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.

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.

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.