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Clinical pathology

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Abstract

Laboratory evaluation of patients presenting with neurological diseases can be challenging due to the numerous assays that may be performed and the non-specific results that are often obtained from routinely performed laboratory tests. However, when coupled with signalment, clinical findings and other ancillary diagnostic tests (e.g. imaging techniques), laboratory tests can be extremely valuable in the accurate identification of a number of conditions that affect neurological and neuromuscular function. This chapter considers standard minimum database, other biochemical assays, serology and microbiology, cerebrospinal fluid analysis.

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Figures

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3.2 Peripheral blood film from a Siamese cat with lysosomal storage disease (mucopolysaccharidosis VI). The cytoplasm of the neutrophil contains several irregularly shaped magenta inclusions, which are present due to an abnormal accumulation of substrate material that would normally be degraded by the deficient lysosomal enzyme. The cytoplasm in normal neutrophils should be virtually colourless or very pale basophilic. (Wright–Giemsa stain; original magnification X50)
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3.3 Spinal fluid from a Muntjac deer with lysosomal storage disease (GM2 gangliosidosis). The mononuclear phagocytes contain linear, eosinophilic cytoplasmic inclusions, which are present due to an abnormal accumulation of substrate material that would normally be degraded by the deficient lysosomal enzyme. (Wright–Giemsa stain; original magnification X50)
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3.4 Buffy coat film from a dog with acute anaplasmosis. The neutrophil on the right contains a round, basophilic, stippled morula (arrowed). (Wright–Giemsa stain; original magnification X100)
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3.5 Peripheral blood films from a dog with acute canine distemper virus infection showing cytoplasmic viral inclusions. Eosinophilic inclusions are visible in the cytoplasm of a reactive lymphocyte (arrowed) and an erythrocyte (top; arrowed) and a neutrophil (bottom; arrowed). These blood films have been stained with Diff-Quik. If they had been stained with Wright–Giemsa, the inclusions would have been expected to be pale aqua. (Original magnification X100)
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3.6 Spinal fluid from a dog with acute canine distemper virus. The large mononuclear cell contains a pink, oval inclusion of viral capsid proteins (arrowed). (Wright–Giemsa stain; original magnification X100)
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3.7 Thromboelastography tracings: normal (green); hypercoagulable (red); hypocoagulable (blue); and secondary fibrinolysis (black). Time is shown on the x-axis; blood clot strength is shown on the y-axis. Hypocoagulable sample: it has taken longer for the blood clot to establish than normal, which is evident by the increased length of the tracing before it bifurcates. In addition, the clot is weaker than normal, which is evident by the smaller distance between the two arms of the tracing following birfurcation, which corresponds with the force required of the torsion wire as it oscillates in the clotting blood sample (modified from ).
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3.9 The ventricular system depicting the neuroanatomical origins of CSF production and sites of absorption (modified from ). Illustration created by Allison L. Wright, MS, CMI, Athens, Georgia, USA.
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3.12 Anatomical landmarks for AO CSF collection. The dog is placed in lateral recumbency with the head flexed at 90 degrees to the neck. This flexion opens up the cerebellomedullary cistern (C) accessible via the AO space. With the head flexed at 90 degrees to the cervical spine the AO joint opens up so that the tap can be performed. The spinal needle is placed perpendicular to the cord and advanced into the cistern situated above the cord. A = wing of the atlas vertebra; B = occipital protruberance. Illustration created by Allison L. Wright, MS, CMI, Athens, Georgia, USA.
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3.13 AO CSF collection. Proper positioning. Close-up demonstrating CSF dripping from the hub of the spinal needle into the collection tube.
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3.14 Anatomical landmarks for lumbar CSF collection. The spinal needle is inserted over the spinous process of L6 and angled forward to access the CSF via the interarcuate space of L5–L6. The dorsal or ventral subarachnoid space at this site can be used to obtain samples. Illustration created by Allison L. Wright, MS, CMI, Athens, Georgia, USA.
