Methodology-related pseudohyperkalemia associated with marked muscle enzyme leakage in a dog.

BACKGROUND: As hyperkalemia may be life-threatening, it is critical to recognize artifactually increased potassium concentrations. Pseudohyperkalemia may occur in myopathies when using the VetScan2 analyzer (VS2), but the degree of pseudohyperkalemia and relationships relative to creatine kinase activity (CK) are unknown. OBJECTIVES: We aimed to determine what degree of muscle enzyme leakage, as reflected by increased serum CK activity, results in cases with falsely elevated potassium concentrations when measured by the VS2. We also sought to establish if a linear relationship exists between potassium concentrations measured by the VS2 and CK activity. METHODS: Serum samples from dogs with increased CK activity and with CK activity within the reference interval and without clinically relevant biochemical alterations were used to create diluted samples having various CK activities. Potassium concentrations and CK activities were determined on VS2 and Cobas c501 (Cobas) analyzers. Wilcoxon signed rank, Bland-Altman, and Passing-Bablok analyses were used to compare potassium concentrations generated by the VS2 and Cobas analyzers. Least squares regression analysis was performed to evaluate if a linear relationship exists between VS2 potassium concentrations and Cobas CK activities. RESULTS: Potassium concentrations from the VS2 were higher (median and standard deviation (SD) = 5.2 +/- 0.46 mmol/L) than those from the Cobas analyzer (4.4 +/- 0.35 mmol/L; P < 0.000), and a positive mean bias was found (mean difference = 0.774 mmol/L; 95% Confidence Interval (CI) = 0.706-0.842; limits of agreement = 0.21-1.34). Passing-Bablok regression showed a positive proportional bias for potassium concentrations on the VS2 compared with paired Cobas results (Slope = 1.328; 95% CI = 1.100-1.500) but did not reveal systematic bias (Intercept = -0.714; 95% CI = -1.46-0.265). Least squares regression analysis showed a poor non-significant relationship (R2 = 0.19) between potassium measured by the VS2 and CK measured by the Cobas analyzer. CONCLUSIONS: A defined threshold value of CK activity at which potassium concentration begins to falsely increase when measured on the VS2 was not established as data widely varied. A poor non-significant relationship between potassium concentrations and CK activities did not allow prediction of the threshold at which falsely increased potassium concentrations would be expected on the VS2.

Impact on canine neutrophil preservation with the addition of bovine serum albumin to K3 -EDTA whole blood samples.

BACKGROUND: Cellular deterioration occurs with blood sample aging, impacting white blood cell (WBC) identification and differential accuracy. This may be exacerbated in samples from patients experiencing inflammation. Previously, bovine serum albumin (BSA) has been shown to improve cellular preservation of blood and other samples, but the effect on cell preservation in canine blood has not been assessed. OBJECTIVES: We aimed to determine the effects of BSA on neutrophil nuclear area when added to potassium ethylene diamine tetra-acetic acid (K3 -EDTA)-anticoagulated canine blood prior to blood smear preparation. We evaluated the impact of inflammatory leukograms, sample storage temperatures (4° and 20°C), and time on outcomes. MATERIALS AND METHODS: Canine K3 -EDTA-anticoagulated blood samples stored at 4° and 20°C were used from unique patients, 10 with and 10 without inflammatory leukograms. Blood smears were prepared from aliquots with or without the addition of 22% BSA at 0, 4, 8, 24, 48, and 72 h. The nuclear area was measured for 25 randomly selected neutrophils per slide using Fiji software. Mixed-effect linear regression modeling was performed (significance: P < 0.05). RESULTS: Nuclear area increased over time with and without added BSA. Both sample storage temperatures and the presence or absence of an inflammatory leukogram significantly impacted neutrophil nuclear area. Samples with added BSA had slightly higher predicted nuclear areas than those without BSA, but this difference was not statistically significant. CONCLUSIONS: BSA did not significantly impact neutrophil nuclear area and did not improve neutrophil preservation in canine blood samples.

