Plasma endotoxin concentration in horses: a methods study

Background: Accurate determination of plasma endotoxin concentration is critical for ex vivo and in vitro cellular and molecular studies of endotoxemia in horses. However, reports are conflicting with respect to anticoagulant, handling, and sample preparation.

Objective:

Objective: The purpose of this study was to determine the effect of blood sample fraction and handling time on measurement of endotoxin concentration in horses.

Methods:

Methods: Whole blood, anticoagulated with 3.8% (0.12 M) sodium citrate (9:1), was collected from 5 healthy horses. Whole blood (WB), platelet-rich plasma (PRP), and platelet-poor plasma (PPP) were spiked with endotoxin (2 EU/mL). Endotoxin-spiked WB samples were centrifuged immediately to generate PRP for measurement. Endotoxin concentration was subsequently measured by Limulus amebocyte assay at 0, 15, 30, 45, and 60 minutes. Assays were performed in triplicate and results were analyzed using Student's t -test, with significance set at P < .05.

Results:

Results: Mean endotoxin concentrations in 2 EU/mL-spiked WB were significantly different from those in PPP at all time points tested. Recovery of endotoxin in PRP generated from WB was significantly diminished after just 15 minutes.

Conclusion:

Conclusion: PRP generated from WB is significantly more reliable than PPP in determining endotoxin concentration ex vivo. Measurement of endotoxin in PRP generated from WB was significantly diminished after 15 min, identifying a time frame within which to process blood samples for endotoxin analysis.     

Comparative clinical study of canine and feline total blood cell count results with seven in‐clinic and two commercial laboratory hematology analyzers

Background: A CBC is an integral part of the assessment of health and disease in companion animals. While in the past newer technologies for CBC analysis were limited to large clinical pathology laboratories, several smaller and affordable automated hematology analyzers have been developed for in‐clinic use. Objectives: The purpose of this study was to compare CBC results generated by 7 in‐clinic laser‐ and impedance‐based hematology instruments and 2 commercial laboratory analyzers. Methods: Over a 3‐month period, fresh EDTA‐anticoagulated blood samples from healthy and diseased dogs (n=260) and cats (n=110) were analyzed on the LaserCyte, ForCyte, MS45, Heska CBC, Scil Vet ABC, VetScan HMT, QBC Vet Autoread, CELL‐DYN 3500, and ADVIA 120 analyzers. Results were compared by regression correlation (linear, Deming, Passing‐Bablok) and Bland–Altman bias plots using the ADVIA as the criterion standard for all analytes except HCT, which was compared with manual PCV. Precision, linearity, and carryover also were evaluated. Results: For most analytes, the in‐clinic analyzers and the CELL‐DYN performed similarly and correlated well with the ADVIA. The biases ranged from ?0.6 to 2.4 × 10⁹/L for WBC count, 0 to 0.9 × 10¹²/L for RBC count, ?1.5 to 0.7 g/dL for hemoglobin concentration, ?4.3 to 8.3 fL for MCV, and ?69.3 to 77.2 × 10⁹/L for platelet count. Compared with PCV, the HCT on most analyzers had a bias from 0.1% to 7.2%. Canine reticulocyte counts on the LaserCyte and ForCyte correlated but had a negative bias compared with those on the ADVIA. Precision, linearity, and carryover results were excellent for most analyzers. Conclusions: Total WBC and RBC counts were acceptable on all in‐clinic hematology instruments studied, with limitations for some RBC parameters and platelet counts. Together with evaluation of a blood film, these in‐clinic instruments can provide useful information on canine and feline patients in veterinary practices.     

Snake envenomation in cats and its detection by rapid immunoassay

Objective To determine the usefulness of a snake venom detection kit (SVDK) in the management of envenomed cats.
Design A clinical study.  

Animals

Twenty-two cats were investigated.
Procedure Cats injected subcutaneously with approximately 0.25 or 1.0 lethal dose (LD) of tiger snake venom or 1 or 4 LD of brown snake venom were observed for clinical symptoms of envenomation at intervals over the ensuring 24 to 48 hours(h). Blood and urine samples were taken at regular intervals and assayed in a quantitative laboratory assay for snake venoms. Selected samples were assayed in parallel in a rapid, semi-quantitative SVDK.
Results The studies showed that it was important to estimate the elapsed time from envenomation to presentation. If this time was less than 8 h, blood was the most appropriate sample and a negative result should exclude serious envenomation. If the elapsed time exceeded 8 h, it was essential that urine be sampled. Venom levels in urine were high at 8 h and approached the level of test sensitivity over 24 to 48 h; however by this time clinical signs were obvious in endangered cats.  

