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Diagnostic cytology

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Abstract

Cytopathology, often abbreviated to cytology, is the microscopic examination of individual cells or groups of cells to identify their origin and any changes characteristic of disease. It is a quick and safe diagnostic tool that can be used to investigate superficial or internal masses, internal organs, lymph nodes and fluids (effusions, joint fluids, cerebrospinal fluid (CSF), bronchoalveolar lavages (BALs), urine, prostatic washes). This chapter deals with general sampling guidelines, routine stains, cytochemical stains, immunocytochemistry, complications and contraindications of cytology, cytological cell types, the approach to slide examination, skin cytology and cytology of lymph nodes. Case examples are included.

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Figures

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21.2 Fine-needle aspiration technique. The mass is immobilized with one hand while the needle is inserted into the lesion and moved to and fro.
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21.3 The centre of large masses is frequently necrotic and inflamed. Hence, during sampling, aspiration from the peripheral areas of the mass should guarantee a more representative sample. (© D Berlato, AHT)
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21.4 Prostatic wash from a dog. The abundant pink amorphous material is lubricant gel, which hampers visualization of cellular details. (Wright–Giemsa stain; original magnification X500)
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21.5 Squash smear preparation. (a) A drop of specimen is placed on one edge of the bottom side. (b) A second slide is gently applied on top and drawn across at right angles.
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21.6 (a–b) These slides are suboptimal for cytological examination. (a) The specimen is insufficiently smeared. (b) The slide contains an excessive amount of material and the smer is too thick. (c) The slide shows an ideal cytological preparation. (Stained with Wright–Giemsa)
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21.7 Line concentration technique. (a) After the spreader slide has been advanced two-thirds of the way along the bottom slide, (b–c) it is lifted upwards in order to concentrate cells at the end of the smear.
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21.8 Impression smear technique for a surgical biopsy specimen. (a) Excess blood is firstly removed from the lesion by blotting the cut surface with a clean swab. (b–c) Several imprints from the mass are obtained by apposing the sample to the slide.
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21.9 Cytological sample stained with Papanicolaou. (Papanicolaou stain; original magnification X600) (Courtesy of Paul Elgert, New York University School of Medicine)
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21.10 Fine-needle aspirates from a mediastinal mass in a dog. Some slides were stained with (a) Diff-Quik and others with (b) Wright–Giemsa. Diff-Quik failed to stain the mast cell granules, making the cytological interpretation more difficult. (Original magnification X500)
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21.12 Direct and indirect techniques for immunocytochemistry. Refer to text for more details. DAB = diaminobenzidine.
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21.13 Immunocytochemistry on a canine pleural mesothelioma. (a) Negative control. (b–c) The plasma membrane of the neoplastic mesothelial cells reacted positively (brown colour) with (b) cytokeratin and (c) Vimentin. (Original magnification X500)
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21.15 Non-degenerate neutrophils from non-septic subcutaneous neutrophilic inflammation in a dog. The nuclei of the neutrophils are lobulated and the chromatin is dense and darkly stained. Low numbers of macrophages are also present in the photograph. (Wright–Giemsa stain; original magnification X500)
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21.16 Degenerate neutrophils from a septic abscess in a cat. The nuclei of the neutrophils are swollen (karyolytic) and have lost the typical lobulation. The chromatin is paler and less condensed. A few intracytoplasmic cocci are seen in a neutrophil. (Wright–Giemsa stain; original magnification X500)
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21.17 Pyknotic and karyorrhectic neutrophils from a sterile abscess in a dog. Pyknotic nuclei are round and the chromatin is condensed and deeply basophilic. The fragmentation of pyknotic nuclei is defined as karyorrhexis. (Wright–Giemsa stain; original magnification X500)
