Alcohol-based Fixative Research Articles

The impact of different fixatives on immunostaining of lung adenocarcinomas in pleural effusion cell blocks.

Cell blocks (CBs) are widely used for biomarker analyses such as immunostaining. Although immunohistochemistry on formalin-fixed paraffin-embedded tissues is standardized, there are multiple preparation methods and fixatives for cytology. Our objective was to investigate the effect of different common fixatives on the immunoreactivity of pleural effusion CBs with metastatic lung adenocarcinomas. This prospective study included 24 malignant pleural effusions from different patients with lung adenocarcinoma. From each case, four identical CBs were fixed in 10% neutral buffered formalin, PreservCyt, CytoLyt, and CytoRich Red (only 17 of the cases), respectively. Samples containing <100 malignant cells were excluded. All CBs were stained with thyroid transcription factor 1 (TTF-1; clones 8G7G3/1 and SPT24), napsin A, claudin 4, CEA, CK7, and epithelial cell adhesion molecule (EpCAM; clones BS14, Ber-Ep4, and MOC-31). The fraction and intensity of stained cells were evaluated. Of the investigated markers, a significant difference in staining proportion was seen for TTF-1 clone 8G7G3/1 and EpCAM clone MOC-31, especially with cases being negative in CytoLyt (33.3% and 83.3% positive, respectively) and PreservCyt (62.5% and 83.3%) whereas being positive in CytoRich Red (76.5% and 94.1%) and formalin (both 95.8%). A significantly weaker intensity of staining was seen for all alcohol-based fixatives compared to formalin for TTF-1 clone 8G7G3/1, napsin A, and EpCAM clone MOC-31, whereas EpCAM clone Ber-Ep4 was significantly weaker only in PreservCyt compared with formalin. Immunocytochemical expression and concordance with formalin-fixed CBs differ depending on the used fixative as well as the antibody and clone, warranting investigation of the reliability of each biomarker for non-formalin-fixed cytology.

The effect of aldehyde fixatives, alcohol-based fixatives, and fixatives combining denaturation and crosslinking on histomorphological, histochemical, and molecular genetic studies during short-term and long-term fixation: a comprehensive analysis

The effect of aldehyde fixatives, alcohol-based fixatives, and fixatives combining denaturation and crosslinking on histomorphological, histochemical, and molecular genetic studies during short-term and long-term fixation: a comprehensive analysis

Expanding panel size in spectral flow cytometry with StarBright Dyes excitable by the ultraviolet (355 nm), violet (405 nm), blue (488 nm) and yellow (561 nm) lasers.

Abstract The recently launched fluorescent dyes from Bio-Rad, StarBright Dyes, deliver tunable brightness and spectral properties, greater stability, improved lot-to-lot reproducibility, and spectral consistency. Specifically designed for multicolor flow cytometry with researchers needs in mind, StarBright Dyes address the common pain points in flow such as brightness, broad emission spectra, staining consistency, and ease-of-use, solving issues of signal resolution when constructing complex panels. These bright dyes have the additional benefit of a unique full spectral profile, making them ideal for inclusion in spectral flow cytometry, when creating multicolor panels. Here we present data of our newest additions to the StarBright range, the StarBright Blue and StarBright Yellow Dyes on a five-laser spectral cell analyzer. Both dye series are bright and allow expansion across the 488 and 561 lasers giving more dye choice for all panel sizes but are particularly useful when large panel design is required. In this study we show, when StarBright Blue and StarBright Yellow Dyes are combined with other members of the StarBright Dye range, polymer dyes and traditional fluorescent dyes, very large immunophenotyping panels can be constructed allowing identification of many peripheral blood subsets. In addition, novel dye combinations can be identified increasing the numbers of dyes and therefore markers identified from one sample. When combined with their ability to be fixed in both PFA based and alcohol-based fixatives, with minimal spectral changes, and to be pre-mixed for up to 33 days, the flexibility of StarBright Dyes means they are ideal for new and expanding existing panels regardless of the type of flow cytometer being used.