Distribution and diversity of dog parvoviruses in wild, free-roaming and domestic canids of Newfoundland and Labrador, Canada.

Some parvoviruses of carnivorans can infect multiple host species. Since many canine parvoviruses were only discovered recently, their host-range is still unexplored. We examined the host distribution and diversity of five dog parvoviruses in four canine populations from Newfoundland and Labrador, Canada, and investigated the potential for these viruses to cross the species barriers. Canine bocavirus 2 (CBoV-2) and the minute virus of canines were detected in stool from free-roaming dogs from Labrador (5/48 [10.4%] and 3/48 [6.3%], respectively) and two different CBoV-2 variants were identified. Canine bufavirus was identified in stool from free-roaming dogs (1/48, 2.1%) and foxes (3/80, 3.8%) from Labrador, but two different variants were observed in the two host species. The variant found in foxes was highly divergent from previously identified strains. Two cachavirus 1 variants, genetically similar to those circulating in other Canadian wildlife, were found in spleens from Newfoundland coyotes (3/87, 3.5%). Canine parvovirus type 2 (CPV-2) was found in stool from free-roaming dogs from Labrador (2/48, 4.2%) and in spleens from Newfoundland coyotes (3/87, 3.5%). Comparing CPV-2 sequences from these hosts to those retrieved from local symptomatic domestic dogs revealed the presence of a highly heterogeneous viral population as detected strains belonged to five different clades. The close relationship between CPV-2a strains from a dog and a coyote suggests the occurrence of viral transfer between wild and domestic canids. The identification of highly related strains with a similar molecular signature characteristic of older CPV-2 strains in free-roaming and domestic dogs suggests a probable common ancestry and that older CPV-2 strains, which have not been identified in dogs since the 1990s, persist in this part of Canada. Follow-up studies should evaluate samples from a larger number of animals and host species to extensively investigate the possible occurrence of cross-species transmission for recently discovered parvoviruses.

Newlavirus, a Novel, Highly Prevalent, and Highly Diverse Protoparvovirus of Foxes (Vulpes spp.).

The genus Protoparvovirus (family Parvoviridae) includes several viruses of carnivores. We describe a novel fox protoparvovirus, which we named Newlavirus as it was discovered in samples from Newfoundland and Labrador, Canada. Analysis of the full non-structural protein (NS1) sequence indicates that this virus is a previously uncharacterized species. Newlavirus showed high prevalence in foxes from both the mainland (Labrador, 54/137, 39.4%) and the island of Newfoundland (22/50, 44%) but was not detected in samples from other carnivores, including coyotes (n = 92), lynx (n = 58), martens (n = 146), mink (n = 47), ermines (n = 17), dogs (n = 48), and ringed (n = 4), harp (n = 6), bearded (n = 6), and harbor (n = 2) seals. Newlavirus was found at similar rates in stool and spleen (24/80, 30% vs. 59/152, 38.8%, p = 0.2) but at lower rates in lymph nodes (2/37, 5.4%, p < 0.01). Sequencing a fragment of approximately 750 nt of the capsid protein gene from 53 samples showed a high frequency of co-infection by more than one strain (33.9%), high genetic diversity with 13 genotypes with low sequence identities (70.5-87.8%), and no geographic segregation of strains. Given the high prevalence, high diversity, and the lack of identification in other species, foxes are likely the natural reservoir of Newlavirus, and further studies should investigate its distribution.

Zinc toxicosis - Associated hemolytic anemia and pancreatic disease in 2 dogs.

