Cytologic interpretation of canine cerebrospinal fluid samples with low total nucleated cell concentration,with and without blood contamination

Background: Cerebrospinal fluid (CSF) is potentially altered by iatrogenic blood contamination at the time of sampling due to the addition of blood‐associated leukocytes and protein. Objectives: The objective of this study was to assess whether protein concentration, neutrophil percentage, and the presence of activated macrophages, reactive lymphocytes, or eosinophils in CSF samples with low total nucleated cell concentration (TNCC) are affected by blood contamination or associated with central nervous system (CNS) disease. Methods: Case records from the Royal Veterinary College Diagnostic Laboratory were searched retrospectively for dogs with CSF having ≤5 TNCC/μL. TNCC, RBC, and protein concentrations; neutrophil percentage; and the presence of activated macrophages, reactive lymphocytes, and eosinophils were recorded. Results of magnetic resonance imaging (MRI) also were recorded as a marker of CNS disease. Results: Of 906 cases evaluated, 106 (12%) had blood contamination (>500 RBCs/μL) in CSF. Protein concentration and neutrophil percentage were significantly higher and the presence of eosinophils was more likely in blood contaminated vs noncontaminated samples. Non‐blood‐contaminated samples with activated macrophages or reactive lymphocytes had higher protein concentrations and neutrophil percentages, and those with activated macrophages were more likely to have a positive finding on MRI. Conclusions: Protein concentration, neutrophil percentage, and the presence of eosinophils are significantly affected by blood contamination in canine CSF having low TNCC. Activated macrophages and reactive lymphocytes are not affected by blood contamination, however, and may be useful in identifying dogs with CNS abnormalities.     

Determination of immunoglobulin A concentrations in the serum and cerebrospinal fluid of dogs: an estimation of its diagnostic value in canine steroid-responsive meningitis-arteritis

Previous studies on canine steroid-responsive meningitis-arteritis (SRMA) suggested that elevation of immunoglobulin A (IgA) concentrations in both serum and cerebrospinal fluid (CSF) is specific for SRMA throughout the different disease stages. Recent studies however have raised concerns about the value of this test. The purpose of this study was to investigate the diagnostic value of IgA concentration testing in paired CSF and serum samples. IgA concentrations of 525 paired canine CSF and serum samples were evaluated. Samples were obtained from dogs with SRMA (n=311) and dogs with miscellaneous conditions (n=214) such as other central nervous system (CNS) inflammatory diseases (n=34), CNS tumours (n=46), idiopathic epilepsy (n=42), intervertebral disc disease (n=46) and non-CNS diseases (n=46). Serum IgA concentrations were significantly higher in dogs with untreated SRMA compared to those with other diseases. IgA CSF concentrations were significantly higher in dogs with SRMA compared to other disease categories, with the exception of inflammatory CNS disease. The sensitivity for IgA concentrations in serum and CSF was 91% with a specificity of 78%. Analysis of 525 paired samples confirmed that IgA concentrations were higher in dogs with SRMA. Calculation of the diagnostic value of IgA concentration confirmed that the test is highly sensitive for SRMA. Testing paired CSF and serum samples for IgA is still recommended for the diagnosis of suspected cases of SRMA.     

Transglutaminase 2: a novel autoantigen in canine idiopathic central nervous system inflammatory diseases

Necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE) and granulomatous meningoencephalomyelitis (GME) are common idiopathic inflammatory central nervous system (CNS) diseases with unknown etiology in dogs. We previously showed that IgG autoantibodies in the cerebrospinal fluid (CSF) of NME cases reacted to unknown brain proteins as well as to glial fibrillary acidic protein (GFAP). In the present report, we evaluated the autoantibodies against transglutaminase2 (TG2) in the canine CNS diseases. CSF samples obtained from dogs with NME (n=19), NLE (n=7), GME (n=11) and miscellaneous CNS diseases (n=12) were subjected. CSFs from 20 healthy dogs were used as controls. Indirect fluorescent antibody test on the canine cerebrum revealed astrocyte-binding IgG in the CSF of NME. After absorption of the CSF with bovine GFAP, the CSF still possessed the reactivity to astrocytes. Double-color staining showed clear colocalization of the autoantibodies and anti-human TG2 rabbit polyclonal IgG. An immunoblot assay against human recombinant TG2 revealed anti-TG2 IgG in the CSF from dogs with NME, NLE and GME. The CSF of canine idiopathic encephalitis cases, notably of NME, tended to show high ELISA OD values against human recombinant TG2 compared to healthy controls. The presence of anti-TG2 autoantibodies in the CSF may contribute to the elucidation of the etiology of canine NME, NLE and GME.     

