SIMULTANEOUS DIFFERENTIATION AND QUANTITATION OF ERYTHROBLASTS AND WHITE BLOOD-CELLS ON A HIGH-THROUGHPUT CLINICAL HEMATOLOGY ANALYZER

After the neonatal period, the presence of nucleated red brood cells ( NRBC) in peripheral blood is indicative of pathology. Despite the clin ical utility of such measurements, automated NRBC counting has hithert o not been available on routine automated blood cell counting analyser s. To address this, an automated method for the analysis of NRBC was d eveloped and incorporated into the Abbott Cell Dyn 4000 (CD4000) haema tology analyser, The system white blood cell(WBC) reagent was specific ally formulated to preserve concomitantly white blood cell (WBC) morph ology, rapidly lyse red blood cell and NRBC membranes, and subsequentl y stain NRBC nuclei with a nucleotide specific fluorochrome dye (I(im et al. 1996a). The fluorochrome itself does not permeabilize the membr ane of intact viable white blood cells. The sample is processed by Bow cytometry and the signals generated from an argon-ion laser light sou rce are analysed. Axial light loss (AxLL), intermediate angle light sc atter (IAS) and red fluorescence (FL3) are used to discriminate betwee n particles of various types. By using these discriminators in a three -dimensional approach, NRBC form a discrete cluster which can easily b e separated from leucocytes and enumerated as a distinct cell populati on during the optical WBC differential analysis, Consequently, accurat e absolute WBC counts and differentials can be obtained even in the pr esence of NRBC, Background 'noise' (both fluorescent and non-fluoresce nt) from platelets, Howell-Jolly bodies, basophilic stippling, RNA fro m lysed reticulocytes, and DNA from leucocyte and megakaryocytic fragm ents are essentially eliminated (I(im ct al, 1996b), While the membran es of intact and viable leucocytes remain impermeable to the passage o f the fluorochrome stain, leucocytes with damaged membranes are permea ble to the dye and generate FL3+ signals. Such cells, which are common ly seen as a consequence of sample ageing as well as in some distincti ve pathologies, are identified by the algorithm (using their AxLL sign al size) and are labelled as non-viable. Moreover, because non-viable leucocytes are retained in the WBC count and differential analyses, th e CD4000 is further able to provide both numerical and graphical data regarding the relative frequency of viable and non-viable components, This additional information can serve as valuable 'decision-drivers' i n the laboratory data review process.