Development and validation of an ELISA to measure regenerating island-derived protein 3E in canine blood.

BACKGROUND: Regenerating island-derived proteins (REG) are upregulated in people with sepsis, pancreatitis, and gastrointestinal diseases. One member of the REG family, namely REG3E, was recently identified in pancreatic tissue and plasma of dogs, with high expression in pancreatitis and sepsis. OBJECTIVES: We aimed to develop and validate an ELISA to measure REG3E concentrations in canine blood. METHODS: An indirect sandwich ELISA was developed using recombinant canine REG3E protein and polyclonal anti-canine REG3E antibodies raised in guinea pigs and rabbits. Antibody specificity was assessed using western blot and mass spectrometric analysis of protein purified from canine plasma. Assay validation included evaluation of dilutional linearity, parallelism, spiking recovery, repeatability and reproducibility, stability, interferences, and comparison of serum and heparinized plasma. RESULTS: Antibodies bound specifically to REG3E with no evidence of cross-reactivity with other proteins. The limit of detection of the ELISA was 15 ng/mL, and the lower limit of quantification was 30 ng/mL. The assay demonstrated good to excellent linearity, dilutional and mixing parallelism, and recovery, with mean observed-to-expected ratios of 104%, 107%, 102%, and 92%, respectively, and no evidence of a hook effect. Coefficients of variation were ≤8.5% for repeatability and ≤14.3% for reproducibility at three different levels. Measurements of REG3E in plasma were not significantly influenced by different storage conditions, freeze-thawing cycles, hemolysis, lipemia, or icterus. There was no significant difference between REG3E concentrations in heparinized plasma and serum samples. CONCLUSIONS: The canine REG3E ELISA has acceptable precision, accuracy, linearity, and reproducibility for the measurement of REG3E in canine plasma and serum.

The effects of selected sedatives on basal and stimulated serum cortisol concentrations in healthy dogs.

Background: Hormone assessment is typically recommended for awake, unsedated dogs. However, one of the most commonly asked questions from veterinary practitioners to the endocrinology laboratory is how sedation impacts cortisol concentrations and the adrenocorticotropic hormone (ACTH) stimulation test. Butorphanol, dexmedetomidine, and trazodone are common sedatives for dogs, but their impact on the hypothalamic-pituitary-adrenal axis (HPA) is unknown. The objective of this study was to evaluate the effects of butorphanol, dexmedetomidine, and trazodone on serum cortisol concentrations. Methods: Twelve healthy beagles were included in a prospective, randomized, four-period crossover design study with a 7-day washout. ACTH stimulation test results were determined after saline (0.5 mL IV), butorphanol (0.3 mg/kg IV), dexmedetomidine (4 µg/kg IV), and trazodone (3-5 mg/kg PO) administration. Results: Compared to saline, butorphanol increased basal (median 11.75 µg/dL (range 2.50-23.00) (324.13 nmol/L; range 68.97-634.48) vs 1.27 µg/dL (0.74-2.10) (35.03 nmol/L; 20.41-57.93); P < 0.0001) and post-ACTH cortisol concentrations (17.05 µg/dL (12.40-26.00) (470.34 nmol/L; 342.07-717.24) vs 13.75 µg/dL (10.00-18.90) (379.31 nmol/L; 275.96-521.38); P ≤ 0.0001). Dexmedetomidine and trazodone did not significantly affect basal (1.55 µg/dL (range 0.75-1.55) (42.76 nmol/L; 20.69-42.76); P = 0.33 and 0.79 µg/dL (range 0.69-1.89) (21.79 nmol/L; 19.03-52.14); P = 0.13, respectively, vs saline 1.27 (0.74-2.10) (35.03 nmol/L; 20.41-57.93)) or post-ACTH cortisol concentrations (14.35 µg/dL (range 10.70-18.00) (395.86 nmol/L; 295.17-496.55); (P = 0.98 and 12.90 µg/dL (range 8.94-17.40) (355.86 nmol/L; 246.62-480); P = 0.65), respectively, vs saline 13.75 µg/dL (10.00-18.60) (379.31 nmol/L; 275.86-513.10). Conclusion: Butorphanol administration should be avoided prior to ACTH stimulation testing in dogs. Further evaluation of dexmedetomidine and trazodone's effects on adrenocortical hormone testing in dogs suspected of HPA derangements is warranted to confirm they do not impact clinical diagnosis.

