Pharmacokinetics and Alterations in Glucose and Insulin Levels After a Single Dose of Canagliflozin in Healthy Icelandic Horses.

Canagliflozin (CFZ) is a sodium-glucose cotransporter-2 inhibitor that has shown promising results as a drug for the treatment of insulin dysregulation in horses. Even though CFZ is used clinically, no pharmacokinetic data has previously been published. In this study, the pharmacokinetics of CFZ after administration of a single oral dose of 1.8 mg/kg in eight healthy Icelandic horses was examined. Additionally, the effect of treatment on glucose and insulin levels in response to a graded glucose infusion was investigated. Plasma samples for CFZ quantification were taken at 0, 0.33, 0.66, 1, 1.33, 1.66, 2, 2.33, 2.66, 3, 3.5, 4, 5, 6, 8, 12, 24, 32, and 48 h post administration. CFZ was quantified using UHPLC coupled to tandem quadrupole mass spectrometry (UHPLC-MS/MS). A non-compartmental analysis revealed key pharmacokinetic parameters, including a median Tmax of 7 h, a Cmax of 2350 ng/mL, and a t1/2Z of 28.5 h. CFZ treatment reduced glucose (AUCGLU, p = 0.001) and insulin (AUCINS, p = 0.04) response to a graded glucose infusion administered 5 h after treatment. This indicates a rapid onset of action following a single dose in healthy Icelandic horses. No obvious adverse effects related to the treatment were observed.

Biological variation of biochemical urine and serum analytes in healthy dogs.

BACKGROUND: Biological variation (BV) of urinary (U) biochemical analytes has not been described in absolute terms, let alone as a ratio of the U-creatinine or fractional excretion in healthy dogs. These analytes are potential diagnostic tools for different types of kidney damage and electrolyte disorders in dogs. OBJECTIVES: We aimed to investigate the BV of specific gravity, osmolality, creatinine, urea, protein, glucose, chloride, sodium, potassium, calcium, and phosphate in urine from healthy pet dogs. METHODS: Blood and urine samples from 13 dogs were collected once weekly for 8 weeks. Samples were analyzed in duplicate and in randomized order. For each sample, U-analyte and serum concentrations were measured, and U-analyte/U-creatinine and fractional excretion (FE) were calculated. Components of variance, estimated by restricted maximum likelihood, were used to determine within-subject variation (CVI ), between-subject variation (CVG ), and analytical variation (CVA ). Index of individuality (II) and reference change values were calculated. RESULTS: CVI for all urine analytes varied between 12.6% and 35.9%, except for U-sodium, U-sodium/U-Cr, and FE-sodium, which had higher CVI s (59.5%-60.7%). For U-protein, U-sodium, U-potassium, U-sodium/U-creatinine, FE-urea, FE-glucose, FE-sodium, FE-potassium, and FE-phosphate II were low, indicating that population-based RIs were appropriate. The remaining analytes had an intermediate II, suggesting that population-based RIs should be used with caution. CONCLUSION: This study presents information on the biological variation of urinary and serum biochemical analytes from healthy dogs. These data are important for an appropriate interpretation of laboratory results.

Cardiac troponin I in healthy Norwegian Forest Cat, Birman and domestic shorthair cats, and in cats with hypertrophic cardiomyopathy.

