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Unroofing, which is the mechanical shearing of a cell to expose the cytoplasmic surface of the cell membrane, is a unique preparation method that allows membrane cytoskeletons to be observed by cryo-electron microscopy, atomic force microscopy, freeze-etching electron microscopy and other methods. Ultrasound and adhesion have been known to mechanically unroof cells. In this study, unroofing using these two means was denoted sonication unroofing and adhesion unroofing, respectively. We clarified the mechanisms by which cell membranes are removed in these unroofing procedures and established efficient protocols for each based on the mechanisms. In sonication unroofing, fine bubbles generated by sonication adhered electrostatically to apical cell surfaces and then removed the apical (dorsal) cell membrane with the assistance of buoyancy and water flow. The cytoplasmic surface of the ventral cell membrane remaining on the grids became observable by this method. In adhesion unroofing, grids charged positively by coating with Alcian blue were pressed onto the cells, thereby tightly adsorbing the dorsal cell membrane. Subsequently, a part of the cell membrane strongly adhered to the grids was peeled from the cells and transferred onto the grids when the grids were lifted. This method thus allowed the visualization of the cytoplasmic surface of the dorsal cell membrane. This paper describes robust, improved protocols for the two unroofing methods in detail. In addition, micro-unroofing (perforation) likely due to nanobubbles is introduced as a new method to make cells transparent to electron beams., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ultrastructural analyses with electron microscopy have provided indispensable information to understand physiology and pathology of the nervous system. Recent advancement in imaging methodology paved the way for complete reconstruction of the neuronal connection map in the central nervous system, which is termed 'connectome' and would provide key insights to understand the functions of the brain. The critical advancement includes serial ultrastructural observation with scanning electron microscopy (SEM) instead of conventional serial sectioning transmission electron microscopy along with specific tissue preparation methods to increase heavy metal deposition for efficient SEM imaging. The advanced imaging methods using SEM have distinct advantages and disadvantages in multiple aspects, such as resolution and imaging speed, and should be selected depending on the observation conditions, such as target tissue sizes, required spatial resolution and necessity for re-observation. Dealing with the huge dataset remained to be a major obstacle, and automation in segmentation and 3D reconstruction would be critical to understand neuronal circuits in a larger volume of the brain. Future improvement in acquisition and analyses of the morphological data obtained with the advanced SEM imaging is awaited to elucidate the significance of whole connectome as the structural basis of the consciousness, intelligence and memory of a subject., (© The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

When dysplasia is identified in a gallbladder, many experts recommend submission of the entire gallbladder for histologic examination. We sought to determine if this practice could be justified. We reviewed 16,611 gallbladder resections over an 8-year period, and identified 17 cases of carcinoma (15 primary and 2 metastatic), 9 cases of high-grade dysplasia, 16 cases of low-grade dysplasia, and 81 cases with atypia, not otherwise specified (NOS). Sixteen (94%) of 17 adenocarcinomas were identified and sampled on the initial gross inspection, and the remaining case was identified and sampled on review of the gross specimen. None of the high- or low-grade dysplasias were identified on gross examination, but all were identified as atypical on the initial slide submitted and correctly graded with the submission of 4 additional slides. Eight (89%) of 9 high-grade, 6 (38%) of 16 low-grade, and 1 (1%) of 81 atypia, NOS, cases were subsequently entirely submitted without identification of any new lesion. We conclude that for cases of dysplasia and atypia, NOS, review of the gross specimen and submission of up to 4 additional sections identify all significant lesions, and submission of the entire gallbladder is not justified.

