Use of hyaluronidase in the comparison between manual and automated hematology analysis with the ADVIA 120 to improve analysis of feline body cavity effusions

Classification of body cavity effusions is an important step in the investigation and diagnosis of disease in cats. Feline inflammatory effusions are often highly proteinaceous and viscous, which can cause clumping of white cells and subsequently inaccurate nucleated cell counts (NCCs) using automated and manual methods. Microscopic assessment of cellularity can also be difficult given the variable thickness of smears and cell clumping, which skews white cell distribution. The ADVIA 120 uses 2 white cell–counting channels, the basophil/lobularity (WBC/baso) and differential/peroxidase channels, which can provide quite different results in highly viscous feline samples and often disagree with smear assessment of cellularity. We investigated the effects of pre-incubation of feline effusion samples with hyaluronidase and its effects on NCCs and cellularity assessment. NCCs were obtained by automated analysis using the ADVIA 120 and by manual counting methods. Agreement was assessed using a Bland–Altman chart. Pretreatment of samples with hyaluronidase resulted in good agreement between the ADVIA basophil channel and manual counting methods in all samples in the study. However, improvements in NCCs after hyaluronidase treatment were significantly greater in clumped samples, and cell distribution of these samples on direct smears was also improved. Therefore, when nucleated cell clumping is observed on a direct smear, pretreatment of the sample with hyaluronidase prior to analysis on an automated analyzer is advised, with the WBC/baso channel displaying the most accurate NCC

Making Way for the Car: Minimum Parking Requirements and Porirua City Centre

Minimum parking requirements (MPRs) mandate that each new development provides enough parking to ensure ample provision at the time of peak demand. This approach tends to oversupply parking above the optimal level, and by bundling parking into the development costs, ensures that parking is free to the user. As a result, land-use and transport decisions are distorted. A case study of Porirua central business district (CBD) was undertaken to investigate the use of MPRs in the New Zealand context, and to assess their impacts on transport and land-use patterns. Findings indicate that MPRs tend to oversupply parking relative to weekly mean and peak occupancies. Land use mapping found that 24 percent of CBD land is allocated to car parking and MPRs were shown to contribute to dispersed development patterns. Stated choice data and a cost recovery model for car parking highlight how free and ample car parking provision favours car driving and has distortionary impacts on travel decisions

Structure and Function of Leukocytes in the Family Macropodidae

Leukocytes play a central role in protecting the body against infectious organisms and their research is essential for understanding the mechanisms of immunity. By studying leukocytes across a range of species, insights are provided into differing strategies employed to ensure resistance to disease. Surprisingly, the structure and function of marsupial leukocytes has received very limited study. Marsupials represent a major evolutionary pathway with distinct differences in reproduction and development from placental mammals. These differences in the life history of marsupials place unique challenges on the immune system, and differences in leukocyte structure and function could be reasonably expected. In this thesis, studies were undertaken to examine the cytochemical, ultrastructural and functional features of leukocytes from species of marsupials, belonging to the family Macropodidae (kangaroos and wallabies). The aim of these studies was to elucidate the characteristics of macropodid leukocytes and to compare and contrast these features with the known characteristics of other mammalian leukocytes. Leukocytes from two species of macropodid, the tammar wallaby (Macropus eugenii) and the western grey kangaroo (Macropus fuliginosis), formed the basis of this study with additional material provided from quokka (Setonix brachyurus), woylie (Bettongia pencillata) and red kangaroo (Macropus rufus). Staining characteristics of cells were examined following reaction with Sudan black B, peroxidase, chloroacetate esterase, naphthyl butyrate esterase, alkaline phosphatase and periodic acid-Schiff. Peroxidase and Sudan Black B reactions were similar to domestic animal species but chloroacetate esterase and naphthyl butyrate esterase were unreliable as markers for macropodid neutrophils and monocytes, respectively. Significant variation in staining for alkaline phosphatase was seen between species of macropodid. Tammar wallabies and quokka demonstrated strong neutrophil alkaline phosphatase activity whereas western grey kangaroos, red kangaroos and woylies contained no activity within their leukocytes. Peroxidase and alkaline phosphatase cytochemistry were also assessed at the ultrastructural level with transmission electron microscopy. This allowed the identification of distinct granule populations within macropodid neutrophils. Two subcellular compartments containing alkaline phosphatase activity were identified within tammar wallaby neutrophils. These were considered equivalent to secretory vesicles and a subpopulation of specific granules. Tubular vesicles containing alkaline phosphatase were also identified within the eosinophils of tammar wallabies. These structures were a novel finding having not been reported previously in the eosinophils of other animal species. In addition to cytochemistry, the general ultrastructure of leukocytes from tammar wallabies and western grey kangaroos were reported. Results were similar to previous reports for other marsupial species. The current body of knowledge was extended by the first detailed description of the ultrastructure of basophils in a marsupial. To assess functional aspects of macropdid neutrophils, flow cytometric assays were performed examining oxidative burst responses and phagocytosis. Reactive oxygen species were generated by neutrophils from tammar wallabies and western grey kangaroos in response to phorbol 12-myristate 13-acetate but not N-formyl-Met-Leu-Phe or opsonised bacteria. Phagocytosis of opsonised bacteria was also measured in neutrophils from tammar wallabies, which was poor in contrast to ovine neutrophils. However, flow cytometric studies were limited by sample preparation. Further optimisation of isolation methods for tammar wallaby leukocytes should be undertaken before dogmatic conclusions are drawn. Overall, the results of this thesis demonstrate that, in the areas examined, the general characteristics of leukocyte structure and function of mammals are present in macropodids. However differences were identified both within and outside of the macropodid group. These differences have important ramifications for the use of ‘model’ species in the study of leukocyte biology in marsupials. The results also provide potentially useful tools for the clinical diagnosis of haematological disease in macropodids and may be of interest to those studying comparative and evolutionary aspects of leukocyte structure and function