HEMATOLOGY AND PLASMA CHEMISTRY COMPARISONS AMONG JUVENILE AMERICAN BLACK BEARS (URSUS AMERICANUS) UNDERGOING REHABILITATION.

The American black bear (Ursus americanus) is an opportunistic and adaptable species with high rehabilitation success rates. Injured, ill, and orphaned bears across the southeastern United States are examined and treated at the University of Tennessee College of Veterinary Medicine followed by rehabilitation at Appalachian Bear Rescue (ABR). Hematology and biochemistry reference ranges exist for healthy adult black bears; however, most bears presenting to ABR are young and of variable health status. Thus, further investigation into the difference of blood values at varying ages and presentations is warranted. ABR records from 1996 to 2022 included 106 bears with completed hematology and plasma biochemistry panels (22 paired samples at intake and release, 84 at intake only). Intake-only samples consisted of 12 neonates (<3 mon old), 64 cubs (3-12 mon), and 30 yearlings (1-2 yr). Bears presented as orphaned neonates (22%), orphaned cubs (45%), malnourished yearlings (24%), and injured/ill (9%) during fall (16%), winter (13%), spring (32%), and summer (39%). Changes in hematology and plasma biochemistry results between intake and release included an increase in hematocrit and glucose. Injured/ill bears presented with higher total leukocyte count (WBC), absolute neutrophils (ABS segs), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (P < 0.05). Positive correlation between ALT, AST, proteins, and blood urea nitrogen and negative correlation between absolute lymphocytes and alkaline phosphatase were noted with age. Both WBC and ABS segs were lower during winter (P < 0.05). Understanding what factors affect juvenile black bear blood values improves clinical expectations and evaluation upon intake, clinical evaluation, and treatment.

Serum steroid and thyroid hormone concentrations in healthy domestic male and female alpacas.

Alpacas are high quality fiber producing animals, kept for production purpose and as pets. Endocrine imbalances from adrenal glands, gonads, or thyroid gland may result in coat abnormalities in domestic animals and affect reproduction. Contrary to many domesticated animals, information on hormone concentrations in alpacas is scarce. The purpose of this study was to provide steroid and thyroid hormone values in domestic alpacas. Blood was collected from healthy male (35 intact, 2 castrated) and female (48 non-pregnant, 3 pregnant) alpacas from local farms in Tennessee. Adrenal, reproductive, and thyroid hormones were analyzed. There were no significant differences in median concentrations of progesterone, estradiol, thyroxine (T4), and triiodothyronine (T3) between intact male and female non-pregnant alpacas. Median concentrations of testosterone, androstenedione, 17-hydroxyprogesterone, and cortisol were significantly higher in intact male alpacas compared to female non-pregnant alpacas. This information provides adrenal, gonadal, and thyroid hormone concentrations in alpacas to help with diagnosis of endocrine disorders.

Validation of a human paper-based blood typing method for use in pet pigs.

BACKGROUND: Evidence-based medical practices for pet pigs are needed. EldonCard is a human blood-typing card shown to be rapid and reliable in identifying blood phenotypes of pet pigs. The objective of this study was to validate EldonCard by determining its reliability, reproducibility, and robustness for its routine use in the clinical setting. KEY FINDINGS: Twenty-four venous blood samples from pet pigs were collected for a prospective in vitro study. Blood genotypes ("EAAA0 " and "EAA00 ") were identified in 15 samples via polymerase chain reaction (PCR). All samples were phenotyped ("A," "Aweak ," and "0" or "-") via EldonCard. Kappa (κ) statistics measured the level of agreement between 2 raters, and between EldonCard and PCR. McNemar's test determined if an association between the blood types and EldonCard or PCR exists, with significance at P < 0.05. Agreement between raters and methods was perfect (60/60 [100%], κ:1, P < 0.001; 15/15 [100%], κ:1, P < 0.001). There was no difference in the proportions of blood groups based on method. SIGNIFICANCE: In conjunction with previous data, EldonCard is a rapid, accurate, reliable, precise, and robust in-clinic blood-typing method for the A0 system of pet pigs. EldonCard is now a validated blood-typing tool for the A0 system of pet pigs and maybe used for pretransfusion screenings and identification of donors and recipients.

