To develop and analytically validate a liquid chromatography–tandem mass spectrometry method for measurement of endogenous trans-4-hydroxy-l-proline concentrations in canine serum and to assess serum trans-4-hydroxy-l-proline concentrations in dogs with chronic hepatitis.
Serum samples obtained from 20 dogs with histopathologically confirmed chronic hepatitis and 20 healthy control dogs.
A liquid chromatography–tandem mass spectrometry method for quantification of trans-4-hydroxy-l-proline concentration was developed and assessed for analytic sensitivity, linearity, accuracy, precision, and reproducibility. Serum concentration of trans-4-hydroxy-l-proline in dogs with chronic hepatitis and healthy control dogs was measured.
Observed-to-expected ratios for dilutional parallelism ranged from 72.7% to 111.5% (mean ± SD, 91.3 ± 19.6%). Intra-assay and interassay coefficients of variation ranged from 2.1% to 3.0% and 3.2% to 5.3%, respectively. Relative error ranged from −2.3% to 7.8%. Trans-4-hydroxy-l-proline concentrations were significantly lower in serum obtained from dogs with chronic hepatitis (median, 0.24 ng/mL; range, 0.06 to 1.84 ng/mL) than in serum obtained from healthy control dogs (median, 0.78 ng/mL; range, 0.14 to 4.90 ng/mL).
CONCLUSIONS AND CLINICAL RELEVANCE
The method described here for the quantification of trans-4-hydroxy-l-proline concentration in canine serum was found to be sensitive, specific, precise, accurate, and reproducible. Dogs with chronic hepatitis had significantly lower serum trans-4-hydroxy-l-proline concentrations than did healthy control dogs, possibly as a result of altered hepatic metabolism of amino acids.
To assess feasibility of the use of a dynamic viscoelastic coagulometer on chicken blood and compare coagulation variables for fresh whole blood and sodium citrate–preserved whole blood as well as effects of 3 coagulation activators on blood from chickens.
Blood samples from 30 hens.
Chickens were allowed to rest undisturbed for 1 hour. A blood sample was collected from an ulnar vein; 1.4 mL was analyzed immediately, and 1.8 mL was mixed with sodium citrate and subsequently recalcified and analyzed. A separate coagulation activator (glass beads, kaolin clay, or tissue factor) was in each of the 2 channels of the analyzer. Chickens were allowed a 1-hour rest period, and another blood sample was collected from the contralateral ulnar vein; it was processed in the same manner as for the first sample, except both channels of the analyzer contained the same coagulation activator.
Compared with fresh samples, citrated samples had higher values for activated clotting time and platelet function and lower clotting rates. Intra-assay coefficients of variation of coagulation profiles for citrated samples were markedly greater than the limit of 10%, whereas values for fresh samples were close to or < 10%.
CONCLUSIONS AND CLINICAL RELEVANCE
Results suggested that use of a dynamic viscoelastic coagulometer on chicken blood was feasible and that analysis of fresh whole blood from healthy chickens provided results with less variability than did analysis of citrated blood. Samples preserved with sodium citrate were associated with significant relative hypocoagulability, compared with results for fresh blood.
To compare ultracentrifugation, precipitation, and membrane affinity chromatography methods for isolation of extracellular vesicles (EVs) from canine plasma samples and to identify suitable reference genes for incorporation into a quantitative reverse transcription PCR assay of microRNA expression in plasma EVs of healthy dogs.
6 healthy Beagles.
Plasma samples were obtained from each dog, and EVs were isolated from 0.3 mL of these samples via ultracentrifugation, precipitation, and membrane-affinity chromatographic methods. Nanoparticle tracking analysis was performed to determine the concentration and size distribution of EVs isolated by the ultracentrifugation method. Expression levels (cycle threshold values) of 4 microRNAs (let-7a, miR-16, miR-26a, and miR-103) were then compared by means of quantitative reverse transcription PCR assay. Three statistical programs were used to identify the microRNAs most suitable for use as reference genes.
