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 degrees C and 25 degrees 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.