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21.18 Fine-needle aspirate from a subcutaneous nodule of a dog with steatitis. Numerous foamy activated macrophages are seen in a background of clear (lipid) vacuoles. Macrophages have abundant cytoplasm containing small clear vacuoles. (Wright–Giemsa stain; original magnification X500)
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21.19 Bronchoalveolar lavage (BAL) sample from a dog with an eosinophilic bronchopneumopathy (EBP). High numbers of eosinophils and a few columnar respiratory epithelial cells are seen in the preparation. (Wright–Giemsa stain; original magnification X500)
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21.20 Urine samples from dogs with urinary tract infection. (a) Cocci and rod-shaped bacteria (bacilli) are visible. (b–c) Round bacteria (cocci) are visible. (b) The bacteria are arranged in a large group, sugggesting spp. (c) The bacteria form chains of variable length, suggesting spp. (Wright–Giemsa stain; original magnification X500)
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21.21 Sample from a mass on the cheek of a cat. (a) Numerous fine negatively stained rods of mycobacteria are seen within the cytoplasm of a macrophage. (Wright–Giemsa stain; original magnification X500). (b) With acid-fast staining, mycobacteria appear as fine purple rods. (Ziehl–Neelsen; original magnification X500)
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21.22 spp. in a pleural effusion from a dog. The filamentous bacterial rods stain lightly basophilic with small purple dots. (Wright–Giemsa stain; original magnification X1000)
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21.23 (a) Pseudohyphae of from a cutaneous lesion in a cat with diabetes mellitus. (Wright–Giemsa stain; original magnification X500). (b) Fungal hyphae stained with periodic acid–Schiff (PAS). (Original magnification X1000). (c) Fine-needle aspirate of a cutaneous granuloma in a dog. Multiple round and deeply basophilic structures are consistent with . (Wright–Giemsa stain; original magnification X1000). (d) Fine-needle aspirate from a nasal lesion in a cat. Multiple round basophilic structures with thick clear capsules are consistent with spp. (Wright–Giemsa stain; original magnification X1000) (b,d, Courtesy of E Villiers)
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21.24 Ear swab preparation from a dog with otitis externa. Numerous peanut-shaped basophilic yeasts consistent with spp. are seen in this sample. (Wright–Giemsa stain; original magnification X500)
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21.25 BAL sample from a young Cavalier King Charles Spaniel with interstitial pneumonia secondary to spp. Multiple trophozoites (white arrow) and a cyst (black arrow) are present. Inexperienced cytologists can easily confuse the trophozoites with platelets. (Wright–Giemsa stain; original magnification X500)
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21.26 BAL sample from a cat with respiratory signs. tachyzoites are seen in the centre of the picture. These are ‘banana-shaped’ bodies with a lightly basophilic cytoplasm and a deeply staining nucleus. (Wright–Giemsa stain; original magnification X1000)
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21.27 organisms in (a) lymph node and (b) bone marrow aspirates from a dog. amastigotes are seen within the macrophages or free in the background. They measure approximately 2–4 µm and are oval with a small purple nucleus and a small rod-shaped kinetoplast. (Wright–Giemsa stain; original magnification (a) X500 and (b) X1000)
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21.28 (a) Fine-needle aspirate from a thyroid mass in a dog. The background is heavily haemodiluted and contains a blood-derived microfilarium (). No thyroid tissue is present in this image. (b) Fine-needle aspirate from a skin nodule in a dog. Several round structures containing small larvae are consistent with ‘egg cells’. (Wright–Giemsa stain; original magnification (a) X500 and (b) X1000) (a, Courtesy of I Lloverals, CV AMVET)
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21.29 Wet faecal preparation from a dog with lungworm infection (). Note the kinked tail typical of this parasite (extremity on the left). (Original magnification X1000)
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21.31 Fine-needle aspirates from a haematoma on the shoulder of a dog. Macrophages display (a) erythrophagia, or (b) contain basophilic amorphous material (haemosiderin) or (c) Rhomboid orange crystals (haematoidin). (Wright–Giemsa stain; original magnification X500)
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21.32 Fine-needle aspirate from a surgical scar of a dog. Fusiform mesenchymal cells represent reactive fibroblasts involved in the wound healing process. (Wright–Giemsa stain; original magnification X1000)
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21.33 The main morphological features of the three cytological tumour cell types: epithelial, mesenchymal and round cells.
