Description of a new biosafe procedure for cytological specimens from patients with COVID-19 processed by liquid-based preparations.

BACKGROUND: Coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and represents the causative agent of a potentially fatal disease. The spread of the infection and the severe clinical disease have led to the widespread adoption of social distancing measures. Special attention and efforts to protect or reduce transmission have been applied at all social levels, including health care operators. Hence, this reports focuses on the description of a new protocol for the safe management of cytological samples processed by liquid-based cytology (LBC) with an evaluation of the changes in terms of morphology and immunoreactivity. METHODS: From March 11 to April 25, 2020, 414 cytological cases suspicious for SARS-CoV-2 were processed with a new virus-inactivating method suggested by Hologic, Inc, for all LBC specimens. RESULTS: The samples showed an increased amount of fibrin in the background. A slight decrease in cellular size was also observed in comparison with the standard method of preparation. Nonetheless, the nuclear details of the neoplastic cells were well identified, and the immunoreactivity of the majority of those cells was maintained. The cell blocks did not show significant differences in morphology, immunoreactivity, or nucleic acid stability. CONCLUSIONS: Despite some minor changes in the morphology of the cells, the results of this study highlight that the adoption of the new protocol for the biosafety of LBC-processed samples in pathology laboratories is important for minimizing the risk for personnel, trainees, and cytopathologists without impairing the diagnostic efficacy of the technique.

In the Era of the Leeds Protocol: A Systematic Review and A Meta-Analysis on the Effect of Resection Margins on Survival Among Pancreatic Ductal Adenocarcinoma Patients.

BACKGROUND AND AIMS: A positive resection margin is considered to be a factor associated with poor prognosis after pancreatic ductal adenocarcinoma resection. However, analysis of the resection margin is dependent on the pathological slicing technique. The aim of this systematic review and meta-analysis was to study the impact of resection margin on the survival of pancreatic ductal adenocarcinoma patients whose specimens were analyzed using the axial slicing technique. MATERIAL AND METHODS: A systematic search in the PubMed, Cochrane, and Embase datasets covering the time period from November 2006 to January 2019 was performed. Only studies with axial slicing technique (Leeds Pathology Protocol or Royal College of Pathology Protocol) were included in the final database. Meta-analysis between the marginal distance and survival was performed with the Inverse Variance Method in RevMan. RESULTS: The systematic search resulted in nine studies meeting the inclusion criteria. The median survival for a resection margin 0 mm ranged from 12.3 to 23.4 months, for resection margin <0.5 mm 16 months, for resection margin <1 mm ranged from 11 to 27.5 months, for resection margin <1.5 mm ranged from 16.9 to 21.2 months, and for resection margin >2 mm ranged from 53.9 to 63.1 months. Five studies were eligible for meta-analysis. The pooled multivariable hazard ratio favored resection margin ⩾1 mm (hazard ratio: 1.32 and 95% confidence interval: 1.03-1.68, p = 0.03). CONCLUSION: Resection margins ⩾1 mm seem to lead to better survival in pancreatic ductal adenocarcinoma patients than resection margin <1 mm. However, there is not enough data to evaluate the effect of oncologic therapy or to analyze the impact of other resection margin distances on survival.

Diagnostic utility of cell block in fine needle aspiration cytology of thyroid gland.