OBJECTIVES: The aims of this study were to assess the potential associations between the serum cardiac troponin I (cTnI) concentration in healthy cats and feline characteristics, systolic blood pressure, heart rate (HR), echocardiographic measurements and storage time; and to compare cTnI concentrations in healthy cats with concentrations in cats with hypertrophic cardiomyopathy (HCM), with or without left atrial enlargement (LAE) and in cats with HCM, to assess potential associations between cTnI concentration and echocardiographic variables. METHODS: Cardiac TnI was analysed using an Abbott ARCHITECT ci16200 analyser in serum from prospectively included healthy Norwegian Forest Cat (NF; n = 33), Birman (n = 33) and domestic shorthair (DSH; n = 30) cats, and from 39 cats with HCM, with or without LAE. RESULTS: In healthy cats, higher cTnI concentrations were found in Birman cats than in NF cats (P = 0.014) and in neutered male cats than in intact females (P = 0.032). Cardiac TnI was positively associated with HR (P <0.0001). In cats with HCM, cTnI concentration was positively associated with left ventricular wall thickness and with left atrial-to-aortic root ratio (all P ⩽0.010). Cats with HCM had higher cTnI concentrations than healthy cats, and cTnI concentrations were higher in cats with HCM and LAE than in those with HCM without LAE (all P = 0.0003). CONCLUSIONS AND RELEVANCE: Breed and sex may affect serum cTnI concentrations in healthy cats. The cTnI concentration increased with increasing severity of HCM.

Prednisolone in Dogs-Plasma Exposure and White Blood Cell Response.

Glucocorticoids such as prednisolone are commonly used in dogs but there is sparse quantitative pharmacokinetic and pharmacodynamic information of this drug in this species. The objective of this study was to quantitatively characterize the concentration-effect relationship for prednisolone in dogs on neutrophil and lymphocyte trafficking and cortisol suppression. Nine beagles, 2-12 years old and part of a group for teaching/research were used in a 4-way crossover experiment including two treatments, active or placebo, administered either per os (PO) or intravenously (IV). Plasma was analyzed for prednisolone and cortisol using ultra-high performance liquid chromatography - tandem mass spectrometry. Leucocyte counts were performed in whole blood. Data was then analyzed by non-linear mixed effect modeling to estimate pharmacokinetic and pharmacodynamic parameters. After administration of prednisolone sodium succinate IV, the typical value (between subject variation) for total body prednisolone clearance was 1,370 ml/h·kg (13.4%). The volumes of the central and peripheral compartment were 2,300 ml/kg (10.7%) and 600 ml/kg (16.0%), respectively. The terminal plasma half-life was 1.7 h. The prednisolone plasma concentration producing 50% of the maximum response was 10 ng/mL (90.3%), 22.5 ng/ml (52.3%) and 0.04 ng/mL (197.3%) for neutrophil, lymphocyte and cortisol response, respectively. The administered dose (1 mg/kg) increased neutrophil and decreased lymphocyte numbers but not over the entire dosage interval of 24 h, due to the short half-life. However, glucocorticoids have a wide range of responses. An anti-inflammatory response due to altered gene transcription might have a longer duration. Future studies on the anti-inflammatory potency together with data presented are needed to optimize future dosage recommendations in dogs.

Hematologic, prostaglandin F2α -metabolite, serum amyloid A, and serum iron changes in horses with experimentally induced endotoxemia.

BACKGROUND: Endotoxemia is a common and severe disease of horses. Most previous studies have monitored changes caused by a bolus dose of endotoxin over short time periods. OBJECTIVES: We aimed to describe inflammatory responses to endotoxin with inflammatory and hematologic markers monitored over a longer time than has been performed in the past using more prolonged endotoxin exposures. METHODS: Escherichia coli O55:B5 endotoxin was administered as a 6-hour continuous intravenous infusion of lipopolysaccharide (LPS) to eight horses. Blood cell counts, and prostaglandin F2α -metabolite (PGM), serum amyloid A (SAA), and serum total iron concentrations were monitored for up to 3 or 6 days. RESULTS: An immediate and severe decrease in neutrophils and monocytes occurred in all horses, which subsequently changed to a moderate to strong neutrophilia and monocytosis that persisted for more than 78 hours postinfusion (PI) of LPS. Lymphocyte and eosinophil numbers decreased gradually and then normalized after 66- and 78-hours PI, respectively. Mild to moderate, biphasic thrombocytopenia occurred. A pronounced, transient increase in PGM occurred between 1 and 7 hours, peaking at 2 hours. Serum amyloid A began to increase after 6 hours PI and remained elevated after 72 hours PI. Serum iron was decreased between 6 and 48 hours. The clinical signs were most prominent during the first 24 hours PI and subsided within 48 hours PI. CONCLUSIONS: Neutrophilia, monocytoses, and high SAA concentrations were present in horses even after the clinical signs had subsided. Serum iron normalized before SAA. Knowledge of these findings is imperative when interpreting laboratory results in horses with possible endotoxin exposure.

