Distribution of Inflammatory Infiltrate in Feline Mammary Lesions: Relationship With Clinicopathological Features.

Inflammation is a frequent finding in feline mammary neoplasms. Recent research suggests that the presence and location of tumour-associated immune cells might play a significant role in the clinical outcome of feline mammary carcinomas. The present study aimed to characterise the overall inflammatory infiltrates in healthy, hyperplastic/dysplastic, benign and malignant lesions of the feline mammary gland, and to evaluate its association with clinicopathological features. Perilesional and intralesional inflammatory foci were evaluated in 307 lesions from 185 queens, and categorised according to its distribution and intensity. The presence, location and density of tertiary lymphoid structures were also assessed. A control group included 24 queens without mammary changes. The presence of intralesional and perilesional inflammatory infiltrate was observed in a majority of the lesions (80.8% and 90.2%, respectively), but differed according to the type of mammary lesion, being more remarkable in malignant neoplasms. Only scarce individual cells were observed in 28.1% of the normal mammary glands. Data analysis revealed statistically significant associations (p < 0.05) between the presence of a more prominent intralesional and perilesional inflammatory reaction and several clinicopathological features associated with worse prognosis, including clinical stage, tumour size, mitotic count, lymphovascular invasion and lymph node metastasis. Furthermore, tertiary lymphoid structures were significantly more frequent in tumours with an infiltrative growth and lymph node metastasis. According to our results, the inflammatory reaction present in different types of feline mammary lesions is associated with the development of more aggressive tumours.

Detection of Lymphoid Markers (CD3 and PAX5) for Immunophenotyping in Dogs and Cats: Comparison of Stained Cytology Slides and Matched Cell Blocks.

Immunolabeling on Romanowsky-stained cytology (RSC) slides can be used, although there is limited evidence of its suitability for phenotyping canine and feline lymphomas. A comparison with matched cell blocks (CB) is missing. Immunolabeling on RSC and CB was compared for lymphoid markers (CD3 and PAX5) in 53 lymphomas and 4 chylous effusions from dogs and cats. The influence of pre-analytical variables (species, time of archive, type of specimens and coverslipping) and the interobserver agreement among the 2 observers was assessed. Fewer CD3+ lymphocytes were identified in RSC, while the PAX5 positivity by RSC and CB had a substantial agreement. Immunodetection of CD3 and the diagnosis of a T-cell population on RSC was more difficult. Lower intensity and higher background were noted in RSC. Immunophenotyping was inconclusive in 54% RSC and 19% CB. The interobserver reproducibility of immunophenotyping on CB was substantial, being higher than in RSC. The immunolabeling performance on the RSC of effusion and feline samples was unsatisfactory. The detection of lymphoid markers, especially membranous antigens in retrospective RSC, is affected by the pre-analytical variables: species, time of the archive, and type of specimens. CB are a more consistent type of sample for immunophenotyping purposes.

Doing more with less: multiple uses of a single slide in veterinary cytology. A practical approach.

Veterinary cytology faced a remarkable evolution in the last 15 years, in part due to increase recognition of the advantages of the cytology by veterinary clinicians. Simultaneously, there has been a growing awareness by the owners about the importance of a complete diagnostic workup aimed at defining a proper treatment protocol. With the extended use of cytology, challenging diagnostic cases are more frequent, and more clinically useful answers are requested. In this scenario, the use of cytology specimens to perform ancillary techniques is a valid approach. Rather than being simply archived, cytology slides can be a valuable source and a good platform to carry out cytochemistry, immunocytochemistry, and molecular techniques. Therefore, several diagnostic techniques can be applied in tiny samples, thus following the "doing more with less" principle. The aim of this approach is to refine the cytologic diagnosis and provide additional prognostic and therapeutic information. Herein, we detailed this principle in veterinary cytology and reviewed the use of cytology specimens for ancillary techniques as a single procedure, i.e., using the whole slide, or multiple procedures, i.e., multiple procedures applied in the same slide.

Needle rinse cell blocks as an ancillary technique: Diagnostic and clinical utility in gastrointestinal neoplasia.

