Effects of time, initial composition, and stabilizing agents on the results of canine cerebrospinal fluid analysis

BACKGROUND: Cerebrospinal fluid (CSF) is considered highly labile, but not all samples are analyzed immediately. Changes in the composition of CSF could potentially affect diagnostic test results and thus influence decisions about patient management. There has been little scientific inquiry into how variables such as time, initial composition, and storage conditions affect results of standard laboratory analysis of CSF. OBJECTIVES: The objectives of this study were to determine the effects of time, protein concentration, and presence or absence of exogenous stabilizing agents on standard CSF analysis results. METHODS: Thirty abnormal CSF samples from 26 dogs were evaluated. Samples were divided into aliquots comprising different treatment groups and stored at 4 degrees C. Total nucleated cell count (TNCC), differential cell count (DCC), and cell morphology were evaluated for all groups; protein concentration was measured for selected groups. Unaltered aliquots were analyzed immediately (T0Hr) and at 2, 4, 8, 12, 24, and 48 hours (T2Hr-T48Hr); aliquots with added fetal calf serum (FCS) or hydroxyethyl starch (hetastarch) were analyzed at T48Hr. RESULTS: Significant time-dependent changes were observed in DCC in unaltered samples. Mononuclear cells deteriorated more rapidly than did neutrophils. Based on microscopic examination and subjective scoring of cell morphology, cells were consistently more degenerate by T24Hr compared with T0Hr. Samples with protein concentrations > or =50 mg/dL were less susceptible to cell deterioration than those with lower protein concentrations. Adding either FCS or hetastarch improved sample stability. CONCLUSIONS: Delayed analysis of canine CSF by 4-8 hours is unlikely to alter diagnostic interpretation, especially for samples with protein concentrations > or =50 mg/dL. The likelihood of misinterpretation is higher for samples with low cellularity or low protein concentration. We provide specific recommendations for adding FCS or hetastarch to samples that will not be analyzed within 1 hour.     

Measurement of myelin basic protein in the cerebrospinal fluid of dogs with degenerative myelopathy

BACKGROUND: Analysis of cerebrospinal fluid (CSF) is part of a routine clinical workup in veterinary patients when neurologic disease is suspected. However, knowledge of particular protein markers of disease in CSF is limited. The concentration of myelin basic protein (MBP) in CSF is used as a biochemical marker in humans to evaluate demyelinating lesions in the central nervous system (CNS). OBJECTIVE: The purpose of this study was to evaluate an ELISA for determination of MBP concentration in the CSF of German shepherd dogs with degenerative myelopathy (GSDM). METHODS: Cross-reactivity of the anti-human polyclonal antibody used in a commercial ELISA (Active MBP ELISA, Diagnostic Systems Laboratories Inc, Webster, TX, USA) was tested with canine MBP by immunoblotting. CSF samples were collected from both the cisterna magna and the lumbar cistern of 8 clinically healthy control dogs and 8 German shepherd dogs clinically diagnosed with GSDM. MBP concentrations were measured in all CSF samples using the ELISA. RESULTS: The mean MBP concentration in CSF from the lumbar cistern of dogs with GSDM (3.13 -/+ 0.46 ng/mL) was significantly higher than that in the cisterna magna (0.70 -/+ 0.06 ng/mL) and from both cisternal (0.47 -/+ 0.07 ng/mL) and lumbar (0.94 -/+ 0.37 ng/mL) samples from control dogs. CONCLUSION: The MBP ELISA has potential as a supplemental test of CSF to diagnose demyelinating disorders in dogs.     

