Artifactual changes in equine blood following storage,detected using the Advia 120 hematology analyzer

Background — Delayed analysis of blood samples may be caused by restricted access to laboratories. Artifactual changes may occur in the measured analytes as a consequence of delayed analysis and may complicate interpretation of the data.
Objective — The purpose of this study was to characterize artifactual changes in equine blood, due to storage, using the Advia 120 hematology analyzer.
Methods — Samples of blood from 5 horses were analyzed using the Advia 120 soon after collection and again after 24 and 48 hours of storage at either 4°C or ambient laboratory temperature (∼24°C).
Results — Delayed analysis of equine blood samples resulted in increased numbers of normocytic hypochromic RBCs, increased numbers of macrocytic hypochromic RBCs, misclassification of granulocytes as mononuclear cells using the basophil reagent method, and pseudothrombocytosis, due to misclassification of ghost RBCs as platelets. The latter artifact was corrected by an amended version of the software. Many of the artifactual changes were identified by morphology flags.
Conclusion — Characteristic changes in cytograms produced by the Advia 120 allowed recognition of artifactual changes in stored equine blood samples. These changes were less pronounced in samples stored at 24°C than at 4°C.     

Canine and feline hematology reference values for the ADVIA 120 hematology system

Background: The ADVIA 120 is a laser-based hematology analyzer with software applications for animal species. Accurate reference values would be useful for the assessment of new hematologic parameters and for interlaboratory comparisons.

Objective:

Objective: The goal of this study was to establish reference intervals for CBC results and new parameters for RBC morphology, reticulocytes, and platelets in healthy dogs and cats using the ADVIA 120 hematology system.

Methods:

Methods: The ADVIA 120, with multispecies software (version 1.107-MS), was used to analyze whole blood samples from clinically healthy dogs (n=46) and cats (n=61). Data distribution was determined and reference intervals were calculated as 2.5 to 97.5 percentiles and 25 to 75 percentiles.

Results:

Results: Most data showed Gaussian or log-normal distribution. The numbers of RBCs falling outside the normocyticnormochromic range were slightly higher in cats than in dogs. Both dogs and cats had reticulocytes with low, medium, and high absorbance. Mean numbers of large platelets and platelet clumps were higher in cats compared with dogs.

Conclusions:

Conclusions: Reference intervals obtained on the ADVIA 120 provide valuable baseline information for assessing new hematologic parameters and for interlaboratory comparisons. Differences compared with previously published reference values can be attributed largely to differences in methodology.     

Evaluation of the ADVIA 120 for analysis of canine cerebrospinal fluid

BACKGROUND: Conventional techniques for canine cerebrospinal fluid (CSF) analysis require large sample volumes and are labor intensive and subject to operator variability. Objective: The purpose of this study was to evaluate the ADVIA120 CSF assay for analysis of canine CSF samples. METHODS: CSF samples collected from 36 healthy control dogs and 17 dogs with neurologic disease were processed in parallel using the automated assay and established manual methods using a hemocytometer and cytocentrifugation. Results for WBC (total nucleated cell) count, RBC count, and differential nucleated cell percentages were compared using Spearman rank correlation coefficients and Bland-Altman bias plots. RESULTS: Correlation coefficients for WBC and RBC counts were 0.57 and 0.83 for controls, and 0.92 and 0.94 for ill dogs, respectively. Coefficients for the percentages of neutrophils, lymphocytes, and monocytes were 0.53, 0.26, and 0.12 for controls and 0.77, 0.92, and 0.70 for dogs with neurologic disease. When data were combined (n=53), correlation coefficients were 0.86 and 0.91 for WBC and RBC counts, and 0.63, 0.43, and 0.30 for neutrophil, lymphocyte, and monocyte percentages. A 9.5% positive bias and 7.0% negative bias were obtained for the ADVIA 120 CSF assay for lymphocytes and macrophages in dogs with neurologic disease with Bland-Altman analysis. A 12.2% positive bias was found for lymphocyte percentage in dogs with neurologic disease. CONCLUSIONS: Manual and automated CSF assays had moderate to excellent correlation for WBC and RBC concentrations, but results were more variable for differential cell percentages. The ADVIA assay may be more useful for assessment of canine CSF with adjustment of cell differentiation algorithms.     