The hazardous effects of formalin and alcoholic fixative in mice: A public health perspective study

Formalin is used for different purposes due to its preservation capability. But continuous exposure to formalin may result various health related issues leading to cancer and death. A new alcohol-based fixative, EMA (ethanol, methanol and acetic acid = 3:1:1) could be a safer option in this regard. To compare the health hazards of formalin and EMA, a total 15 adult male mice were randomly distributed into three groups- exposure groups (formalin and EMA) and control group. The mice were subjected to natural inhalation exposure of the fixatives followed by behavioral depression test (forced swimming test), histopathology and serum biochemical tests. Our results showed that the hazardous effects of formalin were remarkably higher than that of EMA. Formalin exposed group showed severe depression (P < 0.001) in the forced swimming test compared to EMA and control groups. Histopathologically, diffuse lymphocytic infiltrations around the lung alveoli and bronchioles and severe inflammation with accumulation of reactive cells in the cerebral cortex were detected in the formalin exposed group, whereas little or no inflammation with fibrinous exudates in the bronchioles was reported in the EMA group and no inflammatory cells were detected in the cerebral tissues. The serum biochemical analysis of the inflammatory mediators (Interleukin-6 and C-reactive protein) revealed that both significantly (P < 0.001) increased in the formalin exposed group compared to EMA and control groups. These results confer that EMA could be a safer option to reduce health hazards of formalin in the workplace environment.

Intestinal giardiasis in children: Five years’ experience in a reference unit

BackgroundGiardiasis is highly prevalent in children and is often mildly symptomatic. First-line treatment is metronidazole, but treatment failure is not uncommon. We describe a paediatric series, to identify risk factors for treatment failure and to analyse the safety and effectiveness of other treatment strategies. MethodsRetrospective observational study, including children diagnosed with giardiasis from 2014 to 2019. Diagnosis was based on direct visualisation by microscopy after concentration using an alcohol-based fixative, antigen detection and/or DNA detection by polymerase chain reaction in stool. Treatment failure was considered when GI was detected 4 weeks after treatment. ResultsA total of 120 patients were included, 71.6% internationally adopted, median age 4.2 (2.3–7.3) years. Only 50% presented with symptoms, mainly diarrhoea (35%) and abdominal pain (14.1%); co-parasitism was frequent (45%). First-line treatment failure after a standard dose of metronidazole was 20%, lowering to 8.3% when a higher dose was administered (p < 0.001). Quinacrine was administered in 10 patients, with 100% effectiveness. Children <2 years were at higher risk of treatment failure (OR 3.49; 95% CI 1.06–11.53; p = 0.040). ConclusionsIn children with giardiasis, treatment failure is frequent, especially before 2 years of age. Quinacrine can be considered as a second-line treatment. After treatment, eradication should be confirmed.

Formalin fixation for optimal concordance of programmed death-ligand 1 immunostaining between cytologic and histologic specimens from patients with non-small cell lung cancer.