Evaluation of the ADVIA 120 for analysis of canine cerebrospinal fluid

BACKGROUND: Conventional techniques for canine cerebrospinal fluid (CSF) analysis require large sample volumes and are labor intensive and subject to operator variability. Objective: The purpose of this study was to evaluate the ADVIA120 CSF assay for analysis of canine CSF samples. METHODS: CSF samples collected from 36 healthy control dogs and 17 dogs with neurologic disease were processed in parallel using the automated assay and established manual methods using a hemocytometer and cytocentrifugation. Results for WBC (total nucleated cell) count, RBC count, and differential nucleated cell percentages were compared using Spearman rank correlation coefficients and Bland-Altman bias plots. RESULTS: Correlation coefficients for WBC and RBC counts were 0.57 and 0.83 for controls, and 0.92 and 0.94 for ill dogs, respectively. Coefficients for the percentages of neutrophils, lymphocytes, and monocytes were 0.53, 0.26, and 0.12 for controls and 0.77, 0.92, and 0.70 for dogs with neurologic disease. When data were combined (n=53), correlation coefficients were 0.86 and 0.91 for WBC and RBC counts, and 0.63, 0.43, and 0.30 for neutrophil, lymphocyte, and monocyte percentages. A 9.5% positive bias and 7.0% negative bias were obtained for the ADVIA 120 CSF assay for lymphocytes and macrophages in dogs with neurologic disease with Bland-Altman analysis. A 12.2% positive bias was found for lymphocyte percentage in dogs with neurologic disease. CONCLUSIONS: Manual and automated CSF assays had moderate to excellent correlation for WBC and RBC concentrations, but results were more variable for differential cell percentages. The ADVIA assay may be more useful for assessment of canine CSF with adjustment of cell differentiation algorithms.     

Blood–brain‐barrier disruption in chronic canine hypothyroidism

Background: Central nervous system (CNS) manifestations of hypothyroidism have been associated with cerebrovascular complications. Reports of cerebrospinal fluid (CSF) abnormalities are rare in hypothyroid dogs. Objective: The aim of this study was to determine if chronic hypothyroidism causes blood–brain‐barrier (BBB) abnormalities that are detectable using indirect CSF biomarkers. Methods: The study included 18 normal, euthyroid, female mixed‐breed dogs. Hypothyroidism was induced by ¹³¹iodine administration in 9 dogs; 9 served as untreated controls. Evaluations included physical and neurologic examination, complete CSF analysis, serum and CSF protein electrophoresis, measurement of plasma vascular endothelial growth factor (VEGF) and serum S‐100B concentrations, and calculation of CSF albumin quota (AQ) and were conducted at baseline and 6, 12, and 18 months after induction of hypothyroidism. Data were analyzed using repeated measures ANOVA. Results: At baseline, differences between groups were not detected for any variable. Throughout the study, controls dogs remained free of neurologic disease and had test variables that remained within reference intervals. Two hypothyroid dogs developed CNS signs during the study, and evidence of cerebrovascular disease was found at necropsy. At 12 and 18 months, the CSF total protein, VEGF, S‐100B, and fractional albumin concentrations, and AQ were significantly higher (P<.04) in hypothyroid dogs than controls. Among test variables assayed in serum or plasma, the only significant difference was a higher S‐100B concentration in hypothyroid dogs (P=.003) at 18 months. Conclusions: BBB integrity is disrupted in chronic hypothyroidism. Significant increases in CSF concentrations of VEGF and S100‐B in hypothyroid dogs indicate dysfunction in both endothelial and glial elements of the BBB.     

Analysis of radiosensitivity of cancer stem‐like cells derived from canine cancer cell lines