Results indicated that ultracentrifugation was the most stable of all 3 methods for isolating microRNAs from 0.3 mL of plasma. Nanoparticle tracking revealed that EV samples obtained by the ultracentrifugation method contained a mean ± SD of approximately 1.59 × 1010 vesicles/mL ± 4.2 × 108 vesicles/mL. Of the 4 microRNAs in plasma EVs isolated by ultracentrifugation, miR-103 was the most stable.
CONCLUSIONS AND CLINICAL RELEVANCE
The ultracentrifugation method has potential as a stable method for isolating EVs from canine plasma samples with a high recovery rate, and miR-103 may provide the most stable reference gene for normalizing microRNA expression data pertaining to plasma EVs isolated by ultracentrifugation.
OBJECTIVE To determine usefulness of skin turgor and capillary refill time (CRT) for predicting changes in hydration status of working dogs after a 15-minute exercise period.
ANIMALS 9 exercise-conditioned working dogs between 8 and 108 months of age.
PROCEDURES Skin tent time (SkTT; time for tented skin on the forehead to return to an anatomically normal position) and CRT (time for occluded mucous membrane capillary vessels to return to the color visible before occlusion) were measured on dogs in a field setting and by video review. Body weight (BW), SkTT, CRT, and core body temperature were measured before and after a 15-minute exercise period. Exercise challenge was performed on days 1 and 8.
RESULTS Time (day 1 vs day 8) did not significantly affect results; therefore, data were pooled for the 2 trial days. Mean ± SE BW decreased (but not significantly) by 0.83 ± 0.27% after exercise. The SkTT increased significantly (both field setting and video review) after exercise. Correlation between SkTT results for the field setting and video review (r = 0.68) was significant. The CRT decreased (but not significantly) after exercise.
CONCLUSIONS AND CLINICAL RELEVANCE Dogs became mildly dehydrated (mean BW loss, 0.83%) during a 15-minute exercise period, and the mild dehydration was evident as a visually detectable change in skin turgor. Monitoring the SkTT appeared to be a useful strategy for predicting small shifts in hydration status of dogs during exercise. The CRT decreased and was not a significant predictor of a change in hydration status.
OBJECTIVE To develop and characterize flow cytometric assays for detecting IgG bound to canine erythrocytes and bone marrow erythroid precursors.
SAMPLE Blood samples from 20 healthy and 61 sick dogs with (n = 33) or without (28) immune-mediated hemolytic anemia (IMHA) and bone marrow samples from 14 healthy dogs.
PROCEDURES A flow cytometric assay for measurement of IgG on RBCs was developed, and appropriate positive control cells were generated. Analytic and diagnostic performance were characterized. The RBC IgG assay was then combined with density-gradient fractionation of aspirated bone marrow cells and a 2-color process to yield an assay for detecting IgG on nucleated RBCs (nRBCs). Cell sorting and cytologic examination confirmed target cell populations, and anti–dog erythrocyte antigen 1 (DEA1) blood-typing serum was used to generate IgG-positive nRBCs.
RESULTS Within- and between-run coefficients of variation for the RBC IgG assay were 0.1% to 13.9%, and > 90% of spiked IgG-positive RBCs were detected. Diagnostic sensitivity and specificity of the assay for detection of IMHA were 88% and 93%, respectively. Cytologic findings for sorted bone marrow fractions rich in early-, mid-, and late-stage nRBCs from 3 healthy dogs indicated 89% to 98% nRBC purity. After IgG coating with anti-DEA1 blood-typing serum, IgG was detected on nRBCs from DEA1-positive, but not DEA1-negative, healthy dogs.
CONCLUSIONS AND CLINICAL RELEVANCE The developed RBC IgG assay had favorable analytic and diagnostic performance for detection of IMHA in dogs and was successfully adapted to detect IgG on canine nRBCs of various maturation stages. The findings supported the presence of DEA1 on canine nRBCs.
