Canine differential leukocyte counting with the CellaVision DM96Vision, Sysmex XT-2000iV, and Advia 2120 hematology analyzers and a manual method

Background: For differential leukocyte counts, automated blood smear evaluation systems have been too slow or inaccurate to replace or supplement the manual differential count. The CellaVision DM96Vision (DM96V), a new instrument, is an automated image analysis system that is rapid and accurate enough to be used for enumerating human leukocytes and may be useful for analysis of canine blood. Objectives: The aims of this study were to evaluate the performance of the DM96V in differential counting of canine leukocytes, to compare its performance with that of other methods, and to analyze interoperator variability. Methods: Four methods of determining the leukocyte differential count of 108 canine blood samples were compared based on agreement, precision, and errors as well as relative performance. Differential counts were obtained using the DM96V, the manual method, and automated methods performed by the Advia 2120 and Sysmex XT‐2000iV. Results: All leukocyte types were detected by the DM96V and the manual method, and all 4 methods had similar mean and median results in most cases. The automated methods were more precise than either the DM96V or manual method when comparing identification of a single type of leukocyte, especially neutrophils and lymphocytes. However, precision of the automated methods was only fair for monocytes, and the Advia and Sysmex failed to identify basophils. The Advia reported fewer monocytes and eosinophils than did the other methods. Significantly fewer lymphocytes were identified by the manual method than by the Sysmex, Advia, and DM96V. The DM96V occasionally presented duplicate images of the same neutrophils. Conclusions: The CellaVision DM96V is a satisfactory system for facilitating canine differential leukocyte counting. The DM96V differential count was more similar to the manual count than to automated counts, which were more precise but had errors and omissions in detecting some types of leukocytes.     

Characterization of the immunoglobulin A heavy chain gene of the Atlantic bottlenose dolphin (Tursiops truncatus)

Immunoglobulin constant region heavy chain genes of the dolphin (Tursiops truncatus) have been described for IgM and IgG but not for IgA. Here, the heavy chain sequence of dolphin IgA has been cloned and sequenced as cDNA. RT-PCR amplification from blood peripheral lymphocytes was carried out using degenerate primers and a single sequence was detected. The inferred heavy chain structure shows conserved features typical of mammalian IgA heavy chains, including three constant (C) regions, a hinge region between constant region domain 1 (C1) and constant region domain 2 (C2), and conserved residues for interaction with the Fc alpha R1 and N-glycosylation sites. Comparisons of the deduced amino acid sequences of the IgA heavy chain for the dolphin and the evolutionarily related artiodactyl species showed high similarity. In cattle and sheep, as in dolphins, a single IgA subclass has been identified. Southern blot analysis as well as genomic PCR confirmed the presence of multiple IGHA sequences suggesting that IGHA pseudogenes may be present in the dolphin genome.     

Morphology of the lymph nodes in bottlenose dolphin (Tursiops truncatus) and striped dolphin (Stenella coeruleoalba) from the Adriatic Sea

Morphology of the lymph nodes was examined in six bottlenose dolphins (Tursiops truncatus) and three striped dolphins (Stenella coeruleoalba) from the Adriatic Sea. All animals had been found dead in nature. One group of the nodes was taken from the tracheal branching area and was marked as bifurcational lymph node, and the other group was taken from the mesenteric root and was marked as mesenteric lymph node. Microscopic analysis showed that the lymph nodes in both dolphin species were surrounded by a connective tissue capsule comprising smooth muscle cells. The parenchyma of the mesenteric and bifurcational lymph nodes in bottlenose dolphin was divided into the peripherally situated cortex with the lymphatic nodules and diffuse lymphatic tissue, and the centrally situated medulla structured of the medullary cords separated by the medullary sinuses. These lymph nodes structurally correspond to the lymph nodes in the majority of terrestrial mammals. The mesenteric lymph node of striped dolphin also had a peripherally situated cortex and a centrally positioned medulla as the majority of terrestrial mammals. In the bifurcational lymph nodes of striped dolphin, there was a central dense lymphatic tissue with the lymphatic nodules and a peripheral less dense lymphatic tissue structured of the cell cords and sinuses. The bifurcational lymph node in striped dolphin resembled porcine lymph nodes and belonged to the inverse lymph nodes.     

Within-day and between-day variability of transthoracic anatomic M-mode echocardiography in the awake bottlenose dolphin (Tursiops truncatus)

The use of transthoracic echocardiography in dolphins has been limited so far owing to technical and anatomical specificities. Anatomic M-mode (AMM) is a postprocessing echocardiographic technique generating M-mode studies from two-dimensional (2D) cineloops independently of the ultrasound beam orientation. The aim of the present study was to determine the within-day (repeatability) and between-day (reproducibility) variability of AMM echocardiography in awake healthy bottlenose dolphins (BN, Tursiops truncatus). Four adult BN trained to lie in left recumbency at the water surface were involved in the protocol. A total of 96 echocardiographic examinations were performed on 4 different days by a trained observer examining each BN 6 times per day. Video clips of 2D left parasternal long-axis views showing the left ventricle (LV) ventrally and the aortic root dorsally were recorded at each examination and analyzed for AMM measurements in a random order. A general linear model was used to determine the within-day and between-day coefficients of variation (CV). All examinations were interpretable allowing calculation of 10 AMM variables (i.e., end-diastolic and end-systolic ventral and dorsal LV myocardial wall thicknesses as well as LV and aortic diameters, mean aortic diameter, and LV shortening fraction). Most within- and between-day CV values (18/20) were <15%, the lowest being observed for the end-diastolic LV diameter (1.6%). In conclusion, AMM provides a simple non-invasive evaluation of heart morphology and function in the awake BN with good repeatability and reproducibility of the measurements. Further studies are required to determine the corresponding reference intervals.     

Comparison between microscopic and automated differential leukocyte counts in the silver fox (Vulpes vulpes) and the blue fox (Alopex lagopus)

Differential leukocyte (WBC) counts in blood from clinically healthy silver foxes (n=32) and blue foxes (n=37) obtained from an automated hematology analyzer (Technicon H*1 Hematology System) with canine software were compared with microscopic differential WBC counts (M-diff). There was good agreement between the automated differential cell count (A-diff) and the M-diff for neutrophil and lymphocyte percentages. The correlation was lower for monocyte percentages and variable for eosinophil percentages. There was no significant difference between the A-diff and M-diff in either fox species. The A-diff counts were very precise, and may be a good alternative to the traditional M-diff for screening populations of clinically healthy foxes or for studies on stress and animal welfare. Intercept values, however, indicated a constant bias that must be taken into account before interpreting results based on different methods of analysis