A case of canine renal lymphoma of granular lymphocytes with severe polycythemia.

BACKGROUND: Renal lymphoma in dogs is rare and has a poor prognosis. Granular lymphocyte morphology is rarely reported in canine renal lymphoma. Mild to moderate polycythemia is reported in a number of canine renal lymphoma cases. CASE PRESENTATION: A 10-year-old Labrador retriever presented to a university veterinary teaching hospital after a 1-month history of polyuria, polydipsia, and pollakiuria and a 2-week history of abdominal distention, lethargy, and increased respiratory effort. Abdominal ultrasound showed a wedge-shaped to rounded, heterogeneously hypoechoic mass lesion in the left kidney. Cytologic analysis of a percutaneous aspirate of the mass was consistent with lymphoma of granular lymphocytes. Severe polycythemia (hematocrit 0.871) was noted on a complete blood cell count. Clonality analysis identified a clonally rearranged T-cell receptor (TCR) gene and immunohistochemical staining was CD3+, CD79a- and CD11d+, supporting cytotoxic T-cell lymphoma. CONCLUSIONS: To our knowledge, this is the first report of renal cytotoxic T-cell lymphoma with severe polycythemia in a dog. Severe polycythemia and renal cytotoxic T-cell lymphoma are both rare in dogs; this report adds to the body of knowledge on these conditions.

Survey of Institutional Teaching Approaches to Clinical-Year Clinical Pathology Instruction and Comparison with Prior Survey Results.

Teaching approaches to veterinary clinical pathology in the final (clinical) year of veterinary school are often different than those for other specialties. Anecdotally, many schools teach these rotations separately from the routine diagnostic service, but minimal published data are available on this topic or on approaches to teaching and assessment in these rotations. An online survey of 69 veterinary institutions around the world was conducted in 2019. A total of 30 completed surveys were received from 10 countries; 22 completed responses were from North American institutions (73.3%). Survey question categories included information on basic rotations, including microscopy format, personnel involved in instruction, and assessment methods; information on advanced rotations; and challenges and successes with clinical pathology instruction. Data were analyzed and, when appropriate, compared with results from a similar survey conducted in 1997. Formats and content varied greatly among institutions. Several shifts in teaching strategies and rotation format over time were found since the 1997 survey, including increased use of projection microscopy and decreased use of multiheaded microscopy in 2019. More teaching by medical technologists and residents, less teaching by faculty, and a significant increase in the number of students per rotation were seen in 2019 compared with 1997. Several free-text comments referred to challenges related to increasing class size. These data and the comparison with the prior survey highlight common challenges and potential solutions to final-year clinical pathology instruction. Creation of specific, measurable objectives for clinical pathology competence may aid future development and refinement of clinical pathology teaching.

Activation of the nuclear factor-κB pathway during postnatal lung inflammation preserves alveolarization by suppressing macrophage inflammatory protein-2.

A significant portion of lung development is completed postnatally during alveolarization, rendering the immature lung vulnerable to inflammatory stimuli that can disrupt lung structure and function. Although the NF-κB pathway has well-recognized pro-inflammatory functions, novel anti-inflammatory and developmental roles for NF-κB have recently been described. Thus, to determine how NF-κB modulates alveolarization during inflammation, we exposed postnatal day 6 mice to vehicle (PBS), systemic lipopolysaccharide (LPS), or the combination of LPS and the global NF-κB pathway inhibitor BAY 11-7082 (LPS + BAY). LPS impaired alveolarization, decreased lung cell proliferation, and reduced epithelial growth factor expression. BAY exaggerated these detrimental effects of LPS, further suppressing proliferation and disrupting pulmonary angiogenesis, an essential component of alveolarization. The more severe pathology induced by LPS + BAY was associated with marked increases in lung and plasma levels of macrophage inflammatory protein-2 (MIP-2). Experiments using primary neonatal pulmonary endothelial cells (PEC) demonstrated that MIP-2 directly impaired neonatal PEC migration in vitro; and neutralization of MIP-2 in vivo preserved lung cell proliferation and pulmonary angiogenesis and prevented the more severe alveolar disruption induced by the combined treatment of LPS + BAY. Taken together, these studies demonstrate a key anti-inflammatory function of the NF-κB pathway in the early alveolar lung that functions to mitigate the detrimental effects of inflammation on pulmonary angiogenesis and alveolarization. Furthermore, these data suggest that neutralization of MIP-2 may represent a novel therapeutic target that could be beneficial in preserving lung growth in premature infants exposed to inflammatory stress.