Beta-2-microglobulin levels in the cerebrospinal fluid of normal dogs and dogs with neurological disease

BACKGROUND: Cerebrospinal fluid (CSF) analysis is the basis for establishing a diagnosis of central nervous system (CNS) inflammation. However, the information provided by routine CSF analysis is limited. Determination of CSF beta-2-microglobulin (beta2m) concentration has been used diagnostically in humans to identify inflammatory CNS disease; we hypothesized that it may have similar value in dogs. OBJECTIVES: The objective of this study was to measure (beta2m concentration in the CSF of clinically healthy dogs and compare the values to those observed in dogs with inflammatory CNS disease and intervertebral disc disease (IVDD). METHODS: CSF was collected from 10 clinically healthy laboratory dogs and 11 dogs each with inflammatory CNS disease and IVDD. Routine CSF analysis was performed, and (beta2m concentration was measured by ELISA. CSF (beta2m concentration and CSF:serum (beta2m ratio were compared between groups by ANOVA. Linear relationships between CSF total nucleated cell count (TNCC), RBC count, total protein concentration, and (beta2m concentration were assessed by regression analysis. RESULTS: The mean (+/- SD) CSF (beta2m concentration in clinically healthy dogs was 0.36 (+/- 0.05 microg/mL (cisternal) and 0.40 (+/- 0.07 microg/mL (lumbar). Median CSF (beta2m concentration in dogs with IVDD (0.46 microg/mL) and inflammatory CNS disease (0.85 microg/mL) differed from that of controls (0.36 microg/mL; P=.002). The concentration also differed between the 2 disease groups (P=.01). Five dogs with inflammatory CNS disease had CSF:serum (beta2m ratios >1. A correlation was identified between TNCC and (beta2m concentration (r=0.69, P=.0003). CONCLUSIONS: CSF (beta2m concentration is higher in dogs with IVDD and inflammatory CNS disease, with highest values seen with inflammatory disease. This may be attributed in part to the correlation between CSF (beta2m concentration and TNCC, but also may reflect intrathecal immune activation.     

Cerebrospinal Fluid Eosinophilia in Dogs

Background: Marked eosinophilic meningitis or meningoencephalomyelitis (EME) is rarely reported in dogs and the cause is usually undetermined. Long-term prognosis for dogs with cerebrospinal fluid (CSF) eosinophilia is variable.
Animals: Twenty-three client-owned dogs.
Methods: Retrospective case series. Dogs with eosinophilic CSF, defined as total nucleated cell count (TNCC) >3 cells/μL with >20% eosinophils, were identified by a computerized search of all dogs having cisternal and/or lumbar CSF analyzed as part of the diagnostic workup between 1992 and 2007.
Results: TNCC in CSF ranged from 4 to 4,740 cells/μL (median 84 cells/μL, reference range ≤3 cells/μL), with 22 to 95% (median 78%) eosinophils in the differential count. An infectious agent was identified on necropsy in 4 of 23 (17%) dogs ( Cryptococcus neoformans [n = 2], Neospora caninum [n = 1], and Baylisascaris procyonis [n = 1]). Each of these dogs had progressive neurologic deterioration. Sixteen dogs had idiopathic EME. Magnetic resonance imaging (MRI) findings were abnormal in 7 of 13 dogs with EME; 2 dogs had focal lesions and 5 dogs had multifocal lesions. Clinical signs in 12 of 16 (75%) dogs with idiopathic EME resolved with prednisone treatment. Three dogs with acute intervertebral disc herniations recovered after decompressive surgery alone.
Conclusions: Idiopathic EME is a common cause of eosinophilic pleocytosis in dogs. MRI findings are variable. Infectious causes of EME were less common and had a poor prognosis.     