Objectives: Glutaraldehyde (GA)-fixed xenografts are prone to calcification after implantation in humans and there is evidence that immune reaction to the Galα1,3-Galβ1,4GlcNAc-R (α-Gal) antigen may play a part in this process. The objectives of this study were to evaluate the immune response of α1,3-galactosyltransferase knockout (α-Gal KO) mice to bovine pericardium and to evaluate the effect of various anticalcification treatments on bovine pericardium using mouse subcutaneous implantation model., Methods: Bovine pericardial tissues were divided into eight groups according to the method of anticalcification treatments. Prepared tissues were subcutaneously implanted into the α-Gal KO and wild-type mice for 2 months, and anti-α-Gal antibodies were measured at 2 weeks and 2 months after implantation. Explanted tissues were examined by immunohistochemistry and calcium contents of the explanted tissues were measured., Results: Titres of IgM and IgG antibodies in the α-Gal KO mice increased significantly according to the duration of implantation, whereas titres of IgM and IgG antibodies in the wild-type mice increased until 2 weeks after implantation without further increase thereafter. Titres of IgG antibodies measured at 2 months after implantation were significantly higher in the α-Gal KO mice than in the wild-type mice. Immunohistochemistry revealed macrophages surrounding the pericardial tissues irrespective of the mouse type into which the tissues implanted, whereas T-cells could only be observed in the tissues implanted into the α-Gal KO mice. Except the high-concentration GA-treated group, calcium contents of anticalcification-treated groups were all significantly lower or tended to be lower than that of the control group, irrespective of the mouse type. Calcium contents of the control group were significantly higher in the α-Gal KO mice than in the wild-type mice., Conclusions: Bovine pericardium implanted into the α-Gal KO mice caused significant increase in anti-α-Gal antibodies, showed some histologic evidences of chronic rejection and revealed a potential toward more calcification. These findings suggest a possible role of immune response in calcification of xenografts. High-concentration GA fixation alone did not prove to be an effective anticalcification treatment in mouse subcutaneous implantation model. α-Gal KO mouse subcutaneous implantation model might be a feasible animal model for testing efficacy of anticalcification treatments incorporating immunologic approach.

Objective: We evaluated DNA amplificability to achieve a 100% success rate in KRAS mutation testing with dideoxy sequencing using formalin-fixed and paraffin-embedded colorectal cancer tissue samples obtained from a recent clinical trial., Methods: We evaluated the effects of deparaffinization, formalin fixation or storage time, and amplicon size on the amplificability of DNAs extracted from 19 formalin-fixed and paraffin-embedded colorectal cancer tissue samples. We subjected to KRAS mutation analysis 112 samples from metastatic colorectal cancer patients in 31 hospitals enrolled in a Phase II trial of a second-line FOLFIRI (5-fluorouracil+ leucovorin + irinotecan) + cetuximab regimen., Results: Deparaffinization, formalin fixation and storage times did not appear to affect the recovery and amplificability of DNAs. However, amplicon size had a remarkable effect on the amplificability of DNAs. The smaller fragments with a size of ≤278 bp (96-278 bp) were successfully amplified with polymerase chain reaction in all samples tested, whereas the larger fragments with a size of ≥298 bp (298-565 bp) were not amplified. All samples from our clinical trial were successfully analyzed using three sets of primers with the amplicon sizes of 201, 221 and 240 bp, and KRAS mutations in exons 2 and 3 were detected in 49 of the 112 cases (43.8%)., Conclusions: These data suggest that the evaluation of DNA amplificability and amplicon size is important for the success of mutation detection tests such as the KRAS test with dideoxy sequencing using formalin-fixed and paraffin-embedded tissue samples in the clinical setting.

The increasing evidence of trastuzumab efficacy in breast cancer (BC) patients means that an accurate and reproducible evaluation of HER-2 statusis of paramount importance in histological and in cytological samples. Currently, the two main methods used to analyze HER-2 amplification or overexpression are fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Although the two methods are strongly correlated for histological tissue, the evaluation of tumor morphology through FISH may be difficult and fluorescence fades quickly. These limitations can be overcome by chromogenic in situ hybridization (CISH), which can visualize the amplification product along with morphological features. In view of this, in the present study, we analyzed the usefulness of CISH on formalin-fixed, paraffin-embedded (FFPE) BC specimens and investigated whether CISH can be a valid technique in the determination of HER-2 status for fine-needle aspirates (FNAs) processed by liquid-based cytology. The results we obtained in a retrospective series of 111 FFPE BC specimens demonstrated good concordance between CISH and IHC and between CISH and FISH. The former concordance was comparable with that observed between FISH and IHC. When CISH was applied to a prospective series of 53 FNAs, from surgically removed BC, our data showed evidence of a higher concordance of results between liquid-based cytology and the companion FFPE tissues using CISH rather than HercepTesttrade mark. Therefore, CISH analysis, which is avaluable and reproducible alternative to FISH for selecting breast cancer patients for trastuzumab therapy, can lower false-positive immunocytochemistry findings in ThinPrep-processed FNAs.