Unilateral intraocular mastocytosis and anterior uveitis in a dog with subcutaneous mast cell tumors.

A 9-year-old male castrated Scottish terrier was referred to the Radiation Oncology Service at the William R. Pritchard Veterinary Medical Teaching Hospital for palliative radiation therapy of an incompletely excised, recurrent subcutaneous mast cell tumor (MCT) located over the right scapula, and surgical removal of a perianal MCT. Three weeks after initial presentation and prior to the fifth radiation treatment, the patient was presented with cloudiness of the left eye of 3-7 days duration. Ophthalmic consultation revealed 3+ aqueous flare with a dependent, swirling component filling approximately one-third of the anterior chamber. Aqueocentesis was performed under general anesthesia. Cytology revealed mast cells with highly atypical morphology and considered most consistent with neoplasia. The patient died 7 months after pathologic diagnosis of MCT on the right shoulder and 2 months after the cytologic diagnosis of malignant mast cells in the left anterior chamber. To the authors' knowledge, this is the first report of intraocular involvement in a mammal with MCTs, described here as intraocular mastocytosis.

Phagocytic Function and Flow Cytometric Phenotype of Asian Elephant Monocytes.

Optimal veterinary care of managed elephant populations is vital due to the continued decline of wild populations. Appropriate health monitoring and accurate disease diagnosis include hematologic evaluation. Elephant hematology is distinctive in that elephants have high percentages of monocytes in health. Elephant monocytes also have unusual morphology, a feature shared with manatees and rock hyraxes. Manual white blood cell counting is used for elephant hematology, as analyzers are generally inaccurate. The aims of this study were to evaluate basic cell isolation and functional testing protocols for use in elephant monocyte research, and to test several available antibodies via flow cytometry for use in elephant monocyte identification. Peripheral blood samples from five Asian elephants (Elephas maximus) were used. Methods for monocyte isolation and evaluation of phagocytic function were established. Putative lymphocyte and monocyte populations were identified using a scatter on flow cytometry. Antibodies against CD11b, CD11c, CD14, and ionized calcium-binding adapter molecule 1 (IBA1) were tested, with IBA1 showing the highest apparent diagnostic utility in labeling monocytes. Combined flow cytometric scatter and IBA1 positivity appear to identify Asian elephant monocytes. These data provide a methodologic basis for further investigation into elephant monocyte function and immune response to infection.

Associations between urogenital carcinoma and DECA-BDE (BDE-209) among wild California Sea lions (Zalophus californianus) and Steller Sea lions (Eumetopias jubatus).