Lymphocyte subset distribution in steroid responsive meningitis-arteriitis in comparison to different canine encephalitides

Steroid responsive meningitis-arteriitis (SRMA) is a well-known disease in dogs, but the aetiology and pathogenesis are not yet understood. In the peripheral blood an overrepresentation of B cells was found. In the present study we therefore evaluated the distribution of lymphocyte subsets in SRMA in paraffin-embedded tissue sections directly at the lesion sites and compared the results to different canine encephalitides. An intriguing finding was that the B cell/T cell distribution varied depending on the aetiology of the disease: in viral encephalitides, T cells were the predominant cell population in perivascular cuffs, whereas in protozoal and bacterial diseases B cells prevailed. In SRMA an overrepresentation of B cells occurred in meningeal lesions, as already found in the peripheral blood. The distribution of lymphocyte subsets was similar to bacterial and protozoal diseases and was not a unique phenomenon for this specific inflammatory lesion in the canine central nervous system (CNS). Multiple mechanisms seem to be responsible for recruitment and activation of different leukocyte subsets after alteration of the CNS tissue by an environmental factor. A specific finding in SRMA was that the distribution of T and B cells depended also on the lesion site. In contrast to meningeal lesions, in inflamed arteries T cells were the only lymphocyte population found. In these vessels, diffuse infiltration with immunoglobulins was revealed. Inactivated or resting lymphocytes and large granular lymphocytes occurred in each of the diseases examined. These similarities between SRMA and infectious CNS diseases of the dog support earlier suggestions that the disease is somehow triggered by a hitherto unknown environmental factor which leads to the dysregulation of the immune system.     

Clinical, cerebrospinal fluid, and histological data from thirty-four cats with primary noninflammatory disease of the central nervous system.

The purpose of this study was to report the clinical, cerebrospinal fluid (CSF), and histological data derived from a study of 34 cats with noninflammatory central nervous system (CNS) disease, and to report the activities of the enzymes lactate-dehydrogenase (LDH), aspartate transferase (AST), and creatine kinase (CK) in the CSF from 15 cats with a variety of CNS diseases. The cats were part of a study of 61 cats that were admitted to two university clinics because of signs of CNS disease. The most frequent noninflammatory diseases were neoplasia (n = 12) and ischemic encephalopathy (n = 4). The majority of cats with CNS neoplasia had a mild increase in CSF protein concentration (less than 1 g/L [100 mg/dL]), an increased percentage of neutrophils or lymphocytes, and a normal total white cell count. Cats with ischemic encephalopathy (IE) had a mild to moderate increase in CSF protein concentration (< or = 2 g/L [200 mg/dL]) and a mild increase in white cell count (< or = 10 cells/microL) with an increased percentage of lymphocytes. The enzymes LDH, AST, and CK in the CSF were not sensitive indicators of chronic CNS disease. The CSF differential cell count was frequently abnormal when the total white cell count was normal, and blood contamination in the CSF samples was a frequent problem that had to be considered in the interpretation of the results. The history, signalment, and clinical signs, when combined with the CSF findings, were valuable in the diagnosis of noninflammatory CNS disease.     