BACKGROUND: Fine-needle aspirate (FNA) cytology is often the first-choice method for diagnosing gastrointestinal nodular lesions. The FNA material can be converted to histopathology specimens by a needle rinse cell block (NRCB) technique, allowing ancillary studies to refine the cytologic diagnosis. Despite use in human pathology, NRCB has never been applied to canine or feline gastrointestinal neoplasia. OBJECTIVE: This study described NRCB methodology and its diagnostic utility in specific cases of neoplastic gastrointestinal lesions. METHODS: Needle rinses with saline were performed after ultrasound-guided FNAs of two intestinal lymphomas (canine and feline) and a canine gastrointestinal stromal tumor (GIST). The NRCB was prepared using the cell tube block technique and processed for paraffin embedding. Routine immunohistochemistry protocols (using CD3, PAX-5, and Ki-67 for lymphoma cases and vimentin, desmin, S-100, and KIT markers for GIST) were applied to NRCB sections, and the results were compared with matched tissue biopsies. RESULTS: NRCBs with adequate cell numbers, preservation, and good separation of blood were obtained. The diagnosis and immunophenotyping were confirmed in both cases of lymphoma in NRCBs. In the GIST, the immunolabeling of the neoplastic cells in NRCB was completely concordant with the tissue biopsy. CONCLUSIONS: The described methodology is suitable for veterinary settings, having few technical requirements and low invasiveness. The presented cases of gastrointestinal neoplasia highlight the utility of NRCBs as a platform to conduct ancillary studies and refine the cytologic diagnosis.

Comparison of effusion cell block and biopsy immunohistochemistry in mesothelial hyperplasia, mesothelioma, and carcinoma in dogs.

BACKGROUND: Determining the cause of effusions is challenging and might require a biopsy. Whether cell blocks from effusions are representative of biopsies requires investigation. A previously developed immunohistochemical panel aids in the differentiation of hyperplastic and neoplastic mesothelium in canine biopsies but has not been investigated in effusions. OBJECTIVES: The study aimed to assess cell blocks as an alternative to biopsies and determine whether immunohistochemistry helps distinguish hyperplastic mesothelium, mesothelioma, and carcinoma. METHODS: Effusions and biopsies were collected from five dogs with mesothelial hyperplasia (group MH), six with mesothelioma (group M), and five with carcinoma (group C). Immunohistochemistry (IHC) for cytokeratin, vimentin, Wilm's tumor protein 1 (WT1), desmin, glucose transporter 1 (GLUT1), and insulin-like growth factor II mRNA-binding protein 3 (IMP3) was performed. Sections were scored for staining intensity and the percentage of positively stained cells. RESULTS: In paired cell blocks and biopsies, vimentin and WT1 staining were positively correlated for intensity and the percentage of positive cells, although not all paired results were identical. The intensity of IMP3 staining in cell blocks was higher in group M than in group C (P = 0.012), and WT1 staining was higher in group MH than in group C (P = 0.020). For biopsies, the intensity of WT1 staining was higher in group MH than in group C (P = 0.031). In group C, WT1 was negative in all cell blocks and biopsies, and desmin was negative in four of five cases. CONCLUSIONS: IHC results for the cell blocks and biopsies were comparable for potentially useful markers, such as WT1, which helped discriminate between groups. IHC provided additional information, although results were not always definitive. Further studies on a larger population are required.

The cell tube block technique and an immunohistochemistry panel including Wilms tumor 1 to assist in diagnosing cavitary effusions in dogs and cats.