OBJECTIVE To characterize the fecal microbiota of horses and to investigate alterations in that microbiota on the basis of sample collection site (rectum vs stall floor), sample location within the fecal ball (center vs surface), and duration of environmental exposure (collection time).
ANIMALS 6 healthy adult mixed-breed mares.
PROCEDURES From each horse, feces were collected from the rectum and placed on a straw-bedded stall floor. A fecal ball was selected for analysis immediately after removal from the rectum and at 0 (immediately), 2, 6, 12, and 24 hours after placement on the stall floor. Approximately 250 mg of feces was extracted from the surface and center of each fecal ball, and genomic DNA was extracted, purified, amplified for the V1-V2 hypervariable region of the 16S rDNA gene, and analyzed with a bioinformatics pipeline.
RESULTS The fecal microbiota was unique for each horse. Bacterial community composition varied significantly between center and surface fecal samples but was not affected by collection time. Bacterial community composition varied rapidly for surface fecal samples. Individual bacterial taxa were significantly associated with both sample location and collection time but remained fairly stable for up to 6 hours for center fecal samples.
CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that, for horses, fecal samples for microbiota analysis should be extracted from the center of fecal balls collected within 6 hours after defecation. Samples obtained up to 24 hours after defecation can be analyzed with the realization that some bacterial populations may deviate from those immediately after defecation.
OBJECTIVE To assess 2 human ELISA kits for measurement of angiopoietin-1 and -2 concentrations in canine plasma samples, determine whether plasma angiopoeitin-2 concentration differed between septic and healthy dogs, and determine the effect of tumor necrosis factor-α (TNF-α) stimulation on angiopoeitin-2 release from primary canine aortic endothelial cells (pCAECs) in vitro.
ANIMALS 10 healthy dogs and 10 septic dogs.
PROCEDURES Human angiopoietin-1 and -2 ELISAs were used to detect recombinant canine angiopoietins-1 and -2 in canine plasma samples. The angiopoietin-2 ELISA was further validated by use of plasma samples from healthy and septic dogs and supernatants of pCAEC cultures. Associations between plasma angiopoeitin-2 and C-reactive protein (CRP) concentrations were examined.
RESULTS Angiopoeitin-2 but not angiopoeitin-1 was detected in canine plasma samples by the respective ELISAs. The angiopoeitin-2 ELISA had excellent dilutional linearity, parallelism, accuracy, precision, and reproducibility for measurements in canine plasma samples and pCAEC supernatants. Plasma angiopoeitin-2 concentration was significantly higher in septic dogs (median, 25.5 ng/mL) than in healthy dogs (median, 6.7 ng/mL) and was positively correlated with plasma CRP concentration (R2 = 0.60). Stimulation of pCAECs with TNF-α resulted in a significant increase in supernatant angiopoietin-2 concentration.
CONCLUSIONS AND CLINICAL RELEVANCE The tested human angiopoietin-2 ELISA kit was useful for measuring angiopoietin-2 concentrations in canine plasma samples and pCAEC supernatants. Sepsis appeared to increase angiopoietin-2 concentration in dogs in vivo, whereas TNF-α stimulation caused release of angiopoietin-2 from pCAECs in vitro. These findings support the use of angiopoietin-2 as a marker of endothelial cell activation and inflammation in dogs.
OBJECTIVE To characterize spatial release of platinum from carboplatin-impregnated calcium sulfate hemihydrate (CI-CSH) beads by use of an agarose tissue phantom.
SAMPLE 3-mm-diameter beads (n = 60) containing 4.6 mg of carboplatin (2.4 mg of platinum)/bead.
