A COMPARISON OF FLUORIMETRIC AND COLORIMETRIC METHODS FOR THE DETERMINATION OF α-MANNOSIDASE ACTIVITY IN BOVINE PLASMA

This paper compares both manual colorimetric and fluorimetric methods for the assay of acidic alpha-mannosidase activity in bovine EDTA plasma. The effect of variable blood volumes per 10 ml blood collecting tube has been investigated, and the colorimetric assay conditions adjusted (for example, zinc concentration in pre-incubation buffer) to cope with low blood volumes resulting in high EDTA/plasma ratios. We have shown that with non-haemolysed EDTA plasma samples, the two methods give similar results, but for haemolysed samples, the colorimetric method yields significantly lower results than those obtained fluorimetrically. Using the manual fluorimetric method, samples can be assayed more rapidly and simply than by using the colorimetric method. This provides definite advantages in a laboratory receiving variable numbers of samples, or which cannot afford the high cost of automated equipment.     

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

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

Sensitivity of commercial prothrombin time reagents to detect coagulation factor deficiencies in equine plasma

The sensitivity of commercial prothrombin time (PT) tests was assessed based on a dilution series of equine pooled plasma (EPP) (experiment 1) and on 40 equine plasma samples with reduced activity of coagulation factors II, V, VII and X (experiment 2). Two different PT reagents (reagent 1, human placental thromboplastin; reagent 2, recombinant human tissue factor) were used according to the manufacturers' instructions (standard test, PT([ST])) and compared to a modified test procedure (modified test, PT([MT])) using sample dilution and fibrinogen addition. In all samples, sensitivity was lower (P<0.01) when using PT([ST]) with reagent 2 (0.20) than when using either PT([ST]) with reagent 1 (0.65) or PT([MT]) with both reagents (reagent 1, 0.60-0.75, reagent 2, 0.58-0.70, depending on sample dilution). The highest sensitivity was found for PT([MT]) when using a 1:20 sample dilution. In those samples in which at least one coagulation factor activity was decreased (by 20%; n=18), the sensitivity of PT([ST]) with reagent 2 (0.33) was found to be inadequate, in contrast to all other test procedures (0.83-0.94). This low sensitivity corresponded to shorter time intervals between different coagulation activity levels prepared by EPP dilution. The results indicate that adequate sensitivity of PT measurements in equine plasma can be achieved using a standard test procedure as long as a suitable reagent is used.     

Relationship between colloid osmotic pressure and plasma protein concentration in cattle, horses, dogs, and cats.

The relationship between colloid osmotic pressure (COP) and protein concentration was investigated for purified proteins and plasma samples obtained from cattle, horses, dogs, and cats. At equivalent concentrations, bovine albumin exerted a COP that exceeded that of gamma-globulins by a mean factor of 4.4. Similar relationships between COP and protein were observed in the other species. Consequently, for a given total protein concentration, COP was dependent on the albumin/gamma-globulins ratio. A commonly used nomogram for estimating COP from protein concentration, the Landis-Pappenheimer equation, did not provide reliable results for plasma samples from these species.     

Automated counting of nucleated cells in equine synovial fluid without and with hyaluronidase pretreatment

Background: Microscopy is usually used to obtain manual total and differential cell counts in equine synovial fluid. A faster, more precise method is desirable. Objectives: The objectives were to compare an automated impedance method with a manual method for obtaining total and differential cell counts in equine synovial fluid and to evaluate the effect of pretreatment with hyaluronidase on automated results. Methods: Synovial fluid samples (n=48) were collected into EDTA and analyzed within 48 hours. Automated total and differential cell counts were evaluated using a Medonic CA620‐VET hematology analyzer before and after pretreatment for 5–30 minutes with hyaluronidase (final concentration 0.01 mg/mL). A hemacytometer count and microscopic evaluation of a direct smear were used as the reference method. Intra‐assay coefficients of variation (CV) were determined. Results: Thirty‐one of 46 untreated samples and 0/46 hyaluronidase‐treated samples were error‐flagged by the analyzer. Correlation between automated (ANCC) and manual (MNCC) nucleated cell counts in untreated samples (n=15; R²=0.93) and pretreated samples (n=46; R²=0.94) was high, and pseudomedian difference was low. Intra‐assay CVs for samples with medium and high cellularity were significantly lower for ANCC (1.5–2.7%) compared with MNCC (6.1–15.7%) (P<.01). Valid automated differential cell counts were not obtained. Conclusions: Automated total cell counts obtained on the Medonic analyzer correlate well with manual counts in equine synovial fluid; however, pretreatment with hyaluronidase is required to minimize error flags. Automated differential counts are not accurate for synovial fluid.     