Urogenital carcinoma (UGC) is prevalent among California sea lions (Zalophus californianus), while less is known concerning UGC among Steller sea lions (Eumetopias jubatus). Our objective was to investigate associations between UGC and polybrominated diphenyl ethers (PBDEs) among both sea lion species. Twenty-nine California sea lions and 20 Steller sea lions were lethally removed from the Columbia River Basin, Oregon, USA between 2020 and 2021, under Section 120 of the Marine Mammal Protection Act. UGC was diagnosed through gross necropsy and histopathology. Forty PBDE congeners were analyzed in blubber, including BDE-209, a potential carcinogen. Twenty (69 %) California sea lions and one (5 %) Steller sea lion were diagnosed with UGC. All cases were identified as early stage UGC, aside from one California sea lion with more advanced stage UGC. Among California sea lions, associations between PBDEs and UGC were analyzed using logistic regression. In the adjusted model, BDE-209 (log2-transformed) was associated with increased odds of UGC [Odds Ratio (OR): 4.68, 95 % confidence interval: 1.04, 21.0, OR p-value = 0.044). This is the first study to report BDE-209 concentrations in sea lion blubber. The percentages of California and Steller sea lions diagnosed with UGC were higher than expected for wild (non-stranded) sea lions. Our results suggested blubber BDE-209 was potentially associated with UGC in California sea lions in the Columbia River Basin.

Anaplasma phagocytophilum dihydrolipoamide dehydrogenase 1 affects host-derived immunopathology during microbial colonization.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum, human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene (lpda1 [APH_0065]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.

Sablefish (Anoplopoma fimbra Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology.

Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RI's from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Döhle bodies. In mammals and avian species, Döhle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions.

Hematology and Culture Assessment of Cranially Implanted Rhesus Macaques (Macaca mulatta).

The use of percutaneous cranial implants in rhesus macaques (Macaca mulatta) has long been a valuable tool for neuroscience research. However, when treating and assessing these animals, veterinarians are required to make assumptions about diagnostic results due to a lack of research into how these implants affect physiology. Microbial cultures of cranial implant sites show an abundance of colonizing bacteria, but whether these microbes affect animal health and wellbeing is poorly understood. In addition, microbial antibiotic resistance can present significant health concerns for both the animals and the researchers. To help elucidate the relationship between percutaneous cranial implants and blood parameters, complete blood cell counts and serum chemistry results were assessed on 57 nonhuman primates at our institution from September 2001 to March 2017. Generalized estimating equations were used to compare the results before and after an animal's first implant surgery. This modelling showed that cranial implants were a significant predictor of alterations in the number of neutrophils, lymphocytes, and red blood cells, and in the concentration of hemoglobin, alkaline phosphatase, creatinine, calcium, phos- phorus, total protein, albumin, and globulin. Anaerobic and aerobic bacterial cultures were performed to identify bacteria associated with cranial implants. Staphylococcus spp., Streptococcus spp., and Corynebacterium spp. comprised the majority of the aerobic bacterial isolates, while Fusobacterium spp., Peptostreptococcus spp. and Bacterioides fragilis comprised the majority of anaerobic bacterial isolates. Using a Pearson r correlation for statistical analysis, we assessed whether any of these bacterial isolates developed antibiotic resistances over time. Cefazolin, the most frequently used antibiotic in monkeys in this study, was the only antimicrobial out of 41 agents tested to which bacteria developed resistance over time. These results indicate that percutaneous implants are associated with a generalized inflammatory state, multiple bacterial species are present at the implant site, and these bacteria may contribute to the inflammatory response.

Use of an in vitro biotinylation technique for determination of posttransfusion survival of fresh and stored autologous red blood cells in Thoroughbreds.

OBJECTIVE: To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. ANIMALS: 13 healthy adult Thoroughbreds. PROCEDURES: Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4 degrees C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined. RESULTS: Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 microg of streptavidin-PE/1 x 10(6) RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.

Influence of specific management practices on blood selenium, vitamin E, and beta-carotene concentrations in horses and risk of nutritional deficiency.