Two cases of canine zinc toxicosis with pancreatic disease are reported. Both dogs had pigmenturia, pallor, regenerative anemia, an inflammatory leukogram, hyperbilirubinemia, hyperamylasemia, and hyperlipasemia. Zinc toxicosis was diagnosed post-mortem in patient 1 based on the discovery of a metallic gastric foreign object, pancreatic necrosis, nephrosis, and high tissue zinc concentrations. Survey radiographs of patient 2 allowed identification and prompt removal of coins from the stomach with resolution of anemia, hyperamylasemia, and hyperlipasemia 3 days post-gastroscopy. Plasma zinc concentration was markedly elevated in patient 2. Zinc toxicosis should be considered in dogs with concurrent hemolytic anemia and pancreatic disease.

Toxicose par le zinc ­ Anémie hémolytique associée et maladie pancréatique chez deux chiens. Deux cas de toxicose par le zinc avec maladie pancréatique sont rapportés. Les deux chiens présentaient de la pigmenturie, de la pâleur, une anémie régénératrice, une formule leucocytaire inflammatoire, de l'hyperbilirubinémie, une hyperamylasémie et une hyperlipasémie. La toxicose par le zinc fut diagnostiquée post-mortem chez le patient 1 basée sur la découverte d'un corps étranger métallique dans l'estomac, une nécrose pancréatique, une néphrose, et des concentrations élevées en zinc dans les tissus. Des radiographies du patient 2 ont permis l'identification et le retrait rapide de pièces de monnaie de l'estomac avec retour à la normale, trois jours post-gastroscopie, de l'anémie, l'hyperamylasémie, et l'hyperlipasémie. La concentration plasmatique de zinc était significativement élevée chez le patient 2. La toxicose par le zinc devrait être prise en considération chez les chiens avec anémie hémolytique concomitante à une maladie pancréatique.(Traduit par Dr Serge Messier).

Intra- and inter-rater agreement for the detection of band neutrophils and toxic change in horses.

BACKGROUND: The detection of band neutrophils and toxic change via microscopic blood smear review is vitally important, as their presence indicates systemic inflammation. However, in-clinic evaluation of WBC morphology is often limited. OBJECTIVE: We aimed to determine the agreement between expert raters in the detection of bands and toxic change. METHODS: Three board-certified clinical pathologists each evaluated 109 blood smears from horses with acute disease, and 19 control smears from healthy horses. The pathologists determined if bands were present, and if so, the percentage of bands present. They also determined if toxic change was present, and if so, the grade of toxic change. Intra-rater agreement was evaluated using 12 duplicate blood smears. Agreement on the presence of bands between pathologists and an in-clinic hematology analyzer was evaluated. RESULTS: Intra-rater agreement was substantial to almost perfect. Agreement between pathologists for the detection of bands was moderate, but when pathologists agreed bands were present, there was excellent agreement on the percentage of bands and mature neutrophils. Agreement between pathologists for the detection of high-grade, clinically relevant toxic change was fair. When pathologists agreed high-grade toxic change was present, there was fair agreement on Döhle bodies and cytoplasmic basophilia and poor agreement on cytoplasmic vacuolation. Agreement between individual pathologists and the in-clinic hematology analyzer for the indication of bands was fair to moderate. CONCLUSIONS: Consistent identification of bands and toxic change is challenging, even for highly trained personnel. It is, thus,not surprising that in-clinic blood smear evaluation of WBC morphology by non-experts could be inadequate.

Effects of stylet-in versus stylet-out collection of cerebrospinal fluid from the cisterna magna on contamination of samples, sample quality, and collection time.

OBJECTIVE: To evaluate safety of stylet-in and stylet-out techniques for collection of CSF from the cisterna magna and to assess whether there were differences between techniques with regard to contamination of samples, sample quality, and efficiency of collection. ANIMALS: 10 adult purpose-bred research Beagles. PROCEDURES: A prospective crossover study was conducted. Preanesthetic physical and neurologic examinations and hematologic analyses were performed. Dogs were anesthetized, and collection of CSF samples from the cisterna magna by use of a stylet-in or stylet-out technique was performed. Two weeks later, samples were collected with the other sample collection technique. Samples of CSF were processed within 1 hour after collection. RESULTS: Cellular debris was detected in higher numbers in stylet-in samples, although this did not affect sample quality. The stylet-out technique was performed more rapidly. No adverse effects were detected for either technique. CONCLUSIONS AND CLINICAL RELEVANCE: Both techniques could be safely performed in healthy anesthetized dogs. The stylet-out technique was performed more rapidly and yielded a sample with less cellular debris. Both techniques can be used in clinical practice to yield CSF samples with good diagnostic quality.