Comparison of steroid and thyroid hormone concentrations in blood serum and plasma of captive tigers.

Analysis of steroid and thyroid hormones is often performed in blood serum. Occasionally though, plasma samples are submitted in lieu of serum for exotic species such as tigers. However, blood tube anticoagulants may affect hormone values. We compared serum and heparin plasma results for 7 hormones in tigers. Serum and plasma samples were collected from 25 tigers and analyzed for progesterone, 17-hydroxyprogesterone, cortisol, androstenedione, testosterone, estradiol, and thyroxine. Using Lin concordance correlation, serum and heparin plasma measures agreed for all hormones except cortisol. However, Passing-Bablok regression only found agreement between serum and heparin plasma measures for androstenedione, testosterone, and estradiol. Median values between the 2 sample types were significantly (p < 0.05) different for progesterone, 17-hydroxyprogesterone, cortisol, and thyroxine. Our results suggest that, for the aforementioned hormones, serum and heparin plasma values may not always be comparable.

TRANSCUTANEOUS OXYGEN MONITORING IN LOUISIANA PINE SNAKES (PITUOPHIS RUTHVENI).

Hypoxic physiological states may occur during anesthetic events of snakes but accurate monitoring of oxygenation is challenging. Oxygenation levels of nine Louisiana pine snakes (Pituophis ruthveni) were assessed using transcutaneous regional oxygen saturation (rSO2) at the level of the liver (rSO2Liver) and at the halfway point of the body (rSO2Half ). Reflectance pulse oximetry measured SpO2, with a sensor overlying the heart. Values were compared with the venous partial pressure of oxygen (PvO2). Measurements were taken during four phases, simulating an anesthetic event: phase 1, breathing room air; phase 2, while supplied with supplemental oxygen via face mask; phase 3, during ketamine and dexmedetomidine sedation; and phase 4, after receiving atipamezole. There were no significant changes in any oxygenation parameters between concurrent phases, but respiratory rate significantly decreased (P = 0.02) between phases 1 and 2. Strong positive associations were found between both rSO2Liver and rSO2Half when compared with PvO2 irrespective of phase (r = 0.72, r = 0.63 respectively), but not with SpO2 (r = 0.3). Strength of correlation varied with each phase but was uniformly strongest for rSO2Liver. The measurement of rSO2 appears superior compared with traditional pulse oximetry for assessing oxygenation levels of snakes.

Cortisol, progesterone, 17α-hydroxyprogesterone, and TSH responses in dogs injected with low-dose lipopolysaccharide.