The role of pro- and anti-inflammatory cytokines in the pathogenesis of spontaneous canine CNS diseases

Dogs are comparatively frequently affected by various spontaneously occurring inflammatory and degenerative central nervous system (CNS) conditions, and immunopathological processes are a hallmark of the associated neuropathology. Due to the low regenerative capacity of the CNS a sophisticated understanding of the underlying molecular basis for disease initiation, progression and remission in canine CNS diseases represents a prerequisite for the development of novel therapeutical approaches. In addition, as many spontaneous canine CNS diseases share striking similarities with their human counterpart, knowledge about the immune pathogenesis may in part be translated for a better understanding of certain human diseases. In addition to cytokine-driven differentiation of peripheral leukocytes including different subsets of T cells recent research suggests a pivotal role of these mediators also in phenotype polarization of resident glial cells. Cytokines thus represent the key mediators of the local and systemic immune response in CNS diseases and their orchestration significantly decides on either lesion progression or remission. The aim of the present review is to summarize the growing number of data focusing on the molecular basis of the immune response during spontaneous canine CNS diseases and to detail the effect of cytokines on the immune pathogenesis of selected idiopathic, infectious, and traumatic canine CNS diseases. Steroid-responsive meningitis arteritis (SRMA) represents a unique idiopathic disease of leptomeningeal blood vessels characterized by excessive IgA secretion into the cerebrospinal fluid. Recent reports have given sophisticated insights into the cytokine-driven, immune-mediated pathogenesis of SRMA that is characterized by a biased T helper 2 cell response. Canine distemper associated leukoencephalitis represents an important spontaneously occurring disease that allows investigations on the basic pathogenesis of immune-mediated myelin loss. It is characterized by an early virus-induced up-regulation of pro-inflammatory cytokines with chronic bystander immune-mediated demyelinating processes. Lastly, canine spinal cord injury (SCI) shares many similarities with the human counterpart and most commonly results from intervertebral disk disease. The knowledge of its pathogenesis is largely restricted to experimental studies in rodents, and the impact of immune processes that accompany secondary injury is discussed controversially. Recent investigations on canine SCI highlight the pivotal role of pro-inflammatory cytokine expression that is paralleled by a dominating reaction of microglia/macrophages potentially indicating a polarization of these immune cells into a neurotoxic and harmful phenotype. This report will review the role of cytokines in the immune processes of the mentioned representative canine CNS diseases and highlight the importance of cytokine/cytokine interaction as a useful therapeutic target in canine CNS diseases.     

Automated flow cytometric cell count and differentiation of canine cerebrospinal fluid cells using the ADVIA 2120

Background: Microscopic cell counts in cerebrospinal fluid (CSF) are time-consuming and prone to imprecision. The recently introduced automated hematology analyzer ADVIA 2120 offers an automated cell count and differential for CSF in the veterinary software mode based on laser light scatter and absorbance measurements. Objectives: The purpose of this study was to evaluate the precision, linearity, and accuracy of the ADVIA 2120 CSF assay. Methods: Sixty-seven CSF samples were analyzed on the ADVIA 2120 and total nucleated cell counts (TNCC) and RBC counts were compared with the hemocytometer results. In 21 samples with TNCC >5/muL, ADVIA 2120 results were compared with 100-300 cell manual differentials performed on cytocentrifuged preparations. Statistical analysis included Spearman's rank correlation, Passing-Bablok regression, and Bland-Altman analysis. Results: Repeatability (intra-assay) coefficients of variation (CVs) ranged from 4.19% to 25.94%. Interassay CVs ranged from 2.56% to 28.67%. Accurate results within 30% were achieved for TNCC up to 4000/muL. Except for low TNCC, deviation from the expected value was higher (TNCC of 8/muL instead of 4/muL). The following correlation coefficients (r) and biases were achieved compared with the reference method: r=.90 and bias 2.3/muL for TNCC; r=.88 and bias 32.0/muL for RBC counts; r=.86 and bias +/-13.4% for mononuclear and polymorphonuclear cell percentages; r=.88 and bias -6.1% for lymphocyte percentage; r=.56 and bias 19.4% for monocyte percentage; and r=.75 and bias -9.7% for neutrophil percentage. Conclusion: Our results demonstrated that the automated ADVIA 2120 CSF assay generally compares well with reference methods although there are some limitations for the automated monocyte count and for samples with only mild pleocytosis.     