In this paper, we review those pathologic entities that may mimic papillary thyroid carcinoma. These include specimen preparation and handling issues such as decalcification, frozen section, and artifacts occurring following fine-needle aspiration (FNA). Also discussed are true disease entities, including chronic lymphocytic thyroidits, benign papillary hyperplastic lesions, hyalinizing trabecular neoplasm, and post-FNA changes. Both the cytologic and histopathologic preparations containing these changes are reviewed and illustrated; guidelines for distinctions among these entities are provided.

Reproducibility of human papillomavirus (HPV) typing on archived ThinPrep (Cytyc, Boxborough, MA) specimens was evaluated repeating Hybrid Capture II (HCII) (Digene, Gaithersburg, MD) testing after 25 to 40 months (mean, 31.3 months; group 1), 6 to 11 months (mean, 8.4 months; group 2), and 0 to 5 months (mean, 3.5 months; group 3). Another ThinPrep slide was prepared to evaluate cellularity and reproducibility of the cytologic diagnosis. The mean residual relative light units (RLU) calculated for each group showed a strong decrease of RLU values at the second typing (group 1, 21.69%; group 2, 26.47%; and group 3, 32.25% of original values). No residual HPV DNA was shown in group 1 in 8 (13%) of 60 cases or in groups 2 and 3 in 2 (3%) of 60 cases each. These cases were associated mostly with poor cellularity and reproducibility of the initial cytologic diagnosis in the final cytologic examination. Intergroup statistical analysis of mean relative percentages for cases with satisfactory residual cellularity revealed a significant difference only between groups 1 and 3 (P < .05). Although mostly reproducible, HPV typing results by HCII on archived specimens are influenced by material consumption. In addition, results might be affected by some DNA degradation after long-term sample storage.

Tissue microarrays (TMAs) reduce the amount of tissue analyzed with the assumption that protein and gene expression patterns are homogeneous throughout tumors. Many tumor types, including glioblastoma multiforme (GBM), are heterogeneous in many regards, including cell proliferation. We retrospectively compared Ki-67 labeling indices (LIs) determined by whole tissue section (WTS) vs TMA in a series of 50 GBMs from 45 patients. A paired t test indicated that the difference between average LIs obtained from a TMA vs a WTS was not significant (P = .51). There was no correlation between TMA and WTS (r = 0.042; P = .77), indicating that the methods yielded very different results in individual tumors. The Ki-67 LI did not always correlate with the tissue section in an individual tumor; however, when evaluating a large number of tumors on a TMA, the LI range and mean LI were roughly comparable with the LI range and mean LI determined from the WTS.

We examined the feasibility of detecting hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) specimens obtained by thin-needle biopsy (TNB). Specimens obtained by large-needle biopsy (LNB) and unfixed frozen tissue served as controls. A total of 23 biopsy specimens, 13 obtained by TNB and 10 by LNB, from 20 patients with chronic hepatitis C were included in the study. HCV RNA was detected by nested reverse transcription-polymerase chain reaction (RT-PCR). HCV RNA was found in FFPE archival specimens obtained by TNB (11 [85%]) or LNB (7 [70%]). The sensitivity was similar in unfixed tissue in which HCV RNA was found in 7 (88%) of 8 TNB specimens and 6 (86%) of 7 LNB specimens. The detection of HCV RNA did not seem to be affected by storage of the paraffin blocks, the presence of advanced fibrosis and cirrhosis, or fragmentation of the core cylinder. TNB yields enough qualitative suitable material to detect HCV RNA by RT-PCR.