BACKGROUND: Cell blocks and immunohistochemistry (IHC) are increasingly recognized as being complementary tools for cytologic diagnostics, especially for neoplastic diseases. OBJECTIVES: The study aimed to evaluate the utility of cell tube block (CTB) IHC for refining the diagnosis of effusions in dogs and cats. METHODS: Cavitary effusions (n = 25) from dogs and cats classified by cytology as reactive, neoplastic, borderline (suspicious of neoplasia), and chylous were studied. CTB sections were stained with H&E, and immunostained with PAX-5, CD3, pancytokeratin (CK), vimentin, and Wilms tumor 1 protein (WT1) antibodies, according to the cytologic diagnoses. A histologic case series of confirmed normal, reactive, and neoplastic mesothelium and several different carcinomas were included to test the utility of WT1 as a marker of mesothelial cells. RESULTS: CTBs had a layered appearance with reduced background staining. CD3 and PAX5 immunolabeling allowed immunophenotype assessment in all of the lymphoma cases. In carcinomatous effusions, neoplastic cells were CK-positive, WT1-negative, and vimentin-negative (except for two cases). Wilms tumor 1 protein was positive in the nuclei of normal, reactive, and neoplastic mesothelial cells, and ovarian carcinomatous cells. Other carcinomas and lymphomas were negative. CONCLUSIONS: CTBs are valuable tools to assist in making a diagnosis of cavitary effusions in dogs and cats, and WT1 is a promising marker to differentiate mesothelial from carcinomatous cells.

Cell tube block: a new technique to produce cell blocks from fluid cytology samples.

BACKGROUND: Cell blocks are widely used in human cytopathology. Several techniques have been proposed to convert fluid specimens into solid material, which after embedding in paraffin can be used for histochemistry, immunohistochemistry, or molecular testing. In contrast, only in the last few years, have cell blocks begun to be used in the veterinary field. OBJECTIVES: The purpose of the study was to present the production and features of cell tube blocks (CTB) with veterinary liquid samples. METHODS: Liquid samples including cerebrospinal fluid, cutaneous cyst fluid, pericardial and pleural effusions, bronchoalveolar lavage, urine, and blood were centrifuged in a microhematocrit centrifuge. Capillary tubes were broken at the liquid-solid interface and fixed in 10% formalin for 24 hours. After paraffin embedding, sections of CTB were used for different stains including immunohistochemistry. RESULTS: The morphology and cellular detail in CTB sections were comparable to conventional histologic sections and other existing cell block techniques. The use of special stains such as Gram, Giemsa, alcian blue, and periodic acid-Schiff was straightforward, and immunohistochemistry results with antibodies to pan-cytokeratin, PAX-5, and CD3 were considered good. CONCLUSIONS: The CTB method was easily implementable under practice conditions (up to the fixation of the microhemtocrit tube), analogous to surgical biopsy submission for histology. Cell tube blocks can increase diagnostic accuracy when the technique is used in tandem after the cytologic evaluation, and the technique allows storage of fluids. Other advantages of CTB were the simplicity, low cost, and separation of erythrocytes from the nucleated cells, which was helpful in hemodiluted samples.

Cytocentrifuge preparation in veterinary cytology: a quick, simple, and affordable manual method to concentrate low cellularity fluids.

BACKGROUND: Smears prepared by cytocentrifugation, the so-called cytospins, are widely used in human and veterinary cytology. However, the high cost has hampered the availability of commercial cytospin centrifuges in some veterinary clinics and laboratories. Nevertheless, cytospins are important for evaluating fluids with very low cellularity such as cerebrospinal fluid (CSF) or bronchoalveolar lavage fluid (BALF). OBJECTIVES: The aim of this study was to devise and test the use of a low-cost, in-house manual cytocentrifuge to obtain cytospin preparations. METHODS: Twenty-two fluid samples (including CSF and BALF) were collected from dogs and cats. These were processed in a conventional cytocentrifuge and in an in-house, manual centrifuge (salad spinner). The cytospins obtained by the 2 methods were compared by scoring cellularity, number of cells per field, hemodilution, cell preservation, and proportion of ruptured cells. Additionally, cell number and size were compared by morphometry. Differences between the automated and manual method were statistically assessed. RESULTS: The morphology and cellular detail of cytospin preparations produced by both methods were identical. There was an almost perfect agreement for cellularity, number of cells per HPF, hemodilution and cell preservation (kappa ≥ 0.85), and a moderate agreement for the amount of ruptured cells. Cell recovery was comparable (including in CSF and BALF). CONCLUSIONS: The manual cytocentrifuge produced cytospins with similar cell yield as the automated cytocentrifuge. Considering the low cost and portability, this new method should be particularly useful for cytologic diagnosis in small clinics, developing countries, and in field studies.