Validation of an immunoturbidimetric D-dimer assay in canine citrated plasma

Abstract: D-dimer is a neoantigen formed when thrombin initiates the transformation of fibrinogen to fibrin; it is derived from plasmin digestion of cross-linked fibrin. In human medicine, the usefulness of this analyte in diagnosing disseminated intravascular coagulation (DIC) has been assessed in patients fulfilling the clinical and laboratory requirements for this disorder. In canine medicine, the use of D-dimer is relatively new. Detailed studies are needed to understand the relationship between D-dimer concentration in plasma and DIC status in dogs. We validated a D-dimer immunoturbidimetric assay (Tina-quant [a] D-Dimer, Boehringer Mannheim) in canine citrated plasma samples. Intra-assay and interassay variability (coefficient of variation) was 5.63% and 8.82%, respectively. The assay was linear, using 2 samples with low and high D-dimer concentrations (r = .996 and .998). Accuracy was 102.2% and 95.7% based on a recovery study in which 2 samples were assessed. Reference values for D-dimer were established using 70 healthy dogs that were assessed clinically and evaluated on the basis of a complete laboratory workup. The reference range was set between 0.02 and 0.28 μg/mL (chi-square test for normal distribution, P > .05).     

Validation of human haptoglobin immunoturbidimetric assay for detection of haptoglobin in equine and canine serum and plasma

The Incstar(R) SPQ II human haptoglobin (Hpt) (Incstar Corporation, Stillwater, MN) immunoturbidimetric assay was validated for the determination of serum and plasma Hpt concentrations in dogs and horses. The anti-human Hpt antiserum supplied with the assay, displayed monospecificity to both dog and horse serum Hpt by immunoelectrophoresis and Western blotting techniques. The automated immunoturbidimetric assay results correlated well with the cyanmethemoglobin binding assay (r=0.953 for canine serum and r=0.941 for equine serum), and had excellent precision at both high and low serum Hpt concentrations (within run and between run coefficients of variation near or less than 5%). The assay was linear in both species by serial dilution of pooled-high serum with pooled-low serum, saline and with Hpt-free serum. Interference from hemolysis (> 25 mg/dl hemoglobin) and lipemia greater than 100 mg/dl caused a false decrease and false increase respectively in Hpt yield with the immunoturbidimetric assay. The anti-Hpt antibody supplied with the assay kit, once diluted with polymer diluent and stored at 4 degrees C, was stable for up to 6 days and gave consistent results.     

Bone marrow contamination of canine cerebrospinal fluid

Bone marrow cells were identified in cytocentrifuge preparations of cerebrospinal fluid (CSF) obtained by lumbar punctures from two dogs. CSF analyses were characterized by normal or increased total cell counts, normal or increased total protein concentration and the presence of erythrocytes. Hematopoietic cells present included both erythroid and myeloid precursors and, in one case, an erythroblastic island was seen. Peripheral blood smear examination confirmed that the hematopoietic cells observed were not the result of blood contamination. Neither case was associated with a difficult tap procedure, nor with a specific disease process. Contamination may have resulted from actual marrow penetration or from extramedullary hematopoiesis in the dura mater. Accurate interpretation of CSF cytology requires the consideration of possible bone marrow contamination when hematopoietic cells are observed.     

Analysis of neurotransmitter metabolite concentrations in canine cerebrospinal fluid

The concentrations of dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole-3-acetic acid (5-HIAA) in CSF obtained from the cisterna magna of 21 nonneurologically compromised dogs were determined by high-pressure liquid chromatography and electrochemical detection. A rapid method of sample preparation, which involved single filtration through a deproteinizing membrane, was used. Canine CSF obtained in this manner contained 5.78 +/- 0.78 ng of DOPAC/ml, 72.19 +/- 4.09 ng of HVA/ml, and 29.95 +/- 1.67 ng of 5-HIAA/ml. Linear regression analysis between HVA and 5-HIAA yielded a correlation coefficient of 0.4804. The neurotransmitter index, HVA/5-HIAA, was found to be more indicative of the dopaminergic metabolite HVA than the acid metabolite of serotonin, 5-HIAA (correlation coefficient with HVA = 0.5529 vs a correlation coefficient with 5-HIAA = -0.4462). A poor relationship (correlation coefficient = -0.1715) was found to exist between the 2 dopaminergic metabolites DOPAC and HVA in the CSF.     