BackgroundImmunohistochemical staining of programmed death‐ligand 1 (PD‐L1) is used to determine which patients with non–small cell lung cancer (NSCLC) may benefit most from immunotherapy. Therapeutic management of many patients with NSCLC is based on cytology instead of histology. In this study, concordance of PD‐L1 immunostaining between cytology cell blocks and their histologic counterparts was analyzed. Furthermore, the effect of various fixatives and fixation times on PD‐L1 immunoreactivity was studied.MethodsPaired histologic and cytologic samples from 67 patients with NSCLC were collected by performing fine‐needle aspiration on pneumonectomy/lobectomy specimens. Formalin‐fixed, agar‐based or CytoLyt/PreservCyt‐fixed Cellient cell blocks were prepared. Sections from cell blocks and tissue blocks were stained with SP263 (standardized assay) and 22C3 (laboratory‐developed test) antibodies. PD‐L1 scores were compared between histology and cytology. In addition, immunostaining was compared between PD‐L1–expressing human cell lines fixed in various fixatives at increasing increments in fixation duration.ResultsAgar cell blocks and tissue blocks showed substantial agreement (κ = 0.70 and κ = 0.67, respectively), whereas fair‐to‐moderate agreement was found between Cellient cell blocks and histology (κ = 0.28 and κ = 0.49, respectively). Cell lines fixed in various alcohol‐based fixatives showed less PD‐L1 immunoreactivity compared with those fixed in formalin. In contrast to SP263, additional formalin fixation after alcohol fixation resulted in preserved staining intensity using the 22C3 laboratory‐developed test and the 22C3 pharmDx assay.ConclusionsPerforming PD‐L1 staining on cytologic specimens fixed in alcohol‐based fixatives could result in false‐negative immunostaining results, whereas fixation in formalin leads to higher and more histology‐concordant PD‐L1 immunostaining. The deleterious effect of alcohol fixation could be reversed to some degree by postfixation in formalin.

Optimization of Sample Preparation Using Glass Slides for Spectral Pathology.

The clinical translation of Fourier transform infrared (FT-IR) microspectroscopy in pathology will require bringing this technique as close as possible to standard practice in pathology departments. An important step is sample preparation for both FT-IR microspectroscopy and pathology. This should entail minimal disruption of standard clinical practice while achieving good quality FT-IR spectral data. In fact, the recently described possibility of obtaining FT-IR spectra of cells placed on glass substrates brings FT-IR microspectroscopy closer to a clinical application. We have now furthered this work in order to identify two different types of lung cancer cells placed on glass coverslips. Two types of sample preparation which are widely used in pathology, cytospin and smear, have been used. Samples were fixed with either methanol, used in pathology, or formalin (4% paraformaldehyde) used widely in spectroscopy. Fixation with methanol (alcohol-based fixative) removed lipids from cells causing a decrease in intensity of the peaks at 2850 cm−1 and 2920 cm−1. Nevertheless, we show for the first time that using either type of sample preparation and fixation on thin glass coverslips allowed to differentiate between two different types of lung cancer cells using either the lipid region or the fingerprint region ranging from 1800 cm−1 to 1350 cm−1. We believe that formalin-fixed cytospin samples would be preferred to study cells on thin coverslips using FT-IR microspectroscopy. This work presents a clear indication for future advances in clinical assessment of samples within pathology units to gain a deeper understanding of cells/tissues under investigation.

A Novel HPLC-Based Method to Investigate on RNA after Fixation.

RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

Alcohol-Based Fixatives can Better Preserve Tissue Morphology than Formalin

Alcohol-Based Fixatives can Better Preserve Tissue Morphology than Formalin

Next-generation sequencing analysis of endometrial screening liquid-based cytology specimens: a comparative study to tissue specimens

BackgroundLiquid-based cytology (LBC) is now a widely used method for cytologic screening and cancer diagnosis. Since the cells are fixed with alcohol-based fixatives, and the specimens are stored in a liquid condition, LBC specimens are suitable for genetic analyses.MethodsHere, we established a small cancer gene panel, including 60 genes and 17 microsatellite markers for next-generation sequencing, and applied to residual LBC specimens obtained by endometrial cancer screening to compare with corresponding formalin-fixed paraffin-embedded (FFPE) tissues.ResultsA total of 49 FFPE and LBC specimens (n = 24) were analyzed, revealing characteristic mutations for endometrial cancer, including PTEN, CTNNB1, PIK3CA, and PIK3R1 mutations. Eight cases had higher scores for both tumor mutation burden (TMB) and microsatellite instability (MSI), which agree with defective mismatch repair (MMR) protein expression. Paired endometrial LBC, and biopsied and/or resected FFPE tissues from 7 cases, presented almost identical mutations, TMB, and MSI profiles in all cases.ConclusionThese findings demonstrate that our ad hoc cancer gene panel enabled the detection of therapeutically actionable gene mutations in endometrial LBC and FFPE specimens. Endometrial cancer LBC specimens offer an alternative and affordable source of molecular testing materials.