BACKGROUND: This study was conducted to evaluate the diagnostic utility of cell block material in fine needle aspiration (FNA) of thyroid nodules. DESIGN: A total of 242 thyroid fine need aspirations (FNAs) were performed between January 2015 and December 2015. Of those, all consecutive thyroid FNA cases with cell blocks (n = 140) from 129 patients (age: 58.9 ± 12.8 years) are included in this study. Cytology slides and cell blocks are reviewed for adequacy assessment based on the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) and then categorizing them into TBSRTC diagnostic categories. These cases are divided into two groups, combined cytology and cell block (C + CB) and cytology without cell block (C). RESULTS: In the first group (C + CB), a total 140 cases are categorized in TBSRTC as follows: I: 13 (9.3%) cases, II: 78 (55.7%) cases, III: 7 (5%), IV: 16 (11.4%), V: 3(2.2%) and VI: 23 (16.4%). In the second group (C), the cases are classified in TBSRTC as follows: I: 23 (16.4%) cases, II: 70 (50%), III: 7 (5%), IV: 16 (11.4%), V: 3 (2.2%) and VI: 21 (15%). Nondiagnostic rate was 7.1% lower in the first group (C + CB) as compared with second group (C) (First group: 9.3% vs second group: 16.4%, P = .0764). CONCLUSIONS: Combined use of cytology slides and cell block decreases the nondiagnostic rate up to 7.1% as compared with cytology without cell block.

[Paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples].

OBJECTIVE: To investigate paraffin section thickness in immunohistochemical detection of p16 expression in cervical tissue samples. METHODS: p16 immunohistochemical staining was performed in 150 cases of chronic cervicitis, 126 cases of low-grade squamous intraepithelial lesions(LSIL), 96 cases of high-grade squamous intraepithelial lesions (HSIL) and 78 cases of cervical cancer from January 2014 to March 2018 in Zhongshan Boai Hospital. The results of p16 protein expression in paraffin sections with thickness of 2, 3, 4, 5 and 6 µm were compared using Logistic regression analysis. RESULTS: With the increase of slice thickness, the staining of p16 protein in nucleus gradually increased. The thickness of cervical slices in chronic cervicitis and cervical cancer samples had no significant effect on the positive rate of p16 protein(χ2=7.817 and 1.332, both P>0.05), while the thickness of slices in LSIL and HSIL samples had significant effect on the positive rate of p16 protein (χ2=17.688 and 10.182, P<0.05 or P<0.01). The stable and reliable results were obtained when the slices were between 3 and 5 µm thick. CONCLUSIONS: Paraffin sections with thickness of 3.0-5.0 µm are recommended for immnohistochemical staining of p16 protein in cervical tissue samples.

Preparation of Nonhuman Primate Eyes for Histological Evaluation After Retinal Gene Transfer.

To evaluate gene therapy for retinal disorders, appropriate models of the human eye are needed. Nonhuman primate eyes offer significant advantages over rodent eyes. However, current preparation methods have limitations. Here, a protocol is described for histological processing of nonhuman primate eyes after gene transfer. The user dissects unfixed eyes, flattens the globe parts within filter paper, and performs formalin fixation and paraffin embedding. This method obviates the need for harsh fixatives, allowing subsequent immunostaining or in situ hybridization while preserving tissue integrity for histopathological evaluation. Moreover, the straight orientation of the retinal cell layers is ideal for image analysis.

Useful aspects of diagnosis of imprint cytology in intraoperative consultation of ovarian tumors: comparison between imprint cytology and frozen sections.

BACKGROUND: In the intraoperative consultation of ovarian tumors, the histological diagnosis of frozen sections (FS) of large tumors is frequently difficult because of the limited number of tumor samples. The application of imprint cytology (IC), in which samples are obtained from wide areas of the tumors, is useful for intraoperative consultation. However, the useful aspects of IC have not been clearly defined. The present study is a detailed comparison of IC and FS that clearly defines the useful aspects of IC. METHODS: Fifty-five cases of ovarian tumors that were examined using both IC and FS were evaluated. The histological diagnoses consisted of benign (16), borderline (6), and malignancy (33). All of the malignant tumors consisted of various types of carcinoma. RESULTS: Benignity and malignancy were accurately diagnosed by both IC and FS. In the borderline group, the diagnostic accuracy of IC was very low (1/6: 16.6%) compared with FS (4/6: 66.6%). The diagnostic accuracy including benign, borderline, and malignant groups was 90.9% (50/55) for IC and 96.3% (53/55) for FS. Concerning the diagnosis of the types of carcinoma, the overall diagnostic accuracy of IC (25/31: 80.6%) was greater than that of FS (21/31: 67.7%), especially for the diagnosis of clear cell carcinoma (IC, 100%; FS, 80%) and mixed carcinoma (IC, 66.6%; FS, 16.6%). CONCLUSION: The useful aspects of IC in the intraoperative consultation are the diagnosis of benignity or malignancy and the accuracy of diagnosing clear cell carcinoma and mixed carcinoma.