Conclusions

Careful use of the SVDK is a valuable aid in the management of a potentially envenomed cat.     

Artifactual changes in canine blood following storage, detected using the ADVIA 120 hematology analyzer

BACKGROUND: Artifactual changes in blood may occur as a consequence of delayed analysis and may complicate interpretation of CBC data. OBJECTIVE: The aim of this study was to characterize artifactual changes in canine blood, due to storage, using the ADVIA 120 hematology analyzer. METHODS: Blood samples were collected into EDTA from 5 clinically healthy dogs. Within 1 hour after blood sample collection and at 12, 24, 36 and 48 hours after storage of the samples at either 4 degrees C or room temperature (approximately 24 degrees C), a CBC was done using the ADVIA 120 and multispecies software. A linear mixed model was used to statistically evaluate significant differences in values over time, compared with initial values. RESULTS: The HCT and MCV were increased significantly after 12 hours of collection at both 4 degrees C and 24 degrees C, and continued to increase through 48 hours. The MCHC initially decreased significantly at 12-24 hours and then continued to decrease through 48 hours at both temperatures. Changes in HCT, MCV, and MCHC were greater at 24 degrees C than at 4 degrees C at all time points. A significant increase in MPV and a decrease in mean platelet component concentration were observed at all time points at 24 degrees C. Samples stored at 24 degrees C for 48 hours had significantly higher percentages of normocytic-hypochromic RBCs, and macrocytic-normochromic RBCs, and lower platelet and total WBC counts. CONCLUSIONS: Delayed analysis of canine blood samples produces artifactual changes in CBC results, mainly in RBC morphology and platelet parameters, that are readily detected using the ADVIA 120. Refrigeration of specimens, even after 24 hours of storage at room temperature, is recommended to improve the accuracy of CBC results for canine blood samples.     

Canine reference intervals for the Sysmex XT-2000iV hematology analyzer

Background: The laser‐based Sysmex XT‐2000iV hematology analyzer is increasingly used in veterinary clinical pathology laboratories, and instrument‐specific reference intervals for dogs are not available. Objective: The purpose of this study was to establish canine hematologic reference intervals according to International Federation of Clinical Chemistry and Clinical and Laboratory Standards Institute guidelines using the Sysmex XT‐2000iV hematology analyzer. Methods: Blood samples from 132 healthy purebred dogs from France, selected to represent the most prevalent canine breeds in France, were analyzed. Blood smears were scored for platelet (PLT) aggregates. Reference intervals were established using the nonparametric method. PLT and RBC counts obtained by impedance and optical methods were compared. Effects of sex and age on reference intervals were determined. Results: The correlation between impedance (I) and optical (O) measurements of RBC and PLT counts was excellent (Pearson r=.99 and .98, respectively); however, there were significant differences between the 2 methods (Student's paired t‐test, P<.0001). Differences between sexes were not significant except for HCT, PLT‐I, and PLT‐O. WBC, lymphocyte, and neutrophil counts decreased significantly with age (ANOVA, P<.05). Median eosinophil counts were higher in Brittany Spaniels (1.87 × 10⁹/L), Rottweilers (1.41 × 10⁹/L), and German Shepherd dogs (1.38 × 10⁹/L) than in the overall population (0.9 × 10⁹/L). PLT aggregates were responsible for lower PLT counts by the impedance, but not the optical, method. Conclusion: Reference intervals for hematologic analytes and indices were determined under controlled preanalytical and analytical conditions for a well‐characterized population of dogs according to international recommendations.     