BACKGROUND: Selenium or alpha-tocopherol deficiency can cause neuromuscular disease. Beta-carotene has limited documentation in horses. OBJECTIVE: To evaluate the effect of owner practices on plasma beta-carotene concentration and risk of selenium and alpha-tocopherol deficiencies. ANIMALS: Three-hundred and forty-nine adult (≥1 year), university and privately owned horses and mules. METHODS: Cross-sectional study. Whole blood selenium, plasma alpha-tocopherol, and plasma beta-carotene concentrations were measured once. Estimates of daily selenium and vitamin E intake, pasture access, and exercise load were determined by owner questionnaire. Data were analyzed using t tests, Mann-Whitney tests, parametric or nonparametric analysis of variance (ANOVA), Kruskal-Wallis test, Spearman's correlation and contingency tables (P < .05). RESULTS: Nearly 88% of the horses received supplemental selenium; 71.3% received ≥1 mg/d. Low blood selenium concentration (<80 ng/mL) was identified in 3.3% of horses, and 13.6% had marginal concentrations (80-159 ng/mL). Non-supplemented horses were much more likely to have low blood selenium (odds ratio [OR], 20.2; 95% confidence interval [CI], 9.26-42.7; P < .001). Supplemental vitamin E was provided to 87.3% of horses; 57.7% received ≥500 IU/d. Deficient (<1.5 µg/mL) and marginal (1.5-2.0 µg/mL) plasma (alpha-tocopherol) occurred in 15.4% and 19.9% of horses, respectively. Pasture access (>6 h/d) and daily provision of ≥500 IU of vitamin E was associated (P < .001) with higher plasma alpha-tocopherol concentrations. Plasma beta-carotene concentration was higher in horses with pasture access (0.26 ± 0.43 versus 0.12 ± 0.13 µg/mL, P = .003). CONCLUSIONS AND CLINICAL IMPORTANCE: Suboptimal blood selenium and plasma alpha-tocopherol concentrations occurred in 16.7% and 35.5% of horses, respectively, despite most owners providing supplementation. Inadequate pasture access was associated with alpha-tocopherol deficiency, and reliance on selenium-containing salt blocks was associated with selenium deficiency.

What is your diagnosis? Blood smear from an injured red-tailed hawk.

An injured juvenile red-tailed hawk (Buteo jamaicensis) was evaluated at the Veterinary Medical Teaching Hospital at the University of California, Davis. The hawk was quiet, alert, and emaciated, and had a closed comminuted, mid-diaphyseal ulnar fracture. CBC results included heterophilia with a left shift, monocytosis, and increased plasma fibrinogen concentration. The blood smear included rare heterophils containing small, dark blue inclusions approximately 1-2 mum in diameter that ranged from round to coccobacillary in shape and formed variably shaped aggregates; the morphology of the inclusions was suspicious for Chlamydophila or Ehrlichia spp. pathogens. The hawk died, and histopathologic examination of tissues obtained at necropsy found severe multifocal histiocytic and heterophilic splenitis in addition to chronic hepatitis, myocarditis and epicarditis, meningoencephalitis, and airsacculitis. Using immunohistochemistry the presence of Chlamydia/Chlamydophila spp. antigen within multiple tissues was confirmed. Chlamydophila psittaci DNA was demonstrated in whole blood and fresh splenic tissue via real-time PCR. Direct fluorescent antibody staining of air-dried blood smears was positive in rare leukocytes for Chlamydia/Chlamydophila spp. antigen, and immunocytochemical staining of blood smears for Chlamydia/Chlamydophila spp. antigen was focally positive in rare heterophils. These findings may represent the first reported diagnosis of natural avian C. psittaci infection by visualization of organisms in peripheral blood heterophils. Immunocytochemical evaluation of blood smears was valuable in confirming the diagnosis and may be a useful antemortem test to discriminate between bacteria and other inclusions within heterophils.

Development of a technique for quantification of reticulocytes and assessment of erythrocyte regenerative capacity in birds.