Validation of the Sysmex XT-2000iV hematology system for dogs, cats, and horses. II. Differential leukocyte counts.

BACKGROUND: The Sysmex XT-2000iV is a laser-based, flow cytometric hematology system that stains nucleic acids in leukocytes with a fluorescent dye. A 4-part differential is obtained using side fluorescence light and laser side scatter. OBJECTIVE: The purpose of this study was to validate the Sysmex XT-2000iV for determining differential leukocyte counts in blood from ill dogs, cats, and horses. METHODS: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT-2000iV (Auto-diff) and the CELL-DYN 3500. Manual differentials were obtained by counting 100 leukocytes in Wright-stained blood smears. RESULTS: Leukocyte populations in the Sysmex DIFF scattergram were usually well separated in equine samples, but were not as well separated in canine and feline samples. Correlation among the Sysmex XT-2000iV, CELL-DYN 3500, and manual counts was excellent for neutrophil counts (r >or=.97) and good for lymphocyte counts (r >or=.87) for all three species. Systematic differences between the 3 methods were seen for lymphocyte and monocyte counts. The Sysmex reported incomplete differential counts on 18% of feline, 13% of canine, and 3% of equine samples, often when a marked left shift (>10% bands) and/or toxic neutrophils were present. Eosinophils were readily identified in cytograms from all 3 species. Neither the Sysmex nor the CELL-DYN detected basophils in the 7 dogs and 5 cats with basophilia. CONCLUSIONS: The Sysmex XT-2000iV automated differential leukocyte count performed well with most samples from diseased dogs, cats, and horses. Basophils were not detected. Immature neutrophils or prominent toxic changes often induced errors in samples from cats and dogs.

Validation of the Sysmex XT-2000iV hematology system for dogs, cats, and horses. I. Erythrocytes, platelets, and total leukocyte counts.

BACKGROUND: The Sysmex XT-2000iV is a laser-based, flow cytometric hematology system that has been introduced for use in large and referral veterinary laboratories. OBJECTIVE: The purpose of this study was to validate the Sysmex XT-2000iV for counting erythrocytes, reticulocytes, platelets, and total leukocytes in blood from ill dogs, cats, and horses. METHODS: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT-2000iV and the CELL-DYN 3500. Manual reticulocyte counts were done on an additional 98 canine and 14 feline samples and manual platelet counts were done on an additional 73 feline and 55 canine samples, and compared with automated Sysmex results. RESULTS: Hemoglobin concentration, RBC counts, and total WBC counts on the Sysmex were highly correlated with those from the CELL-DYN (r>/=0.98). Systematic differences occurred for MCV and HCT. MCHC was poorly correlated in all species (r=0.33-0.67). The Sysmex impedance platelet count in dogs was highly correlated with both the impedance count from the CELL-DYN (r=0.99) and the optical platelet count from the Sysmex (r=0.98). The Sysmex optical platelet count included large platelets, such that in samples from cats, the results agreed better with manual platelet counts than with impedance platelet counts on the Sysmex. Canine reticulocyte counts on the Sysmex correlated well (r=0.90) with manual reticulocyte counts. Feline reticulocyte counts on the Sysmex correlated well with aggregate (r=0.86) but not punctate (r=0.50) reticulocyte counts. CONCLUSION: The Sysmex XT-2000iV performed as well as the CELL-DYN on blood samples from dogs, cats, and horses with a variety of hematologic abnormalities. In addition, the Sysmex detected large platelets and provided accurate reticulocyte counts.

Hepatozoon canis infection causing a strong monocytosis with intra-monocytic gamonts and leading to erroneous leukocyte determinations.