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3.15 Comparison of leucocyte morphology in 3-day-old canine CSF samples. Aliquot with no preservative. Aliquot with hetastarch preservative. The cells in the unpreserved aliquot are swollen and karyolytic, preventing accurate cell identification and differential leucocyte count. The leucocytes in the aliquot with hetastarch are adequately preserved and recognizable as predominately mature, non-degenerative neutrophils. A single pyknotic neutrophil is also present (arrowed). (Wright–Giemsa stain; original magnification X50)
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3.16 Cytomorphology of a CSF sample collected from a horse with Eastern equine encephalitis virus. Aliquot with no preservative. Aliquot with formalin preservative. The cells in the formalin aliquot are compressed making it difficult to distinguish neutrophils from lymphocytes and to fully observe cellular details. (Wright–Giemsa stain; original magnification X50)
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3.17 Spinal fluid sedimentation chamber. The chamber was constructed from a microscope slide, a piece of filter paper with a hole punched in the centre, the barrel of a 1 ml syringe and two binder clips. The second hole in the filter paper is present for illustration. In the functioning chamber, only the hole directly under the barrel of the syringe is needed.
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3.22 Humidified Petri dish and haemocytometer. The Petri dish is humidified by placing damp, absorbent material (e.g. thin gauze or filter paper) at the bottom of the dish. The handle of a cotton tipped applicator is broken in half and placed on top of the absorbent material. The haemocytometer is loaded with CSF and placed on the two halves of the applicator handle. The lid is then closed. This prevents the CSF sample from evaporating whilst the cells settle into the grid that is etched in the haemocytometer.
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3.23 Haemocytometer grid found in the counting chambers. The cells in the centre large square and the four corner large squares from both chambers are counted (10 large squares in total). The total number of cells in the 10 large squares is equivalent to the number of cells per microlitre.
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3.24 Erythrocytes and leucocytes in a haemodilute CSF sample in one small square within a large corner square of a haemocytometer counting chamber grid. The lines etched in the haemocytometer appear white. Darker squares etched in the ocular Miller’s disc for performing reticulocyte counts are also present. Close-up image showing numerous erythrocytes and two leucocytes. The leucocytes (thick arrow) are stippled and do not transmit as much light as the erythrocytes, which typically appear as biconcave discs. Based on size, the leucocytes are likely to be lymphocytes (mononuclear phagocytes and macrophages are typically larger). A severely crenated erythrocyte (echinocyte; thin arrow) is also present. (Original magnification X20)
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3.25 Spinal fluid from a dog. Small, well differentiated lymphocytes and a minimally vacuolated, large mononuclear phagocyte (M) are visible in this sample. (Wright–Giemsa stain; original magnification X20)
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3.26 Cluster of ependymal cells in a canine spinal fluid sample. Note the eccentric nuclei and accumulation of eosinophilic, granular material in the basophilic cytoplasm. (Wright–Giemsa stain; original magnification X20)
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3.27 Spinal fluid sample from a dog demonstrating pathological haemorrhage. A large mononuclear phagocyte containing multiple phagocytosed erythrocytes (indicative of acute haemorrhage) is visible. The dark blue–green granules above the nucleus are consistent with haemosiderin pigment and suggest chronic haemorrhage. The golden rhomboid crystal is consistent with haematoidin and also indicates chronic haemorrhage. (Wright–Giemsa stain; original magnification X100)
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3.29 Spinal fluid from a dog. The neutrophil nuclei are hyperchromic and have >5 lobulations, which is consistent with hypersegmentation. (Diff-Quik stain; original magnification X50)
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3.30 Spinal fluid from a dog with rabies demonstrating lymphocytic pleocytosis. Several small, well differentiated lymphocytes are present along with a few large mononuclear cells. (Wright–Giesma stain; original magnification X20)