Glial fibrillary acidic protein (GFAP) and anti-GFAP autoantibody in canine necrotising meningoencephalitis

To establish clinical markers for canine necrotising meningoencephalitis (NME) and to elucidate its pathogenesis, glial fibrillary acidic protein (GFAP) and anti-GFAP autoantibodies were measured in the cerebrospinal fluid (CSF) of 32 dogs with NME, 23 dogs with other inflammatory central nervous system (CNS) diseases, 27 dogs with miscellaneous CNS diseases and 25 healthy dogs, including five pugs. The dogs with NME had the highest levels of anti-GFAP autoantibodies. The diagnostic sensitivity and specificity of anti-GFAP autoantibodies for NME were 91 per cent and 73 per cent, respectively. Some of the dogs with NME and the healthy pugs, had high CSF concentrations of GFAP, suggesting a breed-specific fragility of astrocytes. The leakage of GFAP and the development of autoimmunity may be key to understanding the pathogenesis of NME.     

Immunomagnetic isolation of canine circulating endothelial and endothelial progenitor cells

Background: Increased concentrations of circulating endothelial cells (CECs) are thought to be a biomarker of vascular injury in human patients with cardiovascular disease, neoplasia, vasculitis, sickle cell anemia, shock, and sepsis. Immunomagnetic isolation is a technique currently used to enumerate human CECs and can detect low numbers of cells. Objectives: The purpose of this study was to determine whether a standard protocol for immunomagnetic isolation could be used to obtain and enumerate CECs and a subpopulation of endothelial progenitor cells (EPCs) from canine whole blood. Methods: Cultured canine aortic endothelial cells were stained immunohistochemically with von Willebrand factor to verify morphology and number. Using magnetic beads conjugated with anti‐CD146, CECs/EPCs were isolated in culture and in canine whole blood. CD146‐positive cells were stained with fluorescein‐conjugated Ulex europaeus agglutinin 1 (UEA‐1) to confirm endothelial origin and cells were counted manually using a fluorescent microscope. The method was then applied to EDTA‐anticoagulated whole blood samples from 10 healthy client‐owned dogs. Results: The anti‐CD146–coated magnetic beads (>5/cell) bound the cultured canine aortic endothelial cells. Only rare UEA‐1–positive cells were obtained from whole blood, while >85–90% of cultured canine aortic endothelial cells were UEA‐1 positive. The percentage recovery of cultured canine aortic endothelial cells was >86%. CECs in canine whole blood had >8 beads attached to the surface and were 10–40 μm in size. Using immunomagnetic isolation, 43.4 ± 15.6 CECs/mL (range 24–70/mL) were isolated from canine whole blood samples. Conclusions: Immunomagnetic isolation is an acceptable method for enumerating canine CECs/EPCs in whole blood. Further studies are warranted to evaluate the clinical significance of CEC/EPC concentration in different canine diseases.     

Association of cerebrospinal fluid analysis findings with clinical signs and outcome in acute nonambulatory thoracolumbar disc disease in dogs

Background: Cerebrospinal fluid (CSF) pleocytosis recently was associated with the severity of neurologic signs in dogs with intervertebral disc disease (IVDD). Hypothesis/Objectives: To look for an association among CSF cell counts, total protein concentration, and severity of neurologic signs at presentation with outcome in dogs with acute thoracolumbar IVDD. Our hypothesis was that CSF total nucleated cell count (TNCC) and percentage cell types would be associated with the severity of spinal cord damage and therefore with both the presenting clinical signs and the prognosis of affected dogs. Animals: Fifty‐four dogs with acute nonambulatory thoracolumbar IVDD were evaluated. Methods: Retrospective study. Signalment, neurologic grade, CSF TNCC, protein concentration, red blood cells count and differential cell percentages, and short‐ and long‐term outcomes were evaluated. Results: CSF pleocytosis (>5 cells/μL) was present in 54% of dogs and was positively associated with neurologic grade at presentation and with postoperative time to regaining ambulation. Neutrophils were observed most frequently. The percentage of CSF macrophages and macrophage to monocyte ratio were higher (P= .001, for both) in dogs presented without deep pain sensation (DPS) that did not regain ambulation. Receiver operator characteristics curve analysis yielded a cut‐off point of 13% macrophages with a sensitivity and specificity of 100 and 83%, respectively, for prediction of a negative outcome. Conclusions and Clinical Importance: CSF pleocytosis is positively associated with the severity of spinal cord damage in dogs with thoracolumbar IVDD. The percentage of CSF macrophages can be used as a prognostic indicator for regaining ambulation in dogs that have lost DPS.     

