Analysis of feline,canine and equine hemograms using the QBC VetAutoread

Blood samples form 120 consecutive clinical cases (40 cats, 40 dogs and 40 horses) were analyzed on the QBC VetAutoread analyzer and the results compared with those obtained by a Baker 9000 electronic resistance cell counter and a 100-cell manual differential leukocyte (WBC) count. Packed cell volume (PCV), hemoglobin (Hb) concentration, mean cell hemoglobin concentration (MCHC), and platelet, total WBC, granulocytes, and lymphocyte plus monocyte (L+M) counts were determined. Indistinct separation of red blood cell and granulocytes layers on the QBC VetAutoread was observed in samples from five cats (12.5%), two dogs (5%), and one horse. Significantly different (P=0.002) median values for the two methods were obtained for PCV, Hb concentration, MCHC and platelet count in cats; PCV, MCHC, WBC, count and granulocytes count in dogs; and PCV, Hb concentration, MCHC and WBC, granulocytes and platelet counts in horses. Results from the QBC VetAutoread should not be interpreted using reference ranges established using other equipment. Results were abnormal on a limited number of samples; however, when correlation coefficients were low, marked discrepancy existed between values within as well as outside of reference ranges. Spearman rank correlation coefficients were excellent (r=0.93) for PCV and Hb concentration in dogs, and Hb concentration and WBC count in horses. Correlation was good (r=0.80-0.92) for PCV and Hb concentration in cats, WBC count in dogs, and PCV, granulocytes count and platelet count in horses. For remaining parameters, correlation was fair to poor (r=0.79). Acceptable correlations (r>0.80) were achieved between the two test systems for all equine values except MCHC and L+M count, but only for PCV and HB concentration in feline and canine blood samples.     

Clinical evaluation of the CA530-VET hematology analyzer for use in veterinary practice

BACKGROUND: The CA530-VET is a completely automated impedance cell hematology analyzer, which yields a 16-parameter blood count including a 3-part leukocyte differential. OBJECTIVES: The aim of this study was to examine the operational potential of the CA530-VET and its value for use in veterinary practice. METHODS: The analyzer was tested for blood carry-over, precision, and accuracy. Comparison methods included the CELL-DYN 3500, microhematocrit centrifugation, manual platelet (PLT) counting for feline and equine species, and a 100-cell manual WBC differential. Blood samples for comparison of the methods were obtained from 242 dogs, 166 cats, and 144 horses. RESULTS: The carry-over ratio (K) was 0.28% for RBC, 0.59% for PLT, 0.32% for WBC, and 0.18% for hemoglobin (HGB) concentration. Coefficients of variation (CVs) for within-batch precision and duplicate measurement of blood samples were clearly within the required limits, except for duplicate platelet counts in cats (8.7%) and horses (9.5%). The WBC count was in excellent agreement for dogs and horses and RBC count was in excellent agreement for horses. The accuracy of feline WBC counts was not acceptable, with the exception of values at the high end of the range. RBC counts in dogs and cats, and HGB concentration and MCV in all 3 species were sufficiently accurate. The CA530-VET HCT results were in excellent agreement with microhematocrit results in horses but exceeded the maximum allowed inaccuracy for cats and dogs. In all species, PLT counts established mechanically and manually were not in adequate agreement. Large differences were found between the CA530-VET and the manual differential percentage for lymphocytes and "mid-sized cells" (monocytes and basophilic granulocytes). CONCLUSIONS: The CA530-VET can be considered useful for routine canine, feline, and equine blood cell analyses. It should not be considered accurate, however, for PLT counts, feline total WBC counts in the subnormal and normal range, and leukocyte differentials, except for granulocytes.     

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.     

Analysis of canine and feline haemograms using the VetScan HMT analyser

The VetScan HMT is an impedance counter haematology analyser which produces a full blood count and three-part white blood cell differential. The aim of this study was to compare the results generated by the analyser with those obtained by standard methods used routinely in the authors' laboratory. Blood samples from 68 dogs and 59 cats were run on the VetScan HMT analyser and also subjected to reference methods, and the results obtained were compared. Correlation coefficients (feline/canine) were: 0.97/0.99 for haematocrit (Hct), 0.98/0.99 for haemoglobin (Hb), 0.81/0.98 for total white blood cells (WBC), and 0.89/0.97 for granulocyte and 0.65/0.93 for platelet counts. Coefficients for lymphocyte counts were 0.25/0.28 and for monocyte counts were 0.12/0.79. In conclusion, the VetScan HMT performed well on canine samples, showing excellent correlation for canine Hct, Hb, RBC, WBC, granulocyte and platelet counts. For feline samples, although there was excellent correlation for Hct, Hb and RBC, the WBC and three-part white blood cell differential and platelet count should be interpreted with caution as they can be unreliable.     