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21.34 Fine-needle aspirate from a lymph node with a metastatic prostatic carcinoma. Malignant neoplastic epithelial cells are intimately cohesive. The clear line separating the two contiguous epithelial cells represents the tight junction. (Wright–Giemsa stain; original magnification X1000)
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21.35 Ciliated columnar respiratory epithelial cells from the nasal mucosa of a cat. (Wright–Giemsa stain; original magnification X1000)
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21.36 Mesenchymal and epithelial architectural patterns with examples. (Wright–Giemsa stain; original magnifications X100, X500 and X1000) (Illustrations by Paola Monti)
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21.37 Artefactual cells: disruption occurred during smearing. (Wright–Giemsa stain; original magnification X200)
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21.38 Neuroendocrine tumour (phaeochromocytoma): cells occur as bare nuclei embedded in a lightly basophilic background of cytoplasmic material. (Wright–Giemsa stain; original magnification X1000)
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21.39 (a) Fine-needle aspirate from a well differentiated soft tissue sarcoma in a dog. Cells are fusiform with two slim cytoplasmic tails and do not display atypical features. (b) Poorly differentiated sarcoma in a dog. Cells are oval, plump and display moderate anisokaryosis, a high nuclear:cytoplasmic (N:C) ratio and prominent nucleoli. (Wright–Giemsa stain; original magnification X500)
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21.40 Fine-needle aspirate from a canine plasma cell tumour. Cells are discrete with an eccentric nucleus and basophilic cytoplasm. (Wright–Giemsa stain; original magnification X500) (Courtesy of E Villiers)
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21.41 Fine-needle aspirate from an enlarged prostate gland in an entire adult dog. Numerous uniform epithelial cells with small round nuclei and granular chromatin are seen. The cytoplasm contains many clear punctate vacuoles. (Wright–Giemsa stain; original magnification X500)
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21.42 Fine-needle aspirate from a metastatic transitional cell carcinoma in a dog. Nuclear moulding is displayed in the centre of the picture where the nucleus of a cell is deforming the nucleus of a contiguous cell. (Wright–Giemsa stain; original magnification X1000)
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21.43 General criteria of malignancy. (a) Fine-needle aspirate from a transitional cell carcinoma. The neoplastic epithelial cells are arranged in loosely cohesive clusters. (b) Fine-needle aspirate from a hepatocellular carcinoma. Atypical cells are arranged in disorganized clusters. (Wright–Giemsa stain; original magnification X500) (Courtesy of E Villiers)
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21.44 Nuclear criteria of malignancy: (a) multinucleation, (a–b) anisokaryosis and prominent multiple nucleoli, (c–d) atypical mitotic figures. (Wright–Giemsa stain; original magnification X500)
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21.45 Cytoplasmic criteria of malignancy: the cytoplasm contains a large vacuole of secretory material, which deforms and displaces the nucleus to the periphery (signet ring). Canine prostatic carcinoma. (Wright–Giemsa stain; original magnification X1000)
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21.46 Algorithm showing an approach to slide examination, with the main cytological subtypes.
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21.47 Fine-needle aspirate from an epidermal inclusion cyst on the back of a dog. Numerous anucleate squamous epithelial cells are present. (Wright-Giemsa stain; original magnification X1000)
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21.48 Epidermal inclusion cyst. A large polygonal negatively stained structure consistent with a cholesterol crystal is present. (Wright-Giemsa stain; original magnification X1000)
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21.49 (a) Squamous cell carcinoma on the nose of a cat. (b) The neoplastic cells appear well differentiated and have abundant lightly basophilic cytoplasm. Small punctate perinuclear keratohyaline vacuoles can be observed in most of these cells. (Wright–Giemsa stain; original magnification X1000) (a, ©D Berlato, AHT)
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21.50 Fine-needle aspirate of a moderately differentiated SCC. Compared with the cells shown in Figure 21.49b , these cells display a higher N:C ratio and the cytoplasm is more deeply basophilic. Nucleoli are prominent. (Wright–Giemsa stain; original magnification X500)
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21.51 Tadpole epithelial cell from an SCC of a cat. (Wright–Giemsa stain; original magnification X500)
