Role of Ancillary Techniques in Reporting Serous Fluid Cytology - "Redefining Categories, Refining Diagnosis".

Background: The "International System of Reporting Serous Fluid Cytology (TIS)" together with cytomorphology promotes the use of ancillary techniques to resolve difficulties in reporting serous fluid cytology. Objective: To classify serous effusion fluid samples received at our department in line with "TIS", indicating the risk of malignancy (ROM), and directing appropriate usage of ancillary testing. Materials and Methods: Prospective study carried out from October 2021 to September 2022. The study included all pleural, ascitic, and pericardial fluid samples, reported according to 'TIS'. Flow cytometry and immunocytochemistry were ancillary methods utilized to assist in reporting. Cases with available history and convincing correlations didn't require further assessment. Results: A total of 1200 serous effusion samples were evaluated including 604 pleural, 591 ascitic, and 5 pericardial fluid samples. After categorization, there were 23 samples in non-diagnostic (ND, 1.9%), 575 in negative for malignancy (NFM, 47.91%), 44 in atypia of undetermined significance (AUS, 3.66%), 64 in suspicious for malignancy (SFM, 5.33%), and 494 in malignant category (MAL, 41.16%). Ancillary studies were beneficial in the recategorization of 26% (11/44) AUS cases, 29.6% (19/64) SFM cases, and it helped refine tumor characteristics in 35.42% (175/494) cases categorized as malignant. Final ROM calculated for each category: ND 25%, NFM 18.6%, AUS 66.6%, SFM 88%, and MAL 100%. Conclusion: Serous fluid is an easily obtainable sample that can provide opportunities for ancillary testing with clinical implications. In AUS and suspicious category although, diagnostic yield is increased however, a larger number of cases are required to obtain definite results.

In Situ Hybridization (ISH) Combined with Immunocytochemistry (ICC) Co-detection of Phosphorylated EGFR in A431 Cultured Cells.

Antibodies have been commonly used to study protein phosphorylation since the first phospho-specific antibody was described in 1981. Antibodies can be developed so that they specifically recognize phosphorylated areas of particular proteins. In situ hybridization (ISH) is the technique where specific RNA or DNA molecules can be detected in a single cell without the need for antibodies. Using ACD's integrated Co-Detection Workflow (ICW), we have developed a protocol to use phospho-specific antibodies in combination with ISH to show co-localization of EGFR mRNA and EGFR proteins phosphorylated at different sites in tumor cells. Our protocol has been used for multiplexing Y1086 phosphorylated EGFR, Y1068 phosphorylated EGFR, and EGFR RNA in A431 human epidermoid carcinoma cells.

A novel combined fluorescent probe staining method for circulating tumor cell identification.

Background: To develop a novel highly accurate circulating tumor cell (CTC) identification method and to validate its application in cancer diagnostics and/or prognostics. Methods: We verified and validated the combined fluorescent probe staining protocol (combination of three fluorescent probes: Dil, Hoechst 33342, and PY) through CTC and non-CTC (white blood cell) morphological comparison of five tumor cell lines (THP-1, HEC, HEPG2, Eca-109, HeLa) in vitro and 32 patient tumor samples from the Shandong Cancer Hospital and Institute. Wright's Giemsa staining and cluster differentiation 45 (CD45) immunocytochemistry (ICC) staining were used as reference control methods. The association between the developed method and clinicopathology was also investigated. Results: We successfully developed and optimized the protocol, and validated the use of combined fluorescent probe staining for the identification of CTCs in the peripheral blood (PB) of tumor cell lines and tumor patients. Comparable CTC and non-CTC morphologies were observed for combined fluorescent probe staining and Giemsa staining methods in vitro. However, in vivo comparison between the three staining methods revealed that the identified CTCs differed in cell diameter and nucleo-cytoplasmic ratio. In addition, a higher CTC detection rate of 14/32, lower standard deviation (SD), and higher area under the receiver operating characteristic (ROC) curve (AUC) value of 0.844 were noted for combined fluorescence staining. Clinicopathological analysis revealed that CTCs were correlated with platelet levels (P=0.031), but not with age, gender, drinking history, or granule ratio. Conclusions: We developed a combined fluorescent probe staining method with higher CTC identification accuracy than Wright's Giemsa staining, and propose this technique as a novel clinical diagnostic/prognostic tool.

Thyroid lymphoproliferative lesions in Asia.