Hematologic and biochemical abnormalities indicating iron deficiency are associated with decreased reticulocyte hemoglobin content (CHr) and reticulocyte volume (rMCV) in dogs

BACKGROUND: The ADVIA 120 automated hematology system uses low- and high-angle light scatter to determine individual RBC and reticulocyte volume and hemoglobin (Hgb) concentration. Current hematologic and biochemical markers of iron status in the dog are insensitive, and results may be highly variable, especially in the presence of concurrent disease (ie, inflammation, neoplasia). Reticulocyte Hgb content (CHr) has proven useful in detecting early iron deficiency and iron deficiency masked by concurrent disease in human patients. OBJECTIVES: The purpose of this study was to retrospectively investigate the association of low CHr and reticulocyte MCV (rMCV) with hematologic and biochemical abnormalities indicative of iron deficiency in canine patients. METHODS: Reference intervals for CHr and rMCV were established on a population of 362 hematologically-normal dogs using standard methods. CBC and serum biochemical results from 833 dogs at Colorado State University Veterinary Teaching Hospital were retrospectively evaluated. The prevalence of decreased CHr and rMCV values was determined based on the reference intervals. Hematologic (HCT, MCV) and biochemical (serum Fe concentration, percent saturation of transferrin [% sat]) values were compared among dogs with low CHr (n=58), low rMCV (n=50), and control dogs (cohort groups from the initial population) using a Fisher exact test. RESULTS: Reference intervals were 22.3-27.9 pg for CHr and 77.8-100.2 fL for rMCV. Seven percent (n=58) of dogs in the hospital population had low CHr and 6% (n=50) had low rMCV based on the reference values. Dogs with low CHr had significantly lower HCT, MCV, serum Fe, and % sat values than did control dogs. In addition, dogs with low CHr or low rMCV values had a higher frequency of microcytosis, anemia, low serum Fe concentration, and low % sat than did control dogs. CONCLUSION: Low CHr and low rMCV are associated with hematologic and serum biochemical abnormalities indicative of iron deficiency. CHr and rMCV hold promise as noninvasive, cost-effective measures of iron status in the dog.     

Comparison of bovine hematology reference intervals from 1957 to 2006

Background: The most commonly used bovine hematology reference intervals were published in 1965. We found the results from healthy cattle in 2001 differed from those in many ways. Discovery of the original laboratory book used to calculate the 1965 values gave us the opportunity to evaluate whether hematology values of healthy cattle have changed over time. Objective: The purpose of this study was to establish hematology reference intervals for Holstein cows, compare selected hematologic results with similar population data from 1957, and compare these reference intervals with those of other North American veterinary schools and published values. Methods: Reference intervals were developed in 2001 using clinically healthy, bovine leukemia virus‐negative, mid‐lactation Holstein cows. Selected parts of the hemograms and neutrophil:lymphocyte (N:L) ratio were compared with those from healthy, age‐matched Holstein cows evaluated in 1957. Bovine reference intervals were solicited from clinical pathology laboratories in North American veterinary colleges and analyzed for population characteristics and method of analysis. Results: Between 1957 and 2001, mean neutrophil counts increased significantly, whereas lymphocyte, monocyte, and eosinophil counts and hemoglobin concentration decreased significantly. Mean N:L ratio increased significantly to 1.17. Most surveyed laboratories were using the 1965 reference intervals. Two other institutions that had developed reference intervals after 2000 had results similar to ours. Conclusions: Continued use of older bovine hematology reference intervals could lead to misinterpretation of within‐reference neutrophil counts as neutrophilia and under‐recognition of neutropenia, eosinophilia, monocytosis, or lymphocytosis. Use of N:L>1 as evidence of inflammation should be discontinued or used with great caution.     

Hematologic values in calves during the first 6 months of life

BACKGROUND: Age-related changes in hematologic values are known to occur in many species. Few published studies include repeated measurements of hematologic parameters in calves during the first months of life. OBJECTIVE: The aim of the present study was to monitor hematologic values by sequential measurements from birth to 6 months of age in 15 healthy calves of the Norwegian Red breed, and compare the results to reference intervals for adult, lactating dairy cows. METHODS: Fifteen clinically healthy calves were sampled every week during the first 5 weeks of life and every month thereafter until 6 months of age. Hematologic values were measured using the ADVIA 120 hematology system. Reference intervals were determined for 75 healthy adult cows of the same breed. RESULTS: Compared with adult reference intervals, the MCV was lower and the RBC count was higher in calves throughout the investigation period. Hemoglobin concentration stayed largely within the adult reference interval. Mean MCHC was lower than adult values for 5 weeks, then increased and reached adult values by weeks 10-12. The mean lymphocyte count for calves reached adult reference values at weeks 6-8, and the mean monocyte count increased steadily until weeks 14-16. For most leukocytes, interindividual variation was larger during the first 5-8 weeks of life. The mean platelet count for calves was higher than the adult reference interval until weeks 19-21 of age. CONCLUSIONS: Age-specific reference intervals for calves from birth to 6 month of age are needed for RBC count, MCV, MCHC, red cell distribution width, and platelet and lymphocyte counts.     