BACKGROUND: Stress and diseases such as endotoxemia induce cortisol synthesis through a complex biosynthetic pathway involving intermediates (progesterone, and 17α-hydroxyprogesterone (17α-OHP)) and suppression of the hypothalamus-pituitary-thyroid axis. OBJECTIVE: To measure plasma concentrations of cortisol, progesterone, 17α-OHP, and thyroid stimulating hormone (TSH) in dogs experimentally injected with intravenous low-dose lipopolysaccharide (LPS). Our hypothesis was that LPS treatment would elicit a significant increase in cortisol and its precursors, and a significant decrease in TSH concentration. METHODS: Hormone measurements were performed on blood samples left over from a previous investigation (2011) on the effect of low-dose LPS on hematological measurands. Five sexually intact female dogs, none in estrous at the time of the study, were administered saline treatment two weeks prior to LPS treatment. LPS was administered intravenously at a dose of 0.1 µg/kg. Blood was collected before (baseline, time -24 hours) and 3-, 6- and 24-hours post-injection. Mixed model analysis for repeated measures was used, with both treatment and time as the repeated factors. Ranked transformation were applied when diagnostic analysis exhibited violation of normality and equal variance assumptions. Post hoc multiple comparisons were performed with Tukey's adjustment. Statistical significance was defined as p < 0.05. RESULTS: Significant differences relative to baseline values were detected following both treatments. Compared to baseline, dogs had significantly higher cortisol and 17α-OHP at 3-hours, and significantly lower TSH at 3- and 6-hours following LPS treatment. Dogs had significantly lower TSH at 6- and 24- following saline treatment. Though not statistically significant, the trend in progesterone concentrations was similar to cortisol and 17α-OHP, with an increase at 3-hours post-injection followed by a decrease close to baseline following both LPS and saline. Cortisol and 17α-OHP concentrations were higher after LPS treatment than after saline treatment at 3- and 6-hours post-injection, but differences were not statistically significant, and no significant differences between treatments were detected for any other hormone or timepoint. DISCUSSION AND CONCLUSION: Cortisol and its adrenal precursors are released in the bloodstream following a low dose of LPS, while TSH appears to decrease. Similar changes occurred following saline treatment, suggesting that even routine handling and saline injection in conditioned dogs can elicit alterations in the internal equilibrium with subsequent modification of both hypothalamus-pituitary-adrenal and thyroid axes. Changes to adrenal and thyroid hormone concentrations must be interpreted in light of clinical information. Further studies are needed to elucidate mechanisms of adrenal steroidal hormone synthesis and secretion in response to various stressful stimuli in both neutered and intact animals.

Analytic performance evaluation of a veterinary-specific ELISA for measurement of serum cortisol concentrations of dogs.

OBJECTIVE To investigate the precision of an ELISA for measurement of serum cortisol concentration (SCC) in dogs, assess agreement between this ELISA and 2 validated chemiluminescence assays (CLAs), and evaluate the clinical implications of any bias associated with this ELISA when measuring SCC in dogs. DESIGN Evaluation study. SAMPLE 75 stored, frozen serum samples from client-owned dogs. PROCEDURES Enzyme-linked immunosorbent assay precision was evaluated by measuring SCC of pooled serum samples. Agreement with standard methods was evaluated with Spearman rank correlation, Passing-Bablok regression, and Bland-Altman analysis to compare SCCs obtained with the ELISA and the 2 CLAs. An error grid was used to evaluate identified bias. RESULTS Within-laboratory coefficients of variation for pooled serum samples with low, medium, and high SCCs were 21.4%, 28.9%, and 13.0%, respectively. There was a high correlation between ELISA results (for all samples combined) and results of the 2 CLAs (CLA 1, r = 0.96; CLA 2, r = 0.97), but constant and proportional biases between the ELISA and CLAs were present at all concentrations. Clinically important disagreement between ELISA results and CLA results occurred in 16 of 63 (25%) samples, particularly with low and high SCCs. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the rate of clinical disagreement between the ELISA and CLAs was sufficiently high to recommend that equivocal results obtained with the ELISA be confirmed by a reference laboratory. Further evaluation of analytic performance of the ELISA should focus on samples with very high and very low SCCs.

Accuracy of cytology in distinguishing adrenocortical tumors from pheochromocytoma in companion animals.

BACKGROUND: The distinction between adrenocortical tumors and pheochromocytoma can be challenging using clinical findings, diagnostic imaging and laboratory tests. Cytology might be a simple, minimally invasive method to reach a correct diagnosis. OBJECTIVES: The purpose of this study was to assess the accuracy of cytology in differentiating cortical from medullary tumors of the adrenal glands in dogs and cats. METHODS: Cytologic key features of adrenocortical tumors and pheochromocytoma were defined by one reference author. Cytologic specimens from primary adrenal tumors were submitted to 4 cytopathologists who were asked to classify the tumors based on the previously defined key features without knowledge of previous classification. RESULTS: Twenty specimens from histologically confirmed adrenal tumors (Group 1) and 4 specimens from adrenal tumors causing adrenal-dependent Cushing's syndrome (Group 2) were evaluated by the 4 cytopathologists. Accuracy in differentiating cortical from medullary origin ranged from 90% to 100%, with a Kappa coefficient of agreement between cytopathologists of 0.95. CONCLUSIONS: The origin of an adrenal tumor can be easily determined by cytology alone in many cases. However, cytology was not reliable in distinguishing benign from malignant neoplasia. Additional studies are needed to assess possible risks and complications associated with fine-needle biopsy of adrenal tumors in dogs and cats.