Handling of Thyroid FNA Samples During the COVID-19 Pandemic

Handling of Thyroid FNA Samples During the COVID-19 Pandemic

International perspectives: Impact of the COVID-19 pandemic on cytology.

Just about every country in the world is being challenged by a potentially fatal infectious disease known as Coronavirus disease (COVID-19). The etiologic agent is a novel zoonotic coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) that first was detected during an outbreak in Wuhan, China, at the end of 2019.1 Governments around the globe have since responded with lockdowns to facilitate social distancing and declared national emergencies. Given the pathogenicity of this virus, many patients are becoming critically ill and require hospitalization, which is stressing many health care systems. Cytology laboratories also are being impacted by this pandemic in several ways.2 Specimens, especially respiratory samples, may be submitted for cytologic evaluation. Not only are these contagious samples to be handled with strict biosafety measures (eg, following biosafety guidelines from the Centers for Disease Control and Prevention),3 but their cytologic findings may be unique and something we likely have not encountered before.4 Brave cytology staff who are being called to the front line to provide rapid on-site evaluation need to be adequately armed with personal protective equipment (eg, N95 respirator masks). Now is the time to leverage digital imaging technology in order for cytopathologists to perform telecytology remotely, even from home if required. To allow for social distancing, cytotechnologists who are screening cases should be allowed to spread out as much possible. However, there still is much confusion and anxiety among the cytology community, and many questions remain unanswered at this time. Can cytotechnologists screen cases at home? Should air-dried smears be avoided? Are the alcohol-based fixatives typically used in cytology appropriate? Are cytospin preparations that can aerosolize material prohibited? Are cell blocks still required? Should all glass slides be wiped down with disinfectants? Should elective fine-needle aspirations performed by pathologists be cancelled? Must cytotechnologist training move online? We have traveled down this road before with prior outbreaks (eg, influenza A virus subtype H1N1, SARS, Middle East respiratory syndrome [MERS], and the Ebola virus).5 However, there remains much to be learned with COVID-19. Therefore, it is critical for us to gain knowledge from cytology colleagues who have had early experience during the current crisis. Accordingly, we are pleased to share with you timely publications regarding COVID-19 from Italy,6 Taiwan,7 and the United States8 in this issue of Cancer Cytopathology. No specific funding was disclosed. The authors made no disclosures.

Ultrafast Toluidine Blue Staining for Rapid On-Site Evaluation of Cytological Smears

Rapid on-site evaluation (ROSE) is one of cytopathology’s “unique selling propositions.” The quality, speed, and ease of handling of the staining used is a critical factor for the efficacy of the ROSE procedure. Here, we describe a modification of rapid toluidine blue staining that can be performed within 25 s, provides excellent nuclear morphology, and is compatible with subsequent Papanicolaou staining of the slides. Furthermore, exposure to hazardous chemicals is minimized, as no organic solvents other than the alcohol-based fixative and glycerin for temporary mounting and coverslipping are required. We have used this protocol successfully in our ROSE practice and have not observed any discrepancies between toluidine blue- and permanent Papanicolaou-stained slides.

Programmed death ligand 1 expression in EBUS aspirates of non-small cell lung cancer: Is interpretation affected by type of fixation?

