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Cytology refers to the scientific study of cells' structure, function, in situ locale and environment. In this field of veterinary science, cells harvested from various biological fluid samples are examined under the microscope to detect normal and abnormal structures. Therefore, cytology is mainly used to detect disease early, monitor the progression of treatment, detect disease progress, detect cancer, and identify many infectious diseases. This book, "Techniques in Veterinary Cytology," is designed to provide a comprehensive and practical guide to the fundamental principles and basic procedures of veterinary cytology for students, young investigators, veterinary clinicians and other animal science personnel. Though cytology procedures look simple and easy to perform, the experiment results vary depending on the practice, knowledge of the techniques, and, ultimately, the goal of the study, which is what we are looking for. This book is structured to take readers through the essential steps of sample collection, preparation, and analysis. Section A, "Collection of Biological Fluids," covers the collection of various sample types, including blood, milk, fine needle aspirates, urine, and saliva, from a range of animal species. Proper collection techniques are critical for obtaining representative samples and ensuring accurate cytological evaluation. The subsequent sections of this book include processing and analyzing samples. Section B, "Blood," describes the isolation, preparation, and immunostaining of peripheral blood mononuclear cells (PBMCs). Section C, "Milk," details the isolation, staining, and immunostaining of milk somatic cells, providing essential protocols for the study of mammary health. Section D, "Fine Needle Aspirates," guides readers through preparing and staining FNA smears, including Giemsa, Leishman, and immunostaining techniques for identifying proliferating cells. Finally, Section E, "Analysis of Morphology of Cells," introduces Image J, a powerful tool for cell counting and cytomorphological analysis. This section equips the reader with the skills to quantitatively assess cytological samples, enhancing diagnostic accuracy and research capabilities. Each chapter provides step-by-step protocols, helpful illustrations, and troubleshooting tips to facilitate hands-on learning. This manual is an invaluable resource for students, researchers, and practitioners seeking to master the techniques of veterinary cytology.

Background Blood collection from cows and other animals is a routine procedure often performed for diagnostic, research, or health-monitoring purposes. Diagnostic testing, such as hematology, biochemistry, hormone assay, infectious disease testing, and animal pregnancy diagnosis, can be done using blood. Blood samples are required for genetic testing (DNA fingerprinting or parentage determination), pharmacokinetic studies, and immunological studies. Another use of blood collection is monitoring animal health. It is essential to consider that proper techniques of blood sample collection should be followed. Avoid introducing infection to the animal during blood collection. Remember the animal welfare to minimize distress and discomfort to the animal during blood collection. Materials Required • Sterile syringe or vacutainer (usually 16-18 G needle) • Blood collection tubes (BD Science K2 EDTA Vacutainer(R) PLUS 6 mL) for whole blood, serum tubes for serum collection, or other specific tubes depending on the test (Figure 1). • Antiseptic solution (e.g., alcohol or iodine) • Restraint (head gate or halter) • Gloves

Background Milk is known as a whole food for human consumption. It is the first food of the newly born human mammals. Milk contains almost all the nutrients required for the patients, the elderly, pregnant mother and new born baby. There is no doubt that milk plays a significant role in human development. We can prepare many physical forms of milk like cheese, yogurt, ghee, and milk powder. The calcium content is higher in buffalo than in cows. Buffaloes are the second largest source of milk and produce 15 % milk. Higher biological value proteins, fat, soluble vitamins, fatty acids, amino acids, water-soluble vitamins, and calcium are all present in milk. The main antioxidant components of milk and milk products are sulfur, phosphate, zinc, and amino acids like cysteine (necessary to produce collagen). Milk somatic cells are normal cells found in milk in small amounts (epithelial cells), some of which are leukocytes (white blood cells). When mastitis is infected or injured, leukocyte production rises. Milk with a low SC level produces superior milk products that last longer. Better milk management, better cleanliness, and correct nutrition all contribute to a decrease in milk SC, which indicates poor milk production hygiene and a cause of mastitis. Milk somatic cells are made up of both immunological and milk-producing cells. In terms of defense, somatic cells are the second line. During the routine process of milking, these cells are secreted in milk. Somatic cell count is a good tool for evaluating farm cleanliness in bulk milk and is utilized worldwide as an indication of udder health in individual milk. The most prevalent condition affecting dairy buffalo production and their health and well-being is mastitis. The somatic cell count consists of immune cells (75%), such as neutrophils, macrophages, lymphocytes, erythrocytes, and epithelial cells (25%). Investigators have made different attempts to exploit milk somatic cells as a potential non-invasive sample to diagnose early pregnancy udder health in bovines.