Evaluation of three methods for measurement of hemoglobin and calculated hemoglobin parameters with the ADVIA 2120 and ADVIA 120 in dogs, cats, and horses

BACKGROUND: Besides flow cytometric detection of cellular hemoglobin (HGB) concentration, the ADVIA 2120 uses a novel cyanide-free colorimetric method to determine extracellular total HGB concentration. In human samples, the results are equivalent to those of the cyanmethemoglobin method on the ADVIA 120. Cyanide-free HGB measurement has not been evaluated in animal samples. OBJECTIVES: The aim of this prospective study was to compare the 3 methods of HGB analysis on the ADVIA 2120 and ADVIA 120 in blood samples from dogs, cats, and horses. METHODS: Consecutive fresh K(3)EDTA blood samples from 119 dogs, 113 cats, and 151 horses submitted to the Central Laboratory, Department of Veterinary Clinical Sciences, Justus-Liebig University Giessen, were included. A CBC was performed on each sample using the ADVIA 2120 and ADVIA 120. Colorimetric and cellular HGB concentrations and all calculated variables based on HGB measurement were compared using linear regression, Passing Bablok regression, and Bland Altman plots, using the ADVIA 120 as the reference method. RESULTS: In samples from all species, an excellent correlation was found for colorimetric HGB results (r=0.99). HGB measured with the cyanide-free method was overestimated on the ADVIA 2120 compared with the cyanide-based method on the ADVIA 120, with a mean proportional bias of -21.0% (dog), -22.0% (cat), and -19.4% (horse). The correlation of cellular HGB concentration between analyzers was excellent (r=0.99); however, imprecision was higher than for colorimetric methods. Excellent to fair agreement was found for all calculated variables. CONCLUSION: The cyanide-free method of HGB determination is appropriate for use in blood samples from animals, provided the proportional bias is considered.     

Evaluation of a point-of-care hematology analyzer for use in dogs and cats receiving chemotherapeutic treatment

OBJECTIVE: To compare WBC, neutrophil, and platelet counts and Hct values obtained with a point-of-care hematology analyzer with values obtained by a reference method for dogs and cats receiving chemotherapy. DESIGN: Cross-sectional study. ANIMALS:105 dogs and 25 cats undergoing chemotherapy. PROCEDURES:Blood samples were analyzed with a point-of-care hematology analyzer and with an impedance- and laser-based analyzer with manual differential WBC counts. Results for WBC, neutrophil, and platelet counts and Hct were compared. Sensitivity and specificity of the point-of-care analyzer to detect leukopenia, neutropenia, and anemia were calculated. RESULTS: 554 canine and 96 feline blood samples were evaluated. Correlation coefficients for dogs and cats, respectively, were 0.92 and 0.95 for total WBC count, 0.91 and 0.88 for neutrophil count, 0.95 and 0.92 for Hct, and 0.93 and 0.71 for platelet count. Sensitivity and specificity, respectively, of the point-of-care analyzer to detect leukopenia were 100% and 75% for dogs and 100% and 68% for cats; to detect neutropenia were 80% and 97% for dogs and 100% and 80% for cats; to detect anemia were 100% and 80% for dogs and 100% and 66% for cats; and to detect thrombocytopenia were 86% and 95% for dogs and 50% and 87% for cats. CONCLUSIONS AND CLINICAL RELEVANCE:The point-of-care analyzer was reliable for monitoring CBCs of dogs and cats receiving chemotherapy. It had good to excellent correlation for WBC and neutrophil counts and Hct and accurately detected leukopenia, neutropenia, and anemia. Sensitivity of the analyzer for detecting thrombocytopenia was lower but acceptable.