Making Way for the Car: Minimum Parking Requirements and Porirua City Centre

Minimum parking requirements (MPRs) mandate that each new development provides enough parking to ensure ample provision at the time of peak demand. This approach tends to oversupply parking above the optimal level, and by bundling parking into the development costs, ensures that parking is free to the user. As a result, land-use and transport decisions are distorted. A case study of Porirua central business district (CBD) was undertaken to investigate the use of MPRs in the New Zealand context, and to assess their impacts on transport and land-use patterns. Findings indicate that MPRs tend to oversupply parking relative to weekly mean and peak occupancies. Land use mapping found that 24 percent of CBD land is allocated to car parking and MPRs were shown to contribute to dispersed development patterns. Stated choice data and a cost recovery model for car parking highlight how free and ample car parking provision favours car driving and has distortionary impacts on travel decisions

Hematologic characteristics of captive western barred bandicoots (Perameles bougainville) from Western Australia

Background: The western barred bandicoot (Perameles bougainville) is an Australian marsupial species now considered endangered as a consequence of habitat destruction and predation. A recently discovered papillomatosis syndrome is hindering efforts to repopulate this species. Hematology reference intervals have been lacking for P bougainville, preventing optimal interpretation of hematology results from wart-affected and clinically normal animals. Objectives: The purpose of this study was to establish hematology reference values and describe morphologic characteristics of blood cells of healthy western barred bandicoots. Methods: Fifty-nine whole blood samples were collected by jugular venipuncture into EDTA from 47 clinically healthy captive western barred bandicoots at 3 locations on the Western Australian mainland. A CBC was performed using an ADVIA-120 analyzer. Data were compared on the basis of geographic location, sex, age, and lactation status, and reference intervals were calculated. Blood cell morphology was evaluated using light microscopy, and transmission and scanning electron microscopy. Results: Significant differences were found based on sex (RBC indices, fibrinogen), age (% polychromatophilic RBCs), and geographic location (RBC, neutrophil, and lymphocyte counts, MCHC, % polychromatophilic RBCs, fibrinogen). Combined reference intervals were calculated for hemoglobin concentration (122-165 g/L), HCT (0.36-0.49 L/L), and total WBC (2.9-14.9 x 10^9/L), monocyte (0-0.6 x 10^9/L), eosinophil (0-0.9 x 10^9/L), and total plasma protein (47-63 g/L) concentrations. Leukocyte, erythrocyte, and platelet morphology were similar to those of other marsupial peramelid species. Nuclei in neutrophils, monocytes, and eosinophils occasionally had an annular configuration. \ud Conclusions: Reference intervals and blood cell morphology obtained in this study will be useful for the evaluation of laboratory data from ill animals and assist with population health monitoring of western barred bandicoots