In this case report, a Swedish flat-coated retriever was diagnosed with an extensive Hepatozoon canis infection. The dog had a prominent monocytosis (14.0 × 109 /L) with H canis gamonts detected in most monocytes, but none were found in the neutrophils. On the hematology system ADVIA 2120 peroxidase (PEROX) cytogram, most leukocytes were seen as a distinct cell population above the lymphocytes, which indicated that most of the cells were larger than lymphocytes and had weak myeloperoxidase staining. This distinct cell cluster appeared to be of a single cell type but was incorrectly divided by the ADVIA 2120 into lymphocytes, monocytes, and large unstained cells (LUC). The total leukocyte counts on the ADVIA 2120 WBC basophil (BASO) channel were much higher than that on the WBC PEROX count. The WBC BASO cytogram appeared abnormal with two parallel cell populations, so the BASO WBC count was considered erroneous. Polymerase chain reaction and DNA sequencing verified H canis infection. The dog was treated with subcutaneous imidocarb dipropionate (6 mg/kg) injections every other week. Post-treatment hematology analyses indicated that the percentage of parasitized leukocytes decreased from 40% to 5% about 4 weeks after the start of treatment and were not found in any monocytes 6 weeks after the beginning of the treatment. In conclusion, H canis infection in this dog was associated with a strong monocytosis, and gamonts were present in many monocytes, which caused aberrant automated leukocyte counts to occur.

Massive uric acid crystalluria and cylinduria in a dog after l-asparaginase treatment for lymphoma.

A 10-year-old golden retriever bitch was treated for diarrhea and vomiting that lasted about 1 month without a specific diagnosis until a hepatic biopsy provided a histopathologic diagnosis of lymphoma. The dog was referred to the Swedish University of Agricultural Science and treated with one dose of l-asparaginase. The day after chemotherapy, the urine was dark yellow, very turbid, and had large amounts of small amorphous crystals and many casts made of similar appearing material identified by infrared spectroscopy to be 100% uric acid dihydrate. Serum uric acid was elevated at 224 µmol/L (RI 0-59). The dog's illness became worse after chemotherapy. Lymphoma treatment was not continued, and the dog was euthanized 9 days after the l-asparaginase treatment. Among other problems were persistent proteinuria with a urine protein-to-creatinine ratio of 2.3 and severe hypoalbuminemia. Serum protein electrophoresis performed 3 weeks prior to chemotherapy indicated hyperproteinemia (total protein 78 g/L) having a biclonal gammopathy with 35 g/L ß-2 globulins and 11 g/L γ globulins. Despite prominent cylinduria and crystalluria, the patient did not develop azotemia or isosthenuria.

Hemostatic function in Cavalier King Charles Spaniels assessed using thromboelastography.

BACKGROUND: Macrothrombocytopenia is a well-known anomaly in Cavalier King Charles Spaniels (CKCSs), a breed also highly predisposed to develop myxomatous mitral valve disease (MMVD). Thromboelastography (TEG) has been shown to be a valuable instrument for whole blood hemostatic evaluation in dogs and correlates well with different physiologic and pathologic situations. OBJECTIVES: We aimed to assess the influence of macrothrombocytopenia and the severity of MMVD on hemostatic function as measured by TEG. METHODS: Associations between TEG variables (R, K, α, MA, and G) and dog characteristics, heart rates, systolic blood pressures, MMVD severities (healthy, mild or moderate, and severe), echocardiographic variables, platelet variables (platelet count, mean platelet volume [MPV], and plateletcrit), and hematocrits were evaluated in 47 prospectively recruited privately owned CKCSs. Blood samples were analyzed using a computerized thromboelastograph and an Advia 2120 hematology analyzer. RESULTS: Univariable and multiple regression analyses showed an effect of left ventricular (LV) fractional shortening (FS%) on all TEG variables, an effect of LV FS% and age on TEG α, and an effect of LV FS% and MPV on TEG MA and TEG G. TEG MA and G increased with increasing MPV, but the associations were generally weak. No significant differences were detected in the TEG variables between the MMVD severity groups. CONCLUSION: Macrothrombocytopenia and increased LV FS%, of which the latter commonly increases in various positive inotropic states, were both associated with a more hypercoagulable hemostatic system, according to the TEG results, in the present study.