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.     

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.     

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.     

Evaluation of an in-house centrifugal hematology analyzer for use in veterinary practice

OBJECTIVE: To compare CBC results obtained by use of an in-house centrifugal analyzer with results of a reference method. DESIGN: Prospective study. SAMPLE POPULATION: Blood samples from 147 dogs, 42 cats, and 60 horses admitted to a veterinary teaching hospital and from 24 cows in a commercial dairy herd. PROCEDURE: Results obtained with the centrifugal analyzer were compared with results obtained with an electrical-impedance light-scatter hematology analyzer and manual differential cell counting (reference method). RESULTS: The centrifugal analyzer yielded error messages for 50 of 273 (18%) samples. Error messages were most common for samples with values outside established reference ranges. Correlation coefficients ranged from 0.80 to 0.99 for Hct, 0.55 to 0.90 for platelet count, 0.76 to 0.95 for total WBC count, and 0.63 (cattle) to 0.82 (cats) to 0.95 (dogs and horses) for granulocyte count. Coefficients for mononuclear cell (combined lymphocyte and monocyte) counts were 0.56, 0.65, 0.68, and 0.92 for cats, horses, dogs, and cattle, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that there was an excellent correlation between results of the centrifugal analyzer and results of the reference method only for Hct in feline, canine, and equine samples; WBC count in canine and equine samples; granulocyte count in canine and equine samples; and reticulocyte count in canine samples. However, an inability to identify abnormal cells, the high percentage of error messages, particularly for samples with abnormal WBC counts, and the wide confidence intervals precluded reliance on differential cell counts obtained with the centrifugal analyzer.     

Canine complete blood counts: a comparison of four in‐office instruments with the ADVIA 120 and manual differential counts

Background: With more use of bench‐top in‐office hematology analyzers, the accuracy of reported values is increasingly important. Instruments use varied methods for cell counting and differentiation, and blood smears may not always be examined. Objective: The purpose of this study was to compare canine CBC results using 4 bench‐top instruments (Hemavet 950, Heska CBC‐Diff, IDEXX LaserCyte, and IDEXX VetAutoread) with ADVIA 120 and manual leukocyte counts. Methods: EDTA‐anticoagulated canine blood samples (n=100) were analyzed on each instrument. Manual differentials were based on 100‐cell counts. Linear regression, difference plots, paired t‐tests, and estimation of diagnostic equivalence were used to analyze results. Results: Correlations of HCT, WBC, and platelet counts were very good to excellent between all in‐office instruments and the ADVIA 120, but results varied in accuracy (comparability). Hemavet 950 and Heska CBC‐Diff results compared best with ADVIA results and manual leukocyte differentials. HCT and platelet counts on the IDEXX VetAutoread compared well with those from the ADVIA. Except for neutrophil counts, leukocyte differentials from all instruments compared poorly with ADVIA and manual counts. Reticulocyte counts on the LaserCyte and VetAutoread compared poorly with those from the ADVIA. Conclusions: The Hemavet 950 and Heska CBC‐Diff performed best of the 4 analyzers we compared. HCT, WBC, and platelet counts on the LaserCyte had minimally sufficient comparability for diagnostic use. Except for neutrophils (granulocytes), leukocyte differential counts were unreliable on all in‐office analyzers. Instruments with a 5‐part leukocyte differential provided no added benefit over a 3‐part differential. Assessment of erythrocyte regeneration on the LaserCyte and VetAutoread was unreliable compared with the ADVIA 120.     

Comparison of methods for determining platelet numbers and volume in Cavalier King Charles spaniels

OBJECTIVE: The purpose of this study was to compare platelet concentration in cavalier King Charles spaniels (CKCS) measured by different methods commonly used in veterinary hospitals and commercial laboratories. METHODS: Blood samples obtained from 41 CKCS [corrected] were analysed by impedance cell counter, laser cell counter and microscopic estimation. Quantitative buffy coat analysis was performed only on 17 samples, selected from CKCS [corrected] that had low platelet counts detected by cell counters. Platelet counts, platelet estimations and platelet parameters using these different methods were compared. RESULTS: The median platelet number was lower when estimated using impedance cell counter (1363x10(9)/I) with respect to laser cell counter (1723x10(9)/I), microscopic estimation (238x10(9)/I) [corrected] or quantitative buffy coat analyser (292x10(9)/I) [corrected] (P<0.01). Although impedance cell counter, laser cell counter and microscopic estimation were positively correlated, there was no acceptable agreement among methods. CKCS [corrected] with macrothrombocytes in blood smears had significantly lower counts on impedance cell counter, laser cell counter and microscopic estimation. The percentages of CKCS [corrected] with platelet count < 100x10(9)/I [corrected] were 34.1 per cent (impedance cell counter), 26.8 per cent (laser cell counter), 22.0 per cent (microscopic estimation) (not statistically different) and 5.8 per cent (quantitative buffy coat analyser) (P<0.05). CLINICAL SIGNIFICANCE: CKCS [corrected] with macrothrombocytosis have low platelet counts on impedance cell counters, laser cell counters and microscopic estimation. CKCS [corrected] with low platelet counts may have a normal platelet crit detected by a quantitative buffy coat analyser and thus a normal circulating platelet mass.     