Evaluation of equine hemograms using the ADVIA 120 as compared with an impedance counter and manual differential count

BACKGROUND: The ADVIA 120 is an automated laser cell counter widely used in veterinary medicine. Although specific software for equine samples is available and validated, only a few reports have been published comparing the ADVIA 120 with other methods for equine hemogram evaluation. OBJECTIVES: The purpose of this study was to compare the hematologic values and reference intervals obtained on the ADVIA 120 with those obtained on an impedance cell counter and manual differential counts in healthy horses. METHODS: EDTA-anticoagulated blood samples were obtained from 114 clinically healthy horses of various breeds, both sexes, and 2-6 years of age. Samples were stored for up to 12 hours at 4 degrees C and then analyzed on the ADVIA 120 and the Hemat 8. A 100-cell to 200-cell differential leukocyte count was performed by 3 independent observers on May-Grünwald-Giemsa-stained smears. Intra-assay precision of the ADVIA 120 was determined by analyzing 5 replicates each of 10 of the blood samples. RESULTS: Results from the ADVIA were significantly higher than those from the impedance counter for RBC count, total WBC count, hemoglobin concentration, red cell distribution width, MCH, and MCHC, and significantly lower for HCT and platelet count. Significantly higher neutrophil and basophil counts and significantly lower lymphocyte counts were obtained with the ADVIA 120 compared with manual counts. Based on Passing-Bablok regression analysis, RBC and platelet counts were in good agreement between the 2 analyzers; a constant and proportional bias was present for other values. Coefficients of variation for erythrocyte parameters on the ADVIA were <1%, but were higher for platelet (6%), total WBC (2%), differential WBC (4%-30%), and reticulocyte (75%) counts. CONCLUSIONS: Results obtained with equine samples on the ADVIA 120 were comparable with those obtained on an impedance counter; reference intervals differed statistically but overlapped. The ADVIA had poor precision for reticulocyte and differential leukocyte counts such that the latter should always be verified on smears.     

Comparison between manual and automated total nucleated cell counts using the ADVIA 120 for pleural and peritoneal fluid samples from dogs,cats, horses,and alpacas

Background: Analysis of body fluids includes an estimate of total nucleated cell count (TNCC). Automated methods may enhance the accuracy and timeliness of TNCC results. Objective: The purpose of this report was to assess the ability of the ADVIA 120 hematology analyzer to accurately count nucleated cells in pleural and peritoneal fluids from animals, compared with manual counts. Methods: Pleural and peritoneal fluids submitted in EDTA tubes to our laboratory over a 17‐month period were used in the study. TNCC/μL was determined by a manual method, using a hemocytometer, and by an automated method, using the ADVIA 120. Correlation of results was determined by Passing‐Bablok regression, Bland–Altman plots, and Pearson correlation analysis. Results: Samples from dogs (n=36), cats (n=36), horses (n=59), and alpacas (n=11) were analyzed. High correlation in TNCC between methods was found for peritoneal fluid (n=93, r=.959), pleural fluid (n=49, r=.966), and all fluids combined (n=142, r=.960) (P<.001). Variation between methods was greater in samples with TNCCs<1000/μL (r=.62, P<.001). The ADVIA systematically overestimated the number of cells in all fluid samples by 95 cells/μL (confidence interval=19.2–190.5/μL). Conclusion: The ADVIA 120 reliably determines TNCC in pleural and peritoneal effusions and can be recommended for routine veterinary laboratory analysis.     