Inflammatory changes during canine pregnancy.

Pregnancy is considered a pro-inflammatory state that requires physiologic adaptation of the immune system of the mother. The aim of the present study was to study inflammatory and hormonal changes during canine pregnancy. Studies included analyses of peripheral concentrations of the acute phase proteins fibrinogen and C-reactive protein (CRP), the hormones progesterone and insulin-like growth factor I (IGF-I), hemoglobin, and analyses of the total leukocyte numbers and expression of cell surface antigens. Twenty bitches were included in the present study; 12 pregnant bitches and eight non-pregnant control bitches that were followed during the corresponding phase of the oestrous cycle. Blood samples were collected at the day of optimal mating (day 0) and then on days 7, 14, 21, 28 and 42. Progesterone, IGF-I and CRP were analysed in serum and fibrinogen in EDTA plasma. Haematology and leukocyte expression of a panel of inflammation-associated adhesion molecules (CD 11a, CD 18 and CD 49d) were evaluated from EDTA blood. The data were analyzed as repeated-measures data, using a mixed model approach. Progesterone varied with time in both pregnant and control bitches, and IGF-I varied with time in pregnant bitches. Both fibrinogen and CRP increased significantly with time for the pregnant bitches, but no significant change was detected for the control bitches. Increases were seen from day 21. The hemoglobin concentration decreased significantly with time in both pregnant and non-pregnant bitches. The neutrophil and monocyte numbers increased significantly in pregnant but not in control bitches. Pregnancy induced increased granulocyte expression of cell surface marker CD 18, increased monocyte expression of CD 18 and CD 49d, and increased lymphocyte expression of CD 49d. In conclusion, we describe inflammatory changes during canine pregnancy that are manifested as increases in concentrations of CRP and fibrinogen, an increase in neutrophils and monocytes, and in activation of granulocytes, monocytes and lymphocytes. The changes should be taken into account when evaluating concentrations of APPs and WBC in bitches during pregnancy. A variation in IGF-I concentrations was detected during pregnancy.

Plateletcrit is superior to platelet count for assessing platelet status in Cavalier King Charles Spaniels.

BACKGROUND: Many Cavalier King Charles Spaniel (CKCS) dogs are affected by an autosomal recessive dysplasia of platelets resulting in fewer but larger platelets. The IDEXX Vet Autoread (QBC) hematology analyzer directly measures the relative volume of platelets in a blood sample (plateletcrit). We hypothesized that CKCS both with and without hereditary macrothrombocytosis would have a normal plateletcrit and that the QBC results would better identify the total circulating volume of platelets in CKSC than methods directly enumerating platelet numbers. OBJECTIVES: The major purpose of this study was to compare the QBC platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs. METHODS: Platelet counts were determined in fresh EDTA blood from 27 adult CKCS dogs using the QBC, Sysmex XT-2000iV (optical and impedance), CELL-DYN 3500, blood smear estimate, and manual methods. Sysmex optical platelet counts were reanalyzed following gating to determine the number and percentage of normal- and large-sized platelets in each blood sample. RESULTS: None of the 27 CKCS dogs had thrombocytopenia (defined as <164 x 10(9) platelets/L) based on the QBC platelet count. Fourteen (52%) to 18 (66%) of the dogs had thrombocytopenia with other methods. The percentage of large platelets, as determined by regating the Sysmex optical platelet counts, ranged from 1% to 75%, in a gradual continuum. CONCLUSIONS: The QBC may be the best analyzer for assessing clinically relevant thrombocytopenia in CKCS dogs, because its platelet count is based on the plateletcrit, a measurement of platelet mass.

Evaluation of an ELISA for metanephrines in feline urine.