Fine-needle aspiration of cutaneous, subcutaneous, and intracavitary masses in dogs and cats using 22- vs 25-gauge needles.

BACKGROUND: Fine-needle aspiration (FNA) is a common procedure as a diagnostic tool in veterinary medicine. However, it is unclear whether the gauge of the needle affects the quality of cytology. OBJECTIVE: This study compared the quality of cytologic samples obtained via FNA using 22- or 25-gauge needles. METHODS: Fine-needle aspiration was performed on 50 masses (cutaneous, subcutaneous, or intracavitary) obtained from client-owned animals. The size of the needle was randomly assigned using either of the following two sequences: 22-25-22 gauge or 25-22-25 gauge. Samples were evaluated by two board-certified clinical pathologists to assess cellularity, blood contamination, amount of cellular debris, degree of cellular trauma, and the overall ability to make a diagnosis for each sample. RESULTS: No significant difference was detected between the 22- and 25-gauge needle samples for cellularity, whereas a significant difference was present for blood contamination, amount of cellular debris, and degree of cellular trauma. The overall ability to make a diagnosis was not significantly affected by the needle gauge. The degree of cellular trauma was significantly increased in intracavitary samples. CONCLUSIONS AND CLINICAL RELEVANCE: Needle gauge is a contributing factor to FNA sample quality. However, it did not affect the overall ability to make a diagnosis. Samples obtained using 25-gauge needles resulted in less blood contamination yet increased cellular trauma compared to 22-gauge needle samples.

Hematologic reference intervals for Rana sylvatica (Lithobates sylvaticus) and effect of infection with Frog Virus 3 (Ranavirus sp., Iridoviridae).

BACKGROUND: Although the Wood Frog, Rana sylvatica, is used in research on infectious diseases of amphibians, hematologic RIs or response to infection have not been established. OBJECTIVES: The purpose of the study was to determine hematologic RIs for adult Wood Frogs and alterations associated with infection with Frog Virus 3 (FV3, Ranavirus sp.). METHODS: Blood was collected from 40 wild-caught adult Wood Frogs that had been in captivity for 6 months. Complete (Natt-Herrick solution hemocytometry) and differential (Wright-Giemsa-stained smears) WBC, RBC, and thrombocyte cell counts, PCV, and automated total cell counts (WBC+RBC+thrombocytes, Sysmex particle counting) were determined. Concordance correlation coefficients determined agreement between hemocytometric and automated total cell counts. Thirteen frogs were orally infected with a lethal dose of 10(4.43) plaque-forming units of FV3 and terminally sampled 4, 9, or 14 days postinfection (dpi). Pre- and postinfection variables for each frog were compared. RESULTS: Leukocyte morphology was similar to that of other amphibians and mammals. Lymphocytes were the most numerous WBC. PCV and RBC counts were similar to other frogs in the same family. Agreement was good between hemocytometry and automated total cell counts. Infection with FV3 caused neutrophilia, increase in undifferentiated blast-like cells, and reduction in the percentage of basophils. Lymphocytes decreased at 4 and 9 dpi but increased at 14 dpi. From 9 dpi onwards, nuclear deterioration and mild toxic change were present in neutrophils; viral cytoplasmic inclusion bodies were observed in lymphocytes, monocytes, neutrophils, and eosinophils. CONCLUSION: We provide hematology RIs for Rana sylvatica, and report hematologic changes associated with a lethal FV3 infection.

Hematologic reference intervals for Xenopus tropicalis with partial use of automatic counting methods and reliability of long-term stored samples.