Background Fine needle aspiration (FNA) is a minimally invasive diagnostic procedure used to obtain cells from a mass or lesion for examination under a microscope. It’s a valuable tool in diagnosing tumors in canines (Figure 5) and other species of animals, including humans. Advantages of FNA • Minimal invasiveness to the animals. Thus, no sedation of animals is required. • Rapid results - Results are obtained faster as cell processing is not required. • Cost-effective • Risk-free • FNA can be performed under image guidance, such as ultrasound, CT, and MRI, for precise identification of the location of cells inside the tissue. • May be able to differentiate between benign and malignant tumors • Can assess tumor grade • FNA helps monitor treatment response. FNA is helpful as it can help pre-plan the treatment of the dogs. If the tumor is malignant, then the treatment can be given immediately, and if it is benign, then the treatment can be postponed.

Background Urine collection is a fundamental procedure in veterinary medicine, providing valuable insights into an animal’s health status. Urine analysis can detect urinary tract infections, kidney function, and metabolic disorders. Several methods can be employed for urine collection in dogs, each with its advantages and limitations. Always collect urine in clean, sterile and leakproof containers. Procedures of Urine Collection In Animals 1. Free catch or voided urine: Collecting urine as the animal voids naturally. This procedure is non-invasive and easy to perform. However, the collection of voided urine may not be free from contaminants. Contamination risk from external genitalia and dilution or concentration variation in urine composition is common. 2. Catheterization: Inserting a catheter into the urethra to obtain a sample, often used in larger animals or when free catch is not possible. It provides a sterile sample that is suitable for bacterial culture and cytology. However, catheterization is an invasive procedure with the potential for trauma and discomfort to the animals. 3. Cystocentesis: Inserting a needle directly into the bladder through the abdominal wall to collect a sterile sample. Like catheterization, cystocentesis provides a sterile sample, ideal for bacterial culture and cytology.

Background As a non-invasive and relatively stress-free method, saliva holds great potential as a diagnostic sample. It has already been utilized in diagnostic tests for rabies and other diseases in dogs. Saliva secretion is the secretion of salivary glands that maintains homeostasis in the oral cavity, protects against pathogens, and helps digest food. It contains inorganic substances, cells (blood and epithelial cells) and microorganisms. Saliva also contains immunoglobulins, enzymes, hormones, and cytokines derived from serum and bronchial secretions. The saliva of dog is being used to detect rabies virus (Kasempimolporn et al., 2000), parvo virus (Kocaturk et al., 2021), cancer and measure stress levels. Obtaining saliva rapidly, painlessly, and uncomplicatedly can be an easy tool for disease screening. For the identification of serval prognostic biomarkers for various physiological and pathological conductions, saliva proteomics can be very helpful. In humans, saliva contains more than 3000 proteins (Kawas et al., 2012). Several diseases like oral carcinoma, AIDS, hepatitis B, cystic fibrosis, and other diseases like diabetes mellitus. Differential expression of dog saliva proteins compared to human saliva revealed 2532 proteins. Proteins related to apoptosis and adhesion were predominant in the oral proteome of dogs, in addition to proteins related to regeneration and healing processes such as FGF and EGF (Sanguansermsri, et al., 2018).