Much of the reluctance about using cytology specimens rather than histology specimens to assess programmed death ligand 1 (PD-L1) expression for guiding the use of immune modulating drugs in the management of non-small cell lung cancer (NSCLC) is based on the belief that the alcohol-based fixatives favored by cytopathologists might reduce the antigenicity of PD-L1 and lead to artifactually low expression levels and false-negative reporting. Therefore, this study was performed to determine whether there is any difference in PD-L1 expression between endobronchial ultrasound (EBUS)-guided aspirates of NSCLC fixed in alcohol-based fixatives and those fixed in neutral buffered formalin (NBF), the standard laboratory fixative for histology specimens. The expression of PD-L1 was compared in 50 paired EBUS aspirates of NSCLC taken from the same lymph node during the same procedure. One aspirate of each pair was fixed in an alcohol-based fixative, and the other was fixed in NBF. In none of the 50 pairs was there any significant difference, qualitative or quantitative, in the strength, pattern, or extent of PD-L1 expression. In the great majority, the expression was identical, regardless of fixation. There is no evidence from this study showing that the use of alcohol-based fixatives has any effect on the expression of PD-L1 or its interpretation. Notwithstanding the general challenges in accurately assessing such expression in cytology specimens, pathologists should feel able to interpret them with confidence, and clinicians should feel able to rely on the results.

Abstract DP-002: COMPARISON OF POTENTIAL OVARIAN CANCER BIOMARKERS BY MASS SPECTROMETRY-BASED PROTEOMIC ANALYSIS OF RESIDUAL PAP TEST FLUID, CERVICAL SWABS, AND TUMOR TISSUE FROM AN OVARIAN CANCER PATIENT

Abstract Early detection is the key to increased survival for women with ovarian cancer, yet a screening tool has yet to be developed that is adequately sensitive and specific enough for use in the general population. In contrast, screening for cervical cancer by Pap tests has been routinely performed for over 50 years. In the liquid-based Pap test, cells are collected from the cervix and placed into an alcohol-based fixative and then examined for abnormal cells. Since ovarian cancer cells have been observed in Pap tests, we reasoned that ovarian cancer peptide biomarkers may also be present. Our central hypothesis is that proteins shed by ovarian cancer cells can be detected during routine Pap tests by mass spectrometry (MS)-based proteomics. In particular, when collected at the time of cervical cancer Pap test screening, the alcohol-based Pap test fixative and cervical swabs are ideal for biomarker discovery since they are derived from a site near the ovarian cancer (i.e. proteins may be secreted or shed from the tumor and flow through the fallopian tube into the uterus and out the cervical opening). Recently, the fimbria of the fallopian tube have been suggested to be the true precursor to ovarian cancer, strengthening our hypothesis that ovarian cancer proteins will be found in the lower genital tract, perhaps even at early stages. To demonstrate the feasibility of using Pap tests as a biospecimen for proteomics, we previously examined the proteins present in residual Pap test fixative samples from women with normal cervical cytology by MS and described 152 proteins in the “Normal Pap Proteome.” The objective of this study was to identify and compare the proteins from three different sources from the same ovarian cancer patient: (i) the residual Pap test fixative, (ii) a Merocel swab of the cervix, and (iii) the primary ovarian cancer tumor tissue. Proteins were concentrated from the cell-free supernatant of the Pap test fixative or eluted from the swab, and then trypsin digested using the filter-aided sample preparation method. A total protein extract from the patient's tumor tissue was digested by standard in-solution trypsin digestion. The samples were run on 2D-liquid chromatography MS/MS, followed by bioinformatics integration. We identified over 5000 proteins total in the three samples. More than 2000 proteins were expressed in all three ovarian cancer samples, including several known ovarian cancer biomarkers such as CA125. By Scaffold analysis of the Gene Ontology nomenclature of the proteins, we classified the proteins by both cellular localization and biological processes. Additional matched samples from patients will be used to build a library of proteins and peptides that are specific to ovarian cancer for use in the development of targeted MS assays. We conclude that quantification of proteins from Pap test fixatives and cervical swabs will prove to be a rich source of biomarkers for ovarian cancer detection. Citation Format: Kristin L.M. Boylan, Anna C. Rogers, Melissa A. Geller, Peter A. Argenta, Timothy J. Griffin, and Amy P.N. Skubitz. COMPARISON OF POTENTIAL OVARIAN CANCER BIOMARKERS BY MASS SPECTROMETRY-BASED PROTEOMIC ANALYSIS OF RESIDUAL PAP TEST FLUID, CERVICAL SWABS, AND TUMOR TISSUE FROM AN OVARIAN CANCER PATIENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-002.