OBJECTIVE: To develop a reticulocyte classification scheme, optimize an avian reticulocyte staining protocol, and compare the percentages of reticulocyte types with polychromatophil percentage in blood samples from birds. SAMPLE POPULATION: Blood samples from a red-tailed hawk and 31 ill birds. PROCEDURES: A single blood sample obtained from a red-tailed hawk (Buteo jamaicensis) was used to optimize the staining protocol. For optimization of the staining protocol, 4 dilutions of whole blood with new methylene blue stain and 4 incubation times were evaluated. From samples submitted for avian CBCs, EDTA-anticoagulated whole blood samples from 31 ill birds were randomly selected and examined to compare polychromatophil and reticulocyte percentages. Reticulocyte staining was performed in all samples by use of a 1:3 (whole blood to new methylene blue) dilution with incubation for 10 minutes at room temperature (approx 22 degrees C); reticulocytes were assessed as a percentage of 1,000 RBCs by 2 independent observers. In Wright-Giemsa-stained blood smears, a polychromatophil percentage was similarly determined. RESULTS: 4 avian reticulocyte types were defined: ring-form reticulocytes, aggregate reticulocytes, and 2 subcategories of punctate reticulocytes. A reticulocyte-staining protocol was optimized. Interobserver and intraobserver variations in assessment of reticulocyte and polychromatophil percentages were not significant. A strong positive correlation (Spearman coefficient of rank correlation [rho] = 0.978) was identified between the percentage of polychromatophils and the percentage of ring-form reticulocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that quantification of ring-form reticulocytes provides an accurate assessment of erythrocyte regenerative capacity in birds.

Bias due to Preanalytical Dilution of Rodent Serum for Biochemical Analysis on the Siemens Dimension Xpand Plus.

Clinical pathology testing of rodents is often challenging due to insufficient sample volume. One solution in clinical veterinary and exploratory research environments is dilution of samples prior to analysis. However, published information on the impact of preanalytical sample dilution on rodent biochemical data is incomplete. The objective of this study was to evaluate the effects of preanalytical sample dilution on biochemical analysis of mouse and rat serum samples utilizing the Siemens Dimension Xpand Plus. Rats were obtained from end of study research projects. Mice were obtained from sentinel testing programs. For both, whole blood was collected via terminal cardiocentesis into empty tubes and serum was harvested. Biochemical parameters were measured on fresh and thawed frozen samples run straight and at dilution factors 2-10. Dilutions were performed manually, utilizing either ultrapure water or enzyme diluent per manufacturer recommendations. All diluted samples were generated directly from the undiluted sample. Preanalytical dilution caused clinically unacceptable bias in most analytes at dilution factors four and above. Dilution-induced bias in total calcium, creatinine, total bilirubin, and uric acid was considered unacceptable with any degree of dilution, based on the more conservative of two definitions of acceptability. Dilution often caused electrolyte values to fall below assay range precluding evaluation of bias. Dilution-induced bias occurred in most biochemical parameters to varying degrees and may render dilution unacceptable in the exploratory research and clinical veterinary environments. Additionally, differences between results obtained at different dilution factors may confound statistical comparisons in research settings. Comparison of data obtained at a single dilution factor is highly recommended.

Influence of Genetic Background on Hematologic and Histopathologic Alterations during Acute Granulocytic Anaplasmosis in 129/SvEv and C57BL/6J Mice Lacking Type I and Type II Interferon Signaling.

The role of host type I IFN signaling and its interaction with other immune pathways during bacterial infections is incompletely understood. Type II IFN signaling plays a key role during numerous bacterial infections including granulocytic anaplasmosis (GA) caused by Anaplasma phagocytophilum infection. The function of combined type I and type II IFN signaling and their potential synergism during GA and similar tick-borne diseases is a topic of current research investigation. The goal of this study was to evaluate 2 mouse models of absent type I/type II IFN signaling in experimental A. phagocytophilum infection to determine the effects of background strain. Mice lacking both type I and type II IFN receptor signaling (IFNAR-/-/IFNGR-/-) on either the 129/SvEv or C57BL/6J genetic background were evaluated at days 0, 6, 8, and 12 of infection. Pathogen burden in multiple organs was largely similar between strains of infected mice, with few significant differences. Background strain influenced the immune response to infection. Mice of the 129/SvEv strain developed more severe hematologic abnormalities, particularly more severe leukocytosis with marked neutrophilia and lymphocytosis, throughout acute infection. Histopathologic changes occurred in infected mice of both strains and varied in severity by organ. 129/SvEv mice developed more severe pathologic changes in spleen and bone marrow, whereas C57BL/6J mice developed more severe renal pathology. This work highlights the importance of mouse background strain in dictating pathophysiologic response to infection and informs future work regarding the loss of type I and type II IFN signaling on the immune response during GA.