We investigated the use of laser capture microdissection (LCM) to identify differences in gene expression between cell types or regions within the rhesus monkey endometrium. Different cell types were harvested from the two major regions of the endometrium during midsecretory phases (Days 21-23) of adequate artificial menstrual cycles: glandular epithelia (G) or stroma (S) from the functionalis (F) or the basalis (B). Amplification of the cDNA populations (primer-specific adaptors) was used to increase the amount of nucleic acid for further analysis. This single amplification step allowed us to detect the housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase and 18S rRNA) and the cDNA smears in the samples. Using differential display reverse transcription polymerase chain reaction (DDRT-PCR), six fragments were selected, cloned, and sequenced based on their regional and cell type localization. Primer-specific PCR analysis subsequently confirmed the localization of three fragments: F1, highly expressed in the functionalis but not the basalis, was homologous (93% identical) to the human leukotriene B4 receptor BLT2; FS-1, highly expressed only in the stroma of the functionalis, had a 94% homology with an as yet uncharacterized gene (FLJ124360); and BG-1, primarily expressed only in the glandular epithelia of the basalis, showed a 98% homology with an uncharacterized bacterial artificial chromosome clone sequence. These LCM-generated cDNA populations coupled with DDRT-PCR can provide an important avenue for the identification of new or novel gene fragments that display cell type- or region-specific gene expression in the rhesus monkey endometrium.

We evaluated 62fine-needle aspiration (FNA) biopsy specimens of metastatic malignant melanoma (MM) from 62 patients and compared 84 smears with the corresponding tissue sections. Melanin is found more often in FNA smears than in corresponding tissue sections of metastatic MM but is significantly less abundant in smears. Melanin is present more often in liquid-based than in conventional smears. Pseudoinclusions were almost twice as frequent in tissue sections as in corresponding aspirates, regardless of the preparation method. There was no statistical difference for cell type or cytoplasmic features in the tissue sections and aspirates or between the type of preparation and/or stain used. With the exception of melanin, direct comparison of liquid-based and conventional smears showed higher cellularity in the former as the only difference.

Background: Isolation of sperm suitable for ICSI from fresh or frozen-thawed testicular sperm extraction (TESE) can be facilitated by mechanical or enzymatic processing of the samples., Methods: A retrospective multicentre study was initiated to compare these two approaches. Eleven German centres provided data on their TESE cycles performed during the period 1996/1997. Quality of retrieved sperm, fertilization rates of injected oocytes, embryo quality, resulting pregnancy rates and evolution of pregnancies were evaluated., Results: The percentage of cycles with at least some motile sperm available for injection was higher after mechanical preparation. Independent of the preparation method, fertilization rates were higher for motile compared with immotile sperm or elongated spermatids in all groups and in general higher for cryopreserved versus fresh samples. Embryo quality was significantly better after injection of motile sperm for all treatments and in particular after enzymatic versus mechanical processing of biopsies. Pregnancy rates were identical for embryos derived from sperm prepared mechanically or enzymatically from fresh or cryopreserved testicular samples. The abortion rate (32/172, 18.6%) and the rate of multiple implantations (32/140, 22.9%) were not different from results reported in the literature for ICSI using ejaculated sperm., Conclusion: In this retrospective multicentre study, no unequivocal advantage of one over the other preparation method could be identified in 839 ICSI cycles using testicular sperm from 549 patients.

The determination of tumor cell estrogen receptor (ER) expression status by immunohistochemical analysis has become standard practice, yet assay reproducibility has not been assessed adequately. By using a breast cancer tissue microarray, we examined interlaboratory variability in ER reporting. A 2-fold redundant tissue microarray block was created from 29 breast cancers. Unstained slides were distributed to 5 laboratories, and each laboratory immunostained and scored 1 slide for ER. Interlaboratory agreement ranged from moderate to high (overall kappa = 0.54 for 0-3+ grading; overall kappa = 0.84 for negative vs positive assessment of ER status). When 1 observer scored each of the 5 slides, interlaboratory agreement was slightly better (kappa = 0.63 for 0-3+ scoring; kappa = 0.96 for negative vs positive scoring). One laboratory, which had used an antibody and antigen retrieval method different from the others, demonstrated only fair concordance with the other 4 laboratories, but there was substantial intralaboratory interobserver agreement and excellent agreement with an outside observer reviewing the slide stained in that laboratory. The tissue microarray was an efficient and effective tool for identifying variability in ER reporting and should prove valuable in other external quality assurance programs.