Background Peripheral blood mononuclear cells (PBMCs) are a crucial immune system component. Isolating them from whole blood allows for an in-depth study of immune responses, disease mechanisms, and the development of noveltherapies. Lymphocytes, monocytes, and other different  white blood cells (WBCs) possessing circular nuclei make up the cells of PBMCs. These cells can be directly obtained from whole blood using different separating methods. PBMCs can be utilized for multiple purposes, from drug assessment to expression analysis to parentage determination. • Toxicity assessment of novel compounds: Interaction of drugs with PBMCs can help assess the safety of compounds and set appropriate doses for safe consumption. • Comparative analysis: In vitro assessment of diseased vs. healthy PBMCs can provide insights into affected biochemical pathways. The effects of foreign chemical compounds can also be assayed to drive future studies. • Personalized medication: Studying PBMCs can help develop a genetic immune profile, leading to more specificity and effectiveness in personalized medicines. • Occupational disorders: Diseases developed due to the overexposure of certain toxic compounds can also be determined by testing PBMCs in peripheral blood.

Background Peripheral Blood Mononuclear Cell (PBMC) smears are a valuable diagnostic tool in veterinary medicine, offering insights into various animal diseases. By isolating and examining PBMCs— comprising lymphocytes and monocytes, veterinarians can detect hematologic abnormalities, immune responses, and the presence of infectious agents or neoplastic cells. This method is particularly useful in diagnosing viral, bacterial, and parasitic infections, as well as immune-mediated disorders and certain cancers. PBMC smears provide a quick and cost- effective way to assess animal health, aiding disease diagnosis, monitoring, and treatment planning. Following is the utility of PBMC smear in animal disease diagnosis; 1. Identification of abnormalities in WBC morphology 2. Identification of abnormalities in the number of cells like leukosis leukopenia. 3. Certain viral infections, such as canine distemper or feline leukemia virus (FeLV), can cause characteristic changes in PBMCs that are detectable on a smear. 4. Detection of blood parasites like Babesia and Ehrlichia. These parasites are detected using a blood smear; isolation of PBMC from whole blood is not required. 5. Certain types of cancers, like leukemia or lymphoma, can be identified through changes in PBMCs. Abnormal proliferation of lymphocytes or the presence of blast cells can suggest hematologic malignancies.

Background Immunocytochemistry is the immunostaining of a specific cell or its components in a cell smear. It is a technique to visualize a specific protein or an antigen in cells based on an antigen- antibody interaction (Figure 10). The site of antibody-antigen binding can be visualized by color development (enzymechromogen system) or fluorescence. The macrophages can be visualized in the smear of peripheral blood mononuclear cells by immunocytochemistry using antibodies against macrophages. We need to use a light or fluorescent microscope to detect color or fluorescence, respectively.

Background Milk somatic cells are a diverse population of cells found in milk, including epithelial cells, leukocytes (white blood cells), and macrophages. Isolating these cells from milk provides valuable insights into mammary gland health, mastitis detection, and milk quality assessment. Several methods can be employed for milk somatic cell isolation. Among the others, centrifugation is the most common method of cell separation. Filtration, immunomagnetic separation and flow cytometry are the other methods of isolating specific cell populations. In bovines, isolating milk somatic cells is useful in milk quality assessment, mastitis detection, and mammary gland biology research. An increased milk somatic cell count level, particularly neutrophil, indicates mammary gland inflammation. Healthy and mastitis glands can be diagnosed by analyzing the differential counting of milk somatic cells.

Background Staining of milk somatic cells is the oldest method of enumerating cell types in milk by direct microscopic examination. Various stains, namely propidium iodide, methylene blue and hematoxylin-eosin stains, are used. The total milk cell (TMC) pellet obtained above was resuspended gently in 500 μl of PBS 1X (Sigma, MA, USA). Place 20 μl of cell suspension on a clean glass slide (BioGenex, CA, USA) and let it dry at room temperature. Materials and reagents required 1. Positively-charged glass slides 2. Glass coverslips 3. Hot plate 4. Fixatives – Methanol or ethanol 5. Hematoxyline and Eosin stain 6. Phosphate-buffered saline (PBS) 7. Distill water 8. Ethanol for dehydration 9. Xylene 10. Mounting medium (like DBX)