Primary thyroid lymphomas (PTLs) are rare and most commonly present as rapidly enlarging thyroid mass causing obstructive symptoms. Due to worldwide differences in clinical practices related to thyroid malignancy, this review was conducted to compare the clinicopathological and diagnostic modalities related to PTL and their similarities and differences between the Asian and Western countries. Using the search engine PubMed, published data on thyroid lymphomas was collected and reviewed. A total of 18 Asian and 22 Western studies were included. Most of PTLs were B-cell Non-Hodgkin lymphomas (NHL). While mucosa-associated lymphoid tissue (MALT) lymphoma was the commonest (41.1%) among Asians, diffuse large B cell lymphoma (DLBCL) (71.9%) predominated in the Western population. Some rare subtypes of PTL were also identified. Majority of all patients in Asian as well as Western studies presented with early stage (stage I/II) disease. Interestingly, when compared with Asian patients, a larger proportion of patients from the West presented with higher stage (stage III/IV) disease (12.2% vs. 3%). Ultrasonography (USG) and fine needle aspiration cytology (FNAC) in addition to histological examination usually by core needle biopsy and in some by open procedures were used for the diagnosis of PTL in both the cohorts. The various ancillary techniques used were immunocytochemistry (ICC), flowcytometry (FC), immunohistochemistry (IHC), and molecular testing. The use of ancillary techniques for PTL diagnosis was more common in the West compared to Asia and markedly increased the sensitivity of cytology to diagnose PTL. Treatment and prognosis largely depend upon the subtype of PTL and stage at presentation. To conclude, from the available published literature, there is an apparent difference between Asian and Western cohorts in the histological type and stage of presentation of PTL, but the results may be affected by publication and selection bias. Also, advanced ancillary techniques are more commonly adopted in the West.

Immunocytochemistry of cytology specimens for predictive biomarkers in lung cancer.

With a growing number of predictive biomarkers that have emerged in non-small cell lung carcinoma (NSCLC), there has been a paradigm shift in the management of these patients. Of the various predictive biomarker testing methods, immunohistochemistry (IHC) is the most widely available, cost-effective, and commonly used methods. However, most predictive IHC assays are validated primarily on formalin-fixed paraffin-embedded (FFPE) histologic tissue samples and translating these assays to cytologic specimens requires additional and rigorous validation. This is part due to the lack of standardized processing protocols in cytology resulting in a variety of preanalytic variables that can impact the antigenicity of antibodies used for predictive biomarker testing. The review article discusses the various preanalytical and analytical factors that impact immunocytochemistry (ICC) in cytologic specimens and summarizes the current published literature on ALK, ROS1, PD-L1, and other predictive biomarker ICC in cytology.

Investigation of nervous necrosis virus (NNV) replication in vitro using RNA in situ hybridization.

Nervous necrosis virus (NNV) belongs to the genus Betanodavirus of family Nodaviridae. Its genome consists of two RNA segments, RNA1 and RNA2. Several studies have investigated NNV detection by in situ hybridization (ISH), but these have typically focused on the detection of the RNA2 gene. In this study, we localized both RNA1 and RNA2 NNV segments in viral-infected cells by ISH, using labeled RNA probes (RNA-ISH). Also, immunocytochemistry (ICC) assay was carried out for localization of viral particle by targeting the coat protein. Further, viral quantification assays were performed by quantitative RT-PCR and viral infectivity (TCID50) in SSN-1 cells. Viral segments were observed by RNA-ISH at 6 h post infection (hpi), while NNV particles were detected at 24 hpi by ICC. Use of double labeling RNA-ISH revealed the co-expression of the two viral segments in the same area of the cells, while RNA1 was also detected separately. Comparison of the level of viral genomic segments and viral infectivity revealed significantly more copies of RNA1 at each time points than copies of RNA2 and greater NNV titers. The results suggest that RNA1 might be expressed in the early stages of replication, with RNA2 expressed later. The virions then assemble through initially expressed viral genomic segments. Even though infectious particles displayed very efficient packaging, the RNA1 segment was still over-produced.

Development of an in-situ hybridization assay using riboprobes for detection of viral haemorrhagic septicemia virus (VHSV) mRNAs in a cell culture model.

An in situ hybridization (RNA-ISH) assay has been developed and optimized to detect viral haemorrhagic septicemia virus (VHSV), an OIE listed piscine rhabdovirus, in infected fish cells using fathead minnow (FHM) as a model cell line. Two antisense riboprobes (RNA probes) targeting viral transcripts from a fragment of nucleoprotein (N) and glycoprotein (G) genes were generated by reverse transcription polymerase chain reaction (RT-PCR) using VHSV specific primers followed by a transcription reaction in the presence of digoxigenin dUTP. The synthesized RNA probes were able to detect viral mRNAs in formalin fixed VHSV infected FHM cells at different time points post inoculation (pi). To correlate the signal intensity, a time dependent quantitation of the viral mRNA transcript and infectivity titer was done by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and 50% tissue culture infectivity dose (TCID50), respectively, from the infected cells and culture supernatants. Further, we compared the diagnostic sensitivity of ISH assay with immunocytochemistry (ICC). Both the riboprobes used in the ISH assay detected VHSV as early as 6 hpi in the FHM cells inoculated with a multiplicity of infection (moi) of 2. Also, the signal detection in ISH was at an early stage in comparison to ICC, wherein, signal was first detected at 12 hpi. Our results clearly highlight that current ISH assay can be of value as a diagnostic tool to localize and detect VHSV in conjunction with conventional virus isolation in cell culture.

The value of cell block based on fine needle aspiration for lung cancer diagnosis.

