratory device with mathematically based positioning of a frozen tissue block facilitating precise sectioning of large specimens.

Mohs micrographic surgery (MMS) is a treatment method aiming at thorough, personalized eradication of skin cancers by mean of staged excision of tissues surrounding the tumor with complete (100%) histopathological examination of their margins. In many MMS laboratories, the excised tissue is divided, shaped, frozen in a cryostat with a heat extractor and positioned manually (with the block on the object disc) in an articulated cryostat chuck during cutting. However, these activities may be difficult, time-consuming and associated with the risk of imprecise tissue sectioning. Development of a laboratory device allowing for processing of large tissue specimens, with the function of mechanical, mathematically steered positioning of the tissue block surface directly to the microtome knife cutting place, eliminating the need for manual adjustment. The prototype device was designed and manufactured. Its functioning was tested on 513 histological slides produced during 212 operations of skin cancers using MMS. The depth of the first complete sections and the diameter of sections were measured. Complete sections were obtained at an average depth of 81.60 m (min. 20 m, max. 180 m, SD = 29.15), whereas the average diameter of sections was 18.11 mm (min. 4 mm, max. 42 mm, SD = 9.10). The histological processing of large specimens with mathematically based positioning of the tissue surface in relation to the cryotome knife cutting plane is precise, fast and easy. The device can be useful in those MMS centers which continue to employ manual setting of the cryostat chuck or share the cryostat with other users, which prevents fixing the chuck position (including large hospital settings). It may also be helpful in centers using a cryostat with a fixed chuck, for the correction of minimal inaccuracies of its preset position.

Optimization of sample cooling temperature for redox cryo-imaging.

Cryo-imaging techniques have been widely used to measure the metabolic state of tissues by capturing reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. However, NADH and FAD fluorescence is sensitive to changes in temperature, which may result in unreliable redox ratio calculations. Here, the relationship between the measured redox ratio and sample surface temperature was analyzed using a standard phantom solution and biological tissues. The results indicated that a temperature < - 100°C was a suitable cryo-imaging temperature window in which redox ratio measuring was immune to temperature fluctuations. These results may serve as a reference for designing and optimizing redox cryo-imaging experiments for quantitatively mapping the metabolic state of biological samples.

Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography.

Vitreous freezing offers a way to study cells and tissue in a near-native state by cryo-transmission electron microscopy (cryo-TEM), which is important when structural information at the macromolecular level is required. Many cells - especially those in tissue - are too thick to study intact in the cryo-TEM. Cryo focused-ion-beam (cryo-FIB) milling is being used in a few laboratories to thin vitreously frozen specimens, thus avoiding the artifacts and difficulties of cryo-ultramicrotomy. However, the technique is challenging because of the need to avoid devitrification and frost accumulation during the entire process, from the initial step of freezing to the final step of loading the specimen into the cryo-TEM. We present a robust workflow that makes use of custom fixtures and devices that can be used for high-pressure-frozen bulk tissue samples as well as for samples frozen on TEM grids.

A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons.

A close to native structure of bulk biological specimens can be imaged by cryo-electron microscopy of vitreous sections (CEMOVIS). In some cases structural information can be combined with X-ray data leading to atomic resolution in situ. However, CEMOVIS is not routinely used. The two critical steps consist of producing a frozen section ribbon of a few millimeters in length and transferring the ribbon onto an electron microscopy grid. During these steps, the first sections of the ribbon are wrapped around an eyelash (unwrapping is frequent). When a ribbon is sufficiently attached to the eyelash, the operator must guide the nascent ribbon. Steady hands are required. Shaking or overstretching may break the ribbon. In turn, the ribbon immediately wraps around itself or flies away and thereby becomes unusable. Micromanipulators for eyelashes and grids as well as ionizers to attach section ribbons to grids were proposed. The rate of successful ribbon collection, however, remained low for most operators. Here we present a setup composed of two micromanipulators. One of the micromanipulators guides an electrically conductive fiber to which the ribbon sticks with unprecedented efficiency in comparison to a not conductive eyelash. The second micromanipulator positions the grid beneath the newly formed section ribbon and with the help of an ionizer the ribbon is attached to the grid. Although manipulations are greatly facilitated, sectioning artifacts remain but the likelihood to investigate high quality sections is significantly increased due to the large number of sections that can be produced with the reported tool.

Excision of periocular basal cell carcinoma guided by en face frozen section.

We describe a technique for monitoring excision margins in periocular basal cell carcinoma (BCC) using en face frozen sections and report outcomes. We excised periocular BCC with 3mm margins. An outer 1mm sliver of the perimeter of the specimen was mapped and sent for evaluation by en face frozen section. The central tumour mass was processed using routine paraffin sections. A further 3mm level was excised at the site of any affected margin and the outer 1mm sliver was again evaluated by frozen section. We identified 78 patients from November 2003 to July 2009; 67 had primary tumours and 11 (14%) had recurrent BCC of which 52 (66%) were located on the lower eyelid. Growth patterns were nodular (n=34, 43%), infiltrative (n=25, 32%), micronodular (n=12, 16%), and superficial (n=7, 9%). A third of BCC with a clinically nodular appearance showed additional histological patterns including infiltrative and micronodular growth patterns. Of 30 clinically nodular carcinomas, 29 were excised completely with one level, and one required 2 levels of excision for clearance after evaluation by frozen section. Mean follow-up was 23 months (range 2-60). There was one recurrence (1%). Excision of margins guided by en face frozen section is justified by the low rates of recurrence, and it can easily be taught or imported into hospital practice. Clinically nodular BCC have subclinical extensions that can be missed on bread loaf sectioning, which makes the sampling of margins a standard for periocular BCC.