Catecholamines can be used to evaluate neuroendocrine tumors, stress, and potentially pain, but catecholamines degrade rapidly. Their metabolites normetanephrine (NME) and metanephrine (ME) have better stability in urine. In cats, urine sampling in a home environment would be beneficial to reduce effects of clinical stress and simplify sampling. We evaluated a human urine ELISA for analysis of NME and ME in feline urine, and investigated the effects of acidification, cat tray pellets, and storage time at room temperature up to 8.5 h. In 26 feline urine samples, mean NME concentration was 192 ± 80 ng/mL, mean intra- and inter-assay CV was 6.5% and 4.2%, respectively, and spike recovery was 98-101%, but dilutional recovery was unsatisfactory. For ME, mean intra- and inter-assay CV was 10.2% and 4.1%, respectively. Mean urine ME concentration was 32.1 ± 18.3 ng/mL, close to the kit's lowest standard, and spike recovery was 65-90%; the ELISA could not be validated for ME. The stability study, performed for NME on 12 urine samples, did not identify differences between acidified and non-acidified samples, cat tray pellets, or storage time, and no interaction effects. The ME ELISA was not suitable for feline urine; performance of the NME ELISA was acceptable, except for dilution recovery. For analysis of NME, feline urine can be sampled at home using cat tray pellets and stored at room temperature up to 8.5 h without acidification.

ILF2 and ILF3 are autoantigens in canine systemic autoimmune disease.

Dogs can spontaneously develop complex systemic autoimmune disorders, with similarities to human autoimmune disease. Autoantibodies directed at self-antigens are a key feature of these autoimmune diseases. Here we report the identification of interleukin enhancer-binding factors 2 and 3 (ILF2 and ILF3) as autoantigens in canine immune-mediated rheumatic disease. The ILF2 autoantibodies were discovered in a small, selected canine cohort through the use of human protein arrays; a method not previously described in dogs. Subsequently, ILF3 autoantibodies were also identified in the same cohort. The results were validated with an independent method in a larger cohort of dogs. ILF2 and ILF3 autoantibodies were found exclusively, and at a high frequency, in dogs that showed a speckled pattern of antinuclear antibodies on immunofluorescence. ILF2 and ILF3 autoantibodies were also found at low frequency in human patients with SLE and Sjögren's syndrome. These autoantibodies have the potential to be used as diagnostic biomarkers for canine, and possibly also human, autoimmune disease.

Subgroups of canine antinuclear antibodies in relation to laboratory and clinical findings in immune-mediated disease.

BACKGROUND: Autoimmune system diseases in dogs are commonly referred to as systemic lupus erythematosus (SLE), with a positive antinuclear antibody (ANA) test as a hallmark. In human patients, other systemic ANA-positive diseases with overlapping diagnostic features, referred to as SLE-related diseases, are described. OBJECTIVES: The objective of this study was to investigate whether different patterns of ANA reactivity represent different systemic autoimmune diseases in dogs. METHODS: Dogs with serum positive for ANA by indirect immunofluorescence (IIF-ANA, titer > or = 1:100) (n = 56) were identified retrospectively from the patient population at the Department of Small Animal Clinical Sciences, Swedish University of Agricultural Sciences. Dogs were grouped on the basis of ANA staining patterns, and the results of immunodiffusion tests. Clinical, hematologic, serum biochemical, radiologic, and pathologic examinations were described for each group. RESULTS: Dogs with a chromosomal-positive, homogeneous ANA staining pattern (n = 14) had clinical signs involving multiple organ systems; 8 dogs were anemic. Dogs with a speckled IIF-ANA staining pattern (n = 42) primarily had clinical signs of musculoskeletal disorders, fatigue and fever. Precipitating antibodies by immunodiffusion were found only in dogs with a speckled IIF-ANA staining pattern and comprised 4 different subgroups based on antigen specificity. CONCLUSIONS: In dogs with homogeneous IIF-ANA staining, SLE is a probable diagnosis because of the diversity of clinical manifestations and autoantibody reactivity against chromosomal antigens. Dogs with a speckled IIF-ANA pattern may have SLE-related diseases, which, in turn, may be correlated with different immunodiffusion subgroups. These syndromes had overlapping clinicopathologic features, as described for human patients.