Effects of short and long-term alcohol-based fixation on Sprague-Dawley rat tissue morphology, protein and nucleic acid preservation

Safety concerns on the toxic and carcinogenic effects of formalin exposure have drawn increasing attention to the search for alternative low risk fixatives for processing tissue specimens in laboratories worldwide. Alcohol-based fixatives are considered some of the most promising alternatives. We evaluated the performance of alcohol-fixed paraffin-embedded (AFPE) samples from Sprague-Dawley (SD) rats analyzing tissue morphology, protein and nucleic acid preservation after short and extremely long fixation times (up to 7 years), using formalin-fixed paraffin-embedded (FFPE) samples as a comparator fixative. Following short and long-term alcohol fixation, tissue morphology and cellular details in tissues, evaluated by scoring stained sections (Hematoxylin-Eosin and Mallory’s trichrome), were optimally preserved if compared to formalin fixation. Immunoreactivity of proteins (Ki67, CD3, PAX5, CD68), evaluated by immunohistochemistry, showed satisfactory results when the fixation period did not exceed 1 year. Finally, we confirm the superiority of alcohol fixation compared to formalin, in terms of quantity of nucleic acid extracted from paraffin blocks, even after an extremely long time of alcohol fixation.Our results confirm that alcohol fixation is a suitable and safe alternative to formalin for pathological evaluations. There is a need for standardization of formalin-free methods and harmonization of diagnosis in pathology department worldwide.

Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis.

In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 103 or 2 × 104 cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 103 cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genome-based cancer medicine.

Immunocytochemistry for predictive biomarker testing in lung cancer cytology.

With an escalating number of predictive biomarkers emerging in non-small cell lung carcinoma (NSCLC), immunohistochemistry (IHC) is being used as a rapid and cost-effective tool for the screening and detection of many of these markers. In particular, robust IHC assays performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue are widely used as surrogate markers for ALK and ROS1 rearrangements and for detecting programmed death ligand 1 (PD-L1) expression in patients with advanced NSCLC; in addition, they have become essential for treatment decisions. Cytology samples represent the only source of tumor in a significant proportion of patients with inoperable NSCLC, and there is increasing demand for predictive biomarker testing on them. However, the wide variation in the types of cytology samples and their preparatory methods, the use of alcohol-based fixatives that interfere with immunochemistry results, the difficulty in procurement of cytology-specific controls, and the uncertainty regarding test validity have resulted in underutilization of cytology material for predictive immunocytochemistry (ICC), and most cytopathologists limit such testing to FFPE cell blocks (CBs). The purpose of this review is to: 1) analyze various preanalytical, analytical, and postanalytical factors influencing ICC results; 2) discuss measures for validation of ICC protocols; and 3) summarize published data on predictive ICC for ALK, ROS1, EGFR gene alterations and PD-L1 expression on lung cancer cytology. Based on our experience and from a review of the literature, we conclude that cytology specimens are in principal suitable for predictive ICC, but proper optimization and rigorous quality control for high-quality staining are essential, particularly for non-CB preparations.

Beneficial Effect of Heat-induced Antigen Retrieval in Immunocytochemical Detection of Intracellular Antigens in Alcohol-fixed Cell Samples.