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3.31 Spinal fluid from a dog with granular lymphoma. Large lymphoblasts with eccentric magenta granules are visible. (Wright–Giemsa stain; original magnification X50)
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3.32 Spinal fluid from a dog with mixed cell pleocytosis. Three large mononuclear phagocytes (M), two mature non-degenerative neutrophils (N) and one small well differentiated lymphocyte (L) are visible. (Wright–Giemsa stain; original magnification X50)
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3.33 Spinal fluid from a dog with steroid-responsive meningoencephalomyelitis and neutrophilic pleocytosis. Mature non-degenerative neutrophils are present along with a lesser number of larger mononuclear phagocytes (M) and a lymphocyte (L). (Wright–Giemsa stain; original magnification X20)
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3.34 Spinal fluid from a dog with bacterial meningitis. Numerous rod-shaped bacteria are present in both the cytoplasm of the neutrophils and the extracellular background. Close-up view. (Wright–Giemsa stain; original magnification X50 and X100, respectively)
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3.35 Spinal fluid from a Golden Retriever with a very marked eosinophilic pleocytosis (21,000 WBCs/µl). Three eosinophils along with a number of small well differentiated lymphocytes and a highly vacuolated macrophage (right) are present in this non-concentrated sample. (Wright–Giemsa stain; original magnification X50)
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3.36 Spinal fluid from a dog with cryptococcal meningitis. Large organisms with thick, non-staining capsules are visible. Note that the organism in the centre is exhibiting narrow-based budding. (Diff-Quik stain; original magnification X50)
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3.37 Spinal fluid from a dog with fungal meningitis due to aspergillosis. A septate fungal hypha (arrowheads) is surrounded by large mononuclear phagocytes and neutrophils. (Wright–Giemsa stain; original magnification X100) When stained with a special silver stain, the branching fungal hyphae appear black. (Gomori methenamine silver stain; original magnification X100)
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3.38 Aspirate from an extradural feline lymphoma. Large lymphoblasts with subtly indented nuclei, smooth chromatin, indistinct nucleoli and punctate cytoplasmic vacuoles are visible along with a single well differentiated lymphocyte (right). (Wright–Giemsa stain; original magnification X100)
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3.39 Spinal fluid samples containing myelin. Free myelin is visible as a loose aggregate of circular, pink, moth-eaten material. Curvilinear, pink, moth-eaten material is present within a phagocytic vacuole of a macrophage; darker red erythrocytes are visible to the right of the macrophage. (Diff-Quik stain; original magnification X50)
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3.40 Tissue imprint from a dog with granulomatous meningoencephalomyelitis. A large neuron cell body with ill defined borders is visible with a single mature, non-degenerative neutrophil (left) and macrophage (right). (Wright–Giemsa stain; original magnification X50)
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3.41 Tissue imprint from a dog with a meningioma. The cytoplasm of the neoplastic meningeal cells is fusiform and basophilic with ill defined, wispy borders. The oval nuclei exhibit malignant features, such as anisokaryosis (variable nuclear size), open, coarse chromatin and a few prominent nucleoli. (Wright–Giemsa stain; original magnification X50)

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Atlanto-occipital CSF collection

Under general anaesthesia, the dog is placed in right lateral recumbency if the operator is right handed. An area of skin is prepared for a sterile procedure. The head of the dog is flexed at approximately 90 degrees to the cervical spine. The operator palpates the appropriate landmarks (the wings of the first cervical vertebra and the occipital protuberance) which act as a guide for the point of insertion of the needle. The spinal needle is inserted through the skin perpendicular to the spinal column and horizontal to the table surface. Once through the skin and soft tissues, the stylet of the needle can be removed. The needle is then advanced, 1 mm at a time, until a ‘flash’ of fluid appears in the hub of the needle. The fluid is allowed to drip into a sterile polypropylene container until approximately 0.5 ml is collected. (See page 47 in the Manual)

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