Evaluation of factors that affect analytic variability of urine protein-to-creatinine ratio determination in dogs.

OBJECTIVE: To determine whether preanalytic and analytic factors affect evaluation of the urinary protein-to-creatinine (UPC) ratio in dogs. SAMPLE: 50 canine urine samples. PROCEDURES: The UPC ratio was measured to assess the intra-assay imprecision (20 measurements within a single session), the influence of predilution (1:10, 1:20, and 1:100) for urine creatinine concentration measurement, and the effect of storage at room temperature (approx 20°C), 4°C, and -20°C. RESULTS: The coefficient of variation at room temperature determined with the 1:20 predilution was < 10.0%, with the highest coefficients of variation found in samples with a low protein concentration or low urine specific gravity. This variability could result in misclassification of samples with UPC ratios close to the thresholds defined by the International Renal Interest Society to classify dogs as nonproteinuric (0.2), borderline proteinuric (0.21 to 0.50), or proteinuric (> 0.51). A proportional bias was found in samples prediluted 1:10, compared with samples prediluted 1:20 or 1:100. At room temperature, the UPC ratio did not significantly increase after 2 and 4 hours. After 12 hours at room temperature and at 4°C, the UPC ratio significantly increased. The UPC ratio did not significantly change during 3 months of storage at -20°C. CONCLUSIONS AND CLINICAL RELEVANCE: The intra-assay precision of the UPC ratio was sufficiently low to avoid misclassification of samples, except for values close to 0.2 or 0.5. The optimal predilution ratio for urine creatinine concentration measurement was 1:20. A 1:100 predilution is recommended in samples with a urine specific gravity > 1.030. The UPC ratio must be measured as soon as samples are collected. Alternatively, samples should be immediately frozen to increase their stability and minimize the risk of misclassification of proteinuria.

Role of hepcidin in iron metabolism and potential clinical applications.

The relatively recent discovery of hepcidin has stimulated renewed research interest in iron metabolism and iron-related disorders, emphasizing the importance of this hormone in many normal and pathologic processes. Important questions still remain to be answered; however, research to date offers promising diagnostic and therapeutic implications for both humans and veterinary species.

Grey eosinophils in sighthounds: frequency in 3 breeds and comparison of eosinophil counts determined manually and with 2 hematology analyzers.

BACKGROUND: Grey eosinophils (GE) reported to occur in Greyhounds, and occasionally in other breeds, have clear granules, probably due to abnormal staining properties. OBJECTIVES: The aims of this study were to investigate the frequency of GE in Greyhounds and 2 other sighthound breeds, and to assess the capacity of the ADVIA 120 and Sysmex XT-2000iV hematology analyzers to correctly identify GE. METHODS: Blood samples from 20 Greyhounds, 29 Italian Greyhounds, and 24 Whippets were analyzed using the ADVIA and Sysmex hematology analyzers, and blood smears stained with May-Grünwald Giemsa were evaluated microscopically. The frequency of samples with GE detected on smears was recorded for each breed. Manual and automated eosinophil counts were compared using a Wilcoxon signed-rank test. Agreement between methods was assessed using Passing-Bablok and Bland-Altman plots. RESULTS: GE were detected in all 3 breeds: 9/20 Greyhounds (45.0%), 10/29 Italian Greyhounds (34.5%), and 5/24 Whippets (62.5%) with no significant differences in the frequency of GE among the breeds. In samples containing GE, both analyzers underestimated the percentage of eosinophils and occasionally eosinophils were not detected at all. When a novel "GE gate" was used, the percentage of eosinophils reported by the Sysmex was similar to that obtained by manual counting. CONCLUSIONS: GE are found in the blood of sighthounds other than Greyhounds. Hematology analyzers may underestimate the percentage of GE, probably due to their abnormal physical or chemical features. Underestimation is slight and usually clinically insignificant, but occasionally eosinophils are completely misclassified. Using the Sysmex analyzer, a GE gate can be designed to normalize the eosinophil count.