Creatinine concentration in plasma from dog, rat, and mouse: a comparison of 3 different methods

BACKGROUND: There are numerous methods for analyzing creatinine concentration in plasma, including the Jaffé alkaline picrate method in various modifications, enzymatic tests, and chromatographic methods. OBJECTIVE: The purpose of this study was to evaluate whether an enzymatic method could replace a Jaffé method for routine creatinine measurements in plasma from dogs, rats, and mice. The enzymatic method and a compensated Jaffé method were tested against a high-pressure liquid chromatography (HPLC) method, regarded as the gold standard for creatinine measurement. METHODS: Heparinized plasma samples were obtained from 20 beagle dogs, 20 Wistar rats, and 20 CD1-strain mice. The 2 test kits (Roche Diagnostics), Creatinine Jaffé Compensated and the enzymatic Creatinine Plus Version 2 reagent, were used on a Cobas Integra 400. The Jaffé compensated method used a calibration adjustment of 18 micromol/L to correct for the protein matrix in serum and plasma. The HPLC method was an isocratic method using a weak cation-exchange column following protein precipitation. RESULTS: Creatinine concentrations obtained using the enzymatic and the Jaffé methods differed significantly from the results obtained by the HPLC method. For dog plasma, mean values of 61.2, 61.8, and 67.8 micromol/L were obtained by the compensated Jaffé, enzymatic and HPLC methods, respectively. In the rat, respective mean values were 26.7, 21.9, and 23.0 micromol/L, and in the mouse, respective mean values were 14.2, 5.4, and 9.2 micromol/L. CONCLUSION: The enzymatic method can replace the Jaffé method for plasma creatinine determination in dogs, rats, and mice because results from the enzymatic method were closer to HPLC values than were those of the Jaffé method.     

Platelet concentrations and platelet-associated IgG in greyhounds

BACKGROUND: Greyhounds have lower platelet concentrations (PC) than dogs of other breeds have. No underlying cause has been investigated. HYPOTHESIS: We hypothesized that Greyhounds have lower mean PC because of breed variation, not immune-mediated causes. Our secondary hypothesis was that PC is dependent on the method of analysis. ANIMALS: Sixty privately owned Greyhounds in Kansas. METHODS: Blood samples were collected into evacuated glass tubes containing ethylenediamine tetraacetic acid (EDTA). Blood smears were evaluated for platelet clumps. All 60 samples had PC determined by manual, impedance, and buffy coat analyzer methods. Results of the 60 samples were compared with results of samples with (n = 25) and without (n = 35) clumps, and with control dogs. Platelets were assayed for the presence of surface-associated antigen (PSAIgG) by direct immunofluorescence. RESULTS: The mean PC was below that of the control dogs for the impedance method (P < .001). No significant difference in PC was detected between analysis methods or between samples with or without platelet clumps. Three of 60 (5%) of the Greyhounds had PC between 50,000 and 100,000/microL with impedance analysis; no samples had < 100,000/microL via buffy coat analysis. PSAIgG was not identified in any samples. CONCLUSIONS AND CLINICAL IMPORTANCE: The mean Greyhound PC for the impedance method was below the reference interval for control dogs but was not significantly different from PC determined by other methods. An immune-mediated cause for the lower PC was unlikely because no samples had PSAIgG. The decreased PC is most consistent with breed variation. As only 0-5% of samples, depending on analysis method, had PC < 100,000/microL, a Greyhound with a PC < 100,000/microL is not necessarily consistent with breed variation, thus diagnostic testing is indicated.     

Evaluation of a rapid reagent strip test for blood glucose determination in diarrheic calves

Sixty-three blood samples from 10 diarrheic calves were tested for glucose concentration by two methods. Plasma glucose concentration was measured by the conventional glucose-6-phosphate dehydrogenase method in the clinical laboratory, and the results compared to those obtained using a rapid reagent strip test for blood glucose concentration measurement. The rapid reagent strip test result could not be used to make an accurate prediction of the actual plasma glucose concentration as determined by the conventional method, due to the wide variability in actual plasma glucose concentrations corresponding to each rapid test result.     