BACKGROUND: The African frog, Xenopus tropicalis, is widely used in biomedical and toxicologic research. Reference intervals (RI) for hematologic variables, valuable to research and health status assessment, have not been established. OBJECTIVES: The purpose of the study was to determine hematologic RI of X tropicalis, and establish whether automated cell counting can facilitate routine hematologic assessment in frogs. METHODS: Blood from 41 adult healthy X tropicalis was collected via cardiac puncture, and diluted in Natt-Herrick solution. Complete WBC, RBC, and thrombocyte counts (hemocytometry), differential WBC counts (Wright-Giemsa-stained smears), PCV (centrifugation), total protein (refractometry), and automated total cell counts (WBC + RBC + thrombocytes, Sysmex particle counting) were determined. Concordance correlation coefficients calculated the agreement between total cell counts obtained by hemocytometry and automated particle counting, and between total cell counts at collection and after 2 years of storage. RESULTS: Leukocyte morphology was similar to other amphibians and mammals. PCV was similar to other frogs; RBC counts were higher, and MCV was lower than in other frog species. Neutrophils were the most numerous WBC. Agreement was good between hemocytometry and automated cell counts. Subtracting the hemocytometer WBC and thrombocyte counts from the automated total cell count reliably yielded the RBC count. Cellular integrity evaluated 2 years post collection was good, and automated counts were not clinically different from counts at collection. CONCLUSION: We provide hematologic RI for X tropicalis, suggest how automated cell counts may facilitate hematologic assessments of frogs, and establish that blood in Natt-Herrick solution is stable 2 years post collection.

Poorly differentiated cutaneous carcinoma of non-sebaceous origin in a 3-year-old Mongolian gerbil (Meriones unguiculatus).

A 3-year-old female gerbil developed a non-healing skin wound due to a malignant neoplasm. Histology, immunohistochemistry (cytokeratin 19 positive; vimentin, estrogen, and progesterone receptor negative), and electron microscopy (no desmosomes or melanosomes) revealed an undifferentiated carcinoma with pulmonary metastasis. Unlike in previous reports, it did not arise from the abdominal pad's sebaceous gland.

Carcinome cutané d'origine non sébacée peu différencié chez une gerbille de Mongolie âgée de 3 ans(Meriones unguiculatus). Une gerbille femelle âgée de 3 ans a développé une plaie cutanée qui ne guérissait pas en raison d'un néoplasme malin. Des examens histologiques, par immunohistochimie (positif pour la cytokératine 19; négatif pour les récepteurs de vimentine, d'œstrogène et de progestérone) et par microscopie électronique (pas de desmosomes ni de mélanosomes) ont révélé un carcinome indifférencié avec métastase pulmonaire. Contrairement aux rapports antérieurs, il n'était pas causé par la glande sébacée du coussinet abdominal.(Traduit par Isabelle Vallières).

Reliability of 400-cell and 5-field leukocyte differential counts for equine bronchoalveolar lavage fluid.

BACKGROUND: Reliable enumeration of mast cells and eosinophils in equine bronchoalveolar lavage (BAL) fluid is important because small increases in the percentages of these cells support the clinical diagnosis of inflammatory airway disease (IAD). Increases in BAL neutrophils also occur with IAD but are not specific due to overlap between IAD and recurrent airway obstruction (RAO). OBJECTIVES: The objectives of this study were to evaluate the reliability of a standard 400-cell leukocyte differential count and an alternate method evaluating 5 microscopic fields at 500× magnification in equine BAL fluid cytocentrifuged preparations. METHODS: BAL samples from 60 horses with and without pulmonary inflammation were evaluated using 400-cell and 5-field leukocyte differential counting methods. Reliability of enumeration of each leukocyte type was assessed by calculating and comparing intraclass correlation coefficients (ICC). Reliability of mast cell enumeration was further evaluated by comparing ICCs of slides with different cell densities. RESULTS: Reliability was higher for all cell types with the 5-field method; however, overall the difference between methods was not statistically significant. Neutrophil reliability was high (ICC > 0.90) with both methods. Adequate reliability (ICC > 0.85) for mast cells was achieved only with the 5-field method on slides with higher cell density. CONCLUSION: Enumeration of mast cells is unreliable when the standard 400-cell differential counting method is used, whereas the 5-field method on slides with higher cell density reached acceptable reproducibility. Neutrophil percentages were highly reliable with both methods.

