Automated reticulocyte counting: state of the art and clinical applications in the evaluation of erythropoiesis

Abstract

The reticulocyte count reflects the erythropoietic activity of the bone marrow and is thus useful in both the diagnosis of anemias and in monitoring bone marrow response to therapy. Starting in the mid-1990s, automated flow-cytometric analysis has replaced traditional microscopic quantitation of reticulocytes. Reticulocyte analysis now includes measurements mRNA content and the maturity of reticulocytes, cell volume, hemoglobin concentration and content. The immature reticulocyte fraction is a reliable early predictor of hematopoietic engraftment following allogeneic stem cell transplantation, while the reticulocyte hemoglobin content provides an indirect measure of the functional iron available for new red blood cell production over the previous 3-4 days. Especially in anemic newborns, reticulocyte analysis is useful to help clinicians follow erythropoietic changes, to monitor response to recombinant human erythropoietin therapy, to gauge transfusion needs, and to evaluate jaundice. Despite improved accuracy and precision, significant problems still persist in maintaining adequate levels of precision and comparability across different laboratories. In the absence of better laboratory standardization, having a single reference range for the parameters provided by flow-cytometric studies of reticulocytes remains problematic.

Laboratory hematology in the history of Clinical Chemistry and Laboratory Medicine

“…Compared to manual reticulocyte counting, flow cytometry was found to be much more precise and enabled improved reticulocyte enumeration of abnormally low counts [39] . A recent review article confirmed the superiority of automated, flow cytometric methods, in particular methods using fluorescent dyes [40] . However, standardization of the reticulocyte count is not yet satisfactory, meaning that instrument-specific reference ranges are still necessary [40] .…”

Instrument-dependent interference of Howell-Jolly bodies in reticulocyte enumeration

“…In subsequent microscopic evaluation, reticulocytes can be discriminated from mature red blood cells by an intracellular deep blue precipitate. Nowadays, state-of-the-art hematology automatic counters replace the labor intensive microscopic evaluation of reticulocytes with quick and reliable enumeration of reticulocytes in whole blood samples [2]. The technique is based on specific dyes, which mostly are poly nucleotide-specific, thus binding DNA as well as RNA [2].…”

“…Nowadays, state-of-the-art hematology automatic counters replace the labor intensive microscopic evaluation of reticulocytes with quick and reliable enumeration of reticulocytes in whole blood samples [2]. The technique is based on specific dyes, which mostly are poly nucleotide-specific, thus binding DNA as well as RNA [2]. Here, we describe a recently observed case where two out of three state-of-theart hematology analyzers displayed a strong discrepancy with manual counting in reticulocyte enumeration due to the presence of Howell Jolly bodies.…”

“…Beckman Coulter LH 750 stains blood cells with new methylene blue, a colorimetric dye comparable to BCB, and discriminates reticulocytes from other cells by volume, conductivity, and laser light scatter; the latter signal is proportional to the residual RNA within the red cell. This staining is specific for RNA remnants in reticulocytes [2]. In contrast, Sysmex XE-5000 and CELL-DYN Sapphire instruments use fluorescent dyes, staining polynucleotides within cells.…”

“…In contrast, Sysmex XE-5000 and CELL-DYN Sapphire instruments use fluorescent dyes, staining polynucleotides within cells. CELL-DYN Sapphire combines a cyanine dye (Sybr Ii) and narrow angle light scattering to separate reticulocytes from platelets, RBC and nucleated cells [2,6]. Sysmex XE-5000 uses similar techniques, with polymethine dye, and forward light scattering differentiating platelets and RBC/reticulocytes based on cell volume.…”