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21.52 Emperipolesis in an aspirate from a feline SCC. A neutrophil is transiting within the cytoplasm of a squamous epithelial cell. (Wright–Giemsa stain; original magnification X500)
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21.53 Cohesive cluster of uniform basal cells from a trichoblastoma on the neck of a dog. At the edges of these clusters, cells are arranged in rows (palisades). (Wright–Giemsa stain; original magnification X500)
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21.54 (a) Macroscopic appearance of a sebaceous adenoma. These tumours are often raised, multilobulated and hairless. (b) Microscopic appearance of a sebaceous adenoma. Epithelial cells have abundant and heavily vacuolated cytoplasm. Vacuoles are discrete and clear. These cells often exfoliate in tridimensional balls. (Wright–Giemsa stain; original magnification X500) (a, © D Berlato, AHT)
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21.55 (a) Fine-needle aspirate of a perianal gland tumour from a mass on the tail base of an entire male Labrador Retriever. Cells have exfoliated in uniform sheets and display minimal cell size variation. These cells are also known as hepatoid cells given their similarity to (b) hepatocytes. (Wright–Giemsa stain; original magnification X500)
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21.56 Fine-needle aspirate of an anal sac mass in a dog. The mass has yielded a population of uniform epithelial cells with indistinct cytoplasmic margins. An acinoid arrangement is present in the top left corner of the photograph. (Wright–Giemsa stain; original magnification X500)
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21.58 Fine-needle aspirate from an interscapular fibrosarcoma on a cat. Cells are spindle shaped and loosely arranged. They display multiple criteria of malignancy (anisokaryosis, coarse chromatin, prominent nucleoli, multinucleation and mitoses). (Wright–Giemsa stain; original magnification X500)
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21.59 Fine-needle aspirate from a lipoma in a dog. The adipocytes have large cytoplasmic lipid vacuoles and small nuclei. Two small capillaries are seen crossing this aggregate. (Wright–Giemsa stain; original magnification X500)
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21.60 Fine-needle aspirates from a liposarcoma on the hindlimb of a dog. (a) The background of this aspirate contains abundant fat globules. The atypical mesenchymal cells display features of malignancy and are characterized by numerous cytoplasmic lipid droplets. (b) Less vacuolated neoplastic cells often have a pink, finely granular cytoplasm. (Wright–Giemsa stain; original magnification X500)
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21.61 Fine-needle aspirates from a perivascular wall tumour (PWT) on the leg of a dog. (a) The cells are arranged in loose aggregates. The cytoplasm has poorly defined and fringed borders giving a veil-like appearance. (b) Crown cell: this is a multinucleated cell, characterized by nuclei arranged in a circle at the periphery of the cytoplasm. This typical cell is frequently seen in PWTs. (Wright–Giemsa stain; original magnification (a) X500, (b) X1000)
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21.62 Fine-needle aspirates from a cutaneous haemangiosarcoma (HSA) in a dog. (a) The background is heavily haemodiluted. Neoplastic cells are large, plump, spindle shaped and have a high N:C ratio and prominent nucleoli. (b) One of the neoplastic cells contains an engulfed red blood cell (erythrophagia). (Wright–Giemsa stain; original magnification (a) X500, (b) X1000)
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21.63 Fine-needle aspirate of a subcutaneous embryonal rhabdomyosarcoma on the antebrachium of a dog. The cells are large and polygonal and have abundant basophilic cytoplasm, large eccentric nuclei and prominent nucleoli. A few multinucleated cells are also present. (Wright–Giemsa stain; original magnification X500) (Courtesy of G Avallone, University of Milan)
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21.64 Fine-needle aspirate from a subcutaneous myxosarcoma in a dog. The cells are typically aligned in rows owing to the viscosity of the mucinous secretory material. (Wright–Giemsa stain; original magnification X100)
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21.65 Fine-needle aspirate from a melanoma in a cat. Melanocytes contain numerous intracytoplasmic dark green–black pigment (melanin) granules. (Wright–Giemsa stain; original magnification X500)
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21.66 Fine-needle aspirate from an oral amelanotic melanoma in a dog. Large atypical cells display marked criteria of malignancy and a lack of cytoplasmic melanin granules. The diagnosis was confirmed by immunohistochemical staining (Melan A). (Wright–Giemsa stain; original magnification X500)
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21.67 Fine-needle aspirate from a periarticular mass in a Flat Coated Retriever consistent with a histiocytic sarcoma. Neoplastic cells are discrete and display features of malignancy (anisokaryosis, irregular mitosis). (Wright–Giemsa stain; original magnification X1000)