[Rapid frozen sections in neuropathology]. / Der Schnellschnitt in der Neuropathologie.

Neuropathological evaluation of frozen sections requires a) special expertise in neuropathological specimen assessment and neurooncology as well as b) a trustful and open communication culture with the neurosurgeons. In addition to frozen sections, cytological examinations of smear and touch preparations as supporting methods are available to reach a correct diagnosis: these additional methods should therefore be performed whenever possible. Besides evaluation of biopsy specimens, appraisal of resection specimens and resection margin controls are of high clinical relevance. In the case of diffusely infiltrating central nervous system (CNS) neoplasms, in particular gliomas, resection margin control is often not feasible in contrast to other types of solid tumor.

[Indications and limitations of fresh frozen sections in the pulmonary apparatus]. / Indikation und Grenzen des intraoperativen Schnellschnitts im Organbereich Lunge.

Recommendations for the diagnosis of lung tumors almost limit the use of fresh frozen sections to the evaluation of resection margins. In pathology pretherapeutic methods for assessment of clinically suspected lung cancer are favored over intraoperative frozen section diagnosis. For the interdisciplinary management of uncertain lung findings diagnostic methods, such as cytopathology and examination of biopsy material are available. The use of rapid on-site evaluation (ROSE) in cytopathology is limited due to the lack of necessary personnel. Diagnosis of unclear pulmonary lesions or distinction of metastases from primary lung tumors by intraoperative frozen sections is therefore limited to exceptional cases that were not resolved by preoperative biopsies. Such rare cases require a common consensus strategy between thoracic surgeons and pathologists in a preoperative tumor board.

Digital pathology: exploring its applications in diagnostic surgical pathology practice.

There has been a recent upsurge in worldwide attention on digital pathology, which has transformed from static snapshots from camera-equipped microscopes to its modern form that encompasses scanning of whole glass slides with evaluation of histological images on a computer screen, along with management of its accompanying information. Although it has been widely accepted in education and research, its implementation in diagnostic surgical pathology practice is not without challenges in workflow integration, technological infrastructure, pathologist acclimatisation, global standardisation for clinical practice, and cost issues, among others. Nonetheless, early adopters have harnessed its benefits in specific niches, like frozen section services and remote second opinion consultations. Its tremendous potential is worthy of further validation to compare with conventional glass slide evaluation, even while it is already paving the way for advancement into virtual three-dimensional imaging technology, with a glimpse into a possible future digital diagnostic pathology practice.

The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.

There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.

Micromachining tools and correlative approaches for cellular cryo-electron tomography.

A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. This is exacerbated in tomography applications, where the aspect ratio (and thus the apparent specimen thickness) changes considerably during specimen tilting. Cryo-ultramicrotomy is the most obvious way of dealing with this problem; however, frozen-hydrated sections suffer from potentially inconsistent compression that cannot be corrected with certainty, and furthermore, yields of sections that satisfy all of the conditions necessary for tomographic imaging are poor. An alternative approach that avoids mechanical deformations is the use of focused ion beam (FIB) instrumentation, where thinning of the frozen-hydrated specimen occurs through the process of sputtering with heavy ions, typically gallium. Here, we use correlative cryo-fluorescence microscopy to navigate large cellular volumes and to localize specific cellular targets. We show that the selected targets in frozen-hydrated specimens can be accessed directly by focused ion beam milling. We also introduce a novel cryo-planing procedure as a method that could facilitate thinning of large areas of vitreous ice prior to cryo-fluorescence, FIB thinning, and cryo-electron tomography.

A procedure for tissue freezing and processing applicable to both intra-operative frozen section diagnosis and tissue banking in surgical pathology.

Different methods for snap freezing surgical human tissue specimens exist. At pathology institutes with higher work loads, solid carbon dioxide, freezing sprays, and cryostat freezing are commonly used as coolants for diagnosing frozen tissue sections, whereas for tissue banking, liquid nitrogen or isopentane cooled with liquid nitrogen is preferred. Freezing tissues for diagnostic and research purposes are therefore often time consuming, laborious, even hazardous, and not user friendly. In tissue banks, frozen tissue samples are stored in cryovials, capsules, cryomolds, or cryocassettes. Tissues are additionally embedded using freezing media or wrapped in plastic bags or aluminum foils to prevent desiccation. The latter method aggravates enormously further tissue handling and processing. Here, we describe an isopentane-based workflow which concurrently facilitates tissue freezing and processing for both routine intra-operative frozen section and tissue banking and satisfies the qualitative demands of pathologists, cancer researchers, laboratory technicians, and tissue bankers.