Analytical validation of a point-of-care test and an automated immunoturbidimetric assay for the measurement of canine C-reactive protein in serum

C-reactive protein (CRP) is an acute phase protein, which is used to evaluate and monitor the response of the innate immune system to a variety of inflammatory processes in the dog. The purpose of this study was to analytically validate a point-of-care assay (IDEXX Catalyst CRP Test) and an immunoturbidimetric assay (Gentian Canine CRP Immunoassay) for the measurement of serum CRP concentrations in dogs. These 2 assays (Catalyst, Gentian) were compared to a previously validated enzyme-linked immunosorbent assay (Tridelta Development EIA Canine CRP Assay). Linearity, precision, reproducibility, and accuracy were assessed using leftover serum samples. Agreement between assays was assessed using leftover serum samples and serum from clinically healthy dogs. Observed to expected ratios (O/E) for dilutional parallelism were 83.9 to 163.1% and 108.3 to 160.6% for the Catalyst and the Gentian assays, respectively. Coefficients of variation for intra-assay variability ranged from 6.4 to 9.5% for the Catalyst assay and 1.5 to 2.6% for the Gentian assay. Coefficients of variation for inter-assay variability ranged from 3.8 to 18.2% for the Catalyst assay and 4.5 to 5.8% for the Gentian assay. The mean O/E for recovery were 97.9% and 98.5% for the Catalyst and Gentian assays, respectively. Correlations between assays were as follows: Catalyst and Tridelta (R² = 0.76), Gentian and Tridelta (R² = 0.79), and Catalyst and Gentian (R² = 0.98). The Catalyst and Gentian assays are both acceptable for measuring CRP in dog serum, but their results are not directly comparable with the Tridelta assay.     

Proteome analysis of cerebrospinal fluid in healthy beagles and canine encephalitis

We performed proteomics analysis of the cerebrospinal fluid (CSF) of healthy dogs and dogs with meningoencephalitis of unknown etiology (MUE). By comparing two-dimensional electrophoreses (2DE), an upregulated spot was found in MUE dogs. This protein was identified as a neuron-specific enolase (NSE) by analysis with MALDI-TOF mass spectrometry. In comparing dot blots using an antibody against NSE, the NSE levels in the CSF of MUE dogs was significantly higher than that of the controls. NSE is a diagnostic marker of neuroendocrine tumors, brain injury and spinal cord trauma in humans. It seems that the NSE concentration in the CSF is increased by cellular destruction in canine encephalitis. Though elevation of NSE may not be specific in canine encephalitis because the NSE level was increased in other CNS diseases, further study including measurement with serum is necessary.     

Laboratory diagnosis of canine GM2-gangliosidosis using blood and cerebrospinal fluid.

In the present study, laboratory techniques were used to diagnose canine GM2-gangliosidosis using blood and cerebrospinal fluid (CSF) that can be collected noninvasively from living individuals. Lysosomal acid beta-hexosaminidase (Hex) was measured spectrofluorometrically using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl 7-(6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside) as substrates. Main isoenzymes A and B of Hex in leukocytes were also analyzed using cellulose acetate membrane electrophoresis. GM2-ganglioside in CSF was detected and determined quantitatively by using thin-layer chromatography/enzyme-immunostaining method with anti-GM2-ganglioside antibody. In normal dogs, Hex activities could be determined in leukocytes, serum, and CSF and the total activities were markedly reduced in all the enzyme sources in a dog with Sandhoff disease. Electrophoresis of a leukocyte lysate from a normal dog showed that the Hex A and Hex B were not separated distinctively with formation of a broad band, whereas there were no bands in electrophoresis of a lysate from a dog with Sandhoff disease, showing a deficiency in the total enzyme activity. GM2-ganglioside could be detected and determined quantitatively in as little as 100 microl of canine CSE GM2-ganglioside in CSF in a dog with Sandhoff disease increased to 46 times the normal level. In conclusion, the methods in the present study are useful for diagnosis of canine GM2-gangliosidosis. These techniques enable definitive and early diagnosis of canine GM2-gangliosidosis even if tissues and organs cannot be obtained.     