Lymph node aspirate from a California wine-country dog.

A 4-year-old, male Golden Retriever was presented to the Veterinary Medical Teaching Hospital at the University of California-Davis with a history of lethargy, inappetance, and vomiting. The patient had generalized lymphadenomegaly, marked thrombocytopenia, mild anemia, and moderate hypoalbuminemia. Moderate to marked histiocytic inflammation and lymphocytic-plasmacytic reactivity of the mesenteric, left popliteal, and right mandibular lymph nodes were diagnosed cytologically. Many macrophages contained granular to amorphous material of a uniform blue color, occasionally in morula formation, suggestive of rickettsial organisms. Exposure to raw trout was subsequently documented, leading to a presumptive diagnosis of salmon poisoning disease (SPD). The patient responded quickly to doxycycline therapy for the causative agent of SPD (Neorickettsia helminthoeca). SPD should be considered as a differential diagnosis for a canine patient with clinical signs of vomiting, diarrhea, lethargy, and lymphadenomegaly; laboratory findings of thrombocytopenia and hypoalbuminemia; and potential exposure to raw fish from an endemic area. The cytologic finding of rickettsial inclusions within lymph node macrophages is reportedly seen within a majority of SPD cases and can be valuable in supporting a clinical suspicion of SPD, as it was in this case.

Alterations due to dilution and anticoagulant effects in hematologic analysis of rodent blood samples on the Sysmex XT-2000iV.

BACKGROUND: Clinical pathology of rodents is hindered by sample volume limitations. A single whole heparinized blood sample is often submitted for hematologic and clinical chemistry analysis in exploratory research settings, and sample dilution may be required. Published information on the potential impact of sample dilution and heparin use on hematology variables in rodents is sparse. OBJECTIVES: The purpose of the study was to evaluate the effects of sample dilution and of anticoagulant on hematologic analysis of mouse and rat blood samples on the Sysmex XT-2000iV. METHODS: Mice and rats were obtained from various ends of study research projects, and whole blood was collected via terminal cardiocentesis in lithium heparin, and additionally in EDTA when paired samples were obtained from rats. Hematology analytes were measured on the Sysmex XT-2000iV straight and diluted from ×2 to ×5. RESULTS: Significant differences between heparinized samples analyzed straight vs diluted were found for MCV and MCHC, with a bias for several additional variables. Significant differences between paired heparinized and EDTA-anticoagulated samples at each dilution point were found for most variables, with the largest differences found in platelet count. Evidence of platelet clumping presumably due to heparin exposure was noted in numerous samples. CONCLUSIONS: Dilution-induced changes occur in some hematologic variables and may render dilution unacceptable in the exploratory research environment. Many variables, most notably platelet count, differ based on the anticoagulant used, and values from heparinized vs EDTA-anticoagulated samples should not be directly compared.

Acute phase proteins in healthy goats: establishment of reference intervals.