Evaluation of methods of platelet counting in the cat

Platelet counts were performed in 50 cats presented for diagnostic investigation. For each cat, counts were obtained using a manual haemocytometer method and compared with counts obtained by estimation from a stained blood smear, a QBC VetAutoread analyser, a Zynocyte VS/2000 analyser, impedance automated counts on a Baker System using both EDTA and citrated anticoagulated blood, and use of a Zynostain modified counting chamber kit. None of the methods gave high correlation with the haemocytometer counts. The blood smear estimation of platelet counts had the highest correlation (r = 0.776) and was the only method to have reasonable values for both sensitivity and specificity. With the impedance automated counts, citrated anticoagulated blood had marginally higher correlation than EDTA anticoagulated blood, and the time between blood sampling and platelet count determination had no effect on the count obtained. When in-house analyser or impedance automated platelet counts are abnormal or not consistent with clinical findings, the authors recommend that a manual platelet count using either haemocytometry or examination of a blood smear is performed.     

Assessment of three automated assays for C-reactive protein determination in dogs

OBJECTIVE: To determine the characteristics of an automated canine C-reactive protein (CRP) assay and evaluate 2 human CRP assays for use in dogs. Animals-56 client-owned dogs with pyometra and 11 healthy control dogs. PROCEDURES: Samples from 11 dogs with high (> 100 mg/L) or low (< 10 mg/L) CRP concentrations (determined by use of a canine ELISA) were evaluated by use of the automated canine CRP assay. Intra- and interassay imprecision was determined (by use of those 2 plasma pools), and assay inaccuracy was assessed by use of logistic regression analysis of results obtained via ELISA and the automated canine CRP assay. Two automated human CRP assays were used to measure plasma CRP concentration in 10 dogs. RESULTS: By use of the ELISA, mean +/- SD plasma CRP concentration was 96.1 +/- 38.5 mg/L and 10.1 +/- 23.2 mg/L in dogs with pyometra and control dogs, respectively. The automated canine assay had intra-assay coefficients of variation (CVs) of 7.8% and 7.9%, respectively, and interassay CVs of 11.1% and 13.1%, respectively. Results from the automated assay were highly correlated with results obtained via ELISA. The human assay results did not exceed 0.4 mg/L in any dog. CONCLUSIONS AND CLINICAL RELEVANCE: The automated canine CRP assay had less interassay imprecision, compared with the ELISA. The 2 human CRP assays were not suitable for analysis of canine plasma samples. The automated canine CRP assay was more precise than the ELISA for serial evaluations of plasma CRP concentration in dogs.     

Differential leukocyte counts determined in chicken blood using the Cell-Dyn 3500

Background: Automated hematology instruments commonly are used for mammalian blood analysis, but there is a lack of accurate automated methods available for avian leukocyte analysis. Objective: The aim of this study was to validate differential leukocyte counts in blood from chickens using the Cell-Dyn 3500 hematology system and avian-specific software.
Methods: Blood samples were collected in lithium-heparin tubes from 2 groups (n = 84 and n = 139) of laying hens. Manual 200-cell differential counts were done on routinely-stained blood smears, and manual total granulocyte counts (heterophils and eosinophils) were done using an eosinophil stain in a counting chamber. Automated differential counts were done using VET 2.3, a research and development version of avian-specific software for the Cell-Dyn 3500. Results were analyzed using Pearson's correlation and difference plots.
Results: Automated granulocyte counts from the Cell-Dyn were in good agreement with manual granulocyte counts ( r = 0.93 and 0.80 for the 2 study groups). No correlation was found between automated and manual lymphocyte counts. Correlation coefficients for monocyte counts were 0.70 and 0.43. Conclusion: Automated leukocyte results from the Cell-Dyn using VET 2.3 software were not fully accurate. Total granulocyte counts may be of clinical usefulness, but results obtained for other parameters were unreliable.     

Comparison of biochemical values in serum and plasma, fresh and frozen plasma, and hemolyzed samples from orange-winged Amazon parrots (Amazona amazonica)