Characterization and quantification of blood cells from the umbilical cord of dogs

BACKGROUND: Models for the study of hematopoietic stem cells in dogs provide important information for bone marrow transplantation in humans. Recent studies have reported the importance of human umbilical cord blood (UCB) as an alternative to allogenic bone marrow for hematopoietic reconstitution. However, there are no studies on the UCB cells of dogs. OBJECTIVE: The aim of this experiment was to characterize and quantify the blood cells of the umbilical cord of dogs. METHODS: The blood of the umbilical cord of 20 neonatal dogs, delivered at term, with a median gestation time of 58 days, was collected with a 5-mL syringe containing EDTA. Total RBC, WBC, and platelet counts, HCT, hemoglobin (Hgb) concentration, and RBC indices were determined using an automatic cell counter. The differential leukocyte count was determined manually in blood smears stained with May-Grünwald-Giemsa. Reticulocyte percentages were determined on blood smears stained with brilliant cresyl blue and counterstained with May-Grünwald Giemsa. RESULTS: The MCHC and numbers of RBCs, WBCs, neutrophils, and eosinophils in UCB were lower as compared with reference values for the peripheral blood of healthy neonatal and adult dogs; whereas, the MCV and reticulocyte percentages were higher. CONCLUSION: Erythrocyte macrocytosis and hypochromasia in UCB were consistent with marked reticulocytosis and indicative of high erythropoietic activity. The results of this study are an important first step in the characterization of UCB from neonatal dogs.     

Thrombocytopenia in Horses: 35 Cases (1989–1994)

The records of 3,952 equine patients presenting to the Veterinary Teaching Hospital at North Carolina State University College of Veterinary Medicine were evaluated to determine risk factors associated with thrombocytopenia. Of 2,346 horses from which a CBC was obtained, 35 (1.49%) were thrombocytopenic (platelet count < 75,000/μL). A reference population of 189 horses with normal platelet counts (75,000 to 300,000/μL) was also studied. Standardbred horses were at increased risk for thrombocytopenia. but age and gender were not identified as significant risk factors. Horses with infectious or inflammatory diseases were at increased risk for thrombocytopenia. The potential association of clinical and clinicopathologic factors with thrombocytopenia were assessed by reviewing a series of multiple logistic regression models. Clinical and clinicopathologic variables significantly associated with thrombocytopenia in the final model included increased PCV, increased band neutrophil count, increased total WBC, and decreased plasma protein concentration. Increased mature neutrophil count was associated with normal platelet counts. Thrombocytopenic horses were significantly more likely to die or be euthanized than were horses with normal platelet counts. J Vet Intern Med 1996;10:127–132. Copyright © 1996 by the American College of Veterinary Internal Medicine .     

Improved mouse blood smears using the DiffSpin slide spinner

Push smears of mouse blood prepared for differential white blood cell (WBC) determination often have many lysed WBCs, numerous RBC "ghosts", and poor morphology of intact RBCs. The purpose of this study was to compare the quality of peripheral blood smears prepared by 3 different methods and to optimize a technique for mouse blood differential WBC determination. Peripheral blood smears were prepared from blood obtained from clinically normal adult mice and human adults. Differential WBC counts, numbers of lysed WBCs/100 intact WBCs, and RBC morphology were compared in blood smears made using the standard push method with undiluted blood, the push method with blood diluted 1:5 with bovine serum albumin, and in centrifugally-prepared smears made with the DiffSpin Slide Spinner (StatSpin, Norwood, Mass, USA). The number of damaged WBCs in mouse versus human samples using the push method was compared using an unpaired Student's t test. ANOVA was used to compare differences in WBC differential counts and numbers of damaged WBCs among the 3 methods for each species. In addition, unpaired Student's t tests were used to compare each method against the other methods, within species. The number of damaged WBCs/100 intact WBCs was approximately 3 times higher in mouse than in human push smears (P=0.002). There was no significant difference in WBC differential cell counts among the 3 methods in either species. However, compared with both push techniques, a significantly (P <.01) greater number of intact cells was observed with the DiffSpin technique for mouse blood samples (damaged WBC/100 intact cells = 4.4 +/- 2.6 for DiffSpin smears, 9.5 +/- 3.9 for push smears with added albumin, and 31.3 +/- 10.2 for standard push smears). DiffSpin mouse blood smears consistently had better RBC morphology when compared with standard push smears. In conclusion, the DiffSpin Slide Spinner produced optimal smears of mouse blood for WBC differential determination and analysis of RBC morphology.     