Quality control in diagnostic immunohistochemistry: integrated on-slide positive controls.

Standardization in immunohistochemistry is a priority in modern pathology and requires strict quality control. Cost containment has also become fundamental and auditing of all procedures must take into account both these principles. Positive controls must be routinely performed so that their positivity guarantees the appropriateness of the immunohistochemical procedure. The aim of this study is to develop a low cost (utilizing a punch biopsy-PB-tool) procedure to construct positive controls which can be integrated in the patient's tissue slide. Sixteen frequently used control blocks were selected and multiple cylindrical samples were obtained using a 5-mm diameter punch biopsy tool, separately re-embedding them in single blocks. For each diagnostic immunoreaction requiring a positive control, an integrated PB-control section (cut from the appropriate PB-control block) was added to the top right corner of the diagnostic slide before immunostaining. This integrated control technique permitted a saving of 4.75% in total direct lab costs and proved to be technically feasible and reliable. Our proposal is easy to perform and within the reach of all pathology labs, requires easily available tools, its application costs is less than using external paired controls and ensures that a specific control for each slide is always available.

Utility of intraoperative frozen sections for thyroid nodules with prior fine needle aspiration cytology diagnosis.

INTRODUCTION: The objective of this study was to evaluate the role of intraoperative frozen section (IFS) in determining the course of surgery in thyroid nodules with a prior fine needle aspiration (FNA) biopsy diagnosis. In addition, reliability of FNA interpretation to guide surgical management without IFS was investigated. MATERIAL AND METHODS: This is a retrospective study of all patients who had a FNA biopsy, IFS, and final pathology performed on a thyroid nodule over a 9 month period. The extent of surgery at the time of the IFS was recorded. Subsequent change in surgical procedure following the IFS diagnosis was noted in each of the Bethesda diagnostic categories. RESULTS: 55% of the cases were deferred at IFS overall, with 68 and 86% in Bethesda III and IV categories, respectively. Overall, there was a change in management in 6% of cases. CONCLUSIONS: Our study does not support the use of IFS for nodules with prior FNA interpretation of Bethesda II, III, IV and VI as management was not significantly changed. IFS is of value for nodules with prior FNA diagnosis of Bethesda I for interpretation of nodule, and Bethesda V for planning surgery. A confirmatory diagnosis could not be rendered at IFS for lesions with follicular architecture, which comprised most of the cases in Bethesda III and IV.

Design-Based Stereology for Evaluation of Histological Parameters.

Valid quantification of organ volume and total cell numbers are crucial parameters for morphometric studies. The number of a specific cell type cannot be simply deduced from the number of its profiles found in thin tissue sections, as this parameter also depends on cell volume, tissue orientation as well as tissue atrophy. Design-based stereology has become the method of choice for unbiased, reproducible total cell number quantification. Steps described in this protocol include transcardial perfusion of mice, postfixation, and cryoprotection of the region of interest (ROI), followed by the preparation of a systematically and randomly sampled series of thick sections through the entire ROI. Furthermore, it is described how to perform immuno-histochemical staining of such thick cryo-sections, followed by providing a guidance for quantification of the ROI volume, the generation of unbiased virtual counting spaces, and steps to work with these counting spaces to obtain an unbiased estimate of total cell numbers.

Impact of routine cell block preparation on results of head and neck fine needle aspirates.