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21.68 Fine-needle aspirate from a cutaneous histiocytoma in a young dog. Discrete cells show mild size variation and have abundant lightly basophilic cytoplasm, often lighter than the surrounding background. The cells are often compared to fried eggs. (Wright–Giemsa stain; original magnification X500)
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21.69 Fine-needle aspirate from a well granulated mast cell tumour in a dog. Mast cells contain numerous fine purple granules, often obscuring the nuclei. (Wright–Giemsa stain; original magnification X500)
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21.70 Fine-needle aspirates from a poorly granulated mast cell tumour in a dog. (a) Mast cells contain low numbers of dust-like purple granules, often grouped together on one side of the cytoplasm. (b) Mitotic mast cell. (Wright–Giemsa stain; original magnification X500)
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21.71 Fine-needle aspirate from a mast cell tumour in a dog. Several eosinophilic elongated structures are present in the aspirate. These are collagen fibrils and suggest collagenolysis caused by the degranulation of the mast cells. (Wright–Giemsa stain; original magnification X100)
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21.72 (a) Mast cell tumour in the temporal area of a Siamese cat. (b) Mast cells contain rare intracytoplasmic purple granules and have a vacuolated appearance (atypical subtype, previously classified as histiocytic type). (Wright–Giemsa stain; original magnification X1000) (a, © D Berlato, AHT; b, © R Powell, PTDS)
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21.73 Fine-needle aspirate from a plasma cell tumour in a dog. Plasma cells have eccentric nuclei with clumped chromatin and a moderate amount of deeply basophilic cytoplasm, often showing a perinuclear halo. (Wright–Giemsa stain; original magnification X500) (Courtesy of E Villiers)
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21.74 Fine-needle aspirate from a plasma cell tumour in a dog. Plasma cells display marked features of atypia (multinucleation, anisokaryosis, atypical mitosis). One Mott cell containing multiple Russell bodies (immunoglobulins) is also seen (arrowed). (Wright–Giemsa stain; original magnification X500) (Courtesy of E Villiers)
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21.75 (a) Lymphoma in the buccal mucocutaneous junction of a Maltese. (b) Histological examination shows extensive neoplastic lymphoid cells infiltrating the epidermis and dermis. (Haematoxylin and eosin-stain; original magnification X40). (c) Cytological examination of the lesion shows high numbers of small lymphoid cells and small numbers of neutrophils. These lesions are frequently ulcerated and concurrent inflammation is common. (Wright–Giemsa stain; original magnification X500)
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21.76 Fine-needle aspirate from a transmissible venereal tumour on the prepuce of a stray dog. The neoplastic cells have large round nuclei with granular chromatin and a small round nucleolus. The cytoplasm contains typical punctate clear vacuoles. (Wright–Giemsa stain; original magnification X1000)
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21.77 Incidental aspiration of a submandibular salivary gland is not uncommon while attempting aspiration of the submandibular lymph node. Both secretory and non-secretory salivary epithelial cells are present in the smear. Secretory cells have abundant and heavily vacuolated cytoplasm. Non-secretory cells are cuboidal. (Wright–Giemsa stain; original magnification X100)
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21.78 Normal lymph node in a dog. (a) Tissue section. Cortical area containing lymphoid nodules composed mostly of small lymphocytes. (Haematoxylin and eosin stain; original magnification X40). (b) The fine-needle aspirate from the same lymph node is characterized by the prevalence of small lymphocytes. (Wright–Giemsa stain; original magnification X500) (a, Courtesy of L Aresu, University of Padua)
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21.79 Fine-needle aspirate from an enlarged submandibular lymph node from a cat with hypereosinophilic syndrome. Increased numbers of eosinophils and one granulated mast cell are seen together with small lymphocytes. (Wright–Giemsa stain; original magnification X500)
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21.80 Fine-needle aspirate from a mesenteric lymph node of a cat with feline infectious peritonitis (FIP). Increased numbers of macrophages and neutrophils are seen in this preparation, supporting a diagnosis of pyogranulomatous lymphadenitis. (Wright–Giemsa stain; original magnification X500)