Immunohistochemistry and immunocytochemistry (ICC) play an irreplaceable role in research and diagnostics. It is well known that antigen retrieval (AR) can, as a technique, have beneficial outcomes on immunohistochemistry results when using formalin-fixed, paraffin-embedded tissue samples. The main purpose of AR is to break protein crosslinks which are formed during formalin fixation. Although AR was originally designed for formalin-fixed, paraffin-embedded samples, the usefulness of AR in ICC has been described in previous studies. Cytologic samples are often fixed in alcohol-based fixatives which does not lead to the formation of crosslinks. Therefore, alcohol-fixed samples can be successfully immunostained without AR. We investigated the effect of heat-induced antigen retrieval (HIAR) on alcohol-fixed HEK293 cell line samples and patient cytologic samples from thyroid gland obtained by fine needle aspiration technique. We compared indirect 2-step ICC staining results performed according to the protocol with or without HIAR in citrate buffer pH 6 for several antibodies. Utilizing HIAR against intracellular antigens has beneficial effects. Therefore, more diluted antibodies can be used for satisfactory results. However, surface antigens were probably damaged by HIAR treatment. We demonstrated evident changes in cell surface topography after HIAR treatment by atomic force microscopy. Staining specificity of patient samples improves and background staining is reduced, allowing higher dilutions of primary antibody. Improving staining specificity is necessary for accurate diagnostics. Although we have shown the beneficial effect of HIAR for immunostaining intracellular antigens, proper staining protocol should be tested on appropriate controls for individual antibodies.

Abstract B34: Tandem Mass Tag 10-plex isobaric labeling of Pap test proteins: A novel method for the identification of ovarian cancer protein biomarkers by mass spectrometry.

Abstract Early detection is the key to increased survival for women with ovarian cancer, yet a screening tool has yet to be developed that is adequately sensitive and specific enough for use in the general population. A need also exists for a reliable diagnostic method for women who present with an abdominal mass. In contrast, screening for cervical cancer by Pap tests has been routinely performed for over 50 years. In the liquid-based Pap test, cells are collected from the cervical os and placed into an alcohol-based fixative and then examined for abnormal cells. Since ovarian cancer cells have been observed in Pap tests, we reasoned that ovarian cancer peptide biomarkers may also be present. Our central hypothesis is that proteins shed by ovarian cancer cells can be detected in the Pap test fixative by Mass Spectrometry (MS)-based proteomics. The Pap test fixative is ideal for biomarker discovery since it is derived from a site near the ovarian cancer (i.e. proteins may be secreted or shed from the tumor and flow through the fallopian tube into the uterus and out the cervical opening). Recently, the fimbria of the fallopian tube have been suggested to be the true precursor to ovarian cancer, strengthening our hypothesis that ovarian cancer proteins will be found in the lower genital tract, perhaps even at early stages. To demonstrate the feasibility of this approach, we previously examined the proteins present in residual Pap test fixative samples from women with normal cervical cytology by MS, and described 152 proteins in the “Normal Pap Proteome.” (Boylan et al., Clinical Proteomics, 2014). The objective of this study is to discover ovarian cancer biomarkers in Pap test fixative samples by MS-based proteomic techniques. We used isobaric tags to quantify the proteins in liquid Pap test samples from women with ovarian cancer compared to women with benign gynecological disease and healthy women. Proteins were concentrated from “Mock” Pap tests, and samples were trypsin digested utilizing the STrap technique to increase the likelihood that proteins would be recovered. We then performed Tandem Mass Tag™ (TMT) 10-plex labeling of Pap test fixative samples from post-menopausal women: 3 with ovarian cancer, 3 with benign gynecological conditions, 3 healthy, and a Reference Control consisting of pooled samples. The 10 labeled samples were combined and run on 2D LC-MS/MS, followed by bioinformatics integration to discover proteins in Pap test fixatives that are unique and/or differentially expressed in the samples. We identified &amp;gt;3800 proteins total, and 22 proteins that were expressed at least 2-fold higher in all three ovarian cancer samples compared to the controls. We conclude that quantification of proteins from Pap test samples will prove a rich source of biomarkers for ovarian cancer detection. Citation Format: Amy P.N. Skubitz, Somi Afiuni, Kristin L.M. Boylan, Melissa Geller, Peter Argenta, Samantha Hoffman, Timothy Griffin. Tandem Mass Tag 10-plex isobaric labeling of Pap test proteins: A novel method for the identification of ovarian cancer protein biomarkers by mass spectrometry. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B34.