BACKGROUND: Computed tomography (CT)-guided percutaneous lung fine needle aspiration (FNA) is a convenient method to obtain samples from pulmonary lesions. FNA has a lower rate of complications than the use of a core needle biopsy, but is more difficult for the diagnosis of cytological samples. We use cell block (CB) and immunocytochemistry (ICC) to improve the accuracy of cytological diagnoses based on CT-guided percutaneous lung FNA. METHODS: We collected 526 cytological samples obtained using CT-guided percutaneous lung FNA at Shanghai Pulmonary Hospital from May 2015 to October 2015. CBs were created from these samples, and ICC was performed to help the further histological classification and confirmation of tumor as primary or metastatic. An automated Ventana ALK with clone D5F3 was used to identify ALK fusion protein. RESULTS: After assessment of the CBs, 32 (6.08%) diagnoses of suspected malignancy were reduced to 10 (1.90%) such diagnoses (P<0.05), and 161 (30.61%) cases of non-small-cell lung carcinoma (NSCLC) were reduced to 33 (6.27%) cases (P<0.05) after their division into specific subtypes. We also diagnosed eight (1.52%, P<0.05) cases of metastatic carcinoma of the lung that were difficult to diagnose by cytological smear alone. Six (3.73%) of 161 NSCLC cases exhibited ALK rearrangement. CONCLUSIONS: CB and ICC are useful for accurate cytological diagnosis using CT-guided percutaneous lung FNA. These approaches are valuable for providing individualized treatment and prognostic evaluations with minor complications.

Determination of PD-L1 expression in effusions from mesothelioma by immuno-cytochemical staining.

BACKGROUND: Malignant mesothelioma (MM) is an aggressive, fatal tumor. Current therapeutic options only marginally improve survival. Programmed cell death ligand 1 (PD-L1) is a dominant mediator of immunosuppression, binding to programmed cell death 1 (PD-1). PD-L1 is up-regulated in cancer cells, and the PD-1/PD-L1 pathway plays a critical role in tumor immune evasion, thus providing a target for antitumor therapy. Further, a correlation between PD-L1 expression and prognosis has been reported. Studies performed on histological material have revealed expression of PD-L1 in MM, but no study has been performed on MM effusions thus far. METHODS: PD-L1 expression was determined by a commercially available antibody (clone 28-8) in 74 formalin-fixed, paraffin-embedded cell blocks from body effusions obtained at diagnosis from patients with MM. The presence of MM cells was confirmed with CK5/6, calretinin, and EMA and the admixture of macrophages was assessed with CD68. Only cases containing more than 100 tumor cells were assessed. Membranous staining in tumor cells was considered positive. Survival time was calculated from the appearance of the first malignant effusion until death. RESULTS: Reactivity was observed in 23 of 61 (38%) of cases and was classified as ≥1%-5% (n = 9 cases), >5%-10% (n = 4 cases), >10%-50% (n = 4 cases), and >50% (n = 6 cases) positive cells. Survival times did not differ significantly between patients with PD-L1-positive and PD-L1-negative tumors. CONCLUSION: MM effusions are suitable for immune-cytochemical assessment of PD-L1 expression in malignant cells and the results are similar to those reported for histological specimens. Cancer Cytopathol 2017;125:908-17. © 2017 American Cancer Society.

Automated Microfluidic Filtration and Immunocytochemistry Detection System for Capture and Enumeration of Circulating Tumor Cells and Other Rare Cell Populations in Blood.

Isolation by size using a filter membrane offers an antigen-independent method for capturing rare cells present in blood of cancer patients. Multiple cell types, including circulating tumor cells (CTCs), captured on the filter membrane can be simultaneously identified via immunocytochemistry (ICC) analysis of specific cellular biomarkers. Here, we describe an automated microfluidic filtration method combined with a liquid handling system for sequential ICC assays to detect and enumerate non-hematologic rare cells in blood.

Rapid immunocytochemistry based on alternating current electric field using squash smear preparation of central nervous system tumors.

The role of intraoperative pathological diagnosis for central nervous system (CNS) tumors is crucial for neurosurgery when determining the surgical procedure. Especially, treatment of carmustine (BCNU) wafers requires a conclusive diagnosis of high-grade glioma proven by intraoperative diagnosis. Recently, we demonstrated the usefulness of rapid immunohistochemistry (R-IHC) that facilitates antigen-antibody reaction under alternative current (AC) electric field in the intraoperative diagnosis of CNS tumors; however, a higher proportion of water and lipid in the brain parenchyma sometimes leads to freezing artifacts, resulting in poor quality of frozen sections. On the other hand, squash smear preparation of CNS tumors for cytology does not affect the frozen artifacts, and the importance of smear preparation is now being re-recognized as being better than that of the tissue sections. In this study, we established the rapid immunocytochemistry (R-ICC) protocol for squash smears of CNS tumors using AC electric field that takes only 22 min, and demonstrated its usefulness for semi-quantitative Ki-67/MIB-1 labeling index and CD 20 by R-ICC for intraoperative diagnosis. R-ICC by AC electric field may become a substantial tool for compensating R-IHC and will be applied for broad antibodies in the future.