BACKGROUND: To the authors' knowledge, on the basis of sample type, storage condition, or hemolysis, differences in serum and plasma biochemical values have not been evaluated in orange-winged Amazon parrots (Amazona amazonica). OBJECTIVES: The purpose of this study was to compare values for biochemical analytes in serum vs plasma, fresh vs frozen plasma, and nonhemolyzed vs hemolyzed samples in orange-winged Amazon parrots. We also compared differences in serum and plasma yield from whole-blood aliquots. METHODS: Fifteen biochemical analytes were evaluated in paired serum and plasma, fresh and frozen plasma, nonhemolyzed and hemolyzed serum and plasma samples from orange-winged Amazon parrots (n = 10) using a wet reagent analyzer. Hemolysis was assessed qualitatively (visually) and quantitatively (hemoglobin [Hgb] measured spectrophotometrically). Serum and plasma yields from 500-microl whole-blood aliquots were determined from centrifuged samples. RESULTS: Analyte values significantly differed among sample groups, but were still within published reference intervals, with the exception of increases in potassium concentration in markedly hemolyzed serum and plasma samples. Clinically important changes in hemolyzed serum and plasma samples included increases in potassium, phosphorus, and albumin concentrations and lactate dehydrogenase activity. The degree of hemolysis assigned qualitatively did not correlate with quantitative Hgb concentration. A significantly greater yield of plasma (288 +/- 13 microL) than serum (241 +/- 44 microL) was obtained. CONCLUSIONS: Significant differences may occur in different sample types, however, only changes in potassium, phosphorus, albumin, and lactate dehydrogenase values in hemolyzed samples were considered clinically relevant. Lack of agreement between qualitative and quantitative Hgb concentration indicates the unreliability of visual estimation. Based on higher sample yield, and lack of clinically relevant differences from serum, plasma is a better sample choice for clinical chemistry analysis in birds.     

Evaluation of an automated colorimetric assay for the measurement of lipase activity in canine sera.

An automated colorimetric method for determining lipase activity in canine sera was evaluated for precision, linearity and correlation to existing assay methods. The colorimetric method was a commercial reagent that used a series of enzymatic reactions based on the hydrolysis of 1,2 diglyceride by pancreatic lipase. Within-run and between-run coefficients of variation were < 6.8% and < 8.3%, respectively. Linearity was determined to be at least 1366 U/L. Canine serum lipase concentrations attained using the colorimetric method were compared to both titrimetric and dry-film methods for measuring serum lipase activity, resulting in significant (P < or = 0.05) correlation coefficients of 0.92 and 0.77, respectively. Canine serum lipase concentrations measured using the colorimetric assay on 2 different automated analyzers had a significant (P < or = 0.05) correlation coefficient of 0.92. A laboratory reference range using serum samples from 56 healthy dogs (0-561 U/L) was established. There were no significant (P < or = 0.05) differences in mean serum lipase concentrations comparing male and female dogs or comparing young dogs (< or = 3 y) to mature (4-7 y) and older (> 7 y) dogs using this assay. It was concluded that the automated colorimetric assay was a reliable indicator of canine serum lipase activity and offered several advantages, including small sample volume and short analysis time.     

Evaluation of a Rapid Reagent Strip Test for Blood Glucose Determination in Diarrheic Calves

Sixty-three blood samples from 10 diarrheic calves were tested for glucose concentration by two methods. Plasma glucose concentration was measured by the conventional glucose-6-phosphate dehydrogenase method in the clinical laboratory, and the results compared to those obtained using a rapid reagent strip test for blood glucose concentration measurement. The rapid reagent strip test result could not be used to make an accurate prediction of the actual plasma glucose concentration as determined by the conventional method, due to the wide variability in actual plasma glucose concentrations corresponding to each rapid test result.     

Hematology of the sandbar shark, Carcharhinus plumbeus: standardization of complete blood count techniques for elasmobranchs

BACKGROUND: Standardized hematologic methods and reference intervals have not been established for cartilaginous fishes (sharks, skates, and rays) despite the large number of animals displayed in zoos and aquariums worldwide. OBJECTIVE: The focus of this study was to validate CBC methods for sandbar shark (Carcharhinus plumbeus) blood, based on criteria established in human medicine, for the following tests: RBC count, total WBC count, PCV, hemoglobin (Hgb) concentration, and WBC differential percentages. METHODS: Replicate CBCs were performed using blood samples from 5 captive sandbar sharks. Three protocols for RBC and total WBC counts were compared, as were different centrifugation times for PCV determination, and 2 methods for Hgb concentration. Means, minimum and maximum values, and CVs were compared to CAP and CLIA performance guidelines for human tests. RESULTS: Total WBC counts in a diluent modified for elasmobranch blood, Hgb concentration by the cyanmethemoglobin method after removal of nuclei, and WBC differential percentages showed acceptable performance. PCV results were acceptable when tubes were centrifuged for at least 5 minutes. Total RBC counts by all 3 methods exceeded the acceptable error for manual counts of human cells. CONCLUSIONS: Standardized CBC tests can be used as health assessment tools for elasmobranchs. Total RBC counts should be viewed as estimates.     