Cancer stem‐like cells (CSCs) are self‐renewing cells comprising a small subpopulation in tumours, and generate differentiated progeny through asymmetric division. It has been shown that CSCs are resistant to ionizing radiation, and this feature could be one of the mechanisms of tumour recurrence after radiation therapy. Much attention has been focused on to target CSCs; however, difficult of isolating CSCs and lack of knowledge on their radiosensitivity have limited this kind of research in veterinary medicine. In the present study, sphere‐forming cells (SC), cultured using sphere formation method, were isolated from four type of canine tumour cell lines and evaluated if they have CSCs‐like properties by expression of CSCs markers (real‐time polymerase chain reaction) and capacity of tumorigenesis (xenograft transplantation in nude mice), and were assessed radiosensitivity (clonogenic survival assay) and DNA repair kinetics (immunofluorescence staining for p53‐binding protein 1) after X‐ray irradiation in comparison with the corresponding normal adherent culture cells (AC). All SCs were isolated using sphere formation and showed high gene expression of CD133 and tumorigenic ability as compared with AC. All SCs were significantly resistant against X‐ray irradiation as compared with AC. In addition, the amount of DNA double‐strand breaks after X‐ray irradiation were significantly lower in SC compared with the corresponding AC. These results indicate that SC isolated through sphere formation possess CSCs‐like characteristics and CSCs are important factor that affect radiosensitivity in canine tumours. In addition, radioresistance of CSCs may depend on reaction of DNA double‐strand break after X‐ray exposure.     

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.     

Detection of oligoclonal bands in cerebrospinal fluid from German Shepherd dogs with degenerative myelopathy by isoelectric focusing and immunofixation

BACKGROUND: Detection of intrathecal IgG synthesis is important in evaluating inflammatory diseases in the central nervous system. Isoelectric focusing (IEF) is currently the most sensitive method to demonstrate intrathecal IgG synthesis and may have diagnostic value for German Shepherd degenerative myelopathy (GSDM). OBJECTIVE: The objective of this study was to adapt a modified IEF and immunofixation method for the detection of oligoclonal bands in cerebrospinal fluid (CSF) from dogs with GSDM. METHODS: Serum and lumbar CSF samples were collected from 6 German Shepherd dogs clinically diagnosed with GSDM. Samples were also collected from 6 clinically healthy dogs for comparison. The concentration of IgG was measured by quantitative ELISA and the concentration of total protein was measured by the Bradford protein assay. The presence of oligoclonal bands was evaluated by use of modified IEF followed by immunofixation. RESULTS: The concentrations of total protein and IgG, and the IgG/total protein ratio in CSF samples, were not significantly different between GSDM patients and control dogs. Four GSDM patients had oligoclonal bands in their CSF based on IEF-immunofixation. No oligoclonal bands were found in CSF from control dogs. CONCLUSION: The results suggest that the detection of oligoclonal bands by IEF-immunofixation may have diagnostic value for GSDM, and support the idea that humoral immune responses may play a role in the pathogenesis of GSDM.     

The immunophenotype of blood and cerebrospinal fluid mononuclear cells in dogs

Inflammatory neurologic diseases are common in dogs, but establishing a definitive diagnosis often is difficult. Nucleated cell number and type in cerebrospinal fluid (CSF) rarely are suggestive of an etiologic agent. We speculated that CSF leukocyte immunophenotyping would be a useful adjunct in the investigation of canine inflammatory neurologic diseases by yielding more specific etiologic information. The goals of this study were to establish the feasibility of flow cytometric evaluation of individual canine CSF samples and to identify the cell distribution in healthy dogs. The mononuclear cell populations of paired blood and CSF samples from 23 healthy dogs were characterized by labeling of cells with antibodies against CD4, CD8alpha, CD21, and CD14 molecules and by flow cytometric analysis of their expression. The mean proportion of CD4+ and CD21+ cells was significantly higher in blood than in the CSF (P < .002 and P < .001, respectively). In contrast, the mean proportion of CD14+ and CD8a+ cells was not significantly different between blood and CSF (P = .5 and p = .9, respectively). These findings demonstrate differences in the distribution and function of mononuclear cells in the circulating venous and subarachnoid compartments in the dog.     

Radioresistance of cancer stem‐like cell derived from canine tumours

Cancer stem‐like cells (CSCs)/cancer‐initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye‐based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage‐independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct‐4 and CD133 gene than those of adherent‐cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent‐cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent‐cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.     