Time-related changes in equine neutrophils after experimental endotoxemia: myeloperoxidase staining, size, and numbers.

BACKGROUND: Neutrophil myeloperoxidase content is determined by the Advia 2120 hematology system by staining characteristics. Changes in myeloperoxidase staining are shown by location of neutrophils on Advia peroxidase dot plots and as myeloperoxidase index (MPXI). Significant changes in MPXI have been reported during severe inflammation in horses, dogs, and people but conclusions were inconsistent. OBJECTIVES: Infusion of endotoxin was used to initiate an inflammatory stimulus under controlled conditions and over a longer time period than in previous studies to document kinetics of changes in neutrophil numbers, morphology, and myeloperoxidase staining. Identification of consistent time-related changes may allow better interpretation of changes in neutrophil characteristics during inflammation. MATERIALS: Five Standardbred trotting horses received an intravenous infusion over a 6-hour period with Escherichia coli endotoxin. Neutrophil count, MPXI, neutrophil characteristics in Advia 2120 Perox dot plots and neutrophil morphology in blood smears were monitored with repeated sampling for up to 10 days. RESULTS: Endotoxin infusion immediately caused severe neutropenia which converted to neutrophilia 14 hours after start of endotoxin infusion. Neutrophilia was still present 78 hours after start of infusion. Large "giant" neutrophils first appeared in blood smears and Advia Perox dot plots after 36-48 hours. A marked and consistent decrease in MPXI was seen in all horses 6 days (150 hours) after endotoxin exposure. CONCLUSIONS: Endotoxemia caused prominent, time-related changes in equine neutrophil characteristics including emergence of giant neutrophils and markedly decreased MPXI several days after endotoxin infusion.

Reducing error in feline platelet enumeration by addition of Iloprost to blood specimens: comparison to prostaglandin E1 and EDTA.

BACKGROUND: Prostaglandin E1 (PGE1) and Iloprost inhibit platelet aggregation and should prevent or minimize preanalytic error with feline platelet enumeration. OBJECTIVES: The objective was to compare the relative effectiveness in reducing errors in platelet enumeration by adding Iloprost to feline EDTA blood specimens in comparison to adding PGE1 or EDTA alone. In addition, a grading system for platelet aggregation in blood smears was evaluated for effectiveness in predicting prominent errors and compared to ADVIA's PLT-CLM flag. Finally, the use of plateletcrit in feline blood with platelet aggregation was evaluated. METHODS: Blood specimens from 35 cats were included. Blood was collected into EDTA tubes with or without Iloprost or PGE1, and was rapidly mixed. Platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV), and platelet flags were determined with an ADVIA 2120. Manual PLT was performed with a Leucoplate stain. PLT was determined by an IDEXX VetAutoread hematology analyzer (QBC). RESULTS: Neither addition of Iloprost nor PGE1 to EDTA blood specimens completely prevented platelet aggregation. Iloprost-treated specimens had the least severe aggregation. PGE1 was better than EDTA alone. Significant errors in PLT results were consistently identified by the grading system. ADVIA's PLT-CL flag usually predicted significant errors in PLT. QBC PLT results showed high imprecision. Manual PLT error was smaller than ADVIA PLT in EDTA specimens with aggregation. CONCLUSIONS: Adding Iloprost to feline blood specimens improved platelet enumeration accuracy. A grading system for severity of platelet aggregation and usually the ADVIA's PLT-CL alarm predicted specimens with significant errors in platelet enumeration.

Identification of specific antinuclear antibodies in dogs using a line immunoassay and enzyme-linked immunosorbent assay.

