Reticulocyte counting by flow cytometry. A comparison with manual methods

Abstract

The reticulocyte count (RC) is a key diagnostic test in the evaluation, classification, and response to therapy of anemia. The RC, as determined by manual methods, has a frustrating inherent imprecision owing to its binomial counting statistics (i.e., low counts/low precision) and inaccuracy because of inter- and intraobserver variability as to what indeed is a reticulocyte. Fluorescent activated cytometric (FACS) analysis of reticulocytes by thiazole orange (TO) is a rapid, relatively simple, and precise method for counting reticulocytes. The automated method counts 10,000 cells or more vs. 1,000 cells counted by the manual method. Although inherently more precise, the FACS method may be inaccurate owing to the presence of Howell-Jolly bodies, nucleated red blood cells (RBCs), sickled cells, or giant platelets. The RC by FACS is well correlated with the manual method and the reference ranges are similar. A new parameter by FACS, the reticulocyte maturation index (RMI), provides an independent measurement of reticulocyte RNA content. Although the RMI does not correlate with RC either by FACS or manual methods, it does provide an independent parameter of erythropoietic activity and may be useful in predicting bone marrow engraftment or further subclassifying anemias. Determination by FACS of the RC offers significant advantages over manual methods in monitoring a patients erythropoietic response. However, one must be cognizant of potential pitfalls in the method.