Low concentrations of cerebrospinal fluid GABA correlate to a reduced response to phenobarbital therapy in primary canine epilepsy

In this study, we investigated whether pretreatment cerebrospinal fluid (CSF) neurotransmitter concentrations of gamma-aminobutyric acid (GABA) and glutamate (GLU) were correlated with response to phenobarbital treatment in dogs with primary epilepsy. Eleven untreated dogs, 6 males and 5 females, with a median age of onset of seizures of 3 years (range: 0.5-5 years) were selected for therapy based on progressive or serious seizure patterns. The median interval between the first observed seizure and start of phenobarbital therapy was 485 days (range: 101-1,765 days). All dogs were purebred, with the exception of I male dog. Oral phenobarbital was started at 2.5 mg/kg every 12 hours. Trough serum phenobarbital concentrations were measured at 15, 45, 90, 180, 360, 540, and 720 days after the start of treatment. There was no difference in the mean trough serum concentration or in the mean number of seizures recorded between each time period of phenobarbital measurement over the 2-year evaluation. No correlation was found between CSF GLU, GABA, or GLU: GABA ratio and the total number of seizures recorded before or after initiation of phenobarbital therapy. Lower CSF GABA concentration, however, was correlated with a lower seizure frequency difference (the total number of seizures before phenobarbital therapy minus the total number of seizures after phenobarbital therapy for an identical time period of evaluation) and lower percentage reduction in seizures: ([total number of seizures before phenobarbital therapy minus the total number of seizures after phenobarbital therapy] divided by the total number of seizures before phenobarbital therapy) x 100. There was no correlation between CSF GLU and the seizure frequency difference and percentage reduction in seizures. A negative correlation between the CSF GLU:GABA ratio and seizure frequency difference was found. Thus, dogs with an initial lower CSF GABA concentration before phenobarbital therapy did not respond as well as did dogs with a higher CSF GABA concentration.     

Ultrasonographic anatomy of the atlanto‐occipital region and ultrasound‐guided cerebrospinal fluid collection in rabbits (Oryctolagus cuniculus)

Cerebrospinal fluid analyses are important for diagnosis of neurologic problems in rabbits and for translational research projects using rabbits as models. Blind puncture of the cisterna magna is the current standard technique for sampling cerebrospinal fluid in this species. However, the complexity and small size of the cisterna magna and surrounding structures are limitations of this technique. Aims of this prospective, anatomic, pilot study were to (1) describe the normal anatomy of the atlanto‐occipital region, (2) describe ultrasonographic anatomic landmarks, and (3) develop and evaluate a technique for ultrasound‐guided puncture of the cisterna magna for cerebrospinal fluid sampling in rabbits. Thirty healthy rabbits were included and the study was conducted in three stages. Three rabbit cadavers were used for the first stage of the study. Then, the second stage was completed using 13 rabbit cadavers. Finally, the third stage was completed in 14 live rabbits. The ultrasound‐guided puncture performed in 13 cadavers was successful at the first attempt in 10 cases, and at the second attempt in the remaining three cases. In the in vivo study, the ultrasound‐guided puncture was successful in all 14 cases, without signs of complications. Findings supported the use of ultrasound‐guided puncture of the cisterna magna as a safe technique that may be used routinely or when the sample of cerebrospinal fluid cannot be obtained with the blind technique in rabbits.     

Concentrations of minerals in the canine prostatic fluid

Current knowledge about the composition of the prostate fluid in healthy male dogs is limited and restricted to small case numbers. Furthermore, published data often vary significantly regarding sample processing and analytical methods. Therefore, we aimed to provide data on the composition of electrolytes and minerals in the canine prostatic fluid in a larger population (n = 30 dogs/samples) and to compare these results with the existing literature. Concentrations of sodium, potassium and copper analysed in our population were most consistent with those in the literature. Different to this, concentrations of total calcium, magnesium, zinc and inorganic phosphate varied. Whereas magnesium, zinc and inorganic phosphate seemed to depend on the analysis method, total calcium concentrations differed if centrifugation was performed or not. Our results clearly indicate a need for standardization of methods for analysis of seminal plasma components.     