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21.81 Hyperplastic lymph node. (a) Tissue section. Cortical area containing multiple reactive lymphoid follicles composed of two zones: a dark zone with a thin rim of small lymphocytes, and a light middle zone composed of larger lymphoid cells. (Haematoxylin and eosin stain; original magnification X40). (b) Fine-needle aspirate from the same lymph node. The majority of the cells are small lymphocytes with a lower but significant percentage of intermediate to large forms. (Wright–Giemsa stain; original magnification X500) (a, Courtesy of L Aresu, University of Padua)
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21.82 (a) Fine-needle aspirate from an enlarged lymph node from a dog with reactive lymphoid hyperplasia. The majority of the cells are small lymphocytes with a lower percentage of intermediate forms and a large lymphoid cell in the centre of the picture. (b) Fine-needle aspirate from an enlarged lymph node from a dog with plasma cell hyperplasia. Increased numbers of plasma cells are present in the aspirate. This is usually associated with antigen stimulation. (Wright–Giemsa stain; original magnification X500)
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21.84 Lymph node from a dog with lymphoma. (a) Tissue section. Dense and diffuse infiltration of neoplastic lymphoid cells effacing the normal architecture. No appreciable difference between cortex and medulla is present. (Haematoxylin and eosin stain; original magnification X100). (b) The fine-needle aspirate shows that the majority of the lymphoid cells are intermediate to large in size. A mitotic figure is present (arrowed). Frequent lymphoglandular bodies are seen in the background. (Wright–Giemsa stain; original magnification X500) (a, Courtesy of L Aresu, University of Padua)
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21.85 Fine-needle aspirate of a mediastinal mass in a dog with lymphoma. Lymphoid cells are intermediate to large in size and have a moderate amount of lightly basophilic cyotplasm containing small intracytoplasmic purple granules. This is consistent with large granular lymphoma. (Wright–Giemsa stain; original magnification X500)
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21.86 Fine-needle aspirate of a lymph node from a dog with large B-cell lymphoma. There are a few macrophages containing amorphous phagosomes (tingible body: phagocytosed cellular debris). Tingible body macrophages are frequently seen in high-grade lymphoma. They may also be observed in hyperplastic and reactive processes. (Wright–Giemsa stain; original magnification X500)
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21.87 Fine-needle aspirate from an enlarged lymph node of a dog with a small cell lymphoma. The lymphoid cells are small, with a small amount of basophilic cytoplasm, often extending away from the nucleus (hand mirror cells). (Wright–Giemsa stain; original magnification X500)
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21.88 Metastatic epithelial tumours. (a) Aspirate from a mesenteric lymph node from a dog with metastatic intestinal adenocarcinoma. A cluster of epithelial cells with large nuclei is present. (b) Aspirate from a submandibular lymph node from a cat with metastatic squamous cell carcinoma. Three large atypical squamous epithelial cells with vacuolated cytoplasm are present. (Wright–Giemsa stain; original magnification X500) (Courtesy of E Villiers)
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21.89 Fine-needle aspirate of a prescapular lymph node from a dog with metastatic soft tissue sarcoma. Neoplastic cells are plump and spindle shaped. Only low numbers of residual small lymphocytes are seen scattered around in the slide. (Wright–Giemsa stain; original magnification X500)
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21.90 Fine-needle aspirate of a prescapular lymph node from a dog with a metastatic mast cell tumour. Numerous mast cells with heavily granulated cytoplasm are present. (Wright–Giemsa stain; original magnification X200)
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21.91 Fine-needle aspirate of a submandibular lymph node from a dog with a metastatic oral melanoma. Note the presence of cells containing green-black melanin granules. (Wright–Giemsa stain; original magnification X500)
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21.92 (a–b) Fine-needle aspirates from the mass in the ventral region of the neck. In (b) note the extracellular pinkish colloid between the cells. (Wright–Giemsa stain; original magnification (a) X1000, (b) X500)
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21.93 Wet preparation of the urine sediment. (Original magnification X400)
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21.94 Vaginal smear showing a fungal hypha and associated fragments (arrowed). (Wright–Giemsa stain; original magnification X500)
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21.95 Fine-needle aspirate from the left medial iliac lymph node showing lymphocytes and macrophages (*). (Wright–Giemsa stain; original magnification X500)

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