Estimating leukocyte,platelet, and erythrocyte counts in rats by blood smear examination

Background: The CBC is an essential test for assessing the health of rats used in drug development studies. Because of limited blood volume, estimates of cell counts from a blood smear would be valuable when other analytical methods of enumerating cells are not possible or available. Objective: The purpose of this study was to develop a statistical model to accurately estimate WBC, platelet (PLT), and RBC counts in blood smears from rats. Method: Blood smears and quantitative cell counts were obtained from vehicle‐treated male and female Fischer 344 rats (n=65) involved in a variety of studies. The numbers of WBCs, PLTs, and RBCs were estimated in 10 fields in the monolayer of smears using × 20 (WBC) or × 100 (PLT, RBC) objectives. Using a statistical model and the quantitative cell counts obtained on an ADVIA 120 hematology analyzer, formulas were developed to predict the quantitative counts from the estimates. Results: Data were log‐transformed before analysis. A formula was derived using the slope and intercept of the regression line between cell estimates and ADVIA counts to predict WBC, PLT, and RBC counts based only on estimates. A second formula was developed for situations in which limited quantitative analyses may be available, and resulted in even more accurately predicted counts from smear estimates. Conclusion: The formulas developed in this study can be a valuable tool in estimating cell counts from a blood smear when cell counting instruments are not available or when an instrument cell count needs to be verified. These formulas may be useful in the assessment of rat blood in discovery and lead optimization studies.     

Evaluation of a hematology analyzer with canine and feline blood

A semiautomatic electronic blood cell counter (Sysmex F-800:Toa Medical Electronics Europa Gmbh, Hamburg, Germany) was evaluated using canine and feline blood, following the International Committee for Standardization in Hematology protocol (ICSH, 1984). Precision and overall reproducibility were acceptable for all the parameters studied except for the feline platelet count, in which overlapping of erythrocyte and platelet populations prohibited determination of an accurate platelet count. Since carry-over from canine hematocrit values and platelet counts and from feline hematocrit values was unsatisfactory, the use of a blank diluent sample between different analyses was necessary. Linearity of the analyzer was acceptable in the studied range. Thirty canine and feline blood samples were analyzed using the Sysmex F-800 and a manual method. Correlations between both methods were acceptable for all the parameters, except for feline platelet count and erythrocyte indices for both species. In the storage study, red blood cell count and hemoglobin concentration were the parameters with the longest stability (72 hours at 4 degrees C and 25 degrees C) in both species. A statistically significant increase in MCV was obtained at 12 hours post-extraction in canine samples stored at 25 degrees C and at 24 hours in refrigerated samples. Feline leucocyte counts showed a downward trend at 12 hours post-extraction at both temperatures. Canine platelet count decreased significantly at 6 hours post-extraction in samples stored at 4 degrees C. During the evaluation period, Sysmex F-800 was user friendly and appeared well suited for routine canine and feline blood cell analysis.     

A comparison of platelet parameters in EDTA- and citrate-anticoagulated blood in dogs

BACKGROUND: Platelet aggregates are a common artifact in canine blood. Aggregates may affect the accuracy of platelet counts, with important consequences for patient care. OBJECTIVES: The purpose of this study was to determine if platelet counts in dogs were more accurate if blood was collected into citrate instead of EDTA as an anticoagulant. METHODS: Blood was collected from 50 dogs with neoplasia admitted to the oncology service at Cornell University. EDTA and citrate Vacutainer tubes were filled with blood in random order. Platelet counts and parameters (mean platelet volume [MPV], platelet distribution width [PDW], mean platelet component concentration [MPC], platelet component distribution width [PCDW], and automated platelet clump count [APCC]) were determined using an optical-based hematology analyzer (ADVIA 120). Blood smears from each anticoagulated sample were scored visually for platelet aggregates. RESULTS: The median platelet count was significantly lower (median decrease, 27 x 10(9)/L) in citrate-anticoagulated blood compared with EDTA-anticoagulated blood. This was attributed to platelet activation and aggregation: significantly more aggregates were seen in smears of citrate- than of EDTA-anticoagulated blood. Aggregates were typically small and not detected by the analyzer. Also, the MPV and MPC (or density) were significantly higher (median increase, 3 fL) and lower (median decrease, 33 g/L) in citrate-anticoagulated samples, respectively. CONCLUSIONS: Platelets aggregate, likely from activation, when blood from dogs with neoplasia is anticoagulated with citrate for hematology testing, resulting in lower platelet counts. Citrate also yields inaccurate results for MPV and MPC, likely because of inadequate sphering of platelets. Thus, we recommend that citrate not be used as an anticoagulant when accurate platelet counts are desired in dogs.     