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.     

Validation of a commercial human ELISA to measure hyaluronic acid concentration in feline plasma

Our goal was to validate a human hyaluronic acid (HA) ELISA (Hyaluronic acid plus ELISA; TECOmedical Group) for use in feline plasma. Plasma from 5 healthy cats and 5 critically ill cats was used for validation of the assay. Validation methods performed included intra- and inter-assay variability, spike-and-recovery, and dilutional linearity. All measurements were performed in duplicate. The precision study revealed good intra-assay CV of 7.4–8.9%; inter-assay CV was 3.4–4.2%. Extraction efficiency via spiking tests yielded mean recovery of 89.6%. The assay met criteria for acceptable linearity using 3 serial dilutions. Our results demonstrate that this commercial HA ELISA had acceptable analytical performance using feline plasma and could be a useful tool in the veterinary clinical research setting.     

Characteristics of cerebrospinal fluid associated with canine granulomatous meningoencephalomyelitis: a retrospective study

Cerebrospinal fluid of 22 dogs with histologically confirmed granulomatous meningoencephalomyelitis was analyzed, retrospectively. Seventeen dogs had cisternal CSF analysis, 4 dogs had lumbar CSF analysis, and 1 dog had both. For cisternal CSF, the mean +/- SEM total WBC count was 800.8 +/- 300.9 cells/microliter. The WBC differential count was predominantly lymphoplasmacytic cells, but 13 of the 18 cisternal CSF had polymorphonuclear (PMN) cells, and the mean +/- SEM PMN cell percentage was 18.6 +/- 5.3%. The mean +/- SEM total protein content of cisternal CSF was 255.8 +/- 98 mg/dl. Of 5 cisternal CSF pressures measured, 4 were within the normal range. The mean +/- SEM total WBC count and total protein content of lumbar CSF were 533.4 +/- 256.5 cells/mu/microliter and 163.2 +/- 25 mg of protein/dl, respectively. As with cisternal CSF, the WBC differential count of lumbar CSF was predominantly lymphoplasmacytic cells. Of 5 lumbar CSF, 4 contained PMN cells, but the percentage was less than the PMN cell percentage of cisternal CSF. Although variable, the general pattern of CSF abnormality associated with granulomatous meningoencephalomyelitis was different from the CSF abnormalities commonly seen with viral, bacterial, or mycotic encephalitides.     

Significance of surface epithelial cells in canine cerebrospinal fluid and relationship to central nervous system disease

Background: The term “surface epithelium” is used to describe cells, including meningeal, choroid plexus, ependymal, and endothelial cells, that are found in human cerebrospinal fluid (CSF) and are difficult to distinguish cytologically. We hypothesized that the presence of surface epithelial cells in canine CSF was associated with specific diseases of the central nervous system (CNS). Objectives: In this retrospective study the frequency of surface epithelial cells in CSF from dogs with neurologic disease was investigated along with the potential association with age, specific type of CNS disease, and CSF total nucleated cell count (TNCC) and protein concentration. Methods: The frequency of surface epithelial cells in 359 canine CSF samples was analyzed for 5 disease groups: CNS neoplasia, CNS compression, CNS inflammation, idiopathic epilepsy, and miscellaneous diseases. Groups were also combined into those with and without expected meningeal involvement. Association of the presence of surface epithelial cells in CSF with age, disease type, and CSF TNCC and protein concentration was investigated. Results: Surface epithelial cells were found in 27 of 359 (7.5%) CSF samples: CNS neoplasia 2/30 (6.7%), CNS compression 7/64 (10.9%), CNS inflammation 1/39 (2.6%), idiopathic epilepsy 8/124 (6.5%), and miscellaneous diseases 9/102 (8.8%). Significant associations between surface epithelial cell presence in CSF and age, disease type, CSF TNCC, and CSF protein concentration were not found. Conclusions: The presence of surface epithelial cells was not related to a specific disease group or CSF changes in the studied population. Thus, the presence of surface epithelial cells should be interpreted carefully, as it could represent an incidental finding in CSF specimens.     