Background Immunostaining is a technique used to visualize specific proteins or antigens within cells. In milk somatic cells, immunostaining is employed to identify and quantify various cell types, study their functional characteristics, and diagnose diseases. Immunostaining can be used to identify and characterize different cell types based on the expression of specific surface markers. For example, CD45 is a leukocyte marker, while cytokeratins are markers for epithelial cells. In addition to the research on mammary gland biology, immunostaining is a valuable tool for studying the biology of the mammary gland. It can be used to investigate the expression of genes involved in milk production, cell differentiation, disease processes and even pregnancy detection. For example, galectin-3-binding protein (LGALS3BP) is a gene with elevated expression in the PBMCs of early pregnant buffaloes (Sharma et al., 2023). In this chapter, we have investigated the protein expression of LGALS3BP in milk somatic cells to determine if immunopositive cells are present in milk. Apart from this, stem cells in milk can be identified using markers like CD133, CD73, CD44,and other cell surface markers (Coni et al., 2023).

Background During a fine needle aspiration biopsy (FNAB), a small amount of mammary tissue fluid is removed using a fine needle by applying suction, and cells present therein are identified in a thin smear. FNAB has been the most sensitive, quick, and precise tool for the differential diagnosis of malignancy. However, further research is required into the possibility of malignancy in dogs based on cytological evidence and candidate gene expression analysis. This is a noninvasive cancer detection technique and is often used in clinics. Materials and Reagents Required 1. Fixative- cold acetone, ethanol, or aldehyde-based fixative (10% neutral buffered formalin/ NBF or paraformaldehyde). 2. Ethanol – 70%, 90% and 100% 3. Washing buffer (phosphate buffer saline)- PBS 4. Clean glass slides – preferably positively charged 5. 22-gauge needle fitted with 2 ml syringe Procedures Prepare for a dog having a mammary tumor • Clean the site of the mammary tumor with alcohol and allow it to dry. • Keep slides ready, clean, and dry.

Background Staining is a crucial step in the microscopic examination of fine needle aspirates (FNAs) from canine mammary tumors. It enhances the visibility of cellular structures, making it easier to diagnose the tumor type. The cryopreservation of unfixed, air-dried smears of FNA can safely be used for staining up to 33 months, including immunostaining for epithelial and mesenchymal cell markers like cytokeratin and vimentin (Furusawa et al., 2023). Common Staining Techniques 1. Romanowsky Stains: Romanowsky stains are a group of related stains, Dimitri Romanowsky in 1891. It used a mixture of Eosin (an acidic dye) and methylene blue (a basic dye). The original method has since been modified to include additional derivatives of methylene blue, such as azure B, leading to more distinct staining effects (Marshall, 1986). It is widely used in hematology, cytology, and microbiology for staining blood smears, bone marrow smears, and other cell types. These stains are significant for identifying and differentiating cellular components like nuclei, cytoplasm, and intracellular structures in various types of cells. • May-Grunwald-Giemsa stain: A modification of the Romanowsky stain, combined with Giemsa for its ability to differentiate between cell types and structures. It stains nuclei blue and cytoplasm pink. Giemsa stain utilizes the azure dye to stain the azurophilic cellular components purple.

Background Leishman stain (named after W. B. Leishman, a British surgeon) is one of the Romanowsky- type stains. It is a differential stain used to stain the different components of the cells variably. The Leishman stain is one of the best for routine blood staining and can be used for FNAC smear staining. It stains the peripheral blood smear when examining blood film under the microscope (Figure 20). Leishman stain combines Azure B (blue) and Eosin Y (orange) dyes. Azure B has an affinity for DNA and acidic groups in cell nuclei, while Eosin Y has an affinity to basic groupings in cytoplasm. The staining results show crisp blue nuclei while the cytoplasm takes a purple hue. Procedure for Leishman Staining Sample Preparation • Obtain a thin blood smear or FNAC sample. • Air-dry the smear thoroughly. • Flood the slide with methanol for 3-5 minutes for fixation • Air dry the smear.