Spurious,marked leukocytosis in 2 cats with Heinz body hemolytic anemia

Two domestic shorthair cats were presented with anorexia and dehydration following ingestion of caramelized onions. Shared key findings from a CBC (ADVIA 2120), serum biochemistry, and urinalysis included a spurious, marked leukocytosis with discordant basophil (BASO) channel and peroxidase channel WBC counts, normal manual leukocyte counts, mild, non-regenerative anemia with discrepancies between automated and manual reticulocyte counts, an abundance of large Heinz bodies (HBs), and highly irregular scattergrams. Case 1 also demonstrated a markedly elevated mean corpuscular hemoglobin concentration (MCHC) and discrepancies between RBC hemoglobin indices. Spurious leukocyte results were confirmed through re-analysis of samples (including the acquisition of a new sample, use of an alternate analyzer (Sysmex XT-2000iV; Case 1 only), and evaluation of scattergrams and blood films (Cases 1 and 2). Repeatedly discrepant reticulocyte counts were also identified. In both cases, the erroneous BASO WBC counts, discrepancies in reticulocyte counts and RBC indices, and atypical scattergrams were interpreted to result from various effects of the HBs. These cases emphasize the importance of reviewing blood films, interpreting scattergrams, and the usefulness of duplicate methods for determining various measurands on hematology analyzers.     

Equine immune-mediated hemolytic anemia associated with Clostridium perfringens infection

Background: Previous studies have suggested an association between equine immune-mediated hemolytic anemia and clostridial infections or neck abscesses.
Objective: The purpose of this report was to describe and characterize the hematologic abnormalities in a horse with Clostridium -associated immune-mediated hemolytic anemia. We also retrospectively evaluated hematologic abnormalities in 8 horses with clostridial myositis or subcutaneous emphysema.
Methods: A 7-year-old Foreign Warm-Blood gelding was evaluated for anemia and a cervical abscess. CBCs and reticulocyte counts were obtained using an Advia 120 analyzer and evaluation of Wright's-stained smears. All cases of equine Clostridium spp. myositis or subcutaneous emphysema over a 7-year period were identified in a retrospective search of the University of Minnesota Veterinary Teaching Hospital database. Clinical, hematologic, and microbiological findings were recorded.
Results: Clostridium perfringens genotype A was isolated from the neck wound of the gelding. The CBC was characterized by severe regenerative anemia, intravascular hemolysis, and RBC agglutination. A direct Coombs' test was positive. Moderate numbers of spheroechinocytes were observed. The total automated reticulocyte count was 4.5% (56,700/μL), with most reticulocytes having low absorbance (mature reticulocytes). The anemia responded to penicillin and steroidal and nonsteroidal immunosuppressive drugs. Of 8 horses with myositis, all of which involved the cervical region, 5 were anemic, 1 had a positive direct Coombs' test, and 2 had increased numbers of type III echinocytes and spheroechinocytes.
Conclusions: The presence of type III echinocytes or spheroechinocytes may be helpful in diagnosing immune-mediated hemolytic anemia associated with clostridial infections in horses. Automated reticulocyte counts may detect very low levels of reticulocytosis in hemolytic anemia in horses.     

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 ≥.97) and good for lymphocyte counts (r ≥.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.     