BACKGROUND: Fine needle aspiration (FNA) of head and neck masses is a common technique for providing cytology specimens to guide patient management. Cell blocks made from these specimens can be beneficial. Policy at our institution was changed from production of cell blocks only when requested by the pathologist to routine production for all non-parotid gland head and neck FNAs. The program was evaluated in terms of its impact on diagnosis and specimen turnaround time (TAT). METHODS: A retrospective study was carried out using electronic records at our institution. The Intervention group consisted of FNAs obtained in the 15-month period following implementation of routine cell block preparation (n = 391). The Control group consisted of the same specimens obtained in the 15 months prior to implementation (n = 403). The groups were compared with regards to diagnostic distribution into five categories-Unsatisfactory, Negative/Benign, Abnormal, Suggestive of Malignancy, and Malignant. Cytological-histological correlation and TAT were also compared. Chi square and t tests with P < 0.05 threshold were used. RESULTS: There was no difference in diagnostic distribution between the two groups (P = 0.59) and TAT was unchanged (P = 0.74). Cytological-histological correlation was borderline improved in the Intervention group, with fewer false negatives (33.0% Intervention, 44.3% Control, P = 0.050). The cost of the program was estimated at CAD$53.60/cell block, or CAD$16,771/year. CONCLUSION: Implementation of routine cell blocks for head and neck FNAs did not result in a difference in diagnostic distribution or improve case turnaround time despite incurring substantial cost. Correlation with final histology, however, was borderline improved, with fewer false negatives. Diagn. Cytopathol. 2016;44:880-887. © 2016 Wiley Periodicals, Inc.

Achieving consensus for the histopathologic diagnosis of melanocytic lesions: use of the modified Delphi method.

OBJECTIVE: To understand the sophisticated nature of coming to consensus when diagnosing complex melanocytic lesions among a panel of experienced dermatopathologists. METHODS: A total of 240 melanocytic lesions were assessed independently by three experienced dermatopathologists with their diagnoses mapped into one of five Melanocytic Pathology Assessment Tool and Hierarchy for Diagnosis (MPATH-DX) categories: (I) nevus/mild atypia, (II) moderate atypia, (III) severe atypia/melanoma in situ, (IV) T1a invasive melanoma and (V) ≥ T1b invasive melanoma. The dermatopathologists then discussed the cases, using a modified Delphi method to facilitated consensus building for cases with discordant diagnoses. RESULTS: For most cases, a majority of interpretations (two or three of three) agreed with the consensus diagnosis in 95% of Category I, 64% of Category II, 84% of Category III, 88% for Category IV and 100% of Category V cases. Disagreements were typically due to diagnostic threshold differences (64.5%), differing contents on slides even though the slides were sequential cuts (18.5%), and missed findings (15.3%). Disagreements were resolved via discussion of histopathologic features and their significance while reviewing the slides using a multi-headed microscope, considering treatment recommendations, citing existing literature, reviewing additional slides for a case, and choosing a provisional/borderline diagnosis to capture diverse opinions. All experienced pathologists participating in this study reported that the process of coming to consensus was challenging for borderline cases and may have represented compromise rather than consensus. They also reported the process changed their approaches to diagnosing complex melanocytic lesions. CONCLUSIONS: The most frequent reason for disagreement of experienced dermatopathologists was differences in diagnostic thresholds related to observer viewpoints. A range of approaches was needed to come to consensus, and this may guide pathology groups who do not currently hold consensus conferences.

A superior method for cell block preparation for fine-needle aspiration biopsies.