Eosinophilia in a cat with acute leukemia.

A 4-year-old castrated male domestic shorthaired cat with a history of vomiting and anorexia was diagnosed with leukemia with marked hepatic and splenic infiltration and concurrent eosinophilia with marked tissue infiltration. Despite thorough immunocytochemical and immunohistochemical immunophenotyping, the cell lineage of the leukemia was not identified.

Evaluation of ionized and total serum magnesium concentrations in hyperthyroid cats.

Hyperthyroidism can increase the renal excretion of magnesium and thus cause hypomagnesemia in various species. Anaerobically collected blood samples from 15 hyperthyroid and 40 normal, healthy cats were analyzed with an ion-selective electrode analyzer and a serum biochemical analyzer. There was no significant difference in ionized or total serum magnesium concentration between the 2 groups, but there was a significant difference (P = 0.004) in the ratio of ionized to total serum magnesium concentrations between the healthy cats and the hyperthyroid cats with thyroxine (T4) concentrations at or above the median. There was a significant correlation (r = 0.894, P = 0.000) between the ionized and total magnesium concentrations in the hyperthyroid cats. The hyperthyroid cats had a significantly lower (P = 0.003) total serum protein concentration than the healthy cats. A significant negative correlation (r = -0.670, P = 0.006) was detected between the ionized magnesium and logarithmically transformed total T4 concentrations in the hyperthyroid cats, which suggests that the severity of hyperthyroidism may contribute to a decrease in the ionized magnesium concentration.

Validation of the Nova CRT8 for the measurement of ionized magnesium in feline serum.

BACKGROUND: Evaluation of serum magnesium (Mg) concentration is becoming important in human and veterinary critical care medicine. An ion-selective electrode can measure the physiologically active ionized fraction. OBJECTIVES: The purpose of this study was to validate an ion-specific electrode analyzer and assay for measuring ionized Mg in feline serum and to determine a reference interval for this analyte in cats. METHODS: Venous blood samples were collected anaerobically from clinically healthy cats, and the serum was used to validate the analyzer and assay. This included investigating the stability of samples stored at different temperatures, intra- and interassay precision, linearity, analytical sensitivity, and potential interferences from bilirubin, lipemia, hemoglobin, or serum separator tubes. A reference interval was calculated. RESULTS: Serum samples evaluated for ionized Mg concentrations can be stored at 20 degrees C for < or =24 hours, at 4 degrees C for < or =72 hours, and at 20 degrees C for < or =4 weeks, when samples are minimally exposed to air. Intra- and interassay precisions had coefficients of variation (CVs) of 1.23% and 2.02%, respectively. There was good linearity using serum (r = .998; y = -0.0057 + 1.0256x) and manufacturer-supplied aqueous solutions and quality control materials (r = .999; y = 0.0110 + 0.9213x). Apparent analytical sensitivity was at least 0.015 mmol/L. Mean recovery was good for ionized Mg in samples with 1+ icterus (104%), 4+ lipemia (99.3%) and 1-4+ hemolysis (98.6%). There was no significant difference (P = .52) in ionized Mg concentrations in serum collected in tubes containing no additives compared with serum collected in glass separator tubes. The serum ionized Mg reference interval was 0.47-0.63 mmol/L (n = 40). CONCLUSIONS: The Nova CRT8 analyzer and assay provide a precise and reliable method of measuring ionized Mg concentration in feline serum. Strict adherence to sampling techniques, handling, and storage are necessary for reliable results.