High-resolution gel electrophoresis and sodium dodecyl sulphate-agarose gel electrophoresis on urine samples for qualitative analysis of proteinuria in dogs.

The aims of the current study were to assess whether sodium dodecyl sulphate-agarose gel electrophoresis (SDS-AGE) and high-resolution electrophoresis (HRE) can identify dogs with a urinary protein-to-creatinine ratio (UPC ratio) >0.2 and whether HRE can provide preliminary information about the type of proteinuria, using SDS-AGE as a reference method. HRE and SDS-AGE were conducted on 87 urine samples classified according to the International Renal Interest Society as non-proteinuric (NP; UPC ratio: <0.20; 32/87), borderline proteinuric (BP; UPC ratio: 0.21-0.50; 15/87), or proteinuric (P; UPC ratio: >0.51; 40/87). SDS-AGE and HRE were positive in 14 out of 32 and 3 out of 32 NP samples and in 52 out of 55 and 40 out of 55 samples with a UPC ratio >0.20, respectively. The concordance between HRE or SDS and UPC ratio was comparable (κ = 0.59; κ = 0.55). However, specificity (90%) and positive likelihood ratio (7.76) were higher for HRE than for SDS-AGE (56% and 2.16) while sensitivity was lower (73% vs. 94%). The analysis of HRE results revealed that a percentage of albumin >41.4% and an albumin/α(1)-globulin ratio (alb/α(1) ratio) >1.46 can identify samples classified by SDS-AGE as affected by glomerular proteinuria while a percentage of α(1)-globulin >40.8% and an alb/α(1) ratio <0.84 can identify samples classified by SDS-AGE as affected by tubular proteinuria. In conclusion, both SDS-AGE and HRE could misclassify samples with a UPC ratio higher or lower than 0.20. Therefore, UPC ratio must always be determined before conducting these tests. The percentage of albumin and α(1)-globulin or the alb/α(1) ratio determined by HRE can provide preliminary information about the origin of proteinuria.

Identification of neoplastic cells in blood using the Sysmex XT-2000iV: a preliminary step in the diagnosis of canine leukemia.

BACKGROUND: Classification of leukemias requires specialized diagnostic techniques. Automated preliminary indicators of neoplastic cells in blood would expedite selection of appropriate tests. OBJECTIVE: The objective of this study was to assess the capacity of the Sysmex XT-2000iV hematology analyzer to identify neoplastic cells in canine blood samples. METHODS: Blood samples (n=160) were grouped into 5 categories: acute leukemia (n=30), chronic leukemia (n=15), neoplasia without blood involvement (n=41), non-neoplastic reactive conditions (n=31), and healthy dogs (n=43). WBC counts, WBC flags, scattergrams, percentages of cells with high fluorescence intensity, and percentages of cells in the lysis-resistant region were evaluated alone or in combination to establish a "leukemic flag." Sensitivity, specificity, negative (LR-) and positive (LR+) likelihood ratios, and the number of false-negative (FN) and false-positive (FP) results were calculated, and receiver operating characteristic curves were designed for numerical values. RESULTS: Among single measurements and parameters, only the evaluation of scattergrams minimized FN and FP results (sensitivity 100%, specificity 94.8%, LR+ 19.17, and LR- 0.00), although their interpretation was subjective. The more objective approach based on the generation of a "leukemic flag" had a sensitivity of 100%, specificity of 87.0%, LR- of 0.00, and LR+ of 7.67. CONCLUSION: Using a novel gating strategy the Sysmex XT-2000iV may be used effectively to screen canine blood for hematopoietic neoplasia.