Evaluation of a commercially available human C-reactive protein (CRP) turbidometric immunoassay for determination of canine serum CRP concentration

Background: Serum C-reactive protein (CRP) is an acute phase marker in dogs that is useful for the diagnosis and monitoring of inflammatory disease. Rapid, reliable, and automated assays are preferable for routine evaluation of canine serum CRP concentration.
Objective: The aim of this study was to evaluate whether canine serum CRP concentration could be measured reliably using an automated turbidometric immunoassay (TIA) designed for use with human serum.
Methods: A commercially available TIA for human serum CRP (Bayer, Newbury, UK) was used to measure canine serum CRP concentration. Cross-reactivity of antigen was evaluated by the Ouchterlony procedure. Intra-and interassay imprecision was investigated by multiple measurements on canine serum samples and serum pools, respectively. Assay inaccuracy was investigated by linearity under dilution and comparison of methodologies (canine CRP ELISA, Tridelta Development Ltd, Kildare, UK). Then the assay was applied to serum samples from 14 clinically healthy dogs, 11 dogs with neoplasia, 13 with infections, 8 with endocrine or metabolic diseases, and 10 with miscellaneous diseases.
Results: Cross-reactivity between canine serum CRP and the anti-human CRP antibody was found. Intra-and interassay imprecision ranged from 5.2% to 10.8% and 3.0% to 10.2%, respectively. Serum CRP concentration was measured in a linear and proportional manner. There was no significant disagreement and there was linear correlation of the results in the comparison of methodologies, except for a slight proportional discrepancy at low CRP concentrations (<10 μg/mL). Dogs with infections had a significantly higher concentration of serum CRP than did all other dogs, and dogs with neoplasia had a significantly higher concentration of serum CRP than did clinically healthy dogs.
Conclusions: Canine serum CRP concentration can be measured reliably using the commercially available TIA designed for human CRP.     

Cerebrospinal fluid creatine kinase activity in horses with central nervous system disease: 69 cases (1984-1989)

The CSF creatine kinase (CK) activity was determined in 70 CSF samples from 69 horses with CNS disease. Abnormal values (greater than or equal to 1 IU/L) were determined from 32 CSF samples, and normal values (less than 1 IU/L) were found in 38 samples. Increased CK activity was most frequently associated with a diagnosis of equine protozoal myelitis; CK activity was not increased in 11 horses with cervical compressive myelopathy. Other diagnoses, in which CSF CK activity was increased included trauma (n = 1), idiopathic epilepsy (n = 2), botulism (n = 2), articular facet fracture (n = 1), intervertebral disk protrusion (n = 1), and toxemia (n = 1).     

Analytical validation of the Sysmex XT‐2000iV for cell counts in canine and feline effusions and concordance with cytologic diagnosis