Artifactual changes in PCV, hemoglobin concentration, and cell counts in bovine, caprine, and porcine blood stored at room and refrigerator temperatures

BACKGROUND: Blood samples collected from farm animals for hematology testing may not reach the laboratory or be examined immediately upon collection, and in some cases may need to be transported for hours before reaching a laboratory. OBJECTIVE: The objective of this study was to investigate the artifactual changes that may occur in PCV, hemoglobin (Hgb) concentration, and cell counts in bovine, caprine, and porcine blood samples stored at room (30 degrees C) or refrigerator (5 degrees C) temperature. METHODS: Baseline values for PCV, Hgb concentration, and RBC and WBC counts were determined immediately after blood collection from 36 cattle, 32 goats, and 48 pigs using manual techniques. Blood samples were split into 2 aliquots and stored at 30 degrees C or 5 degrees C. Hematologic analyses were carried out at specified intervals during 120 hours of storage. Results were analyzed by repeated measure ANOVA; results at different temperatures were compared by paired t-tests. RESULTS: Compared to baseline values, there were no significant changes in Hgb concentration, RBC count, or WBC count in samples from cattle; in Hgb concentration and RBC count in samples from goats; and in Hgb concentration and WBC count in samples from pigs throughout the 120 hours of storage at both 30 degrees C and 5 degrees C. Significant changes (P <.05) from baseline occurred in PCV after 14 hours of storage at 30 degrees C and after 19 hours of storage at 5 degrees C in cattle and goats; and after 10 hours of storage at 30 degrees C and 14 hours of storage at 5 degrees C in pigs. Significant changes also were observed in Hgb concentration at 96 hours at 30 degrees C and 5 degrees C, and in RBC counts at 48 hours at 30 degrees C and 96 hours at 5 degrees C in porcine samples; and in total WBC counts at 120 hours at 30 degrees C and 5 degrees C in caprine samples. Artifactual changes were more pronounced in the samples stored at 30 degrees C. CONCLUSIONS: At both 30 degrees C and 5 degrees C, blood samples from cattle and goats can be stored for up to 12 hours, while blood samples from pigs can be stored for up to 8 hours without any significant changes in PCV. Blood samples from all 3 species can be stored for more than 24 hours without significant changes in Hgb concentration, RBC count, and total WBC count.     

The importance of manual white blood cell differential counts and platelet estimates in elephant hematology: blood film review is essential

Unique features of elephant hematology are known challenges in analytical methodology like two types of monocytes typical for members of the Order Afrotheria and platelet counts of the comparatively small elephant platelet. To investigate WBC differential and platelet data generated by an impedance-based hematology analyzer without availability of validated species-specific software for recognition of elephant WBCs and platelets, compared to manual blood film review. Blood samples preserved in ethylenediaminetetraacetic acid (EDTA) of 50 elephants (n = 35 Elephas maximus and n = 15 Loxodonta africana) were used. A Mann-Whitney test for independent samples was used to compare parameters between methods and agreement was tested using Bland-Altman bias plots. All hematological variables, including absolute numbers of heterophils, lymphocytes, monocytes, eosinophils, basophils, and platelets, were significantly different (p < 0.0001) between both methods of analysis, and there was no agreement using Bland-Altman bias plots. Manual review consistently produced higher heterophil and monocyte counts as well as platelet estimates, while the automated analyzer produced higher lymphocyte, eosinophil, and basophil counts. The hematology analyzer did not properly differentiate elephant lymphocytes and monocytes, and did not accurately count elephant platelets. These findings emphasize the importance of manual blood film review as part of elephant complete blood counts in both clinical and research settings and as a basis for the development of hematological reference intervals.