Pleural mesothelioma is a rare and highly malignant tumor that has shown increasing incidence. In approximately 90% of patients the tumor manifests initially as recurrent, unilateral, bloody pleural effusions; in the remaining 10% of patients the tumor is detected by chest radiography. As a result, pleural mesotheliomas can be diagnosed by cytologic examination of pleural effusions or transthoracic fine-needle aspirates; the cytologic manifestations of such an examination are described in detail. Additionally, the handling of cytologic specimens, diagnostic problems, and the value and limitations of immunocytochemical and electron microscopic studies and other ancillary techniques in the cytologic identification of pleural mesotheliomas are discussed.

A method for analysis of silicon in tissue was developed to determine silicon content in breast parenchymal and periprosthetic capsular tissues of patients with silicone or saline implants and to compare levels in tissues from normal (nonaugmented) breasts. It is of interest to determine whether increased silicon content in tissues can be associated with morbidity in patients who have received silicone implants. This manuscript addresses the issues involved in analysis of breast tissue samples for silicon and compares silicon levels with tissue histologic findings and patient morbidity. One hundred sixty tissue samples were obtained for silicon analysis from 72 patients during augmentation, capsulectomy with or without replacement mammoplasty, mastectomy, or biopsy procedures and were frozen in acid-washed polystyrene tubes at 220 degrees C until analysis. Samples were thawed, sectioned to approximately 0.1 g (dry weight), and digested in nitric acid before analysis by inductively coupled plasma emission spectroscopy, monitoring emission intensity at 251.6 nm. Tissue silicon levels (breast parenchymal and periprosthetic capsular tissue) in patients with silicone gel implants were much higher (mean, 9,287 micrograms/g, n = 106) than in patients with saline implants (mean, 196 micrograms/g, n = 37) or nonaugmented breasts (mean, 64 micrograms/g, n = 17). Histologic examination was performed on 54 tissue samples stained with hematoxylin-eosin. Tissue samples were rated as to degree of inflammation and calcification, and amount of giant cells, foamy histiocytes, and vacuoles containing a colorless refractory material. Vacuolization and foamy histiocyte ratings correlated significantly with tissue silicon concentration. No correlations were found between tissue silicon concentration and inflammation, calcification, or giant cell rating. Implant age (number of years an implant was in place before sampling) correlated with capsular tissue silicon concentration in patients with intact implants but not in those with ruptured implants. No difference in tissue silicon concentration was found between patients with or without signs or symptoms of morbidity. Using 0.1 g of tissue, the method was linear to 1,000 micrograms/g, and sensitivity was 3.7 micrograms/g. Precision between runs (mean, 5.1 micrograms/g; coefficient of variance, 13.7%; n = 13) was calculated from multiple analyses of a bovine liver standard (National Bureau of Standards, reference material 1577a). Significant biologic variability (21.4% to 52.5%) was seen in tissues with high silicon levels. Paraffin-embedded, formalin-fixed tissues are not amenable to silicon analysis by this method, because of leaching of silicone from the tissues during preparation. Thus only fresh frozen tissue samples were used.

The regional distribution of initial (i.e. most peripheral) lymph vessels of the human skin is unknown. New methods permit a precise systematic determination of the density of initial lymph vessels in every region of the dermis. Based on 202 skin specimens, it was possible to investigate the density of the lymph vessels of the skin derived from head, neck, upper arm, forearm, upper and lower leg. In all regions there seem to be a uniform structured dense subpapillary network and a wide meshed deep dermal network. The lymph vessels are at their most dense in the skin of the head.