Three cases of canine bile peritonitis with mucinous material in abdominal fluid as the prominent cytologic finding

Background: Bile peritonitis is a severe, nonseptic inflammatory response to bile in the peritoneal cavity. It may result from generalized or localized leakage of bile due to spontaneous rupture of the biliary system or as a complication of biliary tract inflammation, obstruction, manipulation, or trauma. Cytologically, bile in abdominal fluid appears as golden-green granular pigment.
Objective: The purpose of this report is to describe the atypical cytologic features of abdominal fluid in 3 dogs with bile peritonitis.
Methods: As part of a diagnostic workup, abdominal fluid was obtained from 3 dogs with bile peritonitis and analyzed. In 2 dogs, fluid bilirubin concentration was determined and Hall's bile stain, Alcian blue-periodic acid-Schiff stain, and Mayer's mucicarmine stain were applied to direct smears of the fluid.
Results: Acellular mucinous fibrillar material in clumps and lakes was the prominent cytologic finding in the abdominal fluid from all 3 dogs. Bile pigment was not observed. Fluid from the 3 dogs contained increased numbers of inflammatory cells, predominantly neutrophils. Total protein concentration ranged from 2.9 to 5.6 g/dL. Fluid total bilirubin concentration was greater than twice that of the concurrent serum bilirubin concentration. Based on results of the special stains, the amorphous material was positive for mucosubstances, but was negative for bilirubin. In all dogs, bile peritonitis originated from a rent in the common bile duct.
Conclusions: Bile peritonitis with fibrillar mucinous material in abdominal fluid has not been described previously in dogs. The material was similar to "white bile" observed in humans and experimentally in dogs as a sequela to extrahepatic biliary tract obstruction. When mucinous material is observed in abdominal fluid from dogs and the fluid bilirubin concentration is greater than twice the serum bilirubin concentration, rupture of the extrahepatic biliary tract should be suspected.     

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.     

Characterisation of transthyretin and retinol-binding protein in plasma and cerebrospinal fluid of dogs

The aim of this study was to investigate differences in concentrations of vitamin A, transthyretin (TTR) and retinol-binding protein (RBP) between plasma and cerebrospinal fluid (CSF) in dogs. RBP was detected using ELISA, and both RBP and TTR by Western blot analysis after separation on SDS-PAGE. Vitamin A was determined by high performance liquid chromatography. RBP and TTR as well as vitamin A were detected in all samples but at substantially lower concentrations in CSF compared to plasma. RBP in dog plasma showed a similar molecular mass to that of humans, whereas canine TTR had a lower molecular mass. Comparison between plasma and CSF showed that both RBP and TTR were of lower molecular mass in CSF. In CSF, RBP and retinol were present at 10-100-fold lower concentrations compared to plasma. Retinyl esters were present only in minute amounts in 5/17 samples. In conclusion, the CSF of dogs compared to humans is significantly different in terms of both quality and quantity of transport proteins for vitamin A.     

Role of prostatic fluid in cooled canine epididymal sperm

In this study, sperms collected from the right and left cauda epididymis were grouped into having canine prostatic fluid (PF) sensitization or not diluted with egg yolk Tris–fructose citrate extender, and stored at 4°C. The necessity of canine PF in cooled preservation was determined by elucidating the sperm quality after the storage. As a result, while there was no difference among all groups up to 48 hr of storage, after storage for 96 hr and more, a significantly lower sperm motility was observed in the group without being sensitized to PF than the groups with being sensitized to PF (p < .05, p < .01). Although sperm abnormality increased in all groups with increased storage time, the group without being sensitized to PF showed significantly higher sperm abnormality than did the groups with being sensitized to PF after storage for 24 hr and more (p < .01). From these findings, we concluded that PF was necessary for the cooled preservation of the canine sperm because these sperms were protected from any effects of low temperatures by being sensitized to PF.