Making Way for the Car: Minimum Parking Requirements and Porirua City Centre

Minimum parking requirements (MPRs) mandate that each new development provides enough parking to ensure ample provision at the time of peak demand. This approach tends to oversupply parking above the optimal level, and by bundling parking into the development costs, ensures that parking is free to the user. As a result, land-use and transport decisions are distorted. A case study of Porirua central business district (CBD) was undertaken to investigate the use of MPRs in the New Zealand context, and to assess their impacts on transport and land-use patterns. Findings indicate that MPRs tend to oversupply parking relative to weekly mean and peak occupancies. Land use mapping found that 24 percent of CBD land is allocated to car parking and MPRs were shown to contribute to dispersed development patterns. Stated choice data and a cost recovery model for car parking highlight how free and ample car parking provision favours car driving and has distortionary impacts on travel decisions.</p

Cytochemistry of leukocytes from the family Macropodidae

The cytochemical staining characteristics of leukocytes from four species of Macropodidae and one species of Potoroidae were studied to investigate the cellular composition of leukocytes within the Macropodidae family and determine markers that may be useful for identifying cell lineage. Blood smears from the tammar wallaby (Macropus eugenii), the western grey kangaroo (Macropus fuliginosis), the red kangaroo (Macropus rufus), quokka (Setonix brachyurus) and a potoroid species, the woylie (Bettongia pencillata) were examined following reaction for Sudan Black B (SBB), peroxidase (PER), chloracetate esterase (CAE), α-naphthyl butyrate esterase (NBE) and alkaline phosphatase (ALP). Similar to domestic animal species, the neutrophils and eosinophils of macropodid species stained for both SBB and PER while monocytes and lymphocytes showed little or no reaction. CAE and NBE, however, were not useful as markers for macropodid neutrophils and monocytes, respectively. Significant variation between species was seen in ALP content. Tammar wallabies and quokkas demonstrated strong ALP activity within their neutrophils and eosinophils. In contrast, western grey kangaroos and red kangaroos contained no ALP activity in any of their leukocytes and staining in woylies was limited to weak reaction within some eosinophils

Sub-cellular localisation of alkaline phosphatase activity in the cytoplasm of tammar wallaby (Macropus eugenii) neutrophils and eosinophils

Alkaline phosphatase (ALP) has been used in studies of neutrophil morphology and function as a marker for identifying different granule populations. In human neutrophils, ALP is found within secretory vesicles, a rapidly mobilisable vesicle population important for upregulating membrane receptors during early activation. Intra-cellular ALP activity in the heterophils of rabbits and guinea pigs, in contrast, is found only in secondary granules. The neutrophils and eosinophils of tammar wallabies (Macropus eugenii) have previously been reported to contain large amounts of ALP activity when stained using routine cytochemical techniques. To define the subcellular location of ALP in this species, cell suspensions were examined using cerium chloride cytochemistry and transmission electron microscopy (TEM). ALP was found in 2 distinct cytoplasmic compartments. One compartment displayed morphology consistent with a subpopulation of secondary granules while a second tubulo-vesicular population appeared similar to the secretory vesicles of human neutrophils. Thin tubular vesicles containing ALP were also identified within the cytoplasm of tammar wallaby eosinophils. Large numbers of ALP-containing vesicles have not been recognised previously in eosinophils and this may represent a novel cytoplasmic compartment. In both cell types, ALP-containing structures showed alteration in morphology following stimulation with N-formyl-Met-Leu-Phe (fMLP) and PMA

Effects of temperature and duration of sample storage on the haematological characteristics of western grey kangaroos (Macropus fuliginosus)

Objective: To investigate the effects of storage duration and temperature on haematological analyses performed on blood from the western grey kangaroo (Macropus fuliginosis). Method: Blood samples from five western grey kangaroos were stored at 4°C, 24°C and 36°C. Each sample was analysed haematologically over a 5-day period. Results: The blood samples maintained optimal stability at 4°C. At this temperature the haematological values remained essentially unchanged for the duration of the study, while samples stored at 36°C and 24°C showed significant changes in some haematological measures by 12 h and 48 h, respectively. Disturbances in leukocyte morphology were evident, to varying degrees, in all samples. Conclusions: Blood samples from macropodids should be tested within 48 h of collection if stored at a room temperature of about 24°C. Where testing is to be delayed for more than 48 h, samples should be refrigerated as soon as possible. Exposure of samples to heat in excess of 24°C should be avoided at all times