Materials and Reagents Required 1. Fixative- cold acetone or methanol 2. Ethanol – 70%, 90% and 100% for rehydration and dehydration 3. Xylene for clearing 4. Washing buffer (phosphate buffer saline)- PBS 5. Hydrogen peroxide – for endogenous peroxidase blocking 6. Normal horse serum or any other serum: 2.5% horse serum or any other serum can be used to block non-specific epitopes. 7. Ki67 mouse primary antibody 8. Secondary antiboy kit - Impress Excel kit (Cat no. MP-7601; Vector Laboratories, NY, USA) or a similar kit from other vendors like Biogenex (Figure 22; left panel). 9. Peroxide substrate kit—Immpact NovaRed (cat no. SK-4850; Vector Laboratories, NY, USA) for developing a red color signal (Figure 22; right panel).

Background Cells are the basic unit of life. The morphology of cells describes their structural framework. It refers to the morphological features of the cell(s). It comprehends the various shapes and sizes in which cells function. Thus, the shape and size of the cell and the number of cells indicate their functional status. Hematological diagnostics requires the proper assessment and valid classification of cells for final diagnosis and prognosis. Clinicians, patients, and diagnosticians are all concerned with achieving a definitive specific diagnosis employing a single investigative test. The utilization of different cytological examinations from different organs gives adequate indicative data that leads to treatment outcomes. What is ImageJ? ImageJ is a public-domain image processing and analysis software that is Java-based and developed by the National Institutes of Health. The software is used to process and analyze scientific images. The key features of ImageJ are 1) the versatility of working with various image formats, 2) extensibility with several plugins, 3) multidimensional data handling capability, and 4) vibrant community, making ImageJ a unique software for image processing and analysis. It can run as an online applet or be downloaded and used as an app.

The cell number of a particular cell type in a total cell population might indicate the physiological or pathological status of the cell smear/FNAC/tissue. ImageJ is a powerful open- source software extensively used for image assessment, including cell counting. In this chapter, the steps to count cells using ImageJ has been discussed. There are two ways to count the cells in a research image using ImageJ. A. Manual Counting Use the “Cell Counter” plugin or simply use the multipoint tool in ImageJ. Open it, select the cell type you want to count, and click on each cell in the image to tally them. Double-click on the multipoint tool. A separate window will open named point tool. One can select the type of marking, color and size of making (Figure 30). Keep clicking on the cell type to be counted. It will record the number of cells counted. If one wants to count the second cell type in the same image, change the counter number and select the size, color and type of click point.

Measuring cell size, diameter, length, distance between cells, area, etc, are important indicators of the identification of cell types and the physiological or pathological state of cells and tissues. ImageJ is a useful program that allows researchers and scientists to work with microscopy images for cytomorphological detailing. The steps to measure cell size are: A. Open Your Image in ImageJ: First, open the image containing the cells you want to measure. B. Set the Scale (Optional): If your image has a known scale (e.g., if you used a microscope with a calibrated scale bar), you can set it inImageJ. This step ensures accurate measurements. Go to Analyze > Set Scale. Enter the known distance (in pixels) and the corresponding physical distance (e.g., micrometers). Click OK. C. Choose the Measurement Tool: ImageJ provides several measurement tools. The most common ones for cell size are: Area Measurement: Use the Polygon Selection Tool to outline the cell, and then go to Analyze > Measure. D. Length Measurement: Use the Straight-Line Selection Tool to measure the length of cell projections (e.g., neurites or filopodia). E. Record Measurements: After selecting the appropriate tool, click and drag to measure the cell area or length. Press’ t’ to record the measurement. Repeat for multiple cells if needed. F. View Results: The measured values will appear in the Results window. You can access the results by going to Analyze > Measure or by opening the Results window directly.