BACKGROUND: Cell block (CB) techniques for fine-needle aspiration biopsies (FNABs) vary. A direct comparison of CB techniques with statistical validation was performed to identify the best method. METHODS: Three CB techniques were compared: 1) FNAB rinsed in saline and clotted with plasma and thrombin (SPT); 2) FNAB rinsed in formalin and clotted with HistoGel (HG); and 3) FNAB rinsed in formalin, centrifuged, and the pellet captured in a collodion bag (ColB). FNAB was performed on 35 random surgical specimens for smears and each CB technique. A randomized blinded review of hematoxylin and eosin-stained CB slides was performed and each case was scored on a scale of 1 to 3 for cellularity, preservation, and architecture and the overall best CB was identified. Significance was determined by the Mann-Whitney U test for nonparametric ordinal data. RESULTS: The mean cellularity score was 1.71 for SPT (standard deviation [SD], 0.89), 1.68 for HG (SD, 0.67), and 3.0 for ColB (SD, 0). The mean preservation score was 1.31 for SPT (SD, 0.58), 1.54 for HG (SD, 0.70), and 2.91 for ColB (SD, 0.37). The mean architecture score was 1.45 for SPT (SD, 0.70), 1.43 for HG (SD, 0.60), and 2.71 for ColB (SD, 0.57). There was no statistical significance noted between SPT or HG when compared for each category. ColB was found to be superior to both SPT and HG when compared for each category (P<.05). The overall best CB was obtained with ColB in 33 of 35 cases (94%), with SPT proving superior in 1 of 35 cases (3%) and HG superior in 1 of 35 cases (3%). CONCLUSIONS: ColB appears to be a superior technique for CB, yielding greater cellularity, preservation, and architecture in the majority of cases. Cancer Cytopathol 2016;124:508-18. © 2016 American Cancer Society.

Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments.

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinized. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.

How Severely Is DNA Quantification Hampered by RNA Co-extraction?

The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes.

Laboratory errors leading to nonmelanoma skin cancer recurrence after Mohs micrographic surgery.

BACKGROUND: Compared with standard surgical excision, Mohs micrographic surgery (MMS) provides superior cure rates for nonmelanoma skin cancer (NMSC). Although cure rates of NMSC approach 99% with MMS, local recurrences occasionally occur. OBJECTIVE: The authors sought to identify histological features during frozen section examination that were associated with local recurrence of NMSC after MMS. MATERIALS AND METHODS: A retrospective chart review was performed of patients undergoing a second MMS procedure to treat locally recurrent NMSC over a 20-month period. Histological slides were reviewed to assess for possible causes of local recurrence. RESULTS: Of 3,169 NMSCs treated, 22 were locally recurrent. Possible causes of recurrence identified after MMS included dense inflammation in the final margin at sites affected by tumor in prior slides (27%), visible remaining tumor (23%), missing epidermal or dermal tissue (23%), and actinic keratosis (4%). One recurrence was possibly explained by incorrect mapping. No abnormality could be detected in 18% of cases. Possible limitations include the small sample size, retrospective design, and the possibility that some patients may have been lost to follow-up. CONCLUSION: Local recurrences after MMS are extremely rare. When recurrences do occur, they can be attributed to errors in histological interpretation or tumor mapping.

Effect of substrate choice and tissue type on tissue preparation for spectral histopathology by Raman microspectroscopy.

Raman spectroscopy is a non-destructive, non-invasive, rapid and economical technique which has the potential to be an excellent method for the diagnosis of cancer and understanding disease progression through retrospective studies of archived tissue samples. Historically, biobanks are generally comprised of formalin fixed paraffin preserved tissue and as a result these specimens are often used in spectroscopic research. Tissue in this state has to be dewaxed prior to Raman analysis to reduce paraffin contributions in the spectra. However, although the procedures are derived from histopathological clinical practice, the efficacy of the dewaxing procedures that are currently employed is questionable. Ineffective removal of paraffin results in corruption of the spectra and previous experiments have shown that the efficacy can depend on the dewaxing medium and processing time. The aim of this study was to investigate the influence of commonly used spectroscopic substrates (CaF2, Spectrosil quartz and low-E slides) and the influence of different histological tissue types (normal, cancerous and metastatic) on tissue preparation and to assess their use for spectral histopathology. Results show that CaF2 followed by Spectrosil contribute the least to the spectral background. However, both substrates retain paraffin after dewaxing. Low-E substrates, which exhibit the most intense spectral background, do not retain wax and resulting spectra are not affected by paraffin peaks. We also show a disparity in paraffin retention depending upon the histological identity of the tissue with abnormal tissue retaining more paraffin than normal.