48 B. D. HEDLEY ET AL. DXH 800 PERFORMANCE EVALUATION FCM reference procedures WBC, PLT, and NRBC enumerations by FCM were performed as previously described (Chin-Yee et al., 2001, 2002; Kaplan, Johnson & Wolfe, 2001). WBC counts were performed in duplicate by stain- ing 100 ll of accurately pipetted whole blood with 5 ll of both CD45-PC5 and CD41-FITC for 15 min at room temperature, protected from the light. Samples with a WBC count of 50 · 109/l were diluted with 5% albumin. Following this, red cells were lysed with ammonium chloride and 100 ll of Flow-Count (Beck- man Coulter) was accurately pipetted to each tube. Samples were kept on ice until the time of analysis. The average of the two reference counts was used. For PLT enumeration, specimens were mixed gently by inversion and 5 ll of whole blood added to 100 ll of PBS/BSA and incubated with 10 ll of CD41/ CD61- FITC for 15 min at room temperature, pro- tected from the light. Samples were diluted to 1 : 1000 and immediately analyzed on the XL-MCL with the stop count set at 50 000 red cells events and a minimum of 2000 PLTs. For PLT counts >500 · 109/ l, specimens were diluted in a larger volume of PBS/ BSA to reduce potential coincidence. NRBC enumeration involved the incubation of 25 ll of whole blood with 10 ll CD45-FITC for 15 min at room temperature, protected from the light. Following this, samples were mixed and incubated for 30 s with 250 ll of an acidic lysing solution contain- ing propidium iodide. To quench this lysing solution excess, 500 ll of alkaline reagent was subsequently added. Samples were then incubated for 5 min at room temperature prior to running on the XL-MCL. Manual WBC differential Two blood ﬁlms were prepared from each sample and independently examined by different technologists. Each technologist performed a 200-cell differential count. The number of NRBCs was expressed per 100 WBCs. Criteria for ﬂagging Specimens that generated ‘P’ ﬂags (partial aspiration) as well as non-numeric results were excluded from the analysis. Samples were examined by manual review to determine the number of NRBCs and if giant PLTs or PLT clumping was present, only samples found to have NRBCs by manual review were consid- ered positive for NRBCs, all others were considered negative. Statistical methods Correlation and regression analysis was performed using Microsoft Excel. An analysis of variance (ANOVA) was performed on the WBC, PLT, and NRBC results to determine whether a signiﬁcance difference between groups was present. If a signiﬁcant difference was found, a Wilcoxon test for nonparametric data was used to determine accuracy. Signiﬁcant P values were values below 0.05, values greater than this were not signiﬁcantly different. Biases were calculated on the both ﬂagged and unﬂagged data. When the ﬂagged data was removed from the analysis the biases and percentage differences were reduced on WBC, PLT, and NRBC data. False positive and false negative rates for PLTs and NRBCs were determined on the LH 780 and DxH 800 and used to determine sensitivity (TP/[TP + FN]) and speciﬁcity (TN/[TN + FP]) of each analyzer vs. FCM. Receiver Operator Curves (ROCs) were generated for the DxH 800 NRBC data vs. FCM. The Area Under the Curve (AUC) was determined from the ROC analysis (Aulesa et al., 2004). The higher the AUC number the better the performance, with 0.50 indicating random performance and 1.00 denoting perfect performance. R E S U L T S The goal of this study was to evaluate the new Beck- man Coulter UniCel DxH 800 cellular analysis system with particular focus on correlation of WBC, NRBC and PLT enumeration to a predicate analyzer, the Coulter LH 780 and FCM reference methods. WBC Samples were accrued in the WBC range of 0.1 to 190.4 · 109/l (n = 247) and were examined to deter- mine the precision and accuracy of the new DxH 800 analyzer. The DxH 800 WBC counts were correlated with the LH 780 and FCM during the study period. 2010 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2011, 33, 45–56