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.     

Feline platelet counting with prostaglandin E1 on the Sysmex XT‐2000iV

Background: The large size of many feline platelets and the high frequency of platelet aggregation often results in falsely low platelet counts in this species. A combination of optical platelet counting to detect even large platelets and the use of prostaglandin E1 (PGE1) to inhibit platelet clumping may increase the accuracy of feline platelet counting. Objective: The objective of this study was to compare platelet counts in feline whole blood samples with and without the addition of PGE1 and using different analytical methods in a clinical setting. Methods: Platelet counts were determined in 10 feline patients in a referral veterinary hospital using 2 sample types (EDTA, EDTA with PGE1) and 2 methods of analysis (optical counting [PLT‐O] and impedance counting [PLT‐I]) on the Sysmex XT 2000 iV analyzer. Results: All PGE1–PLT‐O samples had platelet counts of >200 × 10⁹/L. Mean platelet count using PGE1–PLT‐O (410,256±178 × 10⁹/L) was significantly higher (P<.03) compared with PGE1–PLT‐I (256±113 × 10⁹/L), EDTA–PLT‐O (238±107 × 10⁹/L), and EDTA–PLT‐I (142±84 × 10⁹/L) methods. Depending on the method, platelet counts in 2 to 7 of 10 cats were <200 × 10⁹/L when PGE1‐PLT‐O was not used. A slightly increased platelet count in response to treatment of a feline patient with thrombocytopenia would have been missed without use of PGE1–PLT‐O. Conclusions: Using PLT‐O analysis on EDTA samples containing PGE1 provides higher, and therefore likely more accurate, feline platelet counts in a clinical setting.     

Analytical validation of the Sysmex XT‐2000iV for cell counts in canine and feline effusions and concordance with cytologic diagnosis

Background: The Sysmex XT‐2000iV is a hematology analyzer that combines laser and impedance technology. Its usefulness for determining cell counts in canine and feline intracavitary effusions has not yet been studied. Objectives: The objectives of this study were to evaluate the analytical performance of the Sysmex XT‐2000iV for cell counts in effusions from dogs and cats, and to assess correlation with an impedance counter and concordance with diagnoses based on cytologic findings. Methods: Effusions (43 pleural, 23 peritoneal, 6 pericardial) were analyzed from 32 dogs and 34 cats. Total nucleated cell count (TNCC), HCT, and RBC count were determined on the Sysmex and compared with those obtained on an impedance counter (Hemat 8, SEAC). Imprecision, linearity, and limit of detection were determined for the Sysmex. An algorithm was designed using quantitative and qualitative data from the Sysmex to classify the effusions and the results were compared with diagnoses based on cytologic findings. Results: Intra‐assay and interassay coefficients of variation on the Sysmex were variable. Linearity of TNCC was ≥0.993 for dogs and cats, with the exception of effusions from cats with feline infectious peritonitis, which had delta (Δ) TNC values >3.0. In comparison with the Hemat 8, a proportional error was found for TNCC on the Sysmex. Effusion classification based on the algorithm was concordant with that obtained by cytologic examination in 43/72 (60%) samples. Discordant results usually were due to the misclassification of cells with similar morphology (such as mesothelial and carcinoma cells) in Sysmex scattergrams. Conclusion: The Sysmex XT‐2000iV provides a precise and accurate TNCC and has moderate concordance with cytologic findings for classifying canine and feline effusions. Although microscopic examination of effusions is necessary to achieve an accurate diagnosis, the Sysmex can provide preliminary information that may be helpful to cytopathologists.     

Validation of the Coulter AcT Diff hematology analyzer for analysis of blood of common domestic animals

Abstract: The objective of this study was to compare and assess the agreement between the Coulter AcT Diff hematology analyzer (CAD) and the Bayer Technicon H1 (H1) using blood samples from 391 animals of 4 species. The H1 has been used in veterinary laboratories for many years. Recently, Coulter modified the CAD and added veterinary software for hematologic analysis of feline, canine, and equine samples. A comparison of hemograms from dogs, cats, horses, and cattle was made using EDTA-anticoagulated blood samples. Both instruments were calibrated using human blood products. Performance characteristics were excellent for most values. The exceptions were MCV in canine samples (concordance correlation of .710), platelet counts for feline and equine samples (.258 and .740, respectively), feline and bovine WBC counts (.863 and .857, respectively), and bovine hemoglobin (.876).     