PROCEDURES 18 L of 1% agarose was prepared and poured into 36 containers (10 × 10 × 10 cm), each of which was filled half full (0.5 L/container). After the agarose solidified, 1, 3, 6, or 10 CI-CSH beads were placed on the agar in defined patterns. An additional 36 blocks of agar (0.5 L/block) were placed atop the beads, positioning the beads in the center of 1 L of agar. The experiment was replicated 3 times for each bead pattern for 24, 48, and 72 hours. At these times, representative agarose blocks were sectioned in the x-, y-, and z-planes and labeled in accordance with their positions in shells radiating 1, 2, 3, 4, and 5 cm from the center of the blocks. Agarose from each shell was homogenized, and a sample was submitted for platinum analysis by use of inductively coupled plasma–mass spectroscopy.
RESULTS Platinum diffused from CI-CSH beads at predicted anticancer cytotoxic concentrations for 2 to 5 cm.
CONCLUSIONS AND CLINICAL RELEVANCE Results provided information regarding the spatial distribution of platinum expected to occur in vivo. Agarose may be used as a diffusion model, mimicking the characteristics of subcutaneous tissues. Measured platinum concentrations might be used to guide patterns for implantation of CI-CSH beads in animals with susceptible neoplasms.
OBJECTIVE To investigate the use of canine whole blood (WB) for measurement of ammonia concentration by use of a point-of-care ammonia meter and to compare results of measuring ammonia concentrations in WB, EDTA-anticoagulated WB, and plasma.
ANIMALS 40 client-owned dogs.
PROCEDURES A blood sample (2 mL) was obtained from each dog. One drop of WB was immediately applied to a test strip for evaluation with an ammonia meter. The remainder of the blood sample was placed in an EDTA-containing tube, and 1 drop of EDTA-anticoagulated WB was applied to a test strip. The remaining EDTA-anticoagulated WB sample was centrifuged, and the plasma was harvested and placed on ice. One drop of plasma was applied to a test strip; the remainder of the plasma sample was transported on ice and used for ammonia measurement with a reference laboratory instrument. All samples were tested within 1 hour after sample collection. Results were evaluated to detect significant differences in ammonia concentration.
RESULTS Ammonia concentrations did not differ significantly between WB and EDTA-anticoagulated WB and between plasma samples measured with the meter and reference laboratory instrument. However, median ammonia concentration was significantly higher in plasma than in WB or EDTA-anti-coagulated WB.
CONCLUSIONS AND CLINICAL RELEVANCE Anticoagulant-free WB was a valid sample for measurement by use of the ammonia meter. Plasma samples had higher ammonia concentrations than did WB samples. Results for each sample type should be interpreted by use of specimen- and method-specific reference intervals.
OBJECTIVE To determine repeatability of gait variables measured by use of extremity-mounted inertial measurement units (IMUs) in nonlame horses during trotting under controlled conditions of treadmill exercise.
ANIMALS 10 horses.
PROCEDURES Six IMUs were strapped to the metacarpal, metatarsal, and distal tibial regions of each horse. Data were collected in a standardized manner (3 measurements/d on 3 d/wk over a 3-week period) while each horse was trotted on a treadmill. Every measurement consisted of a minimum of 20 strides from which a minimum of 10 strides was selected for analysis. Spatial and temporal variables were derived from the IMUs. Repeatability coefficients based on the within-subject SD were computed for each gait analysis variable at each week.
RESULTS Most of the temporal and spatial variables had high repeatability (repeatability coefficients < 10), and the repeatability coefficients were consistent among the 3 weeks of data collection. Some spatial variables, specifically the symmetry variables (which were calculated from other variables), had somewhat higher repeatability coefficients (ie, lower repeatability) only in the last week.
CONCLUSIONS AND CLINICAL RELEVANCE With the exceptions of some symmetry variables, which may reflect individual variations during movement, the extremity-mounted IMUs provided data with high repeatability for nonlame horses trotting under controlled conditions of treadmill exercise. Repeatability was achieved for each instrumented limb segment with regard to the spatial relationship between 2 adjacent segments (joint angles) and the temporal relationship among all segments (limb phasing). Extremity-mounted IMUs could have the potential to become a method for gait analysis in horses.