Evaluation of a chromogenic assay to measure the factor Xa inhibitory activity of unfractionated heparin in canine plasma

BACKGROUND: Unfractionated heparin (UFH) has a complex pharmacologic profile that necessitates patient monitoring to prevent inadequate anticoagulation or overdosage and hemorrhage. Factor Xa inhibitory assays (to measure anti-Xa activity) are used to adjust UFH dosage and define safe and effective regimens for specific thrombotic disorders in humans. OBJECTIVE: In this study, the accuracy, linearity, and clinical utility of a chromogenic assay were assessed for monitoring UFH anti-Xa activity in canine plasma samples. METHODS: A commercial assay (Rotachrom Heparin, Diagnostica Stago, Parsippany, NJ, USA) was used to measure anti-Xa activity in canine plasma samples spiked with different concentrations of UFH. Background absorbance and assay linearity were compared for canine and human plasmas. Percentage recovery of UFH anti-Xa activity and intra- and interassay imprecisions were investigated by multiple measurements of canine plasma to which known amounts of UFH were added. The spiked plasma samples also were used to determine the heparin sensitivity of an activated partial thromboplastin time (aPTT) test. RESULTS: Canine plasma samples were assayed at a higher dilution than were human plasma samples (3:8 versus 4:8) to eliminate higher background anti-Xa activity in canine plasma. Using this modification, the recovery of anti-Xa activity in canine plasma was linear (R2 > .9) at concentrations of 0 - 0.75 U/mL UFH. Intra- and interassay imprecisions for plasma samples containing 0.5 U/mL UFH were <10%, whereas samples containing 0.25 U/mL UFH had imprecisions of 13% and 24%, respectively. The anti-Xa activity range of 0.5 - 0.75 U/mL caused prolongation of aPTTs to 1.5 - 2.5 times the assay mean. CONCLUSION: Plasma anti-Xa activity of dogs treated with UFH can be accurately monitored using this commercially available chromogenic assay.     

Evaluation of nephelometry for albumin measurement in serum and cerebrospinal fluid: experiences with an indwelling subarachnoidal catheter system for repetitive cerebrospinal fluid collection in horses.

The measurement of albumin concentrations in cerebrospinal fluid (CSF) and serum for albumin quotient (AQ) calculations in normal horses was performed by 2 methods: 1) total protein measurement, followed by electrophoresis of the samples to obtain an albumin percentage; and 2) albumin immunoprecipitation quantitated by nephelometry. The results of both methods correlated well, and nephelometry was chosen to determine the albumin concentrations in CSF samples obtained from an indwelling subarachnoidal catheter for daily sampling. Because the use of an indwelling catheter to collect repetitive CSF samples is a novel technique, routine cytological CSF analysis was performed along with daily clinical evaluation to ascertain the well-being of the horses. The catheters were placed in 2 horses for periods of 14 and 17 days. One horse exhibited pleocytosis on cytological evaluation of CSF on 2 occasions for a 1-2-day duration; however, the AQ showed a significant increase on only 1 occasion. The other horse had a normal cell count in CSF but showed 2 sudden changes in the AQ value; however, these values remained within the 95% confidence interval for AQ in horses. Albumin quotient values of the second horse were consistently below the lower range of the confidence interval. Results from this study indicate that nephelometry can be used for albumin determination in serum and CSF samples from horses. Furthermore, an indwelling subarachnoidal catheter system can provide serial CSF samples in horses, thus obviating the need for repetitive centesis for serial CSF sampling.