Acute inflammatory processes can trigger increased production of acute phase proteins (APPs) that can be useful biomarkers of inflammation. APPs are diverse and include proteins involved in coagulation, opsonization, iron regulation, and limitation of tissue injury. Haptoglobin, serum amyloid A, and alpha-1 acid glycoprotein have been proposed as useful APPs in goats. APPs can differ markedly by species, therefore species-specific reference intervals and studies are necessary. The objective of this study was to determine species-specific reference intervals for 4 APPs in goats. Haptoglobin, serum amyloid A, lipopolysaccharide binding protein, and alpha-1 acid glycoprotein were measured in in 54 clinically normal adult goats. APPs were measured using goat-specific commercial enzyme-linked immunosorbent assay kits. Results were analyzed by 1-way analysis of variance to compare sexes and breeding status. Reference Value Advisor was used to calculate reference limits according to the IFCC-CLSI guidelines. Only 1 APP was found to vary in healthy animals; serum haptoglobin was increased in lactating animals and decreased in pregnant does in their second trimester when compared with open, nonlactating does. No sex-based differences were seen for any of the APPs measured. We report normal reference intervals for 4 serum APPs that may be useful as disease markers. Haptoglobin should be interpreted with caution in animals with unknown pregnancy status. Further studies are needed to determine whether these APPs are useful biomarkers in goat disease states.

Biochemical and Hematologic Reference Intervals for Aged Xenopus laevis in a Research Colony.

Xenopus laevis, the African clawed frog, is commonly used in developmental and toxicology research studies. Little information is available on aged X. laevis; however, with the complete mapping of the genome and the availability of transgenic animal models, the number of aged animals in research colonies is increasing. The goals of this study were to obtain biochemical and hematologic parameters to establish reference intervals for aged X. laevis and to compare results with those from young adult X. laevis. Blood samples were collected from laboratory reared, female frogs (n = 52) between the ages of 10 and 14 y. Reference intervals were generated for 30 biochemistry analytes and full hematologic analysis; these data were compared with prior results for young X. laevis from the same vendor. Parameters that were significantly higher in aged compared with young frogs included calcium, calcium:phosphorus ratio, total protein, albumin, HDL, amylase, potassium, CO2, and uric acid. Parameters found to be significantly lower in aged frogs included glucose, AST, ALT, cholesterol, BUN, BUN:creatinine ratio, phosphorus, triglycerides, LDL, lipase, sodium, chloride, sodium:potassium ratio, and anion gap. Hematology data did not differ between young and old frogs. These findings indicate that chemistry reference intervals for young X. laevis may be inappropriate for use with aged frogs.

Post-transfusion survival of biotin-labeled allogeneic RBCs in adult horses.

BACKGROUND: Post-transfusion survival of allogeneic RBCs has been reported to be much shorter in horses than in other species. We hypothesized that post-transfusion survival of biotinylated allogeneic equine RBCs would be greater than the survival previously reported from studies using radioactive RBC-labeling techniques. OBJECTIVE: The study objective was to determine post-transfusion survival of N-hydroxysuccinimide (NHS)-biotin-labeled allogeneic equine RBCs transfused into adult horses. METHODS: Horses were adults and included 5 donors and 5 recipients. All horses were blood-typed, and donors were paired with recipients based upon blood type and crossmatch results. Donor blood was collected in a volume of 4 L into citrate phosphate dextrose adenine-1 and stored for 24 hours, labeled with NHS-biotin, and re-infused into recipients. Post-transfusion blood samples were collected at 15 minutes and at 1, 2, 3, 5, 7, 14, 21, 28, and 35 days. Biotin-labeled RBCs were detected by flow cytometry using streptavidin-phycoerythrin. Post-transfusion survival at 24 hours, lifespan, and half-life of biotinylated RBCs were determined. RESULTS: Mean ± SD survival of biotinylated RBCs at 24 hours post-transfusion was 95 ± 24%; the mean lifespan of transfused allogeneic RBCs was 39 days based on calculation of a linear regression survival curve, and mean post-transfusion RBC half-life was 20 days. CONCLUSIONS: Post-transfusion survival of 24-hour stored equine allogeneic RBCs was greater than previously reported but less than that observed for other companion animal species. Mechanisms for the relatively short post-transfusion lifespan of allogeneic equine RBCs remain unknown and warrant further study.