Chapter 16 Reticulocyte Analysis and Reticulocyte Maturity Index

Abstract

The traditional laboratory methodology of reticulocyte analysis for the past century has been the manual microscopic counting of erythrocytes or red cells on supravitally stained blood samples. Although slide-based methods, such as new methylene blue staining, do provide clinically useful information, the lack of precision in these subjective assays is well documented. In particular, the poor precision of manual counting methods does not allow for clinically useful sequential monitoring of patients with low reticulocyte levels or for therapeutic monitoring of drug therapies for either erythropoietic inhibition or enhancement. Methods of flow cytometric reticulocyte analysis for both clinical and experimental applications have been reported over the past years utilizing a variety of fluorescent dyes. Thiazole orange (TO) has distinct advantages over other nucleic acid binding dyes for reticulocyte analysis using commercial multipurpose flow cytometry (FCM) instruments and is currently utilized by the vast majority of clinical laboratories. TO reticulocyte analysis offers the ability to use easily reticulocyte enumeration and a stable, reproducible fluorescence intensity measurement over a 60-min period. Furthermore, the fluorescence signal provides a high quantum yield or good separation of the reticulocyte (positive) from the mature red blood cell (negative) populations.