Background: The Sysmex XT‐2000iV is a hematology analyzer that combines laser and impedance technology. Its usefulness for determining cell counts in canine and feline intracavitary effusions has not yet been studied. Objectives: The objectives of this study were to evaluate the analytical performance of the Sysmex XT‐2000iV for cell counts in effusions from dogs and cats, and to assess correlation with an impedance counter and concordance with diagnoses based on cytologic findings. Methods: Effusions (43 pleural, 23 peritoneal, 6 pericardial) were analyzed from 32 dogs and 34 cats. Total nucleated cell count (TNCC), HCT, and RBC count were determined on the Sysmex and compared with those obtained on an impedance counter (Hemat 8, SEAC). Imprecision, linearity, and limit of detection were determined for the Sysmex. An algorithm was designed using quantitative and qualitative data from the Sysmex to classify the effusions and the results were compared with diagnoses based on cytologic findings. Results: Intra‐assay and interassay coefficients of variation on the Sysmex were variable. Linearity of TNCC was ≥0.993 for dogs and cats, with the exception of effusions from cats with feline infectious peritonitis, which had delta (Δ) TNC values >3.0. In comparison with the Hemat 8, a proportional error was found for TNCC on the Sysmex. Effusion classification based on the algorithm was concordant with that obtained by cytologic examination in 43/72 (60%) samples. Discordant results usually were due to the misclassification of cells with similar morphology (such as mesothelial and carcinoma cells) in Sysmex scattergrams. Conclusion: The Sysmex XT‐2000iV provides a precise and accurate TNCC and has moderate concordance with cytologic findings for classifying canine and feline effusions. Although microscopic examination of effusions is necessary to achieve an accurate diagnosis, the Sysmex can provide preliminary information that may be helpful to cytopathologists.     

Diffuse leptomeningeal histiocytic sarcoma in the cerebrospinal fluid of 2 dogs

Two adult male castrated dogs were evaluated for progressive paraparesis and ataxia. Neurologic examination showed severe ataxia, delayed proprioceptive placement in the pelvic limbs, pain upon palpation of the lumbar spine as well as facial paresis in one dog, and decreased withdrawal reflex of the pelvic limbs in the other dog. Magnetic resonance imaging (MRI) in both dogs showed diffuse meningeal and intramedullary lesions. However, no evidence of a mass was found. Biopsies could not be performed safely due to the location of the lesions. Cerebrospinal fluid (CSF) examination revealed an inflammatory pleocytosis associated with increased protein concentration and numerous large atypical round cells, often multinucleated. Nuclear fragmentation, micronuclei, and rare atypical mitoses were observed. Immunocytochemistry revealed CD1⁺ and CD11c⁺ staining, which, in concert with the morphology confirmed the diagnosis of histiocytic sarcoma (HS). Euthanasia was elected due to poor prognosis. Histopathologic examination showed diffuse spinal and meningeal infiltration with CD18⁺ neoplastic cells, without any evidence of mass formation, which completed the diagnosis of diffuse leptomeningeal HS involving the brain and the spinal cord. Canine central nervous system (CNS) HS has been seldom reported in the literature, with only isolated cases identified on CSF cytology. The cases reported here are remarkable in describing a diffuse CNS leptomeningeal HS associated with neoplastic cells in the CSF of dogs without a tumor mass. These cases emphasize the potential critical importance of CSF analysis in providing an antemortem diagnosis of neoplasia in neurologic patients.     

Prevalence and significance of extracellular myelin‐like material in canine cerebrospinal fluid

Background: Myelin‐like material in canine cerebrospinal fluid (CSF) specimens has been attributed to demyelinating or myelomalacic conditions. In our experience, myelin‐like material is observed frequently, especially in lumbar samples, and in a variety of disease conditions. Objectives: The objective of this study was to determine if there are associations between the presence of myelin‐like material and CSF collection site, body weight, underlying disease, and patient outcome. Methods: Wright–Giemsa‐stained cytocentrifuged specimens of CSF from the cerebellomedullary cistern (n=51) and lumbar cistern (n=47) of 98 dogs with neurologic disease were evaluated retrospectively for the presence and amount of extracellular myelin‐like material. Results were compared based on collection site, body weight, type of neurologic disease, and outcome. Results: Myelin‐like material was observed in 20/98 (20%) samples and was more frequently observed in lumbar (17/47, 36%) than cerebellomedullary samples (3/51, 6%) (P=.0028). Samples from dogs <10 kg were more likely to contain myelin (14/36, 39%) compared with dogs ≥10 kg (5/60, 8%) (P=.0052). Larger amounts of myelin‐like material were observed in CSF from dogs with intervertebral disk disease compared with other diseases (P=.045). No association was found between myelin‐like material and outcome. Conclusion: The association of extracellular myelin‐like material in canine CSF samples with sampling site and body weight suggests it is more often an artifact of collection technique and anatomy rather than the result of neurologic disease. Myelin‐like material in CSF is not associated with a poorer prognosis.     