Circulating antinuclear antibodies (ANA) are commonly present in the systemic autoimmune disease Systemic Lupus Erythematosus (SLE) and in other systemic rheumatic diseases, in humans as well as in dogs. The indirect immunofluorescence (IIF)-ANA test is the standard method for detecting ANA. Further testing for specific ANA with immunoblot techniques or ELISAs is routinely performed in humans to aid in the diagnosis and monitoring of disease. Several specific ANA identified in humans have been identified also in suspected canine SLE but, in contrast to humans, investigation of autoantibodies in canine SLE is mainly restricted to the IIF-ANA test. Our aim was to identify both known and novel specific ANA in dogs and to investigate if different IIF-ANA patterns are associated with different specific ANA in dogs. Sera from 240 dogs with suspicion of autoimmune disease (210 IIF-ANA positive (ANA(pos)) and 30 IIF-ANA negative (ANA(neg))) as well as sera from 27 healthy controls were included. The samples were analysed with a line immunoassay, LIA (Euroline ANA Profile 5, Euroimmun, Lübeck, Germany) and four different ELISAs (Euroimmun). The ANA(pos) dogs were divided in two groups depending on the type of IIF-ANA pattern. Of the 210 ANA(pos) samples 68 were classified as ANA homogenous (ANA(H)) and 141 as ANA speckled (ANA(S)), one sample was not possible to classify. Dogs in the ANA(H) group had, compared to the other groups, most frequently high levels of anti-double stranded deoxyribonucleic acid (dsDNA) and anti-nucleosome ANA. Anti-dsDNA antibodies were confirmed in some dogs with the Crithidia luciliae indirect immunofluorescence test (CLIFT). The frequency of ANA(H) dogs with values above those observed in the healthy group was significantly higher compared to ANA(S) dogs for anti-dsDNA, anti-nucleosome, and anti-histone reactivity. Dogs in the ANA(S) group had, compared to the other groups, most frequently high levels of anti-ribonucleoproteins (RNP) and/or anti-Smith (Sm) antibodies. Reactivity against Sjögren's syndrome related antigens (SS)-A (including the Ro-60 and Ro-52 subcomponents), SS-B, histidyl tRNA synthetase (Jo-1), topoisomerase I antigen (Scl-70), polymyositis-scleroderma antigen (PM-Scl) and proliferating cell nuclear antigen (PCNA) was also noted in individual dogs. In conclusion, by using a commercial LIA and different ELISAs originally developed for detection of human ANA, we identified several specific ANA in serum samples from dogs sampled for IIF-ANA testing. Further, we found that the types of IIF-ANA pattern were associated with reactivity against some particular nuclear antigens.

Insulin-like growth factor I in cats: validation of an enzyme-linked immunosorbent assay and determination of biologic variation.

BACKGROUND: Insulin-like growth factor I (IGF-I) measurements are used in veterinary medicine for diagnosing growth hormone disorders. IGF-I assays are subject to interference by IGF-binding proteins (IGFBP) which may not be efficiently removed by standard extraction methods. Adding excess IGF-II during analysis may improve accuracy. OBJECTIVES: The purpose of the study was to validate a commercial human IGF-I ELISA which uses excess IGF-II for feline samples and to evaluate biologic variation. METHODS: Precision was determined by calculating the coefficient of variation (CV). Accuracy was determined by recovery after removal of IGFBP, addition of IGF-I, and linear dilution after the addition of IGFBP. Biologic variation was determined by repeated sampling in 7 cats. RESULTS: There was interference by IGFBP in the high measuring range, resulting in falsely low IGF-I concentrations. This was overcome by the addition of high concentrations of IGF-II. Untreated serum had a measured/expected ratio of 98-115% compared to serum where IGFBP had been removed. Recovery after the addition of IGF-I was 83-112%. Inter- and intra-assay CVs ranged from 2.4% to 5.0% which is within the minimum acceptance criteria based on biologic variation. The reference interval of IGF-I was wide (90-1207 ng/mL) and there was a significant association between body weight and ln IGF-I (P < .000001). CONCLUSIONS: This human ELISA is suitable for feline samples, but interfering IGFBP can cause falsely low concentrations. It is recommended to dilute samples such that IGF-I is < 28 ng/mL on the standard curve to grant for sufficient IGF-II for binding of interferent IGFBP.