Evaluation of a citrate-based anticoagulant with platelet inhibitory activity for feline blood cell Counts

Abstract: Aggregation of feline platelets in vitro results in difficulty assessing platelet number. A citrate-based anticoagulant containing the platelet inhibitors theophylline, adenosine, and dipyridamole (CTAD; Diatube-H, Becton Dickinson, Oxford, UK) has been developed for use in human platelet studies and heparin assays. To evaluate the efficacy of CTAD in reducing platelet aggregation in feline blood samples, aliquots of blood from 51 cats were anticoagulated with EDTA, CTAD, and for 12 samples, citrate solution. Samples preserved in CTAD had significantly higher (P ≤ .001) platelet counts, as determined by an impedance counter, hemacy-tometer, and smear estimation, than samples preserved in EDTA. In addition, subjective assessment of blood smears showed significantly fewer platelet aggregates (P<.001) in CTAD-treated samples compared with EDTA samples. Although values were similar, automated platelet counts and smear estimates of platelet number were significantly higher (P < .05) and platelet aggregation was significantly less (P < .05) in CTAD samples than in citrate samples. These results suggest that the platelet inhibitory activity of CTAD reduced feline platelet aggregation. Automated total WBC counts in CTAD samples were significantly lower (P<.001) than automated counts in EDTA samples but were similar to manual WBC counts in EDTA samples. Differences in both platelet and WBC counts between CTAD and EDTA or citrate samples were clinically relevant. Mean platelet volume and MCV were significantly lower (P< .05) in CTAD samples than in EDTA samples. No effect was seen on cell morphology or staining characteristics. The anticoagulant CTAD offers an advantage over both EDTA and citrate for feline hematologic analysis, by decreasing pseudothrombocytopenia and pseudoleukocytosis.     

Analytic errors in Sysmex‐generated hematology results in blood from a dog with chronic lymphocytic leukemia

A blood sample from a 14‐year‐old dog was submitted to the veterinary diagnostic laboratory of the University of Milan for marked leukocytosis with atypical cells. A diagnosis of chronic T‐cell lymphocytic leukemia (CLL) was made based on blood smear evaluation and flow cytometric phenotyping. A CBC by Sysmex XT‐2000iV revealed a moderate normocytic normochromic anemia. Red blood cells counted by optic flow cytometry (RBC‐O) resulted in a higher value than using electrical impedance (RBC‐I). The relative reticulocyte count based on RNA content and size was 35.3%, while the manual reticulocyte count was < 1%. The WBC count of 1,562,680 cells/μL was accompanied by a flag. Manual counts for RBC and WBC using the Bürker chamber confirmed the Sysmex impedance results. Finally the manual PCV was lower than HCT by Sysmex. While Sysmex XT can differentiate between RBC and WBC by impedance, even in the face of extreme lymphocytosis due to CLL, RBC‐O can be affected by bias, resulting in falsely increased RBC and reticulocyte numbers. Overestimation of RBC‐O may be due to incorrect Sysmex classification of leukemic cells or their fragments as reticulocytes. This phenomenon is known as pseudoreticulocytosis and can lead to misinterpretation of regenerative anemia. On the other side PCV can be affected by bias in CLL due to the trapping of RBC in the buffy coat, resulting in a pink hue in the separation area. As HGB concentration is not affected by flow cytometric or other cell‐related artifacts it may represent the most reliable variable to assess the degree of anemia in cases of CLL.     

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.     

Automated flow cytometric cell count and differentiation of canine cerebrospinal fluid cells using the ADVIA 2120

Background: Microscopic cell counts in cerebrospinal fluid (CSF) are time-consuming and prone to imprecision. The recently introduced automated hematology analyzer ADVIA 2120 offers an automated cell count and differential for CSF in the veterinary software mode based on laser light scatter and absorbance measurements. Objectives: The purpose of this study was to evaluate the precision, linearity, and accuracy of the ADVIA 2120 CSF assay. Methods: Sixty-seven CSF samples were analyzed on the ADVIA 2120 and total nucleated cell counts (TNCC) and RBC counts were compared with the hemocytometer results. In 21 samples with TNCC >5/muL, ADVIA 2120 results were compared with 100-300 cell manual differentials performed on cytocentrifuged preparations. Statistical analysis included Spearman's rank correlation, Passing-Bablok regression, and Bland-Altman analysis. Results: Repeatability (intra-assay) coefficients of variation (CVs) ranged from 4.19% to 25.94%. Interassay CVs ranged from 2.56% to 28.67%. Accurate results within 30% were achieved for TNCC up to 4000/muL. Except for low TNCC, deviation from the expected value was higher (TNCC of 8/muL instead of 4/muL). The following correlation coefficients (r) and biases were achieved compared with the reference method: r=.90 and bias 2.3/muL for TNCC; r=.88 and bias 32.0/muL for RBC counts; r=.86 and bias +/-13.4% for mononuclear and polymorphonuclear cell percentages; r=.88 and bias -6.1% for lymphocyte percentage; r=.56 and bias 19.4% for monocyte percentage; and r=.75 and bias -9.7% for neutrophil percentage. Conclusion: Our results demonstrated that the automated ADVIA 2120 CSF assay generally compares well with reference methods although there are some limitations for the automated monocyte count and for samples with only mild pleocytosis.