Key words:Blood cell differentiation – Cell-Dyn® 3500 – Flow cytometry – Impedance measurement – Mouse and rat haematology – Reference values
Springer Online Journal Archives 1860-2000
Abstract: The objective of this study was to evaluate the performance of the CELL-DYN® 3500 for rat and mouse blood analysis in a routine environment. The WBC (white blood cells), RBC (red blood cells), PLT (platelets) counts and the WBC differential were determined. In addition, the following aspects were studied: within-run precision, day-to-day precision, bias-free paired difference precision; extended ranges of linearity for RBC, HCT (haematocrit), WBC, PLT; carry-over, the effect of blood ageing, cell stability with different anticoagulants; and the normal ranges, the out of range flagging and some typical pathology cases. The CELL-DYN® 3500 is a multiparameter flow cytometer which counts and differentiates WBC, based on the principle of multi-angle polarised light scatter separation. RBC and PLT are determined by the impedance method. The WBC count is evaluated by both, optical and impedance methods. Reference methods used were according to the ICSH recommendations on blood cell analysis, including manual counts of WBC and platelets, a centrifugal microhaematocrit method and a haemoglobin measurement by spectrophotometry using the WHO haemoglobin standard. All cell counts were compared with the results obtained by our routine blood cell analyser (Contraves AL820), and the WBC differential was compared with the manual microscopic differentiation of the 400 WBC (200 cells differentiated by two technicians). The following coefficients of vartiation were obtained: within-run precision was 1.2% and 2.7% for WBC; 1.0% and 1.0% for RBC; 1.3% and 0.9% for haematocrit; 2.1% and 2.7% for platelets (rats and mice respectively). Day-to-day precision was performed using human tri-level control blood, and the CVs were found to be 〈1.7% for WBC, 〈1.4% for RBC, 〈1.2% for haemoglobin and 〈6.3% for platelets. The following ranges of measurement were found to be linear in the rat: WBC: 0.10–20.20×103/μl; RBC: 0.016–14.3×106/μl; haemoglobin: 0.08–26.8 g/dl; haematocrit: 5.0%–77%; platelets: 14.0–1670.0×103/μl. Equal ranges were observed for mouse blood. Carry-over in rat blood was found to be 0.12% for WBC, 0.05% for RBC, 0.15% for haemoglobin and 0.46% for platelets. In mice, similar carry-over results were obtained. The correlation coefficients (Pearson, correlation coefficient) between the CELL-DYN® 3500 and Contraves AL 820 using linear regression analysis were as follows: 0.988 and 0.997 for WBC; 0.986 and 0.920 for RBC; 0.995 and 0.984 for haemoglobin; 0.958 and 0.85 for haematocrit; 0.958 and 0.963 for platelets, for rats and mice, respectively. Correlation coefficients between the CELL-DYN® 3500 and the manual differential of NEU (neutrophils) and LYM (lymphocytes) were higher than 0.8 in rats and higher than 0.9 in mice. Due to the relatively low absolute counts of MONO (monocytes), EOS (eosinophils) and BASO (basophils), only moderate correlation of methods was found. The CELL-DYN® 3500 was judged to be reliable, accurate and easy-to-use for counting and identifying normal and most of the pathological blood specimens obtained from mice and rats. By using the CELL-DYN® 3500, the time for blood sample analysis can be shortened significantly and provides extensive opportunities to characterise pathological samples.
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