Overestimation of canine albumin concentration with the bromcresol green method in heparinized plasma samples

Albumin concentrations are routinely measured in dogs with bromcresol green (BCG)-binding assays on automated chemistry analyzers. Several variables affect this assay, including the length of reaction time, sample type, and lack of specificity of BCG for albumin. We observed that albumin concentrations measured with BCG appeared higher in heparinized plasma samples in sick dogs. The objective of this study was to determine the effect of anticoagulant and assay procedure on BCG albumin concentrations in clinically ill dogs. We hypothesized that albumin concentrations would be overestimated in heparinized plasma compared with serum because of the combination of heparin and fibrinogen. Furthermore, we hypothesized that the overestimation would be influenced by assay parameters. Blood was collected from 32 clinically ill dogs into tubes containing heparin, citrate, or no anticoagulant. Citrate was chosen to assess the effect of fibrinogen in the absence of heparin. Albumin concentration was measured in all 3 sample types from each dog using 2 different BCG procedures on an automated chemistry analyzer. The BCG procedures (standard and modified) differed in the wavelengths used for absorbance readings (standard, 600/700; modified, 570/505) and the time point at which absorbance was measured (standard, 100 seconds; modified, 40 seconds). In addition, the modified method incorporated a sample blank. Globulin fractions, fibrinogen concentration, and indices of lipemia, hemolysis, and icterus were evaluated for their contribution to the overestimation of albumin concentration in heparinized plasma compared with serum samples. Albumin concentrations were significantly higher (P 相似文献   

Automation of an absorbed enzyme immunoassay for the detection of Mycobacterium paratuberculosis antibodies for an eradication program

As part of an ongoing Johne's disease eradication program by the state of Victoria, Australia, the Victorian Veterinary Pathology Services (VVPS) has been involved in testing over 150000 cattle samples per year for the presence of Johne's-specific antibodies. The Parachek kit (CSL, Victoria, Australia) has been used throughout the project. This method was automated using a Rosys 3300 and a Plato 7 (Dade/Behring Diagnostics) to pipette samples and the Behring ELISA Processor 3 (Dade/Behring Diagnostics) to wash EIA plates, add reagents, reads absorbances and perform data reduction of the results. Automation saved about 15h of labour per day, allowing one staff member to analyse up to 2000 samples per 8h shift compared to 800 samples using a manual protocol. Problems that were encountered include pipetting issues, the reading of the Paracheck endpoint, and excessive packaging of the kit. Maintenance, calibration and control of the analysers were an integral part of the process. VVPS, working in conjunction with CSL, suggested modification to the Parachek kit to make automation of the product more convenient. The approach resulted in a change in reagent volume and a reduction in packaging that reduced the cost of the test by 25%.     

Development of an in vitro, isolated, infected spore testing model for disinfectant testing of Microsporum canis isolates

The isolated infected hair model is a commonly used technique to test the fungicidal efficacy of topical therapies against Microsporum canis. The most commonly used model uses mats of infective hairs, and results from various laboratories have differed. The objectives of this study were to develop a method to produce spores for testing when only mycelial forms were available and to develop a semiquantitative testing method that used only infective spores from hairs, and not pooled hair samples for testing. Ten isolates of M. canis were used in this study. Juvenile guinea pigs were easily infected using mycelial forms of M. canis and large numbers of spores were easily harvested for testing. Eight dilutions of disinfectants were tested. Fungal culture data were evaluated using an endpoint dilution at which there was 100% fungicidal activity, i.e. no growth on the plates. The 10 samples showed identical results. Chlorhexidine and Virkon(R) S were ineffective even when used at x4 the manufacturer's recommended dilution. Lime sulphur (1 : 33), enilconazole (20 microL mL(-1)), and bleach (1 : 10) were consistently effective when used at the recommended dilution. In addition, lime sulphur and enilconazole were 100% fungicidal even when the recommended concentration was diluted 1 : 4 or x4 as dilute as recommended.     

An automated method for the measurement of methaemoglobin in avian blood

An automated method has been developed for the measurement of methaemoglobin (metHb) in blood using a biochemistry analyser. The method was validated using blood collected from red-legged partridges exposed in vitro to increasing concentrations of nitrite in order to obtain different percentages of metHb. Results obtained using the original manual method and those using the new automated technique were compared and no significant differences were found. Intra-day and inter-day variabilities (8.8% and 2.6%, respectively) were acceptable for samples containing high levels (63-81%) of metHb. Methaemoglobin measured in blood samples stored in liquid nitrogen was stable for 10 days, but increased significantly by day 20 in nitrite-treated samples.