Inger Lilliehöök

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.

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.

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.

Clinical and biochemical changes in 53 Swedish dogs bitten by the European adder - Vipera berus

BackgroundEvery year many dogs in Sweden are bitten by Vipera berus, the only venomous viper in Sweden. This prospective study investigated clinical signs, some biochemical parameters, treatment, and progress of disease after snakebite in 53 dogs. Effects of treatment with and without glucocorticoids were evaluated.MethodsAll fifty-three dogs bitten by Vipera berus were examined the same day the dog was bitten and the next day. Two more examinations during 23 days post snake bite were included. Creatinine, creatine kinase (CK), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), alkaline phosphatase (ALP) and bile acid results were followed through 3 to 4 samplings from 34 of the dogs.ResultsAll dogs had variable severity of local swelling in the bite area and 73 per cent had affected mental status. Initial cardiac auscultation examination was normal in all dogs, but six dogs had cardiac abnormalities at their second examination, including cardiac arrhythmias and cardiac murmurs. All dogs received fluid therapy, 36 dogs were given analgesics, 22 dogs were treated with glucocorticoids, and ten dogs were treated with antibiotics. Evidence of transient muscle damage (increased CK) was seen one day after the snake bite in 15 (54%) of 28 sampled dogs. Moderate changes in hepatic test results occurred in 1 dog and several dogs (22 of 34) had transient, minor increases in one or more hepatic test result. No dog died during the observation period as a consequence of the snake bite.ConclusionsSnake bite caused local swelling in all dogs and mental depression of short duration in most dogs. Some dogs had transient clinical signs that could be indicative of cardiac injury and some other had transient biochemical signs of liver injury. Treatment with glucocorticoids did not have any clear positive or negative effect on clinical signs and mortality.

Errors in basophil enumeration with 3 veterinary hematology systems and observations on occurrence of basophils in dogs

BACKGROUND Most automated hematology analyzers cannot detect canine or feline basophils. However, many veterinary laboratories continue to report basophils as part of the automated 5-part differential leukocyte count for dogs and cats. OBJECTIVES The study objectives were to evaluate the performance of the Sysmex XT-2000iV, Advia 2120, and CELL-DYN 3500 hematology analyzers in detecting basophils using blood from dogs, cats, and rabbits with basophilia and to investigate the concurrence of basophilia and other hematologic changes, sex, and breed in dogs. METHODS One or more of the 3 hematology analyzers was used to analyze 11 canine blood samples with prominent basophilia (≥ 5%) based on a manual differential count. In addition, samples from 2 cats and 4 rabbits with basophilia were analyzed with the Advia 2120. Leukocyte cytograms were inspected for the likely location of basophil cell clusters. In a retrospective study of canine patients, reports of hematologic results that included a manual leukocyte differential count were identified using the laboratory information system and examined for the occurrence of basophilia and other hematologic changes, sex, and breed of the dogs. RESULTS Canine basophils were not detected by the Sysmex XT-2000iV or CELL-DYN 3500 analyzers, and neither canine nor feline basophils were detected by the Advia 2120. The Advia was able to detect basophils in rabbits. On the Sysmex cytogram canine basophils were found slightly above or together with neutrophils. On the Advia Perox cytogram canine basophils were located in upper part of the lymphocyte box and in the area of large unstained cells (LUC). Dogs with marked basophilia often had concurrent eosinophilia, and basophilia may be found more frequently in Rottweiler dogs than in other breeds. In 5 dogs with marked basophilia and without eosinophilia, marked thrombocytosis and anemia were noted. CONCLUSIONS Canine basophils were not detected by these automated hematology analyzers, and careful analysis of instrument graphical displays or increased LUC (Advia) may guide the need to examine a blood smear for basophils.

Investigation of hypereosinophilia and potential treatments

Hypereosinophilia is excessive eosinophilia and has been defined in dogs and cats as eosinophils greater than 5 x 10(9)/L (> 5000/microL). Canine breeds with a predisposition to higher eosinophil counts or certain eosinophilic diseases include the Rottweiler, German Shepard, Siberian Husky, Alaskan Malamute, and Cavalier King Charles Spaniel. Two of the more common causes of canine hypereosinophilia are pulmonary infiltrates with eosinophils (PIE) and gastrointestinal disease. The highest eosinophil counts are expected in dogs with pneumonia or PIE. The most common cause of eosinophilia in cats is flea allergy. The greatest eosinophilia occurs in cats with flea allergy, feline asthma, and eosinophilic granuloma. Innovative recent treatments for human patients with asthma have been successful in reducing eosinophil numbers but have had a confusing and disappointing lack of reducing symptoms. The role of eosinophils in many eosinophilic diseases remains a mystery.

A possible systemic rheumatic disorder in the Nova Scotia duck tolling retriever

BackgroundA disease complex with chronic musculoskeletal signs, including stiffness and joint pain, and to which there is a strong predisposition in the canine breed Nova Scotia duck tolling retriever (Toller) has been recognized in Sweden. The aim of this first clinical description of the disorder in Tollers was to describe the clinical manifestations and laboratory findings, as well as to try to identify a possible immune-mediated background of the disease and to show the outcome of treatment in 33 Tollers.MethodsThe study included 33 Tollers with musculoskeletal signs and 20 healthy controls. All the dogs were thoroughly examined and followed for a period of 2 months – 4 years. An IIF-ANA (antinuclear antibody) test and an assay for the presence of antibodies to Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato were performed, as well as some haematology, serum biochemistry and urine tests. Routine radiographic examinations were performed on 11 dogs.ResultsAll the Toller patients showed stiffness and lameness that had lasted for at least 14 days and displayed pain from several joints of extremities on manipulation. Twenty-seven per cent of the dogs also showed muscle pain and 18% different skin symptoms. Seventy per cent of the Tollers with signs of disease displayed a positive IIF-ANA test. Most of the dogs were treated with corticosteroids, with the majority of the dogs (65%) showing good responses. There was no association between the IIF-ANA results and the clinical signs or results of treatment.ConclusionThis paper describes a disorder in Nova Scotia duck tolling retrievers where the clinical signs, ANA reactivity and response to corticosteroids strongly suggest that the disorder is immune-mediated. The findings of this research may indicate a chronic systemic rheumatic disorder.