To evaluate ThinPrep (Cytyc, Marlborough, MA) processing of endoscopic brushing specimens for cytologic examination, ThinPrep slides and direct smears of 29 gastrointestinal (GI) and 22 bronchial brushings were compared. Clinicians prepared the direct smears. The brush was then immersed in CytoLyt (Cytyc) and one ThinPrep slide made. All cases had corresponding biopsies. Smears and ThinPrep slides were screened and reviewed independently. Screening time per case was recorded. All slides were evaluated for cellularity, quality, cellular preservation, and quantity of diagnostic cells. A diagnosis was rendered for each case. Cytologic and histologic diagnoses were correlated. Follow up was obtained for cases with discrepant histologic and cytologic diagnosis. Twenty-three brushings were from esophagus, 5 stomach, 1 duodenum, and 22 lung. An average of 3.6 direct smears (range 2-6) was made for each case. Average screening time per case was 12 minutes for GI direct smear, 15 minutes bronchial direct smear, 4 minutes GI ThinPrep slide, and 9 minutes bronchial ThinPrep slide. ThinPrep slides were superior to direct smears in cellularity, quantity of diagnostic cells, and quality of slides. ThinPrep slides and direct smears showed comparable cellular preservation. The sensitivity of detecting malignancy by biopsy, direct smears, and ThinPrep slides was 81%, 75%, and 75%, respectively. One false-positive diagnosis was made on cytology with both direct smear and ThinPrep slide, a case with radiation atypia. In conclusion, ThinPrep slides are at least comparable to direct smears in cytologic examination of brushings. However, false-positive diagnosis is a possible potential pitfall.

Two hundred forty-two breast fine-needle aspirates prepared by the Cytyc ThinPrep Processor were compared with aspirates prepared by the conventional smear method. Palpable and nonpalpable mammographic breast lesions were aspirated and the first half of the aspirate was submitted for conventional smears and the second half was rinsed into a proprietary fixative and loaded on the ThinPrep Processor for monolayer slide preparation. The matched pairs were diagnosed and analyzed separately in a double-blinded manner and later paired for comparison. Diagnoses correlated exactly in 62% of cases. The diagnosis of fibroadenoma was made in only 4 of 21 cases on ThinPrep (19% correlation). Semiquantitative analysis of several cytologic features indicated potential pitfalls for accurate diagnosis using the ThinPrep Processor. These included loss of background constituents (such as stroma and adipose tissue), decreased cellularity and single ductal epithelial cells, and decreased cytologic detail including size, shape and nuclear texture. The ThinPrep Processor may play a role in breast fine-needle aspiration, but further investigation is warranted before it is used as a sole preparatory method.

Highly enriched and specifically unlabelled human Langerhans cells (LC) from epidermal cell suspensions (ECS) are indispensable for the intensive study of LC function. Discontinuous Ficoll-metrizoate density gradient centrifugation was found to be a satisfactory method of enriching LC from ECS. In contrast to a previous study, however, the majority of LC floated on Ficoll-metrizoate with a density of 1.057 g/cm3 instead of 1.068 g/cm3. The concentration of unlabelled LC can be enriched to as high as 92% from the original 1.8-3.1% LC in ECS. Approximately 40-50% of original LC can be harvested. The procedure was also simplified by using a Schick razor instead of a dermatome to obtain thin epidermal sheets (about 0.25 mm in thickness), omitting the density gradients of 1.089 and 1.10 g/cm3 and using the avidin-biotin complex method to identify LC. LC are non-proliferative cells in in vitro culture systems. The viability of LC dropped to 20-30% after 3 days in the culture medium. It was also observed that LC tended to attach to aggregated keratinocytes in the culture system.

A nationwide multicenter joint study was undertaken to investigate the clinicopathologic characteristics of invasive ductal carcinoma of the breast with a predominant intraductal component, a diagnostic entity proposed in the histologic typing of breast tumors by the World Health Organization in 1981. A total of 368 tumors, of which 218 were studied pathologically between 1983 and 1987 and 150 between 1971 and 1977, were accumulated for the present study from seven medical institutes in Japan. The incidence of such types of tumor was higher in the latter period than in the former, as was that of pure intraductal carcinomas. Seventy of the 368 tumors displayed metastatic nodal involvement and nine tumor deaths were observed from 150 tumors followed up for over 10 years following radical mastectomy, the overall quantity of the extraductal stromal invasion clearly acting on lymph node metastases and patients' outcome. We consider a total stromal invasion of greater than 5 mm to be critical in the prognosis of patients with tumor of this type.