Comparison of flow cytometry with the Sysmex XT2000iV automated analyzer for the detection of reticulated platelets in dogs

Background: Immature (reticulated) platelets (r‐PLT) are not routinely assessed by hematology analyzers, but may be useful in the evaluation of the bone marrow response to thrombocytopenia. Objective: The aim of this study was to compare the Sysmex XT2000iV hematology analyzer with standard flow cytometry for the determination of r‐PLT percentage in dogs. Methods: Blood samples were obtained from 40 healthy dogs, 12 thrombocytopenic dogs, and 6 dogs with normal platelet counts but with disorders associated with increased thrombopoiesis. The percentage of r‐PLT was determined with a FACscan flow cytometer (r‐PLT[F]) using CD61‐phycoerythrin antibody and thiazole orange, and with the PLT‐O channel of the Sysmex analyzer (r‐PLT[S]). Mean platelet volume, platelet distribution width, and platelet large cell ratio were also determined on the Sysmex. Repeatability (intra‐assay precision) and effect of storage were tested for the automated analyzer. Results: The reference interval (mean±1.96 X SD) for r‐PLT(F) was 1.91±1.29% (range 0.78–3.68%) and for r‐PLT(S) was 0.56±0.82% (range 0.11–2.16%). For both flow cytometry and the Sysmex, the patient group had a significantly higher mean percentage of r‐PLT compared with the control group (P<.0001, unpaired Student's t‐tests). Fair correlation (r=0.71; Spearman's regression analysis) was found for r‐PLT results between the 2 methods, and a negative proportional systematic bias of ?6.26 was found for the Sysmex (Bland–Altman analysis). Based on receiver operating characteristic curves and a cut‐off of ≥0.975%, a sensitivity of 94.7% and a specificity of 85.7% were obtained for detecting r‐PLT on the Sysmex, using flow cytometry as the reference method. Blood samples stored at 4 °C and 25 °C had a significant increase in the percentage of r‐PLT after 24 and 48 hours, respectively. Conclusions: The PLT‐O channel of the Sysmex XT2000iV is capable of detecting immature platelets in healthy, thrombocytopenic, and nonthrombocytopenic ill dogs.     

Identification of neoplastic cells in blood using the Sysmex XT‐2000iV: a preliminary step in the diagnosis of canine leukemia

Background: Classification of leukemias requires specialized diagnostic techniques. Automated preliminary indicators of neoplastic cells in blood would expedite selection of appropriate tests. Objective: The objective of this study was to assess the capacity of the Sysmex XT‐2000iV hematology analyzer to identify neoplastic cells in canine blood samples. Methods: Blood samples (n=160) were grouped into 5 categories: acute leukemia (n=30), chronic leukemia (n=15), neoplasia without blood involvement (n=41), non‐neoplastic reactive conditions (n=31), and healthy dogs (n=43). WBC counts, WBC flags, scattergrams, percentages of cells with high fluorescence intensity, and percentages of cells in the lysis‐resistant region were evaluated alone or in combination to establish a “leukemic flag.” Sensitivity, specificity, negative (LR?) and positive (LR+) likelihood ratios, and the number of false‐negative (FN) and false‐positive (FP) results were calculated, and receiver operating characteristic curves were designed for numerical values. Results: Among single measurements and parameters, only the evaluation of scattergrams minimized FN and FP results (sensitivity 100%, specificity 94.8%, LR+ 19.17, and LR? 0.00), although their interpretation was subjective. The more objective approach based on the generation of a “leukemic flag” had a sensitivity of 100%, specificity of 87.0%, LR? of 0.00, and LR+ of 7.67. Conclusion: Using a novel gating strategy the Sysmex XT‐2000iV may be used effectively to screen canine blood for hematopoietic neoplasia.     

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.     

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.     

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.     

Automated differential leukocyte count in horses,cattle, and cats using the Technicon H-1E hematology system

The differential leukocyte counts performed by an automated hematology analyzer, the Technicon H-1E Hematology System, and traditional microscopic method (M-Diff) from blood samples of 129 horses, 40 cattle, and 140 cats were compared. The comparison was repeated after selected subsets of data were created by deleting samples with certain patterns suggesting error with the automated differential cell count (A-Diff). The two methods had good comparison of results for neutrophils and lymphocytes in all three species. Results for equine monocytes correlated moderately well between the two methods and the correlation improved in the selected data set Monocyte results did not compare well for the bovine and feline samples. The A-Diff for feline eosinophils was inaccurate. The A-Diff may be accurate for bovine and equine eosinophils but too few examples of eosinophilia were present in the sample set to prove this. Basophils were too rarely seen in cattle and horses to validate A-Diff accuracy, but basophilia identified by the M-Diff in a cat was not identified by the A-Diff.     

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.