Expression of adhesion and Fcgamma-receptors on canine blood eosinophils and neutrophils studied by anti-human monoclonal antibodies

Commercially available anti-human monoclonal antibodies were tested as markers of adhesion receptors and Fcgamma-receptors on canine neutrophils and eosinophils. Purified populations of eosinophils and neutrophils were incubated with selected monoclonal antibodies and binding was measured by flow cytometry. Most of the anti-human monoclonal antibodies used in this study crossreacted with canine granulocytes and many showed expression similar to that of human granulocytes. The results suggest that the adhesion and Fcgamma-receptors are well conserved among species. Canine eosinophils and neutrophils were simultaneously purified by a two-layer Percoll density gradient centrifugation. The purity of the neutrophil fraction was > or = 97% after lysis of the erythrocytes. In the eosinophil fraction, 27-92% were eosinophils (mean 60%). After purification, the neutrophils and eosinophils were incubated with selected monoclonal antibodies and analysed by flow cytometry. Human anti-CD11b and CD18 antibodies bound intensely to both canine eosinophils and neutrophils. Canine neutrophils did label with anti-CD29, but eosinophils did not. Anti-CD49d bound weakly to both eosinophils and neutrophils. Both anti-CD16 and anti-CD32antibodies labelled neutrophils, but not eosinophils. There was no binding of anti-CD9 to canine neutrophils and the binding of anti-CD9 to canine eosinophils varied between dogs.

Evaluation of an in-clinic Serum Amyloid A (SAA) assay and assessment of the effects of storage on SAA samples

BackgroundAn in-clinic assay for equine serum amyloid A (SAA) analysis, Equinostic EVA1, was evaluated for use in a clinical setting. Stability of SAA in serum samples was determined.MethodsIntra- and inter- assay variation of the in-clinic method was determined. The in-clinic method (EVA1) results were compared to a reference method (Eiken LZ SAA) with 62 patient samples. For samples with SAA concentrations within the assay range of EVA1 (10-270 mg/L), differences between the methods were evaluated in a difference plot. Linearity under dilution was evaluated in two samples. Stability of SAA in three serum pools stored at 4°C and approximately 22°C was evaluated with the reference method day 0, 1, 2, 4, 7, 17 and analysed with a two-way ANOVA.ResultsThe imprecision (coefficient of variation, CV) for the in-clinic method was acceptable at higher SAA concentrations with CV values of 7,3-12%, but poor at low SAA concentrations with CV values of 27% and 37% for intra- and inter-assay variation respectively. Recovery after dilution was 50-138%. The in-clinic assay and the reference method identified equally well horses with low (<10 mg/L) and high (>270 mg/L) SAA concentrations. Within the assay range of the in-clinic method, 10-270 mg/L, the difference between the two methods was slightly higher than could be explained by the inherent imprecision of the assays. There were no significant changes of serum SAA concentrations during storage.ConclusionsThe in-clinic assay identified horses with SAA concentrations of <10 mg/L and >270 mg/L in a similar way as the reference method, and provided an estimate of the SAA concentration in the range of 10-270 mg/L. The imprecision of the in-clinic method was acceptable at high SAA concentrations but not at low concentrations. Dilution of samples gave inconsistent results. SAA was stable both at room temperature and refrigerated, and thus samples may be stored before analysis with the reference method.

Validation of an enzyme-linked immunosorbent assay for measurement of feline serum insulin

BACKGROUND Feline insulin has been measured previously using assays developed for measuring human insulin. As feline insulin differs from human insulin, it is important to validate the assay before use. OBJECTIVES The aims of this study were to validate an ELISA, the Mercodia Feline Insulin ELISA, intended for measuring feline insulin and to determine the stability of feline insulin in serum. METHODS Validation of the ELISA, which uses monoclonal antibodies that recognize both human and feline insulin, included evaluation of coefficients of variation (CVs), patterns of variation, and consistency after dilution and spiking with feline insulin. Stability was evaluated by measuring insulin in feline serum samples stored at 20°C, 2-8°C, and -80°C. RESULTS The intra-assay CV in 14-20 adjacent replicates (excluding position effects) was 2.0-4.2% and the inter-assay CV was 7.6-14%. The systematic and random position effect yielded a CV of 6.2-10%. When 3 feline serum samples were set at fixed positions and analyzed on 8 plates, microplate effects and interaction were significant for all 3 samples. Recovery upon dilution and spiking was 78-105% and 86-126%, respectively. Feline serum insulin concentration was stable for 24 hours at 20°C, for 4 days at 2-8°C, and for 15 months at -80°C. CONCLUSIONS The Mercodia Feline Insulin ELISA can be used for measuring serum feline insulin. Recovery after spiking and dilution was acceptable. As in many ELISAs, intra-assay CV for adjacent replicates was low, whereas the position and between-assay CVs were considerably higher.