High resolution protein electrophoresis of 100 paired canine cerebrospinal fluid and serum

This study was performed to investigate the diagnostic relevance of cerebrospinal fluid (CSF) high resolution electrophoresis. The laboratory technique was applied to 100 paired samples of canine CSF and serum, with paired samples tested during the same analytical run, as recommended in human medicine. Ninety four of the dogs had a neurological disease and 6 healthy dogs served as a control group. A strong linear correlation between CSF total protein concentration and the albumin quota (AQ) was found in the control group and in the inflammatory (infectious or noninfectious), neoplastic, and miscellaneous groups: AQ = 0.015 CSF total protein--0.102, r = 0.990. This correlation suggests that an increased CSF total protein concentration can be an indicator of blood brain barrier dysfunction. The highest median AQ value was found in the aseptic suppurative meningitis group, but no statistical differences were found between this and the other groups. The AQ, calculated with this technique, did not provide any additional information. Moreover, although unexpected, the electrophoretic profiles were not characteristic of any particular disease. In conclusion, this study did not confirm high resolution electrophoresis of paired CSF and serum samples to be a valuable ancillary diagnostic tool for canine neurological diseases.     

Isolation and characterization of primary canine lens epithelial cells

OBJECTIVE: The purpose of this study was to characterize the proliferation and differentiation of primary canine lens epithelial cells (LEC) under standard culture conditions. PROCEDURE: Canine LEC were isolated by mechanical dissection of the canine globe and enzymatic digestion of the lens capsule from fresh lenses. Isolated capsules and cell suspensions were seeded in laminin-coated culture flasks. Canine LEC proliferated and formed monolayers, which could be passaged and maintained for approximately 2 weeks. Cells were characterized morphologically and cell lysates examined for expression of protein markers of epithelial origin and differentiation. RESULTS: Canine LEC exhibit morphologic characteristics of epithelial cells when cultured on laminin/lysine coated flasks. Expression of epithelial cell marker, cytokeratin 5, was highest at passage 1 and diminished with increasing passage number. Expression of gamma-crystallin, a protein found only in differentiated lens fiber cells, increased at passage 6. A laminin/lysine-coated surface supported optimal proliferation of canine LEC. Both an initial seeding density of 1 x 10(5) cells/cm(2) and culture in Dulbecco's modified essential media (DMEM) supplemented with 10% FBS supported a doubling time of less than 48 h in canine LEC. CONCLUSION: This study demonstrates that primary canine LEC retain the characteristics of lens epithelial cells prior to passage 6 under the described culture conditions and represent a suitable in vitro model for investigating lens physiology and cataractogenesis.     

Clinical, cerebrospinal fluid, and histological data from twenty-seven cats with primary inflammatory disease of the central nervous system.

The purpose of this report is to present the clinical, cerebrospinal fluid (CSF) and histological data from 27 cats with inflammatory disease of the central nervous system (CNS). The cats were part of a study of 61 cats admitted to two university clinics over an eight-year period because of signs of CNS disease. The most frequent diseases were feline infectious peritonitis (FIP) (12/27) and suspected viral disease other than FIP (10/27). Typical CSF findings in cats with FIP were a protein concentration of greater than 2 g/L (200 mg/dL) and a white cell count of over 100 cells/microL, which consisted predominantly of neutrophils. In contrast, the CSF of cats with suspected viral disease had a protein concentration of less than 1 g/L (100 mg/dL) and a total white cell count of less than 50 cells/microL. In general, cats with FIP or suspected viral disease were less than four years of age. Neurological signs were usually multifocal in cats with FIP, but focal in cats with suspected viral disease. The CSF findings were variable in five other inflammatory diseases represented. Two cats with protozoan infection had normal CSF total cell counts but abnormal differential counts. The CSF findings were invaluable in differentiating FIP from other causes of inflammatory CNS disease.